IDENTIFICATION OF CONDITION-SPECIFIC ENHANCERS IN SETARIA VIRIDIS

The major objective of this project is to identify and characterize cis-regulatory elements that are active in heat stress responses in Setaria viridis, with an ultimate goal of assessing the feasibility of predicting heat stress tolerance when considering variation within key heat responsive cis-regulatory elements. This goal has been divided into three main aims:Identification of heat stress responsive regulatory elements in SetariaRNA-seq analysis of matched leaf and protoplast tissues in control and heat-stressed conditions to identify differentially expressed genes in heat stress.ATAC-seq analysis of matched leaf and protoplast tissues in control and heat-stressed conditions to identify conditionally accessibly chromatin regions.Identification of conditional enhancers in response to heat stress in Setaria protoplastsPilot experiments and intial optimzation of the conditional STARR-seq assay using a pool of known and predicted heat responsive enhancers and promoters.Large-scale STARR-seq experiment to globally identify conditional enhancers in heat stressed Setaria protoplasts.Candidate conditional enhancer validation using luciferase-based reporter constructs.Predicting plant heat stress tolerance by leveraging natural diveristy in predicted transcription factor binding sites within conditional enhancersOptimization ofexisting maize high-throughput phenotyping assays for a single Setaria genotype.Application of high-throughput phenotyping to 6 geographically separated lines of Setaria to explore effects of variable heat stress responses.Extension of phenotyping regiment to include 5 lines of Setaria with natural variation in previously identified conditional enhancers.