MhNF-YA2 modulates drought tolerance by triggering MhHSP70-3 expression in Malus hupehensis

Drought causes serious water deficit and oxidative damage in plants, and the effect can be alleviated by 70-kDa heat shock protein (HSP70). Nuclear factor Y (NF-Y) participates in drought stress response, but its functional mechanism remains largely unclear. Here, a total of 28 NF-Y members were identified in the apple genome, among which the promoter of MdNF-YA2 had a relatively higher number of drought-responsive cis-acting elements. Given that roots play a vital role in sensing drought and are supplied by apple rootstock, we further isolated the homolog of MdNF-YA2 in Malus hupehensis roots, designated MhNF-YA2. This gene was found to be highly expressed in roots, induced by water loss, and localized in both the nucleus and cytoplasm. Genetic experiments using M. hupehensis hairy roots and Arabidopsis revealed that the suppression of MhNF-YA2 resulted in a higher stomatal conductance and transpiration rate, thereby accelerating water loss and withering of leaves. Meanwhile, it also caused higher reactive oxygen species (ROS) and malondialdehyde (MDA) levels in drought-stressed M. hupehensis. Conversely, the overexpression of MhNF-YA2 enhanced the drought tolerance of both M. hupehensis and Arabidopsis. Furthermore, the qRT-PCR, DAP-seq, yeast one-hybrid and dual-luciferase reporter assays showed that MhNF-YA2 directly activated MhHSP70-3 expression by binding to its promoter under drought stress. Overall, our findings suggested that MhNF-YA2 modulates drought resistance via activating MhHSP70-3 expression.