The Use of the Oxygen

Electrode & Fluorescence

Probes in Simple
Measurements of
Photosynthesis.

By David Walker
With illustrations by Richard Walker
 Robert Hill Institute

University of Sheffield

THE USE OF THE OXYGEN ELECTRODE &

FLUORESCENCE PROBES IN SIMPLE
MEASUREMENTS OF PHOTOSYNTHESIS

By David Walker
with illustrations by Richard Walker

Robert Hill Institute,

The University of Sheffield,
SHEFFIELD, S10 2TN. UK.
Published 1987 by Oxygraphics Limited,
Produced by University of Sheffield Print Unit,
Distributed by Oxygraphics Limited,
9, Canterbury Crescent,
Sheffield, S10 3RW, UK.
and
Packard Publishing Limited,
16, Lynch Down, Funtington, ** New address **
Chichester, West Sussex PO18 9LR
And
Hansatech Instruments Limited,
Narborough Road, Pentney
King’s Lynn, Norfolk PE32 1JL UK

All rights reserved. No part of this

publication may be reproduced, stored
in a retrieval system, or transmitted,
in any form or by any means, electronic,
mechanical, photocopying, recording or
otherwise without the prior permission
of the author.

Copyright 1987 by David Walker.

First published 1987

Second Edition 1988
Second Impression 1990

British Library
Cataloguing in Publication Data
Walker, David
The use of the oxygen electrode and fluorescence
probes in simple measurements of photosynthesis.
1. Photosynthesis 2. Electrodes, Oxygen
I. Title
581.1'3342 QK882

ISBN 1 870 23200 3

Typeset at Robert Hill Institute,
University of Sheffield.
"Where we might soon have fainted

in that Enchanted Ground

but now and then a cluster

of pleasant grapes we found"

 PREFACE

Like most biologists of my generation, I was raised on the measurement of gaseous exchange by Warburg
manometry and indeed my first research of any consequence involved an elegant Warburg apparatus which,
in those days (1953), dominated Harry Beevers' laboratory in the "Peirce Conservatory and Small Animal
House" of Purdue University. Here everything was a masterpiece of time and motion but even at its best,
manometry was scarcely a technique which was likely to win friends. Calibration was frightful, taking
measurements tedious, and cleaning Warburg flasks a pain. Happily, after Purdue and Newcastle my
research took me away from manometry and it was not until 1962 that I was obliged to consider using it
again. By then (with Bill Cockburn, Carl Baldry and Chris Bucke), chloroplasts had been isolated from peas
and spinach which could fix 14CO2 at rates approaching those of the parent tissue. CO2-dependent O2
evolution (except for a report by Arnon and Whatley of very slow rates under anaerobic conditions) had
not, however, been demonstrated. At that time I was at Imperial College, travelling one day a week to the
Biochemistry Department at Cambridge to continue a long-standing co-operation with Robin Hill. It was
he who introduced me to the Clark-type O2 electrode. Soon after we were able to purchase a Gilson
electrode at Imperial College and, immediately it was in operation, to show that our CO 2-fixing chloroplasts
did indeed evolve O2 at equally fast rates.

The switch to polarography was both a delight and a challenge. At one stroke we were freed of the need to
base assays on 14C samples and introduced to the pleasures of continuous measurement. The advantages
of using isotopes were not lost because we could also add or withdraw samples, at will, during an
experiment. Suddenly, manometry seemed to be as old fashioned as Pettenkofer tubes. Oxygen electrodes,
however, proved to be a mixed blessing. Those that we could afford were less than perfect and not really
suited to our needs. It was for this reason that Tom Delieu and I tried to come up with something better. We
have been trying ever since. In spare moments I make crude pencil drawings. Tom somehow contrives to
convert these into material reality. Very often my ideas lack feasibility. Always progress has been painfully
slow. Until recently, electrode design and construction was largely a spare-time occupation. Each prototype
has had to earn its living in our day-to-day work. Nevertheless, by the late 60's we had a Clark-type
electrode which has served us very well. Since 1974 it has been produced commercially by Hansatech.
More recently Tom Delieu joined the A.F.R.C. staff in what is now "The Robert Hill Institute" in the
University of Sheffield. Within the Institute one of our aims is to develop new (and, ideally, simpler) means
of measuring photosynthesis.

The main purpose of this book is to describe some of the equipment which Tom Delieu and I have
developed (often with the help of colleagues in Sheffield and elsewhere). Much of this apparatus is now
available commercially so that anyone who has a mind to do so can put together experimental systems very
similar to the ones described here. Clearly, I am not addressing those who are already familiar with such
apparatus, or those from whom we ourselves learn on an almost daily basis. On the contrary, this was
originally intended to be a beginner's guide but what started its existence as an introductory manual, largely
restricted to the leaf-disc electrode, has grown in a way which has threatened the simplicity of the original
concept. Some of the more complex phenomena (such as oscillations) which can be studied with the help
of the leaf-disc electrode, are barely understood and difficult to explain. New ways of analysing
fluorescence signals have been devised and could not be neglected. There are classic experiments, like the
Hill Reaction, which will continue to be an important part of practical teaching into the foreseeable future
and for this and other reasons a section on aqueous-phase measurements has been added.

Inevitably, there will be errors and incorrect assumptions. In some places I have probably erred on the side
of over-simplification and, in others, complexity may have been compounded by brevity. There is no doubt,
however, that polarographic measurement of oxygen has much to offer those involved in photosynthesis,
whether in teaching or research, and that there is advantage to be derived from simultaneous measurements
of chlorophyll a fluorescence. It is hoped that what follows will constitute a useful practical introduction to
these topics.
David Walker, January 1987.

Second Edition, Second Impression

In the second impression we have endeavoured to correct errors which slipped through in the first
impression and have taken the opportunity to incorporate information concerning the "Leafdisc" software
as an appendix to the main text. We have also added a laminated cover which we trust will be as attractive
its predecessor and more hard-wearing.

David Walker, March 1990.

Acknowledgements: To Tom Delieu who by translating inadequate pencil drawings of apparatus into
splendid mechanical reality made the whole thing possible, to Rick Walker who illuminated an otherwise
dull text, to Richard Leegood, Uli Schreiber and Mirta Sivak for invaluable advice, criticism and
contributions to specific sections. To Barry Osmond for endless warm encouragement, to Christa Critchley,
Robyn Cleland, Simon Robinson and Steve Powles for their participation in Australian measurements, to
Ross Lilley for an excursion, with the apparatus, into a rain forest. To Margaret and John Humby, David
Thody and Richard Poole (of Hansatech Instruments Ltd) for continuing and fruitful co-operation. To John
McAuley for his contribution to the first computer software and to George Seaton who has taken it to its
present state of sophistication, to Anne Dawson, Jackie Rowell and Diane Wright for their work on the
manuscript and to Iris Walkland for the index. To Marney and Shirley Walker for grapes.

Much of the earlier work on electrodes was supported by grants from the Royal Society. Most recently,
work on the automated leaf-disc electrode system has been supported by the Society's Paul Instrument Fund.

My colleague and dear friend, Tom Delieu, died after a brief

illness, on Nov 12 1987 aged 58. Tom was a legendary figure to
everyone who knew him - a master craftsman who could make
almost anything out of almost anything; a man of great warmth
and humour who will be sorely missed not only by his wife and
his family but by his friends and colleagues the world over.
 CONTENTS

Part (A) OXYGEN 1

1. INTRODUCTION 1
2. THE PRINCIPLE OF OXYGEN MEASUREMENT 1
3. ASSEMBLY 2
4. CALIBRATION 3
(a) Experiment 1. Does it work? Checks for function and
the determination of values required for calibration
(b) Calculations relating to volume and calibration
(c) Introducing Carbon Dioxide
(d) Experiment 2. Does it work with a leaf?
(e) Quick Calibration
5. EXPRESSION OF RATES 8
Part (B) FLUORESCENCE 11

6. INTRODUCTION 11
7. PRINCIPLE OF MEASUREMENT 11
8. IS FLUORESCENCE INFLUENCED BY CARBON ASSIMILATION? 12
9. FLUORESCENCE INDUCTION 14
10. INDUCTION 15
11. THE FLUORESCENCE "M-PEAK" 15
12. COMPLEX FLUORESCENCE KINETICS AND OSCILLATIONS 16
(a) Gas Transients
(b) Experiment 3
(c) Oscillations
(d) Factors which Favour Oscillations

13. QUENCHING ANALYSIS 22

(a) Introduction
(b) The "DCMU Method"
(c) Light-scattering
(d) "Light-doubling"
(e) The Pulse-Saturation Method
(f ) Modulated Light
(g) The Procedure
(h) The Analysis
(i) Examples

Part (C) LIGHT SOURCES 30

14.
(a) Slide-Projectors and Heat Filters
(b) A High Intensity Light Source
(c) Light-Emitting Diodes
(d) The Björkman Lamp

Part (D) MAXIMUM EFFICIENCY OF PHOTOSYNTHESIS 33

15. INTRODUCTION 33
16. MEASUREMENT OF QUANTUM YIELD 34
17. THEORETICAL BACKGROUND 34
18. MEASUREMENT OF APPARENT QUANTUM YIELD WITH THE LEAF
DISC ELECTRODE AND THE BJÖRKMAN LAMP 35
19. DOING IT IN OTHER WAYS 36
(a) According to Björkman and Demmig
(i) Introduction
(ii) Photon Yield Measurements
(iii) Leaf Absorptance Measurements
(iv) Further Considerations
(b) Doing it with Chloroplasts and Algae
(c) Doing it in the Dark

Part (E) COMPUTERISED MEASUREMENT OF RATE AS A FUNCTION

OF PHOTON FLUX DENSITY 41

20. UNDERLYING PRINCIPLES 41

(a) Why do it at all?
(b) Roofs and Ceilings
(c) The Roof
(d) The Ceiling
(e) How Steep is the Roof?
(f) Relative and Absolute Quantum Yield
(g) Why Trouble to Automate Measurement?
(h) How Does it Work?
(i) What Are The Drawbacks?
(j) What Are The Advantages?
 21. MEASURING RATE AS A FUNCTION OF PFD 46
(a) Choice of CO2 Concentration etc.
(b) The Unsteady State
(c) The Closed Refrigerator Door Conundrum
22. LIGHT ENHANCED RESPIRATION 47
23. THE "KOK EFFECT" 48
24. IS THERE A RECOMMENDED PROCEDURE? 49
25. HOW TO GET THE MOST OUT OF YOUR DATA 50
26. RATE V PFD UNGILDED 50
27. THE INITIAL SLOPE 50
28. THE SHAPE OF THE CURVE 51
29. ANALYSIS OF STRESS 51
(a) Stress and Performance
(b) Shifting the Arbitrary Ceiling
(c) Shifting the Ceiling and the Perpendicular
30. LUX ET VERITAS - A REMINDER ABOUT LIGHT 53
(a) Measurement
(b) The Nature of Light
(c) The Energy Content of Light
(d) Can We Compare Micromoles with Foot Candles?
(e) Energy, Plants and Man - an Aside About Energy Utilisation
31. ENERGY, PLANTS AND MAN -
AN ASIDE ABOUT ENERGY UTILISATION 55
32. PHOTOINHIBITION 56
(a) What is it?
(b) What Causes Photoinhibition?

Part (F) AQUEOUS PHASE MEASUREMENTS 62

33. INTRODUCTION 62
34. PRINCIPLE 62
35.THE "FLEA" 62
36. CALIBRATION 62
(a) Setting an Arbitrary "Air-line"
(b) Establishing the N2 Line
(c) Residual Current
(d) Establishing a New "Air-line"
(e) Normal Calibration Procedure Summarised
(f) Absolute Calibration
(g) Using Constant Chlorophyll
(h) Calculation of Rate
37. MECHANICAL ISOLATION OF CHLOROPLASTS 67
(a) Conventional Wisdom
(b) Grinding
(c) Squeezing and Filtering
(d) Centrifugation
(e) Resuspension
(f) Grinding Medium
(g) Resuspending and Assay Medium
(h) Purification
(i) Washing
(ii) Percoll
38. SPINACH 72
(a) Why Spinach?
(b) The Origin of Spinach
(c) Growing Spinach
(d) Alternatives and What to Avoid
(e) Varieties
39 HYDROPONIC CULTIVATION OF SPINACH 73
(a) General Procedure
(b) Sowing
(c) Water Culture Solution
(d) Cleaning Tanks
(e) Pests and Sprays
(f) Harvesting
 40. THYLAKOIDS 75
41 .EXPERIMENTS WITH ISOLATED CHLOROPLASTS 75
(a) Experiment 1. The Hill Reaction as an Intactness Assay
(b) Some Practical Points
(c) Experiment 2. The Mehler Reaction
(d) Experiment 3. CO2 and PGA-dependent O2 evolution
(e) Experiment 4. The Requirement for Orthophosphate
(f) Experiment 5. Orthophosphate Inhibition and its Reversal
(g) Inorganic Pyrophosphate
(h) Experiment 6. Carbon Dioxide Dependence
42. PROTOPLASTS 86
(a) Digestion
(b) Pretreatment
(c) Enzymes and Incubation
(d) Isolation
(e) Purification
43. CHLOROPLASTS FROM PROTOPLASTS 88
44. THE RECONSTITUTED CHLOROPLAST SYSTEM 88
(a) The Simplest System
(b) Systems with Additional Stroma
45. EXPERIMENTS WITH THE RECONSTITUTED SYSTEM 89
(a) Experiment 1. CO2-dependentO2 evolution
(b) Experiment 2. PGA-dependent O2 evolution
(c) Experiment 3. Uncoupling
46. SOME FURTHER PRACTICAL CONSIDERATIONS 91
(a) Twin Electrodes
(b) Illumination

Part (G) DEFECTS AND PRECAUTIONS 96

47.
(a) The Sensor
(b) Use
(c) Membrane Application
(d) The Leaf-Disc Electrode
(e) Leaks
(f) Membranes and Electrolytes

APPENDIX 1 PREPARATION OF FERREDOXIN 98

APPENDIX 2 SUPPLIERS 99
 PART A

OXYGEN
1. INTRODUCTION

The measurement of oxygen evolution in a closed system is one of the easiest & cheapest means of
demonstrating, or following, the process of photosynthesis in a leaf. The account which follows is based
on a device designed by Delieu & Walker (1981) & now manufactured by Hansatech Instruments Ltd
Instruments Ltd (Appendix 2). As such, it complements "Notes for Users" supplied with the Hansatech
Instruments Ltd Instruments LD2/3/3. It includes descriptions of simple experiments which can be used
in teaching or for gaining experience with the apparatus. Little or no knowledge of photosynthesis, or
of oxygen measurement by polarography, is assumed.

2. THE PRINCIPLE OF OXYGEN MEASUREMENT

“An easy way of following
In photosynthesis, light energy is absorbed by chlorophyll & used to drive the reduction of carbon
photosynthesis”
dioxide to carbohydrate. The major end-product of photosynthesis in higher (flowering) plants is
usually sucrose. Starch is also often formed as a temporary storage product but both of these carbohydrates (sucrose &
starch) are formed from three-carbon sugar derivatives. For simplicity, all of these substances can be represented by a
purely nominal carbohydrate, CH2O, & the overall process by the equation :-
hv
CO2 + H2O ® CH2O + O2 ........Eqn.2.1
in which the light energy needed to drive this process is represented by "h" (h = Planck's constant & the
Greek letter "v" the symbol used to represent the frequency of light).

Accordingly, if a leaf is enclosed in a chamber & provided with carbon dioxide (or bicarbonate as a
source of carbon dioxide) & then illuminated, oxygen will be evolved. In the Hansatech Instruments
Ltd LD2/3 (Fig. 2.1.), a leaf-disc is used & CO2 is provided in the gas-phase or in the form of sodium
bicarbonate (which dissociates in solution):-

NaCO3 → NaOH + CO2 ........Eqn.2.2

“A temporary storage product”
The oxygen which accumulates in the gas-phase during photosynthesis is then detected, polarographically, by a "Clark-
type" electrode (Clark, 1956). The "regular" Hansatech Instruments Ltd version of the Clark-type electrode (Fig 2.1)
comprises a relatively large (2mm) platinum cathode & a silver anode immersed in, & linked by, an electrolyte. Both
electrodes are set in a plastic (epoxy resin) disc; the cathode at the centre of a dome & the silver anode in a circular groove
(the well, or electrolyte reservoir).

The electrodes are protected by a thin Teflon or polythene membrane which is permeable to oxygen & the purpose of the
dome is to stretch the membrane smoothly over the surface of the platinum cathode & to allow it to be secured in position
by an O-ring. The membrane also traps a thin layer of electrolyte (a solution, which usually contains potassium chloride)
over the surface of the electrodes. A paper "spacer" is usually placed beneath the membrane in order to provide a uniform
layer of electrolyte between anode & cathode.

When a small voltage is applied across these electrodes, so that the platinum is made negative with respect to the silver,
the current which flows is at first negligible & the platinum becomes polarized (i.e. it adopts the externally applied
potential). As this potential is increased to 600-700 mV, oxygen is reduced at the platinum surface, initially to
hydrogen peroxide H2O2 (Fig.2.2) so that the polarity tends to discharge as electrons are donated to oxygen
(which acts as an electron acceptor). The current which then flows is stoichiometrically related
to the oxygen consumed at the cathode.

Figure 2.2. Diagrammatic representation of oxygen electrode reactions.

When a potentiating voltage is applied across the two electrodes, the platinum
(Pt) becomes negative (i.e becomes the cathode), & the silver (Ag)
becomes positive (the anode). Oxygen diffuses through the membrane
& is reduced at the cathode surface so that a current flows through the circuit
(which is completed by a thin layer of KCl solution or other electrolyte).
The silver is oxidised & silver chloride deposited on the anode. The
current which is generated bears a direct, stoichiometric, relationship to the
oxygen reduced & is usually recorded, as a voltage, on a pen-recorder.
 Figure 2.1. Schematic diagram of a gas-phase oxygen
electrode & fluorescence probe.
The leaf-disc, or leaf pieces, are supported on a stainless
steel mesh in a chamber which is located in the middle section
of the apparatus. The O2 sensor (Clark-type electrode) lies
beneath the leaf chamber with its Pt cathode exposed to the
atmosphere within it. The leaf tissue is pressed lightly against
the temperature-controlled roof of the chamber by a foam
disc which also separates it from carbonate/bicarbonate
buffer carried on capillary matting. The leaf is illuminated
through this window which also allows fluorescence to reach
a probe (inserted at an angle of 40 degrees) where it is
monitored by a photodiode. Actinic light is delivered to the
top of the apparatus, by an array of light-emitting diodes, as
shown, or from an appropriate light source such as the
Hansatech Instruments Ltd LS1 or LS2. The photodiode is
protected from the actinic light by optical filter or filters. The
clips which draw the top section on to the middle section (so
that the roof of the leaf chamber is sealed against an O-ring)
& the tubes which carry temperature-controlled water to the
top & bottom sections are not shown. The taps (with luers)
are for calibration & adjustment of the gas phase (after
Delieu & Walker, 1983).

As indicated in Fig. 2.2, the electrolyte in the vicinity of the cathode rapidly becomes alkaline during oxygen
measurements (because of the generation of hydroxyl ions in the cathode reaction) & carbon dioxide can permeate the
membrane & dissolve in this solution causing "drift" in the electrical output. For this reason, electrolytes containing
carbonate ions & buffers (Section 47d) are frequently used for gas-phase measurements in atmospheres containing high
concentrations of CO2. These salts facilitate equilibration between the electrolyte & the gas-phase & enhance electrical
stability. Electrolytes containing 95% ethylene glycol & 5% KCl resist desiccation & may be preferred.

The electrical current generated by the reduction of oxygen at the cathode is usually applied, as a voltage, to a recorder in
the 1-10 mV range or converted into a digital output if the Hansatech Instruments Ltd "Digital Read-out Control Box" is
employed. (The latter also has an output to a recorder which is sufficiently large enough to be monitored in the 10 mV
range). If a computer is to be used (Appendix 3) an output in the voltage range will be required.

3. ASSEMBLY

Take a clean electrode (cleaning is best accomplished by polishing with

fine-grade aluminium oxide paste) & place a drop of electrolyte on the dome
which bears the platinum cathode (A). Place a "spacer"
Applicator approximately 2 cm square on to the drop of electrolyte (B). (The
Shaft "spacer" is used to provide a uniform layer of electrolyte between the
anode & the cathode. Cigarette paper, which is thin & uniform is
often used for this purpose - Caution, avoid the gummed part!). Place
C a similarly sized piece of membrane on top of the spacer & secure the
A Applicator membrane & spacer in position, over the dome, with the O-ring
Cone provided for this purpose (C). The O-ring is best applied with an
Electrolyte applicator (Fig. 3.1). In this case, the O-ring is first placed on the
Cathode O-ring end of the applicator & the stem of the applicator is turned. This
advances the stretched O-ring to the tip of the applicator. If the
Anode Membrane/ applicator is now held by its centre in a vertical position on top of
Spacer the dome & pressed downwards, the O-ring will be transferred to
the dome (D). When the membrane is in position it should be
B O-ring checked for smoothness. If it is greatly wrinkled it should be rejected &
Membrane
Membrane D the procedure repeated.

Spacer Outer At this stage, you may wish to connect the electrode to its potentiating
Paper O-ring
circuit & the recorder. If you breathe directly on to the electrode you
should see an excursion in the recorded signal & you will know that the
electrode is functioning.
 nut

Figure 3.1. The membrane applicator.

This is used as follows. The nut is loosened so that the cone emerges from the cylinder, pushed by the
spring. The O-ring is slipped over the cone & the nut tightened so that the cone retracts into the
spring cylinder, pushing the O-ring downwards as it does so, until it comes to rest on the lip of the cone (as
cylinder shown) in a groove made for this purpose. The applicator is then placed in the position shown & the
cylinder pressed downwards. As the descending O-ring touches the membrane it tends to stretch it
cone
slightly &, as the cylinder continues to move downwards, it pushes the O-ring off the lip of the cone
membrane & over the dome so that the membrane is stretched smoothly over the surface of the dome. The
O-ring
spacer membrane is held in this position as the O-ring contracts on to the neck.
Neck
Electrode disc
Disconnect the electrode & place it, dome upwards, on the bottom section of the chamber (Fig.
KCl solution
2.1). Place the central section of the assembly over the electrode & thread it on to the base.
in well
Avoid over-tightening because this can cause damage to the membrane. Finally, place the top
section of the chamber into position & secure it, in place, by tightening the clips. Reconnect the electrode to its
potentiating circuit. The chamber is normally used with temperature-controlled water circulating through the top &
bottom sections & this should be examined to ensure that there are no air bubbles trapped in these compartments (tilt each
section, starting with the base, so that the water exit assumes a vertical position & repeat until any bubbles are dislodged).
You should ensure, by using filtration if necessary, that the circulating water is clean. You may wish to include a "water
wheel" or similar device in your plumbing (on the exit side) so that you can see, at a glance, that water is flowing freely
at a suitable rate.

4. CALIBRATION
The electrochemical reactions generate a minute 'residual current' even in the absence of oxygen & if accurate calibration
is required the discrepancy between zero oxygen & the electrical zero should be identified by passing nitrogen over the
electrode. Alternatively a small drop of dithionite solution may be placed on the cathode. This consumes O 2 according to
the equation

Na2S2O4 + H2O → NaHSO4 + NaHSO3 .....Eqn.4.1

&, since the reaction goes virtually to completion, this procedure is equivalent
to flushing with oxygen-free gas. Unfortunately, dithionite is not pleasant to use
(CAUTION, DITHIONITE IS CORROSIVE) & it can also cause damage to, or
poisoning of, the membrane. For these reasons it is best avoided. If there is no
alternative, it should be removed from the membrane as quickly as possible by using
an aspirator with a soft tip (to prevent mechanical damage).

In this way, the true zero is established &, if preferred, this can be "backed-off" to
coincide with electrical zero. Once the dithionite solution is removed (or N 2
flushing is stopped), & the cathode is exposed to air, the signal generated by 21%
oxygen (the oxygen content of air) can be determined. In a closed system the
amount of O2 is governed by the partial pressure of O2 & the volume of the
chamber. Thus l ml (1000 l) of air will contain 210 l of oxygen. Similarly, if l ml of
the space, in a 5 ml chamber, is occupied by a leaf-disc the increase in signal
which follows the introduction of a given volume of air into the chamber will be
correspondingly greater. In fact, the difference between the two signals can be
used to determine the volume of a leaf-disc with some accuracy (4b & Fig. 4.2). In
“Flushing with O2 free gas” general, however, although allowances can be made for differences in atmospheric pressure
etc., the instrument was designed for simplicity rather than absolute measurement of oxygen. Although it has proved to
be much more versatile than was originally imagined it was originally intended for the measurement of the oxygen
evolved when a relatively large piece of leaf was illuminated in a relatively small chamber. For most purposes, therefore,
the excursions following the introduction (or removal) of small volumes of air into the chamber (using a gas-tight syringe)
can be taken as the basis of calibration & all that remains to be done is some simple arithmetic (see Fig. 4b). Experiment
1 incorporates several aspects of the calibration procedure but this can be shortened (4e) or extended according to need
& the degree of accuracy sought.
 4(a) Experiment 1. Does it work

Using a 1 ml gas-tight syringe, introduce successive 200 l aliquots of air into the leaf-disc
chamber through one of the two gas-vents. (The other should be kept closed during this
procedure). As each volume of air is pushed into the chamber the reading on the pen-recorder
should rise quickly to a new level & stay there (Fig.4.1). You will see that the initial rise
is fast but that it decreases as it approaches its final level. This is because the rate of
diffusion of oxygen to the detector will become limiting as the difference in concentration
between the atmosphere in the chamber & that at the electrode surface approaches
equilibrium. If equilibrium is not approached within about one minute (i.e. if the response is
sluggish) the electrode may require cleaning. If the signal rises rapidly but then falls equally quickly,
the chamber is leaking & you should check to see that you have remembered to close the other gas-tap,
that the electrode disc is pressed securely against its O-ring, that the O-ring which seals the top of the
chamber to the bottom is in place & that the clips which hold the two parts of the chamber together are
properly closed. If the response seems too slow for the experiments that you have in mind, it can be
speeded by using a thinner membrane, by cutting a hole in the
"spacer" immediately above the cathode or by dispensing with
the spacer altogether. If no spacer is used,
“A large leaf and a small chamber” departure from linearity may sometimes result (see
below).

Figure 4.1. Excursions produced by introducing & removing aliquots of air.

A 1 ml, gas-tight, syringe, with its plunger fully withdrawn (to the 1 ml position)
was attached to an open tap on the leaf chamber & the other tap closed. The
figure illustrates the responses which were observed as the plunger was then
pressed inwards in 200 ml stages & finally withdrawn again in a similar fashion.
Note that each successive addition produced a larger signal (for explanation see
text).

(N.B.) Care should be taken to avoid over-tightening the threaded base of the
chamber which pushes the electrode disc against its sealing O-ring. Once an
effective seal is established, excessive tightening can constrict the layer of electrolyte between the anode & the cathode,
leading to loss of linearity of response. It should also be noted that if this procedure is followed with a leaf already in
position in the chamber, the reading will decline with time because of dark respiration. Even with an empty & properly
sealed chamber there will be a small decline with time as oxygen in the chamber is consumed in the cathode reaction. In
normal use this will be negligible but the actual size of all signals will, of course, vary with the degree of pre-amplification
used & the channel on the pen-recorder which you have selected. If you suspect a slight leak,
subject the chamber to negative pressure by attaching an empty syringe to the chamber &
withdrawing aliquots of air. Clearly the excursions of the pen-recorder will now be negative
& a leak will be indicated by an increase in signal rather than the converse.

You may note, & possibly be alarmed by, the fact that there is an increase in the signal
caused by each successive aliquot of added air. On reflection, however, you will
correctly conclude that this is to be expected because if you attach a 1 ml syringe to the
chamber you effectively increase its volume by 1 ml & every time you push the plunger
further into the barrel of the syringe this effective volume becomes smaller. Accordingly,
200 l added to, say, 4 ml will cause a larger excursion than if the same volume were added
to 5. In normal use, the electrode response is linear because oxygen is being consumed or
produced within a fixed volume rather than one which changes in relation to the position
of the plunger in the syringe.

You may use the data that you have derived from Experiment 1 to plot relationships similar
to those illustrated in Fig. 4.2. This, or individual values, can also be used to calculate the
“Subject to negative pressure” effective volume of the chamber & therefore to relate the electrical output on the chart to the
oxygen evolved by a leaf (4b).

4(b) Calculations relating to volume & calibration

At s.t.p. (standard temperature & pressure) the amount of oxygen in 1 ml of air (containing 21% by volume) is 210 l &,
since 1 mole of gas occupies 22.414 l, this is equivalent to 210/22.414 or 9.37moles. At any other temperature (T), the
amount can be derived by multiplying by 273/(273 + T) so that, for example, at 20C the corresponding value would be
8.73mole per ml. Because the oxygen electrode measures concentration (or, more strictly, activity) it is also necessary,
 for some purposes, to know the effective volume of the chamber itself. This, of course, is variable because the leaf-disc
& any other material enclosed within the fixed volume of the chamber will occupy a significant space. However, the
increase in signal (i.e. the increase in the electrical output) from the electrode circuit can be used to calculate both the
effective volume of the chamber & the linearity of the electrode response. Suppose, for example, that the initial electrode
output (R1) in Experiment 1. was 1.5 mV & that, after the plunger was fully depressed, this reading had increased to 1.8
mV (R2). The effective volume (v) of the chamber can be calculated from the relationship:-

V = R = 1.5 = 5ml ...Eqn.4.2

R2 - R1 1.8 - 1.5

but this only applies when 1 ml of air is introduced.

The general form of this equation:-

V = R1 - [R2 x (1 - volume injected)] ...Eqn.4.3

R2 - R 1

can also be used provided that sufficiently large excursions result from the introduction of volumes of air smaller than 1
ml. This has been done in Fig. 4.2 (using the data from Fig. 4.1). Similar data, derived from the excursions which were
observed when this procedure was repeated after the insertion of plastic discs of 1 ml volume into the chamber, has also
been plotted in Fig. 4.2. This provides a measure of the degree of accuracy which can
be expected & also illustrates the way in which the chamber can be used to measure
the volume of any small body enclosed within it. Normally, of course this "small
body" will be a leaf-disc. Moreover, it will be respiring & therefore consuming
oxygen. The rate of oxygen consumption by a leaf-disc of 10 sq cm area (the
maximum that can be accommodated) will not, however, be so large that the
calibration procedure will be invalidated but, clearly, an appropriate correction for
O2 uptake should be applied. For most purposes, the respiratory loss during the
excursion(s) can be ignored but, if greater accuracy is required, the decrease in signal
due to respiratory uptake of oxygen (during the calibration procedure) can be
measured & a correction applied.

Figure 4.2. Data from Figure 4.1 re-plotted as a function of volume of air added.
Additional readings were obtained following the insertion of a body of 1 ml volume
into the chamber (b) & after the insertion of a second body of equal volume (c). This
indicates the degree of accuracy which can be expected & illustrates the use of the
chamber in measuring volume (i.e. the experimental points permit the
horizontals a, b & c to be drawn at 1 ml intervals, as would be expected).

Now, let us suppose that we had established that the volume of the
leaf chamber was 5.17 ml, that the volume of the 10 sq cm leaf-disc
that we wished to use was 500 l, that the sum total of all other inclusions (supporting grids, capillary
matting etc.) was 1.17 ml & that the insertion of 1 ml of air at 20C caused an excursion of 0.775 mV (as
it might have done in the experiment illustrated in Fig. 4.1, in which the initial reading was 3.1 mV). We
could then have calculated, from Equation 4.1, that the effective volume of the chamber was 4.0 ml. If we
had inserted a leaf-disc with a volume of 500 l & repeated this procedure we would have observed an
excursion of 0.885 mV, from which, with the help of Equation 4.3 (above) we would have correctly
calculated the volume of the leaf. We can also see, however, that we do not need to know the volume of the
leaf itself for purposes of calibration since the size of the excursion must inevitably adjust to the effective volume
of the chamber. Accordingly we may simply equate 8.73 moles of O 2 in l ml of air at 20C with a signal of 0.885
mV & say that l.0 mV = 9.86 moles = approx 10 moles.

The establishment of the relevant volumes does, however, allow us to calculate a more precise initial setting because
each ml of otherwise unoccupied space in the leaf-chamber contains 8.73 moles of O 2. We can, therefore, use
Equation 4.3 to calculate a more desirable setting of the initial reading ("the air line"). For example, if the final effective
volume of the chamber was 3.5 ml & we make the denominator (R 2 - R1) = 0.873, then:-

If sufficiently large 3.5 = R1 ....Eqn.4.4

excursions result 0.873

and R1 = 3.05

Accordingly, if the air-line were re-set, electrically, from 3.1 mV to 3.05 mV the relationship would be 1.0 mV = 10 moles
of O2, precisely.
 4(c) Introducing Carbon Dioxide There is a certain
Having assembled & calibrated the apparatus you are now presumably anxious to see that comfort in being
it will record oxygen evolution from an illuminated leaf. At this stage you must remember safely confused
that photosynthesis requires CO2 & that air contains only about 350 parts per million. This
means that the carbon dioxide content of the closed chamber will not sustain rapid
photosynthesis for long. For example 4 ml of air will contain approximately 4 x 0.35 l = l4 l of
CO2. A healthy spinach leaf would, under favourable conditions, have little difficulty in
maintaining a rate of CO2 fixation in excess of 240 moles/mg chl/hr. This is equivalent to
about 2 moles/min for a disc of l0 sq cm area (containing, 500g chlorophyll). At this rate,
the CO2 consumption would be 2 x 22.4 = 44.8 l per minute - enough to deplete the
chamber of CO2 in less that 20 seconds. Of course, this rate would not be sustained as the
CO2 concentration fell but it points to the need for a CO 2 source, if linear rates are to be
maintained. There are also problems associated with too much CO 2. Although these remain
ill-defined it is clear that high concentrations of carbon dioxide may adversely affect many
cellular processes & it is unlikely that photosynthesis will prove to be exceptional in this respect. Moreover,
high concentrations of CO2 are known to induce stomatal closure. Nevertheless external concentrations in the region of
5%, or less, will apparently force CO2 through closed stomata because of the greatly steepened diffusion gradient. With
photorespiration then largely, if not entirely, suppressed, the rate of consumption of CO 2 within the chloroplast at these
high external CO2 concentrations will, at least in high light, be so rapid that it will prevent CO 2 concentrations reaching
inhibitory levels &, if the leaf is not left for long periods in the dark, will allow reasonable estimates of maximal rates of
photosynthesis to be derived.

Gas mixtures can be generated from carbonate/bicarbonate buffers (which have been allowed, if so
required, to equilibrate with N2) or by the time-honoured method of adding a non-volatile
A “time-honoured acid, such as sulphuric acid (H2SO4), to a known quantity of carbonate in a closed vessel
method” of known volume. The CO2 concentration can then be checked by IRGA (infra-red gas
analysis) or by titration & gas mixtures transferred to the chamber using a gas-tight
syringe. If gas mixtures are available in cylinders (tanks) the chamber can be
flushed, from this source, prior to use. Similarly, gases can be mixed using one of
a number of commercial devices now available for this purpose (e.g. Wösthoff
pumps, Signal blenders, mass-flow controllers).

As will be seen below (Experiment 2), carbon dioxide can also be introduced into the
leaf chamber by the simple expedient of breathing into it. Alternatively it can be
generated within the chamber by adding a small volume (say 0.2 ml) of approximately
molar sodium or potassium bicarbonate solution to capillary matting or coarse filter paper
on the floor of the chamber. The leaf-disc is then prevented from contacting this solution by
making a "sandwich" using the stainless-steel grids & foam (sponge) discs provided (see also 4e).

Air-saturated water is about 10 M with respect to CO2 & although the solubility of carbon dioxide is not affected by pH
as such, an increasing amount of bicarbonate is built up as the pH is increased. Thus, at 0C & pH zero, virtually all of the
CO2 will be present as dissolved gas. From pH 4.0 to 9.0, HCO 3- increases from about 0.2% to about 96.5%, & CO 3--
starts to appear at about pH 7.0 & increases to 97% at pH 12. These relationships are the basis of the carbonate/bicarbonate
buffers which have been used for many years in Warburg manometry. They are less useful for the present purpose
because, although at a given concentration, temperature & pH, each buffer is in equilibrium with a known partial pressure
of carbon dioxide in the gas-phase, they can only be used with reasonable precision if the gas-phase is small or if the
mixtures are pre-equilibrated with the appropriate gas mixture, thereby largely defeating the object of the exercise. Where
less accuracy is required, pure bicarbonate solutions can be used instead (see below).

One mole of carbon dioxide at standard temperature & pressure (s.t.p.)occupies 22.414 litres. At 25C, it expands to fill
the larger space of 24.45 litres (V2 = V1.298/273). Accordingly, 1 millimole of CO 2 (24.45 ml at 25C) in 1 litre of gas
will equal 2.445% & 1% CO2 in such a gas mixture would be 1/ 2.445 = 0.409 mM, or approximately 400 M. At 25C, in
the concentration range of 1 to 100 mM, bicarbonate solutions equilibrate to a more or less constant pH of 8.37 & a
similarly constant ratio of [HCO3-]/[CO2] = 90. Thus if n/400 = 90, n = 3,600 M or 36 mM. This means that if 36.4 mM
bicarbonate is left to equilibrate with an equal volume of air at 25C the final concentration will approximate to 1%.
Equilibration can be speeded by the addition of a little phosphate ion (at the same pH) or carbonic anhydrase. If you are
in a hurry & all you need is a shot of CO2, throw an excess of sodium bicarbonate into distilled water, shake in a half-filled
closed flask & the atmosphere within the flask will soon contain about 5% CO 2.
4(d). Experiment 2. Does it work with a leaf?

An experiment of this nature is illustrated in Fig. 4.3.

Cut a l0 sq cm area disc from a broad leaf, such as spinach, with the cutter provided. To avoid dessication
place it, on damp (not wet) capillary matting supported by the stainless-steel grid provided (use the grid
with the unperforated centre which prevents light shining directly on to the cathode). This is best supported,
in turn, by a sponge disc & a second stainless-steel grid. Close the chamber with the clips but leave both taps
open. Attach a tube to the tap & breathe heavily into the chamber. (In so doing you are taking advantage of the fact
that you are possibly a portable, & almost certainly a peripatetic, CO 2-generator & O2-consumer). Close both taps.
You should have succeeded in replacing air (2l% O2, 0.035% CO2) with expired air (say l4% O2, 5% CO2).

Take
a leaf

Figure 4.3. Simultaneous recording of oxygen

evolution & chlorophyll a fluorescence.
Chart showing CO2-dependent O2 evolution by a
spinach leaf & the changes in chlorophyll a
fluorescence associated with the onset of illumination,
the depletion of CO2 & the addition of CO2 following
depletion. Note that the fluorescence signal should be
shifted to the right by approx 0.6 of the distance
between two vertical divisions in order to correct the
displacement caused by the pens on the chart-recorder.

Offset the signal on the pen-recorder using "back-off" until the trace occupies the lower l0% of the chart. Record the dark
respiration for 2 or 3 minutes (until this reaches a steady O 2 consumption as the temperature within the chamber adjusts
to that of the circulated water).

Switch on the light & allow the leaf to photosynthesise at high irradiance. If the leaf-disc has been taken from the dark or
from low light you will observe an initial lag or induction period (Section 10). Thereafter, the rate should approach
linearity & if illumination is interrupted by about 30 sec to l min darkness you should observe enhanced oxygen uptake
& little or no lag following re-illumination. After some time, the rate of O 2 evolution will decline (abruptly
in C3 species, less abruptly in C4 species). If you then open both taps & displace the atmosphere in the
chamber with more expired air you will observe an abrupt fall in the recorded reading as the oxygen
content is returned to about l4% & (possibly after some temperature re-equilibration) a return to
a higher rate of photosynthesis.

If measured simultaneously, as in Fig. 4.3 the fluorescence signal (see Part B) will fall from an
initial high, often in a fairly complex fashion, to a quasi steady-state value. In this experiment,
as the CO2 within the chamber ran out, there was a change in the fluorescence signal
reminiscent of the change seen in air to CO2-free air gas transients (Section 12a). Following the
re-introduction of CO2, dampening oscillations in fluorescence were observed (see also Fig. 11.1 &
Section 12b).
“Breathe heavily”
4(e). Quick Calibration

Much of the above is intended to illustrate principles & to familiarise the user with the leaf-disc electrode (the LD2/3). If
you are already one of the cognoscenti you may wish to proceed, with less ado, to the quickest possible calibration
procedure. If so continue as follows:-
 (i) Establish an electrically convenient "air-line" by closing the chamber & adjusting the recorder
reading to some arbitrary value in the top half of the chart. Now establish the "N2 line" (Fig. 4.4) by
flushing with nitrogen, or, if none is available, by using dithionite (Equation 4.1). If you must use
dithionite, take care & remove it as quickly & as gently as possible.

(ii) Place a leaf-disc on the stainless steel grid & sponge "sandwich" which presses the leaf against the
temperature controlled roof of the chamber. Close the chamber, thus establishing a second arbitrary
"air-line". This will fall, with time, because of respiratory uptake of oxygen but the required
values can be obtained, as indicated in Fig. 4.4, by extrapolation if the rest of the procedure is not
delayed.

(iii) Introduce air from a gas-tight syringe. This gives the value R 2-R1 needed for the calculation (from
Equations 4.2 & 4.3) of the effective volume of the chamber should you need it for other purposes, &
the increment itself (Fig. 4.3) gives the equivalence between electrical output & moles of oxygen
generated. This, if you are impatient, is all that you need.

If you wish to add a little elegance & the undoubted advantages that derive from running
consecutive experiments on the same absolute scale, calculate the effective volume, multiply it by
the number of micromoles of oxygen in this volume at the temperature being used,
& adjust R1 so that the scale on the recorder gives a convenient direct "read-out".

“Leaf-disc sandwich”
N.B. Grid with entire
centre should be beneath
the first layer of
capillary matting
Figure 4.4. "Quick"
5. EXPRESSION OF RATES calibration - for
explanation see text.
Photosynthesis is often expressed as a rate of CO2 uptake per unit leaf area. This
is convenient, although it can be very misleading if it is used as a basis for
comparison. Thus two plants which display identical rates of photosynthesis per
unit area, may grow (accumulate dry matter) at markedly different rates. This is
readily understood if one is capable of diverting a fraction more of its
photosynthetic product into new photosynthetic machinery than the other. On a
"compound interest" basis this would soon account for the observed differences
in weight. Even so, this sort of observation has led to statements such as "there is no
clear correlation between photosynthesis & growth". Since green plants grow (i.e. accumulate dry weight) almost entirely
as a consequence of photosynthesis, such statements are self-evident nonsense. In the above example there is correlation,
by definition, between photosynthesis per plant & growth. Provided, however, that we do not equate
photosynthesis per unit area with "photosynthesis" per se we can proceed to express it on this basis. Our
arithmetic is then made easier by the thoughtful designer who provided us with a tool which cuts a disc of
precisely 10 sq cm from broad leaved species. (An equivalent approximation can be
made for some narrow-leaved species by attaching individual leaves, in rows to 2 strips
of adhesive tape & then cutting one disc, comprising segments from several leaves. There
are also more sophisticated commercially available devices for measuring the area of
irregularly shaped leaves, or pieces of leaves).

We are, however, immediately confounded by lack of agreement about units. The plant
biochemist may prefer mmoles per mg chl per hr (moles. mg -1chl. hr-1) or (App. 3)
moles. m-2 s-1 whereas the crop physiologist may go for mg CO 2 per sq decimeter per
hour (mg.dm-2.hr-1). Suppressing a sigh, we can (if actual values are not available)
arrive at some approximate equivalence by ignoring the differences between pale &
dark green leaves & settling for an "average" chlorophyll content of 3 mg chlorophyll
per sq decimeter (i.e. 300 g/leaf-disc).
“There is correlation between
photosynthesis per plant & growth”
We must also remember that one mole of CO2 weighs 44 gm (i.e. that 1 mmole weighs 44 g). Accordingly, the rate of
photosynthesis by a 10 cm2 leaf-disc which contained 0.3 mg of chlorophyll & evolved O 2 at 150 moles/hr could be
expressed as:-

150 = 500 µmoles/ mg -1chl/ hr-1 .....Eqn.5.1

0.3
or as

150 x 44 x 10 = 66 mg/ dm-2/hr .....Eqn.5.2

1000
from which we can derive a conversion factor such that

1 µmole/ mg -1 chl/hr-1 º approx 0.13 mg/dm-2/hr-1

Or

1 mg/dm2 / hr-1 º approx 7.6 µmoles / mg -1/hr

It should be noted that we have used 44 (the molecular mass of CO 2) rather than 32 (the corresponding value for O 2 ) in
the above conversion because the older values in the literature were based on CO 2 rather than O2 . Since there is a 1 to 1
** (one to one) relationship between CO2 fixed & O2 evolved, at least in photosynthesis as described by the classic overall
equation (Equation 2.1), we can take this liberty. We multiplied by 10 because 1dm = 1/10 metre = 10 cm & 1 dm 2 = 100
sq cm, whereas our actual measurement was based on a disc of 10 sq cm area. (We divided by 1000 in order to convert g
to mg).

According to Zelitch, we can expect C4 crop species to give rates in the region of 35-65 mg. dm -2. hr-1 (265-475 moles
mg-1chl. hr-1), C3 crop species to give 12-45 (91-335) & woody plants to be in the region of 10(75). These values were
at high illuminance, in air, whereas our athletic spinach leaf which gave the convenient rate of 66(500) must have been
provided with sufficiently high CO2 to suppress photorespiration & to overcome stomatal & boundary layer resistances.
It should be noted that if the leaves contain more chlorophyll (say 0.05 mg/sq cm rather than 0.03 mg/sq cm) the values
& the conversion factors based on chlorophyll would be decreased correspondingly (in this case by 3/5).

To convert moles per disc per hr into moles. m -2.s-1 we are on somewhat safer ground with our oxygen measurements
because we are recording the O2 evolved, in moles, from 10 sq cm & there are 10,000 sq cm in 1m 2. Accordingly, our (10
cm2) spinach leaf-disc which evolved O2 at 150 moles/hr, would give us a value of:-

150 x 10,000 = 41.66 µmoles/m -2/s-1 .....Eqn.5.3

3,600 x 10
&, in the general case, we would simply record the change (in moles) per min (a second is too short for accuracy) &
multiply by 103/60 = 16.67 e.g. 150 x 16.67/60 = 41.66 moles m -2 .s-1
 REFERENCES
Photosynthesis in General
Edwards, G.E. & Walker, D.A. (1983) C3, C4, Mechanisms, & Cellular & Environmental Regulation in Photosynthesis.
Blackwell Scientific Publications Ltd, Oxford. 1-542.
Hall, D.O. & Rao, K.K. (1987) Photosynthesis (4th ed.). Edward Arnold, London. pp 1-122.
Rabinowitch, E.I. (1945 & 1951) Photosynthesis & Related Processes. Volumes 1 & 2. Inter Science New York, 1-2088.
Walker, D.A. (1979) Energy, Plants & Man. Packard Publishing Ltd, Chichester. 1-31.
Zelitch, I. (1971) Photosynthesis, Photorespiration & Plant Productivity. Academic Press, New York, 1-347

Polarography in General

Aiba, S. & Huang, S.Y. (1969) Oxygen permeability & diffusivity in polymer membranes immersed in liquids. Chem.
Eng. Sci. 24, 1149-1159.
Benedek, A.A. & Heideger, W.J. (1970) Polarographic oxygen analyzer response: The effect of instrument lag in the
non-steady state reaction test. Water Res. 4, 627-640.
Berkenbosch, A. & Riedstra, J.W. (1963) Temperature effects in amperometric oxygen determinations with the Clark
electrode. Acta Physiol. Pharmacol. Neerl. 12, 131-143,144-156.
Clark, L.C. Jr (1956) Monitor & control of blood & tissue oxygen tension. Trans. Am. Soc. artif. internal Organs, 2, 41.
Fatt, I. (1976) Polarographic oxygen sensors. CRC Press, Cleveland. 1-278
Gnaiger, E. & Forstner, H., eds. (1983) Polarographic Oxygen Sensors. Springer, Berlin, Heidelberg. 1-370.
Hahn, C.E.W., Davis, A.H. & Albery, W.J. (1975) Electromechanical improvement of the performance of O2 electrodes.
Respir. Physiol. 25, 109-133.
Hitchman, M.L. (1978) Measurement of dissolved oxygen. John Wiley & Sons, New York, pp. 255.
Jensen, O.J., Jacobsen, T. & Thomsen, K. (1978) Membrane covered oxygen electrodes. I. Electrode dimensions &
electrode sensitivity. J. Electroanal. Chem. 87, 203-211.
Keidel, F.A. (1960) Coulometric analyser for trace quantities of oxygen. Ind. Eng. Chem 52, 490-493.
Koltoff, I.M. & Lingane, J.J. (1952) Polarography, Vols. I & II. Interscience, New York & London.
Lucerno, D.N. (1969) Design of membrane-covered polarographic gas detectors. Anal. Chem. 41, 613.
Mergenhagen, D. Schweiger, H.G. (1973) Recording the oxygen production of a single Acetabularia Cell for a prolonged
period. Exp. Cell Res. 81, 360-364.
Oeseburg, B., Kwant, G., Schut, J.K. & Zijlstra, W.G. (1979) Measuring oxygen tension in biological systems by means
of Clark-type polarographic electrodes. Proc. K. Ned. Akad. Wet Ser. C. 82, 83-90.
Weast, R.C. (ed) (1969) Handbook of Chemistry & Physics 50th ed. Chemical Rubber Co., Cleveland,
p 151.

Polarography in Particular

Delieu, T. & Walker, D.A. (1972) An improved cathode for the measurement of photosynthetic oxygen evolution by
isolated chloroplasts. New Phytol 71, 201-225.
Delieu, T. & Walker, D.A. (1981) Polarographic measurement of photosynthetic O2 evolution by leaf discs. New Phytol
89, 165-175.
Delieu, T. & Walker, D.A. (1983) Simultaneous measurement of oxygen evolution & chlorophyll
fluorescence from leaf pieces. Plant Physiol. 73, 534-541.

Never admit to working with

spinach
 PART B

FLUORESCENCE

6. INTRODUCTION

Chlorophyll looks green in white light because it absorbs light in the blue (round about 420 nm) & in the red parts (round
about 660 nm) of the visible spectrum & transmits & reflects in the green. Light can be regarded as a stream of particles,
or parcels, of energy. Each particle (quantum or photon) can bring about a single photochemical event provided that it
carries sufficient energy to drive that specific event. Each quantum of red light which is absorbed by a chlorophyll
molecule raises an electron from a ground state to an excited state & all of its energy is transferred in this process. This
excitation is, essentially, an oxidation. Electron transport is initiated, as the electron is lifted into a higher energy orbital
& a positively charged "hole" is left behind. Absorption of blue light causes even greater excitation (because of the higher
energy content of the blue quantum) but the elevated electron then falls back into the "red orbital" too quickly to permit
useful chemical work. Thus, whatever the quality of the light absorbed, the electron reaches the same energy level more
or less immediately after excitation & all subsequent events derive from this common starting point ("excited state one").

Figure. 6.1. The excitation of chlorophyll by light.

The parallel lines represent energy sub-states or electronic orbitals.
Thus the energy delivered by the absorption of a blue photon (left)
is sufficient to raise an electron to "excited state two" from where it
rapidly returns by a process of radiationless de-excitation,
"cascading" through sub-states, to excited state "one". A photon of
red light (centre) only has enough energy to raise an electron to
excited state "one" but this excited state is sufficiently stable to
permit useful chemical work & is, in effect, the starting point of all
other events in photosynthesis. "Excited state one" can also
dissipate energy by re-emitting light as (deep red) fluorescence.

As indicated in Fig. 6.1 some of the excitation energy of chlorophyll is lost by radiationless de-excitation & some is used
to drive the "chemical" reactions of photosynthesis (ATP synthesis, NADP reduction etc.). Some energy is also dissipated
as fluorescence. This, it should be emphasised, is not reflected or transmitted light. It is light which is created in the leaf,
just as electron transport (electric current) through the filament of an incandescent lamp leads to the emission of light.
Fluorescence (which takes about 10-9 sec to discharge) derives from the lower more persistent "excited state one" rather
than "excited state two" which decays in about 10-13 sec. For this reason chlorophyll
fluorescence is red, regardless of the quality of the exciting light & it is a deeper
(longer wavelength) red than the red absorption peak because of the "Stokes shift"
(the rapid cascade of heat dissipation which occurs within "excited state one") so that
electrons fall to the ground state from the lowest levels of excitation & must
therefore give rise to photons of lower energy content (i.e. longer wavelength light).

7. PRINCIPLE OF MEASUREMENT
The fraction of excitation energy which is dissipated as fluorescence in vivo is very
small (3-5%). In solution this fraction is much larger (up to 30%) & if a solution of
chlorophylls in ethanol, or acetone, is illuminated in a rectangular vessel it will look
green from the front & deep red from the side. (Chlorophyll reflects in the red & the
blue & transmits in the green but, viewed from the side, the retina is no longer
flooded with green photons & the deep red fluorescence emanating from the excited
chlorophyll is readily perceived). Photomultipliers are often used in light
measurements but, in most circumstances, fluorescence from a leaf can be
adequately detected with a photodiode. As in photosynthesis, each photon which falls on the detector initiates one
photochemical event in which an electron is raised to a higher energy level & a positively charged "hole" is created. In
photosynthesis, the "hole" accepts an electron from water (Figs. 8.1 & 8.2) & the electron is passed (via photosystem II
& I) to NADP. In the photodiode, the corresponding electron transport (electric current) is amplified & can be applied, as
a voltage, to a pen-recorder. The main problem is to prevent the detector "seeing" light which is not fluorescence &,
inevitably, a relatively large fraction of the "actinic" light (the light used to drive photosynthesis) will be reflected from
the leaf surface into the detector. Accordingly, the detector is protected by optical filters which, ideally, exclude all of the
reflected actinic light & transmit all of the fluorescence. In practice a compromise is needed because in terms of
 "energy-effectiveness" (Section 6) red light is best for photosynthesis & the peak of chlorophyll a
absorption in the red (about 680 nm) is not far removed from the peak of chlorophyll a fluorescence
(about 685 nm). For this reason blue exciting (actinic) light is sometimes used because it is readily
separated from the fluorescence peak. For many purposes, however, fluorescence signals at longer
wavelengths (i.e. wavelengths about 740 nm, where there is a smaller fluorescence maximum) are
sufficiently strong (& sufficiently similar in quality to those at shorter wavelengths) to allow the
employment of filters which will exclude most of the actinic light & transmit most of the
fluorescence. In many experiments, fluorescence signals resulting from excitation with blue light
& detected at about 695nm are similar to fluorescence resulting from excitation with red light &
detected at about 740 nm.

8. IS FLUORESCENCE INFLUENCED BY CARBON ASSIMILATION.

The short answer to this question is "yes". This has been known, or believed, for many years. The
Fluorescence spectra of pale original concept was one of "gainful employment". In short, if excitation energy could be
& dark leaves - after French "gainfully employed" by a plant to drive photosynthesis, fluorescence would represent some
& Young, 1952 fraction of the wasted energy. If this were so, a broadly reciprocal relationship might be expected
- e.g., if photosynthesis were constrained by some external factor (such as CO 2 limitation) more
energy would be "wasted" & more fluorescence might be seen.

Figure 8.1. The arrangement of carriers in the photosynthetic electron transport

pathway (the Z-scheme).
This is an energy diagram showing the "up-hill" transport (in two stages) from H 2O
to NADP. (A) is linear or non-cyclic electron transport (B) is pseudo-cyclic
(the Mehler reaction - see Section 41c) & (C) is cyclic electron transport.
The site of action of 3 inhibitors (Antimycin A, DBMIB & DCMU,) (see
Trebst, 1980) is indicated & also the action of PMS (or pyocyanine), which
creates its own cycle. The spatial organisation of electron carriers is
outlined in Fig. 8.2.

In recent years this concept has been modified many times. Duysens &
Sweers (1963) formalised & extended the original notion by applying
the name "Q" (for "quencher") to an electron-acceptor in photosystem
II (Figs. 8.1 & 8.2).

The first stable electron acceptor in PSII ("the primary quinone

acceptor") is now called "QA". When QA is oxidised, it can accept
electrons from P680 (the reaction centre in PSII). When fully reduced it can no longer accept electrons which would be
passed, in turn (via QB, the plastoquinone pool & PSI) to NADP & beyond. The probability of a fraction of chlorophyll
a excitation energy being dissipated as fluorescence is therefore increased. We can, therefore, talk about "qQ quenching"
(quenching directly related to the oxidation status of Q A) & its relaxation. We can see that if QA is kept oxidised by
transferring electrons to NADP & finally to CO2 it will continue to "quench fluorescence" whereas, if its reoxidation is
limited, fluorescence will increase as qQ quenching relaxes. For these reasons there are obvious links between the
oxidation status of QA, fluorescence, & carbon assimilation.

Figure 8.2. Organisation of electron carriers in the

membrane.
The same carriers as in 8.1, suggesting the manner in
which they might be organised within the thylakoid
membrane. This figure also indicates the inward movement
of protons from the stroma. The proton gradient,
discharging through the ATPase (Fig. 8.3) drives ATP
formation from ADP & Pi. Note that 2 photons (quanta)
are required in each photosystem to transfer 2 electrons from H 2O to NADP.
Accordingly, a total of 8 are required to transfer the 4 electrons associated with the
release of one molecule of O2 (see "quantum requirement" in Sections D & E).
The decrease in fluorescence yield brought about by electron transfer to Q A (i.e. qQ quenching) is now often referred to
as "photochemical quenching" to distinguish it from other quenching mechanisms (see e.g., Krause & Weis, 1984,
Oxborough & Horton, 1986) of a "non-photochemical" nature. These include quenching by oxidised plastoquinone (q P),
quenching resulting from "state-transitions" (qT) & quenching associated with photoinhibition (q I). The major non-
photochemical quenching mechanism, however, is high energy state quenching, or "q E".

Following an original observation by Murata & Sugahara (1969), work by Krause (for a review see Krause & Weis, 1984
& Barber et al, 1974) has shown that qE is closely related to the proton gradient which develops across the thylakoid
membrane as a result of illumination. No one yet knows precisely how this works but it seems that cation exchange
processes (involving protons & magnesium ions) bring about changes in the ultra-structure of thylakoid membranes
which switch excitation energy dissipation from fluorescence into thermal channels so that energy is lost as heat rather
than light. Light-scattering (see Heber, 1969 & Section 13c) is also related to energisation & may be used to monitor
changes in the proton gradient & the contribution of q E to fluorescence in vivo (see e.g., Sivak et al, 1985). [It should be
noted that, in experiments with isolated chloroplasts, qE may be inhibited by antimycin A although pH is unaffected (see
e.g., Oxborough & Horton, 1986)].

When chloroplasts are illuminated, electron transport brings about movement of protons (hydrogen ions, H +) into the
thylakoid compartment so that a gradient of protons is established between the thylakoid compartment (which becomes
more acid) & the stroma (which becomes more alkaline). According to Mitchell's Chemiosmotic Theory this proton
gradient & its associated membrane potential (the "proton motive force" or "p.m.f.") drives ATP synthesis as the proton
gradient discharges through the ATPase. The proton gradient also exerts a "back-pressure" on electron transport (it is
more difficult to push a proton into a space already full of protons) & this is why electron transport runs faster if it is
"uncoupled" (e.g., by detergents which make artificial holes in the thylakoid membrane, or by compounds which act as
artificial proton sinks - see e.g., Good et al, 1966; McCarty, 1980). In this respect, electron transport is like a railway
engine which could go faster if it were uncoupled from its trucks or carriages. Electron transport will also run faster if it
is "coupled", however. This is not as contradictory as it may at first seem because "coupled", in this sense, means provided
with an ample supply of ADP & inorganic phosphate. This allows the proton gradient to discharge through the ATPase,
bringing about ATP synthesis. Thus both "coupling" & "uncoupling" allow the proton gradient to discharge. It is only in
the absence of ADP, inorganic phosphate, (Pi) or uncouplers that the proton gradient exerts its maximal back-pressure.

This is why qE quenching is affected by CO2 assimilation. If CO2 is being rapidly fixed, ATP consumption will be high
& there will be plenty of ADP to discharge the proton gradient. Conversely, if CO 2 assimilation is limited, ATP
consumption will also be limited, ADP will be in short supply, & the proton gradient will increase. As it increases, there
will be a shift from energy dissipation as light, to energy dissipation as heat. Thus, when the proton gradient is high, qE
will be high & fluorescence will be quenched.

The effect, on fluorescence, of changes in ATP & NADPH consumption can be extremely complex but the more simple
interactions are probably best understood in regard to air CO 2-free air transients (Section 12a).

Figure 8.3. Electron transport linked to proton transfer into the thylakoid
compartment.
The proton gradient across the thylakoid membrane can be dissipated by
transport through the coupling-factor, which facilitates ATP synthesis, or
artificially by uncouplers. CF1 is the ATPase component of the coupling
factor & CFo (Fo in this figure) forms a channel in the membrane
through which protons are transported.
9.FLUORESCENCE INDUCTION
If a leaf is kept for a few minutes in darkness (or low light) & then brightly illuminated, fluorescence rises (in fractions
of a second) to an initial peak & then declines (in seconds or minutes) in a more or less simple fashion to a steady-state
value. Fluorescence induction kinetics (the Kautsky effect 1931) are, however, often much more complicated than this.
The induction curve may be regarded as variable fluorescence superimposed on a "constant" background fluorescence
(Fo). The curve has often been labelled OIDPSMT (Fig. 9.1). On illumination, fluorescence rises immediately (in
picoseconds but limited by the opening time of the shutter) to O (the level of the "constant fluorescence") &, thereafter,
there is an initial rise to a level "I" in the variable fluorescence (Fv), followed by a dip (D), a peak (P) & a fall, via a
quasi-steady-state (S) to a terminal value (T). Sometimes, particularly when 3 or 4 minutes darkness is followed by
re-illumination at very low light intensities, a secondary maximum (M) is seen between S & T (Section 11). In other
circumstances, particularly in high light & high CO2, oscillations may follow. The initial changes (O to P) are very rapid
(about 1 sec) compared with the onset of carbon assimilation &, at least in leaves, there are often additional complexities
within the first few seconds which make this nomenclature inadequate.

Figure 9.1. Fluorescence induction

kinetics.
Schematic representation of fast
(left) & slow (right) fluorescence
kinetics during a dark to light
transition showing the use of the
OIDPSMT terminology (after
Lavorel & Etienne, 1977). Note that
"O" is designated "Fo" in this figure.

The initial fluorescence (Fo) is believed to come from excited chlorophyll a molecules in the antennae of PSII before the
excitons have migrated to the reaction centres. Its level is determined by illumination after a period of darkness in order
to ensure that QA is fully oxidised (otherwise the rise to O cannot be easily separated from the onset of variable
fluorescence & the subsequent rise to I). QA is believed to exist in a state of quasi-equilibrium with plastoquinone to
which it passes electrons via QB & the pause at I, & the dip at D, are thought to reflect imbalances between the rates of
reduction & reoxidation of QA. Thus, from O to I, reduction is thought to exceed oxidation. Thereafter, increased
reoxidation will cause the fluorescence rise to falter at I or even to reverse momentarily, causing the dip (D). In strong
light, P may approach Fm (the maximal fluorescence) & the ratio of Fv/Fo may be 4 to 6. The changes O to P are complete
within one or two seconds & are best followed on a transient recorder. P to S, on the other hand, takes about 10 seconds
& the "M peak" (Section 11), if it develops, may be a minute or so after the initial rise. For this reason some of what occurs
could be called induction fluorescence (the fluorescence associated with induction - see Section 10) rather than
fluorescence induction (the fluorescence induced by a period of darkness). If the period of darkness is extended (i.e., if
the leaf is "dark adapted") the initial rise approaches a maximum value & the subsequent decline is similar to that observed
when illumination in low light is interrupted by short periods of darkness (a minute or so) as in Figure 9.2. In these
circumstances, however, the initial rise is small & fluorescence returns very quickly to its steady-state or terminal value.
The initial rise can be mostly attributed to the rapid reduction of Q A which occurs upon re-illumination.

Figure 9.2. Simple Fluorescence Kinetics Following Re-

illumination After Darkness
The sequence of events is as follows. Upon illumination, Q A is
rapidly reduced, qQ quenching is therefore suppressed &
fluorescence rises to an "immediate" peak. Thereafter, qQ
quenching slowly increases as QA re-oxidises to its steady-state
value. At the same time a proton-gradient builds up across the
thylakoid membrane with a consequent increase in q E
quenching. During this period, both quenching mechanisms
combine to push fluorescence down, from the initial peak (P) to
its steady-state or terminal value (T).
The subsequent fall results from QA becoming more oxidised (as electrons move on to NADP & beyond) & to the
generation of a proton-gradient (the initiation of q E quenching).

Between these two extremes (i.e., very long darkness & relatively short darkness) all manner of complexities may appear
in the fluorescence kinetics. One is the appearance of the "M-peak" (see above & Section 11). It now seems likely that
this particular rise in fluorescence is associated with the termination of the induction period in carbon assimilation
(Section 10).

10. INDUCTION
As noted above (& in Section 4d), there is often a lag between first illumination & the attainment of
maximal photosynthesis, i.e., carbon assimilation does not reach its full rate immediately on illumination
but only after an interval. This lag is often evident after a few minutes darkness. After long periods
(hours) of darkness, the induction period may last for many minutes.

The reasons for induction are complex & still a matter for argument (see e.g. Edwards &
Walker, 1983). More than 50 years ago, when Osterhout & Haas first studied induction
in Ulva they suggested two possibilities. One was, light-activation (&, by implication,
dark deactivation) of catalysts &, the second, depletion (during darkness) of metabolite
pools. These explanations are still valid & there is evidence for both. Light-
activation of enzymes is often so rapid, however, that it is unlikely to make much
contribution to long periods of induction & these are probably best explained in
terms of the known depletion of metabolites such as ribulose bisphosphate (RuBP)
(which often falls to very low concentrations in darkened leaves). Some enzymes,
are activated by their substrates, as well as by light, so that there can be complex interactions
Haas studied induction
between the two principal underlying factors which lead to induction. In leaves, stomatal
opening may also contribute to lags in gaseous exchange following re-illumination after
darkness.

11. THE FLUORESCENCE "M-PEAK"

In Fig. 9.1, the slower changes which follow re-illumination after a period of darkness, include a decline from a peak (P)
to a terminal (T) value via a secondary maximum (M).

Perhaps inevitably, this "M-peak" has meant different things to different observers. One approach (Horton, 1985) is to
describe any secondary maximum, or maxima (because there may be several) as "M-peaks". This has the virtue of
simplicity but it must be immediately emphasised that it can create a pitfall for the unwary if it is assumed that all "M
peaks" are causally related in precisely the same way. A comparison of A & B in Fig. 13.2 shows that fluorescence signals
which are superficially very similar may mask underlying components which are quite different & the same may be true
of "M-peaks" if they are not additionally defined. For example, re-illumination after a dark interval may give rise to a
"CO2-gulp" & an associated "O2 burst" prior to the termination of induction (Section 10). In some circumstances, the
termination of induction may be followed by oscillations. All three events (the gulp & burst, the termination of induction
& oscillations) may give rise to secondary maxima or "M-peaks" but the underlying causes, the relative contributions of
qQ & qE & their timing may differ substantially.

Fig. 11.1 illustrated an "M-peak" of the sort which may sometimes be observed when a leaf is abruptly re-illuminated with
low intensity light following a period of darkness long enough to permit appreciable depletion of metabolites & some
dark-deactivation of enzymes but not so long that the leaf is fully "dark-adapted". This particular "M-peak" was attributed
(Walker, 1981) to the termination of induction (i.e., the onset of more rapid carbon assimilation after an initial lag).
According to this view, QA will be rapidly reduced as soon as the leaf is illuminated because, although it must accept
electrons from excited chlorophyll, it is denied the possibility of reoxidation by the remainder of the electron transport
chain. This is because carbon assimilation has been "switched-off" in the dark & requires time (spent on light-activation
of enzymes & building-up metabolites) to get going again. Some CO 2 assimilation will soon occur, however, & electrons
will also be passed to O2 as an alternative acceptor so that QA oxidation will become detectable within seconds & there
will be a decline in fluorescence from the original peak value as q Q quenching increases in parallel with O2 evolution.
This decline will be reinforced by qE quenching as cyclic & linear electron transport lead to the establishment of a
proton-gradient across the thylakoid membrane (Figs. 8.2 & 8.3) which is not yet being discharged at its maximal rate.
Such a discharge of protons through the ATPase will increase as the flux of metabolites through the Benson-Calvin cycle
(Fig. 12.6) increases & ATP consumption by its PGA & Ru5P kinases (Eqns. 12.2, 12.4) makes more ADP available for
photophosphorylation. In relatively low light, this new drain on the transthylakoid proton gradient will bring about a
significant relaxation of qE quenching (or a significant slowing of the rate of increase of q E) & cause fluorescence to rise
 from S (Fig. 9.1) towards M. Almost immediately further changes will combine to reverse this trend.
Any decrease in the proton gradient will tend to accelerate electron transport as will the increasing
availability of NADP reoxidised in PGA reduction (Fig. 12.3). Associated increases in q Q will once
again push fluorescence downwards from M & qQ will once again be reinforced by an increasing qE
caused by an increasing proton gradient. In higher light (Sivak et al, 1985), the drain on the proton
gradient associated with the termination of induction will have a smaller impact on the fluorescence
kinetics & the associated M-peak will be smaller.

Figure 11.1. "M-Peak" fluorescence kinetics following re-illumination, in very low light, after darkness
The initial events are similar to those in Fig. 9.1 but the subsequent differences relate to the fact that induction has been
established (i.e. that the period of darkness has been long enough to allow metabolite pools to be partially depleted &
enzymes to be inactivated). When the leaf is re-illuminated, therefore, it is not fully prepared for photosynthetic carbon
assimilation at maximum rates, & there is an initial lag. As this lag terminates, some of the newly developed proton-
gradient is discharged in ATP synthesis, qE relaxes & fluorescence starts to rise again. As this happens, electron
transport to CO2 causes a simultaneous increase in the proton gradient so that it pushes fluorescence down from its
"M-Peak". At higher light intensities (Sivak et al, 1985) the M-Peak is lost or modified - after Walker (1981).

12. COMPLEX FLUORESCENCE KINETICS & OSCILLATIONS

12(a) Gas Transients

When a leaf is allowed to photosynthesise for some minutes in air, photosynthesis will often rise to a constant rate &
chlorophyll a fluorescence will fall to a steady-state value. If the gas-phase is then changed in a way which will affect
photosynthesis the fluorescence signal will also respond. The air to CO 2-free air transient provides a good example & one
which can be explained with some degree of confidence. It will be seen (Fig. 12.1) that, in the steady-state, if the
gas-phase surrounding a leaf is changed, abruptly, from air to CO 2-free air, the fluorescence signal will rise quickly &
then fall again, almost as quickly, to a value which (at least in moderate light intensities) is often lower than it was at the
outset.

Figure 12.1. "Gas-transients" in spinach.

This is a facsimile of a chart showing
fluorescence transients in spinach induced
by changes in the gas-phase. Air to CO2-
free air transients alternate with similar
transients in which the O2 was dropped
from 21% to 2% at the same moment as the
CO2 was removed.
 The theoretical interpretation of some of the gas-transients is straightforward but possibly over-simplified. For air to
CO2-free air, it goes as follows. When the leaf is deprived of CO 2, carboxylation of RuBP ceases & so does the
consumption of ATP & the oxidation of NADPH in the following sequence.

RuBP + H2O + CO2 → 2 x 3PGA ......Eqn.12.1

3PGA + ATP → 1, 3DPGA + ADP ......Eqn.12.2

1, 3DPGA + NADPH → G3P + NADP ......Eqn.12.3

Initially, therefore, fluorescence will rise because QA (the primary electron acceptor
in PSII) will become reduced as soon as it can no longer pass electrons to NADP (or
when electron transfer to NADP is diminished). This relaxation of q Q quenching
may be accompanied by an initial relaxation of qE quenching (cf. Fig. 13.2B) A “gas-transient”
because ATP utilisation will continue in the absence of CO 2 so long as there is 3PGA
& Ru5P available for phosphorylation (equations 12.2 above & 12.4 below).

RuBP + ATP → ADP + RuBP ......Eqn.12.4

Any relaxation of qE quenching will reinforce the rise in fluorescence brought about by
the relaxation of qQ. Very quickly, however, QA will arrive at a more reduced state &
fluorescence would level out at this higher value if q E had not already started to
increase & push it downwards again. This increase in qE quenching results from lack
of ADP regeneration (caused by ATP consumption in reactions 12.2 & 12.4) & a
switch to increased cyclic or pseudocyclic electron transport once linear electron
transport to CO2 has stopped. If O2 is decreased to 2% at the same time as the CO 2
is removed (Fig. 12.1), the initial rise in fluorescence is greater because of
decreased transfer of electrons to O2 (which can substitute, albeit less
effectively, for CO2). Accordingly, qQ will relax more quickly & to a
greater extent than it does when CO2 alone is removed. In these
circumstances, qE will also increase less rapidly because the rate of electron transfer to O 2 will
photorespiration
be less rapid in low O2 but the interactions are extremely complex. For example, consumption of
ATP in photorespiration will also be diminished in decreased O 2.

Figure 12.2. Effect of Pi & a phosphate-

sequestering sugar on gas-transients
displayed by a young barley leaf.
From left to right the gas transients were
1000 ppm CO2 (5 min) air (0.5 min) air
(3 min). This sequence was then repeated
(4 times in this illustration). The change
from air to 1000 ppm CO2 (in air)
induced oscillations but, when Pi (50mM)
was fed, as indicated at the end of the first
cycle, the oscillations were rapidly
suppressed. They were equally rapidly
re-initiated & increased when 2-
deoxyglucose (DG) was fed, as indicated,
in the last cycle (from Walker & Sivak,
1985)

The switch back to air from CO2-free air is followed by the reverse of these changes (Fig. 12.1) but the entire transient is
asymmetric. This is because the resumption of linear electron transport (when CO 2 is restored) occurs in the presence of
the high proton-gradient which has developed in CO 2-free air & although the light-generated "pressure" which drives
electrons from chlorophyll to CO2 is unchanged, the "back-pressure" exerted by the proton gradient is larger (Section 8)
& electron transport is consequently slower.
 Gas transients have been used recently (see e.g. Walker & Sivak, 1985) to help
to define the relationship between oscillations & cytosolic Pi status. A chamber
similar to the Hansatech Instruments Ltd LD2/3 was used but the design
allowed young barley or spinach leaves to be fed, through the cut-end of the
leaves, with water or solutions containing Pi or Pi-sequestering sugars (such as
mannose or 2-deoxyglucose - see Chen-She et al, (** not in refs) 1975; Herold
et al, 1976). These enter the cytosol & are phosphorylated, (in a reaction
catalysed by hexokinase) but not further metabolised. This leads to a decrease
in cytosolic Pi. If gas-transients from air to high CO2 (say 1000 ppm) are used,
oscillations (see also Section 12c) will follow (Fig. 12.2) if the light intensity is
sufficiently high or the temperature sufficiently low. Thresholds in CO 2
concentration, light intensity, O2 concentration & temperature can be defined
but these can all be raised by Pi-feeding & lowered by Pi-sequestering sugars
(Walker & Osmond, 1986; Laisk & Walker, 1986, Sivak & Walker, 1986). This
suggests that Pi supply is the common feature in most oscillatory behaviour
because Pi-recycling in sucrose synthesis will tend to be limiting in high light,
low temperature, high CO2 & low O2 (& vice versa). These relationships are
explored more fully in section 12c.

12(b) Experiment 3.

Attach a small soda-lime column to one vent of one of the 3-way taps of the
Hansatech Instruments Ltd LD2/3 & a small pump to the other tap so that when
the pump is switched on it is possible to draw air directly through the chamber
or through the chamber via the soda-lime column. Place a leaf in the chamber
& allow it to photosynthesise, with air being drawn through the chamber by the
pump, until fluorescence has fallen to a steady-state value. Change from air to
CO2-free air by changing the position of the 3-way tap. After 30 seconds change
back to air. Transients similar to some of those illustrated in Fig. 12.1 should
be observed.

12(c) Oscillations

In certain circumstances, photosynthesis can be induced to oscillate (see for example, Figs 12.2, 12.3). These oscillations
can sometimes be seen in the O2 evolution trace but become very much more evident if an electronic "differentiator" is
employed. This converts "change in oxygen" into "rate of change". The fluorescence signal which you record is already
a "rate" measurement &, if photosynthesis does oscillate, associated oscillations in fluorescence become immediately
apparent.

Oscillations (Fig. 12.3) are most readily observed when steady-state photosynthesis is perturbed under conditions which
normally favour rapid rates (e.g. high light, high CO 2). Much remains to be elucidated but there seems to be little doubt
that dampening oscillations are a manifestation of regulatory mechanisms struggling to regain control (& over-reacting)
after being thrown off-balance (see e.g. Sivak & Walker, 1984; 1985). One control mechanism which is thought to be
implicated involves the [ATP]/[ADP] ratio. As we have seen (Section 8), a low [ATP]/[ADP] ratio favours electron
transport because there is then ample ADP to discharge the proton gradient by ATP synthesis. However, a key reaction
in the Calvin cycle is inhibited by low [ATP]/[ADP] ratios (Robinson & Walker, 1979) This is
the reaction in which 3-phosphoglycerate (3PGA) is phosphorylated to give 1,3-
diphosphoglycerate (1,3DPGA).

3PGA + ATP → 1, 3DPGA + ADP .....Eqn.12.5

This is a freely reversible reaction but the equilibrium position favours PGA formation
from DPGA &, in photosynthesis, it has to be pushed to the right by high [PGA] & a
favourably high [ATP]/[ADP] ratio. Carbon assimilation, therefore, must inevitably
strike a compromise between the low [ATP]/[ADP] ratio which favours electron
control by transport (Section 8) & the high [ATP]/[ADP] ratio which favours PGA
[ATP] / [ADP] ratio phosphorylation.
 Figure 12.3. Dampening oscillations in
fluorescence.
This is essentially the same experiment as that
illustrated in Fig. 4.3, except that a comfrey
(Symphytum officinale L.) leaf was used instead of
spinach. Dampening oscillations follow the re-
introduction of CO2 once the CO2 has been
exhausted by photosynthesis in a closed system.

Let us now suppose that steady-state photosynthesis has been attained & we suddenly raise the CO 2 concentration. This
will cause a transient increase in PGA as the high CO2 discharges the ribulose 1,5-bisphosphate pool.

RuBP + CO2 + H2O → 2 x 3PGA .....Eqn.12.6

This, in turn, will be associated with a transient increase in electron transport & O 2 evolution as more PGA is reduced.
Fluorescence will start to fall as the proton gradient & q E increase but this fall will be rapidly overtaken by an increase in
fluorescence as increased ATP consumption (in the phosphorylation of PGA & Ru5P - Eqns. 12.2. & 12.4) makes more
ADP available to discharge the proton gradient. The consequent decrease in the [ATP]/[ADP] ratio will become
unfavourable to PGA phosphorylation & reduction (& therefore to O 2 evolution & CO2 fixation) & the rise in
fluorescence will be reinforced by relaxation of qQ as NADP & QA become more reduced. Each time the [ATP]/[ADP]
ratio becomes unfavourably low, the interruption of PGA phosphorylation will tend to diminish the "waves" of
metabolites transversing the cycle. As the metabolite "waves" subside (aided by discharge to the cytosol), via the
translocator, in exchange for incoming Pi - see Figs. 12.4 & 12.7) the oscillations in fluorescence will also dampen (Fig.
12.3). Because of the intimate relationship between the cytosol & the chloroplast, Pi-supply has a major impact on
oscillatory behaviour. High cytosolic Pi will facilitate ATP formation & the discharge of metabolites to the cytosol,
thereby suppressing oscillatory behaviour. Conversely, any conditions or events which limit Pi-supply (& hence retain
metabolite transients within the stroma) will tend to favour
oscillations (see e.g. Section 12a & Fig. 12.2).

Figure 12.4.
"High cytosolic Pi will facilitate ATP formation & the discharge of
metabolites to the cytosol".

This explanation is consistent with the fact that the oscillations in fluorescence bear a broadly reciprocal, but phase-shifted
relationship, to the oscillation in CO2 & O2 (Fig. 12.5). The phase-shift (the fact that fluorescence changes anticipate O2
& CO2 changes) can be accounted for by the fact that O2 evolution would be tightly linked to the oxidation status of Q A
but that changes in O2 evolution would be preceded by change in q E. Other measurements, such as light-scattering
(Section 13c), suggest that the proton gradient does, in fact, oscillate in advance of O 2 & CO2 (Sivak et al, 1985).
 Figure 12.5. Oscillatory behaviour in spinach showing
relationship between Carbon dioxide fixation,
fluorescence & energisation.
Oscillations were induced by re-illumination in O.55% CO 2, 2%
O2. The fall in fluorescence (Fv) anticipates the rise in CO2
fixation & vice versa. Similarly, the increase in energisation (E),
which is largely responsible for the fall in fluorescence,
anticipates Fv. "E" was defined by q-analysis (Section
13a) & is the difference between maximal & variable
fluorescence (E=qE.(Fv)m = (Fv)m-(Fv)s -
see Eqn 13.7). (After Sivak & Walker 1986).

It should be noted that while oscillatory behaviour may be initiated by sudden illumination following a period of darkness
(a circumstance in which induction will be more or less re-established depending on the duration of the dark interval) it
can also be initiated equally readily by almost any perturbation of steady-state photosynthesis. During induction,
interpretation is complicated by "switching-off" of light-activated enzymes & depletion of
metabolites in the dark whereas, in continuous light, such factors might be expected to make a
much smaller contribution.

12(d) Factors which favour oscillations

(i) Dark to light

(ii) Low light to high light
(iii) Low CO2 to high CO2 (e.g.air 1000 ppm CO2)
(iv) Low temperature high temperature
(v) High O2 to low O2 (e.g.21% 2% O2)
(vi) Low O2 to high O2

Oscillations occur more readily in shade-grown leaves or following illumination of the

under-surface of leaves (Walker & Osmond, 1986). Young cereal leaves, which do not
form much starch, oscillate readily, whereas Soya, which is a massive starch former, is
only induced to oscillate with great difficulty, if at all. Oscillations occur most readily at
low temperatures, in high light & in high CO 2 but the thresholds for light, CO2, etc., below
which oscillations will not normally occur may be lowered or raised by experimental
lowering or raising of cytosolic orthophosphate concentration (Section 12a & Fig. 12.2). In
all of these regards, oscillatory behaviour may be regarded as an over-reaction, in
conditions of limiting phosphate supply, of the regulatory mechanisms which control
photosynthetic carbon assimilation. An analogy which has been used is that of a car being
driven up a road. If the driver is obliged to swerve violently to avoid an accident he will tend
to over-react & his vehicle, following such a perturbation, will proceed down the road in a
series of dampening over-reactions until a straight course is recovered.

Leaves photosynthesise in order to produce the wherewithal for maintaining respiration &
growth. The chloroplast (Section 41e) is an orthophosphate-requiring, triose phosphate-
exporting organelle (Walker & Herold, 1977; Walker & Robinson, 1978) & a balance has to
be struck between the export of triose phosphate to the cytosol (where it is mostly converted
to sucrose) & internal utilisation in the regeneration of ribulose bisphosphate, RuBP, the CO 2
acceptor (Fig. 12.6). Five out of six molecules of triose phosphate formed in the Benson-
Calvin cycle must be consumed in the regeneration of RuBP simply to maintain the status quo
as five C3 compounds (triose phosphates) are re-arranged to give three C5 compounds (ultimately RuBP).

Even more triose phosphate must be consumed internally to allow for the autocatalytic increase in RuBP, at the expense
of triose phosphate, without which photosynthesis could not increase & growth could not occur (Fig. 12.6). Only the
"spare" triose phosphate can be exported or consumed within the chloroplast during starch synthesis (if it occurs).
Export, via the phosphate translocator (Fig. 12.7) is by strict, one-to-one stoichiometric exchange with orthophosphate in
the cytosol. Cytosolic Pi seems to be controlled within fine limits & the utilisation of triose phosphate in sucrose synthesis

4 triose phosphate → 1 sucrose + 4 Pi .....Eqn.12.7

in the cytosol is the principal mechanism of Pi recycling. This allows the synthesis of triose phosphate in the chloroplast
to continue (see Section 41e)

3CO2 + 1 Pi → 1 triose phosphate .....Eqn.12.8

Figure 12.6. The Benson-Calvin cycle as an autocatalytic sequence.

Five out of six C3 molecules (G3P) are re-arranged to give three C5 molecules (RuBP). The sixth is available for feedback
or export to the cytosol via the Pi-translocator as DHAP.

Sucrose synthesis in the cytosol (like starch synthesis in the stroma) is regulated by a complex mechanism involving a
modulator (fructose 2,6-bisphosphate) & is favoured by high concentrations of triose phosphate & low concentrations of
Pi. (see e.g. Cseke et al, 1984; Stitt, 1986)

Orthophosphate enters photosynthetic carbon assimilation through photosynthetic phosphorylation in which

ADP + Pi → ATP ......Eqn.12.9

& it is ADP & Pi which bring about the discharge of the proton-gradient through the ATPase (Section 8). In this way,
Pi-supply embraces both electron transport & photosynthetic carbon assimilation. Much remains to be determined about
the manner in which all of these events interact & are regulated but it is evident that a number of compromises must be
established. The maintenance of these checks & balances is evidently central to regulation & it is these which clearly
cannot cope, without over-reaction, when steady-state photosynthesis is violently perturbed. Hence the oscillations in O2
& CO2 & metabolites, which are most easily followed by measurement of chlorophyll fluorescence & which,
undoubtedly, involve rapid interaction between stroma & cytosol, with Pi acting as a chemical messenger.
 Figure 12.7. The Phosphate Translocator
Located in the inner envelope of the
chloroplast, the Pi-translocator permits a
strict, one-to-one exchange between internal
DHAP & external Pi.

13. QUENCHING ANALYSIS

13(a) Introduction

No contemporary consideration of chlorophyll a fluorescence in its relation to photosynthetic carbon assimilation would
be complete without a mention of q-analysis (the analysis of fluorescence quenching - see e.g. Schreiber et al, 1986). Once
again the underlying concept is that fluorescence emission (in this context) is largely determined by two quenching
mechanisms. As already noted (Section 8) the first of these, Q-quenching (qQ) derives from photochemical energy
conversion at PSII reaction centres such that if the primary acceptor, Q A, is oxidised it accepts electrons (thereby
quenching fluorescence) whereas, if it is reduced, some of the excitation energy is dissipated as fluorescence. Energy is
also dissipated by radiationless de-excitation (e.g. as heat) & an increase in the rate of dissipation via this channel is
believed to be associated with the build-up of the proton gradient across the thylakoid membrane (Figs. 6.1, 8.2 & 8.3),
hence qE or "energy-quenching".

13(b) The "DCMU Method"

The action of the two quenching components q Q & qE can be resolved in experiments
with isolated chloroplasts (Krause et al, 1982) by the addition of the inhibitor DCMU
which blocks the reoxidation of QA by PSI & the rest of the electron transport chain (Fig.
8.2).

Accordingly, if 10 to 15 M DCMU is added to a chloroplast suspension which has been illuminated for some minutes, an
abrupt rise in fluorescence is observed (Fig. 13.1). This relates to the difference in the degree of reduction of QA brought
about by photosynthesis (the balance between reduction by electrons from water & reoxidation by PSI) & the complete
reduction which follows once re-oxidation of Q A is blocked. The fast rise in fluorescence is followed by a slower increase
which is attributed to the decrease in the proton gradient.

Figure 13.1. Fluorescence changes consequent upon the addition of DCMU

to isolated chloroplasts.
The significance of the bi-phasic rise in fluorescence following the addition of
DCMU to intact isolated chloroplasts in the presence of KHCO 3 (2 x 10-5M) is
explained in the text (from Krause et al, 1982).
13(c) Light scattering

Ulrich Heber made a major contribution (Heber, 1969) to photosynthetic measurements when he devised a method of
following changes in pH based on "light-scattering". A weak beam of 535 nm green light is passed through a leaf &
increases in the signal, detected by a photomultiplier, have been related to changes in the thylakoid membrane during
energisation. Fig. 13.2 shows changes in fluorescence & light-scattering during gas-transients (Section 12a). If it is
accepted that light-scattering is an indicator of the proton gradient (some caution is necessary because light-scattering is
also affected by water status) the respective contributions of qQ & qE are immediately apparent. Upon withdrawal of CO2,
QA re-oxidation is constrained & fluorescence rises steeply. This rise is soon over-taken by a fall because, in the absence
of CO2, ATP consumption (& associated ADP generation) no longer discharges the proton gradient whereas cyclic &
pseudocyclic electron transport continue. It is this increase (Fig. 13.2) in the proton gradient (& the associated imposition
of qQ quenching) which pushes fluorescence back down following its initial rise. When CO 2 is restored everything is
reversed.

At higher CO2 concentrations, the behaviour is more complex (e.g. ATP may continue to be consumed at appreciable rates
for some seconds after CO2 is withdrawn so that the proton gradient may fall before it rises). Comparison of Figs. 13.2A
& 13.2B show this situation & emphasises how important "q-analysis" is (see below) because fluorescence kinetics which
are superficially very similar can disguise quite different underlying changes in quenching.

Figure 13.2. Changes in Fluorescence During Gas-transient.

(A) air CO2-free air air transient (cf. Section 12a) showing associated change in qE
quenching as indicated by light-scattering. (B) & (C) as for (A) but involving higher CO 2 concentrations & therefore
more complex kinetics, including the onset of oscillations in (C).

13(d) "Light-doubling"

An alternative approach ("light-doubling") was introduced by Bradbury & Baker (1981) &, in experiments with
protoplasts, Quick & Horton (1984), used modulated light (Section 13f) in demonstrating an acceptable correlation
between qQ determination by the "light-doubling method" & by the "DCMU method". Their procedures (like those of
Dietz et.al.,1985) involved periodic illumination by saturating light, superimposed for brief intervals on the normal
"actinic" (or "driving" light) during photosynthesis measurements. An apparatus, designed by Neil Baker, which
combines a modulated measuring beam & lock-in amplifier detection is available from Hansatech Instruments Ltd
Limited. The rationale behind this approach is that the pulses of saturating light will drive Q A fully reduced (qQ will be
zero) & that the contribution of qE will then be given by subtraction. It also presupposes that brief, intermittent, pulses
will not have a significant impact on other photosynthetic processes.

13(e) The Pulse-Saturation Method

"Q-analysis", as it is now known, has been based largely on pulse-saturation apparatus designed by Schreiber &
manufactured by Walz (Appendix 2). The Schreiber apparatus (Schreiber et al 1986) also utilises a modulated measuring
beam, but one of such low intensity (0.12 mole quanta m -2s-1) that, to all effects & purposes, the leaf is still in darkness
when this is applied. The Schreiber apparatus also employs a novel & sophisticated detection system which "is free of
the switching-on & switching-off artefacts found with conventional lock-in amplifier system".
13(f) Modulated Light

In simple measurements of fluorescence, actinic light is used at one wavelength (e.g. blue) & (red) fluorescence measured
at another (say 685 nm or above). This, in itself, limits the range of experiments which can be undertaken (measurements
in natural light would be precluded) & there are also changes in "constant" fluorescence emission (changes in F 0) which
result from changes in light intensity per se rather than changes in the rate of photosynthesis. These complicate
interpretation & comparison. For all of these reasons, there are advantages in using a modulated measuring beam at an
intensity which is so low that the leaf scarcely notices that it is not in the dark. A "lock-in" amplifier tuned (or "locked-in")
to the same frequency of modulation is then used as an integral part of the fluorescence detector so that it effectively
disregards all changes in fluorescence which are not also modulated. (Such a detector may also be protected by optical
filters to prevent it becoming saturated with reflected actinic light). Modulation can be achieved mechanically by
"chopping" (a "chopper" is essentially a revolving disc fitted with slits) or electrically. Light emitting diodes are easily
modulated by an electrical pulse generator & are used for this purpose in the commercially available apparatus.

13(g) The Procedure

A dark adapted leaf or a suspension of chloroplasts etc., is first illuminated by the extremely weak measuring beam. This
gives Fo, the so-called "dark" or initial fluorescence (Fig. 13.3). A single saturating pulse then establishes (F v)m (Fig.
13.3) the "maximal" variable fluorescence (i.e. the fluorescence seen when all of the quenching mechanisms are at zero).
Thereafter, illumination is started using actinic light of the desired intensity (photon flux density) & pulses of, say, 300
msec duration of saturating light are super-imposed at appropriate intervals (say every 10 to 20 seconds).

At the time of writing, there are incompatibilities between various types of existing commercial apparatus but both the
Schreiber (Walz) & Hansatech Instruments Ltd equipment have, for example, been successfully used with modified
Hansatech Instruments Ltd LD2/3 & DW2 electrode systems (e.g., by bringing the fibre optic which carries the actinic,
saturating & measuring light & collects the fluorescence nearer to the sample).

13(h) "Q" Analysis

The variable fluorescence (Fv) varies between zero at Fo & a maximum, (Fv)m. Each saturating pulse drives QA
fully reduced so that a pulse applied soon after the actinic light is switched on will give a fluorescence peak height
close to that of (Fv)m. Thereafter, such pulses will give smaller fluorescence peaks, largely because
of the development of qE (quenching associated with the proton gradient). If it is assumed that
neither (Fv)m nor Fo change, the rest is a matter of simple but tedious arithmetic best left to a
computer. It is becoming increasingly clear that this assumption is not
valid in all circumstances but this does not detract from the
general usefulness of the procedure & the analysis - it simply
means that you should, as always, proceed carefully & avoid
pitfalls. These relationships, as defined by Schreiber et al
(1986), are as follows (Fig. 13.3).

During induction (Section 9 & 10) the variable

fluorescence (Fv) falls short of the maximum
(Fv)m because of quenching so that

Fv = (Fv)m - q(Fv)m ......Eqn.13.1

In Eqn. 13.1 "q" is the quenching coefficient [q = 1-(1-q E)(1-qQ)]. This can vary between q = 0 (qQ = 0 because QA is
100% reduced & qE = 0 because there is no proton gradient) & q=1 (when variable fluorescence (F v) is fully suppressed).

Following each saturating pulse, fluorescence is pushed to (F v)s because qQ is eliminated

(Fv)s = (Fv + qQ (Fv)s ......Eqn.13.2

(Fv)s fall short of the maximum, (Fv)m, because of the generation of a proton gradient & its associated quenching (q E)
so that:-

(Fv)s = (Fv)m - qE (Fv)m .....Eqn.13.3

Thus, combining Equations 13.2 & 13.3:-

Fv = (fv)m - qE (Fv)m - qQ (Fv)s .....Eqn.13.4

By replacing (Fv)s in 13.4 with 13.3 & rearranging

Fv = (1 - qE) (1 - qQ) (Fv)m .....Eqn.13.5

From Eqn 13.5 four further expressions can be derived

qQ = (Fv)s - Fv ....Eqn.13.6
(Fv)s

QE = (Fv)m - (Fv)s ....Eqn.13.7

(Fv)m

1 - qQ = Fv ....Eqn.13.8
(Fv)s

1 - qE = (Fv)s ....Eqn.13.9
(Fv)m
The two quenching mechanisms (qQ & qE) have already been explained. These bear an inverse relationship to
fluorescence so that emission decreases as quenching increases. Conversely, the two expressions 13.8 & 13.9 bear a direct
relationship to fluorescence so that, as first approximations, (1- q E) follows the lowering of fluorescence yield by the
development of the proton gradient & (1-q Q) reflects the degree of reduction of Q.

Schreiber & Bilger (1986) have recently re-emphasised the problems involved in q-analysis in certain circumstances in
which changes in the initial or "dark" fluorescence (Fo) are regarded as constant, become appreciable. Variable
fluorescence may also fall below the Fo level & some allowance must be made if an analysis is to be attempted along the
lines described in Section 13h. Schreiber & Bilger have defined a quenching coefficient q o & shown that there is a
relationship between qo & qE which, when fully resolved, is likely to provide the basis of an appropriate correction.

Figure 13.3. Q-analysis.

Definition of quenching coefficients etc by
Schreiber et al (1986). F0 is the fluorescence
displayed by a dark-adapted leaf in very weak
modulated light. (Fv)m is the maximal
variable fluorescence first seen when a pulse
of saturating actinic light is applied. Fv is the
variable fluorescence seen, once continuous
actinic lightisapplied & (Fv)s are the peaks to
which this fluorescence is then driven by
pulses of saturating light. The derivation of qQ
& qE quenching is explained in the text.

13(i) Examples.

Like light-scattering (Heber, 1969), q-analysis has allowed a fuller understanding of the manner in which fluorescence is
related to photosynthetic carbon assimilation. A "causal connection" between the M-peak (Section 11) & the onset of
carbon assimilation (the termination of induction) was first proposed by Walker (1981) & subsequently by Ireland et al,
(1984) who used "light-doubling" techniques. Both light-scattering (Sivak et al, 1985) & recent q-analysis have
corroborated this, showing that the M-peak is associated with relaxation of "non-photochemical quenching" (q E) & an
increase in photochemical quenching.
During oscillations (Section 12a) fluorescence anticipates the changes in O2 & CO2. Light-scattering measurements were
consistent with the proposal that the phase-shift was attributable to changes in the proton gradient (Sivak et al, 1985) &
q-analysis (Fig. 12.4) allows a similar conclusion to be drawn.

Quick & Horton (1984) have determined the changes in q Q & qE in experiments with isolated protoplasts &, like
Schreiber et al (1986), have found that the fluorescence induction curve is dominated by q Q at low light intensities
whereas at higher light intensities there is a greater influence of q E. The reduction status of QA in the light is largely
dependent on the balance which is established between electron transfer from H 2O & electron transfer to CO2. Not
surprisingly, therefore, qQ declines at a given light intensity as carbon assimilation declines but this relationship is
affected by other factors & is best seen in high [CO2.] & low [O2.], i.e., when the situation is not complicated by
photorespiration & electron transport to oxygen.

Quenching-analysis has been shown to have important applications in studies on photoinhibition, heat stress, water-stress
etc (for reviews, see Schreiber & Bilger, 1986; Schreiber et al, 1986). Fig. 13.4 for example shows the relationship
between Q-quenching & assimilation rate as the water content of Arbutus unedo was decreased from 100% to 40%.

Figure 13.4.
Correlation between Q-quenching & assimilation rate at
varying relative water contents in leaves of Arbutus unedo.
Fluorescence & rate were measured simultaneously in a leaf
disc electrode at high CO2 concentration. (after Schreiber &
Bilger, 1986).

As already noted, cuvettes & leaf-chambers which will allow q-analysis to be combined with O 2. measurement are not
yet commercially available although much can be achieved (see e.g. Stitt, 1986) with minor modification of existing
apparatus. New prototypes have been designed by Delieu & Walker in the University of Sheffield Research Institute for
Photosynthesis (now the Robert Hill Institute) & it is hoped that these will soon be made commercially available to a
wider public. When incompatibility does exist it lies in the fact that the Schreiber apparatus is partly based on fibre optics.
One optic carries the modulated measuring beam, a second the actinic light, a third the saturating pulses & a fourth the
fluorescence signal from the leaf (or plant material) to the detector. These four optics are combined to form a single,
larger, optic with an effective diameter of approximately 12 mm. Accordingly if this optic is required to illuminate an area
much larger than 12 mm in diameter or to collect fluorescence at a distance from a source it will be more difficult to
achieve light saturation on the one hand or to optimise the fluorescence signal on the other. The Sheffield prototypes
overcome these problems. Fig. 13.5 illustrates signals obtained with the Schreiber apparatus used in conjunction with a
leaf-disc electrode specifically designed for this purpose.
Figure 13.5. Pulse-saturation combined with oxygen measurements in a specifically designed leaf-disc electrode.
Both parts of the figure are facsimiles of chart recordings of oxygen & fluorescence traces.

Left: Oscillatory behaviour induced by abrupt illumination & re-illumination after a brief dark interval. Note oscillations
in the fluorescence maxima elicited by saturating pulses & the inverse relationship between the rate of oxygen evolution
& fluorescence emission.
Right: Decline in photosynthetic oxygen evolution & corresponding decline in fluorescence maxima associated with
depletion of carbon dioxide. Note that re-illumination after a dark interval immediately before this (when carbon dioxide
was nearing depletion) did not induce the oscillatory behaviour seen in the upper facsimile.

An alternative is to use a conventional leaf-disc electrode fitted with an adaptor now manufactured by Hansatech
Instruments Ltd for this purpose. Walz have recently marketed a device designed by Schreiber for the evaluation of the
oxidation status of P700 (Fig. 8.2) so that it is now possible to measure P700 plus O 2 in one system & undertake
Q-analysis plus O2 simultaneously in a parallel experiment.
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 PART C

LIGHT SOURCES

new lamps for old

14(a). Slide-projectors & Heat Filters

One of the easiest ways to illuminate a small area of leaf in a laboratory is to use a slide-projector. Sadly, there is also a
grave danger of inadvertently cooking the leaf unless adequate heat filtration is also used. These days it is possible to buy
excellent "hot" & "cold" mirrors (for example from O.C.L.I. Ltd, High Wycombe, Bucks, U.K.). The former transmit
visible light & reflect radiant heat (near far-red, far-red & infra-red radiation). The latter, if held at 450 to the incident
light will transmit incident long-wave radiation & reflect the visible. Even using various combinations of such filters as
in Fig. 14.1 it is, however, still not easy to stop an illuminated leaf getting hot. This is partly because conventional light
sources often emit much more long-wave radiation than visible radiation & a lot still sneaks through the filters. For this
reason water filters are often inserted into the light path. Water is a good infra-red filter but it must be remembered that,
when it is used in this capacity, its heat absorbing prowess is directly related to its depth. A shallow, flowing, layer of
water will conduct heat away from a surface but it will not absorb as much radiant heat as a deeper one. In leaf chambers
such as the LD2/3 there is a built-in water path which will absorb some of the heat & also help to keep the leaf-disc,
pressed against it, close to the required temperature. Here again, however, it is important to remember that high-intensity
visible light (i.e. light from which all infra-red radiation has been eliminated) will still cause some heating. It is therefore
very difficult to avoid any heating within a leaf as a result of very bright illumination even in a system through which air
is being rapidly circulated. Where heating is thought, or known, to be a problem it is best to make measurements with a
suitable thermocouple in order to ascertain the extent of the problem.

In addition to slide-projectors, & sources such as xenon arc lamps, a number of commercially available light-sources are
now available, some specifically designed for work on photosynthesis. Several firms, for example (e.g. Schott Glass,
Stafford, U.K.) produce light-sources which permit fibre or liquid optics to be attached but few of these allow easy
insertion of additonal optical filters for adjusting light quality. It must also be borne in mind that despite claims to the
contrary, fibre optics do transmit radiant heat as well as visible light & that if the heat coming out of the optic is really
significantly less than that going in, the same will apply to the light.

Hansatech Instruments Ltd now manufacture several light sources, specifically designed for work on photosynthesis.
 14(b) A High Intensity Light Source.

Fig. 14.1. A High Intensity Light Source.

This is a powerful lamp, of a type similar to that
employed in the Research Institute for Photosynthesis
in Sheffield but not commercially available. It
comprises a quartz-halogen source with a built-in
dichroic reflector which preferentially reflects visible
light & therefore allows some heat to escape through the
reflector. A combination of hot & cold mirrors pass the visible
light through a water path & finally through an iris diaphragm
& an electronic shutter. The shutter box also incorporates a
system of mirrors which allows additional lights to be added, or
fluorescence signals to be deflected.

14(c) Light-Emitting Diodes

Light-emitting diodes (LEDs) are becoming increasingly useful as

light sources in photosynthetic applications (Rurainski & Mader,
1980). They are small & do not require high amperage currents. They
do not produce a great deal of heat & the extensive heat dissipation
built into high intensity sources such as that described in Section 14(a) is no longer necessary.
The red versions, which have peak output at 635 nm or above, produce light which is not far
removed from the peak absorption of chlorophyll in the red (Part B) & such LEDs can often be
used (e.g. in fluorescence measurements) without the need to define the light quality by using expensive optical filters.
Similarly, their very rapid "switch-on" time (about 0.25 sec) eliminates the requirement for rapidly opening shutters which
are mandatory when conventional light sources are used as actinic light in the measurement of early
events in fluorescence induction etc. Their output can be varied electrically with little effect on
light quality & their output can be readily modulated electronically for applications involving
lock-in amplifiers. They are relatively cheap, relatively stable, & relatively long-lived.

Figure 14.2 Light Emitting Diodes (The Hansatech Instruments Ltd LH36/2R)

An array of LEDs (Fig. 14.2) which give enough light to support about one third of maximal
photosynthesis by most C3 leaves is currently marketed by Hansatech Instruments Ltd (the
Hansatech Instruments Ltd LH36/2R). This light source was specifically designed for use with
the leaf-disc electrode & will work off mains-electricity or car batteries. It has all of the intrinsic
properties & advantages of LED sources. The field of illumination is not as uniform, nor the output as
high, as that of the Bjorkman lamp (Section 14d, below) but uniformity can be improved (at some cost in output) by
judicious use of diffusing screens & it is a versatile & extremely useful light source for many applications involving the
leaf-disc electrode.

Hansatech Instruments Ltd offer a high intensity LED head (LH36U) which provides a much higher output than those
employed in the LH36. A computer controlled box (LS3) is also available for incorporation into the measurement of
quantum yield (Part D & Appendix 3). The light output is varied by changing the current to the LED's (the relationship is
almost linear). This can be calibrated to give pre-selected photon flux densitites which are digitally displayed. Once
combined with appropriate software it will permit a programme of light intensities to be switched automatically by the
computer.
14(d) The Björkman Lamp

This was designed by Olle Björkman of the Carnegie Institute for use with the Hansatech Instruments Ltd LD2/3 leaf-disc
electrode & is now also marketed by Hansatech Instruments Ltd (the LS2). It is an elegantly simple lamp (Fig. 14.3) which
gives a very uniform field of illumination. It accommodates neutral density filters which slip into slots, thereby providing
a range of light intensities.

Figure 14.3. The Björkman Lamp.

This fits directly on to the top of the Hansatech Instruments Ltd leaf-disc electrodes but may also be used as an actinic
source for a variety of purposes. This makes it particularly suitable for measuring the rate of photosynthesis as a function
of light intensity (see Part D). It is portable, robust & will work off a 12-volt car battery. It does not have a rapid switch-on
time like the LED's (the light source itself is a tungsten-halogen bulb) & (unlike the Hansatech Instruments Ltd LS1) could
not be used for following the earliest changes in induction fluorescence unless it was used in conjunction with a fast
shutter & appropriate optical filters to define the light quality.

Reference

Rurainski, H.J. & Mader, G. (1980) Light-emitting diodes as a light source in photochemical & photobiological work. In:
Methods in Enzymology (San Pietro, A., ed) Vol 69, 667-675. Academic Press. Inc., New York, London.
 PART D

MAXIMUM EFFICIENCY
OF PHOTOSYNTHESIS

15. INTRODUCTION
Few scientific controversies have raged as long, or as fiercely, as that surrounding the
determination of the maximum efficiency of photosynthesis i.e. the relationship between
CO2 fixed (or O2 evolved) & light energy utilised. For many years the minimum quantum
requirement has been taken to be 8, but, during his lifetime, this view was steadfastly
rejected by Otto Warburg who put the value at 4 or even less. Warburg, of course, was a Nobel
laureate, the man who first used spectrophotometry & manometry in biochemistry & the
discoverer of NAD & NADP. In short, his expressed opinion, based on meticulous experiments,
was not to be taken lightly. One who did have the temerity to question the great man's
conclusions, however, was a young American called Robert Emerson who studied in
Warburg's laboratory in Berlin & then returned to Urbana, Illinois, where he was to make
major contributions to photosynthesis (e.g., the Emerson enhancement effect). Emerson found
the requirement to be 8, but Warburg insisted that it was 4 & nothing, seemingly, would bridge
the gap. Eventually Warburg travelled to the United States with a view to undertaking
definitive experiments but, in the end, nothing was resolved. Emerson himself believed that he
wasted years of his life in attempting to arrive at the definitive answer.

Warburg's measurements of quantum yield requirement were made on Chlorella, by

manometry. Over the years, the procedures that he first described in 1923 were greatly
simplified & even, he said, "performed as simple classroom experiments" (Warburg &
Burke, 1950). Nevertheless, it was never an easy matter to obtain low values. The algae had to be carefully grown in well
water or spring water & in experiments undertaken in 1949 "it took us about two & a half months to find out the conditions
under which high efficiencies could be obtained in every experiment". "Only gradually did we come to clarify & identify
the reasons for the irregularities: the sedimentation of the cells in the cultures, the not always adequate shaking of the
manometer vessels, the too few cells in the manometer vessels, the too great time differences between manometric
readings of the two vessels, the damage done to the cells when shaken too long in the dark, the too high intensity of the
measured red-light beam, etc. The experiments themselves took several hours". After all this effort Warburg believed that
he had answered his critics, that high efficiencies could be routinely measured & "that in a perfect nature, photosynthesis
is perfect too". He was wrong. Why he was wrong remains uncertain to this day. Perhaps James Franck (1953) came
nearest to the mark in his erudite discussion of this problem in which he implicated respiratory intermediates in an attempt
to rationalise the results of Warburg & Burke. Here the suggestion was that the lowest values were not a measure of
photosynthetic efficiency at all but of some mixture of respiration & incomplete photosynthesis (reduction of 3-
phosphoglycerate not linked by one-to-one stoichiometry with CO 2 uptake) induced by an unusual combination of
conditions (shaking of the algae in the manometer vessels resulted in only 5% of the algae being illuminated at any one
time, spending 0.1 or 0.2 seconds in bright light & a further second or so in darkness) & compounded
by the sluggishness of manometry ("it has been repeatedly shown that manometry is the most
inadequate technique for following closely the quick rate changes which occur during the
induction period"). Whatever the real explanation of Warburg's low quantum values, Franck was,
however, undoubtedly correct when he said "We do not feel that it is justified to disregard
all other evidence only to make possible an unusual -- explanation of certain brief,
transient anomalies of the gas exchange, which occur under special conditions". Since he
made this statement in 1953 the "other evidence" has come to be regarded as compelling
by almost everyone in the field. Photosynthesis is an imperfect phenomenon in an
imperfect world. In higher plants the quantum requirement might approach 8 but a value
of 4, or less, is inconceivable.

Using the leaf disc electrode, it is now really possible to "perform simple classroom
experiments" to measure apparent quantum yield by the procedures described below.
Using a computerised version of this procedure a value can be obtained in 20 minutes, using
the simplest of apparatus. Absolute values can be derived from the apparent value by applying
a correction obtained by determining light absorption by the leaf disc in an integrating sphere.
4 or 8? For some purposes, however, the apparent values will allow useful comparisons to be made
without this correction.
16. MEASUREMENT OF QUANTUM YIELD

Quantum yield is a measure of the costs of converting light energy into

chemical energy in photosynthesis. It is measured as the number of moles of
O2 evolved (or CO2 fixed) per mole quanta of absorbed photosynthetically
active radiation (PAR; 400 - 700nm). It can be calculated from the initial slope
of the light response curve of photosynthesis & correcting this slope for the
absorbance of the particular plant tissue (usually 80 - 85% of the incident light
between, 400 - 700nm, is absorbed). In healthy plants, quantum yield varies
according to the pathway of photosynthetic carbon metabolism, temperature
& prevailing O2 & CO2 concentrations. In plants exposed to stress, quantum
yield varies with the extent & duration of a particular stress, & whether the
stress is experienced in light or darkness. Unchanging quantum yield may be
equally informative. The slope of the light response curve (left) from which
quantum yield is derived, remained unchanged despite massive changes in
photosynthetic rate brought about by experimental manipulation of cytosolic
Pi (Section 12a). Thus, measurements of quantum yield are useful indicators
of a range of different photosynthetic parameters in healthy & stressed plants.

17. THEORETICAL BACKGROUND

The light dependent production of chemical energy in photosynthesis requires

co-operation of two photochemical acts (Figs. 8.1 & 8.2). Production of one
molecule of NADPH requires removal of 2 electrons from water & the
movement of these through the electron transport chain driven by the two
photochemical acts (one in PSII & the second in PSI), each of which requires
one quantum of light energy (one photon) to function (Fig. 8.1). Thus
production of one mole of NADPH requires the absorption of 4 quantum
moles of light energy. As each electron moves through the electron transport
chain, two protons are pumped to the interior of the thylakoid. Production of one molecule of ATP by noncyclic
photophosphorylation requires movement across the thylakoid of 3 protons. Thus the theoretical stoichiometry of
NADPH, ATP production, electron transport & quantum absorption is

1 NADPH : 1.33 ATP : 2 electrons : 4 quanta absorbed

Removal of two electrons from water produces half a molecule of O 2 so that the molar ratio for O2 evolution in
photosynthesis is

1 O2 evolved : 2 NADPH : 2.66 ATP : 4 electrons : 8 quanta absorbed

A theoretical minimal quantum requirement can also be estimated from the known stoichiometry of the photosynthetic
carbon reduction cycle in C3 plants. In the absence of photorespiration, reduction of one molecule of CO 2 requires 2
NADPH & 3 ATP. Note that there is a shortfall of 0.33 ATP between this estimate & that estimated on the basis of O 2
evolution above. Because the stoichiometry of photosynthetic CO 2 & O2 exchange in vivo is 1, we must account for this
additional ATP requirement, & it is thought to be produced in processes of cyclic photophosphorylation (Fig. 8.1). Thus
the minimum quantum requirement for photosynthetic CO 2 fixation is slightly
greater than 9 quanta absorbed per CO2 fixed.

Quantum yield is the reciprocal of quantum requirement. Accordingly, the

theoretically maximum quantum yield is about 1/9 or 0.111 molecules of O 2
evolved (or CO2 absorbed) per photon of PAR absorbed. This calculation refers to red light which is
used with greater efficiency than light of other wavelengths. Because most plants are exposed to the full
solar spectrum & because it is sometimes more practical to use white light for photosynthetic
measurements, a correction can be applied for the lower efficiency of white light overall. The data of McCree
(1972) suggested that quantum yields in white light could be 25% lower than those in red light, because blue
& green light are less effective than red light (but see Evans, 1986 & below). This would have put the maximum
theoretical quantum yield for C3 plants in white light at about 0.83 (a quantum requirement of about 12). Many
literature values approach this (0.081 in Encelia & Agropyron: Ehlringer & Björkman, 1977; Ehlringer &
Pearcy, 1983) but, more recently, values equivalent to an average quantum requirement of 9.47 have been “the full solar
obtained for more than 30 C3 species (Section 19) in measurements undertaken with the leaf-disc electrode. spectrum”
 In C3 plants, in air, a certain amount of photorespiration takes place, depending on the temperature &
the stomatal control of intercellular partial pressure of CO 2. Photorespiration involves the release of
CO2 from recently fixed carbon, so that net fixation per quantum absorbed is reduced. The
reduction of quantum yield in C3 plants due to photorespiration which was controlled by varying
O2 concentrations was demonstrated by Ehlringer & Björkman (1977). For a variety of reasons,
photorespiration in air tends to increase with leaf temperature & these authors also showed that
quantum yield of C3 plants in air decreases with temperature. All of these complications are
removed when quantum yield is measured at CO2 saturation in the O2 electrode. In C4 plants, carbon
reduction takes place under saturating CO2 concentrations in bundle sheath cells & is insensitive to
O2 concentrations up to air levels. However, the CO2 concentrating process involves preliminary
carboxylation of phosphoenolpyruvate & requires additional energy. The known stoichiometrics of
C4 photosynthesis suggest that 2 NADPH & 5 ATP are required, per CO 2 fixed. This corresponds to
a quantum requirement of 15 quanta per molecule of O 2 evolved, or a quantum yield of 0.067
mol O2 evolved, per mol quantum absorbed. Correcting for red light vs. white light, values
“several intriguing of 0.050 would be expected in air. In practice, the quantum yields of C4 plants are higher than
explanations can be this (0.053-0.065, Ehlringer & Pearcy 1983 & 0.069, Björkman & Demmig, 1987).
advanced” In CAM plants, depending on the pathway of decarboxylation, the quantum yield of
photosynthesis should be marginally less than in C4 plants. However, recent experiments show
that quantum yields of CAM plants are the same as those of C3 plants, when measured in the O 2 electrode. Although
several intriguing explanations can be advanced to explain these discrepancies, no direct evidence in support of them is
currently available.

John Evans (1986) has recently re-examined the dependence of quantum yield on wavelengths. His results support earlier
findings that the quantum yield is lower in the blue than in the red & this is attributed to the difference in the absorption
spectra of the antennae of PSI & PSII (PSII, being richer in chlorophyll b, will absorb more quanta than PSI at wavelength
shorter than 680 nm & vice versa at longer wavelength). This will affect the correction for absorbance & will also lead to
an imbalance between the two photosystems such that at wavelengths below 680 nm, PSI receiving fewer quanta than
PSII, will limit whole-chain electron flow. Evan's results showed a smaller difference between red & white light than
those of McCree (1972). His values of quantum yield for spinach were 0.098 (a requirement of 10.2) & 0.112 (a
requirement of 8.9) for white & red light respectively - a difference of about 12%.

18. MEASUREMENT OF APPARENT QUANTUM YIELD WITH THE LEAFDISC

ELECTRODE & THE BJÖRKMAN LAMP

Quantum yield measurements using the LD2/3 require high sensitivity of

photosynthetic rate measurement at low light intensity, uniformity of
illumination over the leaf & precise control of light intensity. All of these can
be obtained with the Björkman lamp. A 50W bulb is normally most suitable
for quantum yield measurements.

First calibrate the light-ouput with a suitable set of neutral density filters & a
sensitive quantum meter. (A "Skye" quantum meter which can also be supplied
by Hansatech Instruments Ltd (QSH1 & QSM1) (Appendix 2) & which is
specifically designed for use with the LD2/3 is ideal for this purpose). This is
done by inverting the lamp, fitting the top chamber of the leaf disc electrode
(filled with clean water) & placing a disc of white paper on the plexiglass plug
which presses against the leaf disc when the chamber is in use. Take care that Quantum yield as a function
the fan is not obstructed & switch the lamp on. of light quality
Fix the quantum-sensor in one place on the disc of white paper (or move it about carefully if an integrating meter is
available) & insert combinations of filters to give light of appropriate intensities. If the "Skye" sensor is used it fits directly
over the plexiglass plug & integrates the photon flux density over this surface (without need for a paper disc).

It is advisable to have four or more photon flux densities between 0 & 100, 2 or more between 100 & 200 & to have 3 or
more between 200 & 800 mol quanta m-2 s-1, for most purposes.

REPEAT THIS PROCEDURE whenever bulbs are exchanged. Check also the alignment of the bulb (Hansatech
Instruments Ltd LS2 Notes for Users page 3).
Check the light output of the lamp at frequent intervals. If changes in the mains voltage cause significant fluctuations in
light output it may be necessary to use a regulated power supply or car batteries.

TAKE PARTICULAR CARE to ensure that cooling water in the chamber is clear at all times. An in-line filter helps.

Quantum yield measurement is commenced by placing a leaf disc in the chamber just as if it were to be used for
photosynthetic or fluorescence transient measurements. Calibrate the chamber volume, & O 2 content as described
previously, for each leaf disc used (Section 4). For most purposes it is best to measure quantum yields in saturating CO 2
This can be done by using a pad soaked in bicarbonate/carbonate buffer, by breathing into the chamber until a standard,
say 5%, CO2 concentration is attained, or by purging the chamber from a bottle of 5%CO 2, passing the gas through an
aspirator to saturate it with water vapour.

Having charged the chamber with CO2 the leaf disc should be exposed to several cycles of dark, followed by full light
output from the Björkman lamp until a steady rate of photosynthesis is attained & oscillations in O 2 evolution are
minimised (Section 12; Fig. 12.1). If no bicarbonate buffer has been used be sure to recharge the chamber with CO2 after
this procedure. In practice, the chamber filled with 5% CO2 will sustain only about 5 min high rate photosynthesis.
Illumination of leaves which are inadequately supplied with CO 2 may lead to photoinhibition. Similarly, if shade leaves
are used, it is prudent to use lower light intensities (say, 200µmol quanta m -2 s-1) during this conditioning phase. For some
purposes, it is convenient to use this conditioning period to measure room temperature fluorescence transients. In this case
a blue filter may be used for the conditioning light, & removed before commencing the quantum yield measurements.

Alternatively, the Hansatech Instruments Ltd LED light source can be used to elicit the fluorescence responses. The
electrode back-off should be applied to obtain the maximum sensitivity available with recording system.

The conditioned leaf disc should then be kept in the dark for 5-10 min until a steady rate of respiratory O 2uptake is
obtained. Starting with the neutral density filter combination which gives the lowest light intensity selected, a light
response curve is then constructed. Filters should be changed at frequent intervals, but only after a steady rate of O 2
exchange has been established. Experience suggests that a single charge of 5% CO 2 is adequate to complete the linear
portion of the light response curve (to 200 µmol quanta m -2 s-1 approximately). Depending on the photosynthetic capacity
of the species, & on whether or not information is to be sought on saturation characteristics, it may be necessary to
recharge the chamber CO2 supply at higher light intensities. Note that when the chamber is recharged by breathing, some
time for temperature adjustment will be required before steady rates can be obtained.

Rate of O2 evolution is then calculated for each light intensity & the "apparent" quantum yield read from the slope of rate
vs incident quanta. Actual quantum yield requires measurement of transmission & reflectance of the leaf surface presented
to the light in the electrode chamber. This may be done with an integrating sphere & reflectance standards. For most
leaves without reflective hairs or wax, a value of 80-85% absorbance can be assumed. Changes in absorbance following
stress are small so that comparisons of apparent quantum yield, before & after stress treatment, are sufficient to indicate
damage.

19. DOING IT IN OTHER WAYS

19(a) According to Björkman & Demmig

(i) Introduction.

Sections D(18) & E described procedures for measuring quantum yield. Since they were written a paper has been
published by Björkman & Demmig (1987) which has such important implications that the methods used by these authors
are reproduced here in their entirety (Section 19b below). The importance of this work, which was concerned with the
measurement of absolute quantum yield, lies in the remarkable constancy of the values (derived with the leaf-disc
electrode) for plants possessing the same pathway of CO 2 assimilation (Table 19.1). Together, the constancy & magnitude
of the C3 values suggests that they may be the best values yet achieved. Even the most careful previous measurements,
such as those made by Ehleringer & Björkman (1977) & Sharp et al. (1984) would appear to have under-estimated the
real quantum yields (the latter authors obtained a value of 0.087 (11.49) for Helianthus leaves using IRGA at 0.205% CO2
& 21% O2).
 Table 19.1. Quantum Yield (from Björkman & Demmig, 1986).

Pathway No of Species Mean QY Mean QR

C3* 37 0.106 (+ 0.001) 9.43
C4 5 0.0692 (+ 0.004) 14.45

* The values for 2 CAM species were similar to those of the C3 species

If this is the case, the difference may lie in the difficulty in achieving CO 2 -saturation without adversely affecting
measurement by IRGA (because of stomatal closure & loss of sensitivity at high CO 2 concentrations). As noted
previously, the leaf-disc electrode is not affected in this way because, even if high CO 2 induces stomatal closure the
diffusion gradients are such that measurement is not impaired. Similarly, the new values prompt questions about the
magnitude of the data of McCree (1972) which suggested that quantum yields in white light might be 25% lower than
those in red light. Björkman & Demmig used white light & a 25% higher quantum yield in red light (i.e. a quantum yield
of 0.1325 or a quantum requirement of 7.54) would be highly questionable. Recent work by John Evans (Section 17)
suggests, however, that the difference between red & white might be smaller than previously supposed.
All of the leaves that were used were of healthy appearance & had not been subjected to stress. The procedure was then
as follows:-

(ii) Photon Yield Measurements

"Rates of photosynthetic O2 exchange were measured at 25C & CO2 saturation with a system incorporating a leaf disc
electrode (Model LD2/3, Hansatech Instruments Ltd Ltd., Kings Lynn, Norfolk, U.K.) designed by Delieu & Walker
(1981, 1983), & a fan-cooled quartz-lamp housing, designed & built in this laboratory. A similar commerical light source
in now manufactured (Model LS2; Hansatech Instruments Ltd). The light from a quartz-halogen lamp is collimated by 2
condenser lenses & filtered through a hot mirror (OCLI, Santa Rosa, California, U.S.A). This source provides a collimated
light beam at the position of the leaf disc immediately below the lower window in the leaf disc chamber. The lamp (Type
Bellaphot 50W; Osram) was operated at 11.60 + 0.02 VDC, provided by a regulated power supply (Vista Model XRD;
Clifford Industries, Amarillo, California, U.S.A.) that was modified to permit voltage adjustment & to provide accurate
display of the output voltage. The PFD at the leaf position was approximately 600 mol m-2s-1. Desired attenuation of the
light was obtained by inserting in the light beam neutral density screens (made by sandwiching fine-mesh nylon screens
between two 50 x 50 mm slide mounting glasses) &/or neutral density filters (Melles Griot, Irvine, California, U.S.A.).
Optimal uniformity & reproducibility of the light field was obtained when the nylon-mesh screens were placed at a
position normal to the light beam, 1 cm below the lenses, & the neutral density filters were placed close to the top of the
leaf chamber window at 20 to the horizontal plane. The PFD incident on the leaf disc with each of the filter combinations
used in the photon yield measurements was measured at 17 different positions in the light field with a quantum sensor
(Model LI 190SB; Li-Cor, Lincoln, Nebraska, U.S.A), recently calibrated at the factory. The variation in PFD over the
light field was +3 to +6%, depending on the filter combination used. The mean PFD values obtained at the 17 positions
were used for calculations of photon yield. Comparisons made with three other quantum sensors from the same
manufacturer gave values which agreed within +2%. The spectral photon distribution of the light incident on the leaf disc
was measured with a spectral radiometer (Model 1800; Li-Cor).

Polarization voltages & signal readouts from the leaf disc electrodes were provided by a read-out control box (Model
CB1-D; Hansatech Instruments Ltd) or by a laboratory-built unit of similar design, & the signals were displayed on a chart
recorder (Recordall 5000; Fischer Scientific Co., Pittsburgh, Pa., U.S.A.). Temperature control was provided by a
controlled-temperature water circulator, capable of keeping the temperature constant within +0.02C. The influence of
variations in ambient air temperature was minimized by insulation of the lower part of the electrode chamber with a
cylinder of polyurethane foam. The electrode was covered with an electrolyte gel (part no. 77948D; Beckman Instruments
Inc., Fullerton, California, U.S.A.) followed by a 2.5 x 2.5 cm piece of cigarette paper (Rizla) & a teflon membrane of the
same size (part no. 76438, Beckman). This procedure proved superior to others that were tried in terms of ease of
application, electrode signal noise & stability, & useful lifetime of each application. The electrode signal usually reached
adequate stability within 1h after the polarization voltage (0.700 V) had been applied. The electrode output was checked
in an air stream of known O2 pressure (usually 20 kPa) & then in a stream of pure N 2; the residual signal in N2 was usually
** symbol not quite there??? 1% of the signal in air. After adjustment of the signal & calibration of the O 2 electrode, the
signal amplification was increased by a factor of 10, & an appropriate bias signal was applied to give desired sensitivity
for the photon yield measurements. In most cases the signal was adjusted so that the difference between zero & full scale
deflection on the chart recorder was equal to a change in O 2 pressure from 20 to 22 kPa O2.

Rates of O2 exchange at 25C & standard barometric pressure were calculated from the expression P = 68.2.VS/SA where
P is the rate of O2 exchange in mol O2 m-2s-1; V the net volume of the electrode chamber in ml; S the change in the
electrode signal per min caused by the leaf disc; S the signal obtained for each kPa change in O 2 pressure using the same
amplification; & A the exposed leaf area in cm2. The volumes of the two electrode chambers used in this study (with the
electrode in place, & the 3 metal discs, the foam disc & the fibre matting supplied with the chamber present) were 4.44.
& 4.54 ml. To obtain the net chamber volume, any addition of water to the chamber & the volume of the leaf disc were
subtracted form the chamber volume. The leaf area exposed to the light beam (A) was 8.86 cm 2 for both of the electrode
chambers. The total leaf area was 10.0 cm2.

In the measurements at Stanford, CO2-containing air was provided from a gas cylinder containing 20 kPa O 2-, 5 kPa
CO2-(balanced N2). A slow stream of this gas whose flow rate was controlled with a mass flow-controller (Model FC260;
Tylan, Carson, California, U.S.A.) was humidified by passing it through a porous ceramic cylinder immersed in water at
25C & then was passed through the electrode chamber. After placing the leaf disc in the chamber, the flow rate was
adjusted in relation to the expected rate of O2 evolution, so that the rate of O2 exchange could be monitored in an open
flow mode. When a constant signal had been reached, the inlet & outlet valves of the chamber were closed, & the rate of
rise in O2 concentration was then followed in closed mode until it could be ascertained that the slope had reached a
constant value. The leaf disc was then darkened & the rate of O 2 uptake recorded, usually for 4 min. The rate of
O2-evolution measured just before darkening the leaf was then added to the value of O 2 uptake measured in the interval
1 to 4 min after darkening the leaf. The light intensity from the light source was then changed, an open-flow mode was
resumed & when a new constant signal had been reached, the valves were again closed & the cycle repeated. The main
advantage with this alternation between open & closed modes in photon yield measurements is that this procedure avoids
undesirable build-up of O2 & depletion of CO2 while waiting for the establishment of a constant rate. At high rates of O 2
evolution, open-mode measurements are superior to measurements in the closed mode; however, quantative
determinations in the open mode require that the O 2 concentration be measured in the exit gas stream from the leaf disc
chamber, not in the centre of this chamber. Very close agreement was obtained when rates measured in open & closed
modes on the same leaf discs were compared. In general, photon yield measurements were started at a PFD of 125 mol
m-2 s-1 & the PFD was then decreased in steps of approximately 25 mol m -2 s-1 , until the sum of the rate of O2 evolution
in the light & the rate of O2 uptake measred in the subsequent dark period (P + R) was linearly dependent on PFD. The
photon yield on the basis of incident PFD (i) was then calculated as i = (P + R)/PFD. Except for extreme shade leaves
which had very low light-saturated rates, (P + R) was generally a linear function of PFD up to PFD values as high as 75
to 125 mol m-2s-1.

In the photon yield measurements made in Australia, CO 2 was supplied from a 1M sodium bicarbonate buffer or by the
operator blowing a slow stream of air from his lungs through the leaf disc chamber. This procedure was not as satisfactory
as that later employed at Stanford, & many more replicate determinations of the rate of O 2 exchange had to be made. Also,
in these photon yield determinations the rate O 2 uptake in the dark was not measured after each light step; it was usually
only measured after the O2 exchange measurements at lowest light level had been completed.

(iii) Leaf Absorptance Measurements

After completion of the photon yield measurement a 4 cm 2 sample was punched from the leaf disc & placed in the centre
of an Ulbricht integrating sphere with the upper surface facing the light source. The spectral absorptance ( αλ) was
determined at 25 nm intervals from 400 to 750 nm as described by Ehleringer (1981). The absorptance ( α) of the photons
incident on the leaf in the photon yield determinations & in natural daylight was then calculated for the waveband 400 to
700 nm according to the expression

700 700
α = Σ αλ . eλ Σ eλ
400 400

where is the spectral absorbtance of the leaf e the spectral photon emittance of the light source & the wavelength in nm".

(iv) Further Considerations

It will be noted that Björkman & Demmig's calibration procedure was different from that described in Sections 4 & see
Appendix 3 which is based on the difference in signal between air & a second concentration of O 2 rather than the
difference between air & nitrogen.

The relationship P= 68.2 x V x∆ S/S.A contains a value of 68.2. This is because the rest of the equation expresses the rate
as a change in the partial pressure of O2 per minute per sq.cm of leaf. In order to express it in mol O 2 m-2s-1 it is necessary
to multiply by 10,000 (to convert cm 2 to m2), by 1/60 (to convert minutes to seconds) & by 0.4087 (to convert 1 ml of 1%
O2 into mols/ml). This latter value (0.4087) comes from the fact that air (21% O 2 ) contains 9.37 moles of O2 at STP & at
250C this becomes 9.37 x 273/298 = 8.58 moles/ml (Section 4) or 8.58/21 (= 0.4087 for 1% O2).
In Björkman & Demmig's experiments only 8.86 cm 2 of the 10 cm2 leaf-disc was exposed to light because of shading
by O-rings round the circumference of the perspex (plexiglass) roof of the chamber. If a roof without O-rings (or O-ring
grooves) is used, the full 10 cm2 of the disc is illuminated.

Open mode measurements were made by using a second leaf-disc chamber in series with
the first. The second chamber contained an electrode but no leaf & was simply used (in
connection with a sophisticated gas-flow device) to monitor the O 2 in the gas-stream
from the first chamber.

It will be noted that corrections were made for respiratory O 2 uptake whereas this is not
done in the procedures described in Appendix 3. The validity of this subtraction is a
matter of judgement. If "dark" respiratory O2 uptake occurs at an unchanging rate in the
light, subtraction of a constant "dark rate" will not affect the slope of the rate vs light curve
from which the quantum yield is derived. If measured respiratory uptake changes as a function
of light intensity, it will affect the slope (& hence the quantum yield) but it has to be assumed that “sophisticated
the "dark" rate is an accurate measure of the "light" rate & it is by no means certain that this is so. gas-flow”
Either way, the correction is unlikely to have a substantial effect on the quantum yield values,
especially if these are estimated from the slope of the line above the light compensation point. Quantum
yield determination based on points above the compensation point (i.e. on net O 2 evolution) have sometimes been
preferred because, in many tissues, a pronounced "Kok effect" (a change in slope at very low light intensities) may be
observed. This is believed to reflect an interaction between respiration & photosynthesis (Section 23).

19(b) Doing it with Chloroplasts & Algae

Although the leaf-disc electrode was obviously designed with leaf-discs in mind it has been used for other purposes. Zoran
Cerovic (Institute of Botany, University of Belgrade, Yugoslavia) for example, has supported intact spinach chloroplasts
on filter discs & used these as you would a disc. Similarly, Avigard Vonshak (of the Ben-Gurion University of the Negev,
Jacob Blaustein Institute for Desert Research, Israel) has undertaken quantum yield measurements on Spirulina platensis
(on filter discs) before & after photoinhibitory treatment.

19(c) Doing it in the Dark

If we look at Fig. 22.1 it is clear that O2 evolution in the light is replaced by O2 uptake in the dark as photosynthesis is
replaced (or ceases to be masked) by "dark" respiration. Whether or not "dark" respiration occurs in the light as well as
in the dark is a bit like trying to decide if the light inside the refrigerator is on permanently or only when the door is open.
As usual, much depends on what is meant by dark respiration. There is no doubt that if 14C labelled intermediates of the
tricarboxylic (Krebs) cycle are fed to green leaves, the label spreads to all of the other intermediates in the light as well
as the dark (Graham & Walker, 1962). Conversely, it is difficult to conceive that such a major metabolic event as
photosynthesis is without effect on respiration (cf Section 19a(iv)). This, however, is not the place to re-kindle old
controversies & the intention of this section is simply to remind the reader that the leaf-disc can be used for following
respiration as well as photosynthesis. Oxygen uptake in the dark immediately following illumination is faster than it is
after prolonged darkness. This may be due, in part, to increased availability of respiratory substrate but it may also reflect
the operation of control mechanisms. Temperature artefacts must also be considered. These
fall into two categories. One is to do with inadequate temperature control because it is
difficult to avoid any heating of the chamber during bright illumination (Section
14) & a small artefact of this nature (caused by temperature changes in
the sensor) can often be observed if a leaf-disc is replaced by damp
capillary matting. The second is to do with heating of the leaf which is
also difficult to avoid entirely & this might be
expected to affect the rate of respiration. With
due regard to these points, however, respiration
may be measured &, provided it will fit in the
chamber, anything which respires (e.g., a seed or
an insect or a politician) can be monitored.
Alternatively, a slow gas-stream can be passed
straight through a larger vessel, containing
respiring tissues or organisms & then through
a leaf-disc chamber, for measurement of
oxygen. Given the fact that research funding in some
parts of the world is fast approaching a nadir the fact
that polarography of this sort is generally less
expensive than many alternatives may not be
an unimportant consideration in this context.
 REFERENCES

References

Björkman, O. & Demmig, B. (1987) Photon yield of O 2 evolution & chlorophyll fluorescence characteristics at 77K
among vascuolar plants of diverse origins. Planta, 170, 489-504.
Ehleringer, J. (1981) Leaf absorptance of Mojave & Sonoran Desert plants. Oecologia 49, 366-370.
Ehleringer, J. & Björkman, O (1977) Quantum yields for CO 2 uptake in C3 & C4 plants. Plant Physiol. 59, 86-90.
Ehleringer, J. & Pearcy, R.W. (1983) Variation in quantum yield for CO 2 uptake among C3 & C4 plants. Plant Physiol.
73, 555-559.
Evans, J.R. (1986) The dependence of quantum yield on wavelength & growth irradiance. Aust J. Plant Physiol. Vol 14
(1). In press.
Franck, J (1953) Participation of respiratory intermediates in the process of photosynthesis as an explanation of
abnormally high quantum yields. Arch. Biochem. Biophys. 45, 190-229.
Gaastra, P. (1959) Photosynthesis of crop plants as influenced by light, carbon dioxide, temperature, & stomatal diffusion
resistance. Mededelingen van de Landbouhogeschool te Wageningen 59, 1-68.
Graham, D. & Walker, D.A. (1962) Some effects of light on the interconversion of metaoblites in green leaves. Biochem.
J. 82, 554-560.
McCree, K.J. (1972) The action spectrum, absorbance & quantum yield of photosynthesis in crop plants. Agric. Meterol
10, 443-453.
Sharp, R.E., Matthews, M.A. & Boyer, J.S. (1984) Kok effect & the quantum yield of photosynthesis. Plant Physiol. 75,
95-101.
Walker, D.A. & Osmond, C.B. (1986) Photon yield of O2 evolution photosynthesis in vivo with a leaf disc electrode:
correlations between light dependence of steady-state photosynthetic O2 evolution & chlorophyll a fluorescence
transients. Proc. R. Soc. Lond. B 227, 267-280.
Warburg, O. & Burke, D. (1950) The maximum efficiency of photosynthesis. Arch. Biochem. Biophys. 25, 410-442.
 PART E

COMPUTERISED MEASUREMENT OF RATE

AS A FUNCTION OF PHOTON FLUX DENSITY
The previous chapter describes a procedure for the determination of quantum yield in which the leaf-disc electrode is used
to measure oxygen, & light intensity is changed by employing a quartz halogen source & neutral density filters. What
follows is a general description of an automated procedure for the determination of the rate of photosynthesis as a function
of PFD & the importance of this relationship. Details of a computer program used for this purpose are given in Appendix
3.

20. UNDERLYING PRINCIPLES

20(a) Why do it at all?

Photosynthesis is, by definition & in fact, a light driven process. It is self-evident that there is no photosynthesis in the
dark & a good bet that the rate of photosynthesis will increase as the light intensity increases. Thereafter, supposition &
inference must give way to measurement if the relationship between photosynthesis & light is to be properly understood.
Surprisingly, although it has had a great deal of attention, there is still much to be learned about it. What is even more
important is the fact that this relationship is not invariable & that the manner in which it changes can tell us a great deal.

If we plot rate as a function of "light intensity" (more properly "photon flux density") we can learn something about "dark"
respiration & the way that it is affected by light. We can put a value on the light compensation point (the point at which
photosynthetic O2 evolution balances respiratory O2 uptake). The initial slope of the rate v PFD plot gives us a measure
of quantum yield (maximum photosynthetic efficiency). We can, within the scope of our measuring system, arrive at an
approximation of how fast a plant might photosynthesise under given conditions. Hidden within the broad relationship
there is additional information, which can be revealed by appropriate analytical methods (Walker, 1988).

20(b) Roofs & Ceilings

Many houses have a roof & a ceiling. The roof keeps out the weather & is usually pitched at an angle so that rain runs off
it. The ceiling is usually horizontal, serving to isolate the roof space from the room below for purposes of insulation &
aesthetics. Blackman (1905) in particular, sought to define the factors which limit photosynthesis in similar terms & there
is still much to be learned from a comparable exercise.

20(c) The Roof

In the present context light intensity is now usually defined as photon flux density (PFD) & expressed as µmole
quanta.m-2.s-1. In other words it is a rate measurement - expressing the rate of arrival of photons (quanta or "parcels" of
electromagnetic radiation in the visible wavelength range - Section 26) as the number which would be intercepted, by
one square meter of surface, each second. Photosynthetic rate can be expressed in similar terms i.e. as µmoles O 2 (or CO2)
evolved (or fixed) per square meter of leaf surface per second (µmoles.O 2.m-2.s-2). If photosynthesis is measured at
different light intensities the rate of photosynthesis can therefore be plotted as a function of light intensity (or PFD) in
these terms so that one rate can be directly related to the other. The initial slope of this relationship is a measure of
maximal efficiency or quantum (photon) yield. This is the roof of our photosynthetic house. The maximum rate is the
ceiling (Fig. 20.1).

Figure 20.1 Roofs & Ceilings. A typical rate v PFD

plot.
The intercept on the vertical axis is a measure of dark
respiration, that on the horizontal axis is the light compensation
point. The initial slope is a measure of quantum yield & its
reciprocal, quantum requirement. The curve lies within two
constraints, a sloping "roof" & a "horizontal ceiling" (the "true
ceiling" or maximum rate). The roof is a thermodynamic
constant pitched at an angle dictated by the maximal
photosynthetic efficiency (in this instance a nominal quantum
yield value of 0.111). The ceiling represents the absolute
maximum rate of carbon assimilation. For purposes of
comparison, a lower or "nominal" ceiling, imposed
by the rate of carbon assimilation at 800
µmole.quanta.m -2.s-1 is employed.
To some, it will be immediately apparent why the initial slope of the rate v PFD plot is a
measure of efficiency. Others may be helped by an everyday analogy. The efficiency of a
motor vehicle can be expressed in terms of its fuel consumption. One that does 30 miles to a
gallon of fuel would be more efficient than one which did 10. Such efficiency would be most
easily be measured by putting precisely one gallon (or one litre) of fuel into the vehicle's tank
& seeing how far it would go before the tank was empty. If accurate instruments were
available which would measure both the speed (i.e. the rate of progress of the vehicle) & the
rate of fuel consumption, it would also be possible to plot one rate against the other. The initial
slope would then be a measure of efficiency. For example, if the vehicle used 1 gallon of fuel
in one hour & traveled 30 miles during the same period the fuel
consumption efficiency could be expressed as 30 miles per gallon.
The corresponding value for photosynthetic efficiency might be 0.1 molecules of O 2 evolved
(or CO2 fixed) per quantum (or photon) of light absorbed. Leaves & motor vehicles have a
common feature in that these relationships are non-linear in both - i.e. there is a departure in
linearity (a decrease in efficiency) at higher rates, because more & more energy is being
dissipated as heat rather than accomplishing useful work.

Figure 20.2.
Illustrates the distribution of incident light between four layers of chloroplasts
in a leaf which is illuminated from above & the corresponding rate v PFD plots,
assuming a strict Blackman (roof & ceiling) relationship. Curve A, bottom left,
is a simulation of the relationship which would be
observed if the leaf was illuminated from the lower
(abaxial) surface & the departure from the Blackman
relationship consequent upon the differences in
photosynthetic capacity between the chloroplasts in
the 4 layers. Curve B, bottom right, shows the extent
to which this effect would be diminished if the
chloroplasts from the 4 layers were homogenously
distributed in one layer (Terashima, personal
communication).

20(d) The Ceiling

As we press the accelerator in a motor vehicle we increase the rate at which fuel is supplied to the engine & therefore the
rate at which the vehicle can travel. Eventually, however, the engine will reach its maximum capacity for utilising fuel,
no matter how rapidly the fuel is supplied. Green leaves behave in a broadly similar fashion. They will photosynthesise
faster & faster as they are given more & more light but eventually the increment in rate per increment in light will become
imperceptible. A ceiling will have been reached. This ceiling is not set at a finite height like that of a house but will move
upwards or downwards according to environmental factors such as temperature & CO 2 availability (C4 plants, which
incorporate a CO2-concentrating facility, behave differently from C3 plants for this reason). Moreover, the ceiling is not
usually horizontal but tilted upwards at higher PFD's. It does not necessarily follow, however, that the ceiling is really as
tilted as it might seem. A given photosynthetic machine must have a finite capacity under given conditions. Once reached,
under unchanging conditions, there is no reason why this value should be exceeded. This implies a horizontal ceiling. One
reason why it is often not horizontal, is that a leaf usually contains several layers of chloroplasts so that when the
photosynthetic machinery in the upper layer (i.e. the layer facing the incident light) has already reached its maximum
capacity, the chloroplasts in the lowermost layer may still be less than saturated. Moreover, the chloroplasts which are
located in the loafer layers of cells will function like those in a "shade" leaf because they have developed in a shaded
environment (Laisk, 1977; Terashima & Saeki, 1985; Terashima et al, 1986; Terashima & Takenaka, 1986). Complex
regulatory factors , such as those which bring about "long induction" (Rabinowitch, 1956; Walker, 1976), may also
contribute to the tilting of the ceiling. Nevertheless, while recognising this inevitable gulf between theory & practice it is
still useful to regard these limiting factors in these terms. Accordingly we shall visualise the rate v PFD plot as being
constrained by a roof & a ceiling. As we shall see, the pitch of the roof is a thermodynamic constraint & is determined by
the efficiency of the photosynthetic machinery. The ceiling is a metabolic constraint & is determined by the capacity of
the photosynthetic machinery to utilise light energy.
20(e) How Steep is the Roof?

As already noted (Part D) little in biology has occasioned so much argument as the answer to this question. Here we shall
start with the simple premise that photosynthetic electron transport from water to NADP is in general accord with the
Z-scheme of Hill & Bendall (1960) & that Einstein's Law of photochemical equivalence demands that one photon will
initiate one photochemical event. Given that the Z-scheme incorporates two photosystems & that it is necessary to
transport 4 electrons through each photosystem in order to evolve one molecule of O 2

2H2O + 2NADP 2NADPH2 + O2

it follows that the minimal quantum requirement is 8 & that the maximal quantum efficiency (the reciprocal of the
requirement) is 1/8 (or 0.125). This defines the pitch of the roof. If the Z-scheme is an adequate description of
photosynthetic electron transport & if what is measured accurately evaluates its function, a quantum efficiency of 0.125
cannot he exceeded. In practical terms it is also difficult to believe that maximal efficiency can ever be achieved. There
is more to photosynthesis than NADP reduction. Except in the very short term, what is measured embraces cytosolic
events such as sucrose synthesis. For the present, at least, there is much merit in accepting a nominal quantum
requirement of 9 (a quantum efficiency of 0.111). Bjorkman & Demmig (1987) reported an average value of 9.37
(a quantum efficiency of 0.106) for 37 different species.

Figure 20.3. The Z-scheme by analogy.

Two hammer blows (quanta) are needed to project each ball (electron from the
starting point (the ground state) to the net (the electron acceptor). Lifting four balls
from ground to net would require 8 hammer blows.

This important finding not only adds credibility to a value of

about 9 (which is consistent with the underlying biochemistry as
it is presently understood) but also has important implications. Much has been said about the need to improve plant
productivity & the extent to which this might be achieved by changing metabolic processes to advantage. Unless
Bjorkman & Demmig's results are substantially refined by future experience (which seems most unlikely) any such
improvements will not result from greatly improved efficiency. Evolution, it would seem, has made photosynthetic energy
transduction about as efficient as possible, despite the energy losses involved. The molecular biologist seeking
improvement would have little or no genetic variation to start from since each species seems to be about as efficient as
the next. Redesigning plants to advantage by modifying existing mechanisms is feasible. Redesigning de novo, in entirety,
is still in the realms of science fiction. However, if it is not a practical proposition to steepen the pitch of the roof it does
not mean that all hope of improvement is gone. Raising the height of the ceiling is not unthinkable nor is improving the
extent to which photosynthesis approaches the constraints imposed by the roof on the one hand & the ceiling on the other.

20(f) Relative & Absolute Quantum yield

If we plot rate v PFD for incident light the initial slope gives us a measure of relative quantum yield. If we plot rate v PFD
for absorbed light we get absolute quantum yield. Bjorkman & Demmig's (1987) values were corrected for transmission
through the leaf & for reflection from the surface. If measurements are made in white light without this correction the
values for (apparent) quantum yield may be as much as 15% lower &, if absolute values are needed, corrections for
transmission & reflection based on the use of an integrating sphere must be applied. For most purposes, such corrections
may not be necessary. Relative (uncorrected) quantum yield is a valid measurement in its own right. For example, if the
consequences of stress, or experimental intervention, are to be examined it will be the change in quantum yield which will
attract attention &, in most circumstances this change will be the same irrespective of whether or not corrections for
transmission & reflectance are made. When 660 nm light, from LEDs (14c) is used in measurement, the difference
between relative & absolute quantum yield is unlikely to be much more than 10% & changes within this correction (such
as increased reflection or decreased transmission) will be accordingly less, if they occur at all.

20(g) Why Trouble to Automate Measurement?

Another immediate & obvious question is why any one should feel that there is a need for automation. A driver, born &
bred to manual gear changes, may disdain a vehicle with an automatic shift because all automatic shifts incur some loss
of power &, what is much worse, they take some of the decision making out of the driver's hands. Their only obvious
advantage is that they require less skill to operate. Happily, automated measurement of quantum yield has many more
advantages to offer. The non-computerised procedures described in Sections 18 & 19 are not difficult. Anyone can learn
how to do these in an hour or so but they are tedious & call for a great deal of concentration if they are to be done in the
most effective way. Sitting & waiting to change neutral density filters is not the most exciting way of brightening an
otherwise dull afternoon & if your mind wanders to some consideration of the meaning of life or if you try to catch up on
your correspondence you are inviting what could be a modestly disastrous lapse in concentration.

So automation ends all that. You offer a piece of leaf to a machine & it does the rest. You can write that letter, long
overdue, to your mother in Falmouth, Lucknow, Alice Springs, Wurzburg or West Lafayette without fear of error.
Moreover, while computers are not yet renowned for their intelligence, they can do some simple tasks much better than
people.

One such task is split-second timing & this is very important in the present context because it allows the timing of repeated
measurements to be very precise. Reproducibility is of the essence in following the change in quantum yield which results
from experimental intervention. This, indeed, goes to the heart of the automated approach. If you
wish to make an occasional measurement of quantum yield the manual procedure is fine &
has infinite flexibility. In general, however, quantum yield measurement for its own sake
is an esoteric pursuit best left to the expert whose work currently indicates that, in the very
best circumstances, you might expect
your C3 or CAM leaf (Adams et al,
1986) to give a value close to nine. “brightens an otherwise
Like Warburg & Burke before them
dull afternoon”
(1950) Bjorkman & Demmig
(1987) may be proved wrong but
unless we are prepared to reject the
entire basis of contemporary
theory of photosynthetic
electron transport (see e.g.
Figs. 8.1,8.2 & 20.3) it seems unlikely
that they will be far wrong. For many
workers, much more interest lies in the
way in which quantum yield might be
affected by environmental stress or by
experimental intervention of one sort or another.
The entire shape of the rate v PFD plot is also full of information but this
information can only be properly elicited by comparing one species, or variety, or ecotype, with another & by examining
the manner in which it changes with circumstance. Such comparisons demand a great many measurements & minimal
variation in procedure. Here the computer comes into its own, not only because it revels in these simple repetitive tasks,
which would soon make you feel that you were working on an assembly line, but also because of the many analytical
procedures which it can be programmed to undertake.

20(h) How Does it Work?

How, you might ask, does the automated system work? As before (Sections 18 & 19), the leaf-disc electrode is used to
measure photosynthetic oxygen evolution but now a high-intensity LED source is substituted for the Bjorkman lamp (p.
49). As we have already seen (Section 14c) LED arrays have much to recommend them in photosynthesis work. They are
a relatively cool source of light & this simplifies temperature control. They switch on very quickly, facilitating
fluorescence measurement. In the present context they have additional advantages. In order to follow the way in which
the rate of photosynthesis varies in its relationship to PFD it is necessary to measure PFD at the leaf surface as accurately
as possible. Quantum sensors are now technically advanced but it helps if all of the light which is being offered to the leaf
is "photosynthetically active". Red LEDs have peak emission at about 660 nm, close to the red absorption maximum of
chlorophyll at 680 nm. Quantum sensors can be calibrated to measure light of this quality without uncertainties about
what fraction of the incident light is non-photosynthetic. Unlike a tungsten source which, although admirable for many
purposes, delivers more heat than light, LEDs emit very little red light at wavelengths which will not be absorbed by the
photochemical apparatus of the leaf. Another major advantage is that there is a near-linear relationship between light
emitted & current supplied to the LEDs. This means that the computer has a very easy job if it is asked to change PFD by
controlling the electrical supply. A tungsten source is not suitable for this purpose even though the changing quality of
the light which it gives out at different currents can be defined by optical filters. (The quality of LED light does not change
significantly as the electrical supply is varied). The main problem with a tungsten source is not merely the fact that the
relationship between light & supplied current is non-linear but also because it changes, in a very complex way as the
filament changes temperature. The LED also suffers from instability as a result of heating but to a much lesser extent
because the ratio of emitted light to heat is much more favorable than it is for a tunsten or quartz halogen source.

Moreover, it is relatively easy to put together crafty electrical circuitry which incorporates feed-back control so that
changes of this nature can be largely corrected. This then is a large part of the automated measurement but, as we shall
see, much less than the full story. What we do, in effect, at this stage is to offer a leaf-disc to the apparatus & sit back &
do whatever we please while it (with the help of the computer) measures photosynthetic O 2 evolution over a range of
pre-selected PFDs (see 20k).
 20(i) What Are The Drawbacks?

LEDs have many virtues but they were not, in common with almost all other light sources,
developed for photosynthetic work. Existing LEDs (or combinations of LEDs) are not yet
capable of supplying as much light as ace would wish. It is true that they will draw much
more current & give much more light at very low temperatures (e.g. in liquid N,) but this
is not yet a practical proposition. Even so the new Hansatech Instruments Ltd
high-intensity LED source will give about 950 µmole.quanta.m -2.s-1 of red light at
the leaf surface (i.e. after transmission through the water filter which is an integral
feature of the leaf-disc chamber) & this is more than adequate for many purposes.
High intensity LEDs are still relatively expensive, however, & the cost of a
suitable array is about two to three times greater than a more conventional light
source. Secondly the LED array is intrinsically less uniform than (for example)
the Bjorkman lamp, comprising, as it does, separate sources rather than one
source & a combination of lenses designed to yield maximal uniformity. This
drawback is largely offset if it is used in conjunction with a specially designed
“crafty electrical circuitry” quantum sensor (Skye Instruments - see Appendix 2) which effectively
integrates the PFD over the surface of the window against which the leaf-disc
is pressed during measurement. The O2-electrode, of course, measures total O2
evolution from the leaf-disc & it is largely immaterial if some cells receive a little more or a little less
light than their neighbors provided that both incident light & oxygen production measurements are
integrated over the full 10cm2 area of the leaf-disc.

Problems of reproducibility may arise if leaf pieces are used but these can be largely overcome if
care is taken to position such pieces in the same place within the chamber each time a measurement
is made. Within the 0-125 µmole.quanta.m-2.s-1 range (i.e. within the largely linear part of the rate
v PFD plot) much greater uniformity can be achieved by inserting a diffuser between the LEDs &
the top window of the chamber.

20(j) What Are The Advantages?

The trivial advantages have already been touched upon. Every research scientist learns, as an
apprentice, that tedium will be part of his life & that routine & repetitive work will not only
be inescapable but will far exceed those rare moments of insight or
accomplishment which makes it all worth while. That is not to say that
tedium should be acorn like a hair-shirt, in order to mortify the flesh. There
is everything to be said for making research a pleasure because there is
little likelihood that your salary will encourage you to do it for money.
Automated measurement of quantum yield is not only more precise than
manual measurement but a great deal less boring. One particular selection
of PFDs & time intervals, which has proved useful & productive, takes only
16 minutes to run.

This is time enough to think about the purpose of the experiment, to take a
coffee or write the first paragraph of that letter. Nevertheless, these are the
trivial advantages. The real advantages are in data analysis as well as in data
acquisition & storage. First of all, you will certainly wish to plot rate of “every research scientist
photosynthesis as a function of light intensity (PFD). In the manual learns”
procedure, rates have to be derived from a pen-recorder chart & plotted
by hand. This will take you 60 to 90 minutes
(according to your aptitude for mental arithmetic & your dexterity with pen or pencil). The
computer does this virtually immediately. Quantum yield is derived from the initial slope of
this relationship (Section 27 & Appendix 3). The PFD for 50% & 90% of the maximal rate can
be derived, at the press of a button, as can the "light utilisation capacity” etc (Appendix 3).
Plots can be immediately re-drawn on different scales (the computer always selects what it
believes to be the most appropriate scale but, lacking intuition or telepathy it is not to know
what is in your mind). An extremely useful facility is the ability to call up previous data so that
this can be “over-plotted" for purposes of comparison (Fig. 20.4). These & other devices,
which will be described below, extend the versatility & usefulness of measurements
enormously.

PFD for 50% of

“maximal” rate
 Figure 20.4. Rate v PFD for Sun &
Shade leaves of Avocado.
(a) To illustrate both the differences
which can often be observed in the
behaviour of leaves of the some species
if they have been grown in a different
light environment (in this instance,
leaves from outside & inside of the
canopy of a single tree) & the ability of
the computer to facilitate such
comparisons (sec Appendix 3) by
allowing previously stored data to be
"over-plotted", on the same axes, using
any chosen scales.

Automation puts rate v PFD determinations into the category of a routine measurement which can be undertaken by the
non-specialist. It becomes possible, really for the first time, to examine how this relationship changes with time, to make
comparisons which could not otherwise be contemplated & to extract the last jot of information from the results.

21. MEASURING RATE AS A FUNCTION OF PFD

21 (a) Choice Of CO2 Concentration etc.

If appropriate carbonate/bicarbonate buffers are employed the leaf-disc electrode can be used to measure photosynthesis
in concentrations of carbon dioxide close to those in air but the instrument was originally designed for use with saturating
CO2 & there is much to be said for using it in this way for this particular purpose. The most compelling reason is Occam's
razor which, loosely interpreted, ask "why make life more difficult than it is already?". If we wish to examine the manner
in which the rate of photosynthesis responds to increasing light intensity interpretation will be simplified if co-limitation
by other facts is avoided. How much CO2 is needed to achieve optimal saturation in these circumstances will, in the end,
have to be determined by experiment for each & every leaf but 5% is often appropriate. As previously noted (4c) this
concentration is readily achieved by enclosing capillary matting moistened with 1.0 M NaHCO 3 in the chamber or by
using exhaled air or both. If a mixture containing 5% CO 2, 21% O2 & 74% N2 is available on tap it is probably to be
preferred because it can be pre-humidified at a selected temperature & this simplifies everything. The use of bicarbonate
buffers as a CO2-source has implications for calibration which should be borne in mind. High CO 2 can effect the electrode
response & therefore the instrument should be calibrated in the same CO2 concentration as that employed in the assay. If
CO2 is generated within the chamber, O2 can be displaced if the taps to the external atmosphere are not immediately closed
& injection of 1 ml of gas containing (e.g.) 5% CO 2 & (20% O2 rather than 21% O2) during calibration could lead to
corresponding over-evaluation of the O2 subsequently evolved in photosynthesis.

Carbon dioxide at concentrations as high as 5% may induce stomatal closure but the concentration gradient is so steep
that the closed stomata may offer no sensible barrier to gaseous exchange. Photorespiration will also be almost entirely
suppressed because of the competitive nature of the oxygenase reaction (Edwards & Walker, 1983; Walker et al, 1986).
[The enzyme Rubisco (ribulose l,5-bisphosphate carboxylase/oxygenase) which catalyses the carboxylation of ribulose
1,5-bisphosphate (RuBP) in photosynthetic carbon assimilation (Walker et al, 1986) also catalyses
the oxygenation of the same substrate. Carboxylation leads to the formation of two molecules of
3-phosphoglycerate This is used both to regenerate the CO 2 -acceptor (RuBP) & to form the
immediate end products of photosynthesis such as starch & sucrose. Oxygenation yields one molecule
of PGA & one molecule of phosphoglycolate which enters a complicated sequence of reactions (Tolbert,
I971) which finally ensure that some newly fixed carbon which would otherwise be lost is returned to
the chloroplasts. Because oxygen & CO2 compete at the active site of Rubisco, these photorespiratory
reactions are largely repressed by high concentrations of CO 2 or by low concentrations (2%) of O2].

21(b) The Unsteady State

The rate of photosynthesis changes with time. If a leaf is abruptly & brightly illuminated after a
prolonged period of darkness there may be little discernible O 2 evolution at all for the first few
minutes &, after this initial lag or short incubation period, a gradual increase in
“The Unsteady photosynthesis ("long" induction) may continue for many minutes or even hours.
State” Induction may also follow abrupt upward or downward changes in light intensity. The
problems which are experienced in going from prolonged darkness into full light can be
largely avoided by pre-illumination or "wake up". However, prolonged photosynthesis
may result in feed-bad inhibition or other changes in the physiological status of the leaf. It is obviously desirable to avoid
measurement during such changes but the true steady-state may be largely illusory. For the present purpose it is pointless
to measure during the period of negligible photosynthesis which characterised “short” induction but a leaf which has been
too rudely awakened may be as misleading in some regards as one which is still half asleep. This has to do with the impact
of light on "dark" respiration.

21(c) The Closed Refrigerator Door Conundrum.

Does the light in the refrigerator stay on when the refrigerator is closed? Answering this question would not call for
inspired experimental science but the impact of light on "dark" respiration is still more of a matter for argument &
conjecture than definitive statement (Graham, 1980; Graham & Chapman, 1979). If respiratory O 2 uptake remained
unchanged on going from darkness through progressively increasing light intensity it would have no impact on the slope
of the rate v PFD plot from which quantum yield is derived. At present no one knows for sure what does happen in these
circumstances but there is no doubt that photosynthetic carbon assimilation has an impact on cytosolic events & vice versa
(Sivak & Walker, 1986). For example “long” induction can be shortened by feeding Pi & may be largely attributable to
the changes in Pi supply associated with the regulation of sucrose synthesis (Cseke et al, 1984; Stitt et al, 1987) the
principal mechanism for recycling of Pi. Oxygen evolution below the light compensation point can also be influenced by
the "Kok effect" (Section 23) & "light enhanced" respiration (Section 22).

22. LIGHT ENHANCED RESPIRATION

Oxygen uptake is low if measured in a leaf which has been kept in darkness for several hours. After induction (10) & a
period (say 5 minutes) of strong illumination, respiration is much faster (than before) if the leaf is then abruptly darkened.
Thereafter the rate of O2 uptake declines rapidly during the first few minutes of darkness (Fig. 22.1) & then more slowly
until it eventually falls to its original value. As measured with the leaf-disc electrode, part of this “O 2 uptake" is artefactual
& related to the changes in temperature which are an inevitable consequence of strong illumination. Thus black cloth or
dead leaf tissue exhibits a small apparent O2 evolution in the light & a corresponding decrease in the O 2 signal on
darkening. Nevertheless, when due allowance has been made for this artefact, light enhancement of dark respiration can
still be discerned. The nature of this enhancement remains unexplained. In bright light, triose phosphate will pour through
the Pi-translocator (Fig. 12.7, Sections 41g & h) on its way from stroma to cytosol. Once there, it will undoubtedly
influence cytoplasmic events in complex ways because of its impact on metabolite status, ATP/ADP ratios etc. Whether
or not these changes lead to enhanced dark respiration (as the O 2 uptake data suggests) or to a light inhibition of "dark"
respiration, as some believe, is still uncertain. It is clear, however that a “Kok effect" (Section 23) can sometimes be
observed.

Figure 22.1. Light-enhanced “Dark” Respiration.

This figure shows the course of O2 evolution following illumination at zero time. Following an initial lag or induction
period lasting about 3 mins (seen most clearly in the differential, dO2/dt) O2 evolution reached a quasi steady-state of 30
µmoles.O 2.m-2.s-1. Following a brief dark interval of 1 min, photosynthesis was resumed after a scarcely perceptible lag
(differentiation was over +/- 12.5 sec). Finally, after 6.5 min, the leaf was darkened & during the next 3 minutes the
apparent rate of dark O2 uptake declined from -6.84 (at the point that the dotted horizontal coincides with the differential)
to –2.37. It is this light-enhancement of the apparent rate of dark O 2 uptake which contributes to the departure from
linearity, near the light compensation point, in Fig. 23.1.
 23. THE “KOK EFFECT”
The “Kok Effect”
This is named after Bessel Kok who was the first to observe what he
interpreted as an inhibitory effect of light (Section 19a(iv)) on dark respiration
in Chlorella (Kok, 1948; 1949; 1951). Fig. 23.1 shows a departure from
linearity in O2 evolution below the light compensation point which was most
pronounced when measurements were made, firstly in darkness & then in
increasing PFDs, immediately following a period of bright illumination. It
could be argued that this is not a true Kok effect but there is no doubt that it
could be easily mistaken for one even though the relationship is a smooth
curve rather than a discontinuity (see left). In turn, this immediately prompts
other questions. Is the "Kok effect" real or an artefact of measurement? Is it a
universal phenomenon &, if not, why not? Is there only one effect or several?
None of these questions can be properly answered yet but some things are
clear. Firstly the Kok effect is by no means a universal phenomenon. Emerson
(1958) was even sceptical about its existence, stating “The small amount of
positive evidence for the "Kok effect", together with negative results in several
instances of search for confirmatory evidence, leads to the conclusion that a
substantial difference in quantum yield above & below the compensation point
(after Sharp et al, 1984) is not a general phenomenon. If it is something associated with special
conditions, these conditions have yet to be specified. It seems equally possible
that the apparent positive evidence arises from changes in rate of respiration between the dark & light intervals chosen
for calculation of rate of photosynthesis”. Decker (1957), like Van der Veen before him (1949), observed a discontinuity
but preferred the interpretation that dark respiration was constant below the light compensation point & thereafter
increased with increasing light intensity. Sharp et at (1984) found the discontinuity to be present even when respiration
was constant in the dark (i.e. when effects such as those illustrated in Fig. 23.1 could not be advanced in support of an
alternative explanation). Of course, it would be most unwise to seek to reinterpret changes in CO 2 exchange, made in very
different circumstances, in terms of the changes in O2 illustrated in Fig. 23.1.

Figure 23.1.
Rate v PPD in quantum yield range (0-125 µmole.quanta.
m-2.s-1 ) for cape weed Arctotheca candula (L.). Single leaf
pre-illuminated for 5 minutes at high (900 µmole.quanta.
m-2.s-1) moderate (125 µmole.quanta.m -2.s-1) & low light
(12.5 µmole.quanta.m -2.s-1). Measurements were made
consecutively in the order "moderate", "high", "low". Note
the departure from linearity at low PFDs (which is most
marked when measurements are commenced immediately
after pre-illlumination at high PFDs) but otherwise the
reproducibility of the data.

At the same time it must be noted that experimental approach is central to this issue. Sharp et al (1987) avoided the rapid
decline in the rate of CO2 efflux from sunflower leaves which occured in the first 2 hours of darkness (after a 14 hour
photoperiod) & measured respiration in a "relatively stable" subsequent period, reporting values of net photosynthesis
(after as much as 45 minutes in PFDs up to 30 µmole.quanta.m -2.s-1 only when “the rate of respiration in the dark was
similar at the beginning & the end of the experiment”. This was an admirably rigorous attempt to preclude the “temporal
changes in dark respiration” which caused Emerson (1958) & Heath (1969) to question the reality of the Kok effect.
Whether or not anything like normal photosynthesis would occur in such circumstances is another matter. Induction
(Section 10) is still not fully understood (Walker, 1981) but it is clear that it involves light activation of enzymes, build-up
of dark depleted substrates, & the supply of Pi from the cytosol to the chloroplast (Walker, 1976; Sivak, 1987; Sivak &
Walker, 1985, 1986; Walker & Sivak, 1987). If a sunflower leaf grown in relatively high light (900 µmole.quanta.m -2.s-1
in the experiments of Sharp et al, 1984) is illuminated at 30 µmole.quanta.m -2.s-1 (or below) following 2 hours or more of
preceding darkness it is most unlikely that cytosolic pools will become filled during the period of measurement even if
apparent photosynthesis has become constant. The changes in slope reported by Sharp et al (1984) are too large to be
explained in terms of a shift, at high PFDs, from Benson-Calvin cycle activity alone (Fig. 12.6) to Benson-Calvin cycle
plus sucrose synthesis. Nevertheless the values of Sharp et al (1984) for quantum yield of about 0.086 (measured in high
CO2 above the light compensation point) imply that photosynthesis in these circumstances was, for whatever reason,
operating at less than maximal efficiency.
Notwithstanding the continuing uncertainty about the nature of the "Kok effect", results such as those in Fig. 23.1 will
have to be borne in mind in making quantum yield measurements. As in the past, the investigator must face an inevitable
dilemma. If "dark" respiration is to be measured in the steady state, several minutes of darkness will be needed in order
to avoid light-enhanced O2 uptake & several hours, in many species, before the decline in dark respiration, following
illumination, becomes imperceptible. However, even 1-3 minutes darkness will normally be enough to re-establish “short
induction" in photosynthetic O2 evolution (Walker 1981). Moreover, re-illumination at high PFDs after a period of
darkness, often initiates a “burst” (Fig. 22.1) of O 2 (& an associated “gulp” of CO2) within the induction period (Sivak &
Walker, 1985). Opinions about the origin of the O 2 gulp still differ but there is no doubt that 3-phosphoglycerate often
persists in darkness in circumstances in which RuBP falls to near zero, or that pre-formed PGA will serve as an oxidant
in suspensions of isolated chloroplasts. If the Benson-Calvin cycle is “switched off" & if the reduction of PGA exists,
even as a possibility, there is an obvious danger in attributing gas exchange, during induction, to "real" photosynthesis.
Conversely, if induction is avoided by pre-illumination, the possibility of light enhancement of respiration cannot be
disregarded.

No doubt, these problems will continue to pre-occupy those who wish to continue to refine the measurement of maximal
photosynthetic efficiency. If, on the other hand, the initial slope of the rate v PFD plot is primarily of interest as an
indicator of environmental stress or experimental intervention there is much to be said for a simple standard procedure
(e.g. measuring from 125 to darkness after pre-illumination at 125 µmole.quanta.m -2.s-1). In unstressed leaves, this
approach (Appendix 3) can yield reproducible quantum yield values close to those demanded by our present
understanding of photosynthetic electron transport. The methods described here permit such determinations to be made,
routinely, in less than half an hour.

24. IS THERE A RECOMMENDED PROCEDURE?

As usual, there are "horses for courses" & what may be ideal for one leaf may be less than ideal for another. If you take
a previously illuminated leaf & measure its rate of photosynthesis as a function of PFD by making 15-20 measurements
each lasting one minute you will immediately learn a great deal about its physiology. Some would insist that such short
time intervals will not permit the achievement of steady-state photosynthesis & they would be right. On the other hand,
enzymologists (similarly concerned with the determination of rate as a function of substrate concentration) have
traditionally ignored the changes in substrate concentration which occur during measurement. As always,
measurement is rarely non-intrusive (p. 95) & compromise is inevitable. In the present context the use of
a relatively large number of relatively small PFD increments avoids the creation of disturbances,
such as oscillations (Section 12c & Fig. 12.5) which are often associated with major perturbations.
The computer is also programmed to ignore the first 10 seconds of the O 2 signal following a
change in PFD during which time minor perturbations, consequent upon this change, will
tend to subside.

How to ensure "wake-up" on the one hand & induction on the other is more difficult.
After prolonged darkness some leaves display such prolonged induction that the term
"short" induction becomes a misnomer & the observer searches, in vain, for a prince to
kiss this sleeping beauty into wakefulness. Other leaves may wake up quite readily
in the sense that the initial slope is unchanged or largely unchanged if the light
intensity is increased without pre-illumination but longer illumination, or
repeated assay (Fig. 24.1), may bring about pronounced increases in the rate of
photosynthesis at high PFDs. How to ensure “wake up”

Figure 24.1.
To illustrate the usefulness of the automated approach in
following a rapidly changing situation. Six measurements
(3 not shown, for clarity) were made successively on a disc
from a shade avocado leaf harvested the previous day &
kept in darkness overnight. Prior to the first measurement,
the disc was illuminated for 15 minutes in 90
mole.quanta.m-2.s-1 light. The second, third & fourth
measurements were consecutive. Between the fourth & fifth
there were 6 minutes darkness followed by 1 minute in 900
mole.quanta.m-2.s-1 light & between the fifth & sixth a
further 10 minutes darkness & 2 minutes in 900
mole.quanta.m-2.s-1 light.

Such behaviour could be indicative of reversal, by light, of dark inactivation of Rubisco. Whatever its cause it adds to the
difficulties in arriving at an acceptable procedure. The Kok effect & photoinhibition also constitute pitfalls. Prolonged
illumination in high light is the obvious way to ensure that the leaf is fully awake but it can induce light enhanced "dark"
respiration &/or photoinhibition.
As usual, there is no real alternative to being familiar with the idiosyncracies of your leaf. In the absence of such
knowledge the best way of achieving wake-up is probably to pre-illuminate the leaf at the highest PFD that you can
provided that this does not exceed that which the leaf normally experiences in its immediate natural environment. Once
fully awake, the leaf will not usually relapse into deep sleep in 5-10 minutes darkness but this will be sufficient to allow
light enhanced dark respiration to subside. Thereafter the leaf should be pre-illuminated at the highest PFD to be
employed in determining the initial slope (100-125 mole.quanta.m -2.s-1) & measurements made by following the rate of
O2 evolution as the PFD is decreased from this maximal value. Wake-up will be ensured, light enhanced "dark"
respiration will be minimal & will decrease throughout the period of measurement so that, as a percentage error, it will
have least impact where it might otherwise lead to greatest distortion (c.f. Fig. 20.6).

25 HOW TO GET THE MOST OUT OF YOUR DATA

It is one thing to ask a computer to make life easier for you but quite another matter to conjure real science out of what
you might be offered. Measurement is descriptive. Experimental science starts with measurement. Thereafter it is a matter
of asking the right questions. Plants are unable to communicate but that is not a major disadvantage. Ask your friend what
causes your migraine (or his or hers) & see if you get a helpful answer. If, on the other hand, you were able to ask your
friend the right questions, physiologically & biochemically, by perturbation & measurement, you could get the right
answers &, at one fell swoop, rid the world of fearful torment & rejoice in new-found wealth, prestige & appreciation.
Ask a plant the right questions & you are unlikely to be rewarded so well (although if you came up with a really nice new
herbicide you might find yourself in a same league as Croesus, Getty, Gulbenkian & QE2). In any event, asking the
questions is down to you. The only way to get answers from a plant is to make measurements. Measuring rate v PFD is
only one of a large spectrum of measurements currently available (see e.g. Sections 13c & e) but at least you should ensure
that you derive as much benefit from your measurements as subsequent analysis will allow.

26. RATE v PFD UNGILDED

As we have already noted, the rate v PFD plot offers a wealth of information in its simplest form. The intercept on the
vertical (Y) axis is a measure of O2 uptake mostly attributable to "dark" respiration. This value is by no means
invariable & the effect of pre-illumination on "dark" respiration & the relationship between
photosynthesis & respiration & how this varies from species to species, from variety to variety,
according to environmental influences is still one of the major unquantified & uncharted areas of plant
physiology (Graham, 1980; Graham & Chapman, 1979; Graham & Walker, 1962). The intercept on
the horizontal (Y) axis is no less important. This is the light
compensation point. Shade species or shade adapted leaves
“a wealth of of sun species may be expected to have low light
information” compensation points but there is more to it than that. If the
pitch of the roof of the rate v PFD plot is as steep as
thermodynamics permit (i.e. if the quantum requirement is
in the region of 9) a low light compensation point
demands a low rate of dark respiration or a pronounced
Kok effect or both. If the relationship is linear & the light
compensation point remains unchanged despite a change in
quantum requirement (as in photoinhibition - Section 32)
then the change in photosynthesis must also
have affected dark respiration. In short, there is
more to the light compensation point than
immediately meets the eye - it merits evaluation.

27. THE INITIAL SLOPE

As we have seen, the initial slope is of fundamental importance because it is a measure of maximal photosynthetic
efficiency (i.e. of quantum requirement & its reciprocal, quantum yield). Some ways of carrying out the actual experiment
(see "Is there a recommended procedure?" - Section 24) are better than others but how do we recognise the initial slope
when we see it? The human hand guided by the human eye is still as good as anything & the computer programme offers
you a "line" or "rule" to help you draw the line of best fit. If you prefer to put your faith in statistical analysis use a least
squares regression (Appendix 3) but bear in mind that whatever procedure you use will involve subjective judgement &
that there is never a substitute for precise data which will confine errors in judgement. A third possibility is to use a more
sophisticated line-fitting procedure which has been instructed to recognise & ignore departures from linearity above &
below the PFDs at which such departures might be expected to occur.
 28. THE SHAPE OF THE CURVE
Put aside all impure thoughts & contemplate the nature of the rate v PFD curve. In the first place
it is not really a curve at all, certainly not the rectangular hyperbola of enzyme kinetics fame.
Often it looks more like two straight lines joined by a curvilinear region & indeed this would
be expected if its photosynthetic performance is constrained by a thermodynamic roof (Section
20c) & a metabolic ceiling (Section 20d). Let us suppose that this is the case. It would follow
that an efficient or successful leaf would take all that it was offered, i.e. that its rate v PFD curve
would be close to the roof & ceiling. Attempts have been made to quantify the realisation of this
potential which is partly constrained by thermodynamics & partly by metabolic capacity.
Terashima & Saeki (1985), Terashima et. al. (1986) & Terashima & Takenaka (1986) have
suggested the convexitivity of the curve as an index. (When the convexitivity index is 1 there
Quantum Requirement would be an abrupt transition between roof & ceiling, i.e. a Blackman relationship. When the
by “Line” index is zero the curve would be a rectangular hyperbola).

One alternative is "nominal light utilisation capacity". This defines an area bounded by the roof, the ceiling, the horizontal
(X) axis & a perpendicular erected at a convenient, high PFD (800 mole.quanta.m -2.s-1)for measurements made with the
high intensity Hansatech Instruments Ltd LED array). "Nominal light utilisation capacity" is then the area under the rate
v PFD curve expressed as a percentage of the area defined by these constraints (see also Appendix 3). In this analysis the
roof is pitched at an angle equivalent to a quantum requirement of 9 & rises
directly from the origin (Fig. 20.1). Because of dark respiration, no leaf will
ever accomplish zero oxygen exchange at zero PFD so that even if the
convexitivity of the curve were maximal the nominal light utilisation capacity
would be less than 100%. A roof that rises directly from the origin is therefore
arbitrary & it will be immediately obvious that a leaf with a low light
compensation point (LCP) will approach this particular constraint more closely
than one which requires a higher PFD to balance dark respiration. In this regard
"nominal light utilisation capacity" is descriptive rather than carrying
implications of efficiency. In terms of survival strategy a leaf which gets little
light & therefore photosynthesises slowly may be best served by a
commensurately low rate of dark respiration. Conversely, a fast growing
species in full light might easily do best if it can grow rapidly in order to
maximise its light interception & compete most successfully with rival species.
In this situation a very high rate of "dark" respiration & a correspondingly high
LCP might simply be an indicator of rapid metabolism rather than inefficient Convexitivity: Rectangular hyperbola (θ = 0)
utilisation of photosynthetic product. Similar considerations apply to the and Blackman (θ = 0) for identical initial
convexitivity of the curve. A fraction (about 5%) of incident slope and asymptote.
photosynthetically active light is transmitted through leaves. Shade leaves (After Terashima and Saeki, 1985)
characteristically contain high chlorophyll & this has, correctly or otherwise,
been regarded as an attempt by the leaf to maximise light interception. Be this
as it may, there must be an optimal response in a given situation. Utilising as much light as possible in a deeply shaded
environment would have obvious advantages but a point must be reached when excessive investment of valuable
resources in chlorophyll could "cost" more than would be gained by increased light interception. Whether or not
quantification of the rate v PFD relationship on the basis of nominal light utilisation capacity, convexitivity (Terashima
& Saeki, 1985; Terashima et. al, 1986; Terashima & Takenaka, 1986) or whatever will come to assume importance in
ecophysiological work remains to be seen but, on the face of it, it would appear to offer more information than some
alternatives.

29 ANALYSIS OF STRESS
29(a) Stress & Performance

Björkman & Demmig's (1987) work confirmed the tacit assumption that a leaf is unlikely to photosynthesise well if it is
stressed. Much the same can be said of poorly trained athletes, tired executives or ageing professors. Different types of
stress must obviously affect leaves in different ways but little has been done to qualify & quantify stress in terms of the
rate v PFD relationship. Again nominal light utilisation capacity affords some promise of quantification, particularly if
combined with other measurements such as Q-analysis of chlorophyll a fluorescence (Sections 13e & h). Wilting affects
the rate at high PFDs more than at low PFDs (Ben et. al, 1987) thereby leaving the initial slope largely intact. Stresses
which lead directly or indirectly to photoinhibition (Section 32) will affect the initial slope &, eventually, the maximum
rate. Such responses, the manner in which they are imposed, & the form which recovery or acclimation takes if the
inflicted damage is not irreversible, can be quantified in terms of quantum yield, nominal light utilisation capacity.
29(b) Shifting the Arbitrary Ceiling

As we have seen in Section 28 above, "nominal light utilisation capacity" offers a means of quantifiying the extent to
which the photosynthetic & respiratory performance of a leaf allows it to approach a potential defined by thermodynamics
on the one hand & metabolic capacity on the other. This analysis can also be used in a comparative way. Figs. 29.1 &
29.2, for example, show ceilings which have been shifted in order to calculate the nominal light utilisation capacity of a
leaf before & after photoinhibition. If the arbitrary ceiling is drawn for the lower (photoinhibited) curve the nominal light
utilisation capacity is 80% but if it is now raised to the ceiling that would be appropriate for the control (prior to
photoinhibition) the nominal light utilisation capacity is only 54%. The control nominal light utilisation capacity was
83.5% so that photoinhibition has, in this case, decreased the nominal light utilisation capacity from 83.5% to 54%.
Following removal from high light, photoinhibitory damage continued to progress at first, falling a further 12% to 42%
but thereafter maintenance of the leaf disc in low light eventually brought about complete recovery. Fig. 29.2 shows an
intermediary stage in recovery at which time nominal light utilisation capacity had increased from 42% to 62%.

Figure 29.1.
Shade leaf before & after photoinhibition which diminished
the "nominal light utilisation capacity" (i.e. the area under
the rate v PFD plots expressed as a percentage of the area
defined by the roof, ceiling & perpendicular) from 83.5%
to 54%.

Figure 29.2.
As for Fig. 29.1 showing further progression of
photoinhibition to 42% despite removal from bright light &
an intermediary stage in recovery at which the area under
the curve (the nominal light utilisation capacity) has
increased from 42% to 62%.

29(c). Shifting the Ceiling & the Perpendicular.

In some circumstances it may be useful to ask questions such as "What is the nominal light utilisation capacity at low
PFDs?" Figs.29.1 & 29.2 illustrate this point. Fig. 29.3(a) is a rate v PFD plot for a sun leaf & if the perpendicular (see
also Appendix 3) is moved from 800 to 100mole.quanta.m -2.s-1) the area under the curve (the nominal light utilisation
capacity within the constraints defined by the roof, the shifted perpendicular & the lower ceiling) is only 25.75%. When
the same exercise is repeated for a shade leaf off the same tree (Fig. 29.3(b)) the corresponding value is 67%. Comparisons
made in this way, which take into account the rate of photosynthesis, the rate of dark respiration (& therefore the light
compensation point) provide an extremely convenient basis for evaluating the extent to which a leaf is accommodated to
a particular light environment.

Figure 29.3(a).
Sun leaf of avocado showing analysis in terms of nominal
light utilisation capacity in the 100 mole.quanta.m -2.s-1
range. The roof is the same as before but the
perpendicular in both has been moved from 800 to 100
mole.quanta.m-2.s-1 ). The ceiling has been lowered to
coincide with the rate at 100 mole.quanta.m-2.s-1 )
perpendicular. The areas under the curves expressed as a
percentage of the area confined by roof, ceiling &
perpendicular was 25.75%.
 Figure 29.3(b).
Shade leaf of avocado from same tree as that used in the
experiment illustrated in Fig. 29.3(a). Corresponding
analysis, in terms of nominal light utilisation capacity, gave
a value of 67%.

30. LUX ET VERITAS, A REMINDER ABOUT LIGHT

30(a) Measurement

If it is our aim to measure the absolute efficiency of light utilisation in photosynthesis according to the above procedure,
it is necessary to know how much light is absorbed by the leaf. This is equal to the incident light, less the reflected &
transmitted light. Corrections for reflection & transmission can be made by using an Ulbricht or integrating sphere. This
is a sphere coated with a totally reflecting surface (such as a deposit of magnesium oxide) into which light is introduced.
A detector then records the difference in brightness within the sphere when a leaf is substituted for a standard reflecting
surface within the sphere. For all of these measurements (& less accurate ones in which a loss of, say, 15% by reflection
& transmission is assumed) it is necessary to measure incident light. This is not an easy matter, though it has been made
easier by recent advances in instrumentation. Most of the older instruments for measuring radiation were thermocouples,
consisting of a number (a 'pile' of thermocouples - i.e. a "thermopile") of alternate junctions between two dissimilar metals
such as copper & constantan. An increase in temperature, at such a junction, when it is exposed to light, generates a
voltage which is proportional to the difference in temperature between that junction & another which is protected from
the light. Such a device is, in fact, an electric thermometer. Thermistors, which are also electric thermometers (but which
are based on electronic devices which vary in resistance according to temperature) may also be used. Both are stable,
reproducible & independent of wavelength but, because of their intrinsic heat capacity they respond slowly &, at very low
light intensities they are less accurate because of heat gain or loss from the surrounding environment which is not
engendered by incident light. Radiation meters based on thermocouples or thermistors may be calibrated in absolute units
such as watts.m-2 (the unit of "radiant power").

The modern alternative to the thermopile is the photodiode which is a quantum (see Sections 31b & c) sensor. In this
instrument, each photon of light which strikes the surface of the detector releases an electron (thus initiating an electron
flow or electric current). Because such detectors are less sensitive at short-wave visible wavelengths this compensates for
the fact that these (high-energy) wavelengths would otherwise generate more current & this, together with the use of
appropriate filters, allows the construction of what is, in effect, a photon counting meter. Such a sensor, screened by filters
which exclude light at wavelengths below 400 nm & above 700 nm, will record "photosynthetically active photon flux
density" (PPFD). Photon flux density (PFD) is now usually expressed as the number of photons (mole quanta) striking an
area of 1 square metre in 1 second i.e. mole quanta. m -2.s-1 (see Section 30c).

30(b) The Nature of Light

"God said, let Newton be, & all was light". Newton himself suggested that light was comprised of
corpuscles traveling through space at a constant speed & that there were different corpuscles for
different colours. However, Thomas Young's experiments, in which two beams of light were
used to partially cancel each other (producing interference patterns) showed that light had
the characteristics of a waveform. Then, in 1900, Max Planck concluded that
electromagnetic radiation is absorbed or emitted only in discrete bundles or particles of
energy or quanta. (Quanta in the visible-light range are called photons). The particulate
(corpuscular) nature of light was confirmed by Lenard in 1902. He studied the photoelectric effect in
which radiation of a given frequency will cause electrons to be ejected from a metal surface. He found
that there was a threshold, or critical frequency, below which electrons were never ejected. Above this
frequency, increasing intensity only increased the number of electrons ejected, whereas increasing
frequency increased the energy of each electron but not the number. Einstein's explanation, in 1905, of this
effect contributed to his Nobel laureate in 1921. This is that light consists of quanta (or photons) & that,
in the photoelectric effect, a single electron can only acquire the energy of a single photon. Increasing
the energy content of the incident photons (i.e., decreasing the wavelength of the incident
light) will then increase the energy per electron. Increasing the light intensity will
increase the number of photochemical events & therefore the number of electrons
emitted. This, of course, is the basis of the quantum sensor (Section 30a).
 30(c) The Energy Content of Light

Quantum theory implies that light must behave simultaneously as a waveform & a stream of particles. The relationship
between wavelength (λ), frequency (v) & the speed of light (c) can be expressed as

λ (nm) x (per second) = c (nm per second)

where a nanometer (nm) is 1 x 10-9 metres in length & the speed of light, c, is 3 x 1010cm/sec or 3 x 1017
nm/sec.

The energy content (E) of one photon of light can be calculated from the
relationship

E = hv or E = hc
λ
where h is Planck's constant (= 6.625 x 10-34 joule. sec) & v is the frequency
joules are a girl’s best friend (= c/λ).

Thus for red light at 680 nm, which approximates to the red absorption peak of chlorophyll in vivo, the energy content of
one photon is

E = 6.625 x 10-34 x 3 x 1017 nm x 1

Sec 680 nm

= 2.92 x 10-19 joules

Clearly the energy content of one photon is a very small value indeed & for many purposes the "einstein" i.e. the energy
content of Avogadro's number (6.023 x 10 23) of quanta has become the basis of expression. Similarly, the term "quantum
mole" is used to describe Avogadro's number of quanta, just as a "gram molecule" or "mole" refers to Avogadro's number
of molecules. Thus the energy content of 1 quantum mole of red photons (at 680nm)

= 2.92 x 10-190 x 6.023 x 1023

= 176 Kjoules
& that of l mole quantum (i.e. 1 microeinstein)

= 176 Kjoules x 10-6

= 0.176 joules Half an Avogadro
Pear
Accordingly (since 1 joule. sec = 1 watt), it would take

500 = 2840
0.176
mole quanta of 680 nm light to deliver as much radiant power as the photosynthetically active component (wavelengths
between 400 & 700 nm) of full sunlight (approximately 500w. metre -2 s-1) - see Section 30d & Table 30.1.

The "einstein", however, is, in turn, too large a unit for many calculations because full sunlight only delivers a fraction of
an einstein per square metre of surface per sec. Accordingly, "micro-einsteins" (E = 10 -6 einsteins) & "micromole
quanta" are the preferred units & "photon flux density" (PFD) & "photosynthetic photon flux
density" (PPFD) are expressed in mole.quanta.m-2.s-1 (Section 30a).

It follows from the relationship E = hc/ (above) that the

energy content of light is inversely related to its
wavelength. Blue light (at, say, 450 nm) will have a
much higher energy content than red but, as we have
seen in Section 6, this additional energy is not used
in photosynthesis because of the extremely
rapid radiationless decay of the blue excited
state of chlorophyll. This is why, when we
are concerned with energy relations, we think in terms micromoles &
of red rather than blue light. It is the red excited state microeinsteins
which is the "driving force" of photosynthesis.
Even green photons, if absorbed & utilised, give rise to the red excited state of chlorophyll & are therefore just as effective
as red or blue photons. Incident green light is less effective than blue or red because it is more likely to be reflected from
the surface of the leaf or transmitted through it (see also Evans in Section 17).

30(d) Can We Compare Micromoles with Foot Candles?

"Shall I compare thee to a summer's day?" While it is difficult to compare chalk with cheese we are often called upon to
do so. The intensity of light was a matter of interest long before quantum mechanics were thought of & it is not surprising
that nineteenth century man thought in terms of "candle power" just as he thought in terms of "horse power". There is a
great deal in the older literature, which is still of considerable interest today, in which light is not expressed in the units
which are now favoured. It is essential, therefore, that the early terminology is not forgotten. However, it follows from
some of the foregoing (Section 30c) that absolute equivalence between some units is impossible to calculate because the
spectral composition of the light employed cannot be accurately defined. Table 30.1 attempts to compare chalk & cheese
by assuming that visible sunlight has a mean wavelength of 575 nm (whereas, in actuality, its wavelengths range from
400 to 700 nm &, although it contains a large blue, component, its spectral composition will change from hour to hour).
It also ignores the fact that the spectral composition of "electric light", though possibly not too dissimilar from "natural"
light, will vary with the source, the age of the source, the current etc.

Table 30.1 Full Sunlight Expressed in Several Ways.

All of the values in Table 30.1 are measures of flux density at an illuminated surface. Light "intensity", as such, is a
property of the radiating source. For example, a "standard candle" emits a flux of one "candle power" or 4 (= 12.566)
lumens. The corresponding value of light received (or flux density) at the surface of a sphere of 1 foot radius with a
standard candle at its centre is "1 foot candle" (1 lumen/foot 2). If the radius is increased to 1 metre the light is spread more
thinly, the flux decreasing according to the square of the distance. Since 1 metre = 3.2808feet & 3.2808 squared = 10.8,
it follows that 1 foot candle = 10.764 lux (or metre candles or lumens/sq metre). Similarly 1 phot = 1 lumen/sq cm or 10 -4
lux & 1 foot candle = 1,0746 milliphots. In S.I. units the standard candle becomes the "candela" which, similarly acting
as a point source, emits 12.566 lumens. The standard source is now taken as a black-body radiator at the melting point of
platinum & the candela emits one sixtieth of the intensity of 1 sq cm of such a black-body.

Because light is measured (Section 30a) in different ways, & because flux density is expressed in units of amount, time
& area, other variations on this theme may be encountered but translation from one to another should be simply a matter
of arithmetic.

31. ENERGY, PLANTS & MAN - AN ASIDE ABOUT ENERGY UTILISATION

In terms of light power one square metre of the earth's surface (at sea level on the equator) is rated at approximately 1 kw,
of which approximately half (500 w) can be used in photosynthesis. The corresponding mean annual value (24 hours a
day, 365 days a year) varies with latitude, altitude, aspect & cloud cover. In the Red Sea area it is about 300 w, in Australia
& the United States about 200 w & in the United Kingdom about 100 w. In terms of energy consumption, man is also
rated at 100w so that, even in the U.K., he could meet his metabolic energy requirement from 1 square metre, given 100%
efficiency of conversion of incident light energy.

Photosynthetic conversion of light energy to chemical energy may approach 33%. This is based on the calorific content
of sugar (one mole of glucose releases 672 Kcal when burnt in a calorimeter), a minimal quantum requirement of eight
(Section 15), & a value of 176joules (= 42 Kcals) per quantum mole of red light at 680 nm. Accordingly, if it is assumed
that 672/6 (= 112) Kcals would be required to convert CO 2 to "CH2O", the efficiency of conversion would be

112 x 100 = 33%

8 x 42
If the quantum requirement were 10 (see below) instead of 8, the value would be a more realistic 26%.
Since the photosynthetically active component of sunlight accounts for only half of the total “when a chlorophyll
radiation & has an average energy content of about 50 Kcal, this decreases the efficiency molecule absorbs a
to photon”
112 x 100 = 11.2%
10 x 50 x 2
If appropriate allowances are made for transmission, reflection, respiration etc., this
theoretical value falls again to about 5% & this percentage conversion has actually been
approached in the field. (For example, Pennisetum typhoides grown in a closed canopy at
Katherine, West Australia, with a daily energy input of 5,100 Kcal/sq metre, showed a daily
dry weight increase of 54g/sq metre. This is an efficiency of some 4.5%). The best recorded yields in
western agriculture are equivalent to a conversion efficiency of about 1.25% & average yields about 0.4%.

It follows from the above that if the quantum requirement were to increase from 10 to 20 (plants need to be
at peak performance to approach the theoretical maximum of about 9) there would be correspondingly
large decreases in yield. Several types of environmental stress can lead to decreases in quantum yield (i.e.
increases in quantum requirement). Such stresses often involve photoinhibitory damage.

32. PHOTOINHIBITION
32(a) What Is It?

When a chlorophyll molecule absorbs a photon it becomes excited. As we have already seen this
excitation energy is dissipated in a variety of ways. It is dissipated as light (as fluorescence), as heat, or
in the accomplishment of chemical work (photosynthesis & related processes). One such
related process is breakdown of the photochemical apparatus itself. Electric light-sources
can be regarded as the converse of the photochemical apparatus of photosynthesis in the
sense that they convert electrical energy into light. They are not able to do this without
damage to themselves &, despite being protected by inert gas, electric filaments have a
finite life. The photochemical apparatus is also thought to be protected in various ways.
The fact that the rate v PFD plot usually departs from linearity at PFDs a little above
100mole.quanta.m-2.s-1 itself implies the existence of a safe mechanism for diverting
excitation energy into channels of thermal dissipation. This seems to be related (Section
13a) to changes in pH (Krause & Weis, 1984) & associated changes in the organisation
of the thylakoid membrane (Demmig & Björkman, 1987 & Section 13c). In addition, the
thylakoid membranes may need to be kept in a state of more or less continuous repair in order
to cope with the rigours of energy transduction as it converts light-energy into electrical-energy. The
part of the photochemical apparatus which is most
susceptible to photoinhibitory damage is
PSII (Fig. 32.1).

Figure 32.1.
A diagrammatic representation of PSII
& its association with components of the
light harvesting complex in the thylakoid membrane. The “it becomes excited”
oxidising (water-splitting) side gives rise to powerful
oxidising species which are harboured in the D1
polypeptide which binds QB. Photoinhibition involves
damage to this QB binding protein (Courtesy of Jim Barber).

Precisely which part of PSII is damaged (& why) is still a matter for argument & conjecture but there is no doubt that one
component of this reaction system undergoes more or less continuous replacement in the light or that inhibition of its
synthesis (by agents such as chloramphenicol) can exaggerate photoinhibitory damage. The component which turns over
rapidly is a 32-kDa protein (i.e. one with a molecular weight of 32 kg) which is located in the reaction centre of
photosystem II & binds QB, the plastoquinone which constitutes the secondary electron acceptor of PSII (Fig. 8.2).
Damaged reaction centres are still capable of trapping excitation energy &
quenching fluorescence but the excitation energy is preferentially dissipated as
heat, rather than initiating photosynthetic electron transport so that both the rate of electron transport,
& the rate of fluorescence emission are diminished. (The Q B-binding protein is normally involved in
the transfer of electrons from the primary electron acceptor, Q A, in PSII to the plastoquinone pool. It
is also often referred to as the "atrazine-binding protein" because this is the site of action of herbicides
such as atrazine, which block the transfer of electrons from PSII to plastoquinone & therefore to the
rest of the electron transport system). New protein synthesis is involved in maintaining the integrity of
P680 (the PSII reaction centre) implying that damage to the Q B-binding protein cannot be brought
about in situ but rather that the damaged protein has to be replaced, in the thylakoid membrane,
by newly synthesised protein. Such replacement can be regarded as an inevitable part of
photosynthetic electron transport just as tyre replacement is an inevitable part of road
transport. On a perfect road the rate of tyre replacement is normally so low that it
would not be apparent to a casual observer but if a vehicle is pushed to its limits,
as in Grand-Prix racing, or if a tyre is stressed by rough terrain, replacement will
become more frequent thereby diminishing
transport efficiency.

“Pushed to it’s
limits”
PSII (courtesy of J. Barber)

In some circumstances, failure to effect adequate

repair may even lead to complete failure &
breakdown. Much the same is true of this repair of P 680 (or, to be specific, the
repair of the QB-binding protein within that reaction centre of PSII). It is in such
circumstances that "photoinhibition" becomes apparent. A decrease in variable
fluorescence can be detected together with a decline in the rate of photosynthesis which
is often most marked at low light intensities - i.e. there is a decrease in the initial slope of the
rate of photosynthesis v PFD plot (Fig. 32.2, p. 92) from which quantum yield is calculated.
Accordingly, photosynthetic efficiency & quantum yield decrease whereas the quantum requirement (the reciprocal of
quantum yield) increases. As photoinhibition progresses, even the rate of photosynthesis at higher PFDs may also become
markedly depressed.

32(b) What Causes Photoinhibition?

Damage to P680 is almost certainly an inescapable aspect of photosynthetic energy transduction but it may only manifest
itself as photoinhibition when repair (of the QB-binding protein) fails to keep pace with wear & tear. Photoinhibition is
often initiated, in contemporary laboratory practice, by strongly illuminating a leaf at temperatures between 5 & 10C, for
about 1 to 3 hours. Not all leaves will show the marked decline in quantum yield & maximum rate which characterises
photoinhibition as a result of such treatment. Much depends upon the species & the environment in which it has been
grown. "Sun species" (i.e. those which normally grow in non-shaded environments) are often very resistent to
photoinhibition. On the other hand, if they have been obliged to grow in the shade, they may be more susceptible to
photoinhibitory damage. Similarly, "shade species" (i.e. those which do well in shaded environments or normally grow
in the shade) are more likely to show photoinhibitory damage if they are brought from deep shade in to bright
light. The role of temperature in this relationship is complex. Shade species are not
noted for high rates of photosynthetic carbon assimilation &
some have been demonstrated to lack the enzymic capacity
to maintain high rates of carbon cycle activity.

Particularly at low temperatures it would

seem evident that such plants would not be
able to dissipate high levels of excitation
energy, via carbon assimilation, at rates
which would avoid photoinhibition. This
notion is also strengthened by the fact that photoinhibition “Particularly at low
may be induced by bright illumination in CO 2-free atmospheres temperatures”
(Powles et. al, 1984).
Although photosynthetic carbon assimilation may help to protect against photoinhibition this is only one part of a larger
story, however. Tolbert, as long ago as 1971, suggested that photorespiration might dissipate excess photosynthetic
energy (photorespiration consumes substantially more ATP & NADP than photosynthesis as such) & Osmond &
Björkman (1972), Björkman (1981) & Osmond (1981) have developed this concept in detail. It has also been
demonstrated that 1% O2 in N2 (which suppresses photorespiration as well as photosynthetic carbon assimilation) is very
effective in inducing photoinhibition in bright light. While there is little doubt, therefore, that a combination of
photosynthesis & photorespiration can partially protect the thylakoid membranes from photodamage it does not follow
that these are the sole channels of energy dissipation normally available to the leaf. Sequestration of cytosolic Pi, by
feeding mannose or 2-deoxyglucose (Section 16), can bring about marked depression in the rate of photosynthesis without
affecting quantum yield (Walker & Osmond, 1986) & phosphate feeding is finally unable to protect against
photoinhibition even though it initially increases the rate of carbon assimilation & may delay the onset of a decline in
quantum yield (Walker, Osmond & Cleland, 1988).

Figure 32.2.
Photoinhibition in Helleborus niger without loss in
maximum rate.A leaf-disc Helleborus niger growing in
deep shade(2 mole.quanta.m-2.s-1) was assayed at 20C
before & after 2.5 hours in 10,000 mole.quanta..m -2.s-1 at
8C. The initial slope is affected (i.e. the quantum yield has
fallen significantly from 10.5 in the control to 24 following
photoinhibition) but there has been no decline in rate at
the highest light intensities.

Krause & Cornic (1987) have drawn attention to the fact that in experiments by Powles et. al (1984) photoinhibition only
occurred when CO2 was depleted below a critical limit of about 50 bar. At this concentration of CO 2 Phaseolus vulgaris
was only able to maintain 40% of its rate of photosynthesis in air, implying that at least 60% of the excitation energy was
safely dissipated in ways other than carbon assimilation. A mechanism for safe dissipation has been suggested recently
by Krause & Behrend (1986), Krause & Laesch (1987a,b) & Weis et. al (1987). It is related to qE quenching (Section 8)
& is believed to be associated with acidification of the intrathylakoid space as a result of light-driven proton transport.
Related changes in the ultrastructure of the membranes are are thought to lead to an increase in the rate of thermal
dissipation. Björkman & Demmig (1987) have drawn attention to the possibility that the violaxanthin-zeaxanthin cycle
may play a specific role in bringing about changes in the rate of thermal dissipation. For much the same reasons it would
be a mistake to leap to the conclusion that photoinhibition of shade leaves in strong light is attributable to their inability
to fix CO2 at high rates. Some shade leaves (see below) can support rates of photosynthesis which are equal to their "sun
grown" counterparts. Even if they photosynthesise more slowly, the limiting factor is as likely to be the rate of electron
transport to NADP as the removal of electrons from NADPH2. To an extent, reoxidation of PSI by O2 will also provide
an alternative safe means of energy dissipation if the mechanisms for detoxification of super-oxide radicals do not become
overloaded. Finally it might be supposed that the switch from assimilatory dissipation to thermal dissipation might occur
as readily (or more readily) in shade leaves. All of this, together with the role of low temperature in photoinhibition,
suggests that, although inadequate CO2 assimilation may sometimes contribute to damage, the crucial factor is more likely
to be the strain imposed on (already inadequate) repair of the Q B-binding protein or on replacement of the damaged
protein within the thylakoid membrane. If, on the other hand, the upper limit of "safe" dissipation of excitation energy is
normally about 60% of the energy used in CO 2 fixation, factors affecting CO2 reduction (such as the availability of
cytosolic Pi) will increase in importance as external constraints (such as low temperatures) bring CO 2 fixation below the
40% level in bright light.

Figure 32.3. Photoinhibition in Helleborus niger.

Sun leaf taken from full Australian summer light, shade
leaf from 2 mole.quanta..m-2.s-1 . Both leaf discs were
assayed before & after the same "photoinhibitory"
treatment (4.5 hours at 1000 mole.quanta..m -2.s-1 &
8C). The shade leaf now showed loss of rate at high
PFDs (not seen after shorter periods of photoinhibition
- c.f. Fig. 32.1) & even greater decline in quantum
yield. Conversely the sun leaf photosynthesised faster.
Figure 32.3 bears on these relationships. Leaves from Christmas Rose (Helleborus niger) growing on the campus of the
Australian National University in Canberra, performed in a very similar way (in regard to quantum yield & maximal rates)
whether they were taken from full sun or from an immediately adjacent site in deep shade (0.5%, or less, of full sun). As
expected, the shade leaf was strongly photoinhibited by strong light 1000 mole.quanta.m -2.s-1 (at 80C) whereas the sun
leaf showed a significant increase in rate after such treatment & a decrease only after very prolonged exposure (36 hours
or more) to high light & low temperature. In the early stages of photoinhibition, however, there was often no decrease in
maximal photosynthetic rate of the shade leaf despite a decline in quantum yield & clearly, in these circumstances, the
leaf's capacity for energy dissipation cannot have been much inferior to that of its "sun" counterpart. Similarly, following
photoinhibition & subsequent overnight exposure to very low light, the shade leaf not only showed complete recovery but
an increase in rate which brought it very close to the performance of the "sun" leaf. Such results are best explained by a
rate of repair which is so rapid in the sun leaf, despite chilling, that it can increase its rate of photosynthesis (see Fig. 32.3)
during a treatment which is markedly deleterious to the shade leaf. The rate of repair in the shade leaf on the other hand
is not sufficiently rapid to allow it to cope with the continuing damage caused by high light at low temperature but, once
returned to low light & normal temperature, its capacity for CO 2 fixation not only returns but increases (Fig. 32.4).

Figure 32.4.
Photoinhibition (1000mole.quanta..m -2.s-1 at 8C)
& recovery from photoinhibition (2mole.quanta..
m-2.s-1 at 25C) in Helleborus niger.

Improvement in the rate at which shade leaves of sun species photosynthesise following long term exposure to higher light
is referred to as "acclimation" (see, for example, Anderson & Osmond, 1987) &, in some species, will undoubtedly
involve new synthesis of enzymes concerned in carboxylation as well as changes to the photosynthetic apparatus. In
Christmas Rose as we have already noted, the difference in photosynthesis between sun & shade leaves was small & the
increase in rate during the high-light illumination of the sun leaves could be reasonably placed into the category of "long
induction" (Rabinowitch 1956). This gradual increase in rate can be shortened by orthophosphate feeding (Walker &
Sivak 1986) & must, in part at least, relate to the availability of cytosolic Pi. It seems likely that similar changes in rate
would also be set in train in the shade leaf but that these would be masked, by photoinhibitory damage so long as strong
illumination at low temperatures was continued. Such short-term responses to sun & shade are important because many
plants exist in unstable & changing environments to which they must adjust. In addition, the short-term changes
themselves may well be part of the complex signalling process by which a plant perceives that it is in a "permanently"
changed environment & should, if it can, proceed to long-term adjustment. Such adjustment would eventually
manifest itself as "acclimation".

Although it is still possible to go to parts of S.E. Asia & find new genera, let alone a new species,
taxonomy is so comprehensive that it can offer authoritative identification & descriptions of most
of the plants that we normally encounter. By comparison, our
knowledge of the physiology of plants is virtually non-existent. In
many respects the physiology of the green leaf is dominated by
photosynthesis but there has not yet been any serious attempt to
evaluate the photosynthetic behaviour of most species. This is
largely because it has been such a difficult thing to do until now.

As a result of recent advances in molecular biology, our need to

“measurement is rarely non-intrusive”
improve our knowledge of plant physiology has assumed new
practical importance. Genetic engineering allows the possibility of
transferring a desirable ability from species to species. The
identification of what is desirable resides in improved
evaluation of physiological performance. Procedures such as
those described above, when used together with techniques such as
Q-analysis (Sections 13e & h) can make a significant first
contribution to such sorely needed evaluation.
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Krause, G.H. & Laasch, H. (1987b) Energy-dependent chlorophyll fluorescence quenching in chloroplasts. Correlation
with quantum yield of photosynthesis. Z. Naturforsch.C 42(5), 581-584.
Osmond, C.B. (1981) Photorespiration & photoinhibition: some implications for the energetics of photosynthesis.
Biochim. Biophys. Acta 639, 77-98.
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141-148.
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 PART F

AQUEOUS-PHASE MEASUREMENTS

33. INTRODUCTION

Although sections A-E inclusive have been concerned with measurements of O 2 in the gas-phase, polarographic
measurement of O2 is more usually undertaken in the aqueous phase & is currently such an every-day technique that
familiarity will largely obviate the need for explanation. For completeness, however, some details & descriptions will be
given.

34. PRINCIPLE

The principle is the same as before (Section 2) except that the rate of diffusion of O2 through water is slower (by 3 orders
of magnitude) than it is through air & this normally necessitates stirring of the solution, or suspension, if rapidity of
response is required (as it will be in any study of kinetics). Stirring is usually achieved by the use of a revolving magnet
(below) & a magnetic follower or "flea" within the vessel containing the reaction mixture. (If a single microcathode
detector is used the rate of consumption of O2 at the electrode surface may be so small that stirring may not be necessary).

35. THE "FLEA"

Most reaction mixtures are small in volume (1 to 3 ml) & the magnetic followers which are
employed are accordingly small in size & easily lost. Like real fleas, they can be immobilised by
pressing them into a bar of wet soap but are more normally given safe keeping on a bar magnet.
They can be readily made out of glass capillary tubing & thin iron wire, although
an element of skill is required & a good flea is therefore a thing to be cherished.
A "good" flea is one which is completely sealed but without conspicuously
heavy glass beads at either end. It is precisely the right length (i.e., a shade
shorter than the diameter of the reaction vessel) & encloses (for buoyancy) about “a good flea is a thing
three to four times as much air as iron. It must be pleasing to the eye on the basis to be cherished”
of these criteria but it must also live up to promise by spinning freely & evenly in
the reaction mixture. Uneven stirring can generate electrical "noise" on pen-recorder traces. Some
electrode circuits incorporate electronic filters to limit this effect & a small capacitor (say 10 F) is
often put across the recorder terminals for the same purpose. However, unless extra sensitivity is
required, (greater amplification can obviously contribute to greater "noise") time invested in
procuring a good flea & a smoothly revolving magnet (avoid sparking electric motors) is often
effect of stopping a rewarded by essentially "noise-free" traces.
stirrer
36. CALIBRATION.
36(a) Setting an Arbitrary "Air-line"

If a pen-recorder with a 10 inch (25 cm) chart is used, calibration normally starts by establishing electrical zero & then
adjusting the electrical output of the O2 sensor until, in distilled water, it draws a line across the chart about three quarters
way from zero (Fig. 36.1). Clearly, the precise electrical value will depend upon the electrode & its circuitry. With a
standard Hansatech Instruments Ltd electrode & CB1 control box with the "output" switch set to x1, a 1v full-scale setting
would be appropriate. If the stirrer is stopped, the trace should immediately fall towards zero but return, equally readily,
to its starting point once stirring is resumed. (This is a consequence of limiting diffusion of oxygen in unstirred solution
- Section 35, above).
 36(b) Establishing the N2 Line

If a few crystals of dithionite are dropped from a spatula into the stirred water the trace should fall, rapidly at first & then
more slowly, to near zero (Fig. 36.1). This is because O 2 is consumed according to Eqn. 36.1:

Na2S2O4 + O2 + H2O → NaHSO4 + NaHSO3 .......Eqn. 36.1

Alternatively, (& preferably) the empty chamber can be flushed with N 2 gas (at the operating temperature). Both
procedures establish the so-called "N2 line" (i.e. the line that the pen traces in a N2 atmosphere - Delieu & Walker 1972).

36(c) Residual Current

If the electrode were perfect the N2 line would coincide with the electrical zero of the recorder
but there is always a small "residual current" (Section 36c, & Fig. 36.1), which should not exceed
10%, at most, of the "air-line" (see 36d below). Some residual current can be diminished by
careful cleaning & by periodically storing electrodes in a dessicator. (Many electrodes are
embedded in epoxy resins & therefore tend to take up a certain amount of water. This can usually
be eliminated by dry storage. For this reason it is good practice to store a spare electrode in silica
gel & to alternate this, on a weekly basis, with one that has been in use).

A large residual current is an indication of malfunction &, if it cannot be corrected by cleaning

Response of electrode to
N2, O2 and air
or desiccation, the electrode will have to be discarded or returned to the manufacturer for repair.
(Some manufacturers will replace or refurbish damaged electrodes for less than the price of a
new electrode). Malfunction is also indicated by a ring of crystals forming around the junction
between an electrode & the epoxy resin in which it is sealed, reflecting the difficulties involved
in sealing one material (platinum) to another (epoxy resin) over a range of temperatures.
Electrolyte (which usually contains KCl) penetrates this junction & subsequently crystallises.
The crystals fracture the seal & eventually give rise to an unacceptably high residual current.
Microcathode electrodes sealed in glass are less vulnerable to damage of this sort than those with
cathodes sealed in epoxy resins but are likely to be more expensive (sometimes very much more
expensive) than those based on resins.

36(d) Establishing a New "Air-line"

Once the "residual current line" or "nitrogen line" has been established either by adding
dithionite or, ideally, by flushing the empty reaction chamber with nitrogen, calibration can
proceed (Fig.36.1). If dithionite has been used, the vessel & the electrode should be washed
repeatedly (about 10 times) with distilled water. This is most easily done with a wash-bottle &
an aspirator which is used to remove the added water as soon as the vessel is full. The
aspirator tube should be fitted with a narrow flexible tip so that the membrane of electrodes
inserted into the base of the chamber is not damaged & the "flea" is not swallowed. Finally, try to ensure that “the
distilled water which has been shaken with air (at the operating temperature) is added. This
establishes the "air-line". The air-line can be set electrically to any arbitrary value (Delieu &
flea is not swallowed”
Walker, 1981) but there is merit in using a convenient
& constant value. For example, if 2 ml is the preferred volume & 20C the
preferred temperature, it makes life easier if the difference between the N 2 line &
the air-line is set at 5.6 divisions of the chart recorder paper. This is because
air-saturated water at 20C contains approximately 0.28 moles of O 2 per ml (Table
“it makes life 36.1) & each large division of the chart will then be equivalent to 0.1 mole of O 2.
easier” Of course, if recording is started without displacing the air-line, the O 2 trace will
soon reach the top of the chart. So it also helps (if O 2 evolution is to be recorded)
to displace the air-line (following calibration) to the foot of the chart using the
"back-off" control on the O2 electrode "box". At one time, a 1.0 mV recorder was
almost mandatory but, over the years, advances in signal amplification have made it
possible to record at larger voltages & also to use a computer rather than a pen-recorder.
 Table 36.1. The Oxygen Content of Air Saturated Water

It should be noted that while air-saturated water is a very convenient standard for calibration it only serves as a first
approximation &, although it is ideal for comparative work, absolute calibration necessitates a different approach. This is
because other solutes affect the solubility & hence the "activity" (or concentration of O 2) in media (Section 36g) which
are commonly employed in work with isolated organelles such as chloroplasts. One method of absolute calibration is to
generate O2 in the medium by the action of catalase on pre-calibrated hydrogen peroxide (see Delieu & Walker, 1972 &
Figure 36.1).

36(e) Normal Calibration Procedure Summarised

The above procedure (Section 36a-d) is summarised in Fig. 36.1.

Figure 36.1. Calibration Summarised.

(i) With water in the vessel set an arbitrary

"air-line" by adjusting the electrical output of the
electrode until the recorder pen is near the top of
the chart.
(ii) Add dithionite crystals (or flush empty vessel
with N2 gas) to establish the "N2 line".
(iii) If dithionite has been used, wash the
chamber several times with distilled water.
(iv) Add water, saturated with air at the
operating temperature, to obtain a true "air-line".
(v) Adjust this true air-line electrically so that it
is a convenient multiple of the number of moles
of O2 per ml of water (e.g. at 20C there are 2.8
moles/ml therefore if a reading of 5.6 is selected
as the calibrated "air-line" & a reaction mixture
of 2 ml is used, each division of the chart will be
equivalent to 0.1 moles of O2).
36(f) Absolute Calibration

Additional checks on calibration & linearity of response may be carried out as follows:

(i) A small quantity of catalase is added to a reaction mixture which is allowed to equilibrate in the closed vessel.

(ii) Hydrogen peroxide is standardized by adding it to an acidic solution of potassium iodide & titrating the iodine released
with sodium thiosulphate (freshly prepared, e.g., from a newly opened BDH analytical ampoule or standardized, in turn,
against an iodine solution). Sodium thiosulphate (Na 2S2O3) is a reducing agent by virtue of the following reaction (Eqn.
36.2)

2S2O3 = → S4O6 = + 2e- ......Eqn.36.2

It will therefore reduce iodine to iodide:

I2 + 2e- → 2I- ......Eqn.36.3

so that

2Na2S2O3 + I2 → NaS4O6 + 2NaI ......Eqn.36.4

&, since hydrogen peroxide reacts with iodide in acid solution as follows:

2I- + H2O2 + 2H+ → I2 + 2H2O ......Eqn.36.5

& because H2O2 also yields O2, by catalatic dismutation, according to equation 36.6:

2H2O2 → 2H2O + O2 .......Eqn.36.6

the relationships between thiosulphate, hydrogen peroxide & oxygen evolved are 1 ml of 0.01 N Na 2S2O3 5moles H2O2
2.5moles O2.

The titration procedure (Vogel, 1961) is to add 0.1-2 ml of hydrogen peroxide solution to a mixture containing 0.5 ml of
10% KI & 2 ml of H2SO4 (in a total of 4.5 ml) to which one drop of 1% ammonium molybdate solution is added (as a
catalyst) & then to titrate after allowing 3 minutes for the release of iodine to be completed. Thiosulphate is added until
the solution is a pale straw colour, at which point two or three drops of starch glycollate are also added. As the titration is
continued a green colour develops which is followed by the appearance of the characteristic blue, of the iodine-amylose
complex immediately prior to the colourless end point. Titration of the H 2O2 used in the experiment illustrated in Fig.
36.2 gave a value of 0.1345 moles for 0.2 ml of H 2O2 calculated from a mean titration value of 5.38 ml of thiosulphate.
When 2 l aliquots were subsequently added to 2 ml of deionized water at 20C in the electrode vessel, a mean recorded
value of 0.1380 moles was obtained. Fresh, "10-volume" commercial H 2O2is approximately molar, so that 1 l of such a
solution would yield approximately 0.5 moles of oxygen by catalatic dismutation (Equation 36.6).

(iii) Small aliquots of standardized H2O2 are added (in sequence) to the vessel, using a microsyringe. The recorder
registers the O2 evolution following each addition & a series of uniform steps are obtained (Fig. 36.2) indicating both the
extent & the linearity of the response. If this method is used with catalase in distilled water at 20C, the oxygen recorded
corresponds very closely to that obtained by using the value of 0.28 moles in setting the air-line. With other aqueous
solutions the recorded values will depend on the effect of the solutes on the activity of oxygen within the solution at a
given concentration. For example, Fig. 36.2 illustrates the traces obtained using H 2O2 & catalase in twin vessels
containing either 2 ml of deionized water or 2 ml of resuspending medium (Section 37g). It will be seen that each
electrode gave virtually the same response in each case but that the response in water was about 11% smaller than that in
the assay medium.
 Figure 36.2.
Serial additions of H2O2 (0.25 moles in 2l) to duplicate 2 ml reaction mixtures
containing water & catalase (left) & resuspending medium & catalase (right). The
"real" values obtained with water were, on average, 11% lower than the "apparent"
values obtained with assay medium (after Delieu & Walker, 1972).

For some purposes, absolute calibration is inescapable but it should be noted that the catalase method is tedious &
exacting. Not only is it necessary to determine, with great precision, the concentration of H2O2 in the solution added, but
it is also important to prevent bubble formation during dismutation & to add the peroxide solution (inevitably containing
excess dissolved oxygen) in very small but very accurate aliquots. For most purposes, therefore, relative calibration
(Section 36e) is to be preferred in conjunction with a correction factor which can be derived (as in Fig.36.2) for any given
solution & temperature.

If isolated chloroplasts are at hand, an additional check can be applied by using a ferricyanide solution, freshly prepared
from clean crystals & protected from strong white light. With such a solution, & washed chloroplasts (free of endogenous
electron donors such as ascorbate) which do not exhibit appreciable dark or post-illumination O 2 uptake, 1 mole of O2 is
evolved for every 4 moles of ferricyanide added (Section 41a). Except under well-defined conditions, however, the
possible errors are substantial (see e.g. Whitehouse et. al., 1971) & the effort involved is no less considerable than the
peroxide-catalase calibration.

36(g) Using Constant Chlorophyll

If a prolonged series of experiments is envisaged, there is also merit in working at a constant & pre-defined chlorophyll
concentration. Chlorophyll is readily determined by adding 50 l of chloroplast suspension to 20 ml of 80% acetone (16
ml of acetone plus 4 ml of H2O) & quickly filtering or centrifuging (avoid evaporation or exposure to bright light). The
optical absorbance (1cm light-path) of the chlorophyll solution at precisely 652 nm (Bruinsma, 1961) is then multiplied
by 100/9 to give g chlorophyll per ml of original suspension (check the calibration of the spectrophotometer as
necessary). Alternatively, the reciprocal of the absorbance x 9 approximates to the number of l of chloroplast suspension
required to give 100 g of chlorophyll (Walker, 1980). The amount of chlorophyll to be used will, of course, vary with the
nature of the experiment but 100 g/ml is about the upper limit for light saturation in a conventional O 2 electrode vessel.

36(h) Calculation of Rate

An advantage of using constant chlorophyll in a series of experiments is that it eliminates a variable & that it also
facilitates both comparisons (within & between experiments) & the determination of rates.

Table 36.2. Rate for a mixture containing 0.1 mg chlorophyll.

Angle 1cm=1 min 1cm = 0.05µmoles

68 150 300
66 135 270
64 123 246
54 82 164
51.5 75 150
50 71 142
Thus the angle of the trace (of change in O2 against time) can be measured (using a protractor) & multiplied by an
appropriate factor. For example in Fig. 36.3 the angle θ is 680 & tan θ = 2.5. Thus if time & ∆ O2 have been recorded on
the same scales as in Fig. 36.3 (in which the vertical scale is 0.1 of the horizontal scale), the value 2.5 will be a measure
of the number of moles of O2 evolved in 10 minutes. This, in terms of O 2 per hour could become 15 (2.5 x 6) & on a 1
mg of chlorophyll basis (since there was only 0.1 mg of chlorophyll in the reaction mixture) would become 150 (15/0.1).
Had the same trace been recorded at 2 cm/min, the angle θ would have been 51.5, the tangent 1.25 & this value would be
a measure of the O2 evolved in 5 rather than 10 minutes so that the final rate (1.25 x 12. x 0.1) would remain the same
(Table 36.2.)

Figure 36.3. Calculation of rate.

In this figure the vertical scale has been electrically set to
display the change (∆) in oxygen in one tenths of moles
according to the procedures described in Section 36. The
horizontal scale has been selected on the chart recorder to
display time in minutes & the reaction mixture (a suspension of
intact chloroplasts engaged in CO2-dependent O2 evolution &
displaying a characteristic lag) contain 100 µg of chlorophyll.
Traditionally, rates have been expressed as µmoles O 2 per mg
chlorophyll per hour & the rate here is readily seen to be 150
µmoles oxygen mg -1 Chl.hr-1 from the fact that 0.5µmole of O 2
was evolved in 2 min by chloroplasts containing 100 µg
chlorophyll (i.e. 0.5 x 60). Alternatively, tables can be compiled
0.1 x 2
(see Table 36.2) based on the tangent of the angle θ. These save
on arithmetic &, once correctly compiled, eliminate errors in
subsequent calculations.

Table 36.2 is part of a larger table (for all angles) based on the relationship tan θ x n = rate of O2 evolution expressed in
µmoles mg -1 Chl. hr-1 where

N = 60 x Chl (mg)
time x oxygen
Thus, if chart speed (in minutes per cm) is 1, oxygen (in µmoles per cm) is 0.1 & the reaction mixture contains 0.1 mg
chlorophyll the rate for an angle of 68 (tan θ = 1.25) would be

2.5 x 60 x 0.1 = 150

1 x 0.1
Pocket calculators make tables of this sort largely superfluous but, once prepared &, particularly for work at constant
chlorophyll, they can remove the tedium & minimise the errors of computation.

37. MECHANICAL ISOLATION OF CHLOROPLASTS

37(a) Conventional Wisdoms

For the last two decades, mechanical separation of fully competent chloroplasts (i.e. those capable of achieving light-
dependent CO2 fixation of the same order of magnitude as the parent tissue) has, in general, only been achieved with
spinach & with peas. Even then, this has meant using "good" spinach (Section 38) or "good" peas. It is not possible "to
make a silk purse out of a sow's ear". Equally it is not possible to isolate chloroplasts, which will display high rates of
photosynthesis, from spinach containing large quantities of starch, or calcium oxalate, or from leaves, which are anything
other than healthy & actively growing. For many purposes, therefore, there has really been no alternative to "good"
spinach.
 More recently, very respectable chloroplasts have been isolated mechanically from a limited number of
additional species & chloroplasts have been enzymically separated (Section 42) from poor spinach & from
notoriously difficult species (like sunflower) which have not yet proved susceptible to the mechanical
approach.

Mechanical isolation involves disrupting the leaf but not the chloroplast (Section 37b, below & Fig.
37.1) in a grinding medium. An appropriate medium (Robinson, Cerovic & Walker, 1986) always
contains an osmoticum (usually 1/3 M sugar or sugar alcohol) to prevent swelling or shrinking. It
may also contain a buffer to maintain pH, an anti-oxidant (such as ascorbate) & a variety of other
additives (some included for traditional or ritualistic reasons or on the principle of "why change a
winning team?"). Following maceration of the leaf, the chloroplasts are separated as quickly &
gently as possible by filtration & centrifugation & then resuspended in another medium containing
the same, alternative, or additional goodies (Fig. 37.1).

37(b) Grinding

Chloroplasts can be prepared by pounding leaves in a pestle & mortar & this has much the same
therapeutic value as kneading dough but it is hard work & difficult to do in a reproducible manner.
In an increasingly sophisticated world it also smacks of alchemy & is usually despised. If you like old
things, however, don't forget to chill the pestle & mortar, use a small quantity of grinding
medium & pound very vigorously for a minute or two, preferably on the floor. “a respectable chloroplast”
All manner of motor-driven homogenisers offer a greater degree of ease & control. Details of procedure vary with the
blender &, at the outset, there is no alternative to trial & error. Clearly, there should be sufficient medium to cover the
leaves. The medium is best frozen to a slush (the consistency of melting snow) because the latent heat of melting then
increases its cooling capacity by an order of magnitude. Similarly, the mechanical resistance of the slush tends to hold the
leaves against the blades during the first moments of blending.

If you have a choice, use a blender which will mash your leaves into a coarse "brei" within 10 sec. A
really good homogeniser like a Polytron (Kinematica GmbH, CH-6000 Kriens, Luzern,
Switzerland) will do this in under 5 seconds. Continued homogenisation will increase the
yield but also increase the damage to the chloroplasts. A bad blender or homogeniser is one
which causes a lot of frothing. Cavitation effects seem to damage fragile chloroplast envelopes,
possibly by smearing nasties (from the vacuole & cell wall) on active surfaces (Leegood et.
al., 1981; Robinson et. al., 1979; Walker 1971, 1980). It often helps to stack the leaves &
slice them with a sharp knife prior to blending but don't be tempted, by admiration of skilled
chefs, to use a chopping action. This causes detrimental bruising of the leaf tissues.
“it smacks of alchemy”
Spinach responds well to 30 minutes pre-illumination in cold water. For some reason, never yet established, this actually
increases yield as well as activity. Spinach will also store beforehand & good chloroplasts have been prepared from
spinach stored in the dark at 5C, in a crate, for as long as a month. Conversely, pea leaves often deteriorate very rapidly
as soon as they are harvested & it therefore makes sense, if working with peas, to emulate the North American practice
of cutting the corn cob only when the water in the pot is already boiling. Once grinding is at an end, the chloroplasts
should be separated from the brei in the shortest possible time.

37(c) Squeezing & Filtering

Have a beaker (400 ml is a good size) & a couple of thicknesses of cheese-cloth or muslin chilled & waiting. "Hairy" cloth
is better than nylon, mira cloth etc. Spread it across the top of the beaker & decant the contents of the blender into it.
Gather up the corners of the cloth & twist & squeeze with the fingers so that the juice is expressed from the brei. Filtration
through additional layers of muslin or a sandwich of muslin & cotton wool is useful & the juice may be squeezed directly
on to such a "sandwich" stretched across a second beaker. Ideally this filter sandwich should be rinsed, immediately prior
to use, with a little cold grinding medium & this time there should be no squeezing so that the chloroplast suspension will
simply drain through under gravity. As quickly as possible the filtered suspension should be decanted into chilled
centrifuge tubes. With practice, these can be adequately "balanced" by eye but use a balance to equalise them prior to
centrifugation if you are in any doubt about your ability to do this successfully.

37(d) Centrifugation

Like grinding, this is often a matter of trial & error until you know, by experience what your centrifuge will accomplish.
Ideally, at this stage of the preparation (Fig. 37.1), you should have about 160 ml of suspension equally distributed
between four 50 ml centrifuge tubes. These should be put into a swing-out centrifuge with sufficient acceleration &
deceleration to go from rest to about 5000 rpm (& back to rest) in about 90 seconds. Alternatively, you may prefer to
accelerate to about 1500 rpm & maintain this speed for 1 min before deceleration. Much longer centrifugation may be
imposed upon you by the characteristics of your centrifuge but, if a short spin will give you a sufficiently large pellet for
your needs, do not even contemplate prolonging spinning for longer than absolutely necessary.
This will increase the yield only at the expense of quality &, unless you can contrive to use a great many chloroplasts in
a short time, you will probably discard most of those that you have prepared anyway. If you have access to a refrigerated
centrifuge you should use it at (or just below) zero degrees but since the entire centrifugation in a one-stage conventional
chloroplast preparation can take as little as 90 seconds it possible to avoid undue warming, even in hot climates, by
pre-chilling tubes & holders to be used in an unrefrigerated bench centrifuge.

A swing-out centrifuge with new, or carefully treated centrifuge tubes, is better than an angle-head with old tubes. This
is because the sedimenting chloroplasts migrate to the outer wall of the tube in an angle-head &, particularly if the wall
has become roughened by harsh cleaning, the effect can be rather like pulling a balloon across a concrete floor with a large
weight on it. You should avoid washing centrifuge tubes with detergent & indeed it is best, if you can afford it, to set aside
a set of tubes & glassware which is never exposed to detergent but simply rinsed in distilled water after use. Depending
on the centrifuge, some advantage may also derive from shortening the sedimentation path - i.e. by filling the tubes to one
half, or less, of their capacity. If you are likely to be using chloroplasts routinely & wish to secure the best preparation
possible, time invested in optimising centrifugation would be well spent because it is one of the most critical parts of the
whole procedure.

Figure 37.1. Diagrammatic representation of mechanical

preparation of isolated chloroplasts.

Starting top left.

1. The leaves are pre-illuminated whilst floating on cold water (for
half an hour or so).
2. They are then shaken dry and sliced, not chopped, on a board
using a sharp knife.
3. They are blended in a domestic blender (or, preferably a polytron)
and the brei squeezed through muslin into a chilled beaker.
4. The suspension is filtered through a sandwich of cotton wool in
muslin. Following filtration, the suspension is decanted into
centrifuge tubes and centrifuged (preferably in the cold, in a swing-
out centrifuge).
5. Finally the supernatant is decanted and the chloroplast pellet is
ready for resuspension.

37(e) Resuspension

Chloroplast pellets are sticky, so that, at the end of centrifugation, the supernatant can be decanted by pouring
confidently into a beaker without fear of undue loss. The inside of the tube can then be wiped with a tissue & the
chloroplasts are ready for resuspension. For some purposes, the chloroplasts may need to be washed but most
procedures are more or less the same until this stage.

If it is a one-stage (no second centrifugation) preparation you may wish to rinse the surface of the pellet with a little
chilled resuspending medium poured carefully down the side of the tube & then decanted. After that, resuspension is
a matter of choice. Ulrich Heber, still probably the unofficial world record holder for "best" chloroplasts, prefers to
add a millilitre or two of medium to each tube &, standing in a cold room, agitating the contents with a trembling
motion of his hands. Others prefer to dislodge the pellet with a small soft paint-brush or by sucking the suspension in
& out of a pipette. Personally, I prefer the "Bob Whatley" method, which is to use a small piece of cotton-wool
soaked in chilled medium & pushed to & fro with a glass rod. This requires a modicum of skill but it is gentle resuspending
& thorough. Ruptured chloroplasts also tend to adhere selectively to the cotton wool. using cotton
wool & a
Resuspension for any purpose is best achieved by using small quantities (2 or 3 ml per tube) of resuspending glass rod
medium (Section 37g) until the pellet is homogenous. Otherwise you are likely to find yourself pursuing pieces
of dislodged pellet through a larger volume of medium with little hope of uniform resuspension. Following this step, the
volume can be adjusted to whatever is desirable. Relatively large quantities (say 40 ml/tube) of medium will be preferred
if you wish to wash the chloroplasts & recover them in a second spin, whereas relatively small quantities are best if you
wish to use your chloroplasts as only one constituent in a conventional reaction mixture containing 50-100 g chlorophyll
(Section 41). You may also find some advantage in storing your chloroplasts as a pellet until immediately prior to use
(Jensen & Bassham, 1965) - a procedure which may diminish loss of essential ions, such as K +, to the medium.
 37(f) Grinding Medium

Chloroplasts contain three main compartments, each surrounded by a membrane. The

thylakoid membrane houses the chlorophyll, other pigments & electron carriers.
Protons driven, by electron transport, into the compartment bounded by the thylakoid
membrane, discharge through the ATPase (Section 41 & Fig. 41.3) into the stroma. The
stroma contains the enzymes involved in carbon assimilation & the stroma is contained,
in turn, by a double envelope. Movement of inorganic ions & metabolites into the space
between the two envelopes appears to occur very freely but the inner envelope
incorporates the phosphate translocator which controls the principal movements of
metabolites into & out of the chloroplast. This basic structure, which has only become clear in
the last 2-3 decades, has many implications for chloroplast isolation & the nature of the grinding
medium (see e.g. Edwards & Walker, 1983).

Firstly, the chloroplast is a fairly fragile organelle. If it contains starch, centrifugation can be a bit like swinging a paper
bag full of potatoes round your head - there is a real risk of the bag bursting. For this reason, it is easier to isolate intact
chloroplasts from peas or spinach early in the day before diurnal starch accumulation is well advanced. Otherwise the
envelopes are reasonably resistant to mechanical shock but very susceptible to osmotic damage. This is because, like any
semi-permeable membrane, the chloroplast envelopes will permit entry of water from a hypotonic solution (i.e. one with
a lower osmotic pressure as a consequence of containing fewer solute molecules than the stroma). As already noted
(Section 37a), it is necessary to employ an osmoticum in sufficient concentration to make the grinding medium & the
chloroplast isotonic (i.e. of equal osmotic pressure) so that there is neither swelling nor shrinking of the chloroplasts
during separation. For many years, chloroplasts were isolated by Arnon & Whatley in "tris-NaCl" (i.e. in a medium
containing trishydroxymethylaminomethane, as the buffer, & NaCl as the osmoticum). There were sound reasons for
doing this. Arnon & Whatley preferred an inorganic osmoticum to a sugar in order to eliminate the possibility of
substrate-driven ATP formation in their pioneering studies on photophosphorylation. "Tris" was simply one of a very
limited range of relatively inert buffers readily available to the plant biochemist in the 1950's. For a variety of reasons,
these chloroplasts (which played a major role in furthering our understanding of photosynthesis), were very imperfect
when it came to CO2 fixation. Increased rates followed a return to Hill's practice of using one third molar sugar as the
osmoticum & to other procedures adopted at that time (Walker, 1964). Sucrose will serve very well as an osmoticum &
considering the fact that domestic sugar is both cheap & pure it is surprising that it is not more widely used to make up
large quantities of medium which frequently go down the sink within a very short time of being prepared. Originally
(Walker, 1964), sorbitol was preferred to sucrose, glucose & fructose for much the same reason as Arnon & Whatley used
salt - i.e., because it was less likely to be metabolised. By now, the use of sorbitol has become time-honoured (& the author
wishes that he had negotiated a percentage with the manufacturers) but other sugar alcohols (e.g. manitol) or sugars (e.g.
glucose) are equally effective & sometimes to be preferred (sorbitol, for example, will interfere with some molybdate
assays for inorganic phosphate & is clearly inappropriate in such circumstances). Ficoll (a sucrose polymer) which gives
a relatively viscous solution at an appropriate osmolarity, has also been used as an osmoticum & seems to affect the shape
of the isolated chloroplast. It may be used to advantage in spectroscopic studies in which stirring is precluded &
sedimentation under gravity needs to be minimised.

Choice of pH & buffer has also varied over the years. It is possible to prepare active chloroplasts from spinach in sorbitol
alone, & for many purposes almost any pH between 5.5 & 8.5 will suffice. Inorganic phosphate was used in the first
preparations of active chloroplasts from peas (Walker, 1964) but was superseded by "tricine" (N-
Tris(hydroxymethyl)methylglycine) following Norman Good's (1966) outstanding gift to the scientific community of a
new range of "zwitterion" buffers such as "Hepes" (N-2-Hydroxyethyl-piperazine- N'-2-ethanesulphonic acid). In turn,
these have been mostly restricted to use in resuspension & assay because of their cost, whereas inorganic pyrophosphate,
which also serves as a chelating agent, has been used extensively.

An anti-oxidant is often included & ascorbate is the usual choice. Isoascorbate once also threatened to become mandatory
but has no special virtue & was originally preferred to the isomer only because it was freely available in the U.K. in the
1960's as the sodium salt. Originally both Mg ++ & Mn++ were believed to be advantageous & were added in the presence
of EDTA to prevent precipitation by phosphate. Surprisingly, their omission can be detrimental although it is difficult to
judge why. Protective agents such as Bovine Serum Albumin may also be added. BSA affords some protection against
phenolics but may also affect the charge distribution on the envelope in a beneficial way. Relatively inactive chloroplasts
from "difficult" species like sunflower can function very well in a "reconstituted system" (Section 44) suggesting that the
"difficulty" lies in adsorption of deleterious materials on the envelope whereas the internal stroma & thylakoids remain
undamaged (Delaney & Walker, 1976).
A traditional grinding medium (Cockburn et. al., 1968) would therefore look something like this:

Sorbitol (330 M)
Na ascorbate (2 mM)
Na pyrophosphate (10 mM) at pH 6.5 (adjusted with HCl)
MgCl2 (5 mM)

but it should again be emphasised that only the osmoticum is really vital & none of the quantities are really critical. In
short, there is no need to waste time ensuring that the sorbitol is 0.33 rather than 0.32 or adjusting the pH to within 0.1 of
a unit.

Whether or not this medium is still the most useful for general purposes is quite another matter. One which can be
recommended is that of Cerovic & Plesnicar (1984) which contains

Sorbitol (340 mM)

KCl (0.4 mM)
EDTA (0.04 mM)
Hepes-KOH (2.0 mM) at pH 7.8

but it should be noted that in order to secure best advantage from this "low ion" & other media (Nakatani & Barber, 1977)
it should be employed throughout (i.e., as both grinding & washing medium). Another extremely interesting innovation
is that of Robinson (1986) who has used 200 mM KCl as the osmoticum rather than a sugar or sugar alcohol. This is
arguably nearer to the in vivo osmoticum & helps to maintain the high K content which appears to be necessary for
optimal activity (Kaiser et.al., 1980).

37(g) Resuspending & Assay Medium

One suitable medium (RS1) contains

Sorbitol (330 mM)
EDTA (2 mM)
MgCl2 (1 mM)
MnCl2 (1 mM)
Hepes-KOH (50 mM) at pH 7.6
BSA (0.2%)

Again, this is a traditional mixture & it is difficult to say what benefit derives from the inclusion of the MgCl 2, the MnCl2
& the chelating agent (EDTA) which they, in part, make necessary (but see Edwards et. al., 1978). An osmoticum is
essential & it is convenient to store the chloroplasts at or about the pH that will be used in the reaction mixtures (see
Section 41). Tradition, however, is hard to escape &, if you are preparing chloroplasts for the first time, you would be
advised to use a well established procedure initially. Once you know that you can prepare chloroplasts from "good"
spinach (Sections 37a & 38) which will support CO2-dependent O2 evolution (at 20C) at rates of about 100 moles -1 O2
mg Chl-1 hr-1 (Section 36f & Fig. 36.3) you will know that you have mastered the simple art of preparation & are licensed
to take as many liberties as you wish with the system. The rate of CO 2-dependent O2 evolution is really the only criterion
for full photosynthetic competence (the category "A" chloroplasts of Hall, 1972) & for any serious work a rate of about
100 is almost mandatory & a rate of 200, or more, most desirable. Apart from anything else, isolated chloroplasts do
deteriorate both during use & during storage & it is possible to do so much more, so quickly, with "good" chloroplasts
rather than "bad". Similarly, if you come up with some totally new observation you will feel much more confident that it
will be "real" rather than artefactual, if it is obtained with active chloroplasts.

Many different resuspending media are used for specific purposes. As already noted (in 37f above) chloroplasts prepared
in "low ion" grinding medium are resuspended in low ion grinding medium. In Robinson's (1986) procedure 200 mM KCl
may also be substituted, for sorbitol in the resuspending medium (RS1).

37(h) Purification

(i) Washing.

Chloroplasts prepared by the simplest one-spin procedures are very active &, once the procedures have been mastered &
solution etc made ready, it is possible to go from intact leaf to chloroplast suspension in about 15 minutes. For general
purposes such chloroplasts are also very pure. The extent of contamination by other cellular constituents, such as
mitochondria, etc., is minimal but it does exist. For example, such chloroplasts will readily evolve O2 in the dark if given
hydrogen peroxide. The contaminating catalase which is largely responsible for this reaction (Eqn. 41.7, Section 41c)
comes from peroxisomes which are ruptured during grinding. Apparently this enzyme then becomes loosely adsorbed to
the chloroplast envelope. Washing, i.e. resuspension of the original pellets in relatively large quantities (roughly
equivalent to the original volume of grinding medium) of a washing medium, followed by a second centrifugation, will
remove a large fraction of such contamination. Washing media can contain almost anything in addition to the osmoticum
which (with the possible exception of K+ - see 4f) is the only really essential component. Grinding medium will suffice
or resuspending medium if it is essential or affordable. Two washes can be better than one for some purposes but it should
be borne in mind that all of the handling procedures carry the risk of mechanical damage to the chloroplast envelope &
that prolonged washing may also lead to undesirable losses (e.g. leaching of K + etc). Similarly, ageing (itself still a very
ill-defined process) will continue to occur at an accelerated rate from the moment that the chloroplast is separated from
its cellular environment.

(ii) Percoll.

A number of procedures involving silica-sol gradients have been reported (see e.g. Morganthaler et. al., 1974; Price et.
al., 1979) but the following, based on a method introduced by Mills & Joy (1980) is quick & cheap (because it does not
call for a great deal of Percoll). All it involves, is resuspending the initial pellets (37e) in about 6ml of medium,
transferring this to two 50ml glass centrifuge tubes, & introducing a 4ml "cushion" (below the chloroplast suspension) of
4 parts by volume of "Percoll" (a modified silica gel from Pharmacia) in 6 parts by volume of resuspending medium. This
cushion of 40% (v/v) Percoll may be put into position by using a syringe or Pasteur pipette. After centrifugation at 1700g
for 1 minute, followed by deceleration without braking, the intact chloroplasts are pelleted whereas the fragmented
chloroplasts & other cell debris & organelles are retained at the interface between the Percoll & the added chloroplast
suspension. The supernatant & the Percoll layer are aspirated & the pellet resuspended. Purification can be achieved
within 15 minutes in this way & gives cleaner & more active preparation. Inevitably, it is not done without cost because
the yield is only 30-40% of that in the original pellet, indicating that some intact chloroplasts must be retained at the
Percoll interface & thereby causing concern that this procedure might select in favour of particularly dense, as well as
intact, chloroplasts. There is also one report that Percoll can be inhibitory (Stitt & Heldt, 1981) although this inhibition
(which is not in line with general experience) could be overcome by dialysis. More sophisticated gradients have also been
successfully employed e.g. by Mourioux & Douce (1981).

38. SPINACH
38(a) Why Spinach?

Spinach (Spinacia oleracia L.) is undoubtedly a trying &, some

might even say, a perverse plant but it could be argued that its
God banishes
use has done at least as much to further our understanding of spinach from
photosynthesis as that of its distant cousin Chlorella or its C4 heaven
counterparts, sugar cane & maize. Why, we might ask, was
spinach chosen to play such a major role? Part of the answer lies
in the fact that spinach is a horticultural crop of importance in
Europe, particularly in the Netherlands. European traditions die
hard in the United States & there too it has been readily
available to the plant biochemist who wished to buy it
from his or her corner store. No doubt much of
plant science is still totally inadequate
because most European & North
American scientists remain lamentably uninformed about tropical species but the fact remains that spinach is largely a
traditional choice, reflecting the origin of its users. This, of course, is only part of the story. These same scientists would
have had even less difficulty in laying their hands on cabbage or lettuce. However, the former is never used & the latter
does not easily yield fully competent CO2-fixing chloroplasts & has only rarely been first choice. The fact is that spinach
also has some intrinsic virtues. It is soft (& therefore readily disrupted) but, most of all, it is largely free of phenolics &
other inhibitory compounds which are prevalent in the majority of cultivated species. Having said that, no one in their
right mind would choose to work with it because it is, or can be, so very difficult to grow.

38(b) The Origin of Spinach

The genus Spinacia belongs to the family Chenopodiaceae. Compared with a really ancient crop like Amaranthus edulis,
(which was grown in Mexico from about 4800 BC & from which the Aztec emperor Montezuma received an annual
tribute of 200,000 bushels of grain), spinach is a relative newcomer but it was mentioned in Arab & Chinese writings as
being consumed in & before the 10th century & it was grown in Spain as early as the 12th century. It is not a tropical
species but, contrary to fairly widely held views, it can do well in warm climates & is believed to have originated in Iran,
or that immediate area (Arab, isfinâj; Pers, isfânâj).
 38(c) Growing Spinach

There is a famous book called "Growing Madder" which describes the cultivation of Madder (Rubia
tinctorum) which was the source of the once important red dye alizarin. Its title would also probably be an apt
description of the feelings of anyone who has set out to grow spinach for scientific purposes. One reason is that
spinach is a "long-day" plant i.e. one which flowers or "bolts" when the daylength to which it is exposed exceeds a
critical length. The critical daylength varies with the cultivar &, especially in the Netherlands, considerable effort has
been put, by plant breeders, into the production of cultivars which are best suited to
sowing at different times of the year. In general, however, the critical daylength is
about 10 hours & in the north of the United Kingdom, where summer days can be as
long as 21 hours, there is little hope of growing spinach in the summer without it
"bolting". At worst, this will result in virtually no leaf. Such spinach will give rise to a leaf about as
large as a thumb nail & then, presumably feeling fulfilled, will flower & die. Conversely, optimal growth
is only achieved at or about the critical daylength, & in fairly high light. Again in the U.K.,
this means that the daylength must be artificially shortened in the summer & that both light
Spinach flowers in long days intensity & daylength must be artificially augmented in the winter (Fig. 39.1). Nearer the
equator, spinach can be grown in the field for most, or even all, of the year but it remains a
crop which requires adequate irrigation & plenty of nitrogen. Ironically, in many countries in which it would undoubtedly
grow well it is virtually unknown. "Spinach" may be available in local markets but it will almost certainly turn out to be
a totally different species (see Section 38d) which will not easily yield actively competent CO2-fixing chloroplasts.

38(d) Alternatives & What to Avoid

It has to be said, immediately, that there is no real alternative to spinach. This is not only because spinach chloroplasts
are usually so much better than those prepared mechanically from other species but because, by now, there is such a huge
scientific literature on spinach chloroplasts that there would be no point in looking elsewhere unless there was a very good
reason for so doing. Very good chloroplasts can be prepared from pea (Pisum sativum) leaves but they are different, in
some important ways from spinach chloroplasts (see e.g. Stankovic & Walker, 1977; Robinson & Wiskich, 1976) & rarely
match those from Spinacia in terms of activity. Recently, good chloroplasts have been prepared from sugar beet leaves
(Robinson, 1983).

Many other species have been called "spinach" but they are best avoided & their chloroplasts will certainly not compare
with those from spinach if prepared by the procedures described here. "Perpetual spinach", for example is a cultivar of
Beta vulgaris & "New Zealand spinach" is Tetragonia expansa. Neither is a sensible choice.

38(e) Varieties

Some varieties of spinach have become fashionable from time to time & have become widely used in consequence.
Perhaps the most famous is Yates Hybrid 102 alias U.S. National Hybrid 74 from the Ferry Morse Seed Company, P.O.
Box 100, Mountain View, California 94042. "Virtuosa" from Rijk Zwaan, P.O. Box 40-2678 ZG Burg, Crezeelaan
40-2678 KX De Lier, Holland is particularly well suited to water culture. Growing conditions are often more important
than varietal differences, however, & the prospective user should not turn to alternatives unless locally available spinach
consistently fails to match the rates reported in the literature.

Both Robinson (1983) who used Pisum sativum, var. Massey Gem, in Australia & Cerovic & Plesnicar (1984) who used
var., Mali provensalac, in Jugoslavia reported exceptionally good rates but, again, locally available
varieties might well have much to offer.

39. HYDROPONIC CULTIVATION OF SPINACH

39(a) General Procedure

At what is now the Robert Hill Institute in Sheffield, we have had more than 10 years experience
of growing spinach in water culture. While we feel that we could comfortably use another
century of experience without even nearing perfection, we have devised a system (Fig.
39.1) which works well. Seeds are sown in polystyrene drinking cups with a
perforated base. Regrettably, while such cups are cheap & readily obtainable,
there is no great call for cups with holes & the perforations have to be done by
hand. The cups are disposable but can be used more than once for economy.
They drop into holes in polystyrene supports which are available commercially
(e.g. from Accelerated Propagation Ltd, Vines Cross, Heathfield, Sussex, U.K.) for supporting
more conventional plastic plant pots (which may be preferred in some systems). If the
polystyrene support is now supported in turn by the edges of a tank fashioned out of wood,
plastic or whatever, & lined with polythene sheet, the bottoms of the cups will dip by a
a cultivated spinach
centimetre or two into a water-culture solution. This is aerated using a compressor & glass tubing (lying on the floor of
the tank) fitted with capillary air outlets. A pump raises the level of solution in the tank to that of a large "over-flow"
through which the solution returns to a reservoir, below the tank. The pump (activated by a time-clock) raises the level in
the tank to that of the main over-flow for an hour or so twice a day. When the pump is switched off the solution falls,
through small holes made near the top of the over-flow pipe, to a level just below the cups. This ensures that the contents
of the cups are moistened twice daily & also that the cups can drain freely back into the tank. The roots can also grow
through the perforations in the floor of the cups into the solution. This device ensures that the hypocotyl region of the
seedling is kept relatively dry (& therefore healthy) but that, at the same time, the developing plant is kept well irrigated
& well supplied with inorganic nutrients.

Figure 39.1. Detail of hydroponic system for spinach.

For full explanation, see text. Plants are grown in polystyrene drinking
cups and these provide an indication of the size of the nutrient tanks, which
can be of any convenient length. The width is dictated by the size of the
commercially available supports in which the cups are suspended over the
tanks. Natural light is augmented by 400W Wotan lamps (metal
halide with disprosium from: Wotan Lamps Ltd, Wotan House, 267
Merton Road, London SW18 5JS, UK). In the UK, summer
daylength necessitates day-shortening to prevent flowering and
this is achieved by using an electrically operated, spring tensioned
blind which runs the length of the tank and is controlled by a time
clock to give an 11 hour day.

39(b) Sowing

Two or three seeds are sown on to a mixture, in each cup, of vermiculite (heat-expanded mica), perlite (alumino-silicate
growing medium) & peat, in roughly equal proportions. The number of seedlings in each cup is then decreased to one by
hand "weeding". If small leaves are preferred, seeds can be sown broad-cast, in a similar mixture, in large well-drained
trays about 12 cms deep.

39(c). Water Culture Solution.

A solution such as the following (Table 39.1) is appropriate

Table 39.1. Water Culture Solution for Spinach etc.,

Volume (ml) per 20 litre
nutrient solution made up
Solution Stock concentration with distilled water
KNO3 1M 120
Ca(NO3)2 1M 80
MgSO4 1M 40
KH2PO4 1M 20
MgCl2 1M 80
Trace elements (B,Mn,Zn,Cu,Mo) 20
NaFe-EDTA (3.86 gm/250 ml) 20

Trace elements Quantity (mg) in 250 ml H2O

H3BO3 715
MnCl2.4H2O 452
ZnSO4.7H2O 55
CuSO4.7H2O 20
NaMoO4.2H2O 7.25
Commonly available mixtures (such as "Solufeed" from I.C.I. Plant Protection Division, U.K. Department, Bear Lane,
Farnham, Surrey, GU9 7UB, U.K.) have much to recommend them because they are time-saving, relatively inexpensive
& minimise errors.

39(d) Cleaning Tanks

A good standard of hygiene will obviously minimise disease. Regular sterilisation of tanks with a 5-10% hyperchlorite
solution & periodic replacement of the plastic sheeting is advisable.

39(e) Pests & Sprays

The worst pests are usually your colleagues who, unless properly trained from a tender
age, are likely to decimate a growing crop without prior consultation or use plants
which you had reserved for an epoch-making experiment. Other natural disasters will
inevitably & invariably occur at the worst possible times so, if you are not the sole user
& sole cultivator, be sure to keep your glasshouse in order.

Happily, spinach is not greatly troubled by other pests (at least in temperate climates)
except greenfly (aphids). The use of pesticides should always be avoided because
even those which are supposedly specific will almost certainly affect some aspect of Biological control
photosynthesis & many a researcher has had occasion to curse a well-meaning but
un-informed sprayer. Biological control is fine & ladybirds (lady bugs, Coccinelliidae
sp.) in both the adult & larval form are rapacious devourers of aphids. Sadly, they are so
good that they have a tendency to eat themselves out of house & home & the temptation to start breeding aphids to feed
ladybirds is one which is probably best resisted. As always, much can be done by good husbandry. The faster the turnover
the less likely you are to be troubled by problems of all kinds. Minimise cross infections by growing young plants some
distance away from older plants if this is a practical possibility. Try to achieve a pest-free house by limiting access. If all
else fails, fumigate with nicotine but bear in mind that nicotine is lethal & that while the spinach will recover in a matter
of days you might easily die if you enter a glasshouse during fumigation.

39(f) Harvesting

If someone has always used young spinach leaves to good effect it is of no avail to attempt to persuade them that older
leaves may, almost certainly, be equally useful. There is also merit in using young plants as a means of maintaining
healthy stock (Section 39b). Otherwise it is a matter for compromise. Depending on light & temperature a spinach plant
can reach maturity in about 6 weeks from sowing. For the first couple of weeks it will be really too small to use. In weeks
3-5 it will be in the "grand period of growth", sometimes increasing in size in an almost alarming manner. Accordingly,
if you need a lot of spinach you will either have to settle for older leaves or devote an inordinate amount of time to growing
it.

40. THYLAKOIDS

If intact chloroplasts are resuspended in media which has been diluted 10-fold, they swell & burst within about 1 min.
The stroma is almost entirely released to the medium & the thylakoids or "free-lamellar" fraction may be recovered by
centrifugation as before. These so called "broken chloroplasts" are ideal for experiments on photophosphorylation,
electron transport etc in which the intact envelope might prevent access to an electron acceptor or co-factor. There is
everything to be gained, in fact, by preparing "broken chloroplasts" from intact chloroplasts rather than homogenising
leaves in low osmolarity medium. This latter procedure ensures that the thylakoids will be exposed to other cellular
components during isolation. Conversely the intact envelope will protect the thylakoids until they need to be "unwrapped"
by osmotic shock.

41. EXPERIMENTS WITH ISOLATED CHLOROPLASTS

The number of experiments which have been carried out with isolated chloroplasts is legion & it might be reasonable to
assume that such experiments will continue to be carried out into the forseeable future.

Here we are concerned specifically with experiments based on O2 measurements & much of what follows simply offers
the reader an opportunity to become acquainted with chloroplasts &, if he or she so wishes, to repeat one or two (now
ancient) experiments which have contributed to our understanding of photosynthesis. The real classic, of course, is the
Hill Reaction (Hill, 1937). When Robert Hill first isolated chloroplasts in the 1930's the events of the second world war
which were to lead to the eventual availability of radioactive isotopes were still just around the corner & nearly twenty
years were to elapse before the invention (by Clark in 1956) of the membrane-
covered O2 electrode. Many electron acceptors or co-factors which we now take
for granted were either not yet described or not commercially available. Against
this background, Hill offered his chloroplasts iron, in the form of ferric
potassium oxalate, as an electron acceptor & followed O2 evolution in a
"spectro-colorimeter" by measuring the change in absorption spectrum as
the evolved oxygen combined with muscle haemoglobin to give
oxyhaemoglobin.

Today, ferricyanide (rather than ferric potassium oxalate) is often used as a "Hill
oxidant" &, in Experiment 1, we shall combine nostalgia with present day
practicability & use the Hill reaction with ferricyanide as a measure of
chloroplast "intactness".

41(a) Experiment 1. The Hill Reaction as an Intactness Assay.

Although ferricyanide is a widely used Hill oxidant it does not cross the intact chloroplast envelope at an appreciable rate
(Lilley et. al., 1975) & will therefore only react freely with thylakoids. Happily, this makes it a very convenient
material for determining the percentage intactness of the chloroplast envelope. Clearly, if you
had succeeded in preparing 100% intact chloroplasts & you illuminated them in the
presence of ferricyanide you would not, in theory, expect to see any O 2 evolution.
Conversely, if you were then to subject these same chloroplasts to osmotic shock, so
that their envelopes would rupture, the ferricyanide would have ready access to the
thylakoid membranes & O2 evolution should be observed. Preparations approaching
100% intactness are rare, so some O2 evolution is almost always seen but, since intact
chloroplasts are readily made 100% envelope-free, comparison of the rates of O 2
evolution observed before & after osmotic shock can be used as an intactness assay.

This experiment also demonstrates the increase in the rate of electron transport associated
with uncoupling (see also Sections 41c & 45c). As electrons from water are passed
through the photosynthetic electron transport system, protons are also "picked up"
from the outside of the thylakoid compartment & released into the internal space or
lumen. This is because some of the electron transport chain can accept or donate
electrons whereas others can only accept or donate hydrogen & therefore require both protons (H +) & electrons (e-)
- see Edwards & Walker (1983)

2H+ + 2e- → H2 ......Eqn.41.1

Chlorophyll, for example, when excited by light of an appropriate wavelength, will emit an electron thereby creating a
positively charged hole which can accept an electron from water. Further along the electron transport chain, this electron
is offered to plastoquinone (see Fig. 41.2 & also Part B for further detail). While gracefully accepting this proffered
electron, plastoquinone simultaneously grabs a proton from the outside of the thylakoid membrane in order to make up
the hydrogen atom that it needs in order to become a fully fledged plastoquinol.

Figure 41.1. The Hill Reaction.

Ferricyanide was used as the oxidant or electron acceptor. (Details of the reaction mixture
are given in Table 41.1). Note the acceleration caused by the addition of NH 4Cl as an
uncoupler & the difference in rate between "whole" & "broken" chloroplasts which can be
used to evaluate "intactness" (see text).
 Subsequently, plastoquinol in its turn offers a hydrogen atom to a cytochrome (in the cytochrome b,
cytochrome complex). Cytochromes, however, can only handle electrons &, such is the structure
of the thylakoid membrane, that the rejected proton is discarded at the inner surface.
This is an essential & important feature of photosynthetic electron transport
because, according to Mitchell's Chemiosmotic theory, the protons which
accumulate within the thylakoid space then discharge through the ATPase into the
stroma (Part B) & it is the movement of protons through this sluice gate which
drive the formation of ATP in the process of photophosphorylation (Eqn.41.2).

ADP + Pi → ATP + H2O .........Eqn.41.2

Needless to say, this account is a gross over-simplification of what really occurs. Proton
gradients are established & can, e.g., be easily observed with a pH electrode if thylakoids are
illuminated in an unbuffered mixture containing an appropriate "cofactor" of cyclic electron
transport, such as pyocyanine. (If ferricyanide is used as an electron acceptor there is a change
in pH associated with its reduction [Eqn. 41.4]. This in itself has nothing to do with the
establishment of a proton gradient). Pyocyanine returns the electron that it receives to an earlier point in the electron
transport chain & this rapid cycling of electrons very quickly establishes a measurable proton gradient. However, the
movement of H+ implies a separation of electron charge &, although charge separation undoubtedly occurs & may be
regarded as an integral part of photophosphorylation, biological membranes are not thought to be capable of
accommodating large electrical imbalances. Movement of Cl- ions into the lumen & Mg++ ions out of the lumen will tend
to lessen these imbalances. Similarly, the mechanism by which ATP formation is driven by
the discharge of the proton gradient is still unknown. Nevertheless, the concept in its most
simple form is still very useful & one which allows us to understand why electron transport
goes faster if it is either coupled to photophosphorylation OR uncoupled from it. The picture
which then unfolds is as follows. As protons accumulate within the thylakoid space they exert
a back-pressure. It becomes more & more difficult to discharge protons into this space as the
proton concentration increases, just as it becomes more & more difficult to blow air into a
balloon as it expands. Accordingly, electron transport will slow down. If, on the other hand,
the proton gradient is free to discharge through the ATPase (Part B, Fig. 8.3) i.e. if there is
adenosine triphosphate an adequate supply of ADP & Pi, electron transport (now coupled to photophosphorylation)
will accelerate. Similarly, if it is uncoupled, i.e., if an "uncoupler" provides another means of
(ATP) discharging the proton gradient, electron transport will again increase. The most easily
understood uncoupling action is that caused by detergents. These simply make holes in the lipid/protein membranes of
the thylakoid through which protons can escape (Part B. Fig. 8.3). Ammonium chloride, which is used in this experiment,
works differently. Free ammonia (NH3) crosses the thylakoid

NH3 + H+ → NH4+ ...........Eqn.41.3

membrane & is reprotonated (Equation 41.3) in the thylakoid compartment, thereby acting as a proton sink.

The experiment itself is based on two reaction mixtures which are identical, except in the order of addition of the reactants
(Table 41.1). Simply varying the order of addition in this way, gives two reaction mixtures which contain either
chloroplasts which are all ruptured or chloroplasts which maintain the
same degree of intactness as before.

Figure 41.2. Consequences of Illumination.

The pigments & electron transport chain are housed in the thylakoid
membrane. On illumination, electrons are donated to excited chlorophyll in
photosystem II (PSII). The transfer of electrons to photosystem I (PSI) via
plastoquinone (PQ) requires protons that are taken up from the stroma &
released into the thylakoid compartment. The stromal pH rises & Mg 2+ moves
as a counter ion. [ATP] is formed from [ADP] & [Pi] as H + is discharged
through the ATPase in photophosphorylation.
 Table 41.1. Reaction Mixture for Intactness Assay.

INTACT RUPTURED
l.0ml RS1 x 2 0.8ml H2O
0.8ml H2O 0.1ml chloroplasts
0.1ml chloroplasts 1.0 ml RS1 x 2
10µl D,L-glyceraldehyde (2M) 10µl D,L-glyceraldehyde (2M)
10µl K Ferricyanide (0.5M)

The reaction mixtures contain the resuspending medium listed at the beginning
of Section 37(g) (the BSA is optional). It is added at double strength (RS1 x 2).
That on the left is diluted to single strength prior to the addition of chloroplasts
which therefore experience no change in osmotic pressure when they are added
to it. On the right, chloroplasts are added to water so that they experience a
9-fold dilution which causes them to swell & rupture almost immediately. In
practice, they are usually left for l min at this osmolarity in order to allow them
all to burst. Double strength resuspending medium is then added in equal volume
so that the two reaction mixtures are finally identical except for the intactness of
the chloroplasts. D,L-glyceraldehyde is added to inhibit CO2 fixation.

To obtain fast & more linear rates (i.e. to avoid any retardation caused by the build-up of a proton gradient) NH 4Cl (say
10 l of a 0.5 M solution) is added to each after a minute, or two in order to uncouple electron transport. The degree of
uncoupling (i.e. the difference between the coupled & uncoupled rates) is also an indicator of competence. Ferricyanide-
dependent O2 evolution by well-coupled chloroplasts may increase in rate by a factor of 10 or more, following the addition
of an uncoupler. Percentage intactness is calculated from the relationship

% intacness = (A - B) x 100
A
in which A is the rate after osmotic shock & B is the rate prior to osmotic shock.

An ancillary to this experiment is to add a smaller amount of carefully made up ferricyanide solution. (Take a large crystal
of potassium ferricyanide & wipe off surface ferrocyanide with a tissue. Dissolve in an appropriate volume of water). The
Hill reaction with this oxidant (electron acceptor) will go virtually to completion (i.e. until nearly all of the ferricyanide
has been reduced). This adequately demonstrates the need for an acceptor &, depending on how you wish to look at it,
will verify the accuracy of calibration (Section 36) or establish the stoichiometry of ferricyanide reduction by water
(Equation41.4).

2H2O + 4Fe+++ → 4Fe++ + 4H+ + O2 .....Eqn.41.4

41(b) Some practical points

The above experiment has become a routine first step in work with intact chloroplasts & it is always worth doing in a
teaching context. It demonstrates Hill's classic experiment while also introducing the concept of uncoupling (an aspect of
photosynthesis which would not have even come to that great man's attention for another twenty years). It also
illustrates one or two useful practical details. The first concerns the use of double strength media. This is a
very convenient device because it can be used to avoid large unintentional decreases in osmotic
pressure. For example, if you wish to work in a final volume of 2 ml, you would simply ensure that
the total volume of all of the other additives was less than 1 ml & then add H 2O to bring up the total
to 1 ml followed RS 1 x 2 to a final volume of 2 ml (adding the chloroplasts last!) Each additive
would, of course, make its own slight contribution to the osmotic pressure but these contributions are
small & can usually be safely disregarded.

Subsequent addition (e.g., the addition of NH4Cl in Experiment 1) should be made in small volume to
avoid a significant effect on the calibration. Ten microlitres added to 2 ml is less than a 1% increase in
volume & is therefore of little consequence.

When all of the constituents have been added to a reaction mixture, the vessel is "sealed" by a
plunger. The depth to which the plunger enters the reaction vessel is dictated by a threaded collar
which should be adjusted by hand until the mixture just enters the capilliary at the top of the cone.
vessel “sealed” by Further additives, in small volumes, can be made with a micro-syringe via this neck & the collar
plunger should always be subsequently readjusted. Particularly if samples are being withdrawn, care should
be taken to ensure that the mixture always just enters the capillary. If a bubble of air is left at
the top of the cone its oxygen will slowly equilibrate with the rest of the mixture & since it
will contain more O2 than there is in the solution, this could give rise to errors in any work
which demands a very high degree of precision.

The chlorophyll content should not exceed 100 g/ml if light saturation is necessary & smaller
amounts (consistent with convenient rates) are to be preferred. Large quantities of active
chloroplasts can produce O2 at such high rates that bubbles will form, again giving rise to
inaccuracies. A polished, semi-circular cross-section, reflector (which rests against the back
of the water-jacketed vessel) will help to shield the contents from laboratory light when the
vessel is not illuminated & help to maximise illumination when it is.

Ideally, the water circulating through the jacket should be filtered & contain an agent (such
as "Myacide AS", Boots Biocides Group, Chemical Marketing, The Boots Company PLC,
Nottingham, NG2 3AA, U.K.) to inhibit algal & bacterial growth.

In Experiment 1, D,L-glyceraldehyde is used (Stokes & Walker, 1972) to inhibit CO 2 fixation

because, unless great care is taken to exclude it, there will be enough dissolved CO 2 present
to allow CO2-dependent O2 evolution to start in the mixture which still contains the intact
chloroplasts. Glyceraldehyde is a "nice" inhibitor in the sense that it appears to be reasonably
specific for the reductive pentose phosphate pathway (i.e. it inhibits CO 2 fixation but does
not affect electron transport or photophosphorylation). Glycoaldehyde can be used for the
same purpose (Sicher, 1984) other inhibitors (cyanide is an obvious example) & electron
acceptors (methyl viologen is more poisonous than cyanide) kill people as well as chloroplasts Inhibition of PGA-dependent
& should always be used with extreme caution. Yet others (e.g., DCMU) are only rinsed with O2 by D, L-glyceraldehyde.
difficulty from the electrode vessel (a detergent which is not, in itself, desirable may have to be Continuous line O2 points
used) & if washing is ineffective can carry over into subsequent reaction mixtures.
CO2
Some of Good's buffers (e.g Hepes) are photoxidised in white light in the presence of flavin containing mixtures & red
light may be preferred if there is any possibility of this occurring (see Section 41c).

Washing, between experiments, is essential to good work. The vessel should be aspirated & flushed with distilled water
at least ten times (don't swallow the flea & don't forget to wash the plunger).

If microsyringes are used, always bear in mind that the floor of the electrode vessel is covered by a delicate membrane
which should not be damaged.

41(c) Experiment 2. The Mehler Reaction.

Before we leave the Hill reaction entirely we ought to remind ourselves of one special version of it. This is named after
Mehler (1951) & involves O2 uptake rather than O2 evolution (Figs. 41.3 & 41.4). Mehler reagents (MR in Eqns. 41.5 &
41.6) are Hill oxidants which accept electrons from water but are then autoxidised by molecular oxygen in reactions which
lead to hydrogen peroxide formation (Equation 41.5)

H2O + MR → MRH2 + ½O2 ......Eqn.41.5

MRH2 + O2 → MR + H2O2 ......Eqn.41.6

In the presence of catalase, hydrogen peroxide undergoes rapid dismutation to water & O 2 (Equation 41.7)

H2O2 → H2O + ½O2 ....Eqn.41.7

O2 evolution & O2 consumption are then equal so that no change is

observed but, if catalase is removed by washing, or inhibited by azide,
the Mehler reaction gives rise to a net O2 uptake. Flavin
mononucleotide (FMN) was used as the Mehler reagent in Figs. 41.3
& 41.4 but it should be noted that flavin compounds (including FMN,
FAD & flavoproteins) will, bring about the photoxidation of
commonly used buffers such as Hepes (Yamazaki & Tolbert, 1970; Andreae, 1955)
&, if this is to be avoided it is necessary to use red, rather than blue or white light.
 Figures 41.3 & 41.4. The Mehler Reaction.
Figure 41.3 (left) compares the Hill Reaction (with ferricyanide as oxidant), with a Mehler Reaction in which FMN
(0.04moles) was used as the Mehler reagent. It will be seen that the rates were similar & that both were accelerated by
the addition of NH4Cl (20 mmoles) as an uncoupler. In Figure 41.4 (right) several Mehler reagents (all 0.1 moles except
ferredoxin which was 0.006 moles) are compared. Note that the rates did not change appreciably until nearly all of the
detectable oxygen in solution had been consumed.

The Mehler Reaction is a bit more time consuming to set up than the Hill Reaction because of the probable presence of
catalase in the chloroplast preparation but it can be a lot of fun. Catalase is not a chloroplast enzyme but the normal
mechanical preparative procedures rupture the peroxisomes in which it is normally located & smears their catalase on the
chloroplast envelopes. Washing (Section 37h(i)) will remove about 10% of this contamination &, if these washed intact
chloroplasts are osmotically shocked & spun again, there should be another five-fold decrease in
catalase. [If you wish to see how effective your washing is, add a little hydrogen
peroxide (about 10moles/ml of reaction mixture) to your chloroplasts &
record the rate of O 2 evolution in the dark]. The last traces of catalase are
difficult, if not impossible, to remove but what can't be cured need not,
in this case, be endured because a little (0.5 moles/ml) cyanide or azide
“it just takes a smidgeon” ("it just takes a smidgeon") will inhibit what is left. Without inhibitor, nice
steady-state relationships can be established in which light-generated H 2O2 formation &
catalatic dismutation of H O (Eqn. 41.7) come into balance (Fig. 41.5). The manganous ion (Mn ++, added as MnCl )
2 2 2
accelerates the Mehler reaction & an oxidation product, possibly manganic ion (Mn +++) will reoxidise H2O2. This can
result in post- illumination O2 evolution (Walker et.al., 1970).

Figure 41.5.
Establishment of steady-state between O2 uptake & catalatic
dismutation of water (Eqn. 41.7). Commercial catalase (5units) was
added at the outset to both reaction mixtures which contained FMN as
the Mehler reagent. In this sort of experiment, the level at which the first
steady-state is established can be raised or lowered by lowering or
increasing the light intensity (after Whitehouse et al, 1971).
The reaction mixture for the Mehler Reaction is much the same as before (Table 41.1) but the order of addition will be
unimportant since you will be using chloroplasts which have already been osmotically shocked (see above). Obviously
you will not need a "Hill oxidant" (like ferricyanide) but you will require a "Mehler reagent" which is also an electron
acceptor but one which, alone or in combination with another, will undergo autoxidation by molecular oxygen (Eqn.
41.5). Methyl viologen (which is the active component of the weed-killer "Paraquat") is a very good Mehler reagent but
it is also horrendously toxic & should NOT IN ANY CIRCUMSTANCES BE PIPETTED BY MOUTH. (Many have
survived ingestion of small quantities of cyanide but paraquat poisoning has been almost invariably fatal. Moreover, it
can induce a proliferation of cells in the lung which is associated with, or brings about, death after 2 or 3 days; so it can
be an extremely unpleasant way to go). For these reasons its use is not recommended. Flavin coenzymes (such as FMN
or FAD) are O.K. although they should not, as already noted above, be used in light containing blue wavelengths.

The Mehler Reaction, like the Hill Reaction will, of course, respond
to uncouplers & to the presence of ADP + Pi. This can be used to
demonstrate so-called "photosynthetic control" (West & Wiskich,
1968) in which ADP (added to a mixture already containing Pi)
stimulates the rate of O2 evolution by discharging part of the proton
gradient (c.f., the action of uncouplers, Section 41c above). The O2
consumed during the period of accelerated O 2 uptake (Fig. 41.6) can
be used to calculate a P/O ratio, which in this instance (Whitehouse
et. al., 1971) was about 1 (i.e., about 1 mole oxygen/0.2 moles of
ADP added).

Figure 41.6. "Photosynthetic control" in the Mehler Reaction.

Both reaction mixtures contained KCN (1mole), to inhibit residual
catalase, & orthophosphate (2 mole) "A" contained FMN (0.1 mole)
& "B" contained methyl viologen (0.1 mole). ADP was added as
indicated.

41(d) Experiment 3. CO2 & PGA-dependent O2 evolution.

This is another golden oldie. Neither quite so venerable nor epoch-making as the Hill Reaction but certainly important in
its own right. The mixture is very much the same as before except that the chloroplasts are kept intact & either CO 2 or
PGA are used as electron acceptors.

Reaction Mixtures

CO2-dependent PGA-dependent
RS1 x 2 (1.0ml) RS1 x 2 (1.0ml)
H2O (0.7ml) H2O (0.7ml)
0.2M NaHCO3 (0.1ml) 200mM PGA (10µl)
chloroplasts (0.1ml) chloroplasts (0.1ml)
1.0 mM Pi (0.1ml) 1.0 mM Pi (0.1ml)

It will be seen that upon illumination (Fig. 41.7), CO 2-dependent O2

evolution does not immediately reach maximal rate (c.f., ferricyanide
reduction, Fig. 41.1) but only after a lag or induction period. The
underlying causes are partly light-activation of enzymes but mostly
autocatalytic build up of carbon cycle intermediates which have become
depleted in the dark (Edwards & Walker, 1983). For this reason the
addition of PGA shortens or eliminates the lag - a predictable result but
one which nevertheless caused the author to rush naked into the street
crying "Eureka" when he did the experiment for the first time.
 Figure 41.7. Carbon dioxide-dependent & PGA-dependent oxygen evolution.
This figure shows the lag or induction period which is characteristic of CO 2-dependent
O2 evolution by chloroplasts & leaves (c.f., Fig. 37.3). The shortening of the lag by PGA
is typical but the rate with PGA is usually slower than the rate with CO 2. In any case,
although the reaction mixture containing PGA contained no added bicarbonate, it had
not been made CO2-free & hence the O2 evolution observed would be partly CO2-
dependent stimulated by PGA. In the complete absence of CO 2 (or in the presence of
D,L-glyceraldehyde), PGA-dependent O2 evolution soon ceases.

The discerning reader will also note that the second part of this experiment is not, strictly speaking,
PGA-dependent O2 evolution but a mixture of PGA-dependent & CO 2-dependent because although
bicarbonate is not added there will still be some CO2 in the various solutions. In fact, if CO2 is rigorously
excluded, PGA-dependent O2 evolution soon falls in rate for reasons that have yet to be certainly established.
Concomitant oxidation of RuBP accumulating in the absence of CO 2 may be a contributory factor but the
decline in rate also occurs in the presence of D,L-glyceraldehyde which is believed to block the cycle at the PGA-dependent
transketolase steps (Stokes & Walker, 1972). oxygen evolution
The first demonstration of rapid CO 2-dependent O2 evolution under aerobic condition (Walker
& Hill, 1967) added the second dimension to the photosynthetic capacity of the isolated
chloroplasts. (Originally, Hill could only detect O 2 if he provided his chloroplasts with
an artificial oxidant &, when Arnon & Whatley first demonstrated CO 2-dependent O2
evolution under anaerobic conditions the rates were extremely low). Observations such
as these following on the pioneering work of Arnon, Whatley et. al., (see Arnon, 1967
for a review) appeared to confirm the supposition, in doubt since Hills 1930's
experiments, that the chloroplast was a self-sufficient photosynthesising organelle.
Subsequent experiments, which you are also invited to repeat (Experiments 4 & 5
below), made it clear, however, that the chloroplast is also a "Pi-consuming, triose
phosphate-exporting", organelle & that in this & other regards photosynthesis is, after all,
really a cellular affair.

During the course of CO -dependent O evolution it is easy, for purposes of

2 2
Photosynthesis and phosphate: demonstration, to make some additional points. One is simply to introduce a brief dark
a cellular affair interval (Fig. 41.8). It is always reassuring to see that the chloroplasts know when they
are in the dark & instructive to observe that there is no lag when illumination is resumed
(built-up intermediates do not decline sufficiently rapidly to reinitiate the induction period if the dark interval is limited
to a duration of about 1 minute). Changes in light intensity will also be seen to affect the rate.

Figure 41.8. Carbon dioxide-dependent oxygen evolution.

To show the effect of light intensity & temperature on the induction period. Note the near
absence of a lag following re-illumination after a dark interval.
41(e) Experiment 4. The Requirement for Orthophosphate.

When CO2 fixation by isolated chloroplasts was first demonstrated in the 1950's (Allen et. al., 1955; Arnon, 1967) &
when rates were improved in the 60's (Walker, 1964) chloroplasts normally failed to incorporate radioactivity into
sucrose. This was commonly regarded as a malfunction caused by isolation, particularly since key enzymes of sucrose
synthesis were found to be associated with chloroplasts prepared in non-aqueous media. Eventually, however, it become
clear that sucrose synthesis was a cytosolic event. Two of the experiments (Experiments 4 & 5) which made it clear that
chloroplasts imported orthophosphate & exported triose phosphate are described here. The reaction mixture is the same
as that used for CO2-dependent O2 evolution in Experiment 2 except that no orthophosphate (Pi) is included. When the
mixture is illuminated, photosynthesis normally starts, as usual because there is enough endogenous Pi to support some
CO2 fixation. Nevertheless, the rate soon declines until it is commensurate with the internal Pi recycling associated with
starch synthesis (Edwards & Walker, 1983). At this stage, rates can be readily restored by the addition of Pi. If small
quantities of Pi are used, it will be seen that there is a stoichiometric relationship between added Pi & extent of the
restoration in rate (such that 1 Pi yields 3 O2). This is consistent with the formation of triose phosphate according to
Equation 41.8.

Figure 41.9. The Requirement for Orthophosphate.

In Pi-deficient chloroplasts, photosynthetic O2
evolution falls to a value commensurate with starch
formation. The addition of Pi then gives approximately
3 molecules of O2 per molecule Pi. In a reaction
mixture containing no Mg++ there is no response to
added PPi unless Mg++ is added. External hydrolysis
then leads to Pi formation & a stoichiometry of 6
molecules of O2 per molecule PPi.

(Sum) 3CO2 + H2O + Pi → triose phosphate + 3O2 .....Eqn. 41.8

41(f) Experiment 5. Orthophosphate Inhibition & its Reversal

The orthophosphate requirement (Experiment 4) for photosynthesis by intact chloroplasts has a very sharp optimum. This
can be readily demonstrated (Fig. 41.10) by using the same reaction mixture as that in Experiment 4 but including
different amounts of Pi to give final concentrations between 0.5 & 10 mM. It will be seen that the initial lag is extended
as the Pi concentration is increased until the onset of O 2 evolution is delayed more or less indefinitely as it is in Fig. 41.11.
At any time, this Pi inhibition may be immediately & fully reversed by the addition of PGA or triose phosphate. Slower
reversal follows the addition of pentose monophosphates. When first performed (Cockburn et. al., 1967), experiments of
this sort led to the suggestion that there must be obligate exchange between external Pi & internal triose phosphate
(Walker & Crofts, 1970). This was confirmed by direct measurement (Heldt & Rapley, 1970) & the concept of the
phosphate translocator became a reality. Seen in this way, triose phosphate is the principal product of the
photosynthesising chloroplast & photosynthesis is therefore severely constrained if it is supplied with inadequate Pi.
Figure 41.10. Inhibition by orthophosphate of photosynthetic
carbon assimilation in isolated spinach chloroplasts.
Kinetics of CO2-dependent O2 evolution in the presence of
increasing quantities of exogenous Pi. The characteristic
induction period is shortest in the absence of added Pi but
photosynthesis then soon declines. Small increments in [Pi]
lengthen the lag & either increase the rate or leave it unchanged.
Larger quantities of Pi cause lag extension & rate depression. Pi
values (mM) are recorded against each trace & the figures in
parentheses are the rates in mol mg-1 Chl. hr-1 (at 20C). The
broken line shows the extent of alleviation of Pi inhibition (at [Pi]
of 5 mM) by the inclusion of 10 mM PPi.

Conversely, if there is too much Pi in the external medium, triose phosphate which would have otherwise been used
to regenerate the CO2 acceptor (RuBP), is "pulled out" of the chloroplast & the induction period (which, in less
extreme circumstances, largely reflects autocatalytic build-up of cycle intermediates) is prolonged indefinitely.

Externally added metabolites such as triose phosphates then reverse the Pi inhibition either by inhibiting Pi
entry, or by entry instead of Pi, or both. In vivo, sucrose synthesis from triose phosphates recycles
orthophosphate & there is now good evidence that there is an intimate relationship between events in the
cytosol & photosynthetic carbon assimilation in the stroma of the chloroplast. In vitro, photosynthesis
by isolated chloroplasts is only possible to the extent that the reaction mixture can imitate, in a
limited way, the contribution that is made by the cytosol.

Figure 41.11. Orthophosphate inhibition

& its reversal.
On the left, complete inhibition by 10 mM
Pi. On the right, the kinetics of reversal of
inhibition following the addition of 2
moles of DHAP, R5P, PGA & FDP
(fructose 1,6-bisphosphate).

41(g) Inorganic Pyrophosphate

In the history of chloroplast isolation, inorganic pyrophosphate was once an enigma & therefore deserves a mention in
this context. The improvements in rate brought about by Walker (1964) still fell short of the magic 100 moles per
milligram chlorophyll per hour which, at that time, was regarded as a convincing rate for reasonably competent
chloroplasts. Rates in excess of about 25 were only rarely obtained at first & then only when catalytic amounts of
Benson-Calvin cycle intermediates were added to the reaction mixtures (Appendix 3). As we have seen (in Fig. 41.7) PGA
dependent oxygen evolution occurs without an appreciable lag but much smaller (i.e. catalytic quantities) of PGA or triose
phosphate will also abolish induction. Rates of 100 were finally obtained by Bucke et. al., (1966) with such additions but,
at about the same time, Jensen & Bassham were able to report that they could routinely obtain as high, or even higher
rates, without recourse to additives. The procedures which Jensen & Bassham (1965) used were, in all essentials, exactly
the same as those previously introduced by Walker (1964) except in one important detail.
 That is they included inorganic pyrophosphate in the reaction mixtures. It took much longer to understand why the
presence of pyrophosphate was so beneficial. Similarly, it was very difficult to understand why inorganic PPi would, in
some circumstances, act like 2 molecules of orthophosphate as it does in Figure 41.9 whereas excess
pyrophosphate does not inhibit in the same way as orthophosphate (Figs. 41.10 & 41.11) but will even afford
a degree of protection against orthophosphate inhibition (Fig. 41.10). After a great deal of work, & a great deal
of head scratching, everything became clear. Orthophosphate inhibition & its reversal by Benson-Calvin cycle
PPi intermediates on the one hand, & the shortening of induction by Benson-Calvin cycle intermediates on the other, led
to the concept that there was "a direct obligatory exchange between orthophosphate (outside) & sugar phosphate
(inside) phosphates (Walker & Crofts, 1970). In the hands of Heldt & Rapley (1970) this concept became the
experimental reality of the "Phosphate Translocator". The nature of orthophosphate inhibition was now clear because, if
isolated chloroplasts were suspended in a medium containing greater than optimal orthophosphate, the lag or induction
period would be extended - because of enforced export of internal triose phosphate. This is why isolated chloroplasts show
such a sharp orthophosphate optimum. If they are offered too little Pi they become phosphate deficient, if they are offered
too much Pi the autocatalytic build-up of intermediates which would otherwise terminate the induction period cannot
occur, or is significantly delayed. In the presence of inorganic pyrophosphate, however, the orthophosphate optimum is
greatly broadened. It is broadened for two reasons. The first is that PPi cannot, itself, cross the chloroplast envelope but
can compete with orthophosphate at the phosphate translocator. For this reason it alleviates orthophosphate inhibition.
The second advantage which comes with the use of PPi was remarkably fortuitous. Chloroplasts were invariably prepared
at this time in media containing magnesium ions. The intact chloroplast contains an inorganic pyrophosphatase & since
some chloroplasts are invariably ruptured, small amounts of pyrophosphatase are released to the external medium. The
substrate for this extremely active pyrophosphatase is magnesium pyrophosphate whereas the anionic form of PPi is
inhibitory. Put all this together (for a review see Edwards & Walker, 1983) & you have a situation in which inorganic
pyrophosphate in the external medium acts, to an extent, like an artificial cytosol. Firstly, it ameliorates the inhibitory
action of orthophosphate by competing with the phosphate translocator in much the same way as would triose phosphates
& PPi in the cytosol. Secondly, the limited hydrolysis of PPi in the reaction mixtures (catalysed by inorganic
pyrophosphatase released from ruptured chloroplasts) brings about cycling of orthophosphate at about the same rates as
sucrose synthesis would bring about recycling of orthophosphate in the in vivo situation.

It is for these reasons that, if you simply wish to obtain the best possible rates of CO 2-dependent oxygen evolution with
isolated chloroplasts, it is a good idea to include Mg ++ & PPi in your reaction mixtures. By this simple device you will,
with good chloroplasts, routinely obtain rates in excess of 100 moles.mg -1chl.hr-1. If, for any reason, you do not wish to
use PPi then you will only obtain the best rates, as a matter of course, if you happen to get the orthophosphate
concentration in your reaction mixtures almost exactly right. The optimum is about 0.2 to 0.5 mole but it does vary from
preparation to preparation.

In retrospect, it is clear that the improvements in the rates of photosynthesis displayed by isolated chloroplasts which took
place in the 1960's were only due in part to the improvements in the methods of isolation. The treatment of the
chloroplasts, once isolated, was equally important. Learning how to treat the chloroplasts properly proved enormously
important because it not only led to the recognition that the chloroplast was an orthophosphate-consuming triose
phosphate- exporting organelle but also to the elucidation of the phosphate translocator & the realisation that the cytosolic
regulation via Pi has a major impact on photosynthesis.

41(h) Experiment 6. Carbon Dioxide Dependence.

This, of course, is what photosynthesis is all about & it is certainly worth demonstrating & it also carries some hidden
messages (Fig. 41.12). Again the mixture is as before (Experiment 2) but without added bicarbonate. There should be
sufficient endogenous CO2 to allow O2 evolution to commence but, after a few minutes, the rate will approach zero. If
CO2 is then added, there will be an immediate resumption of O 2 evolution. If addition of CO2 is deferred, however, the
rate of O2 evolution following addition of bicarbonate will be diminished. (Much the same sort of response is seen if the
addition of Pi is deferred when chloroplasts are running out of phosphate as in Experiment 4, Section 41d). Anything, in
fact, which limits CO2 fixation during intense illumination will bring about photoinhibitory damage.

Figure 41.12. Carbon dioxide dependence.

The original experiment by Walker & Hill (1967). On the
left, CO2-dependent O2 evolution. On the right the same
experiment without added bicarbonate until its addition as
indicated.
 42. PROTOPLASTS
Although one major aspect of research in photosynthesis has rested heavily on chloroplasts, there is additional
information to be derived from protoplasts (Edwards & Walker, 1983). Moreover, you may wish to look at chloroplasts
from species other than spinach or peas because you are interested in that particular species rather than features of the
photosynthetic process which are common to most species. If this is the case you may find that the only realistic option
currently available to you is to prepare chloroplasts from protoplasts (Section 43).

42(a) Digestion.

The separation of protoplasts from leaves is based on enzyme digestion of the cell walls & cellular membranes
(Edwards et. al., 1976; 1978; 1979), (Huber & Edwards, 1975), (Kanai & Edwards, 1973a; 1973b; 1973c).
The procedure is a compromise in much the same way as mechanical disruption is a compromise. Thus
ineffective & slow digestion will give too few protoplasts to be useful in most experiments. Conversely,
if very effective digestion is allowed to go on for too long, everything may be digested or, alternatively,
digestion of vital components may proceed to an extent that function is damaged. Much, of course, depends on
the enzymes that are used in the digestive process, the nature of the leaf, the conditions of incubation etc, etc, but
the guiding principle should be to use the gentlest & shortest treatment which will give you a reasonable & active
yield.

42(b) Pretreatment.

Here the trick is to make the tissue as accessible to the enzymes as possible without causing undue damage
in the meantime. Some leaves will readily part with their lower epidermis if this is pulled away with a pair
of forceps but if you wish to become a good stripper you will need patience & nimble fingers. Alternatively
you might wish to try rubbing the cuticle with carborundum or a toothbrush (an electric toothbrush has distinct
potential). Cereals & spinach respond well to cutting into fine strips (& this may be best done under 0.5M
sugar or sugar-alcohol solution). If you have to recourse to cutting, do not economise on razor blades because
each preparation will consume several, if the best results are to be obtained. Vacuum infiltration or shaking
“a good stripper” during incubation can sometimes help but not, apparently, with slices of cereal leaves.
42(c) Enzymes & Incubation

There is no one mixture which can be recommended in all circumstances. Some of the better enzymes are costly or
difficult to obtain. (If price is a consideration you may also wish to recover your enzyme after use). Each new species will
probably respond most readily to a particular cocktail of enzymes (e.g. Rohament P pectinase is particularly good with
sunflower leaves). The following is worth trying at the outset:

2% cellulase (source-Trichoderma viride)

0.3% pectinase (Macerozyme R-10, source-Rhizopus sp.)

Sorbitol (0.5 M)
CaCl2 (1 mM)
Buffer (20 mM) at pH 5.5
Bovine serum albumin (defatted) 0.05% - may help

(For sunflower try 0.5% Rohament P instead of MacerozymeR-10 & for pea try 0.5% Macrozyme R-10 plus 0.25%
Rohament P)

Incubation is best carried out in a shallow suspension under low light at 25-30C & should not be prolonged for more than
2 to 3 hours at the most (unless a lower temperature is used).

42(d) Isolation.

On returning after 2 - 3 hours or so to your digesting leaves you will find, if you are lucky, that protoplasts will start to
fall out of the leaf material if the incubating dish is gently rocked from side to side Fig. 42.1. If this is not the case you
must either wait a little longer or try a different mixture. If things have gone well, decant the protoplast suspension & wash
additional protoplasts out of the leaf debris with a solution of sorbitol (0.5 M) & CaCl 2 (1 mM) & MES (5 mM at pH 6.0).
Pour through nylon mesh (a plastic tea-strainer is ideal & may be used to support finer mesh if necessary). C3 & CAM
debris is best retained by mesh with 1 mm & 200 m openings respectively. C4 bundle sheath strands are usually resistant
to digestion & can be collected on sieves with 80 m holes. The filtrate is then spun in 15 x 90 mm glass tubes at 100 g for
5 minutes & the supernatant discarded (or stored frozen in saturated NH 4Cl prior to recovery of the cellulase). Do not spin
longer or harder because you are now faced with the need to resuspend the protoplast pellet in a solution of 0.5M sucrose,
1.0mM CaCl2 & 5 mM MES (at pH 6.0) & this is best accomplished, without damage, by gentle agitation of a loosely
packed pellet to which a few drops of this mixture has been added.
 Figure 42.1. Isolation and Purification of Protoplasts.

Starting top left:

1) Leaves are very finely sliced using an extremely sharp knife or

new razor blades (42b).
2) Leaf slices are incubated with digestive enzymes.
3) Dish is rocked to dislodge protoplasts.
4) Protoplasts are decanted through a tea-strainer.
5) Leaf slices (plus those retained in the strainer) are washed with
medium from a Pasteur pipette to dislodge more protoplasts.
6) Dislodged protoplasts are combined, recovered by centrifugation and
resuspended in new medium by gentle shaking.
7) The resuspended protoplasts are over-layered with 0.5M sucrose
and 0.4M sucrose + 0.1M sorbitol resuspending medium from a
Pasteur pipette.
8) After centrifugation, intact protoplasts are recovered, with a
Pasteur pipette, from the interface between the two over-layered
media.

42(e) Purification

Add a further 5 ml of the 0.5 M sucrose resuspending solution (42d, above) to the protoplasts in
the centrifuge tubes & then, on top of this suspension, layer a further 2 ml of a second mixture
containing 0.4 M sucrose, 0.1 M sorbitol & CaCl 2 & MES as before. Finally the whole sandwich
can be topped off with a further 1 ml of a third solution containing 0.5 M sorbitol & CaCl 2 & MES
as before.

The tubes are now spun at 250 g for 5 minutes. The intact protoplasts rise & collect at the interface
between the two upper solutions from where they can be recovered by gentle aspiration with a
Pasteur pipette. If necessary, (i.e. if the protoplasts do not float or if inspection of the "chloroplast"
pellet under the microscope shows it to contain lots of protoplasts), the density of the medium can
be increased by increasing the concentration of the osmoticum the addition of dextran (5-10% w/v
dextran T20 or T40).

Once recovered from the gradient, the protoplasts should be diluted 10-fold in:

Sorbitol (0.4 M)
CaCl2 (1 mM)
MES (20 mM) at pH 6.0

& recovered from this medium by centrifugation at 100 g for 5 minutes. A similar medium
containing sucrose rather than sorbitol is good for prolonged storage.

C3 mesophyll protoplasts are often assayed in a medium somewhat similar to that used for
chloroplasts (Section 41, Experiments 1-4) but containing

Sorbitol (0.4 M)
Tricine-KOH (5 mM) at pH 7.5,
NaHCO3 (10 mM)
CaCl2 (5 mM)

This Ca++ is added to prevent clumping & to inhibit CO 2 fixation by chloroplasts released from
ruptured protoplasts. The protoplasts may be resuspended in this medium if they are to be used
immediately.
For C4 tissues, the procedures are similar but the crude extract is filtered through 500 m & 80 m sieves. The bundle sheath
protoplasts are retained on the 80 m mesh & may be resuspended in:

Sorbitol (0.5 M)
CaCl2 (1 mM)
MES (5 mM) at pH 6.0.

The mesophyll protoplasts can be purified in the same way as C3 protoplasts (above), with 10% (w/v) T20 dextran added
to the flotation medium to compensate for the fact that C4 protoplasts tend to be more dense than C3 (or, ideally, by
increasing the molarity of the entire gradient, e.g., to 0.6 M with respect to sucrose & sorbitol - Day et. al., 1981).

43.CHLOROPLASTS FROM PROTOPLASTS

One reason for preparing protoplasts is that this may be the only way, yet known, of isolating
chloroplasts from certain species. For example, considerable effort was once put into the
mechanical isolation of chloroplast from sunflower (Delaney & Walker, 1976) & all
manner of protective agents were used to prevent damage by phenolics. Although the
thylakoids & the stroma from these chloroplasts functioned well, the intact chloroplasts
themselves supported CO2-dependent O2 evolution at rates which were only 10% of those displayed by those
from spinach, suggesting that irreversible damage had been inflicted on the chloroplast envelope (Section 37b).
Certainly, chloroplasts from sunflower protoplasts can match the performance of those from spinach. No one
ADP
really knows what makes the difference but the cavitation effects produced by mechanical blenders could be a good
guess. Certainly the rapid movement of a blender blade through a solution will leave a momentary partial vacuum in its
wake & this together with consequent frothing seems to cause the damage by depositing a variety of deleterious
compounds on to the chloroplast envelope. Conversely, the separation of chloroplasts from protoplasts is a relatively
gentle process. The principle is simple. C3 protoplasts are about 30-40m in diameter & they will therefore not pass
unbroken through 20 m holes. Accordingly a piece of nylon mesh with pores of this diameter is held over the tip of a 1ml
plastic disposable syringe with a plastic collar fashioned out of a 1 ml automatic pipette tip. The syringe tip itself is usually
cut short in order to widen its aperture. A protoplast suspension is then drawn into the syringe & expelled through the
mesh two or three times. If this suspension is centrifuged at 250 g for 1 min a chloroplast pellet is obtained & this can be
washed & re-centrifuged if so desired.

It should be noted that Edwards et. al., (1978) found a requirement for chelation in their work with isolated chloroplasts
from wheat & sunflower. At pH 7.8 with 0.5 mM Pi in the assay medium sunflower chloroplasts performed best in the
presence of 10 mM EDTA & 5 mM PPi. Wheat chloroplasts, like those from pea leaves (Robinson & Wiskitch, 1977;
Stankovic et. al., 1977) are inhibited by PPi & stimulated by a mixture of PPi & ADP (at about 0.2 mM).

44.THE RECONSTITUTED CHLOROPLAST SYSTEM

This was an invention of Whatley et. al. (1956) which was largely neglected until it was
re-invented, with greatly improved activity, by Stokes & Walker (1971). Since then it
has been again largely neglected but is, arguably, still a system of great potential. One
of its best features is that it is often possible to obtain an active reconstituted system
from less-than-perfect intact chloroplasts & its main recommendation is that it is
accessible to intervention in a way which is otherwise precluded by the chloroplast
envelope.

In its simplest form, the reconstituted chloroplast system (Lilley & Walker, 1979) is
simply intact chloroplasts which have been osmotically shocked in a reaction mixture
- i.e. a mixture of thylakoids & stroma in the same proportions (though not the same
relative "concentrations") as in the parent chloroplasts. More sophisticated versions
have also been used in which the thylakoid & stromal fractions have been separated &
the stroma has been subjected to some degree of concentration or fractionation before
reconstitution. In all cases, however, some degree of augmentation is necessary. This is because
there are changes in concentration of key components such as NADP, ADP & ferredoxin as a
consequence of envelope rupture. In the intact chloroplast these are held in intimate contact with
the thylakoid membranes, as constituents of the stroma, by the chloroplast envelopes. In the
reconstituted system the relative dilution (i.e. the concentration of each of the soluble
components in the vicinity of the insoluble thylakoid membranes) is partly made good by
their addition as purified, or partly purified, components.
44(a) The simplest system.

This is prepared from intact, fully competent, CO2-fixing chloroplasts according to the procedures listed in Section 37 or
43. Reaction mixtures (2 ml in total) could contain, for example,
H2O (0.8 ml)
Chloroplasts (0.1 ml)
RS1 x 2 (1.0 ml) (Section 4g)
Ferredoxin (about 40-50 g)
ADP (0.4 moles)
NADP (0.1 moles)
Substrate (e.g. PGA, 3 moles)
KCl (10 mM)
Dithiothreitol (5 mM)

As in Experiment 1 (Section 41a) the water should be added before the chloroplasts &, to be certain, one minute should
be allowed for the chloroplast to swell & burst as a result of the ensuing osmotic shock.

44(b) Systems with additional stroma.

Several possibilities exist here. One is to osmotically shock a larger batch of chloroplasts in a centrifuge tube & then
recover the thylakoids by centrifugation. This allows the re-addition of a much larger amount of stroma. Inevitably,
however, an upper limit will eventually be reached. This limit can then be raised by using concentration-dialysis of the
chloroplast extract (Lilley & Walker, 1979). Reconstitution from partially purified fractions has also been achieved
(Slabas & Walker, 1976).

45. EXPERIMENTS WITH THE RECONSTITUTED SYSTEM

Because of the dilution involved in preparation, the concentration of endogenous substrate is low & substrates need to be
added in order to initiate a reaction. This, of course, is what gives the system its usefulness because it is possible to add
substrates which would not readily penetrate the chloroplast envelope.

45(a) Experiment 1. CO2-dependent O2 evolution.

Figure 45.1. Carbon dioxide dependent oxygen evolution in the reconstituted system.
O2 evolution commences on illumination at zero time & ceases when the added NADP (0.1
mole) is reduced. It recommences on the addition of RuBP or, after a delay (see text) if a
pentose monophosphate such as R5P is added.

The reaction mixture is that listed in Section 44a above, together with NaHCO 3 (10 mM) & an additional
substrate. The latter can be RuBP, or one of the pentose monophosphates of the Benson-Calvin cycle (Ru5P,
Xu5P or R5P) as in Fig. 45.1, or FBP. If any of these is not added at the outset, O 2 evolution will still
commence because the system will carry out a Hill Reaction in which the added NADP will serve as the
oxidant. This O2 evolution soon ceases as the NADP is reduced but can be reinstated by the addition of RuBP etc.
If a pentose monophosphate is added there is a lag before O 2 evolution starts &, with FBP, a larger lag. These lags
can be largely abolished by the addition of creatine phosphate & its kinase which, together, constitute an artificial
ATP generating system (Eqn. 45.1) suggesting that most of the lag is attributable to an unfavourable ATP/ADP ratio.

CPK
ADP + CP → C + ATP ...........Eqn.45.1

This concept is explored in a second experiment below.

 45(b) Experiment 2. PGA-dependent O2 evolution

Everything is done in precisely the same way as in Experiment 1 but no bicarbonate is added. Once the initial O 2
evolution has ceased, PGA (2 mM) is added & O2 evolution immediately recommences. If a small quantity (say 0.2
to 2 moles) of any of the following is added:

ADP,
Ru5P, Xu5P or R5P,
glucose (plus hexokinase)

Figure 45.2. PGA dependent oxygen evolution.

As for Experiment 1 (above) but with PGA as substrate. O 2 evolution can be interrupted
by any additive (such as R5P, see text) which will serve as an ATP sink. The
interruption is lengthened (broken line, right) if the quantity of additive is increased.
The interruption is eliminated (broken line, left) if CP + CPK is added as an artificial
ATP generator.

O2 evolution quickly ceases (Fig. 45.2) but restarts spontaneously after an interval which is
proportional to the amount of the additive. Conversely, the additives have no effect if creatine
phosphate (CP) & its kinase (CPK) are also present in the mixture.

The explanation of all of this lies in the reaction catalysed by PGA kinase (Eqn. 45.2).
This is freely reversible but needs to be pushed in the "photosynthetic" direction (i.e.
towards the formation of glycerate 1,3 bisphosphate) by a favourable ratio of reactants
to product,

PGA + ATP → GBP + ADP ....Eqn.45.2

Accordingly, the addition of ADP, or any system which consumes ATP (thereby producing ADP) such as the reaction
catalysed by Ru5P kinase (Eqn. 45.3).

Ru5P + ATP → RuBP + ADP .....Eqn.45.3

or hexokinase (Eqn. 45.4)

glucose + ATP → glucose 6-phosphate + ADP ......Eqn.45.4

Conversely, CP + CPK, if present in sufficient quantity, will restore the ATP/ADP ratio & suppress the transitory
inhibition of O2 evolution (Robinson & Walker, 1979; Carver, Hope & Walker, 1983). This is an important experiment
because it illustrates the need for balance in metabolic turnover & the way in which "feed-back" (or in this case,
"feed-forward") can work.
45(c) Experiment 3. Uncoupling.

We have already seen how uncoupling increases the rate of electron transport in the Hill Reaction (Section 41a,
Experiment 1). With the reconstituted system we can see another facet of this situation (Fig. 45.3).

The mixture is as before (Section 45a, Experiment 1, above) & when O2 evolution has ceased, following the initial
reduction of NADP, PGA is added & O2 evolution is resumed.

In a sense, this is also a Hill reaction but it is the "natural" Hill reaction for which ATP is required (Eqns. 45.5 & 45.6)

PGA + ATP → GBP + ADP .......Eqn.45.5

GBP + NADPH → DHAP + NADP .......Eqn.45.6

2NADP + 2H2O → 2NADPH + O2 .....Eqn.45.7

For this reason, when an uncoupler is added, O 2 evolution ceases. If ATP is then added, O2 evolution is at first resumed
at a faster pace because the reaction then no longer depends on ATP which is generated within the system (from ADP &
Pi) by photophosphorylation (Section B8) & the uncoupled rate of electron transport to NADP is faster than before (c.f.,
41a Experiment 1).

Figure 45.3. Uncoupling.

PGA-dependent O2 evolution, unlike the Hill Reaction (41a) or the Mehler
Reaction (41c) is ATP-dependent. Adding an uncoupler (UC) therefore
inhibits rather than causing an acceleration (c.f., Figs. 41.1 & 41.3).
Evolution is restored by ATP but the restoration is short-lived unless CP +
CPK are present or are added. This is because ADP accumulates (in the
uncoupled situation) as ATP is consumed & the unfavourable ATP/ADP
ratio becomes inhibitory (c.f., Fig. 45.1).

This new rate is not maintained, however, because, as the added ATP is consumed, ADP increases until the now adverse
ATP/ADP ratio starts to inhibit the PGA kinase reaction (Eqn. 45.5) by mass action. If CP + CPK is added, a fast rate is
again restored as this ATP regenerating system (Eqn. 45.1) restores a favourable ATP/ADP ratio.

46. SOME FURTHER PRACTICAL CONSIDERATIONS

In all of the foregoing (Part F), oxygen evolution has been followed polarographically & all that is required is a single
electrode vessel which lends itself to illumination (such as the Hansatech Instruments Ltd DW1 & DW2). Distinct
advantages derive, however, from employing twin electrodes.
 46(a) Twin electrodes

Chloroplasts have a relatively short "bench-life" &, however active on separation, will suffer
changes in activity during use. There is much to be said, therefore, for using two electrode
vessels so that experiment & control can be carried out simultaneously. This means that
really subtle differences can be regarded with more confidence. Secondly, oxygen
electrodes are still not as reliable or stable as pH electrodes &, like any sort of apparatus,
they are not immune to malfunction. "Trouble-shooting" is a great deal easier with two
electrodes because components can be switched between each system until that
responsible for the malfunction is identified. Inevitably, malfunction has also been known to
occur during an experiment & there is a natural law (Sod's Law, or more properly "The
Conservation of Chagrin") which will ensure that, if this does occur, it will coincide with a
spinach famine. If, therefore, you have just used the last spinach, or you are leaving the next
day on vacation, & only need to do one more vital experiment in order to win fame &
fortune, it is best to use two electrodes for the same reason that you would probably prefer
to see two engines on each wing.

“you are leaving the next day 46(b) Illumination

on vacation”
Figure 46.1 was first published in 1972 & it illustrates a twin electrode system (Section
45a, above) in which slide projectors were used as light-sources. At the time, there was no cheap & reliable alternative &
indeed even today, because of mass production, there is still a lot to be said for slide projectors if there is no particular
need for anything more sophisticated. Some alternatives have already been mentioned in Part C & if an electrode system
such as the Hansatech Instruments Ltd DW2 is used, a purpose built light-source & a fibre or liquid optic is almost
mandatory. If you use fibre optics bear in mind that, even with the best, there is a significant loss of light. Similarly, do
not be fooled by claims that fibre optics give "cold" light - they act as heat filters only to the extent that they do not
transmit 100% of all that they are offered & in many circumstances it is wise to insert an appropriate heat filter before the
optic, such as a wide band hot mirror as those supplied by O.C.L.I. (Part No 6022001) in order to protect it & to diminish
what heat is transmitted. Liquid optics transmit light more effectively than fibres but they are expensive &, at the time of
writing, cannot be branched.

Figure 46.1. A twin electrode system.

Two electrode vessels, each mounted on its stirrer are sited within
similar compartments of a matt-black E-shaped screen (this is open
above, but together with the shading offered by the mirrors &
relatively subdued laboratory lighting, provides a sufficient
approximation to darkness within the reaction vessel for most
purposes). Intense illumination is provided by two quartz-iodine slide
projectors shining through spherical, water-filled flasks which serve
as additional lenses & heat filters. Further filters (see text) are sited
in apertures in the screen immediately in front of the electrode
vessels. These apertures are closed by a shutter with
two openings which align with the holes in the
screen as the shutter is pulled to the right,
allowing simultaneous onset of illumination. As the shutter
opens or closes it actuates a micro-switch so that this event is
automatically recorded.

New light sources are now coming onto the market which are based on light emitting diodes (Part C) & the brightness of
LED's continues to be improved in manufacture. The red ones are a particularly good source of photosynthetically active
light, they are relatively stable, produce little heat & switch on very quickly.
 REFERENCES

General References

Arnon, D.I. (1967) Photosynthetic activity of isolated chloroplasts. Physiol. Rev., 47,317.
Edwards, G.E., Huber, S.C. & Gutierrez, M. (1976) Photosynthetic properties of plant protoplasts. In: Microbial & Plant
Protoplasts (J.F. Peberdy, A.H. Rose, H.J. Rogers & E.C. Cocking, eds). Academic Press, New York, pp 299-322.
Edwards, G.E. & Walker, D.A. (1983) C3, C4, Mechanisms, & Cellular & Environmental Regulation of Photosynthesis.
Blackwell Scientific Publications Ltd, Oxford. pp 1-542.
Kalberer, P.P., Buchanan, B.B. & Arnon, D.I. (1967) Rates of photosynthesis by isolated chloroplasts. Proc. Nat. Acad.
Sci (Wash.) 57,1542-1549.
Leegood, R.C., Edwards, G.E. & Walker, D.A. (1981) Chloroplasts & Protoplasts. In: Techniques in Bioproductivity &
Photosynthesis (Coombs, J & Hall, D.O. eds) Pergamon Press Oxford, pp 92-109
Lilley, R.McC., Fitzgerald, M.P., Reinits, K.G. & Walker, D.A. (1975) Criteria of intactness & the photosynthetic activity
of spinach chloroplast preparations. New Phytol. 75, 1-10.
Price, C.A. Bartolf, M, Ortiz, W & Reardon, E.M. (1979) Isolation of chloroplasts in silica-sol gradients. In:
Methodological Surveys in Biochemistry Plant Organelles (Reid, E., ed) Vol. 9. Ellis Horwood, Chichester, pp 25-37
Robinson, S.P., Cerovic, Z.G. & Walker, D.A. (1986) Isolation of intact chloroplasts -General principles - criteria of
integrity. Methods in Enzymology. In press. *** must be in print now
Truesdale, G.A. & Downing, A.L. (1954) Solubility of Oxygen in Water. Nature, Lond. 173, 1236.
Vogel, A.I. (1961) Textbook of Quantitative Inorganic Analyses. Longman, London. pp 1-926.
Walker, D.A. (1964) Improved rates of carbon dioxide fixation by illuminated chloroplasts. Biochem. J. 92, 22c-23c.
Walker, D.A. (1971) Chloroplasts (& Grana) - Aqueous (including high carbon fixation ability). In: Methods in
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Walker, D.A. (1980) Preparation of higher plant chloroplasts. In: Methods in Enzymology (San Pietro, A. ed) Vol 69.
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Specific References

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Andreae, W.A. (1955) The photoinduced oxidation of manganous ions. Arch. Biochem. Biophys.55, 584-586.
Baldry, C.W, Walker, D.A, Bucke C. (1966) Calvin-cycle intermediates in relation to induction phenomena in
photosynthetic carbon dioxide fixation by isolated chloroplasts. Biochem J.101, 642-646
Bjorkman, O. & Demmig, B. (1987) Photon yield of O 2 evolution & chlorophyll fluorescence characteristics at 77K
among vascular plants of diverse origins. Planta 170,489-504.
Bruinsma, J. (1961) A comment on the spectrophotometric determination of chlorophyll. Biochim. Biophys. Acta. 52,
576-578.
Bucke, C, Walker, D.A. Baldry C.W. (1966) Some effects of sugars & sugar phosphates on carbon dioxide fixation by
isolated chloroplasts. Biochem J. 101,636-641
Bucke, C, Baldry, C.W., W ‘alker, D.A. (1967) Photosynthetic carbon dioxide fixation by isolated chloroplasts in
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photosynthetic phosphorylation in a reconstituted chloroplast system. Biochem J. 210, 273-276.
Cerovic, Z.G. & Plesnicar, M. (1984) An improved procedure for the isolation of intact chloroplasts of high
photosynthetic capacity. Biochem J. 223, 543-545.
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Cockburn, W., Baldry, C.W. & Walker, D.A. (1967) Photosynthetic induction phenomena in spinach chloroplasts in
relation to the nature of the isolating medium. Biochim. Biophys. Acta 143, 606-613.
Cockburn, W., Baldry, C.W. & Walker, D.A. (1967) Some effects of inorganic phosphate on O 2 evolution by isolated
chloroplasts. Biochim. Biophys. Acta 143, 614-624.
Cockburn, W., Walker, D.A. & Baldry, C.W. (1968) Photosynthesis by isolated chloroplasts. Reversal of orthophosphate
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chloroplasts from Zea mays. Aust. J. Plant Physiol. 8, 21-29.
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Delieu, T. & Walker D.A. (1972) an improved cathode for the measurement of photosynthetic oxygen evolution by
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Light-induced cation uptake into intact chloroplasts is driven by an electrical potential difference. Plant Physiol. 73,
164-174.
Edwards, G.E., Robinson, S.P., Tyler, N.J.C. & Walker, D.A. (1978) Photosynthesis by isolated protoplasts, protoplast
extracts & chloroplasts of wheat. Influence of orthophosphate, pyrophosphate & adenylates. Plant Physiol. 62, 313-7.
Edwards, G.E., Robinson, S.P., Tyler, N.J.C. & Walker, D.A. (1978) A requirement for chelation ion obtaining functional
chloroplasts of sunflower & wheat. Arch. Biochem. Biophys., 190, 421-433.
Edwards, G.E., Lilley, R.McC. & Hatch, M.D. (1979) Isolation of intact & functional chloroplasts from mesophyll &
bundle sheath protoplasts of the C4 plant Panicum miliaceum Plant Physiol. 63,821-827.
Gibbs, M. & Calo, N. (1959) Factors affecting light induced fixation of carbon dioxide by isolated spinach chloroplasts.
Plant Physiol. 34, 318-323.
Good, N.E., Winget, G.D., Winter, W., Conolly, T.N., Izawa, S. & Singh, R.M.M. (1966) Hydrogen ion buffers for
biological research. Biochemistry 5, 467-477.
Hall, D.O. (1972) Nomenclature for isolated chloroplasts. Nature New Biol. 235,125-126.
Heldt, H.W. & Rapley, L. (1970) Specific transport of inorganic phosphate, 3-phosphoglycerate &
dihydroxyacetonephosphate, & of dicarboxylates across the inner membrane of spinach chloroplasts. FEBS Letters 10,
143-148.
Hill, R. (1937) Oxygen evolved by isolated chloroplasts. Nature Lond., 139, 881-882.
Hill, R. (1939) Oxygen production by isolated chloroplasts. Proc. Roy Soc. B: 127, 192-210
Huber, S.C. & Edwards, G.E.(1975) An evaluation of some parameters required for the enzymatic isolation of cells &
protoplasts with CO2 fixation capacity from C3 & C4 grasses. Physiol. Plant. 35, 203-209.
Jensen, R.G. & Bassham, J.A. (1965) Photosynthesis by isolated chloroplasts. Proc. Nat. Acad.Sci (Wash) 56, 1095-1101.
Kaiser, W.M., Urbach, W. & Gimmler, H. (1980) The role of monovalent cations for photosynthesis of isolated intact
chloroplasts. Planta 149, 170-175.
Kanai, R. & Edwards, G. (1973) Enzymatic separation of mesophyll protoplasts & bundle sheath cells from leaves of C4
plants. Die Naturwissenschaften 60, 157-158.
Kanai, R. & Edwards, G. (1973) Separation of mesophyll protoplasts & bundle sheath cells from maize leaves for
photosynthetic studies. Plant Physiol. 51,1133-1137.
Kanai, R. & Edwards, G. (1973) Purification of enzymatically isolated mesophyll protoplasts from C3, C4 &
Crassulacean Acid Metabolism plants using an aqueous dextran polyethylene glycol two-phase system. Plant Physiol. 52,
484-490.
Larkum, A.W.D. & Wyn Jones, R.G. (1979) Carbon dioxide fixation by chloroplasts isolated in glycinebetaine. A
putative cytoplasmic osmoticum. Planta 145, 393-394.
Leegood, R.C, & Walker D.A. (1980) Autocatalysis & light activation of enzymes in relation to photosynthetic induction
in wheat chloroplasts. Arch Biochem Biophys 200, 575-582
Lilley, R.McC. & Walker, D.A. (1979) Studies with the reconstituted chloroplast system. In: Encyclopedia of Plant
Physiology - Photosynthesis. (Gibbs, M. & Latzko,E. eds) Vol. II. New Series, Springer-Verlag Berlin, Heidelberg, New
York, pp 41-52.
Ludwig, L.J. & Whitehouse, D.G. 1970. Oxygen evolution in the dark following illumination of chloroplasts in the
presence of added manganese. FEBS Letters 6, 281-284.
Maury, W.J., Huber, S.C. & Moreland, D.E. (1981) Effects of Mg ++ on intact chloroplasts. II Cation specificity &
involvement of the envelope ATPase in (sodium) potassium/proton exchange across the envelope. Plant Physiol. 68,
1257-1263.
Mehler, A.H. (1951) Studies on reactions of illuminated chloroplasts. I Mechanism of the reduction of oxygen & other
Hill reagents. Arch. Biochem. Biophys. 33, 65-77
Mehler, A.H. (1951) Studies on reactions of illuminated chloroplasts. II Stimulation & inhibition of the reaction with
molecular oxygen Arch. Biochem. Biophys. 34, 339-51.
Mills, W.R. & Joy, K.W. (1980) A rapid method for isolation of purified, physiologically active chloroplasts, used to
study the intracellular distribution of amino acids in pea leaves.Planta 148, 75-83.
Morganthaler,J-J., Price,C.A., Robinson,J.M.& Gibbs,M.(1974) Photosynthetic activity of spinach chloroplasts after
isopycnic centrifugation in gradients of silica. Plant Physiol. 54, 532-534
Nakatani, H.Y. & Barber, J. (1977) An improved method for isolating chloroplasts retaining their outer membranes.
Biochim. Biophys. Acta 461, 510-512.
Mourioux, G. & Douce, R. (1981) Slow passive diffusion of orthophosphate betwen intact isolated chloroplasts &
suspending medium. Plant Physiol. 67, 470-473.
Robinson, S.P. (1977) Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in
exchange endogenous adenine nucleotides.Plant Physiol.59,422-427.
Robinson, S.P. (1983) Isolation of intact chloroplasts with high CO 2 fixation capacity from sugarbeet leaves containing
calcium oxalate. Photosynthesis Research 4, 281-7.
Robinson, S.P. (1986) Improved rates of CO 2-fixation by intact chloroplasts isolated in media with KCl as the osmoticum.
Photosynthesis Research 10, 93-100.
Robinson, S.P. & Downton, W.J.S. (1984) Potassium sodium & chloride content of isolated intact chloroplasts in relation
to ionic compartmentation in leaves. Arch. Biochem. Biophys. 228, 197-206.
Robinson, S.P., Edwards, G.E. & Walker, D.A. (1979) Established methods for the isolation of intact chloroplasts. In:
Methodological Surveys in Biochemistry Plant Organelles (Reid,E.,ed) Vol. 9. Ellis Horwood, Chichester, pp 13-24
Robinson, S.P. & Walker, D.A. (1977) Pyrophosphate inhibition of carbon dioxide fixation inisolated pea chloroplasts by
uptake in exchange for endogenous adenine nucleotides. Plant Physiol.I59, 422-427
Robinson, S.P. & Walker, D.A. (1979) The control of 3-phosphoglycerate reduction in isolated chloroplasts by the
concentrations of ATP, ADP & 3-phosphoglycerate. Biochim. Biophys.Acta. 545,421-433.
Robinson, S.P. & Wiskich, J.T. (1976) Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic
amounts of adenine nucleotides. Plant Physiol. 58, 156-162.
Robinson, S.P. & Wiskich, J.T. (1977) Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts
by uptake in exchange for endogenous adenine nucleotides. Plant Physiol. 65, 291-297.
Sicher, R.C. (1984) Glycolaldehyde inhibition of photosynthetic carbon assimilation by isolated chloroplasts &
protoplasts. In: Advances in Photosynthesis Research (Sybesma,C. ed.) Martinus Nijhoff/ Dr. W .Junk Pubs. Vol.III. p
413-416.
Slabas, A.R. & Walker, D.A. (1976) Enzymic reconstitution of photosynthetic carbon assimilation. Pentose phosphate-
dependent O2 evolution by illuminated envelope free chloroplasts from Spinacia oleracea Arch. Biochem. Biophys. 175
590-597.
Stankovic, Z.S. & Walker, D.A. (1977) Photosynthesis by isolated pea chloroplasts. Some effects of adenylates &
inorganic pyrophosphate. Plant Physiol. 59, 428-432.
Stitt, M. & Heldt, H.W. (1981) Physiological rates of starch breakdown in isolated intact spinach chloroplast. Plant
Physiol. 68, 755-761.
Stokes, D.M. & Walker, D.A. (1971) Phosphoglycerate as a Hill oxidant in a reconstituted chloroplast system. Plant
Physiol. 48, 163-165.
Stokes, D.M. & Walker D.A. (1972) Photosynthesis by isolated chloroplasts. Inhibition by DL-glyceraldehyde of carbon
dioxide assimilation. Biochem. J. 128,1147-1157.
Stokes, D.M., Walker, D.A. & McCormick A.V. (1972) Photosynthetic oxygen evolution in a reconstituted chloroplast
system. In: Progress in Photosynthesis.(Forti, G., Avron, M. & Melandri, A., eds) Proc. II Int. Cong. on Photosynthesis,
Stresa 1971, W. Junk, NV Pub, The Hague p 1779-1785.
Walker, D.A. (1976) CO2 fixation by intact chloroplasts: photosynthetic induction & its relation to transport phenomena
& control mechanisms. In: The Intact Chloroplasts (Barber,J. ed) Chapter 7, Elsevier, Amsterdam pp 235-278.
Walker, D.A. & Hill, R. (1967) The relation of oxygen evolution to carbon assimilation with isolated chloroplasts.
Biochim. Biophys. Acta 131, 594-596.
Walker, D.A., Baldry, C.W. & Cockburn, W. (1968) Photosynthesis by isolated chloroplasts, simultaneous measurement
of carbon assimilation & oxygen evolution Plant Physiol. 43,1419-1422.
Walker, D.A., Ludwig, L.J. & Whitehouse, D.G. (1970) Oxygen evolution in the dark following illumination of
chloroplasts in the presence of added manganese. FEBS Letters 6, 281-284.
West, K.R., & Wiskich J.T. (1968) Photosynthetic control by isolated pea chloroplasts Biochem J. 109, 527-32
Whatley, F.R., Allen, M.B., Rosenberg, L.L., Capindale, J.B. & Arnon, D.I. (1956) Photosynthesis by isolated
chloroplasts. V. Phosphorylation & carbon dioxide fixation by broken chloroplasts. Biochim. Biophys. Acta 20, 462-468.
Whitehouse, D.G., Ludwig, L.J. & Walker, D.A. (1971) Participation of the Mehler reaction & catalase in the oxygen
exchange of chloroplast preparations.J. Exp. Botany 22, 772-791.
Yamazaki, R.K. & Tolbert, N.E. (1970) Photoreactions of flavin mononucleotide & a flavoprotein with zwitterionic
buffers. Biochim. Biophys. Acta. 197,90-92.
 PART G

DEFECTS AND PRECAUTIONS

47(a) The Sensor.

Most O2 sensors have a platinum or gold cathode which is electrically joined to an anode (usually silver) by an electrolyte
(a solution containing potassium chloride). The cathode is then polarised (Part A). All of this constitutes a basically
unstable situation. The best electrodes have cathodes which have been very carefully sealed into lead glass which has a
coefficient of expansion very similar to that of platinum. Other electrodes are often sealed in "Araldite" or a similar epoxy
resin. These are very good for what they cost but they do have a very limited life, partly because they find themselves in
an unstable situation (both electrically & physically) & partly because they are mostly maltreated by their users who are
usually too preoccupied with paying the rent & solving the mysteries of the universe to pay them the attention that they
deserve. Epoxy resins gradually take up water. Seals between cathodes & resin gradually deteriorate. Electrolyte
penetrates into crevices, crystallises & widens fissures. The underlying electrochemistry virtually ensures that cathodes
will become changed by deposition of other metals, oxidised or poisoned. Contacts or leads become broken. So, if you
have an electrode at all, you must resign yourself to the fact that, if it is relatively cheap it won't last & that if it is relatively
expensive you probably won't be able to afford it. Thereafter, there are things that you can do which will help.

47(b) Use.

Do not be tempted to leave a "good" electrode unchanged. You will almost certainly be too idle to change it daily but you
should certainly change it weekly. Have two electrodes. At the end of a week, clean the one that has been in use & put it
in a desiccator (a plastic box containing silica gel will do). Weekly cleaning should not involve more than polishing the
platinum surface with aluminium oxide (polishing grade) on dampened felt or cotton wool. Set up the replacement
electrode & let it polarise overnight or over the weekend. The silver may need cleaning periodically if the layer of chloride
has become contaminated. With the Hansatech Instruments Ltd electrode disc, for example, this is best done by subjecting
it to a reverse current. This can be done with a simple apparatus comprising an electrode (a silver rod), a 3V D.C. supply
(2 x 1 1/2V batteries), 50% saturated sodium sulphate solution, & the electrode disc. The electrode disc is partially
submerged in the solution (enough to cover the silver ring but not the connector). A negative (3V) potential is applied to
the anode & the rod is used to complete the circuit. Hydrogen is thereby generated at the silver ring, dislodging particles
& reducing the oxidised surface layer. A 2 hour treatment is generally sufficient.

Even a modestly priced electrode may cost as much as a reasonable camera. It is unlikely that you dip your camera in
sea-water every week & leave it to dry on the bench. You are obviously obliged to use an electrolyte but don't leave your
electrode drying out on the bench & then expect it to go on working as well as ever, month after month.

47(c) Membrane Application

Use an effective applicator to apply the membrane & ensure that it is correctly in place (neither slack, crinkled nor
over-stretched) & that, if it depends on an O-ring, that the O-ring is the correct size & has not perished. Use a lens or a
microscope to check that you have put the membrane on correctly. If a membrane is too loose or stretched too tightly,
some movement is more or less inevitable & consequent changes in the electrolyte path will lead, or contribute to, "drift"
(i.e. the air-line will change, at an unacceptable rate, in an open chamber or vessel). If a "spacer" (Part A) is not used with
the Hansatech Instruments Ltd electrode & if an over-tight O-ring therefore narrows the electrolyte layer, lack of linearity
may be experienced.

47(d) The Leaf-disc Electrode.

Because this is mostly used in saturating CO2 & measures in the gas-phase, it is important to use a modified electrolyte
(e.g., one part saturated KCl solution, one part 0.4M borate buffer at pH 9.0 & two parts 1.0M sodium bicarbonate
solution previously adjusted to pH 9.0 by the addition of equimolar sodium carbonate - Delieu & Walker, 1981) or to
allow adequate equilibration (hydroxyl ions are generated in use & CO 2 will dissolve in the electrolyte - this does not
matter but it should be allowed for).

47(e) Leaks.

The leaf-disc electrode was designed to measure changes in oxygen in a closed space. If the chamber leaks, it will not
stop the electrode working but there will be a decrease in accuracy at best, & total malfunction at worst. The ports & taps
can leak & should be checked-out individually. Start with a proven electrode disc in place & put a disposable gas-
tightsyringe directly into each port (rather than the taps). Check that each port has its correct O-ring & that it is tightly
threaded. Similarly, the O-ringagainst which the sensor seals should be clean, in good condition & properly positioned
before the electrode is held firmly in position by tightening the base. Moving either of the syringe plungers should then
cause a positive or negative excursion to a new, more or less unchanging value (Section 4). If the chamber seals, repeat
the procedure with first one tap & then both taps in position. Each tap should be pushed firmly into the luer on the port
before its locking-nut is tightened. Check that the large O-ring which makes the seal between the chamber itself & its
upper water-jacket is of the correct size & that it is adequately compressed when the clips which pull the two parts together
are tightened.

47(f) Membranes & Electrolytes

There are lots of both (see e.g., Sections 2 & 47d) & they are almost as difficult to recommend, in general terms, as are
plays to theatre-goers. One which has been used successfully with the leaf-disc electrode contains: 0.2M KCl, 66mM Na 2
HPO4, 0.6M KNO3 + 70mM NaOH at pH 11.2 (with HCl) (N.B. THIS IS CAUSTIC). It has the advantage of speeding
stabilisation on applying a polarising voltage. Most manufacturers provide their own membranes & these are always first
choices but you may find (as did Bjorkman & Demmig, 1987) that a Hansatech Instruments Ltd electrode worked best,
in their hands, with a Beckman membrane (part no. 77948) & electrolyte (part no. 76438). (Our own experience has been
different but the difference could be in the manner in which the membrane is applied - accordingly you should use what
suits you best.) In general, thinner membranes are to be preferred (see Appendix 2) if rapidity of response is required &
more robust membranes if resistance to damage is more important.

PICTURE OF AN O2 ELECTRODE

There was a little girl & she had a little curl

right in the middle of her forehead.

& when she was good, But when she was bad
she was very, very good. she was horrid.

References

Bjorkman, O. & Demmig, B. (1987) Photon yield of O 2 evolution & chlorophyll fluorescence characteristics at 77K
among vascular plants of diverse origins. Planta 170 489-504.
Delieu, T. & Walker, D.A. (1981) Polarographic measurement of photosynthetic oxygen evolution by leaf discs.New
Phytol. 89 165-178.
Osmond, C.B. (1986) Research in South-East Asia. Nature 320: 307.
 Appendix 1

Preparation of Ferredoxin
Preparation of Ferredoxin

If you tire of electrodes & wish to remind yourself of what bucket biochemistry is like, use the spinach that you have
accumulated in your glass-house deepfreeze (every good glasshouse should have one) to prepare ferredoxin. The
procedure involves the preparation of a crude homogenate from spinach leaf & the use of diethylaminoethyl cellulose to
absorb the ferredoxin in it. The crude ferredoxin is eluted from the cellulose, partially fractionated by ammonium sulphate
precipitation & finally passed through a second DE cellulose column. At this stage of purity it is good for experiments
with the reconstituted chloroplast system (Section F12).

Procedure

Day 1
Prepare the buffer solutions & equilibrate the DE52 cellulose.

A. Prepare 2 litres of 1 M KH2PO4 at pH 7.5. Use this in the preparation of the following solutions:
i) 15 litres of grinding medium (20 mM KH 2PO4 at pH 7.5),
ii) 1 litre of 20 mM KH2PO4/0.2 M NaCl at pH 7.5,
iii) 1 litre of 20 mM KH+PO4/0.8 M NaCl at pH 7.5.
iv) 10 litres of 20 mM KH2PO4 at pH 7.5 (for cellulose equilibration)
B. Wash 125 g of Whatman's DE52 cellulose with 5 litres of distilled water. This is best done by suspending the cellulose
in about 2 litres of water & pouring it on to a number 1 filter paper in a large buchner funnel, using the rest of the water
to wash the cellulose on the filter paper. Finally wash with 1 litre of 1 M KHPO 4 at pH 7.5 followed by 10 litres of 20
mM KH2PO4 at pH 7.5.
C. Resuspend the washed cellulose in about 200 ml of grinding medium, & leave this, together with all other solutions,
in the refrigerator overnight.
D. Leave the equipment required on Day 2 in the refrigerator overnight (homogenizer, centrifuge bottles, chromatography
columns, etc).

Day 2
A. Use frozen spinach from which the mid-ribs have been removed. Slice the spinach with a sharp knife & homogenize
in grinding medium using about 200 ml medium for every 200 g of spinach.
B. Squeeze the homogenate through a double layer of muslin into an ice-cooled conical flask.
C. Centrifuge the filtrate at 16,000 g for 10 mins.
D. To the supernatant, add solid NaCl 6 g/litre & adjust the pH to 7.5 via NaOH.
E. Add DE52 slurry (about 40 ml/litre) & stir briefly, then leave to stand in the cold, for at least ½ hour.
F. Decant off the liquid & pour the slurry into a 3 cm (wide) column, & pack the DE52 slowly, wash the column with
buffer containing 0.2 M NaCl. Do not run the column too quickly as the fine cellulose is easily dried out. Wash until the
eluate is clear.
G. When the eluate is clear, elute the ferredoxin with the high salt buffer. A red-brown band begins to descend, & is
collected as it emerges from the column.
H. Add ammonium sulphate slowly to the collected solution to give a 50% w/v solution (0.5 g ammonium sulphate to 1
ml of solution). The pH is adjusted to 7.5 with NaOH & the solution left to stand for 30 min.
I. Centrifuge the suspension at 10,000 rpm for 30 min & retain the supernatant.
J. The supernatant is dialysed against 4 litres of 20 mM phosphate buffer at pH 7.5 for 12 hours (overnight).

Day 3
A. Pack a column (small Pharmacia K9/15) with DE52, KEEP A LOW FLOW RATE! Load the dialysate on the column,
then wash with 0.2 M NaCl in buffer, then slowly elute with 0.8 M NaCl in buffer.
B. Estimate the concentration by measuring the O.D. in a 2 mm light path at 420 nm.

420 O.D. X 1.22 = mg Fd ml-1

0.2
C. Store under liquid nitrogen & dialyse before use (store in high salt for long periods).

N.B. All operations must be carried out in the cold. Speed is essential from homogenization until the first addition of
DE52. Delay at this stage results in a poor yield of ferredoxin.
 Appendix 2

Suppliers

Oxygen Electrodes.

The measurements described in this manual are largely based on Hansatech Instruments Ltd equipment (Appendix 4).
This is because much of the apparatus now commercially available from this firm was originally designed & constructed
by myself & Tom Delieu, specifically for photosynthetic work. The "leaf-disc" electrode & related apparatus is not
available elsewhere. Hansatech Instruments Ltd equipment includes electrodes, control boxes, cuvettes, light sources,
fluorescence accessories, etc., computing software & this manual & is available from Hansatech Instruments Ltd
Instruments Limited, Narborough Road, Pentney, Kings Lynn, Norfolk, PE32 1JL, U.K.

Other aqueous-phase O2 electrodes, not specifically designed for photosynthesis, (& accessories) may be obtained, for
example from:-

Yellow Springs Instrument Co., Box 279, Yellow Springs, OH 45387., U.S.A.
(U.K. Agent: Clandon Scientific, Lysons Avenue, Ash Vale, ALDERSHOT, Hampshire, GU12 5QR).
Beckman Instruments, Inc., Altex Scientific Div. 2350 Camino Ramon, San Ramon, CA 94583., USA
(U.K. Agent: Analytical Instruments, Progress Road, Sands Industrial Estate, HIGH WYCOMBE, Bucks, HP12 4JL).

A gas-phase electrode which can be recommended, is that supplied by Draeger Medical Ltd, Hertfordshire House, Wood
Lane, Hemel Hempstead, Hertfordshire.

Miscellaneous

(a) Algal & Bacterial Inhibitors

Boots Ltd, Nottingham, NG2 3AA, U.K.

(b) Chart Recorders

Fischer Scientific Co., Pittsburgh, Pa., U.S.A.
Rikedenki-Mitsui, Oakcroft Road, Chessington, Surrey, KT9 1SA, U.K.

(c) Dextran
(T20-Avg. MW 20,000)
(T20-Avg. MW 40,000)
U.S. Biochemicals Corp., 21000 Miles Parkway, Cleveland, OH 44128, U.S.A.
(Dextran T20 & T40)
Pharmacia Fine Chemicals, Uppsala, Sweden. (Dextran T40)

(d) Enzymes
Cellulase from Trichoderma viride (contains cellulose & hemicellulose
degrading enzyme)
As Onozuka R10 & RS
Yakult Biochemical Co. Ltd., Enzyme Products, 8-21 Shingikancho, Nishinomiya, Japan.
As Cellulysin
Calbiochem-Behring Corp., Hoechst UK Ltd., Hoechst House, Salisbury Road,
Hounslow, Middx. TW4 6JH, U.K.
Calbiochem-Behring Corp., P.O. Box 12087, San Diego, CA 92112, U.S.A.
As Onozuka R10
Unwin & Co. Ltd., Prospect Place, Welwyn, Hertfordshire, U.K.
As Meicelase
Meiji Seika Kaisha Ltd., 8-2 Chome Kyobashi, Chuo-Ku, Tokyo, Japan.
Pectinase from Rhizopus sp. (contains polygalacturonase)
As Macerozyme R10
Yakult Biochemical Co. Ltd. (see above)
Unwin & Co. Ltd. (see above)
As Macerase
Calbiochem-Behring Corp. (see above)
Pectinase from Aspergillus sp.
As Extractase PC
Fermco Biochemics, Inc., 2638 Delta Lane, Elk Grove Village, IL 60007, U.S.A.
As Rohament P
Rohm GmbH, D-6100 Darmstadt, Kirschenallee, Postfach 4242, West Germany.
As pectolyase Y23-Pectinase from Aspergillus japonicus (active components
polygalacturonase, pectin lyase & an unidentified protein factor).
Seishin Pharmaceutical Co. Ltd., Noda, Chiba, Japan.

(e) Glasshouse Lamps

Wotan Lamps Ltd, Wotan House, 267, Merton Road, London, SW18 5JS, U.K.

(f) Heat Filters

OCLI (Europe), Central Way, Hill End Industrial Estate, Dunfermline, Fife, KYL 5JE, U.K.
OCLI, Santa Rosa, California, U.S.A.

(g) Light Meters/Quantum Sensors

Skye Instruments Ltd, Unit 6, Ddole Industrial Estate, Llandrindod Wells, POWYS, Wales. LD1 6DF
Li-Cor, Lincoln, Nebraska, U.S.A.

(h) Light Sources

Phillips Lighting Division, (U.K. Agent: Photographic Lighting U.K.,
Magnetic Motors Complex, Water Lane, Leeds, LS11 5PR, U.K.)
Schott - U.K., Drummond Road, Astonfields Industrial Estate, Stafford, ST16 3EL, U.K.
West German Agents: Schott Glaswerke, Works Wiesbaden, PO Box 130367, D-6200, Wiesbaden 13, West Germany.

(i) Mass Flow Controllers

Chell Instruments Ltd, Tudor House, Grammar School Road, North Walsham,
Norfolk, NR28 9JH, U.K.
Tylan, Carson, California, U.S.A
(U.K. Agents: Epak Electronics Ltd, Pool House, Bancroft Road, Reigate, Surrey,
RH27AP, U.K.)

(j) Membranes
Beckman Ltd. (see above)
Draeger Medical Limited, Hertford House, Wood Lane Hempstead, Hertfordshire, U.K.
General Electric Company, Membrane Products Operation, Medical Systems Division,
1, River Road, Schenectady, New York, N.Y. 12345, U.S.A.
Hansatech Instruments Ltd Instruments Ltd. (see previous page)
Yellow Springs Instrument Co. (see above)

(k) Nylon mesh

Henry Simon, Ltd., P.O. Box 31, Stockport, Cheshire, SK3 ORT, U.K.
Tetko, Inc., Precision Woven Screening Media, 420 Saw Mill River Road,
Elmsford, NY 10523, U.S.A.

(l) Optical Filters

OCLI (Europe), Central Way, Hill End Industrial Estate, Dunfermline, Fife, KYL 5JE, U.K.
West German Agents: Oriel GmbH, Darmstadt, West Germany
Balzers, Northbridge Road, Berkhamsted, Herts.
Schott - U.K., Drummond Road, Astonfields Industrial Estate, Stafford, ST16 3EL, U.K.
Corning, Optical Products Department, Corning Glass Works, Corning NY 14830, U.K.

(m) Optics
Melles Griot, 15, South Street, Farnham, Surrey, GU9 7QU, U.K.
Melles Griot, Irvine, California, U.S.A.
Ealing Beck Limited, Greycaine Road, Watford, WD2 4PW, U.K.

(n) Percoll
Pharmacia G.B., Prince Regent Road, Hounslow, Middlesex, TW31NE U.K.

(o) Polystyrene Supports

Polystyrene Supports, Accelerated Prop. Ltd, Vines Cross, Heathfield, Sussex, U.K.

(p) Polytron
Kinematica GmbH, Ch-6000 Kriens, Luzern, Switzerland.

(q) Spinach Seeds

Ferry Morse Seed Company, P.O. Box 100, Mountain View, California 94042, U.S.A.
Rijk Zwaan, P.O. Box 40-2678 ZG Burg, Crezeelaan 40-2678 KX De Lier, Holland.

(r) Stabilised Power Supplies

Clifford Industries, Amarillo, California, U.S.A.

(s) Water Culture Solution

I.C.I. Plant Protection Division, UK Department, Bear Lane, Farnham, Surrey, GU9 7UB, U.K.

DIFFERENTIATOR
Differentiation of the O2 signal can be achieved by concentrating a 2-10 F capacitor (in series) followed by a 30 resistor
(in parallel) between the output of the O2 electrode box & the input of a recorder. If such a device is used to differentiate
a change of lv/sec, it will allow "change" in O2 to be recorded as "rate of change" (dO2/dt) in a mv range. Pre-amplification
of the O2 signal prior to differentiation will improve the signal to noise ratio.