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Figure 1. 

The workflow of glycoprotein enrichment and identification in poplar developing xylem. (a) Enrichment and recognition of the glycoproteins. Crude proteins were extracted from poplar developing xylem, and the glycoproteins were bound to ConA, PNA, WGA and jacalin lectin affinity columns. The eluted glycoproteins were digested by trypsin and the peptide segments were further recognized through mass spectrum analysis. (b) Validation of the eluted glycoproteins. The glycoproteins separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were stained using Coomassie brilliant blue (CBB) staining and Pierce Glycoprotein Staining kit (detecting sugar moieties of the glycoproteins), respectively.

Figure 2. 

Verification of N- and/or O-glycosylation in PtSOD and HAD proteins. (a), (b) Analysis of PtSOD or PtHAD gene expression by RT-PCR in their transgenic Arabidopsis plants. AtActin2 serves as a control gene. (c), (d) Western blot analysis of PtSOD1-Flag and HAD-Flag protein levels in corresponding transgenic plants usig anti-FLAG antibody. Coomassie brilliant-stained Rubsico large subunit proteins indicate the loading amount of each sample on SDS-PAGE gels as control. (e), (f) Migration analysis of PtSOD and PtHAD proteins on SDS-PAGE gels. Protein extracts with/without the digestion of PNGase F (N) or O-glycosidase (O) were separated on 10% SDS-PAGE gels and followed by immunoblotting with anti-FLAG antibody.

Figure 3. 

Functional classification and localization of the identified glycoproteins. (a) Functional classification of glycoproteins identified from Populus developing xylem. Please refer to Supplemental Table S1 for detailed analysis. (b) Subcellular localization of the identified glycoproteins predicted by Plant-mPLoc, ngLOC, ProtComp9.0, WoLF PSORT and YLOC.

Figure 4. 

Expression profiles of 25 genes encoding the glycoproteins in different poplar tissues using RT-qPCR analysis. Different tissues included phloem (PH), xylem (XY), young leaf (YL) and mature leaf (OL). The expression of PtActin2 was used as an internal control. Data are means ± standard error of three technical replicate results.

Figure 5. 

Identified glycoproteins are proposed to be involved in wood formation that includes cambia cell division, cell differentiation and expansion, SCW synthesis and PCD.