AtMPK1/AtMPK2 Interact with AtVQ25 to Enhance Its Protein Stability and Coordinately Regulate Salicylic Acid-Mediated Leaf Senescence

Abstract

Leaf senescence is a pivotal developmental program in plants, precisely regulated by diverse signals including salicylic acid (SA). The Arabidopsis VQ protein AtVQ25 has been previously characterized as a positive regulator of SA-mediated leaf senescence, functioning through interaction with AtWRKY53 to relieve the transcriptional self-repression of AtWRKY53 at its own promoter. However, the upstream regulatory mechanisms governing AtVQ25 itself remain elusive. In this study, a yeast library screening was performed, and the mitogen-activated protein kinases AtMPK1 and AtMPK2 were identified as interacting partners of AtVQ25. The direct physical interaction was validated by yeast two-hybrid (Y2H), luciferase complementation imaging (LCI), pull-down, and co-immunoprecipitation assays (Co-IP). Furthermore, AtVQ25 was shown to be directly phosphorylated by AtMPK1/AtMPK2, which enhanced its protein stability and retarded its degradation, thereby positively modulating leaf senescence progression. Genetic analyses revealed that the function of AtVQ25 in SA-mediated leaf senescence depends on the functional integrity of AtMPK1/AtMPK2. Collectively, these findings establish AtMPK1/AtMPK2 as upstream interactors of AtVQ25 that coordinate SA-mediated leaf senescence through phosphorylation-dependent enhancement of AtVQ25 protein stability, providing novel insights into the upstream regulatory circuitry of VQ proteins.