Suppressing GhCKX optimizes the trade-off between photosynthesis and water use efficiency in cotton mature leaves...

Studies on drought stress physiology have primarily focused on the newest fully expanded leaves, in which drought reduces cytokinin (CTK) content, thereby disrupting water balance and photosynthesis. Mature (old) leaves usually exhibit greater sensitivity to drought than the newest fully expanded ones. However, the impact of higher endogenous CTK levels on photosynthetic rate (A_N) and water use efficiency (WUE) in drought-stressed mature leaves remains underexplored. To address this, a drought experiment was performed using wild-type (WT) cotton and two CYTOKININ DEHYDROGENASE (GhCKX) suppression lines with elevated CTK levels. Here, suppressing GhCKX optimized the trade-off between A_N and intrinsic WUE (iWUE) in mature leaves during drought, since not only higher A_N but also higher iWUE were observed in transgenic lines than the WT under drought. This optimization was driven by shifts in the relationships of mesophyll conductance (g_m) and maximum carboxylation rate (V_cmax) to stomatal conductance (g_s), specifically higher g_m/g_s and V_cmax/g_s ratios, resulting from larger enhancements in g_m and V_cmax than in g_s, caused by up-regulated CTK. Moreover, higher g_m in drought-stressed transgenic lines than drought-stressed WT resulted from thinner cell wall, shorter distance between adjacent chloroplasts, and larger ratio of chloroplast area to mesophyll area facing the intercellular airspace; and their superior V_cmax was attributed to increased Rubisco content and initial Rubisco activity. This study reveals that promoting endogenous CTK simultaneously enhances A_N and iWUE in mature leaves under drought via the coordination of photosynthetic constraints, offering a sustainable strategy to improve the drought resistance of crops.