Location: Plant Gene Expression Center2018 Annual Report
The long-term goal of this project is to identify genetic pathways useful for adaptation of crop plants to stressful and/or novel environmental conditions. This project investigates two aspects of post-transcriptional regulation associated with the plant circadian clock, namely regulation of protein activity via protein-protein interactions and control over alternative splicing of transcripts. Based on previous knowledge, these mechanisms are predicted to be associated with key plant signaling networks governing responses to temperature signals. Specifically, during the next five years we will focus on the following objectives. Objective 1: Dissect the circadian clock regulatory network underlying time to maturity at the genetic and molecular levels, particularly in C4 crops including maize and sorghum. • Subobjective 1A: Investigate whether SbG1 contributes to the timing of maturity in sorghum. (Harmon) • Subobjective 1B: Investigate the effect of sbgi mutants on expression of flowering time and circadian clock genes. (Harmon) • Subobjective 1C: Identify protein partners of SbGI and ZmGI proteins. (Harmon) Objective 2: Establish the relationship between alternative transcript splicing and the circadian clock control responses to heat and cold at the genetic and molecular level in C4 crops including maize and sorghum. • Subobjective 2A: Define the impact of sic mutants on low temperature-sensitive alternative splicing within the Arabidopsis transcriptome. (Harmon) • Subobjective 2B: Test whether specific clock-associated splice variants occurring in sic-3 alter circadian clock responses to temperature cues. (Harmon) Objective 3: Identify genetic variation in circadian clock genes to enhance agronomic performance in the field for maize and sorghum in response to temperature variation and/or change in latitude. • Subobjective 3A: Test whether GI genes contribute to yield in maize and sorghum under field conditions with sustained suboptimal temperatures.
Objective 1 will study flowering time, leaf senescence, and time to maturity in SbGI mutants. Flowering time will be days from sowing to boot stage and days from sowing to anthesis/stigma exertion. Leaf senescence will be scored visually from flowering until harvest maturity. Time to maturity will be days to hard dough stage in maturing seeds. Also, expression of flowering time and circadian clock genes will be compared between sbgi mutant and normal plants to test if SbGI contributes to regulation of these genes. Quantitative polymerase chain reaction will be used to determine relative transcript levels in leaves of plants exposed to short or long day photoperiods. Proteins interacting with the SbGI and ZmGI proteins will be identified through testing of candidate proteins and large-scale library screening with yeast two-hybrid. Objective 2 will identify splice variants accumulating at low temperature in the reference sic-3 allele but not wild type plants by RNA sequencing (RNA-seq). All alternative splicing events from circadian clock genes significantly changed in sic-3 according to RNA-seq will be validated with reverse transcription-polymerase chain reaction. In addition, experiments will test whether splice variants of circadian clock transcripts found in sic-3 impair circadian clock function at low temperatures. Two complementary transgenic approaches will be used: 1) ectopic overexpression of a splice variant in wild type plants, which tests whether high levels of a specific splice variant interfere with clock function, and 2) silencing of splice variant expression in sic-3, which tests whether removal of a splice variant suppresses circadian clock defects in this mutant. Objective 3 will test whether GI genes participate in response pathways required for optimal plant growth and yield under high and low temperatures. SbGI and ZmGI mutants will be tested for flowering time, as in Objective 1, and yield-associated traits, including total dry matter (weight of panicle/ear before threshing), grain yield (total seed weight per panicle/ear) and 100 seed weight. Harvest index also will be calculated as the ratio of grain yield to total dry matter. Different temperature conditions will be provided by growing plants at Gill tract in Albany, California, a comparatively cool site, and at University of California, Davis, a comparatively hot site. This objective will also determine whether SIC genes contribute to low temperature tolerance of maize and sorghum. sic mutants will be tested for low temperature sensitivity during germination by calculating percent germination at 26°C, 22°C, 16°C, and 12°C. Also, mutant plants will be evaluated for the flowering time, maturity, and yield traits as described above. If available gi or sic mutations do not sufficiently reduce gene function, CRISPR-Cas9 genome editing can be used to generate mutant alleles. If conditions at the Albany and Davis fields are too severe to score traits, greenhouse space is available for these studies.
This report documents progress for project 2030-21000-049-00D, which started March 2018 and continues research from project 2030-21000-039-00D, "Characterizing Circadian Regulatory Networks in Grain Crops to Establish Their Role in Development and Abiotic Responses." The new project has the goal of understanding gene function to discover how grain crops resist environmental stresses and control developmental transitions. To begin this project, maize and sorghum plants were grown during the summer of 2018 to identify, cross, and increase the necessary mutant lines. Samples of mutant and normal Arabidopsis plants were collected for analysis of the effects of temperature on transcriptome-wide gene expression and transcript structure.