Research Project: Conserved Genes and Signaling Networks that Control Environmental Responses of C4 Grain Crops

Location: Plant Gene Expression Center

2021 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. One approach in this effort is to discover genes acting to control plant growth and flowering. Growth and flowering are both highly sensitive to environmental conditions. Also, flowering 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, 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 dictating flowering time and growth in 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 (FY) 2021 brought field trials with the goal of testing how the gigantea mutation impacts sorghum flowering time for Sub-objective 1A. These trials showed gigantea mutants reach flowering an average of 26 days later than nonmutant plants, which represents a substantial delay in sorghum development. These observations agreed with previous tests in the greenhouse. Along with this work, expression of flowering-related genes was tested in gigantea mutant and nonmutant plants for Sub-objective 1B. As observed previously, the pattern of expression for several key flowering time genes that initiate flowering and promote flowering-related development was substantially disrupted by the gigantea mutation. The changes caused by the gigantea mutant correlate with the observed late flowering phenotype in field and greenhouse trials. This information addresses goal of Sub-objective 1B to understand the underlying cause of gigantea mutant-caused alterations in growth and development. Sub-objective 1C hypothesized GIGANTEA protein interacts with regulatory proteins and these GI-protein complexes control time to maturity and other central aspects of abiotic responses. An experiment to identify GIGANTEA interacting proteins discovered several proteins in sorghum and maize that may form protein complexes with it. Further testing in future years will verify these observations. Progress for Objective 2 was made in understanding factors underlying cold sensitivity in the Arabidopsis thaliana sickle mutant. A goal of Sub-objective 2A is to test whether specific alternatively spliced transcripts cause this cold sensitivity. Previous work identified alternatively spliced transcripts reaching high levels in the sickle mutant. The hypothesis is that some of these aberrant transcripts contribute to the cold sensitivity phenotype. Two different alternatively spliced transcripts were selected, from genes called CCA1 and LHY, and the DNA from these transcripts was put into constructs for expression in Arabidopsis thaliana. These constructs were transformed into plants. Several independent plant lines carrying the transgenic construct were identified based on herbicide resistance. Preliminary analysis of these lines showed no consistent effect of the transgenic construct on plant flowering time or development. Next, homozygous progeny of these lines will be tested again for changes in cold sensitivity like that of the sickle mutant, as well as for alterations in flowering time and development. Progress for Objective 3 was made in testing if gigantea genes contribute to yield in maize and sorghum. For Sub-objective 1A, the FY 2021 field trials also were intended to test yield traits in gigantea mutant and nonmutant plants. The plants grown in Albany, California, did not produce seeds, precluding these tests at this location. Seed bearing panicles were collected from plants at the Davis location for analysis, which will take place when the material from the FY 2021 field trial is available at the end for the season. Progress was made in characterizing sorghum sickle mutant lines. Tests for Sub-objective 3B were preliminary field trials and seed germination evaluation under different temperature treatments. In the field, a sickle mutant displayed stunted growth and reduced leaf width. Contrary to expectation, this sickle mutant had enhanced root growth at a cool temperature (16° Celsius (C)) compared to a warm temperature (26°C). Also, seeds from the sickle mutant had higher germination rates at the cool temperature. These findings suggest the sorghum sickle mutant affects growth and development, possibly enhancing aspects of these processes in cool temperatures. Further tests are needed to confirm these preliminary findings.

Accomplishments

Review Publications
Abdul-Awal, S., Chen, J., Xin, Z., Harmon, F.G. 2020. A sorghum gigantea mutant attenuates florigen gene expression and delays flowering time. Plant Direct. 4(11). Article e00281. https://doi.org/10.1002/pld3.281.
Lai, X., Bendix, C., Zhang, Y., Schnable, J.C., Harmon, F.G. 2021. 72-hour diurnal RNA-seq analysis of fully expanded third leaves from maize, sorghum, and foxtail millet at 3-hour resolution. BMC Research Notes. 14. Article 24. https://doi.org/10.1186/s13104-020-05431-5.
Lai, X., Bendix, C., Yan, L., Zhang, Y., Schnable, J., Harmon, F.G. 2020. Interspecific analysis of diurnal gene regulation in panicoid grasses identifies known and novel regulatory motifs. BMC Genomics. 21. Article 428. https://doi.org/10.1186/s12864-020-06824-3.
Li, Z., Zhu, A., Song, Q., Chen, H.Y., Harmon, F.G., Chen, Z. 2020. Temporal regulation of metabolome and proteome in photosynthetic and photorespiratory pathways contributes to maize heterosis. The Plant Cell. 32(12):3706-3722. https://doi.org/10.1105/tpc.20.00320.