2012 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 system 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.
Progress was made under all objectives. Under objective 1, existing Arabidopsis circadian mutants were found to lack strong changes in their responses to osmotic or salt stress. Therefore, the plant stresses studied in objective 1 were expanded to include high temperature to tie the project to global climate change, because high temperature changes both plant growth and its regulation by the circadian clock. The genes needed for plants to sense and respond to a warmer environment are not well defined. To identify genes involved in responses to elevated temperature, a mutant screen was initiated in Arabidopsis and more than five novel mutants were identified. Under objective 2, the DNA sequence of the promoter regions for two circadian clock genes harboring conserved regulatory elements were cloned into reporter constructs, which will be used to make transgenic plants for the study of conserved regulatory elements that control circadian gene expression in maize. Under objectives 3 and 4, genetic studies continued with maize mutants, which knock out expression of circadian clock genes in order to fully establish where the circadian clock regulates maize growth and development. Furthermore, additional mutants were identified in other likely circadian clock genes and these were preliminarily characterized for miss-regulation of growth.
Analysis of circadian rhythm genes in maize. Interruption of the maize tocl1 gene with a transposon knocks down expression of the gene. A transposon insertion in the tocl1 gene was identified by ARS scientists at the Plant Gene Expression Center, its position confirmed, and its effect on gene expression established. In the mutant line, tocl1 gene expression is substantially reduced, which confirms this is an authentic loss-of-function mutant. This research provides a novel genetic resource for the study of the genes that control drought responses in maize and, ultimately, the effects of global climate change on crop production.
Characterizing the response to temperature signals in circadain rhythms. Arabidopsis mutants with altered response to high temperature cues were identified and were preliminarily characterized. ARS scientists at the Plant Gene Expression Center carried out a screen of a mutagenized Arabidopsis population and identified at least five unique mutants that respond differently from wild type to a high temperature cues. Future analysis of these mutants will help to define the regulatory systems governing the response of the circadian clock to temperature signals and, ultimately, the aspects of growth and development under clock control. This research provides a basic understanding of how plants respond to warmer environments and is relevant to the impact of global climate change on plant development.
Characterizing the genes, controlling flowering time in maize. The two maize gigantea paralogs in the maize genome have distinct roles in the maize circadian system. ARS scientists at the Plant Gene Expression Center, Albany, California, showed that a mutant in giganea2 did not cause an obvious change in flowering time, while previous work showed earlier flowering behavior in mutants without normal activity of the homologous gene gigantea1. This analysis demonstrates the two gigantea genes are not fully redundant with respect to regulation of flowering. This research defines the regulatory systems governing the timing of flowering in maize and provides an understanding of how maize plants respond to environmental conditions.