Location: Plant Gene Expression Center
2021 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 on LIGULELESS NARROW (LGN) and NARROW ODD DWARF (NOD). 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 carried out coimmunoprecipitation (CoIP) using 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.
Previous work identified a quantitative trait locus (QTL) that rescues the Lgn-R mutant phenotype. We named this gene Sympathy for the Ligule (SOL). SOL is an ortholog of the Arabidopsis gene ENHANCED DISEASE RESISTANCE4 and thus links the Lgn-R phenotype to a response to disease. The phenotype of Lgn-R is dependent on the genotype of SOL. With the B73 version of SOL, Lgn-R mutants are small with narrow leaves and fail to make ears. With the A619 version of SOL, Lgn-R mutants appear near normal. Previous work had also suggested that nod-1 mutants show signs of autoimmunity. Genes that were increased in expression in the nod-1 mutant were involved in stress and immunity.
We evaluated a double mutant between Lgn-R heterozygotes and nod-1 in B73 and A619. In the B73 inbred, the single Lgn-R heterozygote has a visible phenotype with narrow, liguleless leaves and few tassel branches. nod-1 in B73 is bushy with small leaves and no reproductive parts. The double mutant in B73 is an enhancement of nod-1, but not changing the overall mutant architecture. This result suggests that Lgn-R acts as a modifier of nod-1. In the A619 inbred, the single Lgn-R heterozygote is nearly the same as normal siblings in terms of plant height and leaf width. In A619, the nod-1 mutant is a dwarf, but with normal leaves and architecture. The double mutant (nod-1/nod-1; Lgn-R/+) has a synergistic phenotype, being a fraction of the size of either single mutant, highly tillered, and failing to make reproductive parts. The double mutant appears similar to Lgn-R homozygotes. This result suggests that the lack of functional NOD protein overcomes the presence of one copy of functional LGN protein and the rescuing modifier SOL. It also suggests that the Lgn-R rescuing modifier SOL does not rescue nod-1.
We further analyzed these genotypes by carrying out a transcriptomic and a metabolomic analysis in collaboration with an ARS group in Gainesville, Florida, and the University of Wisconsin. We analyzed all four genotypes; the normal sibling, the single mutants, and the double mutant. Five replicas of each were assayed for the metabolomics and three replicas for the transcript analysis. These analyses revealed widespread induction of pathogen defenses and severe disruption of primary and secondary metabolism in the double mutants. The data suggest that LGN and NOD act in overlapping pathways to regulate immune signaling, physiology, and development in 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 which 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. Given that FUN is a conserved protein of unknown function, we are hoping that a combination of genetics, RNA sequencing (RNAseq), protein localization, and identification of protein interactors will help us understand its function. Interestingly, the ortholog in rice is tandemly duplicated which may explain why a rice phenotype has not yet been described.
Genetic crosses suggested that fun is not in the jasmonic acid (JA) pathway as is Tasselseed5 (Ts5) and other feminized tassel mutants. Indeed, fun is not rescued by adding JA unlike Ts5, ts1, and ts2. During this last year, we sent tassels to the Danforth Center for hormone measurements. This analysis included auxin, JA, cytokinin and abscisic acid (ABA). Brassinosteroids and gibberellic acid (GA) were not included in the assays. Only ABA levels were significantly altered and are increased in the mutant. This result may connect to the fact that, according to the maize protein database, FUN protein is very high in developing kernels, a tissue where dormancy is regulated by ABA. We are following up on these results by carrying out protein analysis on endosperm and embryos to confirm the maize protein database analysis. If it is true that FUN accumulates to much higher levels in developing kernels, we will use this tissue for protein work. In order to rule out GA or brassinosteroids, we sent seed to a collaborator who has a system to analyze GA responses in maize and will send tissue to a company for brassinosteroid measurements.
Another method to try and understand the function of FUN is to examine the differentially expressed genes through transcriptome analysis. In a project funded by the National Science Foundation (NSF), the transcriptome of fun was included along with the classic liguleless mutants and the inbred B73 to which all the mutants had been introgressed. We found a number of genes that are connected to ABA and none to brassinosteroids or JA.
We purified our FUN antibody and repeated the immunolocalizations. Now three different researchers have produced the same results with FUN accumulating on the abaxial surface of floral organs. The signal is absent in the null mutants. This localization suggests a long-distance signaling system to regulate the sex of the flower.
In order to determine the proteins that FUN interacts with, we did a yeast two hybrid screen with the Hybrigenics company. 155 interacting proteins were identified that provide tantalizing pathways. We hope to carry out co-immunoprecipitation experiments to determine which of these 155 proteins we should follow up on. If the FUN protein is abundant in developing kernels, we will use that tissue for experiments. Otherwise, we will use three to four week old seedlings.
We analyzed and cloned a third gene that has feminized tassels. We named the dominant mutant Terminal ear2, as it appears similar to the recessive mutant, terminal ear1. Terminal ear2 (Te2) mutants make extra leaves at an increased rate of leaf initiation. The leaves occasionally have missing midribs or missing margins. The tassels are variably feminized, depending on inbred. The mutation does not transmit through the female gametophyte, suggesting a role for the gene in female gametogenesis. We cloned the gene by its chromosomal position and found it encodes an Auxin Response Factor (ARF) transcription factor. The mutation is in a domain thought to be important in ARF-ARF protein binding. With collaborators who are carrying out the biochemistry, the mutation appears to stabilize the protein. Interestingly, we cloned another gene that is also involved in auxin signaling. Mutant leaves also have missing midribs and missing margins but fewer leaves are initiated. This mutation, called Hoja loca1, encodes an AUX-IAA protein. AUX-IAA proteins are negative repressors and bind to ARFs to prevent transcription of target genes. In the presence of auxin, AUX-IAA proteins are degraded and ARF transcription factors initiate transcription. The mutation in Hoja loca1 is in its degron, the domain that targets the protein for degradation. Thus, we have identified an interesting point of balancing regulation; by stabilizing the AUX-IAA protein, leaves fail to initiate, but by stabilizing the ARF, we have an increased rate of leaf initiation.
Accomplishments
1. Maize mutants reveal the role of auxin signaling in leaf initiation. Leaves are the site of photosynthesis, the transfer of energy from the sun to the plant. Increasing the rate of leaf initiation has the potential to reduce carbon dioxide levels, as well as increase useful biomass. ARS scientists in Albany, California, identified a maize allele that increases the rate of leaf initiation such that mutant plants have 30% more leaves at maturity. Knowing the identity of the gene and the mechanism of action provides the potential to use this mutation in breeding of other species.
Review Publications
Hake, S., Kramer, F.J., Lunde, C., Koch, M., Kuhn, B.M., Ruehl, C., Brown, P.J., Hoffman, P., Go'Hre, V., Pauly, M., Rami'Rez, V. 2021. A mixed-linkage (1,3;1,4)-ß-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide. Plant Physiology. 185:1559–1573. https://doi.org/10.1093/plphys/kiab009.
Hake, S.C., Du, Y., Lunde, C., Li, Y., Jackson, D., Zhang, Z. 2021. Gene duplication at the Fascicled ear1 locus controls 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.
