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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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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.
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pone-0004854-g003: 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.

Mentions: The nucleocytoplasmic exchange of lectin plays a role in response to osmotic stress in yeast cells [33]. Amino acid sequence analysis indicated that VER2 does not contain a signal sequence for subcellular targeting. The different subcellular localization patterns of VER2 in response to vernalization and devernalization indicated that post-translational modification of VER2 might be involved in regulating its intracellular targeting. To address this possibility, total proteins from vernalized and devernalized materials were analyzed by 2-D gel electrophoresis and immunoblotted with anti-VER2 antibody. Continuous signal spots were apparent in wheat samples vernalized for 3 weeks (Figure 3A). Nevertheless, only a single spot was detected in devernalized plants (Figure 3B). Compared with the predicted isoelectric point (pI) of 6.6 (http://us.expasy.org/tools/pi_tool.html) of VER2, the signal detected from vernalized plants (Figure 3A) showed an acidic shift. In the devernalized sample, the immunoblotting signal was at the approximate expected pI of VER2, which suggested that vernalization could induce post-translational phosphorylation modification of VER2.


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)

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.
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Related In: Results  -  Collection

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

pone-0004854-g003: 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.
Mentions: The nucleocytoplasmic exchange of lectin plays a role in response to osmotic stress in yeast cells [33]. Amino acid sequence analysis indicated that VER2 does not contain a signal sequence for subcellular targeting. The different subcellular localization patterns of VER2 in response to vernalization and devernalization indicated that post-translational modification of VER2 might be involved in regulating its intracellular targeting. To address this possibility, total proteins from vernalized and devernalized materials were analyzed by 2-D gel electrophoresis and immunoblotted with anti-VER2 antibody. Continuous signal spots were apparent in wheat samples vernalized for 3 weeks (Figure 3A). Nevertheless, only a single spot was detected in devernalized plants (Figure 3B). Compared with the predicted isoelectric point (pI) of 6.6 (http://us.expasy.org/tools/pi_tool.html) of VER2, the signal detected from vernalized plants (Figure 3A) showed an acidic shift. In the devernalized sample, the immunoblotting signal was at the approximate expected pI of VER2, which suggested that vernalization could induce post-translational phosphorylation modification of VER2.

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
Related in: MedlinePlus