Progress 09/01/04 to 08/31/06
Outputs
AIM 1: Positionally clone and characterize CDS, a potential circadian clock component. The gene affected by the short period mutant, cds2, has been identified by positional cloning. Its expression and function within the circadian system are currently being characterized. AIM 2: Study the strategies used by the clock to differentially control subsets of output genes and pathways. The circadian clock plays a pervasive role in the temporal regulation of plant physiology, environmental responsiveness, and development. In contrast, the phytohormone auxin plays a similarly far-reaching role in the spatial regulation of plant growth and development. Seventy years ago, Went and Thimann noted that plant sensitivity to auxin varied according to the time of day, an observation which they could not explain. We have prepared and submitted work that explains this puzzle, demonstrating that the circadian clock regulates auxin signal transduction. Using genome-wide transcriptional
profiling, we found many auxin-induced genes are under clock regulation. We verified that endogenous auxin signaling is clock regulated with a luciferase-based assay. Exogenous auxin has only modest effects on the plant clock, but the clock controls plant sensitivity to applied auxin. Significantly, we found both transcriptional and growth responses to exogenous auxin are gated by the clock. Thus the circadian clock regulates some, and perhaps all, auxin responses. As a consequence, many aspects of plant physiology not previously thought to be under circadian control may show time-of-day specific sensitivity, with likely important consequences for plant growth and environmental responses. In addition, the circadian-regulated bHLH transcription factor CRB2 is being further characterized with plans to begin preparation of a manuscript by the end of the year. The project is now complete and terminated.
Impacts
The long-term goal of this proposal is to better understand the molecular basis of circadian rhythms in plants and how these daily rhythms affect plant physiology by enabling the plant to anticipate and react to daily and seasonal changes in the environment. Circadian clocks regulate many essential cellular processes in organisms ranging from cyanobacteria to humans. In higher plants, many major metabolic pathways and developmental decisions (e.g. timing of the transition to flowering) are subject to circadian regulation. Although a plant was the first organism used for the scientific study of circadian clocks, little is known about the plant clock relative to the circadian systems of cyanobacteria, Drosophila, and mammals. Using Arabidopsis thaliana as my model system for agriculturally and economically important crop species, I am investigating the make-up of the central circadian clock mechanism and the ways in which the clock controls different output pathways to
regulate physiological and developmental processes.
Publications
- Covington MF and Harmer SL (2006) The circadian clock regulates auxin signaling and responses in Arabidopsis. Submitted
- Harmer SL, Covington MF, Blasing OE, Stitt M (2005) Circadian regulation of global gene expression and metabolism. In: Hall A, editor. Endogenous Plant Rhythms: Blackwell Synergy. pp. 133-165.
- Nozue, K, Harmer SL, Covington MF, and Maloof JN (2006) The circadian clock controls plant growth through transcriptional regulation of phytochrome signaling components. Submitted
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Progress 01/01/05 to 12/31/05
Outputs
Plants use an internal clock to efficiently coordinate endogenous processes with environmental rhythms. To better understand the physiological relevance of circadian regulation and the mechanism behind it, we investigated the circadian transcriptome of Arabidopsis using Affymetrix ATH1 arrays. Over 10% of the genes detectably expressed in seedlings are clock-regulated at the steady-state mRNA level. Pathways and processes with an over-representation of circadian-regulated genes include primary and secondary metabolism, specific types of transcription factors, aspects of post-translational modification, and hormone biosynthesis and signaling. To further investigate the relationship between the clock and plant growth and development, we focused on interactions between the clock and the plant hormone auxin. The expression of an unexpectedly large proportion of auxin-signaling components and auxin-responsive genes was found to be circadian, with genes that promote auxin
signaling (ARFs) out of phase with antagonists (Aux/IAAs) and auxin-inactivating enzymes (Group II GH3s). We then demonstrated that the synthetic auxin-responsive promoter DR5 confers dawn-phased circadian expression on a luciferase reporter gene. DR5::LUC+ expression is induced following exogenous treatment of plants with auxins. Furthermore, the acute induction of DR5::LUC+ by exogenous IAA is gated by the clock, demonstrating that plant responsiveness to exogenous auxin is under circadian regulation. This induction is greatest when the circadian-regulated abundance of transcripts encoding two ARFs are at peak levels and those encoding six AUX/IAAs and three IAA-amido conjugating enzymes (Group II GH3s) are at their lowest levels, suggesting that rhythmic auxin signaling and/or rhythmic inactivation of auxins may contribute to circadian regulation of auxin responses. Given the ability of auxin to promote cell elongation, this circadian regulation may have a role in phenomena such as
circadian control of hypocotyl elongation. These findings illustrate the power of a genomics approach to studying circadian mechanisms.
Impacts
The long-term goal of this proposal is to better understand the molecular basis of circadian rhythms in plants and how these daily rhythms affect plant physiology by enabling the plant to anticipate and react to daily and seasonal changes in the environment. Circadian clocks regulate many essential cellular processes in organisms ranging from cyanobacteria to humans. In higher plants, many major metabolic pathways and developmental decisions (e.g. timing of the transition to flowering) are subject to circadian regulation. Although a plant was the first organism used for the scientific study of circadian clocks, little is known about the plant clock relative to the circadian systems of cyanobacteria, Drosophila, and mammals. Using Arabidopsis thaliana as my model system for agriculturally and economically important crop species, I am investigating the make-up of the central circadian clock mechanism and the ways in which the clock controls different output pathways to
regulate physiological and developmental processes.
Publications
- Covington, M.F., Straume, M. & Harmer, S.L. 2005, In preparation.
- Harmer, S.L., Covington, M.F., Blasing, O., Stitt, M. 2005. Circadian regulation of global gene expression and metabolism. In: Endogenous Plant Rhythms, ed. A. Hall and H.G. McWatters, Blackwell Publishing.
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Progress 01/01/04 to 12/31/04
Outputs
During the first three months of this project, I have been focusing the determination of physiological pathways regulated by the circadian clock. In collaboration with Dan Kliebenstein to identify compounds whose abundance is clock-regulated, we previously identified an unknown compound that cycles robustly with a peak level of abundance in the evening. We have now tentatively identified this compound by LC-MS and are in the process of confirming the identity of this secondary metabolite. Following this verification, we will utilize our vast circadian microarray data to analyze the temporal regulation of the metabolic pathway responsible for this compounds biosynthesis. In the meantime, I have been completing the analysis of the data from the circadian microarray experiment that I performed in the Harmer lab at UC-Davis. We have identified several interesting physiological pathways that are clock-regulated such that many (or sometimes all) components of the pathway
are transcribed at the same time of day. Using our microarray data, I have also identified a number of putative promoter elements involved in conferring circadian regulation. I have tested and confirmed that two of these elements can, in fact, be used to drive circadian rhythmicity. Until now, only one discrete promoter element had been proven to be involved in circadian regulation. I am in the process of assembling all of my recent findings into a manuscript with a planned submission date of January 2005. I am also working on finishing up a book chapter on circadian clocks in plants that I am co-writing with Stacey Harmer and Mark Stitt. The planned publication date is early/mid 2005.
Impacts
The long-term goal of this proposal is to better understand the molecular basis of circadian rhythms in plants and how these daily rhythms affect plant physiology by enabling the plant to anticipate and react to daily and seasonal changes in the environment. Circadian clocks regulate many essential cellular processes in organisms ranging from cyanobacteria to humans. In higher plants, many major metabolic pathways and developmental decisions (e.g. timing of the transition to flowering) are subject to circadian regulation. Although a plant was the first organism used for the scientific study of circadian clocks, little is known about the plant clock relative to the circadian systems of cyanobacteria, Drosophila, and mammals. Using Arabidopsis thaliana as my model system for agriculturally and economically important crop species, I am investigating the make-up of the central circadian clock mechanism and the ways in which the clock controls different output pathways to
regulate physiological and developmental processes.
Publications
- No publications reported this period
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