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 doesn’t 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 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
* Some phenotypes as phyB mutants (hypocotyl length)
* Involved in phyB signaling
* 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
* transcriptional regulation of gene expression by the circadian clock,
* Daylength measurement is mediated by
* 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
implicated in clock function in many organisms
e.g. cryptochromes
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
WT
toc1
LD
SD (24 and 21): CO accumulation during dark
LD: CO accumulation also during dawn and dusk
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)