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The mechanism by which CML23/24 affects the circadian clock

Final Report Summary - CIRCADIAN CLOCK (The mechanism by which CML23/24 affects the circadian clock)

The Mechanism by which Calcium affects the Circadian Clock


Due to the fact that the rotation of the earth results in daily light and temperature cycles, organisms have evolved a 24 h circadian clock that allows them to anticipate these changes. In plants circadian rhythms control many biological processes like gene expression, timing component of photoperiodism and ethylene production1,2. Also, it has been demonstrated that a correctly tuned circadian clock confers substantial increases in plant productivity and that a circadian clock is essential for the ability of plants to respond to abiotic3,4 and biotic5 stresses.
At it simplest, the genetic structure of the circadian network in Arabidopsis can be separated into three components: a central genetic oscillator that maintains rhythmicity in the absence of external clues; entrainment pathways, through which information from the external environment such as light or temperature are communicated to the oscillator; and outputs pathways which relay the temporal information to physiological and metabolic processes. The central genetic oscillator is thought to be made of at least two interlocking loops6. The morning loop comprises genes that are expressed in the morning. Light activates the expression of CIRCADIAN CLOCK ASSOCIATE1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), two closely related Myb-like transcription factors which activate the expression of PSEUDORESPONSE REGULATORS 7 and 9 (PRR7/9), transcriptional repressors that feedback to repress CCA1/LHY. CCA1/LHY also represses the evening-expressed gene TIMINING OF CAB1 (TOC1) which along with GIGANTEA (GI) forms the evening loop. The evening loop is connected back to the morning loop by TOC1 repression of CCA17. Since the first model of the oscillator was proposed8 it has become clear that circadian-outputs may modulate inputs to the clock6,9,10 and that like in animals11 a loop of cytosolic signalling molecules within the plants circadian clock is present. Dr Webb´s lab proposed that cyclic adenosine diphosphate ribose (cADPR) is required to drive circadian oscillation of cytosolic-free Ca2+ [Ca2+]cyt and also modulate the nuclear transcriptional feedback loop of the Arabidopsis circadian oscillator12. This new pathway provides a paradigm shift in circadian biology because demonstrates that circadian clocks contain rhythm generating loops made up of signalling molecules in addition to the transcriptional and translational feedback loops12,13,14,15,16.
In many organisms, changes in [Ca2+]cyt levels are perceived by calmodulin (CaM). The Arabidopsis genome contains 7 CaM and 50 CAM-like (CML) genes. CMLs transduce cytosolic Ca2+ signals through binding of Ca2+ to four EF hands to expose hydrophobic protein interaction domains that allow CML to act as an intermediate in Ca2+-mediated protein. Previous results obtained in Dr Webb’s lab have identified genes encoding the CML23 and CML24 as Ca2+-signalling components that regulate the circadian network16 in a manner independent of the NO production16. However, we have a poor understanding of the mechanisms by which CML23/24 and moreover [Ca2+]cyt regulates circadian clock gene expression because a target gene in the plant circadian clock has not yet been identified to provide a mechanistic explanation.

Results and conclusions

During this project to analyse the role of CML23 and CML24 in circadian signalling and therefore establish theirs mechanism of action, different approaches were undertaken.
We have demonstrated that CML23 and CML24 participate in the control of the circadian clock in a Ca2+-dependent manner using the Ca2+-antagonists (sucrose and nicotinamide) that we have found to abolish circadian [Ca2+]cyt signals.
To discover pathways that are affected by CML23/24 we have performed a transcriptional analysis using microarray in the cml23 and cml24 loss-of-function plants. Preliminary data show that no major changes in transcript amplitude of circadian and non-circadian targets occur which supports the idea of the CML23 and 24 acting as [Ca2+]cyt sensors through protein-protein interaction after Ca2+ binding. To corroborate this hypothesis we are on the process to determine the proteins that interact with CML23 and CML24. To do this, we have made CamV35S:CML23::MYC, pCML23:CML23::MYC, CamV35S:CML24::FLAG and pCML24:CML24::FLAG stable transgenic lines of Arabidopsis and the pull down analysis is being carried out. Interactors that bind both CML23 and CML24 will be priority for further investigation. Also, to gain understanding of how CML23 and CML24 might regulate the Arabidopsis circadian clock the epistatic relationships between them and the clock genes is being analysed. We have crossed the circadian clock mutants lhy-21, elf3-4, cca1-11, toc1-2, che-1, che-2, elf4-1, ztl-3, gi-11 and lux-4 into cml23-2 cml24-4 to make triple mutants. Lines have been selected by PCR are being analysed by measuring circadian rhythms of leaf movement. We hypothesise that non-additive effects of the cml23-2 cml24-4 double mutants with the circadian mutants will be indicative of a functional relationship between CML23/24 and the target genes.
In addition to understanding the pathways where CML23 and 24 act, we wished to examine the localisation of the CML23 and CML24 and whether their cellular location is regulated by the circadian clock. As a first step, Arabidopsis Col-0 and cml23-2 cml24-4 double mutant were transformed to obtain the stable transgenic lines carrying Camv35S::CML23:RFP and/or Camv35S::CML24:GFP. Preliminary data using confocal scanning laser microscopy show co-localisation of CML23 and CML24 in cytosol and nucleus. Also, the stable transgenic lines carrying pCML23::CML23:RFP and/or pCML24::CML24:GFP were made. These lines are being used to determine the localisation of CML23 and CML24 and the effect of circadian timing, biotic challenge and Ca2+ agonists on tissue and sub-cellular localisation.
In order to identify the mechanism by which [Ca2+]cyt regulates circadian clock gene expression the following experiments were carried out. To validate an analysis of transcriptomic data sets where artificial [Ca2+]cyt oscillations where impose to Col-0 Arabidopsis plants and therefore identify those candidate circadian clock genes regulated by [Ca2+]cyt, we have measured the expression of the circadian clock genes (CCA1, LHY, PRR3, PRR5, PRR7, PRR9, GI, TOC1, CCA1 HIKING EXPEDITION (CHE), EARLY FLOWERING3 (ELF3), ELF4, LUXARRHYTHMO (LUX), and ZEITLUPE (ZTL)) by qPCR in Col-0 plants treated by mastoparan and N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) which we have identified to be the most effective Ca2+ agonists appropriate for circadian studies. We have found that the circadian clock gene CHE and also that PRR family are the only known circadian clock genes that respond to [Ca2+]cyt. This breakthrough discovery identifies targets for [Ca2+]cyt, suggesting a mechanism by which [Ca2+]cyt affects the circadian clock and a tempting hypothesis that CML23/24 act as regulatory intermediaries in the pathway by which circadian [Ca2+]cyt oscillations regulate CHE and PRRs transcript abundance. To further investigate if CHE, PRRs and CML23/24 affect the circadian clock acting in the same pathway, we determined the effect of high [Ca2+]cyt on CHE and PRRs gene expression by qPCR in cml23-2 cml24-4 double loss-of-function mutants, as previously described. The data suggest that CHE and PRR´s are not acting upstream of CML23/24, because an increase in [Ca2+]cyt leaded the same change in gene expression when compared with Col-0.
All these findings provide a new insight concerning the function of CML23 and 24 in the circadian clock and also provide the first approach to unveil how [Ca2+]cyt regulates the genes that form part of the circadian clock. This is important for an understanding of circadian clock in plants and others organisms. It also has wider implications, because it allows us to dissect the little understood processes by which Ca2+ regulates gene expression in general.

Socio-economic importance

There is considerable opportunity to translate this underpinning science to agricultural relevance since it is likely that new components involved in the control of the length of the growing season and regulators of the geographical distribution of crops will be identified. A better knowledge of circadian network will make possible a more efficient selective plant breeding for enhanced crop performance and the development of new biofuel crops and crops for industry, mitigating the effects of global environmental change and extending the geographical range of cultivation.


1Dood et al 2005 Science 309, 630-633; 2Thain et al 2004 Plant Phys. 136, 3751-3761; 3Dood et al Plant J. 48, 962–973; 4Eriksson and Webb 2012 Curr Opin Plant Biol 14, 731–737; 5Wei et al 2011 Nature 470, 110-114; 6Gardner et al 2006 Biochemical J 397, 15–24; 7Gendron et al 2012 PNAS 109, 3167-3172; 8Alabadí et al 2001 Science 293, 880-3; 9Más 2005 Int. J. Dev. Biol. 49, 491-500; 10McClung 2006 Plant Cell 18, 792-803; 11Ikeda et al 2003 Neuron 38, 253-263; 12Dodd et al 2007 Science 318, 1789-1792; 13Love et al 2004 Plant Cell 16, 956– 966; 14Xu et al 2007 Plant Cell 19, 3474-3490; 15Dalchau et al 2010 PNAS 107, 13171-13176; 16Hubbard et al in preparation