Mechanistic Insights into Cytokinin-Regulated Leaf Senescence in Barley: Genotype-Specific Responses in Physiology 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 (<i>Hordeum vulgare</i> L.) cultivars: Carina, Lomerit, and Bursztyn, focusing on responses to exogenous benzyladenine (BA) and inhibition of endogenous CK biosynthesis via the mevalonate (MVA) pathway using lovastatin (LOV). Bursztyn, 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. Lomerit (winter) showed an intermediate phenotype, combining moderate natural resistance to senescence with clear responsiveness to BA. CK application suppressed SAG12 cysteine protease accumulation in all cultivars, serving as a marker of senescence and N remobilization, stabilized photosystem II efficiency, preserved photosynthetic proteins, and alleviated oxidative stress without promoting excessive energy dissipation. 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. Moreover, LOV-based inhibition of the MVA pathway of CK biosynthesis 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 methylerythritol phosphate (MEP) pathway-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.