Location: Plant Gene Expression Center
2019 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, research continued in Albany, California, on Liguleless narrow (LGN) and narrow odd dwarf (NOD). Researchers published a paper in The Plant Cell on the modifier of LGN, which we call Sympathy for the ligule (SOL). Following up on the discovery that LGN was found in a complex with NOD, we confirmed this interaction with additional tests. We evaluated a double mutant between Lgn-R and nod in the A619 inbred background which provides mild single mutant phenotypes. These were grown for a couple of seasons in the greenhouse and field. The double mutant resembles nod in the severe inbred and suggests that a modifier to nod is likely activated by the Lgn-R mutation. RNA was collected from individuals of each genotype (wild type, nod single mutant, Lgn-R single mutant, double mutant) and RNAseq analysis carried out. We are now analyzing the data.
In conjunction with the RNAseq analysis, we also carried out Genotype by sequencing (GBS) analysis of nod mutants that segregate for different inbred backgrounds. In this experiment we crossed nod in A619 to nod in B73 and selfed the F1. Crosses were also made with nod in Mo17 to nod in B73. The F2 were grown last summer and scored for height, leaf width, and tiller number. DNA preps were sent to a company for genotyping. We have the data back and are presently analyzing it.
Both Lgn-R and nod have phenotypes that are reminiscent of auto-immunity. The phosphoproteome of Lgn-R and RNAseq of nod support this hypothesis. To determine if they are more resistant to pathogens, we sent seed to an ARS scientist in North Carolina to grow in the field. The plants were infected with Southern Leaf Blight. The mutants we sent were backcrossed into the Mo17 inbred, as our mutants in the B73 inbred are very severe and die in hot temperatures. Unfortunately, Mo17 is resistant to Southern Leaf Blight and B73 is sensitive. Thus, our material in Mo17 showed no sign of resistance above the control plants. Lgn-R was assayed in B73 with the Mo17 SOL modifier. It is possible these plants are more resistant, but Lgn-R mutants mature faster than wild type, confusing senescence with disease susceptibility.
For Objective 2, we published a manuscript in Communications Biology describing the cloning of Tasselseed5. A University of California Berkeley graduate student carrying out research in the ARS lab finished his thesis on the other mutant under study, feminized upright narrow (fun). The thesis focuses on double mutant analysis which suggests that fun operates in the brassinosteroid pathway and not the jasmonic acid pathway. We will bulk seed this summer to send for hormone analysis.
To understand in more detail the role of FUN, we developed an antibody. With the help of a former postdoctoral scholar we were able to purify the antibody such that a single band was detected in wild type and not in the mutant. The band is of the correct size. This experiment confirms that indeed we have identified the correct gene. The antibody will be used for immunolocalizations in shoots and tassels to determine if there are specific tissues where FUN accumulates. Other experiments will identify interacting partners through co-immunoprecipitation. Proteins identified through this method will be compared with the list of interactors obtained through yeast, two hybrid experiments that already took place in 2018.
For Objective 3, we carried out a number of RNAseq experiments with collaborators. One set of experiments examined 1 millimeter tassels of eight genotypes. These tassels were just beginning to initiate branches. Another experiment used laser capture to isolate the cells in the axil of the branch in 4 genotypes. A postdoctoral scholar in the lab, funded by his own National Science Foundation postdoctoral fellowship, carried out RNAseq in developing maize tassels and developing sorghum panicles.
Accomplishments
1. Maize inbreds vary in their immunity response. Genotype and environment determine crop productivity. By growing the maize mutant, Liguleless narrow-R, in different environments and different genetic backgrounds, a modifier was identified that rescues Lgn-R from heat-induced fatality. ARS researchers in Albany, California, in collaboration with scientists at University of California (UC) Berkeley and UC Davis, identified the modifier as a homolog of an Arabidopsis gene called ENHANCED DISEASE RESISTANCE4 (EDR4). Similar to EDR4, the maize modifier is increased in RNA expression levels when leaf tissue is treated with pathogen-associated molecular patterns (PAMPs) such as fungal chitin or bacterial flagellin. Finding a modifier that functions in immunity supports the hypothesis that LGN plays a role in immunity in addition to its role in development.
2. Bioinformatic tools to identify genes in sorghum panicle development. Sorghum is an important crop for animal fodder, human food and biofuels, however, it does not have the genetic resources found in its close relative, maize. ARS scientists at Albany, California, in collaboration with University of California, Berkeley scientists, prepared RNA transcriptome libraries from individual sorghum panicles and individual maize tassels over a time course of development. Many of the genes identified by mutants in maize have orthologs in sorghum that follow a similar expression profile. A database tool developed by the project can quickly identify the time of expression for any maize or sorghum genes and provide a developmental stage for libraries of unknown developmental timing.
Review Publications
Lunde, C., Kimberlin, A., Leiboff, S., Koo, A., Hake, S.C. 2019. Tasselseed5 overexpresses a wound-inducible enzyme, ZmCYOP94B1, that affects jasmonate catabolism, sex determination, and plant architecture in maize. Communications Biology. 2:114. https://doi.org/10.1038/s42003-019-0354-1.
Richardson, A., Hake, S.C. 2108. Drawing a line: grasses and boundaries. Plants. 8(1):4. https://doi.org/10.3390/plants8010004.
Anderson, A.A., St. Aubin, B., Abraham-Juarez, M.J., Leiboff, S., Shen, Z., Briggs, S.P., Brunkard, J.O., Hake, S.C. 2019. The second site modifier, Sympathy for the ligule, encodes a homolog of Arabidopsis enhanced disease resistance4 and rescues the liguleless narrow maize mutant. The Plant Cell. 31:1829-1844. https://doi.org/10.1105/tpc.18.00840.
