Dynamic N-glycoproteome analysis of maize seedling leaves during de-etiolation using Concanavalin A lectin affinity chromatography and a nano-LC-MS/MS-based iTRAQ approach.

KEY MESSAGE: The identification of N -glycosylated proteins with information about changes in the level of N -glycosylation during de-etiolation provides a database that will aid further research on plant N -glycosylation and de-etiolation. N-glycosylation is one of the most prominent and abundant protein post-translational modifications in all eukaryotes and in plants it plays important roles in development, stress tolerance and immune responses. Because light-induced de-etiolation is one of the most dramatic developmental processes known in plants, seedlings undergoing de-etiolation are an excellent model for investigating dynamic proteomic profiles. Here, we present a comprehensive, quantitative N-glycoproteomic profile of maize seedlings undergoing 12 h of de-etiolation obtained using Concanavalin A (Con A) lectin affinity chromatography enrichment coupled with a nano-LC-MS/MS-based iTRAQ approach. In total, 1084 unique N-glycopeptides carrying 909 N-glycosylation sites and corresponding to 609 proteins were identified and quantified, including 186 N-glycosylation sites from 162 proteins that were significantly regulated over the course of the 12 h de-etiolation period. Based on hierarchical clustering analysis, the significantly regulated N-glycopeptides were divided into seven clusters that showed different N-glycosylation patterns during de-etiolation. We found no obvious difference in the enriched MapMan bincode categories for each cluster, and these clustered significantly regulated N-glycoproteins (SRNPs) are enriched in miscellaneous, protein, cell wall and signaling, indicating that although the N-glycosylation regulation patterns of these SRNPs might differ, they are involved in similar biological processes. Overall, this study represents the first large-scale quantitative N-glycoproteome of the model C4 plant, maize, which is one of the most important cereal and biofuel crops. Our results greatly expand the maize N-glycoproteomic database and also shed light on the potential roles of N-glycosylation modification during the greening of maize leaves.

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

In C4 plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by reversible phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dephosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the reversible phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C4 photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C4PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C4PPDK activity in response to changes in lighting.