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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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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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pone-0004854-g009: Location patterns of VER2-S33G and VER2-T209A mutations fused to GFP.(A) VER2-S33G mutation. (B) VER2-T209A mutation. Bars, 20 µm.

Mentions: The amino acid sequence analysis of VER2 showed predicted serine and threonine phosphorylation sites and Yin-Yang sites, which have potential reciprocal dynamic phosphorylation and O-GlcNAc modification possibility (http://www.cbs.dtu.dk/services/NetPhos/) (http://www.cbs.dtu.dk/services/YinOYang/). The serine at site 33 and threonine at site 209 of the amino acid sequence are predicted Yin-Yang sites. Phosphorylation on these two sites is important for protein functioning. We generated transgenic Arabidopsis overexpressing mutated VER2 of S33G and T209A to observe the compartmentalization of fluorescence of GFP. The location pattern of mutant VER2-S33G is different from that of VER2. VER2S33G was predominantly targeted to punctate structures, and very weak location signaling could be seen in nuclei and cytoplasm (Figure 9A). Transgenic Arabidopsis plants expressing VER2-T209A showed no obvious differences in GFP fluorescence distribution (Figure 9B).


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)

Location patterns of VER2-S33G and VER2-T209A mutations fused to GFP.(A) VER2-S33G mutation. (B) VER2-T209A mutation. Bars, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004854-g009: Location patterns of VER2-S33G and VER2-T209A mutations fused to GFP.(A) VER2-S33G mutation. (B) VER2-T209A mutation. Bars, 20 µm.
Mentions: The amino acid sequence analysis of VER2 showed predicted serine and threonine phosphorylation sites and Yin-Yang sites, which have potential reciprocal dynamic phosphorylation and O-GlcNAc modification possibility (http://www.cbs.dtu.dk/services/NetPhos/) (http://www.cbs.dtu.dk/services/YinOYang/). The serine at site 33 and threonine at site 209 of the amino acid sequence are predicted Yin-Yang sites. Phosphorylation on these two sites is important for protein functioning. We generated transgenic Arabidopsis overexpressing mutated VER2 of S33G and T209A to observe the compartmentalization of fluorescence of GFP. The location pattern of mutant VER2-S33G is different from that of VER2. VER2S33G was predominantly targeted to punctate structures, and very weak location signaling could be seen in nuclei and cytoplasm (Figure 9A). Transgenic Arabidopsis plants expressing VER2-T209A showed no obvious differences in GFP fluorescence distribution (Figure 9B).

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