Research Project: Characterization of Plant Architectural Genes in Maize for Increased Productivity

Location: Plant Gene Expression Center

2022 Annual Report

Objectives
The long-term goal of our research is to identify genes that regulate plant architecture in maize. We recently positionally cloned four genes that were defined by mutant phenotype. The phenotypes affect multiple aspects of architecture including leaf shape, internode length, tassel branching and sex determination. The phenotypes vary depending on inbred background. Of the four genes, one encodes a plasma membrane bound protein, one encodes a kinase, a third encodes an enzyme and the fourth is a conserved gene of unknown function. In order to connect their interesting phenotypes to mechanism we are identifying interacting partners, carrying out RNAseq and conducting metabolomic analysis. The work will increase our dataset from four genes to entire pathways. We will then combine knowledge of these pathways to transcription factor targets that are being developed in collaboration with others. This combined information will provide a network of connectivity that could be useful for breeding. For example, if we hope to change leaf angle, we can ask which genes appear to function solely in leaf angle and not also in leaf width or tassel branching. If we are selecting for improved abiotic stress, we can examine our network and see what genes are likely to have large or small effects. Objective 1: Dissect gene networks that regulate leaf architecture and internode elongation in maize to provide targets for breeding more productive maize. Subobjective 1A. Identify proteins that interact with NOD (NARROW ODD DWARF) and confirm the interaction, in vivo and in vitro. Subobjective 1B. Identify proteins that are phosphorylated by LGN and carry out transcriptome analysis. Subobjective 1C. Map modifiers of nod that are responsible for the inbred differences. Objective 2: Characterize genes that regulate tassel branching and sex determination in maize for higher yields. Subobjective 2A. Prove identity of Tasselseed5 (Ts5) gene by obtaining a revertant allele and by overexpressing the gene. Subobjective 2B. Map the modifiers that differentiate Ts5 in Mo17 compared to B73. Subobjective 2C. Obtain additional feminized upright narrow (fun) alleles and carry out RNAseq analysis. Objective 3: Determine coordinated and independent pathways that regulate leaf, inflorescence, and internode development in maize for enhanced productivity.

Approach
For Objective 1, we hypothesize that NARROW ODD DWARF (NOD), a plasma membrane localized protein known to function in calcium signaling, is an essential protein in plants with a role in development and immunity. We are using proteomics to identify interacting partners and testing these interactions with biochemical and genetic experiments. We also hypothesize that LIGULELESS NARROW (LGN) is critical, given the severe mutant phenotype when it is not able to phosphorylate other proteins. We will determine the targets of this kinase and determine how and when it interacts with NOD. We have antibodies to both of these proteins that function in westerns and in Co-immunoprecipitation. Both NOD and LGN mutants are distinct in different inbreds. We mapped a modifier to LGN and plan to identify the modifiers for NOD. We hypothesize that there are distinct loci responsible for the ligule defects and other loci responsible for the auto-immunity defects. The modifiers will be identified using genotyping by sequencing (GBS) methods. We also have the possibility of mapping the modifiers by crossing to recombinant inbred lines in the GBS method doesn’t work. For objective 2, we hypothesize that Ts5 encodes an enzyme in the jasmonic acid (JA) pathway. We will obtain a revertant of Ts5 using ethyl methyl sulfonate (EMS). If this doesn’t work, we will verify its function by following JA metabolites during wounding. We also plan to overexpress the gene in Brachypodium and determine the effect on plant development. Ts5 is completely feminized in the Mo17 inbred and it is mild in B73. We crossed Ts5 to the recombinant B73 Mo17 inbred lines (IBM) and identified 10 major quantitative trait locus (QTL). We combined this data with an RNA sequencin (RNAseq) experiment that identified the differentially expressed genes between Ts5 and normal tassels. Four genes were identified and we will obtain mutants in these genes to examine their function. The fun mutant is also feminized, but may not be in the JA pathway. From analysis of double mutants, we hypothesize it is in the brassinosteroid (BR) pathway. We are determining the BR levels and will analyze an RNAseq dataset to explore this hypothesis. Because FUN is a gene of unknown function, additional alleles will be useful for understanding the domains. These will be obtained by EMS screens. For Objective 3, we will combine our different datasets into a network analysis. We hypothesize that few genes function in only one tissue and will determine the overlap in the tassel network and leaf network. This analysis may lead to genes that are not yet identified by a mutant phenotype and would be worth study in the future.

Progress Report
In support of Objective 1, ARS researchers in Albany, California, submitted a manuscript on LIGULELESS NARROW (LGN) and NARROW ODD DWARF (NOD) with colleagues from the University of Wisconsin and ARS colleagues in Gainesville, Florida. LGN encodes a receptor-like kinase and NOD encodes a membrane-bound protein that may function in Calcium signaling. To identify NOD interacting proteins, we used a polyclonal antibody that specifically interacts with NOD. We identified a number of proteins that interact with NOD, but focused our attention on LGN given that we had a mutant phenotype for LGN and an antibody. The manuscript includes the analysis of the protein interaction between LGN and NOD carried out with three different methods. In addition to phenotypic measurements during development and at maturity, RNA sequencing (RNA-seq) analysis and a metabolomic analysis were carried out. The unbiased profiling approaches to define molecular phenotypes of the single and double mutants revealed that all three genotypes reprogram maize biology to promote stress and pathogen defense responses at the cost of reducing resources allocated to growth and development, an illustration of the “growth-defense trade-off” hypothesis. Fine-scale analysis reveals that the specific genes or metabolites induced or repressed in B73 and A619 are not easily predicted from one background to the next. This could be a result of upstream genetic modifiers that reorient signal transduction from NOD and LGN to the nucleus, or could be a consequence of differences in genetic regulatory elements downstream of NOD and LGN. The latter hypothesis is well-supported by the literature: genes involved in specialized metabolism, stress responses, and pathogen defense are rapidly evolving and highly variable within populations, but are typically induced by a core set of common signal transduction pathways (stress signaling “hubs). Overall, these findings demonstrate the critical importance of investigating how even severe mutant phenotypes can be differentially expressed in distinct genetic backgrounds, especially in agricultural species with the incredible genetic diversity of maize. For Objective 2, we continued work on the feminized upright narrow (FUN) mutant. The fun phenotype encompasses two different research interests, feminization of the tassel and upright leaf angle. Unlike the well characterized liguleless mutants that lack ligule and auricle, fun mutants still retain the ligule but only lack the auricle. The missing auricle causes the leaves to be more upright. The tassel is a mixture of male and female flowers, but short and less branched, features that are consistent with feminization. Given that FUN is a conserved protein of unknown function, we anticipate a combination of genetics, RNAseq, protein localization, and identification of protein interactors will help us understand its function. In order to determine the proteins with which FUN interacts, we did a yeast two hybrid screen. 155 interacting proteins were identified that provide tantalizing pathways. We carried out co-immunoprecipitations using our antibody, sent the samples to a facility at the University of California, Davis, for mass spectrometry analysis, and are presently analyzing the results. Proteins that are identified by both yeast two hybrid and co-immunoprecipitation will be the focus of additional investigation. Most feminized maize mutants are defective in jasmonic acid (JA) biosynthesis and can be corrected by the addition of JA. The Tasselseed5 mutant that we identified is such a mutant. In a small scale experiment, we added JA to fun mutants and controls and quantified the feminization on mature tassels. It appears that JA does not rescue the feminization of fun. This discovery is supported by hormone analysis that does not detect changes in JA. We will repeat the JA experiment this summer in the corn field where we can get larger numbers of individuals.

Accomplishments

Review Publications
Richardson, A., Cheng, J., Johnston, R., Kennaway, R., Conlon, B., Rebocho, X., Kong, H., Scanlon, M., Hake, S.C., Coen, E. 2021. Evolution of the grass leaf by primordium extension and petiole-lamina remodeling. Science. 374(6573):1377-1381. https://doi.org/10.1126/science.abf9407.
Richardson, A., Hake, S. 2022. The power of classic maize mutants: Driving forward our fundamental understanding of plants. The Plant Cell. 34(7):2505-2517. https://doi.org/10.1093/plcell/koac081.
Du, Y., Lunde, C., Li, Y., Jackson, D., Hake, S., Zhang, Z. 2021. Gene duplication at the Fascicled ear1 locus controls the fate of inflorescence meristem cells in maize. Proceedings of the National Academy of Sciences (PNAS). 118(7). Article e2019218118. https://doi.org/10.1073/pnas.2019218118.