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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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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.
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pone-0004854-g006: 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.

Mentions: In epidermal cells of young leaves of transgenic Arabidopsis overexpressing VER2-GFP, VER2-carrying nuclear and perinuclear structures were observed to change direction randomly and move within the cell (Figure 6A). The proportion of observed cells with nuclear motility is about 10%. In vein cells, nuclei showed axial migration with a velocity of approximately 30 µm/min (Figure 6B), which is much faster than nuclear movement in Arabidopsis root hairs (<10 µm/min) [35]. However, nuclear migration in 35S::GFP transgenic Arabidopsis plants could not be observed under the same conditions. Therefore, VER2-GFP could alter the motility and position of nuclei in transgenic plants. Overexpression of VER2 was suggested to facilitate nuclear motility. Punctate-targeted VER2-GFP was also observed with a mobile pattern in leaf epidermis and veins (Figure 6C,D), and mobile punctate signals could fuse together. Nuclei are reported to move along microtubules [36]. To examine whether perinuclear-distributed VER2-GFP is associated with cellular microtubules or moves passively with the nucleus, we treated Arabidopsis epidermis with the cytoskeleton-destabilizing agent propyzamid (5 µM). The propyzamid was dissolved in DMSO and diluted to working concentration to incubate Arabidopsis leaves. As a control, DMSO treatment alone did not alter the location pattern of VER2-GFP (Figure 7A), but treatment with propyzamid destroyed the perinuclear tubular extension of VER2-GFP (Figure 7B). Thus, VER2 overexpressed at the perinuclear region is associated with microtubules.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004854-g006: 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.
Mentions: In epidermal cells of young leaves of transgenic Arabidopsis overexpressing VER2-GFP, VER2-carrying nuclear and perinuclear structures were observed to change direction randomly and move within the cell (Figure 6A). The proportion of observed cells with nuclear motility is about 10%. In vein cells, nuclei showed axial migration with a velocity of approximately 30 µm/min (Figure 6B), which is much faster than nuclear movement in Arabidopsis root hairs (<10 µm/min) [35]. However, nuclear migration in 35S::GFP transgenic Arabidopsis plants could not be observed under the same conditions. Therefore, VER2-GFP could alter the motility and position of nuclei in transgenic plants. Overexpression of VER2 was suggested to facilitate nuclear motility. Punctate-targeted VER2-GFP was also observed with a mobile pattern in leaf epidermis and veins (Figure 6C,D), and mobile punctate signals could fuse together. Nuclei are reported to move along microtubules [36]. To examine whether perinuclear-distributed VER2-GFP is associated with cellular microtubules or moves passively with the nucleus, we treated Arabidopsis epidermis with the cytoskeleton-destabilizing agent propyzamid (5 µM). The propyzamid was dissolved in DMSO and diluted to working concentration to incubate Arabidopsis leaves. As a control, DMSO treatment alone did not alter the location pattern of VER2-GFP (Figure 7A), but treatment with propyzamid destroyed the perinuclear tubular extension of VER2-GFP (Figure 7B). Thus, VER2 overexpressed at the perinuclear region is associated with microtubules.

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