Quantitative iTRAQ-based proteomic analysis of phosphoproteins and ABA-regulated phosphoproteins in maize leaves under osmotic stress

Abstract

Abscisic acid (ABA) regulates various developmental processes and stress responses in plants. Protein phosphorylation/dephosphorylation is a central post-translational modification (PTM) in ABA signaling. However, the phosphoproteins regulated by ABA under osmotic stress remain unknown in maize. In this study, maize mutant vp5 (deficient in ABA biosynthesis) and wild-type Vp5 were used to identify leaf phosphoproteins regulated by ABA under osmotic stress. Up to 4052 phosphopeptides, corresponding to 3017 phosphoproteins, were identified by Multiplex run iTRAQ-based quantitative proteomic and LC-MS/MS methods. The 4052 phosphopeptides contained 5723 non-redundant phosphosites; 512 phosphopeptides (379 in Vp5, 133 in vp5) displayed at least a 1.5-fold change of phosphorylation level under osmotic stress, of which 40 shared common in both genotypes and were differentially regulated by ABA. Comparing the signaling pathways involved in vp5 response to osmotic stress and those that in Vp5, indicated that ABA played a vital role in regulating these pathways related to mRNA synthesis, protein synthesis and photosynthesis. Our results provide a comprehensive dataset of phosphopeptides and phosphorylation sites regulated by ABA in maize adaptation to osmotic stress. This will be helpful to elucidate the ABA-mediate mechanism of maize endurance to drought by triggering phosphorylation or dephosphorylation cascades.

Figure 1. ABA content in maize ABA-deficient mutant vp5 and wild-type Vp5 leaves under normal conditions (control) or 8 h osmotic stress (OS).

Values are means ± SE (n = 5).

Figure 2. iTRAQ 4-plex labeling and LC MS/MS workflow of identifying phosphorous proteins in leaves of maize ABA mutant vp5 and wild-type Vp5 seedlings under osmotic stress (OS).

Figure 3. Z-scores frequency distribution of differential peptides in maize wild type Vp5 and mutant vp5 under osmotic stress.

iTRAQ ratios between osmotic stress (OS) and controls for each run were converted to z-scores to normalize the data. Positive z-score values represent proteins up-regulated by OS and negative values represent proteins down-regulated by OS. Z-scores between −0.9 and 0.9 indicates proteins not significantly altered, between ±0.9 and 1.96 moderately altered, and ≥1.96 and ≤−1.96 significantly altered ≥2-fold during osmotic stress (>95% confidence).

Figure 4. The distribution of differentially phosphorylated proteins in maize response to osmotic stress.

The 160 proteins identified were classified according to their known or predicted cellular component, molecular function, biological process, and signaling pathway. Left, Vp5; right, vp5.

Figure 5. Venn diagram showing the number of proteins with significant changes of phosphorylation levels in maize vp5 and Vp5 leaves exposed to osmotic stress.