An Expanding Universe of Circadian Networks

In Higher Plants
Vidushi Rastogi
Roll No. 5014
Chairman: Dr. R.C. Bhattacharya
Why at all a plant should have a precise clock?

The flower that you hold in your

hands was born today and already
it is as old as you are. 
~Antonio Porchia, Voces, 1943
"Living organisms have adapted to
the daily roation of the earth on its
axis."
“The Circadian Clock is a plant’s best
friend in a spinning world.”
Eriksson and Millar , 2003
What is a Circadian Rhythm?
Circadian rhythms are the subset of biological rhythms with a
period of ~24 h
Franz Halberg in 1959 coined the term circadian from the Latin
words “circa ”, about and “dies”, day.
The Cir. Rhythms are endogenously generated & self
sustaining, so they persist under constant environmental
conditions.
A true Circadian Rhythm persists in absence of environmental
time cues, termed Zeitgebers.
Zeitgebers entrain endogenous timing system to a period of 24h
, precisely corresponding to the exogenous period of the earth’s
rotation.
Fundamental Parameters of Circadian
Rhythm-

Periodicity
Entrainability
Temperature Compensation
What is a Circadian Clock?

The set of components that underlies all the

properties of a circadian rhythm.
 Build of a Circadian Clock

Input Pathways
Oscillator
Output Pathways
Linear Conceptual Model of a simple Circadian System

McClung,2001
Model of an Arabidopsis circadian clock

XII

IX

III

VI

Alabadi et al , 2001
Hayama & Coupland 2003, 2008
 Arabidopsis Circadian Clock :The components

Input Pathway
In Arabidopsis the Cir. Clock is entrained by:
1. Cryptochromes – e.g. the blue light entrains via
CRY1,CRY2.
2. Phytochromes –e.g. the red light entrains by
PhyA,PhyB,PhyD & PhyE.

ZTL protein interacts with both PhyB & CRY1.

PhyB expression is regulated by a circadian oscillator
Another gene that modulates photoperception &
transduction is ELF3
ELF3 & PhyB interact
Core Oscillator:
Consists of transcriptional
loops of negative feed back 12
inhibition.
The Candidate Components: 9 3
CCA1
6
LHY
TOC1
Core Feed-back Mechanism is based on reciprocal
regulation:

A. 2 morning expressed MYB transcription factors

CCA1 & LHY.
B. Evening based Pseudo Response Regulator TOC1.
 The Mechanism

CCA1 LHY CCA1 LHY

EE

TOC1

Dawn Dusk
The Mechanism

X X

CCA1 LHY
CCA1 , LHY are CCGs & shown to be oscillating at both
mRNA & protein levels.

Daily changes in chromatin structure also regulate

transcription of clock genes.

A rhythmic pattern of histone acetylation at the TOC1

promoter precedes the peak of TOC1 expression
suggesting the presence of clock controlled changes in
chromatin structure.
Additional phase specific feedback loops:

Morning loop
Expression of two TOC1 homologs PRR7 & PRR9 is directly
activated by CCA1 & LHY.
PRR7 & PRR9 are partially redundant to inhibit expression of
CCA1 & LHY.
Evening Loop
Induction of TOC1 by an evening phased component(GI).
GI is light inducible & negatively regulated by TOC1 , LHY &
CCA1.
The Output Pathway:
The PTM of proteins by circadian signals suggest the
presence of intracellular signaling pathways that couple the
circadian oscillator to the regulation of cellular physiology.

Ca2+ based signaling pathways may form part of at least one

of the output pathways. The whole plant cytosolic free
[Ca2+ ] oscillates within a circadian period.
Regulated Protein Turnover with in the Circadian
Oscillator:

Arabidopsis circadian oscillator includes Post Translational

Regulation of protein levels & function.

Stability of many clock components is regulated by proteasomal

degradation.

The proteasomal degradation of TOC1 & its homolog PRR is

mediated by interaction with F-box protein Zeitlupe.
Light dependent interaction between ZTL & GI stabilizes ZTL &
TOC1 protein levels through out the day.

TOC1 is further stabilized by PRR which directly binds to TOC1

preventing its interaction with ZTL at the beginning of the night.

Protein phosphorylation also regulates activity & abundance of

clock components . Over-expression of CK2 beta subunit leads
to altered Cir. rhythm probably by modifying the
phosphorylation status of oscillator components.
 Typical reporters are unsuitable for circadian studies-

Even though mRNA abundance oscillates in response to clock gated

transcription , the reporter activity is too stable to allow turnover
within a circadian cycle. The accumulation of reporter activity
obscures the underlying rhythm in transcription.
 LUC protein is stable & accumulates over time but LUC
activity is unstable.

Activity over time requires translation of new LUC protein &

provides reliable assessment of LUC transcription.

Measurement of LUC transcription is non-destructive ,

quantitative & allows both temporal & spatial resolution of gene
expression in real time in-vivo .
The study
A set of 5’ upstream DNA sequences with varied length of
the CCA1 gene were fused to the LUC gene.

3 types of CCA1::LUC fusions were constructed.

 When the intensities of bioluminescence were monitored in LD
for the cells carrying CCA1::LUC , they showed a robust rhythm
with a period of ~ 24 h.

Although the maximum intensity varied among these

constructs ,in every case the bioluminescence profiles were well
synchronized with peaks at dawn every day.

This is consistent with the fact that CCA1 is a typical e.g. of

circadian controlled morning gene in plants.

This also suggested that the promoter region of CCA1 is

sufficient to generate the oscillation.
 The result was reproducibly confirmed with another independent
cell line(separately cultured under essentially the same conditions).
RNA was prepared from cells & subjected to Northern analysis.

This helped in direct detection of transcripts that was derived

from the endogenous CCA1 gene.

The diurnally oscillated profile of bioluminescence was well

coincident with that of the intrinsic transcript of CCA1.
 The experiment was repeated with APRR1(TOC1).

The bioluminescence intensities peaked at the evening. This was

well consistent with the fact that APRR1 is a typical e.g. of Cir.
controlled evening gene.

The established cell lines , equipped with such bioluminescence

reporters , might provide us with an advantageous means to
characterize the plant circadian clock .
The Phase Specific Oscillation
 How Many Clocks ?

Multiple oscillators exist in multicellular plants

Evidence taken from :

Multiple rhythms running with different periods (Internal

Desynchronisation) demonstrated in Phaseolus coccineus
(Mayer, Sadleder,1972) & Chenopodium rubrum(King.1975).

Demonstration of 2 oscillators with in a single cell of the

dinoflagellate Gonyaulax polyhedra (Roenneberg et al , 1994)
 Its Not Just About the Time
Cir. Clock allows an organism to anticipate rhythmic changes in
envt & hence provides adaptive advantage.

Cir. Clock increases reproductive fitness.

Escape from light hypothesis-posits an advantage in phasing

sunlight sensitive cellular events to the night.

Cir. Clock controls PGR.

Cir. Clock sparks plant’s ability to survive freezing weather.

One can target the expression of one’s favorite gene to particular
time of the day. e.g. mRNA abundance of CAT2 & CAT3 peaks
at dawn & dusk respectively.(Phase Angle Markers)
-Zhong & McClung , 1996

Evening specific promoters have been defined for the

Arabidopsis genes encoding a glycine rich RNA-binding protein
(AtGRP7) & a germin-like protein (AtGER3).
- Staiger,Apel,Trepp , 1999

Cir.Clock orchestrates not only the expression of protein coding

genes but also the rhythmic oscillation of introns, intergenic
regions & non coding RNAs.
- Mas & Yanovsky , 2009
 Path to be traversed

We have to assemble the components into a coherent molecular

model.

Cir. Regulation should be included in climate models in order to

obtain better results.

We have to understand (if) how the plant circadian clock could

assist with increasing agricultural output for both food as well as
demand for biofuels.
 II have
have millions
millions of
of
clocks
clocks to
to keep
keep time.
time. II
flower
flower only
only when
when II
expect
expect Sun
Sun &&
How come u
Pollen…
Pollen…
people always
flower at the
same time?