Root-specific cytokinin catabolism promotes Nicotiana root growth and resilience to drought stress

Root-specific CKX overexpression enhances root architecture, improves drought resilience, and upregulates stress-responsive gene expression, thereby promoting  growth, seed yield, and overall stress tolerance in tobacco under drought stress. Drought stress is a global challenge that reduces crop yields, and is intensified by climate change. Cytokinins (CKs) play a crucial role in plant drought responses which are regulated by enzyme activity and genetic factors. To reveal the physiological effects of cytokinin catabolism on the root architecture under water stress conditions, we perform root-specific ectopic overexpression of the cytokinin-degrading CKX gene using WRKY6 gene promoter in tobacco. The overexpression lines show up to 18% increased plant height, > 30% increase in root length, and over twofold root biomass compared to wild-type (WT) plants. Enhanced root architecture in CKX lines is associated with extent of cytokinin catabolism, which improves nutrient uptake and water absorption, thereby supporting better plant growth. The CKX lines also demonstrate improved drought resilience in both soil- and hydroponic-based systems. Under water stress, the overexpression lines display superior growth, timely flowering, more than a 70% increase in seed formation, and over 27% higher seed germination compared to the WT plants. The CKX lines also maintain better electrolyte balance, higher chlorophyll retention, and increased antioxidant activity. Temporal global gene expression profiling under drought stress in tobacco revealed substantial variations in the number of differentially expressed genes (DEGs), with 6,975 differentially expressed genes after 8 h treatment. Among these DEGs, at least 21 genes were consistently upregulated across all drought stress time points. RT-PCR validation of key drought-responsive genes, viz. NtDREB3, NtP5CS, NtERD, NtLEA5, and NtLTP1 in CKX overexpression lines under water deficit suggest their role in drought adaptation. These findings on the hormonal regulation of root growth could be useful in developing drought-resilient crops.