Location: Plant Gene Expression Center
2023 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
This is the final report for project 2030-21000-052-000D, “Characterization of Plant Architectural Genes in Maize for Increased Productivity” which expired in 2/24/2023, and has been replaced with project 2030-12210-003-000D, “Enhancing Crop Resilience to Biotic and Abiotic Stress Through Understanding the Immune and Microbiome Signaling Mechanisms”. For additional information, see the new project report.
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 RNA sequencing (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.
In service of Objective 1, we characterized narrow odd dwarf (nod) and Liguleless narrow (Lgn), two pleiotropic maize mutants that both encode plasma membrane proteins, cause similar developmental patterning defects, and constitutively induce stress signaling pathways. To investigate how these mutants coordinate maize development and physiology, we screened for protein interactors of NOD by affinity purification. LGN was identified by this screen as a strong candidate interactor, and we confirmed the NOD-LGN molecular interaction through orthogonal experiments. We further demonstrated that LGN, a receptor-like kinase, can phosphorylate NOD in vitro, hinting that they could act in intersecting signal transduction pathways. To test this hypothesis, we generated Lgn-R;nod mutants in two backgrounds (B73 and A619) and found that these mutations enhance each other, causing more severe developmental defects than either single mutation on its own, with phenotypes including very narrow leaves, increased tillering, and failure of the main shoot. Transcriptomic and metabolomic analyses of the single and double mutants in the two genetic backgrounds revealed widespread induction of pathogen defense genes and a shift in resource allocation away from primary metabolism in favor of specialized metabolism. These effects were similar in each single mutant and heightened in the double mutant, leading us to conclude that NOD and LGN act cumulatively in overlapping signaling pathways to coordinate growth-defense tradeoffs in maize.
In service of Objective 2, we characterized the mutant fun1. The feminized upright narrow1 mutant (fun1) is a unique recessive mutant that affects both sex determination as well as leaf development. fun1 tassels are unbranched and feminized, producing perfect flowers with viable silks that can be fertilized and set seed. These phenotypes are reminiscent of the jasmonic acid (JA) biosynthetic mutants in maize such as tasselseed1 that also display feminized tassels. Direct measurements of several stress hormones such as ethylene, ABA and JA in fun1 tassels showed that all three are greatly downregulated in both fun1 alleles compared to wildtype, while levels of other stress signaling molecules such as salicylic acid are unchanged. fun1 leaves lack auricles and have a narrow upright habit but have normal ligules, distinguishing them from other maize leaf mutants that affect auricle development. The role of stress hormones during leaf development is unclear, and the fun1 mutant offers a unique opportunity to understand how this pathway intersects with reproductive development.
fun1 was cloned by a combination of chromosome walking and expression profiling and encodes a highly disordered hydrophilic protein that may have signaling function during embryogenesis. fun1 is primarily expressed in developing seeds starting at two weeks after pollination, but is found a much lower levels in developing shoots and tassels. Two ethyl methyl sulfonate (EMS) induced fun1 alleles were sequenced and have mutations resulting in premature stop codons. Moreover, clustered regularly interspaced short palindromic repeats (CRISPR) mediated frame shift mutations generated in Setaria cause seedling lethality under high temperatures, demonstrating that fun1 may have an essential function. Several fun1 overexpressors were produced in Setaria virids that displayed enhanced primary branching and increased seed yield under long days and high temperatures. Based on these phenotypes we hypothesize that fun1 plays an essential role during embryonic stress response, and that activation during the floral phase offers a protective effect under non-ideal conditions. The lack of this pathway during the floral phase may result in an absence of stress mediated hormones such as ABA and JA, both of which are required for sex determination in maize.
In service of Objective 3, we explored the establishment of a boundary between the meristem and differentiating lateral organ. In maize (Zea mays), evidence suggests a common gene network functions at boundaries of distinct organs and contributes to pleiotropy between leaf angle and tassel branch number, two agronomic traits. To identify developmental regulators and their sub-networks at the nexus of these two traits, we used regulatory network topologies derived from specific developmental contexts to guide multivariate genome-wide association analyses. In addition to defining network plasticity around core pleiotropic loci, we identified new transcription factors that contribute to small trait effects in canopy architecture, likely through redundancy and dose-dependency with interconnected gene family members. Results demonstrate the power of informing statistical genetics with context-specific gene networks to pinpoint small effect genetic loci and their cis-regulatory components, which can be used to fine-tune plant architecture for crop improvement.
Accomplishments
