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.

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 behaviour Extreme day-night temperature difference: 57 oC (-48 oC to 9 oC, Montana, 1972) Typical day-night fluctuation: ~10 oC 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: 399-412. Image: NASA.

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

The circadian oscillator Most circadian clocks are transcription-translation feedback loops Gene A Gene B Protein A Protein B 12 24 36 48 Transcript abundance The feedback loop results in rhythms of transcript abundance of the two genes Time (hours) Gene A Gene B Reciprocal feedback loop Negative feedback step Speed of biochemical reactions adds a rate constant Simple biological oscillator

Morning Loop Evening Complex The circadian oscillator is a complex network of interlocking feedback loops Different clock components are expressed at different times of day The oscillator includes transcriptional and post-transcriptional processes (not all are shown here) Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier.. Data from DIURNAL database: http://diurnal.mocklerlab.org/

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

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 Gene 1 TF Specific gene promoter sequences may underlie specific circadian phases of transcription TF TF Gene 2 TF Gene 3 Night Gene 1 TF Gene 2 Gene 3 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.

CCA1-ox = arrhythmic transgenic line Plants with a functioning circadian clock that matches the environment grow larger T20 = 10 h light, 10 h dark T24 = 12 h light, 12 h dark T28 = 14 h light, 14 h dark (wildtype) Col-0 = wildtype CCA1-ox = arrhythmic transgenic line T24 = 12 h light, 12 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: 630-633. Reprinted with permission from AAAS.

The circadian clock controls multiple aspects of plant biology Molecular Biology: ~30% of the Arabidopsis thaliana transcriptome oscillates with a 24 period Circadian rhythms of gas exchange = wildtype = arrhythmic mutant (CCA1-ox) Physiology: Stomatal opening and closing are under the control of the circadian oscillator Time (h) Relative transcript abundance 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: 2110-2113 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: 630-633. Reprinted with permission from AAAS.

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 Wildtype Circadian clock mutant (gigantea) Plants grown under long days: 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: 63-71 and Amasino, R. (2010). Seasonal and developmental timing of flowering. Plant J. 61: 1001-1013.

The circadian clock gives plants a fitness advantage Arabidopsis thaliana mutant lines with endogenous circadian period: 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 Endogenous period ~20 h ~28 h Environment (= 20 h) (= 28 h) 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: 630-633. Reprinted with permission from AAAS.

Time-course analysis is used to study circadian rhythms in plants The plant is then transferred to conditions of constant light (or dark) and temperature, where circadian-regulated biological process will ‘free run’ The plant is first grown in cycles of light and dark Biological process Time (h) Light Dark 12 24 36 48 Phase = time of peak relative to subjective dawn Period 12 24 36 48 60 72 84 96 Amplitude 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

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, working long 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.

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 Luciferase bioluminescence imaged from Arabidopsis seedlings Placing LUCIFERASE under the control of a promoter with a circadian rhythm allows the rhythm to be monitored. 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: 1075-1087.

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 cca1/lhy mutants exhaust starch reserves at night and enter starvation 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: 9458-9463.

The oscillator, environmental signalling and metabolism form an integrated network Central Oscillator CCA1 PRR7/5/9 GI TOC1 NAD+ Light Temperature Chloroplasts Photosynthesis Sugar Redox ATP/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: 293-300 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: 414-421.

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) 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: 4674-4677.

Circadian gating changes the sensitivity of signalling responses at different times of day Example: Circadian gating of light input into the circadian clock Conceptual Model: Experimental Data: Response Blue light Red light Strong response to light Identical light stimulus Gate open Very weak response to light Gate closed 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: 333-349, 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: 1305-1316.

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: 550-558.

CO induces FT expression, which stimulates the floral transition A molecular model to explain photoperiodic control of flowering time in Arabidopsis In long days the peak of CO mRNA is during the light, so the CO protein can accumulate The expression of CO is controlled by the circadian clock, with peak expression ~12 hours after dawn 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: 550-558.

The circadian clock and photoperiodic flowering pathways are broadly conserved 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: 1231-1244 by permission of Oxford University Press.

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 Light-dependent circadian clock regulation H. vulgare Ppd-H1 HvPRR37 T. aestivum Ppd-D1 PRR3/7 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: 1135-1152.

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

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 does plant circadian regulation contribute to ecosystem dynamics? Can we use our knowledge of circadian biology to increase crop production? What are the molecular bases of circadian gating? How did circadian oscillators evolve?