Large-scale analysis of phosphorylated proteins in maize leaf

Abstract

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.

Phosphoproteomic analysis of the non-seed vascular plant model Selaginella moellendorffii

“…Phosphosites with high confidence of localization were categorized into pSer (86.2%), pThr (13.3%), and pTyr (0.5%). The relative distribution of the three phosphorylated residues is consistent with previous reports for different flowering plant species [45,46]. As Ser/Thr kinases are commonly encoded in plant genomes, more frequent Ser and Thr phosphorylation events are expected.…”

“…In addition, phosphopeptide enrichment procedures, such as immobilized metal ion affinity chromatography (IMAC), are necessary to reduce the complexity of proteolyzed lysates for mass spectrometry analysis. IMAC is based on affinity purification through metal complexation with the phosphate group in phosphopeptides [40] and it has been adopted in Phosphoproteomic analysis in different plant systems [41-46]. …”

Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

“…Large-scale phosphoproteomic analyses have been conducted on several plant species, including Arabidopsis thaliana 454647484950, rice4851, Medicago truncatula 5253, soybean ( Glycine max L.)4954, canola ( Brassica napus )49, maize555657 Brachypodium distachyon 58 and wheat2659. However, further research on large-scale mining of the phosphorylation sites in starch biosynthetic enzymes has not been performed.…”

Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity

“…In addition to the phosphorylated Thr-527 found in every spot, phosphorylated Thr-309, Ser-506, and Ser-528 were also identified (Table I). Among the four identified phosphorylation sites, Thr-527 and Ser-528 have been identified in maize (Casati et al, 2005;Bi et al, 2011) and Arabidopsis (Reiland et al, 2009). Phosphorylation at the other two sites has not been reported previously.…”