Genome-Wide Signatures of Antioxidant Regulation Reveal Mechanisms of Cold-Stress Memory and Yield Stability in Barley

Cold stress poses a major constraint to barley productivity, particularly in temperate and high-altitude regions. To uncover the genetic and biochemical basis of heritable cold resilience, a diverse panel of 139 barley accessions was evaluated over three successive generations under control and cold stress conditions in field environments. Phenotypic screening revealed significant reductions in all agronomic traits in the first generation, accompanied by elevated activities of all antioxidant enzymes. In subsequent generations, the yield penalty under stress diminished while antioxidant activities remained high, indicating the emergence of transgenerational acclimation. Genome-wide association studies (GWAS) identified 28 significant marker–trait associations across seven traits and three stress-test intervals. Chromosome 2H emerged as a key hub for redox control, harboring a stable locus at 3. 55 Mb repeatedly linked to GSH content in multiple generations, while chromosome 1H carried a recurrent signal at 13. 45 Mb associated with grain yield stability. Candidate genes underlying these regions encode cation/H + antiporter, ABC transporter B family protein, fructokinase-2 gene family, and germin-like protein, suggesting a network that integrates signaling, membrane dynamics, and transcriptional regulation of antioxidant metabolism. Using qRTPCR, gene expression trends illustrate that cold tolerance involves a temporally ordered cascade beginning with ionic and transport homeostasis (CPA and ABCB), followed by metabolic adjustments (FRK2), and culminating in oxidative and structural defense (GLP). The consistent and statistically significant upregulation across these functional categories in the tolerant genotype supports a model in which early activation of membrane transporters primes downstream protective mechanisms, conferring superior acclimation capacity under cold stress. A graphical abstract illustrating antioxidant-mediated transgenerational cold-stress memory in barley