Exploring Gene Expression and Epigenetic Regulation in Sunflower Under Drought Conditions.

This thesis investigates the transcriptional and epigenetic mechanisms contributing to drought responses in sunflower (Helianthus annuus), combining gene expression profiling with the development of an optimized chromatin immunoprecipitation workflow. Two inbred lines with contrasting drought tolerance, DF-AB-2 (tolerant) and AB-OR-8 (sensitive), were subjected to controlled drought and recovery treatment under greenhouse conditions. Six candidate genes involved in abscisic acid (ABA) biosynthesis and signaling were selected through RNA-seq datasets and pathway-based curation for developing markers of drought stress. RT-qPCR revealed strong genotype-dependent expression patterns, with the tolerant line exhibiting higher induction of key ABA-responsive genes under drought. A major methodological outcome of this work was the development of an improved protocol for chromatin immunoprecipitation (ChIP) from frozen sunflower leaves, overcoming the challenges posed by high phenolic content and viscous lysates. The optimized methods enabled efficient chromatin fragmentation and specific immunoprecipitation of H3K4me3-chromatin enriched regions. ChIP-qPCR detected clear enrichment of H3K4me3 at two drought-responsive loci, suggesting promoter-proximal enrichment of this chromatin mark associated to active transcription. Comparison with earlier non-optimized assays demonstrated that the new protocol provided substantially improved sensitivity and specificity. Together, these results show that drought adaptation in sunflower involves coordinated transcriptional activation of ABA-related genes and is associated with H3K4me3 deposition at drought-responsive promoters. The optimized ChIP workflow establishes a methodological foundation for future genome-wide epigenomic studies and supports the development of multi-omics approaches for improving the knowledge on drought resilience in sunflower.