Comparative transcriptomics reveals genotype-specific mechanisms of saline-alkali tolerance in highland barley

Saline-alkali stress limits crop establishment and productivity, particularly in arid and semi-arid regions. Highland barley ( Hordeum vulgare L. var. nudum Hook. f) is an important cereal crop on the Qinghai-Tibet Plateau, but the molecular mechanisms underlying its tolerance to saline-alkali stress remain poorly understood. To investigate the molecular basis of genotypic variation in saline-alkali tolerance, the tolerant genotype L86 and sensitive genotype L38 were exposed to saline-alkali stress for 24 h. Results indicated that stress inhibited seedling growth in both genotypes, although L86 maintained significantly greater shoot elongation than L38. RNA sequencing identified more differentially expressed genes (DEGs) in L86 than in L38, largely because of the greater number of upregulated genes in L86. Functional enrichment analysis showed that upregulated DEGs in L86 were preferentially involved in glutathione metabolism; stilbenoid, diarylheptanoid, and gingerol biosynthesis; phenylpropanoid and flavonoid biosynthesis; starch and sucrose metabolism; and several amino acid metabolic pathways. Genes encoding glutathione S-transferases, phenylpropanoid- and flavonoid-related enzymes, β-amylase, and trehalose-6-phosphate phosphatase displayed contrasting expression patterns between the two genotypes, suggesting genotype-dependent regulation of antioxidant defense, secondary metabolism, and carbon allocation. In total, 189 transcription factor genes from 35 families were identified. L86 showed broader transcriptional activation, particularly among members of the ERF, MYB, WRKY, bHLH, and NAC families, whereas downregulated transcription factors predominated in L38. In contrast, photosynthesis- and light-harvesting-related genes were commonly downregulated in both genotypes. Quantitative real-time PCR analysis of selected genes generally confirmed the RNA-sequencing results. The greater early-stage tolerance of L86 was associated with broader transcriptional activation and coordinated regulation of antioxidant defense, secondary metabolism, carbohydrate metabolism, amino acid metabolism, and transcription factor networks. These findings provide candidate genes and regulatory pathways for further functional investigation and the genetic improvement of saline-alkali tolerance in highland barley.