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

2. WHAT IS A CIRCADIAN CLOCK?
* Rhythmicity in behavior, physiology and biochemistry of organisms
* Some rhythms persist even without environmental changes
endogenous control: circadian clock
circadian rhythms – 24 h period
* 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

3. > additional regulatory layers for many genes
RHYTHMIC OUTPUTS
c) plants: leaf movements, cell elongation rates, stomatal aperture, CO2 assimilation, Calvin cycle,
ethylene production, hypocotyl elongation…
* Gene expression:
> additional regulatory layers for many genes
a) cyanobacteria: 80% of genes are CCGs > the clock regulates the transcriptional machinery?
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

4. 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
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

5. 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

6. * when overexpressed: > arrhythmicity under constant light or dark
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?

7. 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
LHY/CCA1 bind to
“evening element” in
toc1 promoter for repression
evening

8. OTHER COMPONENTS a) PIF3 b) ELF3
* 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
* 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
* 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
* 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

9. c) ZEITLUPE (ZTL) d) GIGANTEA (GI)
* 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
* 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?

10. LIGHT INPUT STUDIES ELF3/GI function?
* 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?

11. 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

12. 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
* post-transcriptional regulation by light
* transcriptional regulation of gene expression by the circadian clock,
* important information has been provided from several studies of the flowering-time gene CONSTANS
CO mutations cause delayed flowering under long days (LDs)

13. 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:
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
* 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

14. * CO isolated as a delayed flowering mutant under LDs
MOLECULAR BASIS OF SEASONAL TIME MEASUREMENT IN ARABIDOPSIS
(Yanovsky and Kay, Nature 2002)
* 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
* 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
* 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

15. * 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

16. SD LD 1. CO-EXPRESSION IN TOC1 VS. WT PLANTS
SD (24 and 21): CO accumulation during dark
LD: CO accumulation also during dawn and dusk
toc1
SD 21 and LD: CO expression – and flowering like WT
SD 24: CO accumulation advanced: high during dusk – early flowering
No changes in level of expression or curve shape

17. Co accelerates flowering through direct activation of FT expression: same mechanism here?
2. FT-EXPRESSION IN TOC1 VS. WT PLANTS SD LD WT
SD (24 and 21): no FT accumulation
LD: FT accumulation during illumination
toc1
SD 21 and LD: FT expression like WT
SD 24: FT accumulation during illumination

18. 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
In SD of 30 h CO expression shifted towards daytime
Upregulation of FT
Reduction in flowering time

19. CO-ox grown in SD transferred to constant
FT mRNA levels high but still rhythmic in LD > not only due to CO amounts
CO overexpressors
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
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?
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

20. the effect of cry2 or phyA is through CO
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

21. CO expression and flowering time
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
TOC1
Light (PHYA/CRY2) + high CO expression
high FT expression
flowering

22. 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
* Light
Activates phytochrome through conformational changes
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

23. 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

24. PHYB TYPICAL RESPONSES
a) Reduced chlorophyll in red-light grown seedlings
b) Increased petiole length
> reduced sensitivity to day length, like phyB mutants
c) Not much induction of flowering under LDs v. SDs
> Phy amounts normal
Srr1 mutants deficient in phyB signaling wt
srr1

25. 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

26. 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

27. 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

28. 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)