Group-C/S1 bZIP heterodimers regulate MdIPT5b to negatively modulate drought tolerance in apple species

Cytokinins play a central role in delaying senescence, reducing oxidative damage and maintaining plant growth during drought. This study showed that the ectopic expression of ProRE-deleted MdIPT5b, a key enzyme involved in cytokinin metabolism, increased the drought tolerance of transgenic Malus domestica (apple) callus and Solanum lycopersicum (tomato) seedlings by maintaining cytokinin homeostasis, and thus maintaining redox balance. Under restricted watering regimes, the yields of transgenic tomato plants were enhanced. Heterodimers of C/S1 bZIP are involved in the cytokinin-mediated drought response. The heterodimers bind the ProRE of MdIPT5b promoter, thus directly suppressing gene transcription. Single C/S1 bZIP members could not independently function as suppressors. However, specific paired members (heterodimers of MdbZIP80 with MdbZIP2 or with MdbZIP39) effectively suppressed transcription. The α-helical structure is essential for the heterodimerization of C/S1 bZIP members and for synergistic transcriptional suppression. As negative regulators of drought tolerance, suppressing either MdbZIP2 or MdbZIP39 alone does not improve the expression of MdIPT5b and did not increase the drought tolerance of transgenic apple callus. However, this could be achieved when they were co-suppressed. The suppression of MdbZIP80 alone could improve MdIPT5b expression and increase the drought tolerance of transgenic apple callus. However, these effects were reversed in response to the cosuppression of MdbZIP80 and MdIPT5b. Similar results were also observed during delayed dark-induced senescence in apple leaves. In conclusion, the apple C/S1 bZIP network (involving MdbZIP2, MdbZIP39 and MdbZIP80) directly suppressed the expression of MdIPT5b, thus negatively modulating drought tolerance and dark-induced senescence in a functionally redundant manner.