Adaptation of highland barley to drought stress: from phenotypic analysis to physiological and molecular mechanisms

Drought severely limits crop growth and yield. Highland barley (Hordeum vulgare L.), a naturally stress-tolerant crop, serves as an ideal model for investigating the molecular and physiological mechanisms underlying plant drought adaptation. In this study, two highland barley varieties, Xi-La 22 (XL22) and Zang-Qing 17 (ZQ17), with different degrees of drought tolerance, were used to investigate the mechanism of drought tolerance in highland barley. Compared with XL22, ZQ17 exhibited significant reductions in the biomass and root/shoot ratio, and the increase in ion leakage and malondialdehyde content under drought stress. Natural drought experiment further confirmed that ZQ17 had a higher fatality rate and water loss rate than XL22, indicating that XL22 had higher drought tolerance. Drought induced significant increase in H2O2 and O2.− levels in both barley varieties, especially in ZQ17. Moreover, compared with XL22, more distribution of H2O2 in chloroplasts of ZQ17 might lead to greater degradation of photosynthetic protein complexes (PSI, PSII, LHCII trimers), thereby reducing photosynthetic capacity. The activities of glutathione reductase and glutathione peroxidase and content of reduced glutathione and ascorbate acid were markedly higher in XL22 compared with ZQ17 under drought stress. RNA-seq results showed many genes related to reactive oxygen species (ROS) scavenging, osmotic adjustment (LEA, HSP, aquaporins), hormone signaling and transcription factors (TFs) were specifically up-regulated in XL22. Weighted gene co-expression network analysis (WGCNA) further identified key modules and clarified core hub genes in XL22, mainly including genes in bZIP, AP2/ERF, bHLH transcription factor (TF) families and ABA signaling pathway, which help maintain high ROS scavenging capacity, root/shoot ratio and photosynthetic performance. This study reveals that drought-tolerant highland barley maintains antioxidant activity, photosynthetic complex integrity, hormone signaling, and drought-responsive TF activation, providing insights for barley germplasm screening.