Location: Plant Gene Expression Center2022 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.
ARS researchers in Albany, California, made progress toward identifying genes for improvement of C4 cereal crop performance under stressful environmental conditions. One approach in this effort is to discover genes acting in control of plant growth and flowering. Both these activities are highly sensitive to environmental conditions. Flowering is also the source of grain production. Manipulation of genes responsible for growth and flowering can be used to adjust timing of seed production to avoid predictable stressful conditions, like periods of hot or cold weather. Another approach is identification of genes contributing to plant processes that provide broad abiotic stress resistance. This category of genes can be tools 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 determining flowering time and extent of growth for sorghum plants. This objective studies a sorghum line with a predicted knockout mutation in the gigantea gene that was developed in prior years of this project. Fiscal year 2022 (FY22) brought the identification of an additional gigantea mutant allele. For Sub-objective 1A, a second round of field trials this year evaluating how the gigantea mutation impacts sorghum flowering time again showed gigantea mutants have a substantial delay in development. Sub-objective 1C hypothesized GIGANTEA protein interacts with regulatory proteins and these GI-protein complexes control time to sorghum development. FY22 brought the discovery of previously unknown activity for sorghum GIGANTEA protein. This activity is physical interaction with specific proteins responsible for detection of blue light signals. An exciting component of this activity is its regulation by light, which hints at a mechanism whereby GIGANTEA protein controls the activity of blue light-responsive proteins. Further testing will investigate how this activity integrates into sorghum flowering and growth. Progress for Objective 2 was made in understanding factors underlying cold sensitivity in the Arabidopsis thaliana (Arabidopsis) sickle mutant. A goal of Sub-objective 2A is to test whether certain alternatively spliced transcripts cause this cold sensitivity. Previous work 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. Also, two different alternatively spliced transcripts, from genes called CCA1 and LHY, were made to reach high levels in transgenic Arabidopsis plants. Consistent with preliminary analysis, these lines exhibited no consistent effect of on plant flowering time, development or cold sensitivity. These observations indicate a different mechanism may explain the cold sensitivity of the sickle mutant. Progress for Objective 3 was made in characterizing sorghum sickle mutant lines. Further experiments with this sickle mutant confirmed higher germination rates at a cool temperature (16°C) compared to a warm temperature (26°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. Field trials in FY22 advanced backcrossing of potential maize sickle mutant lines to enable testing of these mutant lines for effects comparable to those observed in sorghum.
Marshall, C.M., Harmon, F.G. 2022. Impact of the sickle mutant and temperature on the structure of transcripts and RNAs from Arabidopsis thaliana. BMC Research Notes. 15. Article 110. https://doi.org/10.1186/s13104-022-05963-y.