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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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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.
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pone-0004854-g004: 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.

Mentions: To test whether the O-GlcNAc modification of proteins is affected by vernalization, which could be regarded as one kind of special environmental stress, we detected the variation of O-GlcNAc modified proteins at the global level in response to vernalization and explored the possibility of VER2 binding with O-GlcNAcylated proteins because of the carbohydrate specificity of VER2 to N-acetylglucosamine. We detected O-GlcNAc-modified proteins in nonvernalized, vernalized and devernalized wheat plants using the monoclonal antibody CTD110.6 originally produced to specifically recognize Ser- or Thr-O-GlcNAc-modified proteins in animal cells [34]. Western blot analysis showed that vernalization increased the global level of O-GlcNAc-modified proteins as compared with nonvernalization; furthermore, devernalization decreased the level of O-GlcNAc-modified total proteins, with only a few proteins detected (Figure 4A, B). We then immunoprecipitated VER2 from vernalized and devernalized extracts of wheat plumules using the anti-VER2 antibody. The antibody CTD110.6 was used to detect O-GlcNAc-modified proteins in immunoprecipitates. A protein band of approximately 35 kDa was detected in vernalized plants. In contrast, no signal was detected in devernalized materials (Figure 4C). Neither vernalized nor devernalized plants showed O-GlcNAc-modified signals corresponding to VER2. Figure 4D shows that VER2 immunoprecipitated with O-GlcNAc-modified proteins in vernalized but not devernalized plants.


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)

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.
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Related In: Results  -  Collection

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pone-0004854-g004: 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.
Mentions: To test whether the O-GlcNAc modification of proteins is affected by vernalization, which could be regarded as one kind of special environmental stress, we detected the variation of O-GlcNAc modified proteins at the global level in response to vernalization and explored the possibility of VER2 binding with O-GlcNAcylated proteins because of the carbohydrate specificity of VER2 to N-acetylglucosamine. We detected O-GlcNAc-modified proteins in nonvernalized, vernalized and devernalized wheat plants using the monoclonal antibody CTD110.6 originally produced to specifically recognize Ser- or Thr-O-GlcNAc-modified proteins in animal cells [34]. Western blot analysis showed that vernalization increased the global level of O-GlcNAc-modified proteins as compared with nonvernalization; furthermore, devernalization decreased the level of O-GlcNAc-modified total proteins, with only a few proteins detected (Figure 4A, B). We then immunoprecipitated VER2 from vernalized and devernalized extracts of wheat plumules using the anti-VER2 antibody. The antibody CTD110.6 was used to detect O-GlcNAc-modified proteins in immunoprecipitates. A protein band of approximately 35 kDa was detected in vernalized plants. In contrast, no signal was detected in devernalized materials (Figure 4C). Neither vernalized nor devernalized plants showed O-GlcNAc-modified signals corresponding to VER2. Figure 4D shows that VER2 immunoprecipitated with O-GlcNAc-modified proteins in vernalized but not devernalized plants.

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