Phosphoproteomic And Acetylproteomic Analysis Of Terminal Buds Of Populus Simonii×P. Nigra During Dormancy

Although there has been considerable progress made towards understanding the molecular mechanisms of bud dormancy, the roles of protein phosphorylation in the process of dormancy regulation in woody plants remain unclear. We used mass spectrometry combined with TiO2phosphopeptide-enrichment strategies to investigate the phosphoproteome of dormant terminal buds (DTBs) in poplar(Populus simonii×P. nigra). There were161unique phosphorylated sites in161phosphopeptides from151proteins;141proteins have orthologs in Arabidopsis, and10proteins are unique to poplar. Only34sites in proteins in poplar did not match well with the equivalent phosphorylation sites of their orthologs in Arabidopsis, indicating that regulatory mechanisms are well conserved between poplar and Arabidopsis. Further functional classifications showed that most of these phosphoproteins were involved in binding and catalytic activity. Extraction of the phosphorylation motif using Motif-X indicated that proline-directed kinases are a major kinase group involved in protein phosphorylation in dormant poplar tissues. This study provides evidence about the significance of protein phosphorylation during dormancy, and will be useful for similar studies on other woody plants.To better understand the role that reversible phosphorylation plays in woody plant ribosomal P-protein function, we initiated a phosphoproteomic investigation of P-proteins from Populus dormant terminal buds. Using gel-free (in-solution) protein digestion and phosphopeptide enrichment combined with a nanoUPLC-ESI-MS/MS strategy, we identified six phosphorylation sites on eight P-proteins from Populus dormant terminal buds. Among these, six Ser sites and one Thr site were identified in the highly conserved C-terminal region of eight P-proteins of various P-protein subfamilies, including two P0, two P1, three P2, and one P3protein. Among these, the Thr site was shown to be novel and has not been identified in any other organisms. Sequence analysis indicated that the phosphothreonine sites identified in the C-terminus of Ptr RPP2A exclusively occurred in woody species of Populus etc. The identified phosphopeptides shared a common phosphorylation motif of (S/T)XX(D/E) and may be phosphorylated in vivo by casein kinase2as suggested by using Scansite analysis. Furthermore, phylogenetic analysis suggested that divergence of P2also occurred in Populus, including Type Ⅰ and Type Ⅱ. To the best of our knowledge, this is the first systematic phosphoproteomic and phylogenetic analysis of P-proteins in woody plants, the results of which will provide a wealth of resources for future understanding and unraveling of the regulatory mechanisms of Populus P-protein phosphorylation during the maintenance of dormancy.The N-terminal protein processing mechanism (NPM) represents a common protein modification process of eukaryotes, and involves the co-translational processes of N-terminal Met excision (NME) and N-terminal acetylation (Nα-acetylation). To reveal the NPM in poplar, we investigated the Nα-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TO2enrichment of acetylated peptides. We identified58N-terminally acetylated (Nα-acetylated) proteins. Most proteins (47,>81%) are subjected to Nα-acetylation following the N-terminal removal of Met, indicating that Nα-acetylation and NME could represent a common NPM of poplar proteins. Based on N-terminal features of these acetylated proteins, the NME in poplar was found to be similar to the NME process observed in yeast and humans. The poplar genome encodes cytosolic or organelle-targeted methionine aminopeptidase (MetAP), and NME of these identified poplar proteins is mediated by three cytosolic poplar MetAPs (PtrMetAP1A, PtrMetAP2A and PtrMetAP2B). A novel member of MetAP1s was identified and named Ptr MetAP1E because of its divergence with other known MetAP1s. We found that poplar possessed the entire N-terminal acetyltransferase (Nat) system composed of six Nats (NatA-F), however, one extended Nat system was also found in poplar, probably arising from multiple gene duplication events. Based on a comparison of these recognized substrate motifs of Nats found in yeast and humans with our extraction of substrate motifs, we predict that NatA and NatB represent the major Nats that acetylate the identified poplar proteins. This result reveals that poplar possesses analogous NPMs to those observed in eukaryotes.