Location: Plant Gene Expression Center
2020 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
Researchers in Albany, California, made progress toward identifying genes for improvement of C4 cereal crop performance under stressful environmental conditions. In this effort, one approach is to discover genes acting in pathways that control plant growth and flowering. Growth and flowering are a focus because these developmental processes are highly sensitive to environmental conditions and flowering, in particular, is the source of grain production. Manipulation of genes responsible for growth and flowering can be used to adjust the timing of seed production to avoid predictable stressful conditions, such as periods of hot or cold weather. Another approach is identification of genes contributing to plant processes that provide broad resistance to abiotic stresses. This category of genes can be a tool to increase the overall stress resistance capacity of plants to create crops that can withstand unpredictable stresses. Together, these two types of genes will be useful tools to breed and engineer abiotic stress-resistant cereal crop varieties.
Progress for Objective 1 was made in defining the role of the gigantea gene in dictating flowering time and growth in sorghum plants. This objective studies a sorghum line with a predicted knockout mutation in the gigantea gene. An initial goal of Sub-objective 1A is development of mutant gigantea lines suitable for testing of phenotypes. Progress toward this goal in fiscal year 2020 was backcrossing of mutant germplasm to remove the initial high mutational load. The backcrossed gigantea mutant germplasm from Sub-objective 1A enabled progress in Sub-objective 1B. The goal of Sub-objective 1B is to test whether the gigantea mutant changes expression of genes involved in determining flowering time and genes needed for the activity of the circadian clock. The experiments for fiscal year 2020 were the first round of tests for expression of several key flowering time genes that initiate flowering and promote flowering-related development. The pattern of expression for these genes was substantially disrupted by the gigantea mutation. In particular, the gigantea mutant shows much lower expression of floral activator genes. This discovery predicts a significant change in flowering time in the mutant. The same mutant and normal plant samples were also tested for expression of fundamental circadian clock genes that produce circadian rhythms. Expression of these circadian clock genes in the gigantea mutant was very different from the normal plants. The conclusion is the gigantea mutant most likely interferes with normal circadian clock function. Future work will be directed at confirming the predictions from these gene expression experiments by replication of gene expression tests and further study of growth and flowering time in the gigantea mutant. Progress for Sub-objective 1C involved completion of a set of DNA constructs for identifying proteins that make contact with the gigantea protein in plant cells. These DNA constructs will be used in future experiments to discover how sorghum gigantea performs its biochemical function.
Progress on Objective 2 was made in understanding the underlying cause of cold sensitivity in the Arabidopsis thaliana sickle mutant. An initial goal of Sub-objective 2A is to identify how the sickle mutant changes alternative splicing of transcripts on a transcriptome-wide scale. Transcripts are types of RNA that encode for proteins. Progress toward this goal involved following up on predictions from prior computational studies with wet lab experiments designed to confirm predicted sequence structures of RNA species. The specific goal was to confirm and provide independent support for the unexpected finding of a high concentration of RNA types known as lariats in the sickle mutant. Lariats are a byproduct of transcript production that are normally removed to avoid disruption of this process. The wet lab work this year confirmed the presence of unusual amounts of lariats in the sickle mutant. This discovery shows the sickle mutant possibly blocks removal of lariats and this feeds back to change transcript splicing. The conclusion is the sickle gene has a critical role in core RNA metabolic processes. Progress also was made on Sub-objective 2B to test whether removal of excess lariats in the sickle mutant eliminates the cold sensitivity phenotype. DNA constructs were made to express a lariat degrading enzyme in sickle plants and these were transformed into Arabidopsis thaliana plants. With further confirmation, these discoveries indicate that fundamental steps in RNA metabolism are a potential point of weakness for crop plant resistance to low growing temperatures.
Progress for Objective 3 was made in addressing whether inactivation of the gigantea gene or sickle genes negatively impacts yield in maize and sorghum under field conditions under suboptimal temperatures. To achieve the goals of this objective, the development of sickle mutant germplasm in maize and sorghum is needed because the initial germplasm source carries a high mutational load. Progress for Sub-objective 3B involved crossing previously identified sorghum and maize plants carrying potential sickle mutations to non-mutant plants for backcrossing.
Accomplishments
1. Evolutionary conservation of gene expression networks in three C4 crop species. For optimal productivity, crop plants must prepare for and adapt to daily changes in light availability and air temperature. Researchers in Albany, California, conducted a study that used next generation RNA sequencing and computational biology techniques to discover ways this is accomplished at the gene expression level in maize (corn), sorghum, and foxtail millet, which are C4 crop plants. The goal was to find shared aspects of gene expression networks because shared aspects of networks represent evolutionarily conserved features that are expected to be the most fundamental regulatory mechanisms. With this approach, this study identified major regulatory networks and DNA sequences that are predicted to generate conserved patterns of gene expression contributing to daily environmental adaptation in these C4 crop species. These findings will inform breeding and engineering efforts to adapt cereal crops to novel growth conditions.
