Comparative phosphoproteomic analysis of microsomal fractions of Arabidopsis thaliana and Oryza sativa subjected to high salinity.

Plants respond to salt stress by initiating phosphorylation cascades in their cells. Many key phosphorylation events take place at membranes. Microsomal fractions from 400 mM salt-treated Arabidopsis suspension plants were isolated, followed by trypsin shaving, enrichment using Zirconium ion-charged or TiO(2) magnetic beads, and tandem mass spectrometry analyses for site mapping. A total of 27 phosphorylation sites from 20 Arabidopsis proteins including photosystem II reaction center protein H PsbH were identified. In addition to Arabidopsis, microsomal fractions from shoots of 200 mM salt-treated rice was carried out, followed by trypsin digestion using shaving or tube-gel, and enrichment using Zirconium ion-charged or TiO(2) magnetic beads. This yielded identification of 13 phosphorylation sites from 8 proteins including photosystem II reaction center protein H PsbH. Label-free quantitative analysis suggests that the phosphorylation sites of PsbH were regulated by salt stress in Arabidopsis and rice. Sequence alignment of PsbH phosphorylation sites indicates that Thr-2 and Thr-4 are evolutionarily conserved in plants. Four conserved phosphorylation motifs were predicted, and these suggest that a specific unknown kinase or phosphatase is involved in high-salt stress responses in plants.