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ARS Home » Pacific West Area » Albany, California » Plant Gene Expression Center » Research » Research Project #425045

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

Location: Plant Gene Expression Center

2018 Annual Report

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.

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
This is the final report for project 2030-21000-039-00D, which has been replaced by new project 2030-21000-049-00D, "Conserved Genes and Signaling Networks that Control Environmental Responses of C4 Grain Crops." For additional information, see the new project report. Progress was made on all objectives. The project advanced existing mutants and developed new mutant lines for testing in Objectives 1 and 2. Seeds for new transposon insertion mutants were obtained through a Cooperative Research and Development Agreement and from the public Maize Genetics Cooperation. The new lines represented potential mutant alleles for several clock-associated genes, including gigantea 1 (gi1), gigantea 2 (gi2), late elongated hypototyl-like1 (lhyl1), late elongated hypototyl-like2 (lhyl2), timing of cab-like1 (tocl1), timing of cab-like2 (tocl2), pseudo-response regulator73, early flowering3-like1, and early flowering3-like2. Over the course of the project, these mutant alleles were moved into several homogenous inbred backgrounds to create lines for testing whether the activity of circadian clock genes is influenced by the extensive genetic diversity present in maize. Analysis of gi1 and gi2 mutant lines showed these genes play a role in circadian clock activity, which is related to Objective 1. In addition, mutants in these genes produce changes in flowering time, growth, and sensitivity to the leaf disease southern leaf blight, which is related to Objective 2. Observations also indicated that the gi1 and gi2 genes act together, instead of separately. Artificially high (overexpression) or low lhyl1 gene expression changed circadian clock function, which is related to Objective 1. Additionally related to Objective 1, large-scale analysis of gene expression revealed identical patterns of fluctuating daily expression in agronomically important genes from three different grain crops: corn, sorghum and millet. Genes in these sets participate in critical processes including photosynthesis, responses to stress, responses to hormones, and control of development, and findings related to Objective 2. This analysis allowed prediction of all the genes important for circadian rhythms, which revealed far more potential regulatory complexity in corn than known for the model plant Arabidopsis thaliana. Objective 3 focused on identification of tocl2 and tocl1 mutant alleles then establishing experimental and phenotypic parameters for testing the drought response of tocl2 and tocl1 mutant lines. Preliminary drought treatments of tocl2 mutant lines indicated these plants are more sensitive to this stress. Researchers also discovered that drought modifies daily regulation of drought-responsive and circadian clock genes in maize and soybean. A collaboration with Brazilian researchers compared the genome-wide gene expression behavior of maize and soybean plants exposed or not to water limitation. Findings included discovery of regulatory crosstalk between the soybean circadian clock and drought stress-signaling pathway. The findings from this project are expected to have a positive impact on engineering and breeding efforts to modify maize growth by use of differentially active versions of the circadian clock-associated genes. Additionally, the findings in this project are potentially useful for improvement of related grass species such as sorghum, sugarcane, and millet.

1. Flooding tolerance in soybean plants utilizes aspects of drought-response pathways due to alteration of gene structure. ARS scientists in Albany, California, joined with Brazilian researchers to identify mechanistic aspects of flooding tolerance in soybean plants by analyzing differences in gene expression and gene structure between flooding tolerant and flooding sensitive soybean lines. This study found that drought stress-signaling pathways responded to flooding in roots of tolerant plants, but not sensitive plants, and genes with different expression between the two varieties had unique regulatory sequence composition. This study shows that soybean lines have gained tolerance to flooding through co-option of drought stress response mechanisms and highlights that plants engage multiple stress pathways to endure stressful environmental conditions. These discoveries provide novel avenues for improving stress tolerance in a wide variety of crop plants, as well as soybean.

Review Publications
Nakayama, T.J., Rodrigues, F.A., Neumaier, N., Marcolina-Gomes, J., Molinari, H.B., Santiago, T.R., Formighieri, E.F., Basso, M.F., Farias, J.R., Emygdio, B.M., De Oliveira, A.C., Campos, A.D., Borem, A., Coleman-Derr, D.A., Mertz-Henning, L.M., Nepomuceno, A. 2017. Insights into soybean transcriptome reconfiguration under hypoxic stress: Functional, regulatory, structural, and compositional characterization. PLoS One. 12(11):e0187920.
Benedix, C., Marshall, C., Harmon, F.G. 2015. Circadian clock genes universally control key agricultural traits. Molecular Plant. 8(8):1135-1152.