2008 Annual Report
1a.Objectives (from AD-416)
Objective 1: Define the contribution of the circadian clock to plant osmotic and salt stress responses using Arabidopsis as an initial model system.
Objective 2: Characterize the contribution of the circadian clock to transcriptional control networks in cereals, using Oryza sativa as a model.
Sub-objective 2.A.: Define the circadian transcriptome of Oryza sativa.
Sub-objective 2.B.: Identify cis-regulatory elements upstream of co-expressed circadian genes.
Objective 3: Determine the function of maize photoperiodism genes identified as naturally occurring alleles in maize recombinant inbred lines.
Objective 4: Assess the feasibility of high-throughput screening of maize seedlings or plants for circadian phenotypes as a prelude to screening large RIL populations for circadian quantitative trait loci (QTL).
1b.Approach (from AD-416)
Maize is an important crop as well as a model organism for other cereals such as sorghum, barley, rice and wheat. Our long term goal is to identify and characterize the activity of maize genes involved in plant production including tolerance to stressful growth conditions and regulation of flowering time. Recent work in model systems demonstrates that the circadian regulation of physiological activities is required for optimal plant growth and for tuning of responses to environmental cues. A comprehensive understanding of the circadian system in cereals is lacking; therefore, this proposal seeks to define the maize circadian system and assess the circadian oscillator’s contribution to important agronomic traits. Known circadian mutants will be tested for their response to salt and osmotic stress. Genes under circadian regulation in cereals will be identified by expression profiling, and this information used to computationally predict regulatory DNA elements that contribute to circadian gene expression. Reverse genetic approaches will evaluate the role of candidate photoperiodism genes in determining the timing of maize flowering. Maize inbreds and recombinant inbred lines will be analyzed for natural variation in overt circadian rhythms. DNA sequences, genes, mutants, and inbred lines identified here provide two types of tools: a better understanding of fundamental processes in environmental responses and targets that can be used to improve crop productivity. Formerly 5335-21000-025-00D (4/08).
The endogenous circadian clock controls plant growth, timing of flowering, and potentially stress responses by organizing daily and seasonal changes in plant physiology. Thus, a comprehensive understanding of the circadian system in crop plants holds great potential to provide novel avenues for optimization of important agronomic traits and to ultimately improve crop production. The fundamental goal of the research group is to define the maize circadian system to assess the impact this system has on key agronomic traits in cereals.
Working in the model plant Arabidopsis thaliana the research group examined with both genetic and molecular techniques the role played by a key component of targeted protein degradation, CULLIN 1 (CUL1), to learn the importance of this process in the generation and maintenance of circadian rhythms. This significant work published in Plant Journal showed a critical functional link between the activity of CUL1 and a well-defined core circadian clock component. This clock protein serves in part to set the length of the period for the circadian clock. As a result, the laboratory’s work illuminates one aspect of the molecular system involved in determining the length of clock period. Clock period affects important traits like flowering time and biomass accumulation, so this work provides additional genes other scientist can explore as experimental targets for alteration of crop flowering time and yield. The emphasis of NP301.2 on identification and mapping of important agronomic traits is addressed with this work. Since little is known of the circadian system outside Arabidopsis, the research group initiated a novel research program directed at investigating the circadian clock in maize. This is a replacement project for 5335-21000-025-00D. This new project will extend the work initiated by the previous project. The Project Plan describing this work was conceived, written and submitted to Office of Scientific Quality Review (OSQR) for peer review. This proposal was well received by the OSQR panel as it was scored 6.0 (out of 7.0), which corresponds to the proposal needing only minor revision. This Project Plan will strengthen collaborations made previously with fellow ARS scientists, as well as providing experimental support for the Cooperative Research and Development Agreement (CRADA) with Pioneer - DuPont Company established previously through project 5335-21000-025-00D. In addition, this year the CRADA with Pioneer Hi-Bred delivered its first set of transposon insertion lines harboring putative null mutations in candidate circadian clock genes. If these lines prove to be null mutants, future work with them will be the first analysis of circadian clock mutants in maize. Overall, the research done by this group will provide critical insight into the molecular and genetic basis of fitness and environmental response in cereal crops. Circadian clock genes will serve as future targets for genetic improvement of key agronomic traits and can be used by breeders as molecular markers for these traits. This work directly relates to the NP301, Comp2 mandate concerning identification and mapping of important agronomic traits.
Receipt and initial characterization of the first putative maize transposon mutants provided by a CRADA with Pioneer Hi-Bred - a DuPont Company. After assembling full-length gene sequence for the CASEIN KINASE BETA 3 (CKB3) gene, and based on this information the ARS scientists in the Plant Gene Expression Center in Albany, CA subsequently received two potential ckb3 null mutants with transposon insertions in this gene. The laboratory assembled the full-length gene sequence for the CASEIN KINASE BETA 3 (CKB3) gene and, based on this information, the laboratory received from Pioneer Hi-Bred two potential ckb3 null mutants with transposon insertions in this gene. Analysis of the ckb3 mutant lines and others received through the CRADA will provide critical insights into the molecular mechanism of the circadian clock in cereals. This work addresses the NP 301 Action Plan, Component 2, Problem Statement 2C: Genetic Analysis and Mapping of Important Traits.
Exploration of the role targeted protein degradation plays in the plant circadian clock. Genetic and molecular analysis by ARS scientists in the Plant Gene Expression Center in Albany, CA of an Arabidopsis mutant with abrogated targeted protein turnover revealed striking changes in circadian behavior. The work demonstrated that a protein necessary for degradation of a specific circadian clock protein is a key part of the plant oscillator mechanism and, thus, a gene that may contribute to circadian behavior in crop plants. These findings were published in the high impact journal Plant Journal, because they expand the understanding of the molecular components of the plant circadian clock. This work addresses the NP 301 Action Plan, Component 2, Problem Statement 2C: Genetic Analysis and Mapping of Important Traits.
5.Significant Activities that Support Special Target Populations
Harmon, F.G., Imaizumi, T., Gray, W.M. 2008. CUL1 Regulates TOC1 Protein Stability in the Arabidopsis Circadian Clock. Plant Journal. 55(4):568-579.
Para, A., Farre, E.M., Imaizumi, T., Pruneda-Paz, J., Harmon, F.G., Kay, S.A. 2007. PRR3 Is a Vascular Regulator of TOC1 Stability in the Arabidopsis Circadian Clock. The Plant Cell. Published on November 30, 2007; 10.1105/tpc.107.054775.