Genome-Wide Identification of the Potato MAPKKKK Gene Family Reveals StMAPKKKK1 as a Positive Regulator of Salt and Drought Tolerance

Abstract Mitogen-activated protein kinase kinase kinase kinases (MAP4Ks/MAPKKKKs) reside at the apex of MAPK signalling cascades, yet their roles in potato abiotic stress responses remain unexplored. Here, we identified eight StMAPKKKK genes via genome-wide identification in potato. StMAPKKKK1 protein was strongly induced by salt and drought stress and localized to the plasma membrane and cytoplasm. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and split-luciferase complementation (SLC) experiments show that StMAPKKKK1 physically interacts with StMAPKK2. To assess its function, we generated StMAPKKKK1- overexpressing (OE) and RNA interference (RNAi) lines in potato cultivar 'Atlantic' . OE lines exhibited improved growth, with higher plant height, biomass, and leaf number, whereas RNAi lines were more sensitive than non-transgenic (NT) controls under both salt and drought stress conditions. Higher activities of ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), as well as proline and total chlorophyll contents, along with lower levels of hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) were observed in OE lines. In contrast, RNAi lines showed opposite trends for all above-mentioned parameters. These results indicate alleviated oxidative damage, enhanced osmotic adjustment, and better pigment retention. This study presents the first functional characterization of the StMAPKKKK family in potato and establishes StMAPKKKK1 as a positive regulator of salt and drought tolerance, acting at least partially through interaction with StMAPKK2 and activation of the antioxidant defense system.