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How developmentally male gametophyte is perturbed by heat stress has remained largely unexplored. The long-term goal of our research is to elucidate the molecular events that are responsible for the reduced yield during pre-fertilization events under heat stress. Recently, we identified two critical pathways affected by heat stress during tetrad stage of pollen development resulting in reduced pollen germination and yield. Both pathways are required for normal pollen development and/or pollen viability. Genes of both pathways are potential candidates for stress resilience. However, not all pollen developmental stages respond in the same way when impacted by heat stress. Because male gametophyte development events are largely conserved among cereals, an opportunity exists to translate this knowledge to other economically important crops. The goal of the proposed work is to fill a significant gap in our understanding on how heat stress perturbs developmentally each stage of pollen formation and ultimately affects productivity in maize. Therefore, it is necessary to elucidate the most critical stages of plant development and reproduction, to understand the underlying molecular stress responses, and to use the knowledge generated with the aim of developing heat stress tolerant crops.The proposed work aims to identify the genetic factors that need to be tightly regulated during normal pollen development but are perturbed when plants experience heat stress. What role do epigenetic factors play in the regulation of these heat-sensitive genes? Elucidating the molecular basis of pollen development under stress will help frame the model needed for improving the resilience of pollen viability and hence yield when plants experience heat stress during this highly sensitive developmental window.Objective 1: Elucidate how heat stress affects pollen development gene networks using transcriptome analysis. 1.1. Transcriptome analysis to identify heat stress responsive pollen gene networks in maize. 1.2. Integration of time series analysis and co-expression-based predictions to identify pollen gene networks that are sensitive to heat stress during pollen microsporogenesis and microgametogenesis. Objective 2: Integrate transcriptional regulation with genome-wide bisulfite sequencing and histone methylation (H3K27me3) analysis of each pollen developmental stage to identify critical genes and networks that determine stress adaptability. 2.1. Perform whole genome bisulfite analysis in each pollen developmental stage in maize under optimal and heat stress conditions to correlate with the gene expression dataset. 2.2. Perform whole genome analysis of H3K27me3 modifications in developing pollen grains under optimal and stressful conditions and integrate the data with transcriptome and bisulfite dataset. Objective 3: Functionally characterize a set of candidate genes that are misregulated during heat stress and contribute to stress resilience. 3.1. Determine the role of a set of candidate maize genes in controlling pollen development and fertilization using CRISPR/Cas9 and overexpression lines

Begcy Padilla, K.; Barbazuk, WI, BR.
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