Transcriptomic and functional characterization reveals CsHAK5;3 as a key player in K⁺ homeostasis in grafted...

Grafting can improve the salt tolerance of many crops. However, critical genes in scions responsive to rootstock under salt stress remain a mystery. We found that pumpkin rootstock decreased the content of Na⁺ by 70.24 %, increased the content of K⁺ by 25.9 %, and increased the K⁺/Na⁺ ratio by 366.0 % in cucumber scion leaves. RNA-seq analysis showed that ion transport-related genes were the key genes involved in salt stress tolerance in grafted cucumber. The identification and analysis of the expression of K⁺ transporter proteins in cucumber and pumpkin revealed six and five HAK5 members, respectively. The expression of CsHAK5;3 in cucumber was elevated in different graft combinations under salt stress and most notably in cucumber scion/pumpkin rootstock. CsHAK5;3 was localized to the plasma membrane, and a yeast complementation assay revealed that it can transport K⁺. CsHAK5;3 knockout in hairy root mutants decreased the K⁺ content of leaves (45.6 %) and roots (50.3 %), increased the Na⁺ content of leaves (29.3 %) and roots (34.8 %), and decreased the K⁺/Na⁺ ratio of the leaves (57.9 %) and roots (62.9 %) in cucumber. However, CsHAK5;3 overexpression in hairy roots increased the K⁺ content of the leaves (31.2 %) and roots (38.3 %), decreased the Na⁺ content of leaves (17.2 %) and roots (14.3 %), and increased the K⁺/Na⁺ ratio of leaves (58.9 %) and roots (61.6 %) in cucumber. In conclusion, CsHAK5;3 in cucumber can mediate K⁺ transport and is one of the key target pumpkin genes that enhance salt tolerance of cucumber grafted.