CIRCADIAN RHYTHMS IN PLANTS

* WHAT IS A CIRCADIAN CLOCK?

* CLOCK OUTPUTS
* CLOCK INPUTS
* THE CENTRAL OSCILLATOR
* OTHER COMPONENTS
* SEASONAL RHYTHMS
* EVOLUTIONARY RELATIONSHIPS

WHAT IS A CIRCADIAN CLOCK?

* Rhythmicity in behavior, physiology and biochemistry of organisms
* Some rhythms persist even without environmental changes
circadian rhythms 24 h period
endogenous control: circadian clock
* Anticipation of rhythmic changes in the environment
changes in the physiological state that provide them with an adaptive advantage

RHYTHMIC OUTPUTS - PROCESSES REGULATED BY THE CLOCK

* de Mairan, 1727: rhythmic leaf movements in constant darkness
* most of current knowledge based on rhythmic cellular or physiological processes:
a) mammals: digestion, regulation of body temperature, hormone secretion, time of sleep onset
b) cyanobacteria: photosynthesis, nitrogen fixation, cell division

RHYTHMIC OUTPUTS
c) plants: leaf movements, cell elongation rates, stomatal aperture, CO2 assimilation, Calvin cycle,
ethylene production, hypocotyl elongation
* Gene expression:
a) cyanobacteria: 80% of genes are CCGs > the clock regulates the transcriptional machinery?
> additional regulatory layers for many genes
b) Arabidopsis: 6% are CCGs, peaking at all phases of day and night

CCG clusters

* photosynthesis-related genes
* photoreceptor genes and downstream signaling components
* photoprotective pigments
* chilling resistance, cold and drought
* carbon allocation, nitrogen and sulfur assimilation
* flowering, cell elongation
* 25% of the CCGs are totally uncharacterized!!!

very conserved motif, evening element, present 46 times in the promoters of 31 genes

CLOCK RESETTING CLOCK INPUTS

* circadian clocks must be
reset so that internal time matches local time (entrainment)> signals such as light, temperature,
and nutrient availability
* Light is the main factor regulating plant development and physiology:
array of photoreceptors for optimal function in both different light intensities and qualities
* two classes of photoreceptors for establishing period length

1) Phytochromes A-E
* PhyA main in dark-grown seedlings; rapidly degraded in light
* PhyB-E more light stable; phyB main in light-grown seedlings
2) Cryptochromes 1-2 (blue, UV-A region)

maintenance of circadian period length

under a whole range of light conditions

THE CENTRAL OSCILLATOR

1) CCA1 and LHY

* MYB-like DNA-binding domains of high similarity - TFs

2) TOC1
* atypical response regulator (of His-kinase)
*C-terminus similarity to the CONSTANS family of TF
mediate pt-pt interactions and nuclear localization

THE CENTRAL OSCILLATOR

1) CCA1 and LHY
* when overexpressed: > arrhythmicity under constant light or dark
> reduction in mRNA levels: negative feedback loop
* loss of either gene affects periodicity but doesnt abolish rhythmicity: overlapping functions

* both mRNA and PT levels oscillate, peaking at dawn

2) TOC1
* mutations: period shortening independently of light, but still rhythmicity > other factors
* model for a feedback loop involving LHY, CCA1 and TOC1 based on:
a) Toc1 expression oscillates peaking during early evening, opposite to CCA1 and LHY
b) TOC1 expression low in LHY or CCA1 overexpressors > transcriptional repression by CCA1/LHY?
c) TOC1 expression high in lhy/cca1 double mutants
d) In TOC1 mutants CCA1/LHY expression very low > TOC1 positive regulator of LHY/CCA1?

MODEL FOR A FEEDBACK LOOP OF LHY, CCA1 AND TOC1 EXPRESSION

Acute induction of lhy/cca1 by light

> resetting of the phase of oscillator?

morning

TOC1 participates in
LHY/CCA1 activation the
next morning

evening

LHY/CCA1 bind to
evening element in
toc1 promoter for repression

OTHER COMPONENTS
a) PIF3
* PAS-domain-containing bHLH transcription factor
* interacts with phyB and binds to a number of phy-regulated promoters (e.g. CCA1 and LHY)
* TOC1 binds PIF3 > interaction necessary for LHY and CCA1 activation?

b) ELF3
* Interacts with PHYB
* arrhythmia only under constant light, while functional clock under constant darkness
not required for clock function in the absence of light
* low [CCA1 and LHY mRNA] and high [TOC1 mRNA]
* loss-of-function mutants

consistent with a negative role of CCA1/LHY in TOC1 regulation

ELF3 required for TOC1 promotion of lhy/cca1 transcription?
elf3 oscillates with the same phase as TOC1
* clock hypersensitive to light in the night > causing arrhythmia

* Seems to gate the light input to the oscillator, protecting it from the light signal at particular times of the day
* ELF3 might gate the light input at dusk so that the circadian clock is reset by the light-on signal

c) ZEITLUPE (ZTL)
* long period for cab/other CCGs, which dependent on fluence rate > light input to the clock
* interacts with PhyB and CRY1
* PAS domain + kelch domain for pt-pt interactions + F-box > targeting of pts to the proteasome?
* Transcript levels dont oscillate

d) GIGANTEA (GI)
* Shortens period of gene expression rhythms
* less severe effect in darkness than light, and dependent on fluence rate > light input to the clock
* Gi transcript levels oscillate

* Involved in phyB signaling

* Some phenotypes as phyB mutants (hypocotyl length)

* Others are opposite: late flowering vs. early flowering phyB mutants

* Different roles in different developmental stages?

* Branch of phyB signaling?

LIGHT INPUT STUDIES

* organisms held in constant darkness or dim light are treated with a brief pulse of light
change in phase of the oscillator

If the pulse of light:

a) during day: small change in phase

b) during dark: significant phase delays

ELF3/GI function?

MODEL FOR A FEEDBACK LOOP INVOLVING LHY, CCA1, AND TOC1

1) PHY and CRY as photoreceptors

2) LHY, CCA1 and TOC1 negative feedback loop

3) LHY, CCA1 repress expression of TOC1, their positive regulator

4) Generation of circadian rhythms, including that of CO for flowering time

5) ELF3 gates the light signals, resetting it at dawn

since itself a CCG, this allows cycling even at constant light
6) ZLP and GI also act on light input

SEASONAL RHYTHMS
* In plants: formation of flowers at the most appropriate times of the year to ensure reproductive success

* Controlled by changes in day length (photoperiodism) monitored by the circadian clock.

* Normally, Arabidopsis flowers more rapidly in LD (summer) than SD conditions (winter)
misregulation or mutation of genes implicated in clock function disrupts this response

* Daylength measurement is mediated by

* transcriptional regulation of gene expression by the circadian clock,

* post-transcriptional regulation by light

* important information has been provided from several studies of the flowering-time gene CONSTANS
CO mutations cause delayed flowering under long days (LDs)

EVOLUTIONARY ASPECTS
* No conservation of clock components among organisms > clocks have arisen multiple times
e.g. cryptochromes

implicated in clock function in many organisms

arose independently from the DNA repair enzyme photolyase in an example of repeated evolution

* Some common features:

a) transcriptional-feedback loop that generates a circadian oscillation in the level of one or more critical clock components
this level defines the phase of the clock at any point
b) presence of PAS protein domains (protein interactions) in critical clock components
* mutations in single genes never eliminate rhythmicity > redundant functions
* plants

* more sophisticated light-entrainment strategy

several photoreceptors that fine-tune the clock to different light conditions

* primary driving force for clock evolution: "flight from light, to set light-sensitive processes to occur at night
cell division in the alga Chlamydomonas occurs during the dark phase
genes encoding enzymes involved the synthesis of UV-protective compounds peak just before dawn in plants

MOLECULAR BASIS OF SEASONAL TIME MEASUREMENT IN ARABIDOPSIS

(Yanovsky and Kay, Nature 2002)

* Many signaling and clock components identified, but unknown how information integrated

* CO isolated as a delayed flowering mutant under LDs

* Expression of CO regulated by clock and photoperiod, with high CO during the day in LD but not in SD

* CO promotes flowering through the induction of the flowering time gene FT

* FT peaks when CO expression is high and illumination > so CO activity regulated by light?

* In gi, lhy and elf3 mutants CO levels correlate with flowering time alterations
mutants affected also in light signaling > difficult to distinguish between circadian and light effect

* How does CO promote flowering through FT?

* How to distinguish between the circadian and light effect?
* toc1 mutant chosen for approaching these questions
pseudo response regulator closely associated with the central oscillator
when mutated: period shortening of all studied output genes even in complete darkness
early flowering under SD of 24 h
normal flowering under SD of 21 h, that match the endogenous period of the mutant

1. CO-EXPRESSION IN TOC1 VS. WT PLANTS

SD

LD

WT

SD (24 and 21): CO accumulation during dark

LD: CO accumulation also during dawn and dusk

toc1

SD 24: CO accumulation advanced: high during dusk early flowering

No changes in level of expression or curve shape
SD 21 and LD: CO expression and flowering like WT

Co accelerates flowering through direct activation of FT expression: same mechanism here?

2. FT-EXPRESSION IN TOC1 VS. WT PLANTS

SD

WT

LD

SD (24 and 21): no FT accumulation

LD: FT accumulation during illumination
SD 24: FT accumulation during illumination

toc1
SD 21 and LD: FT expression like WT

If effects on CO and FT expression due only to the period length defect of the mutant
similar changes in WT under SD of 30 h (10L and 20D)?
(in SD24 FT and TOC peak in WT at 10 h after the onset of light)
3. CO AND FT EXPRESSION IN WT PLANTS

Upregulation of FT
In SD of 30 h CO expression shifted towards daytime
Reduction in flowering time

CO overexpressors

FT mRNA levels high but still rhythmic in LD > not only due to CO amounts
Light regulation of CO function and/or independent clock regulation of FT?

4. FT AND CO EXPRESSION IN CO-OX, WT, CRY2 AND PHYA MUTANTS

CO-ox grown in SD transferred to constant

light: FT expression high
dark: FT expression low despite high CO expression
CO function is light dependent; mechanism?

Cry2 mutants flower late in LD

* CO expression like in WT
* FT expression abolished

phyA mutants flower late under certain conditions

* Some minor changes in CO mRNA
* FT expression abolished

5. KINETICS OF LIGHT EFFECT ON FT mRNA LEVELS

Plants grown in SD transferred at dusk (when CO starts rising) to constant light or darkness

Light induces very rapid (4h) induction of FT in WT

Not in cry2 or phyA
Direct effect of light rather than indirect effect
in clock entrainment

light induction of FT expression abolished in co mutants

the effect of cry2 or phyA is through CO

CONCLUSIONS
* circadian clock
CO expression and flowering time
co-regulated by

* direct light signaling through CRY2 and PHYA

(CO protein levels - activity - other interacting molecules?)

TOC1

Light (PHYA/CRY2) + high CO expression

high FT expression

flowering

The Arabidopsis SRR1 gene mediates phyB signaling and is required for
normal circadian clock function
(Staiger et al. 2003, Gen. Dev.)

* Light input to the oscillator through phytochromes and cryptochromes

Activates phytochrome through conformational changes
* Light

Affects phytochrome subcellular localization

Affects their ability to interact with signaling partners

* Many signaling components downstream of PHY

best characterised: interaction of phyB with PIF3 to activate e.g. CCA1

* Here: srr1(sensitivity to red light reduced)

a new Arabidopsis mutant altered in

* multiple outputs of the clock

* phyB light signaling

1. SRR1 IS IMPAIRED IN PHYB SIGNALING

a) Screening for reduced sensitivity to light:

hypocotyl elongation not suppressed

as much as WT by light

b) Effect specific for red-light: phyB mediated?

test for phyB typical responses

PHYB TYPICAL RESPONSES

a) Reduced chlorophyll in red-light grown seedlings

b) Increased petiole length

c) Not much induction of flowering under LDs v. SDs

> reduced sensitivity to day length, like phyB mutants
> Phy amounts normal

Srr1 mutants deficient in phyB signaling

wt srr1

2. SRR1 ALSO ACTS INDEPENDENTLY OF PHYB

dark

Red-light

White-light

Double mutants srr1/phyB

a) In red light srr1 hypocotyls length = phyB
Same signaling pathway
b) In white light srr1 has an additive effect

different signaling pathway

3. SRR1 REQUIRED FOR MULTIPLE OUTPUTS OF THE CIRCADIAN CLOCK

Flowering time regulated also by the circadian clock:

> Clock affected in srr1?

Plants transferred to constant light

> All CCGs tested had shorter periods 2-3 h shorter
> Also the period of leaf movement
Defect in light input or oscillator?

Plants transferred to constant darkness

> Oscillation of cab:luc had shorter periods (2-3 h)

srr1 required for normal oscillator function

4. SRR1: A CONSERVED NUCLEAR/CYTOPLASMIC PROTEIN

* no recognizable domains
* Putative nuclear localization sequence
SRR1-GFP fusion: SRR1 found both in cytoplasm and nucleus

5. THE SRR1 TRANSCRIPT IS INDUCED BY LIGHT

* mRNA levels constant across the circadian cycle
* Induced by red but not far-red light phyB signaling

6. CONCLUSIONS
* srr1: role in phyB signaling and in regulation of circadian clock (like ELF3 and GI)
* elf3: arrhythmia in light but remains rhythmic in darkness > light input to clock
* srr1 circadian phenotype both in light and darknes > required for normal oscillator function
* elf3 interacts with phyB in vitro > Interaction between srr1 and phyB?
* srr1 homolog even in human
* Lack of conservation among clock components, but some common features
srr1 involved in some of these?
(regulated nuclear translocation, phosphorylation and negative feedback loops)