Rhythms of Life: The Plant Circadian Clock

Transcription

1 Rhythms of Life: The Plant Circadian Clock Somers, D.E. (1999). The physiology and molecular bases of the plant circadian clock. Plant Physiol. 121: 9-20.

2 Living on a rotating planet is biologically stressful Over a 24 hour period there is large variation in environmental conditions including temperature, light intensity, humidity and predator behavior Extreme day-night temperature difference: 57 o C (-48 o C to 9 o C, Montana, 1972) Typical day-night fluctuation: ~10 o C each day (central Japan) See Kudoh, H. (2016). Molecular phenology in plants: in natura systems biology for the comprehensive understanding of seasonal responses under natural environments. New Phytol. 210: Image: NASA.

3 Continuous darkness Light- Dark cycles Circadian clocks are biological oscillators with a ~24 hour period Inactive Active - Higher levels of wheel running activity at night - In continuous darkness these rhythms persist but with a ~ 23 hour period Figure from Li, J.-D., Burton, K.J., Zhang, C., Hu, S.-B. and Zhou, Q.-Y. (2009). Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei. Am. J. Physiol. 296: R824-R830, used with permission. Image source: Mylius.

4 Circadian clocks control many aspects of human physiology High alertness Best coordination Melatonin secretion stops Rise in blood pressure Lowest body temperature Fastest reaction time Greatest muscle strength Highest blood pressure Highest body temperature Melatonin secretion starts Deepest sleep Deepest sleep Bowel movements suppressed Image source: Addicted04

5 Plant circadian biology has a long history De Marian (1729) Observation botanique of Mimosa pudica: Illustration of sleep movements in Medicago, from Charles Darwin (1880) The Power of Movement in Plants sensitive to the Sun and daylight: the leaves & their peduncles fold themselves away & contract around sunset, in the same way they do when the Plant is touched or shaken. Image sources: H. Zell, Charles Darwin Power of Movement in Plants

6 Architecture of the circadian clock

7 Principles of operation of plant circadian clocks Aspect of the Circadian System Circadian oscillator Entrainment pathways Output pathways Circadian gating Biological Function Generate a rhythm with a ~24h period within the cell Synchronize the oscillator with the external time of day so that the clock stays accurate Communicate temporal information from the oscillator to other parts of the cell Adjust the sensitivity of entrainment and output pathways depending on the time of day

8 Interconnected parts of the circadian system Environmental Inputs Circadian gating of entrainment and outputs Gene Entrainment pathways Circadian oscillator Output pathways Rhythms in: - transcription - physiology - biochemistry

9 The circadian oscillator Most circadian clocks are transcription-translation feedback loops The protein encoded by Gene A activates Gene B The protein encoded by Gene B represses Gene A Gene A Protein A Protein B Gene B

10 Gene transcript abundance The circadian oscillator Protein A The feedback loop results in rhythms of transcript abundance of the two genes Gene A Gene B Gene A Protein B Gene B Time (hours) Reciprocal feedback loop Negative feedback step Speed of biochemical reactions adds a rate constant Simple biological oscillator

11 An early model for the functioning of the circadian clock in Arabidopsis This is one of the first structures proposed for the plant circadian clock Protein A Gene A Activation (out of date!) Suppression Gene B Protein B It is an oscillator with activation and suppression feedback (compare with previous slide) The main genes involved are TOC1, LHY and CCA1 The model is out of date (TOC1 actually suppresses CCA1), but provides an example of oscillator structure From Alabadı, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P. and Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293: Reprinted with permission from AAAS

12 Reciprocal repression between CCA1 and TOC1 at the core of the circadian clock Overexpression of CCA1 suppresses circadian oscillations of TOC1 CCA1 and LHY bind to the promoter of TOC1 Overexpression of TOC1 suppresses circadian oscillations of CCA1 The CCT domain of TOC1 is required for it to bind the CCA1 promoter From Alabadı, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P. and Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293: Reprinted with permission from AAAS. Gendron, J.M., Pruneda-Paz, J.L., Doherty, C.J., Gross, A.M., Kang, S.E. and Kay, S.A. (2012). Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor. Proc. Natl. Acad. Sci. USA 109:

13 The current model of the circadian oscillator is a complex network Note TOC1 is a suppressor of CCA1/LHY (compare with previous model) Morning Loop Evening Complex Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: with permission from Elsevier..

14 Normalised transcript abundance Different clock components are expressed at different times of day Network models indicate connections between components, but lack temporal information about clock function This example shows how three clock genes are activated at different times of day/night Time (h) CCA1 PRR9 LUX Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: with permission from Elsevier.. Data from DIURNAL database:

15 The circadian clock also includes post-transcriptional processes Chromatin remodeling: -e.g. promoter of TOC1 has a clock-controlled pattern of histone 3 (H3) acetylation that affects TOC1 expression Control of protein stability by proteasome: -e.g. ZTL is involved in dark-dependent degradation of the TOC1 protein. Phosphorylation: -e.g. Casein Kinase 2 (CK2) phosphorylates CCA1 and LHY Cytosolic signaling molecules: -e.g. circadian rhythms of cadpr and Ca 2+ in the cytosol regulate the dynamics of the oscillator See Más, P. (2008) Circadian clock function in Arabidopsis thaliana: time beyond transcription. Trends Cell Biol. 18: for review

16 Period (h) Period (h) The circadian oscillator is temperature-compensated PRR7, PRR9 and FBH1 have roles in temperature compensation PRR7/PRR9 knockdown changes period in response to temperature change 22 o C 28 o C Temperature ( o C) Col-0 wild type maintains a ~24 period across a range of temperatures, i.e. is temperature compensated Wild type FBH1-ox FBH1-ox Salomé, P.A., Weigel, D. and McClung, C.R. (2010). The role of the Arabidopsis Morning loop components CCA1, LHY, PRR7, and PRR9 in temperature compensation. Plant Cell. 22: ; Nagel, D.H., Pruneda-Paz, J.L. and Kay, S.A. (2014). FBH1 affects warm temperature responses in the Arabidopsis circadian clock. Proc. Natl. Acad. Sci. USA 111:

17 Time of day (24 h clock) Why is entrainment required? The time of dawn and dusk is different every single day 08:00 06:00 04:00 Time of sunrise 02: :00 18:00 16:00 14: Day of year Time of sunset Location: Bristol, UK N, W

18 Measurements Why is entrainment required? The period of the circadian oscillator is approximately 24 h and there is natural variation between plants Measurements of circadian period in three wild type strains of Arabidopsis thaliana There is variation within and between strains, but all have a period of approximately 24 hours Circadian period (h) Reprinted from Swarup, K., Alonso-Blanco, C., Lynn, J.R., Michaels, S.D., Amasino, R.M., Koornneef, M. and Millar, A.J. (1999). Natural allelic variation identifies new genes in the Arabidopsis circadian system. Plant J. 20:

19 Several environmental signals entrain the circadian oscillator Red light (phytochrome photoreceptors) Blue light (cryptochrome photoreceptors) Sugars produced by photosynthesis Temperature fluctuations Circadian oscillator

20 Phytochromes and cryptochromes provide light input to the circadian clock phya mutant Wild type cry1 mutant Wild type phyb mutant Wild type cry2 mutant Wild type Fluence rate of red light (µmol m -2 s -1 ) Fluence rate of blue light (µmol m -2 s -1 ) From Somers, D.E., Devlin, P.F. and Kay, S.A. (1998). Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science. 282: Reprinted with permission from AAAS

21 Circadian clocks regulate plant cells by controlling gene expression Some circadian clock proteins are transcription factors that regulate sets of genes with a circadian rhythm Example: a daytime transcription factor Day TF TF TF Gene 1 Gene 2 TF Gene 3 Night TF Gene 1 Gene 2 Gene 3

22 Specific gene promoter sequences may underlie specific circadian phases of transcription These cis elements occur with high frequency in promoters of transcript sets with certain circadian phases Indicates that the circadian clock regulates different subsets of genes with different circadian phases through particular clockcontrolled promoter motifs Covington, M.F., Maloof, J.N., Straume, M., Kay, S.A. and Harmer, S.L. (2008). Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development. Genome Biology. 9: 1-18.

23 The importance of circadian rhythms in plant biology

24 Plants with a functioning circadian clock that matches the environment grow larger (wildtype) Col-0 = wildtype CCA1-ox = arrhythmic transgenic line T24 = 12 h light, 12 h dark T20 = 10 h light, 10 h dark T24 = 12 h light, 12 h dark T28 = 14 h light, 14 h dark From Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 309: Reprinted with permission from AAAS.

25 Relative transcript abundance The circadian clock controls multiple aspects of plant biology Molecular Biology: ~30% of the Arabidopsis thaliana transcriptome oscillates with a 24 period Physiology: Stomatal opening and closing are under the control of the circadian oscillator Circadian rhythms of gas exchange = wildtype = arrhythmic mutant (CCA1- ox) Time (h) From Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.-S., Han, B., Zhu, T., Wang, X., Kreps, J.A. and Kay, S.A. (2000). Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science. 290: and from Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 309: Reprinted with permission from AAAS.

26 The circadian clock controls multiple aspects of plant biology Growth: Hypocotyl elongation is clock controlled Development: Photoperiod is one of the environmental factors controlling flowering Plants grown under long days: Wildtype Circadian clock mutant (gigantea) Reprinted with permission from from Dowson-Day, M.J. and Millar, A.J. (1999). Circadian dysfunction causes aberrant hypocotyl elongation patterns in Arabidopsis. Plant J. 17: and Amasino, R. (2010). Seasonal and developmental timing of flowering. Plant J. 61:

27 The circadian clock gives plants a fitness advantage Arabidopsis thaliana mutant lines with endogenous circadian period: Endogenous period Environment (= 20 h) (= 28 h) ~20 h ~28 h Competition experiments: When the endogenous period matches the external light-dark cycles, plants perform better in terms of: survival biomass (dry and fresh weight) chlorophyll content From Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 309: Reprinted with permission from AAAS.

28 Investigating the circadian clock in the laboratory

29 Biological process Time-course analysis is used to study circadian rhythms in plants The plant is first grown in cycles of light and dark The plant is then transferred to conditions of constant light (or dark) and temperature, where circadian-regulated biological process will free run Light Dark Light Dark Time (h) Time (h) The time that would have been dark is referred to as subjective night, and is sometimes indicated by grey bars on circadian time-courses

30 Biological process Circadian rhythms have a number of measurable properties Subjective dawn occurs every 24h after lights on Period Phase = time of peak relative to subjective dawn Amplitude Time (h) Period = time to complete one full cycle Phase = the time at which a particular point of cycle occurs (e.g. the peak) Amplitude = the displacement of the oscillation from the center point

31 Common methods for studying circadian rhythms: Transcript analysis Individual transcripts can be monitored or Circadian rhythms of the whole transcriptome can be studied with microarrays or RNA sequencing Light harvesting complex transcripts Photosystem I and II transcripts This transcriptome analysis found that many photosynthesis genes have circadian rhythms From Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.-S., Han, B., Zhu, T., Wang, X., Kreps, J.A. and Kay, S.A. (2000). Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science. 290: Reprinted with permission from AAAS.

32 Non-invasive measurement techniques benefit the study of circadian rhythms Measurements of a biological property need to be made frequently (e.g., hourly) over several days Destructive sampling to obtain RNA or protein is inconvenient: Substantial quantities of plant material required, long working hours, opportunities for human error Non-invasive and automated measurement techniques have been developed Destructive sampling is sometimes essential to monitor rhythms of transcripts, proteins or metabolites

33 9 days old (subj. day) 8 days old (subj. night) 8 days old (subj. day) Leaf position (pixels) Common methods for studying circadian rhythms: Leaf movement Some plants have a pulvinus at the base of the leaf which drives movement of the leaves In Arabidopsis, leaf movements are part of rhythmic patterns in growth Automated video imaging and image analysis allows quantification of leaf movements From Hicks, K.A., Millar, A.J., Carré, I.A., Somers, D.E., Straume, M., Meeks-Wagner, D.R. and Kay, S.A. (1996). Conditional circadian dysfunction of the Arabidopsis early-flowering 3 mutant. Science. 274: Reprinted with permission from AAAS. Image credits: Vojtěch Zavadil; K.Hubbard unpublished

34 Common methods for studying circadian rhythms: Bioluminescence imaging Expression in plants of an enzyme from fireflies called luciferase causes plants to emit light when provided with the substrate luciferin O 2 Light Luciferin ATP LUCIFERASE Oxyluciferin AMP Plant genome LUCIFERASE transgene

35 Common methods for studying circadian rhythms: Bioluminescence imaging Placing LUCIFERASE under the control of a promoter with a circadian rhythm allows the rhythm to be monitored. Luciferase bioluminescence imaged from Arabidopsis seedlings The plant emits circadian rhythms of light that can be detected with a very sensitive camera. Millar, A.J., Short, S.R., Chua, N.H. and Kay, S.A. (1992). A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell. 4:

36 Common methods for studying circadian rhythms: Bioluminescence imaging LUCIFERASE is placed under the control of the rhythmic CAB2 promoter Around 8000 seedlings from a mutagenized population were tested, to identify components of the circadian clock This allowed identification of circadian clock components (here, TOC1) Mutant with short circadian period (toc1-1) Wild type seedlings From Millar, A., Carre, I., Strayer, C., Chua, N. and Kay, S. (1995). Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science. 267: Reprinted with permission from AAAS.

37 Bioluminescence from individual pixels Advanced methods for studying circadian rhythms: Rhythms in individual tissues 1. Use a super sensitive camera with close up lens to monitor luciferase Luciferase bioluminescence from single Arabidopsis leaf Wenden, B., Toner, D.L.K., Hodge, S.K., Grima, R. and Millar, A.J. (2012). Spontaneous spatiotemporal waves of gene expression from biological clocks in the leaf. Proc. Natl. Acad. Sci. USA. 109:

38 Advanced methods for studying circadian rhythms: Rhythms in individual tissues 2. Split luciferase into two fragments, and give one a tissue-specific gene promoter This half of luciferase is only expressed when the clock promoter is active The complete protein is only assembled when both promoters are active This half of luciferase is only expressed in the specific tissue type Reprinted by permission from Macmillan Publishers Ltd: Endo, M., Shimizu, H., Nohales, M.A., Araki, T. and Kay, S.A. (2014). Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature. 515:

39 Advanced methods for studying circadian rhythms: Rhythms in individual tissues 2. Split luciferase into two fragments, and give one a tissue-specific gene promoter One half of luciferase: Vascular SUC2 promoter One half of luciferase: Circadian TOC1 promoter 1 mm This was used to show the circadian clock in vascular tissue is dominant to that of other cell types in leaves Reprinted by permission from Macmillan Publishers Ltd: Endo, M., Shimizu, H., Nohales, M.A., Araki, T. and Kay, S.A. (2014). Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature. 515:

40 Advanced methods for studying circadian rhythms: The value of mathematical modeling How do you unravel how this functions? It s totally non-intuitive! Large number of interconnected clock components Multiple feedback loops, both positive and negative Each protein and transcript has its own unique rate of synthesis and breakdown Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: with permission from Elsevier..

41 Advanced methods for studying circadian rhythms: The value of mathematical modeling Building mathematical simulations of the clock has: Provided new information about how clock components interact Provided explanations for why the clock is so complex Provided new information about how the clock is entrained to the environment Demonstrated the power of mathematical and systems biology approaches for biological research

42 The circadian clock and plant metabolism

43 The circadian clock has extensive control of plant metabolism Almost all metabolic pathways include at least one enzyme that is under circadian transcriptional control Each square = One circadianregulated transcript Color of square = phase of expression Reprinted from Farré, E.M. and Weise, S.E. (2012). The interactions between the circadian clock and primary metabolism. Curr. Opin. Plant Biol. 15: with permission from Elsevier.

44 Relative Expression Level Relative Expression Level Many metabolic pathways have physiologically appropriate phases of maximal transcript abundance 1.6 Subjective time of day: Dawn Dusk Dawn Dusk Dawn Chlorophyll Biosynthesis Time in continuous light (h) Chlorophyll biosynthesis genes peak just before dawn to anticipate light availability Starch catabolism genes peak around dusk Starch Catabolism Time in continuous light (h) Data from DIURNAL database:

45 Circadian clock mutants have Data shown for a prr9/7/5 triple mutant grown in 12h light: 12h dark cycles altered metabolite levels in lightdark cycles Increase in Shikimate suggests changes in secondary metabolism Increase in Citric Acid Cycle intermediates Fukushima, A., Kusano, M., Nakamichi, N., Kobayashi, M., Hayashi, N., Sakakibara, H., Mizuno, T. and Saito, K. (2009). Impact of clock-associated Arabidopsis pseudo-response regulators in metabolic coordination. Proc. Natl. Acad. Sci. USA 106:

46 Carbohydrate degradation at night is temporally controlled The rate of starch degradation is related to the length of the night, so that the plant only exhausts starch reserves just before the end of the night 12hL:12hD cycles (normal growth condition) Early night imposed Graf, A., Schlereth, A., Stitt, M. and Smith, A.M. (2010). Circadian control of carbohydrate availability for growth in Arabidopsis plants at night. Proc. Natl. Acad. Sci. USA 107:

47 Relative Expression Level cca1/lhy mutants exhaust starch reserves at night cca1/lhy mutants: -Accumulate 20% less starch than wildtypes -Degrade starch 35% faster than wildtypes at night -Exhaust starch reserves 3-4 hours before the end of the night -Express starvation genes before the end of the night wildtype cca1-11/lhy-21 Graf, A., Schlereth, A., Stitt, M. and Smith, A.M. (2010). Circadian control of carbohydrate availability for growth in Arabidopsis plants at night. Proc. Natl. Acad. Sci. USA 107:

48 The chloroplast has circadian rhythms of gene expression that are controlled by the nucleus psbd = Chloroplast gene TF Circadian oscillator SIG5 SIG5 imported into chloroplast SIG5 = Nuclear gene cytosol SIG5 Gene chloroplast chloroplast From Noordally, Z.B., Ishii, K., Atkins, K.A., Wetherill, S.J., Kusakina, J., Walton, E.J., Kato, M., Azuma, M., Tanaka, K., Hanaoka, M. and Dodd, A.N. (2013). Circadian control of chloroplast transcription by a nuclear-encoded timing signal. Science. 339: Reprinted with permission from AAAS.

49 Primary metabolites regulate the activity of the circadian oscillator Application of external sucrose restores circadian rhythms in wildtype seedlings grown in the dark This is dependent on the presence of the oscillator component GIGANTEA Dalchau, N., Baek, S.J., Briggs, H.M., Robertson, F.C., Dodd, A.N., Gardner, M.J., Stancombe, M.A., Haydon, M.J., Stan, G.-B., Gonçalves, J.M. and Webb, A.A.R. (2011). The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose. Proc. Natl. Acad. Sci. USA 108:

50 The oscillator, environmental signalling and metabolism form an integrated network Environmental Signalling Light Temperature Central Oscillator CCA1 PRR7/5/9 GI TOC1 Chloroplasts Photosynthesis Sugar Redox ATP/NAD + Metabolism NAD + Mitochondria Redox ATP/NAD + Image based on Farré, E.M. and Weise, S.E. (2012). The interactions between the circadian clock and primary metabolism. Curr. Opin. Plant Biol. 15: and Haydon, M.J., Hearn, T.J., Bell, L.J., Hannah, M.A. and Webb, A.A.R. (2013). Metabolic regulation of circadian clocks. Semin. Cell Devel. Biol. 24:

51 μg emitted/g fresh weight/hr The circadian clock regulates production of volatile compounds There are circadian rhythms of volatile emissions: Petunias use the emission of volatile compounds to attract nocturnal pollinators such as hawkmoths Fenske, M.P., Hewett Hazelton, K.D., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A. and Imaizumi, T. (2015). Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proc. Natl. Acad. Sci. USA 112:

52 The circadian clock regulates production of volatile compounds Many genes required for volatile production are under transcriptional control of the oscillator: Circadian transcriptional control Volatile compounds Fenske, M.P., Hewett Hazelton, K.D., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A. and Imaizumi, T. (2015). Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proc. Natl. Acad. Sci. USA 112:

53 Circadian mutants of Petunia do not emit volatile compounds at night Transgenic overexpression of PvLHY abolishes nocturnal production of volatiles W115 = wildtype line #37* = PvLHY overexpressing line Fenske, M.P., Hewett Hazelton, K.D., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A. and Imaizumi, T. (2015). Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proc. Natl. Acad. Sci. USA 112:

54 Plants are more resistant to herbivory when their circadian rhythms are phased with rhythms of insects When In Phase the plants resist herbivore attack Insects Plants Entrainment conditions Free run (constant dark) Insects Plants When Out of Phase the plants are vulnerable to herbivores Goodspeed, D., Chehab, E.W., Min-Venditti, A., Braam, J. and Covington, M.F. (2012). Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior. Proc. Natl. Acad. Sci. USA 109:

55 Plants produce jasmonates during the subjective day to deter herbivores Insects feed during the subjective day Jasmonates accumulate during the subjective day Jasmonate accumulation induces herbivore defense mechanisms e.g. production of toxic secondary metabolites Goodspeed, D., Chehab, E.W., Min-Venditti, A., Braam, J. and Covington, M.F. (2012). Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior. Proc. Natl. Acad. Sci. USA 109:

56 The circadian clock provides timing information to control photoperiodic flowering

57 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Day length (hours) Many plants use photoperiod as a signal to sense seasonal changes Winter = short photoperiod (~7 h of light) Summer = long photoperiod (~15 h of light) Cambridge, UK Wheat (Triticum aestivum) flowers in the spring when the days are getting longer Image source: anthere

58 Photoperiod sensitive plants induce flowering under either long or short days Long Day Plants e.g., Wheat 24 hours Short Day Plants e.g., Rice 24 hours Flash Critical night length Flash Critical night length (Note: flowering of some species e.g. tomato is photoperiod insensitive; these are referred to as day-neutral plants)

59 Flowering time is a highly regulated event which centers on FLOWERING LOCUS T (FT) Photoperiod Age (autonomous pathway) Gibberellin Cold winters (vernalization pathway) Induction of FT expression in leaves FT protein moves to the shoot apical meristem via the phloem Floral integrator genes induced in meristem, and flowering is initiated

60 The external coincidence model explains photoperiodic induction of flowering time in long days = CONSTANS = FT First proposed by Bünning (1936). Model redrawn from Imaizumi, T. and Kay, S.A. Photoperiodic control of flowering: not only by coincidence. Trends Plant Sci. 11:

61 No. of leaves at flowering The zinc finger transcription factor Long Days (16hL: 8hD) CONSTANS is the circadian Short Days (8hL: 16hD) CONSTANS is required for floral induction in long days in Arabidopsis dependent regulator WT (Ler) co-2 Zn ion The zinc finger is a DNA interaction motif found in all kingdoms of life Data: K. Hubbard (unpublished). Image source: Thomas Splettstoesser

62 A molecular model to explain photoperiodic control of flowering The expression of CO is controlled by the circadian clock, with peak expression ~12 hours after dawn time in Arabidopsis In long days the peak of CO mrna is during the light, so the CO protein can accumulate CO protein is unstable in the dark due to COP1 activity so it doesn t accumulate and FT is not induced CO induces FT expression, which stimulates the floral transition Model redrawn from Imaizumi, T. and Kay, S.A. Photoperiodic control of flowering: not only by coincidence. Trends Plant Sci. 11:

63 The photoperiodic flowering pathway is broadly conserved between Arabidopsis and crops Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of flowering time in temperate cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58: by permission of Oxford University Press.

64 A small change to the model also explains flowering in short day plants such as rice Long Day Plants: CO activates FT expression Short Day Plants: CO represses FT expression Model redrawn from Song, Y.H., Shim, J.S., Kinmonth-Schultz, H.A. and Imaizumi, T. (2015). Photoperiodic flowering: Time measurement mechanisms in leaves. Annu. Rev. Plant Biol. 66:

65 Circadian gating

66 Circadian gating: General concept The regulation of other cell signaling pathways by the circadian clock is known as circadian gating It is a fundamental way that circadian clocks regulate plant cells During gating, the clock acts as a valve on the response of the plant to the environment, so the same environmental cue causes a different strength response depending on the time of day At some times of day the gate is open and the signal passes through. At other times of day the gate is closed and the signal cannot pass through

67 Circadian gating: General concept General principle: an example of light signaling Response Gate open Strong response to light Identical light stimulus Gate closed Very weak response to light

68 Circadian gating acts upon circadian entrainment and environmental signaling pathways 1. The circadian clock gates the light and temperature signals that entrain the circadian clock 2. The circadian clock gates signaling pathways that regulate the responses of plants to the environment

69 1. Circadian gating of light input to If the circadian clock responded identically to light at every time of day, it would be reset to dawn continuously and be unable to provide a measure of time the circadian clock The clock regulates its own sensitivity to light, so the way it responds to light depends on the time of day Red light Blue light Figure reprinted from Hotta, C.T., Gardner, M.J., Hubbard, K.E., Baek, S.J., Dalchau, N., Suhita, D., Dodd, A.N. and Webb, A.A.R. (2007). Modulation of environmental responses of plants by circadian clocks. Plant Cell Environ. 30: , redrawn from Covington, M.F., Panda, S., Liu, X.L., Strayer, C.A., Wagner, D.R. and Kay, S.A. (2001). ELF3 modulates resetting of the circadian clock in Arabidopsis. Plant Cell. 13:

70 CBF2 transcript level 2. Circadian gating of environmental response pathways Example: The circadian clock gates responses of plants to a cold environment *** *** *** *** *** *** *** ---- More sensitive to cold when transferred to cold at this time Less sensitive to cold when transferred to cold at this time Fowler, S.G., Cook, D. and Thomashow, M.F. (2005). Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol. 137:

71 Change in hypocotyl length (%) 3. Circadian gating of environmental response pathways Example: The circadian clock gates shade avoidance in Arabidopsis Hypocotyl Seedling hypocotyls elongate rapidly in the shade to overtop their competitors Time in constant light (h) Seedlings are most sensitive to simulated shade around subjective dusk Reprinted by permission from Macmillan Publishers Ltd: Salter, M.G., Franklin, K.A. and Whitelam, G.C. (2003). Gating of the rapid shade-avoidance response by the circadian clock in plants. Nature. 426:

72 The potential for crop improvement using circadian-dependent traits

73 Circadian clock genes are associated with agronomic traits Species QTL/locus Gene in species Arabidopsis homologue Role/Trait Eudicots P. sativum HR/QTL3 HR ELF3 Circadian clock function, flowering time, light response Monocots O. sativa - OsPRR1 TOC1 - OsPRR37 PRR3/PRR7 Flowering time Ef7/hd17 OsELF3-1 ELF3 Light-dependent circadian clock regulation H. vulgare Ppd-H1 HvPRR37 PRR3/PRR7 Flowering time T. aestivum - Ppd-D1 PRR3/7 Flowering time Z. mays - ZmGI1 GI Flowering time and growth regulation Table based on Bendix, C., Marshall, Carine M. and Harmon, Frank G. (2015). Circadian clock genes universally control key agricultural traits. Mol. Plant. 8:

74 Case study 1: A slower clock was selected for during the domestication of tomato Two QTLs were identified, one of which mapped to a homologue of an Arabidopsis light signaling protein (EID1) Plants with the phase delay mutation were late flowering and had higher chlorophyll content in long days, indicating a competitive advantage Reprinted by permission from Macmillan Publishers Ltd: Muller, N.A., Wijnen, C.L., Srinivasan, A., Ryngajllo, M., Ofner, I., Lin, T., Ranjan, A., West, D., Maloof, J.N., Sinha, N.R., Huang, S., Zamir, D. and Jimenez-Gomez, J.M. (2016). Domestication selected for deceleration of the circadian clock in cultivated tomato. Nat Genet. 48:

75 Case study 2: The role of Ppd-H1 in photoperiodic responses of barley Barley (Hordeum vulgare): - Long-day plant, i.e., it requires day lengths in excess of a critical minimum to flower. - There are also varieties that are insensitive to day length Photoperiod sensitive Igri x Mapping population generated Photoperiod insensitive Triumph Ppd-H1 locus identified Image source: Craig Nagy

76 Ppd-H1 is a homologue of the Arabidopsis PRR7 oscillator gene Phenotypes of homozygous Ppd-H1 (left), heterozygous (middle), and homozygous ppd-h1 (right) plants At PRR7 is a pseudo-response regulator that has 50% overall similarity to Hv Ppd-H1 From Turner, A., Beales, J., Faure, S., Dunford, R.P. and Laurie, D.A. (2005). The Pseudo-Response Regulator Ppd-H1 provides adaptation to photoperiod in barley. Science. 310: Reprinted with permission from AAAS. Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: with permission from Elsevier..

77 Day length (hours) Winter barley (Ppd-H1) is photoperiod sensitive and flowers in early spring Cambridge, UK Lebanon Autumn Winter Spring Summer Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Winter barley (Ppd-H1) Flowering is induced when days get longer than ~13 hours (i.e. in the spring), resulting in early harvest Sowing Harvest Harvest occurs before heat of summer See Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of flowering time in temperate cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58:

78 Day length (hours) Spring barley (ppd-h1) is photoperiod insensitive and flowers late Cambridge, UK Lebanon Autumn Winter Spring Summer Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Spring barley (ppd-h1) Sowing in spring avoids frost damage Sowing Flowering fails to be induced when days get longer than ~13 hours (i.e., in the spring), therefore crop grows throughout the summer Harvest See Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of flowering time in temperate cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58:

79 Photoperiod insensitive landraces of barley are more common in northern Europe Autumn Winter Spring Summer North = spring barley (ppd-h1) Sowing Harvest Spread of barley northwards from the fertile crescent Insensitive to photoperiod so flowers late and grows throughout the summer. Avoids the winter frost South = winter barley (Ppd-H1) Sowing Harvest Sensitive to photoperiod so flowers in the spring when days get longer. Avoids the heat of summer. Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of flowering time in temperate cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58: by permission of Oxford University Press.

80 Summary and Future Perspectives

81 Summary of current understanding of circadian rhythms in plants Circadian rhythms are molecular time keeping mechanisms that synchronize multiple processes with 24 hour light-dark cycles The circadian oscillator is a complex feedback loop primarily based on rhythms of gene expression Investigating circadian rhythms requires novel experimental approaches to capture temporal dynamics The circadian clock controls metabolism and key developmental transitions The circadian clock is broadly similar in crop plants, and represents a target for agronomic optimization

82 There are many big questions left in plant circadian biology Is the oscillator specialized in different cell types, and do these oscillators communicate with each other? How did circadian oscillators evolve? What are the molecular bases of circadian gating? How does plant circadian regulation contribute to ecosystem dynamics? Can we use our knowledge of circadian biology to increase crop production?