Srr1 is a conserved nuclear/cytoplasmic protein in Arabidopsis that mediates phytochrome B signaling and is required for normal circadian clock function. Srr1 mutants show reduced sensitivity to red light, indicating impaired phyB signaling. They also have shorter circadian periods for clock-controlled genes and leaf movements in both light and dark, demonstrating that Srr1 is necessary for the normal oscillator function. Srr1 is induced by red light and may interact with phyB to regulate light input and negative feedback loops within the central circadian clock mechanism.

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

2. * 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

3. 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…

4. * 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

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. 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?

7. 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

8. 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

9. 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

10. 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?

11. 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

12. 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,

13. 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. cryptochromes
arose 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

14. 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

15. * 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?

16. 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 flowering
No changes in level of expression or curve shape
WT
SD (24 and 21): CO accumulation during dark
LD: CO accumulation also during dawn and dusk

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
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
(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)?

19. 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?
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

20. 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. 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

22. 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

23. 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

24. a) Reduced chlorophyll in red-light grown seedlings
b) Increased petiole length
PHYB TYPICAL RESPONSES
srr1
wt
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

25. 2. SRR1 ALSO ACTS INDEPENDENTLY OF PHYB
Double mutants srr1/phyB
dark 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

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
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

28. 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?