Comparative phospho-proteomics analysis of salt-responsive phosphoproteins regulated by the MKK9-MPK6 cascade in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades are involved in the salt stress response in plants. However, the identities of specific proteins operating downstream of MAPKs in the salt stress response remain unclear. Our studies showed that mkk9 and mpk6 null mutant seedlings are hyposensitive to salt stress. Moreover, we showed that MPK6 was activated by salt stress, indicating that the MKK9-MPK6 cascade mediated the salt stress response in Arabidopsis. To identify phosphoproteins downstream of the MKK9-MPK6 cascade in the salt stress response pathway, we performed two-dimensional electrophoresis (2-DE) with Pro-Q phosphoprotein staining and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to identify phosphoproteins induced by salt treatment in mkk9, mpk6, and wild-type seedlings. Phosphorylation of 4 proteins, including Rubisco activase (RCA), plastid ribosomal protein S 1 (PRPS1), plastid division protein (FtsZ2-2), and tortifolia2 (TOR2), was found to be regulated by activation of MKK9-MPK6 cascade. Further Phospho-proteomics analysis of MKK9DD mutant seedlings revealed that RCA phosphorylation was up-regulated as a result of MKK9 activation. The finding that the MKK9-MPK6 cascade functions in the salt stress response by regulating phosphorylation of RCA, PRPS1, FtsZ2-2, and TOR2, provides a novel insight into the MAPK-related mechanisms underlying the salt stress response in plants.