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Research Project: Characterizing Circadian Regulatory Networks in Grain Crops to Establish their Role in Development and Abiotic Responses

Location: Plant Gene Expression Center

2016 Annual Report


Objectives
The circadian clock in crop plants controls important performance traits including growth, timing of flowering and stress and pathogen responses by coordinating daily and seasonal changes in physiology. The long term goal of this project is to define at the genetic and molecular levels the circadian system in grain crops, including corn, and its impact on agronomic traits. This project will use genomic, genetic, and molecular methods to identify and characterize the circadian system in corn, utilizing resources and tools in corn and other model plant systems as appropriate. The circadian system genes identified will provide gene targets for enhancing crop performance and adaptation to global climate change. The objectives of the project are: Objective 1: Identify and characterize genes required for circadian rhythms in grain crops. Objective 2: Identify and characterize genes required for circadian clock-regulated developmental processes in grain crops. Objective 3: Analyze the contribution of the circadian system to drought responses in grain crops.


Approach
The genes required for circadian rhythms in maize remain uncharacterized. The goal of Objective 1 is to identify and/or construct mutants in candidate genes to define the genes that participate in the core circadian oscillator. Subsequent analysis of mutants will establish the function of their gene products to understand the molecular nature of the maize circadian oscillator. The hypotheses to be tested are: Mutations in circadian clock genes will alter circadian clock-driven transcription; Additional mutant alleles in clock genes can be identified and constructed using publicly available germplasm collections; and, Regional mutagenesis with the Ds transposon will create additional gi2 knockout alleles. Work in model plants demonstrates that the circadian system is deeply imbedded in regulatory networks that control growth and developmental processes. Whether such a regulatory system exists in maize remains an open question. The goal of Objective 2 is to investigate whether circadian regulation is an important contributor to maize growth and development by studying circadian clock mutants. The hypotheses to be tested are: Maize circadian clock genes are involved in regulation of maize flowering time; gi functions within the genetic networks known to control maize flowering time; The gi and tocl1 genes underlie known flowering time QTL; gi activity contributes to the timing of the juvenile to adult transition; and, Maize clock genes participate in regulation of growth. Specific core circadian oscillator genes play important roles in the responses of model plants to drought stress, in part through regulation of phytohormone signaling. The goal of Objective 3 is test whether drought stress and phytohormone responses in maize depend on the activity of circadian clock genes. The hypotheses to be tested are: The tocl1 gene is involved in maize drought responses; and, The tocl1 gene contributes to ABA responses in maize.


Progress Report
Progress was made on all Objectives. For all Objectives, we have been assigning function to corn genes to discover genetic networks that provide grain crops with resistance to environmental stress or control of developmental transitions. This year we continued to develop existing mutants and to identify new mutant lines for testing. For Objectives 1 and 2, we combined small-scale studies of single gene mutants in corn with large-scale studies of gene expression and genome sequences of several grain crops. Single gene analysis showed that interference with gi1 and gi2 gene function in corn negatively impacts circadian clock activity, which accompanies previously identified changes in flowering time and growth. Artificially high expression (overexpression) of the lhyl1 gene also changes circadian clock function. These observations relate to Objective 1. Overexpression of the lhyl1 gene also delays flowering time, retards photosynthesis, interferes with floral organ development, and inhibits growth, which relates to Objective 2. Separately, large-scale analysis of gene expression allowed discovery of identical patterns of fluctuating daily gene expression for many of the same genes from three different grain crops: corn, sorghum and millet. The types of genes with this shared expression behavior comprise sets for agronomically important physiological processes including photosynthesis, responses to stress, responses to hormones, and control of development. These observations support the hypotheses being tested in Objectives 2 and 3. Another large-scale study undertaken this year was investigation of grain crop genomes to predict all the genes likely to be important for generating fluctuating daily gene expression patterns produced by the circadian clock. This analysis revealed far more potential regulatory complexity in corn than known for the model plant Arabidopsis thaliana. Newly identified genes found in this analysis will be investigated in the future to gain a better understanding of how circadian rhythms are generated in grain crops, which is related to Objective 1. For Objective 2, our collaboration with an ARS scientist in Raleigh, North Carolina, confirmed that corn mutants in gi1 are more resistant to the leaf disease southern leaf blight, while gi2 mutants are more sensitive to this disease. The impact of these discoveries is that focusing engineering and breeding efforts on beneficial versions of the gi1 and gi2 genes could develop more southern leaf blight-resistant corn varieties. For Objective 3, the contribution of the tocl2 gene to drought responses is currently being evaluated for the second time with field-based drought studies of two different mutations in the tocl2 gene. The overall impact of our work is a clearer understanding of where circadian clock-directed regulation in corn makes contributions to important developmental processes and stress responses.


Accomplishments
1. Gene regulation by circadian clock transcription factors establishes metabolic changes in corn hybrids. The majority of corn grown in the United States is hybrid to capture the growth advantage provided by hybrid vigor, yet the molecular causes of hybrid vigor remain poorly understood. In collaboration with a scientist at University of Texas at Austin, ARS scientists in Albany, California demonstrated that corn hybrids use gene regulation by the circadian clock to achieve increased metabolic activity in seedlings. They hypothesize that maintenance of this early-established, metabolic advantage throughout the life of the plant contributes to greater yield in mature hybrid plants. This discovery holds the promise of engineering and/or breeding vigor-associated metabolic traits into inbred corn lines by manipulating the activity of the circadian clock.


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Review Publications
Gomes, J.M., Rodrigues, F.A., Fuganti-Pagliarini, R., Nakayama, T.J., Reis, R., Farias, J.B., Harmon, F.G., Nepomuceno, A.L. 2015. Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day. PLoS One. 10(9):e0139051.