CIRCADIAN RHYTHMS IN PLANTS

* WHAT IS A CIRCADIAN CLOCK?* CLOCK OUTPUTS * CLOCK INPUTS* THE CENTRAL OSCILLATOR * OTHER COMPONENTS* SEASONAL RHYTHMS * EVOLUTIONARY RELATIONSHIPS

* 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

* Some rhythms persist even without environmental changes

endogenous control: circadian clock

circadian rhythms – 24 h period

RHYTHMIC OUTPUTS - PROCESSES REGULATED BY THE CLOCK

* de Mairan, 1727: rhythmic leaf movements in constant darkness

* Rhythmicity in behavior, physiology and biochemistry of organisms

WHAT IS A CIRCADIAN CLOCK?

* Anticipation of rhythmic changes in the environment

changes in the physiological state that provide them with an adaptive advantage

b) Arabidopsis: 6% are CCGs, peaking at all phases of day and night

* 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!!!

CCG clusters

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

* Gene expression:

> additional regulatory layers for many genes

a) cyanobacteria: 80% of genes are CCGs > the clock regulates the transcriptional machinery?

RHYTHMIC OUTPUTS

c) plants: leaf movements, cell elongation rates, stomatal aperture, CO2 assimilation, Calvin cycle, ethylene production, hypocotyl elongation…

* circadian clocks must be

reset so that internal time matches local time (entrainment)> signals such as light, temperature, and nutrient availability

CLOCK RESETTING – CLOCK INPUTS

* 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

2) Cryptochromes 1-2 (blue, UV-A region)

* PhyA main in dark-grown seedlings; rapidly degraded in light

* PhyB-E more light stable; phyB main in light-grown seedlings

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

2) TOC1

* mutations: period shortening independently of light, but still rhythmicity > other factors

1) CCA1 and LHY

* when overexpressed: > arrhythmicity under constant light or dark

* both mRNA and PT levels oscillate, peaking at dawn

* loss of either gene affects periodicity but doesn’t abolish rhythmicity: overlapping functions

> reduction in mRNA levels: negative feedback loop

* 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

evening

morning

LHY/CCA1 bind to “evening element” in

toc1 promoter for repression

Acute induction of lhy/cca1 by light > resetting of the phase of oscillator?

TOC1 participates in LHY/CCA1 activation the

next morning

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?

* 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

b) ELF3

* Interacts with PHYB

* loss-of-function mutants

* arrhythmia only under constant light, while functional clock under constant darkness

not required for clock function in the absence of light

* clock hypersensitive to light in the night > causing arrhythmia

* low [CCA1 and LHY mRNA] and high [TOC1 mRNA]

ELF3 required for TOC1 promotion of lhy/cca1 transcription?

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

elf3 oscillates with the same phase as TOC1

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?

* 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?

* Transcript levels don’t 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

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

b) during dark: significant phase delays

If the pulse of light:

a) during day: small change in phase

ELF3/GI function?

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

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

1) PHY and CRY as photoreceptors

2) LHY, CCA1 and TOC1 negative feedback loop

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

* Normally, Arabidopsis flowers more rapidly in LD (summer) than SD conditions (winter)

misregulation or mutation of genes implicated in clock function disrupts this response

* important information has been provided from several studies of the flowering-time gene CONSTANS

CO mutations cause delayed flowering under long days (LDs)

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

* Daylength measurement is mediated by* post-transcriptional regulation by light

* transcriptional regulation of gene expression by the circadian clock,

EVOLUTIONARY ASPECTS

* No conservation of clock components among organisms > clocks have arisen multiple times

* Some common features:

this level defines the phase of the clock at any point

a) transcriptional-feedback loop that generates a circadian oscillation in the level of one or more critical clock components

b) presence of PAS protein domains (protein interactions) in critical clock components

* plants

* mutations in single genes never eliminate rhythmicity > redundant functions

* more sophisticated light-entrainment strategy

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

e.g. cryptochromesarose independently from the DNA repair enzyme photolyase in an example of repeated evolution

implicated in clock function in many organisms

* 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

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

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

* CO isolated as a delayed flowering mutant under LDs

(Yanovsky and Kay, Nature 2002)

* 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

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

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

* 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

* How does CO promote flowering through FT?

* How to distinguish between the circadian and light effect?

1. CO-EXPRESSION IN TOC1 VS. WT PLANTS

SD LD

toc1

SD 21 and LD: CO expression – and flowering like WT

SD 24: CO accumulation advanced: high during dusk – early floweringNo changes in level of expression or curve shape

WTSD (24 and 21): CO accumulation during darkLD: CO accumulation also during dawn and dusk

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

2. FT-EXPRESSION IN TOC1 VS. WT PLANTS

SD LD

WTSD (24 and 21): no FT accumulationLD: FT accumulation during illumination

toc1

SD 21 and LD: FT expression like WT

SD 24: FT accumulation during illumination

If effects on CO and FT expression due only to the period length defect of the mutant

3. CO AND FT EXPRESSION IN WT PLANTS

In SD of 30 h CO expression shifted towards daytimeUpregulation of FT

Reduction in flowering time

(in SD24 FT and TOC peak in WT at 10 h after the onset of light)

similar changes in WT under SD of 30 h (10L and 20D)?

CO overexpressorsFT 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?

light: FT expression high

CO-ox grown in SD transferred to constant

dark: FT expression low despite high CO expression

CO function is light dependent; mechanism?

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

Cry2 mutants flower late in LD

* CO expression like in WT

* FT expression abolished

phyA mutants flower late under certain conditions

* FT expression abolished

* Some minor changes in CO mRNA

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

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

CO expression and flowering time co-regulated by

* circadian clock

* direct light signaling through CRY2 and PHYA

Light (PHYA/CRY2) + high CO expression high FT expression flowering

TOC1

The Arabidopsis SRR1 gene mediates phyB signaling and is required for normal circadian clock function

* Light input to the oscillator through phytochromes and cryptochromes

* Light

Activates phytochrome through conformational changes

Affects phytochrome subcellular localization

Affects their ability to interact with signaling partners

(Staiger et al. 2003, Gen. Dev.)

* 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

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

test for phyB typical responses

a) Screening for reduced sensitivity to light:

hypocotyl elongation not suppressed as much as WT by light

a) Reduced chlorophyll in red-light grown seedlings

b) Increased petiole length

PHYB TYPICAL RESPONSES

srr1wt

Srr1 mutants deficient in phyB signaling

> reduced sensitivity to day length, like phyB mutants

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

> Phy amounts normal

2. SRR1 ALSO ACTS INDEPENDENTLY OF PHYB

Double mutants srr1/phyBdark Red-light White-light

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

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

SRR1-GFP fusion: SRR1 found both in cytoplasm and nucleus

6. CONCLUSIONS

* srr1: role in phyB signaling and in regulation of circadian clock (like ELF3 and GI)

* 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

* 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

(regulated nuclear translocation, phosphorylation and negative feedback loops)

srr1 involved in some of these?