[7]. O-GlcNacylation, similar to O-phosphorylation, is a dynamic post-translational modification at serine or threonine residues of nuclear and cytoplasmic proteins [8]. O-GlcNAc transferase (OGT) and neutral b-N-acetylglucosaminidase are responsible for the addition and removal of O-GlcNAc [9]. In animal cells, O- GlcNAc has been well characterized and shown to play functional roles in stress response [10], cell cycle progression [11] and cellular motility [12]. Nucleocytoplasmic proteins modified by O-GlcNAc include transcription factors [13], vesicular trafficking proteins [14], cytoskeletal proteins and nuclear pore proteins [9]. O-GlcNAcylation and O-phosphorylation of proteins have a reciprocal relation [15]. The two translational modifications also operate concomitantly on proteins [16]. Protein O-GlcNAc modification has been proposed to play a critical role in plant development [17]. Two OGT enzymes, SECRET AGENT (SEC) and SPINDLY (SPY), identified in Arabidopsis, show high similarity with animal OGT [18–20]. Nevertheless, the involvement of O- GlcNAc in the physiological process and cellular regulation is largely unknown in plants. The limitation of detection methods, to a great extent, has resulted in an impediment to the research on O- GlcNAc modification. We previously reported that transcription of VER2 is induced by vernalization and jasmonate acid in wheat and that a jacalin-like domain exists at the C terminus [21]. Vernalization refers to the process of prolonged cold inducing the promotion of flowering in plants. Significant progress has been made in understanding the molecular mechanisms of vernalization in the model plant Arabidopsis [22–25]. Winter wheat is one of the major food crops requiring vernalization for the development of flowering organs. The molecular mechanisms for cereals responding to vernalization differ from that for Arabidopsis [26,27]. Methylation and acetylation modification of histones were shown to control epigenetic memory of winter in Arabidopsis [28–31]. Compared with the well-elucidated molecular and biochemical mechanisms of vernalization in Arabi- dopsis, those for winter wheat remain to be further elucidated. In the current study, we characterized the lectin activity of VER2 and analyzed variation of protein O-GlcNAc modification at the global level in response to vernalization. The subcellular location and dynamics of the overexpressed VER2-GFP fusion protein was investigated in transgenic Arabidopsis. Our data suggest that O-GlcNAc glycosylation participates in the vernalization response in wheat, and phosphorylation of VER2 is necessary to mediate O-GlcNAc signaling and its compartmentalization induced by vernalization. Results Lectin activity analysis of VER2 To confirm the lectin activity of VER2, 1 mM isopropyl-b-D- thiogalactoside (IPTG) was used to induce the expression of GST- VER2 fusion protein in Escherichia coli BL21. A protein band matching the expected size of GST-VER2, at approximately 58 kDa, was visible at 3 h after incubation (Figure 1A, lane 2). The fusion protein was affinity purified and the eluted VER2-GST fusion protein was cleaved with thrombin to release VER2 protein (Figure 1B, lane 2). The purified VER2 was able to agglutinate rabbit erythrocytes at a minimum concentration of 1.5 mg/ml. On the basis of the jacalin-like domain at the C terminus of VER2, carbohydrate-binding specificity was detected with mannose, galactose, N-acetyl-D-glucosamine, glucose, sucrose, fructose, malt- ose and albumin egg. These sugars were tested in hapten inhibition assay to analyze their inhibitory effect on agglutination of 2% rabbit erythrocyte suspension. As shown in Table 1, the agglutination activity of VER2 was readily inhibited by N-acetyl-D-glucosamine VER2 and O-GlcNAc Signaling Figure 1. Purification of VER2 protein. (A) Induction of GST-VER2 recombinant protein in E. coli by use of IPTG. Lane 1, cell lysate before the addition of IPTG; Lane 2, cell lysate at 5 h after IPTG induction. The position of induced GST-VER2 is marked by an arrow. Lane M, molecular weight markers. (B) Purification of recombinant VER2 protein. Lane 1, the purified GST-VER2 fusion protein; Lane 2, thrombin digestion of GST-VER2 to GST and VER2; Lane 3, the purified VER2. Lane M, molecular weight markers. The gels were stained with coomassie brilliant blue. doi:10.1371/journal.pone.0004854.g001 and galactose, with the lowest inhibition concentration of 3.1 mM and 6.25 mM, respectively, and VER2 interacts more specifically with N-acetyl-D-glucosamine. In contrast, mannose could not inhibit the agglutination. Fructose, glucose and maltose were less sensitive. Sucrose was 8 times less sensitive than galactose, which suggests that the carbohydrate binding sites of VER2 are more adaptive to monosaccharide than disaccharide. The glycoprotein albumin egg also had an inhibitory effect because of the N-acetyl-D-galactose ligands on its molecular structure. Immunocytochemical localization of VER2 in response to vernalization in wheat Previous in situ hybridization results showed that vernalization induces the mRNA expression of VER2 [21]. Here, protein immunocytochemistry analysis was used to determine the spatial Table 1. Comparison of the carbohydrate-binding specificities of VER2. Sugar/Glycoprotein Galactose Glucose N-Acetyl-D-glucosamine Mannose Sucrose Fructose Maltose Albumin Egg MIC 6.25 mM 25 mM 3.1 mM .100 mM 50 mM 12.5 mM 25 mM 6.25 mg/ml Solutions containing purified VER2 and simple sugars were preincubated for 1 h at room temperature, then rabbit erythrocytes suspension was added, respectively, and the agglutination was evaluated after 1 h. MIC: The lowest concentration of sugars or glycoproteins at which the inhibition of the agglutination was visible. doi:10.1371/journal.pone.0004854.t001 PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4854