植物生物钟ppt课件.ppt

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 behavior,See Kudoh, H. (2016). Molecular phenology in plants: innatura systems biology for the comprehensive understanding of seasonal responses under natural environments. New Phytol. 210: 399-412. Image: NASA.,Extreme day-night temperature difference: 57 oC (-48 oC to 9 oC, Montana, 1972)Typical day-night fluctuation: 10 oC each day (central Japan),Circadian clocks are biological oscillators with a 24 hour period,- 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.,Inactive,Active,Circadian clocks control many aspects of human physiology,Image source: Addicted04,Plant circadian biology has a long history,Illustration of sleep movements in Medicago, from Charles Darwin (1880) The Power of Movement in Plants,Image sources: H. Zell, Charles Darwin “Power of Movement in Plants”,De Marian (1729) Observation botanique of Mimosa pudica:,“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.”,Architecture of the circadian clock,Principles of operation of plant circadian clocks,Interconnected parts of the circadian system,Circadianoscillator,Entrainmentpathways,Output pathways,Gene,Rhythms in: - transcription- physiology- biochemistry,Environmental Inputs,Circadian gating of entrainment and outputs,The circadian oscillator,Most circadian clocks are transcription-translation feedback loops,Gene A,Gene B,Protein A,The protein encoded by Gene A activates Gene B,The circadian oscillator,0,12,24,36,48,Gene A,Gene B,Protein A,Gene transcript abundance,The feedback loop results in rhythms of transcript abundance of the two genes,Time (hours),Gene A,Gene B,An early model for the functioning of the circadian clock in Arabidopsis,From Alabad, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Ms, P. and Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293: 880-883. Reprinted with permission from AAAS,This is one of the first structures proposed for the plant circadian clockIt is an oscillator with activation and suppression feedback (compare with previous slide)The main genes involved are TOC1, LHY and CCA1The model is out of date (TOC1 actually suppresses CCA1), but provides an example of oscillator structure,Gene A,Protein A,Gene B,Protein B,Activation(out of date!),Suppression,Reciprocal repression between CCA1 and TOC1 at the core of the circadian clock,From Alabad, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Ms, P. and Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293: 880-883. 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: 3167-3172.,Overexpression of CCA1 suppresses circadian oscillations of TOC1CCA1 and LHY bind to the promoter of TOC1,Overexpression of TOC1 suppresses circadian oscillations of CCA1The CCT domain of TOC1 is required for it to bind the CCA1 promoter,The current model of the circadian oscillator is a complex network,Evening Complex,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.,Morning Loop,Note TOC1 is a suppressor of CCA1/LHY (compare with previous model),Different clock components are expressed at different times of day,Network models indicate connections between components, but lack temporal information about clock functionThis example shows how three clock genes are activated at different times of day/night,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/,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 expressionControl 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 LHYCytosolic signaling molecules:e.g. circadian rhythms of cADPR and Ca2+ in the cytosol regulate the dynamics of the oscillator,See Ms, P. (2008) Circadian clock function in Arabidopsis thaliana: time beyond transcription. Trends Cell Biol. 18: 273-281 for review,The circadian oscillator is temperature-compensated,Temperature (oC),Col-0 wild type maintains a 24 period across a range of temperatures, i.e. is temperature compensated,PRR7/PRR9knockdown changes period in response to temperature change,Salom, 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: 3650-3661; 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: 14595-14600.,PRR7, PRR9 and FBH1 have rolesin temperature compensation,Why is entrainment required?,The time of dawn and dusk is different every single day,Time of sunrise,Time of sunset,Day of year,Time of day (24 h clock),Location: Bristol, UK51.4500 N, 2.5833 W,Why is entrainment required?,The period of the circadian oscillator is approximately 24h and there is natural variation between plants,Circadian period (h),Measurements,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: 67-77.,Measurements of circadian period in three wild type strains of Arabidopsis thalianaThere is variation within and between strains, but all have a period of approximately 24 hours,Several environmental signals entrain the circadian oscillator,Circadianoscillator,Red light(phytochrome photoreceptors),Blue light(cryptochrome photoreceptors),Sugars produced by photosynthesis,Temperature fluctuations,Phytochromes and cryptochromes provide light input to the circadian clock,cry1 mutant,cry2 mutant,Fluence rate of blue light(mol m-2 s-1),Wild type,Wild type,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: 1488-1490 Reprinted with permission from AAAS,Fluence rate of red light(mol m-2 s-1),Wild type,phyB mutant,Wild type,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,Night,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 phasesIndicates that the circadian clock regulates different subsets of genes with different circadian phases through particular clock-controlled 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.,The importance of circadian rhythms in plant biology,Plants with a functioning circadian clock that matches the environment grow larger,From Dodd, A.N., Salathia, N., Hall, A., Kvei, E., Tth, 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,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., Kvei, E., Tth, 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,Development:Photoperiod is one of the environmental factors controlling flowering,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,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:survivalbiomass (dry and fresh weight)chlorophyll content,Arabidopsis thaliana mutant lines with endogenous circadian period:,From Dodd, A.N., Salathia, N., Hall, A., Kvei, E., Tth, 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.,Endogenous period,Environment,Investigating the circadian clock in the laboratory,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,The time that would have been dark is referred to as subjective night, and is sometimes indicated by grey bars on circadian time-courses,Time (h),Circadian rhythms have a number of measurable properties,60,72,84,96,Biological process,Period,Amplitude,Phase = time of peak relative to subjective dawn,Time (h),Period = time to complete one full cyclePhase = the time at which a particular point of cycle occurs (e.g. the peak)Amplitude = the displacement of the oscillation from the center point,Subjective dawn occurs every 24h after lights on,Common methods for studying circadian rhythms: Transcript analysis,Individual transcripts can be monitored,Circadian rhythms of the whole transcriptome can be studied with microarrays or RNA sequencing,or,Photosystem I and II transcripts,Light harvesting complextranscripts,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. Reprinted with permission from AAAS.,This transcriptome analysis found that many photosynthesis genes have circadian rhythms,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 daysDestructive sampling to obtain RNA or protein is inconvenient: Substantial quantities of plant material required, long working hours, opportunities for human errorNon-invasive and automated measurement techniques have been developedDestructive sampling is sometimes essential to monitor rhythms of transcripts, proteins or metabolites,Common methods for studying circadian rhythms: Leaf movement,From Hicks, K.A., Millar, A.J., Carr, 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: 790-792. Reprinted with permission from AAAS. Image credits: Vojtch Zavadil; K.Hubbard unpublished,Leaf position (pixels),0,24,48,72,96,120,144,Automated video imaging and image analysis allows quantification of leaf movements,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,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,LUCIFERASE,Luciferin,O2,ATP,Light,Oxyluciferin,AMP,LUCIFERASE transgene,Plant genome,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.The plant emits circadian rhythms of light that can be detected with a very sensitive camera.,Luciferase bioluminescence imaged from Arabidopsis seedlings,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.,Common methods for studying circadian rhythms: Bioluminescence imaging,From Millar, A., Carre, I., Strayer, C., Chua, N. and Kay, S. (1995). Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science. 267: 1161-1163. Reprinted with permission from AAAS.,LUCIFERASE is placed under the control of the rhythmic CAB2 promoterAround 8000 seedlings from a mutagenized population were tested, to identify components of the circadian clockThis allowed identification of circadian clock components (here, TOC1),Mutant with shortcircadian period (toc1-1),Wild typeseedlings,Advanced methods for studying circadian rhythms: Rhythms in individual tissues,1. Use a super sensitive camera with close up lens to monitor luciferase,Bioluminescence from individual pixels,Luciferase bioluminescencefrom 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: 6757-6762.,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 l