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.

Quantitative Proteomic Analyses Identify ABA-Related Proteins and Signal Pathways in Maize Leaves under Drought Conditions

“…As described previously (Hu et al, 2015), the vp5 mutant has reduced amounts of ABA because of a deficiency in ABA biosynthesis (Robichaud et al, 1980). Homozygous recessive kernels ( vp5 / vp5 ) lack carotenoids, causing white endosperm and embryos, which are easily distinguishable from the yellow wild-type kernels ( Vp5 /–).…”

“…According to our previous description (Hu et al, 2015), Vp5 and vp5 seeds were germinated on moistened filter paper after being surface-sterilized for 10 min in 2% hypochlorite and then rinsed in distilled water. After germination for 2 days, both vp5 and Vp5 seedlings were cultured in Hoagland's nutrient solution in a light chamber (day 28°C/night 22°C, relative humidity 75%) under 400 μmol/m 2 /s photosynthetically active radiation with a 14/10 h (day/night) cycle.…”

“…According to our previous description (Hu et al, 2015), total proteins from the second newly expanded leaf of the maize seedlings were extracted according to the following procedure. Approximately 0.5 g of fresh leaves from each biological replicate were ground into a fine power in liquid N 2 in a mortar and further ground in 4 ml SDS buffer (30% sucrose, 2% SDS, 100 mM Tris-HCl, pH 8.0, 50 mM EDTA-Na 2 , 20 mM DTT) and 4 ml phenol (Tris-buffered, pH 8.0) in a 10 ml tube, followed by the addition of 1 mM phenylmethanesulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Cocktail (one tablet/10 ml; Roche, Basel, Switzerland) to inhibit protease and phosphatase activity, respectively.…”

“…Maize mutant viviparous - 5 ( vp5 ) is deficient in ABA biosynthesis and suffers photo-bleaching of leaves under normal light conditions (Robichaud et al, 1980; Hable et al, 1998), and has a much reduced ABA content in maize plants compared with wild-type Vp5 plants (Hu et al, 2015). Thus, the mutant vp5 and its wild-type Vp5 are ideal materials in which to identify key drought-response proteins regulated by ABA in maize plants.…”

Proteomic Analysis Reveals a New Benefit of Periodic Mechanical Stress on Chondrocytes

“…The following criteria were required to consider a protein for further phospho RS analysis: phospho RS score >50 and phospho RS site probabilities >75% (Protein Discoverer 1.4). Changes with either a ratio >1.20 or <0.83 and p <0.05 were considered significant in proteomic analysis [22-25]. …”