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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

2015 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
Significant progress was made for Objectives 1 and 2. For Objective 1, the transposon insertions in late elongated hypototyl2 (lhyl2) gene were further moved into homogenous inbred backgrounds during the 2015 winter and summer nurseries to develop lines for future testing. In addition, homozygous mutant lines were made in the summer nursery to confirm disruption of lhyl2 gene expression. Transgenic RNA interference (RNAi) lines were made this year that knock down expression of both late elongated hypototyl1 (lhyl1) and lhyl2; thus, these RNAi lines represent the double mutant state. Although produced for a different project, these RNAi lines will be tested in this project’s experiments to complement analysis of the single lhyl2 transposon insertion lines. Potential insertions in timing of cab-like1, timing of cab-like2 (tocl2), pseudo-response regulator73, early flowering3-like1, and early flowering3-like2 received last year were found to be difficult to cross out of the parental line and work continues on introgressing these to multiple inbreds. For Objective 2, the effort to make the double mutant of the gi1-m1 and gi2-m1 mutant alleles, which disrupt expression of the only two maize gigantea (gi) genes, will be complete at the close of the 2015 summer field season. Initial phenotyping of the gi1-m1 gi2-m1 double mutant plants for flowering time and growth is currently underway. Families carrying dwarf9, a dominant dwarf mutant affected in developmental responses controlled by the phytohormone gibberellin, and gi1-m1 are expected to yield a double mutant of these two alleles by the end of 2015. In collaboration with another ARS researcher, gi1-m1 and gi2-m1 mutant lines were tested for altered susceptibility to southern leaf blight, northern leaf blight and gray leaf spot, which are three important foliar diseases for maize. For Objective 3, the existing tocl2-m1 mutant allele was fully backcrossed into the A632 inbred backgrounds and homozygous families will be available at the end of the 2015 summer nursery. Two additional tocl2 mutant alleles were further introgressed into the A632 and W22 inbreds to provide additional alleles for testing of tocl2 gene activity. These independent alleles will be important to confirm the work with the tocl2-m1 allele.

1. A simple method to genetically transform Setaria viridis (foxtail millet). C4 photosynthesis is more efficient than C3 photosynthesis due to partitioning of components into different cell types. Understanding the mechanism and development of C4 photosynthesis in monocot grasses is important to improve C4 crop yield and to engineer C4 photosynthesis in other cereal crops like rice. The important food and fuel crops, corn, sugar cane, sorghum, and millet perform highly efficient C4 photosynthesis. Setaria viridis or foxtail millet is a relative of cultivated millet and an emerging model plant for study of C4 photosynthesis in grasses. ARS researchers at Albany, California, together with researchers from the Brazilian Enterprise for Agricultural Research in Brasilia, Brazil, developed a rapid technique to genetically transform Setaria viridis. This method directly simplifies genetic improvement of millet and will advance genetic and genomic studies of other important food and fuel crops like sugarcane, sorghum, and corn.

Review Publications
Rodrigues, F.A., Fuganti-Pagliarini, R., Gomes, J.M., Nakayama, T.J., Molinari, H.C., Lobo, F.P., Harmon, F.G., Nepomuceno, A.L. 2015. Daytime soybean transcriptome fluctuations during water deficit stress. BMC Genomics. 16:505. doi:10.1186/s12864-015-1731-x.
Martins, P.K., Nakayama, T.J., Ribeiro, A.P., Brio Da Cunha, B.A., Nepomuceno, A., Harmon, F.G., Kobayashi, A.K., Molinari, H.C. 2015. Setaria viridis floral-dip: A simple and rapid Agrobacterium-medicated transformation method. Plant Biotechnology. 6:61-63.
Harmon, F.G., Thines, B., Wu, A., Filo, J., Eliason, E. 2015. Gibberllin driven growth in elf3 mutants requires PIF4 and PIF5. Plant Signaling and Behavior. 10(3):e992707). doi: 10.4161/15592324.2014.992707.