Drought poses a significant threat to global crop productivity and food security. In this study, we aimed to elucidate the impact of drought on transcriptional regulation and alternative splicing in barley (Hordeum vulgare), and to determine whether these transcriptomic alterations correlate with changes in hormonal profiles. We hypothesized that drought stress induces extensive reprogramming of gene expression, including alternative splicing events, and that these molecular responses are accompanied by tissue-specific shifts in hormone levels, ultimately underpinning adaptive responses in both leaves and roots. To test this, we performed RNA-seq and comprehensive hormone profiling on leaves and roots sampled at 25 days after planting under both optimal and drought conditions. Our analysis identified over 6,655 differentially expressed genes, with a substantial subset exhibiting differential alternative splicing. In leaves, drought primarily downregulated photosynthesis-related genes while upregulating pathways involved in water stress and abscisic acid (ABA) signaling. In contrast, roots displayed broader metabolic adjustments and significant isoform switching. Hormone analysis revealed marked ABA accumulation, particularly in roots, alongside organ-specific modulation of jasmonates and auxins. A limited assessment of the rhizosphere microbial community revealed low transcript abundance, underscoring the primacy of intrinsic plant responses. Collectively, these findings provide valuable insights into the multilayered adaptive strategies of barley under drought stress.

Cell and Molecular Sciences James Hutton Institute Dundee DD2 5DA Scotland UK

Information and Computational Sciences James Hutton Institute Dundee DD2 5DA Scotland UK

Institute of Biology Biotechnology and Environmental Protection Faculty of Natural Sciences University of Silesia in Katowice Jagiellońska 28 Katowice 40 032 Poland

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany The Czech Academy of Sciences Olomouc Czech Republic

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