Qualitative analysis of N-linked glycoproteome in senescent flag leaf of rice

Abstract

N-Glycosylation is one of the most ubiquitous protein modifications. However, the qualitative analysis of N-glycoproteome during leaf senescence has not been studied yet. In this study, N-glycoproteins were identified in senescent flag leaf of rice through hydrophilic interaction chromatography enrichment and liquid chromatography–tandem mass spectrometry strategy. A total of 381 N-glycoproteins and 479 N-glycosylated sites were identified. In which, 40% of glycoproteins contained signal peptide and 40% had transmembrane domain. Meanwhile, α helix and coil/loop accounted for 1.5% and 98.5% of all N-glycosylated sites. Motif-X analysis suggested that [NxT], [NxS] and [NS] were significantly conserved and enriched. During leaf senescence, proteolysis-related proteins were predominately N-glycosylated by gene ontology analysis. Meanwhile, most identified glycoproteins were enriched in photosynthesis and N-glycan biosynthesis pathways by Kyoto Encyclopedia of Genes and Genomes analysis. Protein–protein interaction analysis showed that N-glycoproteins formed interaction networks to function in the metabolism process of energy substances (carbohydrates, lipids and amino acids), the proteolysis process, and the protein glycosylation process. Among 381 N-glycoproteins, 183 proteins were involved in various and famous senescence-related biological processes including energy substance metabolism (70), proteolysis (44), photosynthesis (17), protein glycosylation (24), reactive oxygen species scavenging (14), transcriptional regulation (6), senescence-associated genes (2) and hormone response (6), suggesting the important role of protein N-glycosylation in leaf senescence. Additionally, ten glycoproteins were found to have been well-studied in senescent process of rice. The results provided a novel insight into the significant involvement of protein N-glycosylation in leaf senescence.