RNA-seq of hvsnac1.d (D) during control (C) and drought stress (D)

Drought stress can damage crop growth and lead to a decline in yield, thereby affecting food security, especially in regions vulnerable to climate change. SNAC1 (Stress-responsive NAC1), the NAC transcription factor family member, plays a crucial role in stomatal movement regulation. Effective regulation of stomatal movement is essential for protecting plants from water loss during adverse conditions. Our hypothesis revolves around altering HvSNAC1 activity by introducing a point mutation in its encoding gene, thereby influencing stomatal dynamics in barley. Two TILLING mutants, each harboring missense mutations in the NAC domain, exhibited higher stomatal density after drought stress compared to the parent cultivar 'Sebastian'. These mutants also demonstrated distinct patterns of ABA-induced stomatal movement compared to the 'Sebastian'. To delve deeper, we conducted a comprehensive analysis of the transcriptomes of these mutants and the parent cultivar 'Sebastian' under both optimal watering conditions and ten days of drought stress treatment. We identified differentially expressed genes (DEGs) between the mutants and 'Sebastian' under control and drought conditions. Furthermore, we pinpointed DEGs specifically expressed in both mutants under drought conditions. Our experiments revealed that the cis-regulatory motif CACG, previously identified in Arabidopsis and rice, is recognized by HvSNAC1 in vitro. Enrichment analysis led to the identification of the cell wall organization category and potential target genes, such as HvEXPA8 (Expansin 8), HvXTH (Xyloglucan endotransglucosylase/hydrolase), and HvPAE9 (Pectin acetylesterase 9), suggesting their regulation by HvSNAC1. These findings suggest that HvSNAC1 may play a role in regulating genes associated with stomatal density, size, and reopening.