Local adaptation shapes drought responses in Lithuanian barley: An integrated targeted and transcriptomic analysis

Breeding of new and improved drought-tolerant varieties remains the most effective strategy for mitigating the impact of drought on agriculture, though it is hampered by the complexity of drought-response genetics within and between species. Here, we evaluated previously unexplored Lithuanian barley ( Hordeum vulgare L.) cultivars for PEG-6000–induced drought tolerance and interrogated underlying mechanisms using targeted candidate-gene assays and whole-transcriptome sequencing. Moderate drought stress screening based on free proline content and leaf relative water content (RWC) revealed that the cultivars ‘Ema DS’ and ‘Rusnė DS’ were the most phenotypically contrasting among the Lithuanian varieties. Targeted candidate gene expression assays revealed that the expression patterns of drought-responsive hub transcription factors previously proposed based on non-European cultivars (e.g., HvWRKY42 , HvCBF6 , HvDRF1.3 ) could not explain the physiological and morphological phenotypic differences observed among the Lithuanian barley cultivars. Transcriptome sequencing under severe drought conditions further confirmed that these hub genes did not play a central role in drought tolerance in Lithuanian barley. Instead, we identified 185 new significantly differentially regulated genes, 23 of which lack both annotation and prior study in abiotic-stress contexts, suggesting locally evolved mechanisms of drought adaptation. Notably, transcriptomic analysis revealed that the drought-tolerant cultivar 'Rusnė DS' and the drought-sensitive cultivar 'Ema DS' adopted contrasting strategies under severe stress: 'Rusnė DS' was predominantly enriched in genes related to cell wall remodeling and carbohydrate hydrolysis, whereas 'Ema DS' preferentially activated kinase signaling and glycolytic processes. Examination of the long-term field yield-climate relationship confirmed the biological relevance of these laboratory-defined findings.