1.

INTRODUCTION

The measurement of oxygen evolution in a closed system is one of the easiest and

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 and Walker

(1981) and now manufactured by Hansatech (Appendix 2). As such it complements

“Notes for Users” supplied with the Hansatech LD2. 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

In photosynthesis, light energy is absorbed by chlorophyll and used to drive the

reduction of carbon 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 and

starch) are formed from three-carbon sugar derivatives. For simplicity, all of these

substances can be represented by a purely nominal carbohydrate, CH20, and the

overall process by the equation:-

in which the light energy needed to drive this process is represented by “hv” (h =

Planck’s constant and the Greek letter “v” the symbol used to represent the frequency

of light).

Accordingly, if a leaf is enclosed in a chamber and provided with carbon dioxide (or

bicarbonate as a source of carbon dioxide) and then illuminated, oxygen will be

evolved. In the Hansatech LD2 (Fig. 2.1.), a leaf-disc is used and CO2 is provided in

the gas-phase or in the form of sodium bicarbonate (which dissociates in solution):-

The oxygen which accumulates in the gas-phase during photosynthesis is then

detected, polarographically, by a “Clark-type electrode (Clark, 1956). The “regular”

Hansatech version of the Clark-type electrode (Fig 2.1) comprises a relatively large

(2mm) platinum cathode and a silver anode immersed in, and linked by, an

“an easy way of following

photosynthesis”

“a temporary storage product”

NaHCO3 NaOH + CO2 ........Eqn. 2.2

electrolyte. Both electrodes are set in a plastic (epoxy resin) disc; the cathode at the

centre of a dome and 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 and the purpose of the dome is to stretch the membrane

smoothly over the surface of the platinum cathode and to allow it to be secured in

position by an O-ring. The membrane also traps a thin layer of electrolyte.

Figure 2.1. Schematic diagram of a gas-phase oxygen electrode and 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 LS1 or LS2. The

fluorescence probe is a photodiode 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) are not shown. The taps (with luers)

are for calibration and adjustment of the gas phase (after Delieu and Walker, 1983).