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

Xing L, Li J, Xu Y, Xu Z, Chong K - PLoS ONE (2009)

Bottom Line: Overexpressed VER2 accelerated nuclear migration.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.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Molecular Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing, China.

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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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.
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pone-0004854-g002: 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.

Mentions: Previous in situ hybridization results showed that vernalization induces the mRNA expression of VER2 [21]. Here, protein immunocytochemistry analysis was used to determine the spatial and temporal expression patterns of VER2 in response to vernalization and devernalization. Devernalization treatment is a valuable control system of vernalization at the morphological or physiological level. The specificity of the antibody prepared with purified VER2 was detected by western blot analysis before immunocytochemical labelling. The anti-VER2 antibody specifically recognized a protein band at the expected molecular weight for VER2 (Figure S1) in wheat plants vernalized for 3 weeks. Furthermore, the labelling signal from the anti-VER2 antibody was consistent with the labelling results for antibody prepared with a synthesized polypeptide of VER2 [32]. Vernalized, devernalized and non-vernalized wheat plants were used for immunolabeling. Labeling signals were detected in shoot apical meristem and young leaves of plumules vernalized for 3 weeks (Figure 2A to C). Compared with nuclei indicated by hematoxylin staining in young leaves (Figure 2D), VER2 protein was shown to target predominantly to potential nuclear structures. In devernalized plants, immunolabeling signals were detected only in cytoplasm (Figure 2E), with no signal detected in nonvernalized plants (Figure 2F) or in the negative control (Figure 2G) under the same conditions. As well, immunolabeling signals were not detected in plants vernalized for 1 and 2 weeks (data not shown).


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

Xing L, Li J, Xu Y, Xu Z, Chong K - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654674&req=5

pone-0004854-g002: 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.
Mentions: Previous in situ hybridization results showed that vernalization induces the mRNA expression of VER2 [21]. Here, protein immunocytochemistry analysis was used to determine the spatial and temporal expression patterns of VER2 in response to vernalization and devernalization. Devernalization treatment is a valuable control system of vernalization at the morphological or physiological level. The specificity of the antibody prepared with purified VER2 was detected by western blot analysis before immunocytochemical labelling. The anti-VER2 antibody specifically recognized a protein band at the expected molecular weight for VER2 (Figure S1) in wheat plants vernalized for 3 weeks. Furthermore, the labelling signal from the anti-VER2 antibody was consistent with the labelling results for antibody prepared with a synthesized polypeptide of VER2 [32]. Vernalized, devernalized and non-vernalized wheat plants were used for immunolabeling. Labeling signals were detected in shoot apical meristem and young leaves of plumules vernalized for 3 weeks (Figure 2A to C). Compared with nuclei indicated by hematoxylin staining in young leaves (Figure 2D), VER2 protein was shown to target predominantly to potential nuclear structures. In devernalized plants, immunolabeling signals were detected only in cytoplasm (Figure 2E), with no signal detected in nonvernalized plants (Figure 2F) or in the negative control (Figure 2G) under the same conditions. As well, immunolabeling signals were not detected in plants vernalized for 1 and 2 weeks (data not shown).

Bottom Line: Overexpressed VER2 accelerated nuclear migration.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.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Molecular Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing, China.

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.

Show MeSH