S. Mackenzie, PI, University of Nebraska M. Fromm, coPI, University of Nebraska B. Yu, coPI, University of Nebraska A. Lorenz, coPI, University of Nebraska D. Wang, collaborator, University of Nebraska J.-J. Riethoven, collaborator, University of Nebraska
Mitochondria and chloroplasts serve as bioenergetic focal points of the cell, compartmenting the metabolic underpinnings of cellular function. Likewise, they act as key environmental stress sensors, and are vital to processing plant defense responses. Yet, their integration to a systems model for the plant cell has been complicated by inability to perturb their functions with adequate specificity. This research team has implemented an elegant approach to alter mitochondrial and chloroplast properties that focuses on manipulation of a single nuclear gene, MSH1 (MutS homolog 1). Loss of MSH1 function produces organelle changes to condition distinct plant growth phenotypes without altering genotype of the plant. These include dramatic changes in growth rate, flowering time, reproduction, chloroplast development, and biotic and abiotic stress responses. it is postulated, as the central hypothesis to this research, that the msh1-associated organelle alterations cause heritable, programmed changes to the plant epigenome. The planned investigations capitalize on recent key observations involving MSH1, and implement cross-species comparative analysis in transcript profile-based dissection of emergent phenotypes. The project tests for msh1 effects on small RNA and DNA methylation changes, using Arabidopsis as an initial model for future tests in sorghum and soybean. The collaboration combines expertise in organelle biology, quantitative genetics, epigenetics and next-gen sequence technologies to investigate the influence of organelle signals on the plant epigenome. These studies address major unanswered questions in biology regarding the mode of trans-generational transmission of stress signals and the role of organelles as stress sensing components of the plant cell.
Broader impacts. Since Darwin's time, geneticists have speculated about the underlying basis of hybrid vigor and trans-generational adaptations to stress. While more recent evidence has suggested an epigenetic nature to these phenomena, direct investigation of the process linking bioenergetic or environmental sensing with epigenetic programs in the cell has generally been intractable. The system we investigate may prove invaluable in addressing these processes. Consequently, this research could have important implications for the plant breeding process and for our understanding of plant defense and adaptation. These studies will incorporate high school and undergraduate students in summer research activities, as part of our program to encourage careers in science research.
MSH1 is a gene found in all plant species. Disruption of this gene results in dramatic changes in plant growth, flowering time, and stress responses. Crossing plants that have undergone these growth changes with the original, unmodified line produces progeny that display enhanced growth vigor and yield. During this funding period, we were able to make three important advances in our understanding of these dramatic growth changes. 1. Loss of MSH1 results in reproducible patterns of change in genome-wide cytosine methylation patterns. These changes result in increased methylation of the genome in the msh1 mutant, and hypermethylated pericentromeric CHG occurs in association with changes in growth phenotype, with most severely altered plants showing greatest increases in pericentrometic hypermethylation patterns. Our analysis suggests that it is non-CG methylation that confers the changes in phenotype that are essential to creating plant vigor. 2. We have observed changes in small RNA expression, predominantly in 24-nt RNAs in association with CHH methylation changes. These observations appear to be consistent with observations of graft transmissibility of the msh1 growth effects. Both CHG and CHH methylation changes involve transposable element expression, suggesting that pericentromeric TEs influence gene expression to condition developmental reprogramming. 3. Similar altered growth effects are observed when we down-regulate MSH1 in sorghum using RNA interference. In the case of sorghum, subsequent crossing results in enhanced above-ground biomass and yield, with growth changes subsiding by Generation 5. The consistent and heritable range in phenotypes, graft transmissibility and decline in effect by Generation 5 all support our contention that the growth changes that are created by msh1 are epigenetic. In addition, this work has had broader impacts. We have developed new data analysis strategies for methylome analysis. These include the introduction of information theory and cluster analysis to identify key regions of the genome that may be associated with phenotypic changes. We have applied for patents in association with the methylome information derived, and the outcome of these studies may be of value for commercialization of MSH1 technologies in agriculture. We have trained one postdoctoral research associate, Dr. Yingzhi Xu, who is now working at Dow AgroScience, and two plant breeding student who will complete their degrees in the coming two years.
