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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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Confocal images of VER2-GFP fusion protein and GFP overexpressed in leaf epidermis and veins in Arabidopsis.At least 6 independent transgenic plants expressing VER2-GFP and GFP were analyzed. GFP fluorescence is shown in the green channel. Propidium Iodide (PI) fluorescence is shown in the red channel. (A) Localization of GFP alone in epidermal cells. (B) Localization of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. (C) An enlarged image of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. Nuclear and perinuclear distributed VER2-GFP was labeled with an arrow. (D), (E) and (F) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (D) was stained with PI and shown in (E). The emerged image of (D) and (E) was shown in (F). (G), (H) and (I) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (G) was stained with PI and shown in (H). The emerged image of (G) and (H) was shown in (I). (J), (K) and (I) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (J) was shown in (K). The emerged image of (J) and (K) was shown in (L). (M), (N) and (O) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (M) was shown in (N). The emerged image of (M) and (N) was shown in (O). n, nucleus; cw, cell wall. Bars, 10 µm.
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pone-0004854-g005: Confocal images of VER2-GFP fusion protein and GFP overexpressed in leaf epidermis and veins in Arabidopsis.At least 6 independent transgenic plants expressing VER2-GFP and GFP were analyzed. GFP fluorescence is shown in the green channel. Propidium Iodide (PI) fluorescence is shown in the red channel. (A) Localization of GFP alone in epidermal cells. (B) Localization of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. (C) An enlarged image of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. Nuclear and perinuclear distributed VER2-GFP was labeled with an arrow. (D), (E) and (F) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (D) was stained with PI and shown in (E). The emerged image of (D) and (E) was shown in (F). (G), (H) and (I) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (G) was stained with PI and shown in (H). The emerged image of (G) and (H) was shown in (I). (J), (K) and (I) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (J) was shown in (K). The emerged image of (J) and (K) was shown in (L). (M), (N) and (O) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (M) was shown in (N). The emerged image of (M) and (N) was shown in (O). n, nucleus; cw, cell wall. Bars, 10 µm.

Mentions: We generated 35S::VER2-GFP and 35S::GFP transgenic Arabidopsis plants as a model system to determine the precise subcellular location pattern and dynamics of VER2 and further understand its function. GFP alone was observed in the cytoplasm and nucleus of leaf epidermal cells in 35S::GFP transgenic plants (Figure 5D). However, VER2-GFP fluorescence showed aggregates with tubular extension, as well as punctate signals in cytoplasm (Figure 5B, C). To further confirm the nuclear targeting of VER2 as shown in Figure 2, Arabidopsis epidermis was stained with propidium iodide (PI) to indicate the position of nuclei. VER2-GFP was localized in the nucleus as well as perinuclear region in tubular structures (Figure 5D to F), or only at the perinuclear region in some epidermal cells (Figure 5G to I). Vein cells were arranged in a regular pattern and could be captured in clear bright-field images to confirm nuclear and perinuclear tubular distribution of VER2-GFP (Figure 5J to O).


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)

Confocal images of VER2-GFP fusion protein and GFP overexpressed in leaf epidermis and veins in Arabidopsis.At least 6 independent transgenic plants expressing VER2-GFP and GFP were analyzed. GFP fluorescence is shown in the green channel. Propidium Iodide (PI) fluorescence is shown in the red channel. (A) Localization of GFP alone in epidermal cells. (B) Localization of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. (C) An enlarged image of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. Nuclear and perinuclear distributed VER2-GFP was labeled with an arrow. (D), (E) and (F) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (D) was stained with PI and shown in (E). The emerged image of (D) and (E) was shown in (F). (G), (H) and (I) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (G) was stained with PI and shown in (H). The emerged image of (G) and (H) was shown in (I). (J), (K) and (I) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (J) was shown in (K). The emerged image of (J) and (K) was shown in (L). (M), (N) and (O) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (M) was shown in (N). The emerged image of (M) and (N) was shown in (O). n, nucleus; cw, cell wall. Bars, 10 µm.
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pone-0004854-g005: Confocal images of VER2-GFP fusion protein and GFP overexpressed in leaf epidermis and veins in Arabidopsis.At least 6 independent transgenic plants expressing VER2-GFP and GFP were analyzed. GFP fluorescence is shown in the green channel. Propidium Iodide (PI) fluorescence is shown in the red channel. (A) Localization of GFP alone in epidermal cells. (B) Localization of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. (C) An enlarged image of VER2-GFP fusion protein in epidermal cells. The arrowheads point to punctate distribution of VER2-GFP. Nuclear and perinuclear distributed VER2-GFP was labeled with an arrow. (D), (E) and (F) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (D) was stained with PI and shown in (E). The emerged image of (D) and (E) was shown in (F). (G), (H) and (I) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in leaf epidermal cells. The nucleus in (G) was stained with PI and shown in (H). The emerged image of (G) and (H) was shown in (I). (J), (K) and (I) Enlarged images of fluorescence of nuclear and perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (J) was shown in (K). The emerged image of (J) and (K) was shown in (L). (M), (N) and (O) Enlarged images of fluorescence of perinuclear distributed VER2-GFP in vein cells. Transmitted light image of (M) was shown in (N). The emerged image of (M) and (N) was shown in (O). n, nucleus; cw, cell wall. Bars, 10 µm.
Mentions: We generated 35S::VER2-GFP and 35S::GFP transgenic Arabidopsis plants as a model system to determine the precise subcellular location pattern and dynamics of VER2 and further understand its function. GFP alone was observed in the cytoplasm and nucleus of leaf epidermal cells in 35S::GFP transgenic plants (Figure 5D). However, VER2-GFP fluorescence showed aggregates with tubular extension, as well as punctate signals in cytoplasm (Figure 5B, C). To further confirm the nuclear targeting of VER2 as shown in Figure 2, Arabidopsis epidermis was stained with propidium iodide (PI) to indicate the position of nuclei. VER2-GFP was localized in the nucleus as well as perinuclear region in tubular structures (Figure 5D to F), or only at the perinuclear region in some epidermal cells (Figure 5G to I). Vein cells were arranged in a regular pattern and could be captured in clear bright-field images to confirm nuclear and perinuclear tubular distribution of VER2-GFP (Figure 5J to O).

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