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. 2009;4(3):e4854.
doi: 10.1371/journal.pone.0004854. Epub 2009 Mar 16.

Phosphorylation modification of wheat lectin VER2 is associated with vernalization-induced O-GlcNAc signaling and intracellular motility

Lijing Xing  1 , Juan Li, Yunyuan Xu, Zhihong Xu, Kang Chong
Affiliations

Phosphorylation modification of wheat lectin VER2 is associated with vernalization-induced O-GlcNAc signaling and intracellular motility

Lijing Xing et al. PLoS One. 2009.

Abstract

Background: O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins mediates stress response and cellular motility in animal cells. The plant lectin concanavalin A can increase nuclear O-GlcNAc levels and decrease cytoplasmic O-GlcNAc levels in T lymphocytes. However, the functions of O-GlcNAc signaling in plants, as well as the relation between plant lectins and O-GlcNAc in response to environmental stimuli are largely undefined.

Methodology/principal findings: We describe a jacalin-like lectin VER2 in wheat that shows N-acetylglucosamine and galactose specificity. Immunocytochemical localization showed VER2 expression induced predominantly at potential nuclear structures in shoot tips and young leaves and weakly in cytoplasm in response to vernalization. In contrast, under devernalization (continuous stimulation with a higher temperature after vernalization), VER2 signals appeared predominantly in cytoplasm. 2-D electrophoresis, together with western blot analysis, showed phosphorylation modification of VER2 under vernalization. Immunoblot assay with O-GlcNAc-specific antibody revealed that vernalization increased O-GlcNAc modification of proteins at the global level. An O-GlcNAc-modified protein co-immunoprecipitated with VER2 in vernalized wheat plants but not in devernalized materials. The dynamic of VER2 was observed in transgenic Arabidopsis overexpressing the VER2-GFP fusion protein. Overexpressed VER2 accelerated nuclear migration. Immunogold labeling and indirect immunofluoresence colocalization assay indicated that VER2-GFP was targeted to the secretory pathway.

Conclusions/significance: O-GlcNAc signaling is involved in the vernalization response in wheat, and phosphorylation is necessary for the lectin VER2 involving O-GlcNAc signaling during vernalization. Our findings open the way to studies of O-GlcNAc protein modification in response to environmental signals in plants.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Purification of VER2 protein.
(A) Induction of GST-VER2 recombinant protein in E. coli by use of IPTG. Lane 1, cell lysate before the addition of IPTG; Lane 2, cell lysate at 5 h after IPTG induction. The position of induced GST-VER2 is marked by an arrow. Lane M, molecular weight markers. (B) Purification of recombinant VER2 protein. Lane 1, the purified GST-VER2 fusion protein; Lane 2, thrombin digestion of GST-VER2 to GST and VER2; Lane 3, the purified VER2. Lane M, molecular weight markers. The gels were stained with coomassie brilliant blue.
Figure 2
Figure 2. Immunocytochemical localization of VER2, showing labeling signals in shoot apex and young leaves.
Sections were probed with anti-VER2 antibody followed by a goat anti-rabbit alkaline phosphatase (AP)-conjugated secondary antibody. (A) Plants were vernalized for 21 days. VER2 is predominantly targeted to potential nuclear structures. Weaker labeling was detected in cytoplasm. (B) An enlarged image showing labeling signals in shoot apical meristem. (C) An enlarged image showing labeling signals in young leaves. (D) Showing hematoxylin stained nuclei of young leaves. (E) Plants were first vernalized for 21 days, then devernalized. Signals were dispersed in the cytoplasm. (F) No immunocytochemical signal detected in nonvernalized plants. (G) Negative control performed by omitting the first antibody. Bars, 20 µm.
Figure 3
Figure 3. Immunoblot analysis of VER2 separated on 2-D electrophoresis and dephosphorylation of VER2 by phosphatase in vernalized and devernalized wheat plants.
(A) VER2 was separated into 3 main spots with pI = 5.6, 6.1 and 6.7, respectively, in vernalized wheat plants. (B) VER2 was detected as one spot with pI = 6.7 in devernalized wheat plants. (C) VER2 was immunoprecipitated and then treated with protein phosphatase for the time shown in vernalized and devernalized plants. (D) Immunoblot analysis showing the difference of migration distance of VER2 in vernalized and devernalized materials. The samples were separated by electrophoresis for enough time until the marker band with the molecular weight of 25 kDa shifted to the forefront of the 8 cm gel.
Figure 4
Figure 4. Detection of O-GlcNAc-modified proteins and their association with VER2 in vernalized and devernalized wheat plants.
(A) SDS-PAGE results stained with coomassie blue. (B) Immunoblot analysis of O-GlcNAc-modified proteins in nonvernalized, vernalized and devernalized plants with O-GlcNAc site-specific antibody CTD110.6. Tubulin was immunoblotted as a loading control of total proteins with anti-tubulin polyclonal antibody. (C) Proteins from vernalized and devernalized wheat plants were immunoprecipitated with anti-VER2 antibody and detected with anti-VER2 and CTD110.6 antibodies, respectively. An O-GlcNAc modified protein with a molecular weight of about 35 kD was identified in VER2 immunoprecipitates from vernalized materials; no blotting signals for O-GlcNAcylated proteins were detected in devernalized materials. (D) VER2 was identified in O-GlcNAc immunoprecipitates from vernalized plants but not from devernalized plants. M, molecular weight markers; NV, nonvernalized; V, vernalized; DV, devernalized. IP, immunoprecipitate; WB, western blotting.
Figure 5
Figure 5. Confocal images of VER2-GFP fusion protein and GFP overexpressed in leaf epidermis and veins in Arabidopsis.
At least 6 independent transgenic plants expressing VER2-GFP and GFP were analyzed. GFP fluorescence is shown in the green channel. Propidium Iodide (PI) fluorescence is shown in the red channel. (A) Localization of GFP alone in epidermal cells. (B) Localization of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. (C) An enlarged image of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. Nuclear and perinuclear distributed VER2-GFP was labeled with an arrow. (D), (E) and (F) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (D) was stained with PI and shown in (E). The emerged image of (D) and (E) was shown in (F). (G), (H) and (I) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (G) was stained with PI and shown in (H). The emerged image of (G) and (H) was shown in (I). (J), (K) and (I) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (J) was shown in (K). The emerged image of (J) and (K) was shown in (L). (M), (N) and (O) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (M) was shown in (N). The emerged image of (M) and (N) was shown in (O). n, nucleus; cw, cell wall. Bars, 10 µm.
Figure 6
Figure 6. Motility of VER2-GFP fusion protein in Arabidopsis leaf epidemis and veins.
(A) VER2-GFP involved nuclear movement in epidermal cells. PI-stained cell wall was shown in the red channel. (B) VER2-GFP involved nuclear movement in vein cells. (C) Movement of punctate-located VER2-GFP in leaf epidermal cells. (D) Temporal dynamics of VER2-GFP targeted to punctuate structures in vein cells, showing merged images of fluorescence and corresponding transmitted images. n, nuclei; cw, cell wall. Bars, 20 µm.
Figure 7
Figure 7. Perinuclear-distributed VER2-GFP is associated with microtubules in transgenic Arabidopsis.
(A) Epidermal cells treated with DMSO (1%) for 30 min. (B) Epidermal cells treated with PPM (5 µM) for 30 min. Bars, 20 µm.
Figure 8
Figure 8. Immunogold labeling of VER2-GFP and colocalization of VER2-GFP with VSRAt in Arabidopsis leaves.
(A)–(D) Immunogold labeling of VER2-GFP. Sections were incubated with anti-GFP antibody (A to C) or preimmune serum (D) followed by gold-conjugated secondary antibody. Gold particles accumulate on ER (A) Golgi apparatus (B) and PVC (C). No specific labeling was found in the control image (D). ER is indicated with arrows. Bars, 0.2 µm. (E) VER2-GFP fusion signals with ring structures. (F)–(H) Colocalization of VER2-GFP with VSRAt. The anti-VSR-marked prevacuolar organelles are shown in red channel. Endogenous VSR was detected by indirect immunofluorescence labeling. Fixed leaves were stained with anti-VSR antibody followed by a TRITC-labeled anti-rabbit antibody (F). GFP signals indicated punctate distribution of VER2-GFP in green channel observed from the fixed leaves (G). Merged image of (F) and (G) is shown as (H). g, Golgi; cw, cell wall. Bars, 20 µm.
Figure 9
Figure 9. Location patterns of VER2-S33G and VER2-T209A mutations fused to GFP.
(A) VER2-S33G mutation. (B) VER2-T209A mutation. Bars, 20 µm.
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