Mechanistic Insights into Cytokinin-Regulated Leaf Senescence in Barley: Genotype-Specific Responses in Physiology, Photosynthesis, and Protein Stability

Cytokinins (CKs) are central regulators of leaf senescence, yet their cultivar-specific functions in cereals remain insufficiently understood. Here, we examined dark-induced senescence (DIS) in three barley (Hordeum vulgare L.) cultivars: Carina, Lomerit, and Bursztyn, focusing on responses to exogenous benzyladenine (BA) and inhibition of endogenous CK biosynthesis via the mevalonate (MVA) pathway. Bur-sztyn, a winter cultivar, displayed a previously uncharacterized stay-green phenotype, characterized by delayed chlorophyll and protein degradation and reduced sensitivity to BA with respect to chlorophyll retention. In contrast, Carina (spring) senesced rapidly but exhibited strong responsiveness to BA. BA application suppressed SAG12 accumulation, stabilized photosystem II efficiency, preserved photosynthetic proteins, and alleviated oxidative stress without promoting excessive energy dissipation. Pho-tosystem I antenna proteins were inherently more stable than PSII components, suggesting distinct degradation trajectories. Although BA only partially mitigated the decline in net CO2 assimilation, it sustained ribulose-1,5-bisphosphate regeneration, supported electron transport, and stabilized Rubisco and Rubisco activase. Lovastatin treatments revealed that endogenous CK contributions to senescence delay were most pronounced in Lomerit, moderate in Bursztyn, and negligible in Carina, indicating genotype-specific reliance on MVA- versus MEP-derived CK pools. Collectively, these findings identify Bursztyn as a novel genetic resource for stay-green traits and demonstrate that BA delays DIS primarily by maintaining photosynthetic integrity and redox balance. The results highlight distinct regulatory networks shaping CK-mediated senescence responses in cereals, with implications for improving stress resilience and yield stability.