Comparative analysis of phytohormone-responsive phosphoproteins in Arabidopsis thaliana using TiO2-phosphopeptide enrichment and mass accuracy precursor alignment.

Protein phosphorylation/dephosphorylation is a central post-translational modification in plant hormone signaling, but little is known about its extent and function. Although pertinent protein kinases and phosphatases have been predicted and identified for a variety of hormone responses, classical biochemical approaches have so far revealed only a few candidate proteins and even fewer phosphorylation sites. Here we performed a global quantitative analysis of the Arabidopsis phosphoproteome in response to a time course of treatments with various plant hormones using phosphopeptide enrichment and subsequent mass accuracy precursor alignment (MAPA). The use of three time points, 1, 3 and 6 h, in combination with five phytohormone treatments, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellic acid (GA), jasmonic acid (JA) and kinetin, resulted in 324,000 precursor ions from 54 LC-Orbitrap-MS analyses quantified and aligned in a data matrix with the dimension of 6000 x 54 using the ProtMax algorithm. To dissect the phytohormone responses, multivariate principal/independent components analysis was performed. In total, 152 phosphopeptides were identified as differentially regulated; these phosphopeptides are involved in a wide variety of signaling pathways. New phosphorylation sites were identified for ABA response element binding factors that showed a specific increase in response to ABA. New phosphorylation sites were also found for RLKs and auxin transporters. We found that different hormones regulate distinct amino acid residues of members of the same protein families. In contrast, tyrosine phosphorylation of the G alpha subunit appeared to be a common response for multiple hormones, demonstrating global cross-talk among hormone signaling pathways.