Location: Plant Gene Expression Center
2023 Annual Report
Objectives
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.
Approach
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.
Progress Report
This is the final report for Project 2030-21000-049-000D, Conserved Genes and Signaling Networks that Control Environmental Responses of C4 Grain Crop, which has been replaced by new project 2030-21210-001-000D, Developmental and Environmental Control Mechanisms to Enhance Plant Productivity. For additional information, see the new project report.
Progress was made on all objectives. Overall, genes that define the timing of growth and flowering were identified in this project. These genes provide tool for biotechnology approaches that aim to adjust when seed production occurs to avoid predictable periods of hot or cold weather, thereby improving yield.
Objective 1 sought to define the role of the gigantea gene in determining flowering time of sorghum plants. This objective identified two sorghum lines with knockout mutations in the gigantea gene. Several years of field trials evaluating how the gigantea mutation impacts sorghum flowering time showed gigantea mutants have a substantial delay in development, which is evident as later appearance of the flag leaf in the whorl of the plant. Along with the phenological change in the gigantea mutant, tests of expression of several key flowering time genes that initiate flowering and promote flowering-related development showed substantial disruption in mutant plants. In particular, the gigantea mutant shows much lower expression of floral activator genes, which matches the flowering time defect exhibited by the mutant. Also, expression of key circadian clock genes in the gigantea mutant was much different from normal plants. The conclusion is the gigantea mutant most likely interferes with normal circadian clock function as well as regulation of the initiation of flowering. In addition, this project discovered physical interaction between the GIGANTEA protein and specific proteins responsible for detection of blue light signals. An exciting aspect of this interaction is its regulation by light, which hints at a mechanism whereby GIGANTEA protein controls the activity of blue light-responsive proteins and/or signaling networks. Testing in future years will investigate how this activity of GIGANTEA integrates into sorghum flowering. Work in this area will also be extended to analysis of GIGANTEA regulation of plant growth based on results from Objective 3 (see below).
Objective 2 sought an explanation for cold sensitivity in the Arabidopsis thaliana (Arabidopsis) sickle mutant to better understand low temperature tolerance mechanisms. On question addressed was whether aberrant accumulation of certain alternatively spliced transcripts cause cold sensitivity. Transcriptome-wide analysis (RNA sequencing) and evaluation of candidate transcripts identified alternatively spliced transcripts reaching high levels in the sickle mutant. The RNA sequence data sets associated with this study were released to the public through the National Center for Biotechnological Information. To test this hypothesis, two different alternatively spliced transcripts, from genes called CCA1 and LHY, were made to reach high levels in transgenic Arabidopsis plants. These lines exhibited no consistent change in plant flowering time, development or cold sensitivity. These observations indicate a different, currently unknown, mechanism likely contributes to the cold sensitivity of the sickle mutant.
Objective 3 studied mutants gigantea and sickle for their effect on the relative effect of temperature on maize and sorghum productivity. Both the maize and sorghum gigantea mutants were shorter as seedlings and mature plants at both the low and high temperature field sites. These findings show gigantea plays a critical role in defining plant stature, but its contribution is not influenced by local temperature conditions. Similarly, no field site-specific difference was found in performance of the maize and sorghum mutant lines. Instead, seeds from gigantea mutants had consistently less mass. As indicated above, future work will investigate the biochemical, molecular, and genetic role of the GIGANTEA gene in regulation of sorghum growth, with sorghum serving as a representative of C4 cereal grasses. Analysis of seed germination for the sickle mutant found higher germination rates at a cool temperature (16 degrees C) compared to a warm temperature (26 degrees C). These findings suggest the consequences of interfering with sickle gene function affect growth and development, possibly enhancing aspects of these processes in cool temperatures. However, field trials of sorghum and maize sickle mutant lines did not reveal alterations in mature mutant plants at either field site. These findings suggest sickle contributes to processes in germinating and young seedlings, but appears not to be important for activities in mature cereal grass plants.
Accomplishments
