The GhMAP3K62-GhMKK16-GhMPK32 kinase cascade regulates drought tolerance by activating GhEDT1-mediated ABA accumulation in cotton

INTRODUCTION: Drought is the principal abiotic stress that severely impacts cotton (Gossypium hirsutum) growth and productivity. Upon sensing drought, plants activate stress-related signal transduction pathways, including ABA signal and mitogen-activated protein kinase (MAPK) cascade. However, as the key components with the fewest members in the MAPK cascade, the function and regulation of GhMKKs need to be elucidated. In addition, the relationship between MAPK module and the ABA core signaling pathway remains incompletely understood. OBJECTIVE: Here we aim to elucidate the molecular mechanism of cotton response to drought, with a focus on mitogen-activated protein kinase (MAPK) cascades activating ABA signaling. METHODS: Biochemical, molecular and genetic analysis were used to study the GhMAP3K62-GhMKK16-GhMPK32-GhEDT1 pathway genes. RESULTS: A nucleus- and membrane-localized MAPK cascade pathway GhMAP3K62-GhMKK16-GhMPK32, which targets and phosphorylates the nuclear-localized transcription factor GhEDT1, to activate downstream GhNCED3 to mediate ABA-induced stomatal closure and drought response was characterized in cotton. Overexpression of GhMKK16 promotes ABA accumulation, and enhances drought tolerance via regulating stomatal closure under drought stress. Conversely, RNAi-mediated knockdown of GhMKK16 expression inhibits ABA accumulation, and reduces drought tolerance. Virus-induced gene silencing (VIGS)-mediated knockdown of either GhMAP3K62, GhMPK32 or GhEDT1 expression represses ABA accumulation and reduces drought tolerance through inhibiting stomatal closure. Expression knockdown of GhMPK32 or GhEDT1 in GhMKK16-overexpressing cotton reinstates ABA content and stomatal opening-dependent drought sensitivity to wild type levels. GhEDT1 could bind to the HD boxes in the promoter of GhNCED3 to activate its expression, resulting in ABA accumulation. We propose that the MAPK cascade GhMAP3K62-GhMKK16-GhMPK32 pathway functions on drought response through ABA-dependent stomatal movement in cotton.