Comparative Phospho-proteomics Analysis Of The Downstream Phosphoproteins Of Arabidopsis MKK9-MPK6 Cascade In Salt Stress Response

High soil salinity induces osmotic stress, ionic stress, and secondary oxidative stress in plant cells, eventually affecting plant growth and development. Therefore, in order to adapt to the environment, plants have evolved molecular mechanisms to perceive and respond to various biotic and abiotic stresses. Protein phosphorylation by kinases is an important post-translational modification that regulates cellular signal transduction in response to changes in the environment. In eukaryotes, activation of mitogen-activated protein kinase (MAPK) signaling cascades is a general mechanism through which external stimuli, including biotic and abiotic stresses, are translated into cellular responses. 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. Previous studies demonstrated that activation of MKK9 enhanced the sensitivity of transgenic seedlings to salt stress. However, it is unclear how MKK9 and downstream MAPKs regulate the response to salt stress.Our studies showed that mkk9 and mpk6 null mutant seedlings are both hyposensitive to salt stress. The fresh shoot weight of mkk9 and mpk6 null mutant seedlings was significantly heavier than that of the wild-type seedlings. Moreover, the result of in-gel kinase assay showed that MPK6 was activated by salt stress, indicating that the MKK9-MPK6 cascade mediated the salt stress response in plants and negatively regulated shoot growth under conditions of salt stress.To identify phosphoproteins downstream of the MKK9-MPK6 cascade in the salt stress response pathway in Arabidopsis, 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 mutants, and wild-type seedlings. Twenty salt-responsive phosphoproteins were identified with a high level of confidence in Arabidopsis wild-type seedlings. Analysis of the subcellular localization of proteins with salt-induced phosphorylation revealed that most were localized in the chloroplast, cell membrane, and nucleus. To identify phosphoproteins regulated by the MKK9-MPK6 cascade during the response to salt stress in plants, we compared Pro-Q stained 2-DE gel images of proteins from mkk9, mpk6 mutants and wild-type seedlings after salt treatment. Phosphorylation of 4 proteins was found to be regulated by activation of the MKK9-MPK6 cascade:Rubisco activase (RCA), plastid ribosomal protein S 1 (PRPS1), plastid division protein (FtsZ2-2), and tortifolia2 (TOR2).To further explore proteins downstream of the MKK9-MPK6 cascade in vivo, we investigated changes in phosphoprotein expression after MKK9 activation in mutant seedlings with active MKK9 (MKK9DD), mutant seedlings with inactive MKK9 (MKK9KR), and MKK9DD/mpk6 crossed seedlings. Twenty-four of the phosphoproteins induced by MKK9 activation were identified by MALDI-TOF MS. Furthermore, RCA phosphorylation was also up-regulated in MKK9DD seedlings after DEX treatment, but up-regulated to a much lesser degree by DEX treatment in MKK9DD/mpk6 seedlings. These results indicate that RCA is a downstream phosphoprotein of the MKK9-MPK6 cascade. In vitro phosphorylation results showed that MPK6 can phosphorylate Ftsz2-2 in vitro, but can not phosphorylate RCA.Taken together, these findings indicate that the MKK9-MPK6 cascade functions in the salt stress response by regulating phosphorylation of RCA, PRPS1, FtsZ2-2, and TOR2, providing novel insight into the MAPK-related mechanisms underlying the salt stress response in plants.