Title : A novel functional stay-green barley cultivar reveals genotype-specific cytokinin dependence during leaf senescence
Abstract:
Leaf senescence determines the duration of photosynthetic activity and constitutes a major factor limiting carbon assimilation and yield stability in cereal crops. Although cytokinins are established antagonists of senescence, the extent to which their protective effects depend on genotype and endogenous cytokinin metabolism remains poorly understood. Here, we combined physiological and molecular analyses to dissect cytokinin-mediated regulation of dark-induced senescence in three barley (hordeum vulgare l) cultivars exhibiting contrasting senescence behavior.The winter cultivar Bursztyn displayed a previously uncharacterized functional stay-green phenotype, retaining chlorophyll and protein content while preserving photosynthetic competence during senescence. In contrast, the spring cultivar Carina underwent rapid chlorophyll loss and photosynthetic decline but exhibited pronounced responsiveness to exogenous cytokinin treatment, whereas Lomerit showed an intermediate phenotype. Application of Benzyladenine (BA) attenuated senescence-associated deterioration across all cultivars by maintaining PSII photochemical efficiency, sustaining electron transport, and suppressing excessive non-regulated energy dissipation. Importantly, delayed senescence was accompanied by preservation of photosynthetic machinery integrity, including rubisco, rubisco activase, PsbA, PsaB, and light-harvesting antenna proteins. Despite only partial recovery of net CO2 assimilation, BA maintained ribulose-1,5-bisphosphate regeneration capacity and stabilized photosynthetic performance. Furthermore, cytokinin treatment completely suppressed accumulation of the senescence-associated cysteine protease SAG12, indicating inhibition of proteolytic processes linked to nitrogen remobilization. Comparative analysis of endogenous cytokinin biosynthesis using lovastatin-mediated inhibition of the mevalonate pathway prior the onset of senescence revealed marked genotype-specific differences in cytokinin dependence. Endogenous cytokinin pools contributed most strongly to delayed senescence in Lomerit, moderately in Bursztyn, and only marginally in Carina, suggesting differential reliance on mevalonate- and methylerythritol phosphate-derived cytokinin biosynthesis pathways. Collectively, our findings demonstrate that cytokinin-mediated stay-green phenotypes are underpinned by maintenance of photosynthetic integrity rather than chlorophyll retention alone. The identification of Bursztyn as a novel source of functional stay-green traits in barley highlights distinct regulatory networks governing senescence progression and provides a promising framework for developing climate-resilient cereal cultivars with enhanced photosynthetic longevity and improved yield stability.
