CIRCADIAN RHYTHMS IN PLANTS

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

2. * 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 WHAT IS A CIRCADIAN CLOCK? * Rhythmicity in behavior, physiology and biochemistry of organisms

3. RHYTHMIC OUTPUTS * 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 • * 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 c) plants: leaf movements, cell elongation rates, stomatal aperture, CO2 assimilation, Calvin cycle, ethylene production, hypocotyl elongation… very conserved motif, “evening element”, present 46 times in the promoters of 31 genes

4. * 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 * 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 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

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 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. Acute induction of lhy/cca1 by light > resetting of the phase of oscillator? TOC1 participates in LHY/CCA1 activation the next morning LHY/CCA1 bind to “evening element” in toc1 promoter for repression MODEL FOR A FEEDBACK LOOP OF LHY, CCA1 AND TOC1 EXPRESSION morning evening

8. 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 * 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 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? * clock hypersensitive to light in the night > causing arrhythmia

9. 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? 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

10. If the pulse of light: a) during day: small change in phase b) during dark: significant phase delays ELF3/GI function? 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

11. 5) ELF3 gates the light signals, resetting it at dawn • since itself a CCG, this allows cycling even at constant light 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 6) ZLP and GI also act on light input

12. * 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) 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

13. * 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 * more sophisticated light-entrainment strategy * plants • 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 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

14. * 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 MOLECULAR BASIS OF SEASONAL TIME MEASUREMENT IN ARABIDOPSIS (Yanovsky and Kay, Nature 2002) * Many signaling and clock components identified, but unknown how information integrated

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

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

18. 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 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)

19. 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 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?

20. Light induces very rapid (4h) induction of FT in WT Not in cry2 or phyA light induction of FT expression abolished in co mutants • 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 • Direct effect of light rather than indirect effect in clock entrainment

21. * 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 CONCLUSIONS Light (PHYA/CRY2) + high CO expression high FT expression flowering

22. * 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 * multiple outputs of the clock * Here: srr1(sensitivity to red light reduced) a new Arabidopsis mutant altered in * phyB light signaling The Arabidopsis SRR1 gene mediates phyB signaling and is required for normal circadian clock function (Staiger et al. 2003, Gen. Dev.)

23. 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 1. SRR1 IS IMPAIRED IN PHYB SIGNALING

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

25. a) In red light srr1 hypocotyls length = phyB Same signaling pathway b) In white light srr1 has an additive effect different signaling pathway 2. SRR1 ALSO ACTS INDEPENDENTLY OF PHYB dark Red-light White-light Double mutants srr1/phyB

26. 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 3. SRR1 REQUIRED FOR MULTIPLE OUTPUTS OF THE CIRCADIAN CLOCK Flowering time regulated also by the circadian clock: > Clock affected in srr1?

27. 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 4. SRR1: A CONSERVED NUCLEAR/CYTOPLASMIC PROTEIN * no recognizable domains * Putative nuclear localization sequence • 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: 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)