Cytokinin mediates age-dependent drought response by regulating the removal reactive oxygen species in apple

Plants exhibit age-dependent drought responses during juvenile-to-adult phase transition. However, the specific regulatory molecular mechanisms remain unknown. In this study, juvenile apple plants exhibited better drought tolerance than adult apple plants because of the age-dependent changes in root vitality, cytokinin (CK) levels, and redox status in roots. The study uncovers CYTOKININ OXIDASE/DEHYDROGENASE5 (MdCKX5) as a negative regulator of drought tolerance in apple roots. Silencing of MdCKX5 caused CK accumulation thereby enhancing drought tolerance by increasing root vitality and preventing the accumulation of reactive oxygen species. In contrast, overexpression of MdCKX5 reduced drought tolerance in apple roots. Yeast one-hybrid, dual-luciferase and electrophoretic mobility shift assays revealed that apple transcription factor MdSPL1 directly binds to the promoters of MdCKX5 thereby repressing its expression. Overexpression of MdSPL1 in apple roots increased the CK content thereby enhancing drought tolerance. Further analysis revealed that MdMYB23, a positive CK-responsive gene, interacts with MdSPL1 to alleviate the repression of MdCKX5 expression by MdSPL1. In general, drought stress significantly downregulated MdMYB23, thereby activating the MdSPL1-mediated repression of MdCKX5 transcription by releasing the MdSPL1. This phenomenon led to enhanced drought tolerance in juvenile apple roots by increasing the CK levels. However, adult apple roots lost the capacity to activate this cascade. These findings provide new insights into the molecular mechanisms of CK-mediated age-dependent drought tolerance during the vegetative phase change in apples.