Figure 7. Perinuclear-distributed VER2-GFP is associated with microtubules in transgenic Arabidopsis. (A) Epidermal cells treated with DMSO (1%) for 30 min. (B) Epidermal cells treated with PPM (5 mM) for 30 min. Bars, 20 mm. doi:10.1371/journal.pone.0004854.g007 mitogenetic activity towards T lymphocytes [3]. Mechanisms for plant lectins presenting mitotic activity need to be further elucidated. VER2 involved in intracellular motility may mediate O- GlcNAc signaling In mammalian cells, lectins mediate glycoprotein transport along the secretory pathway. The transmembrane mannose- binding lectin ERGIC-53 homologous to leguminous acts as a receptor for sorting glycoproteins in the secretory pathway [56,57]. The lectin domain of ERGIC-53 is responsible for sorting and transporting glycoproteins from the ER to Golgi apparatus [58]. The biochemical and cell biological mechanisms of lectins in the intracellular secretory pathway in plants are still unknown. N- linked and O-linked carbohydrates act as targeting signals for glycoprotein sorting by lectins from the Golgi apparatus to plasma membrane [59]. Glycoproteins with galactose, N-acetylglucosa- mine and N-acetylgalactosamine oligosaccharide chains were detected in the Golgi apparatus and plasma membrane [60]. In plant cells, cargo proteins are sorted to vacuoles or another intracellular trafficking pathway in the trans-Golgi network [61]. Proteins destined for vacuoles require sorting signals in polypep- tides. N-terminal propeptide (NTPP) and C-terminal propeptide (CTPP) are vacuolar sorting determinants in plants [62]. The amino acid sequence of NPIRL is the conserved motif for vacuolar sorting of sporamin [63] and barley aleurain [64]. The CTPP of a barley lectin is necessary for its vacuolar sorting [65]. VSR1, an intermediate compartment for vacuolar sorting of cargo proteins, interacts with the NTPP but not CTPP of cargo proteins [66]. Amino acid sequence analysis of VER2 does not show a signal peptide, NTPP or CTPP. Therefore, the mobile transport clusters of VER2-GFP do not denote the transport of VER2 itself as a vacuolar storage component. By using dynamic fluorescence and indirect immunofluorescence co-localization analysis, we demonstrate that VER2 and O-GlcNAc Signaling VER2 is associated with vacuolar trafficking. Although vernalization induced O-GlcNAc modification of many proteins, no more proteins were detected binding with VER2. The result also supports that VER2 does not act as a sorting receptor in the secretory pathway. Vesicle trafficking proteins could be modified with O-GlcNAc in animal cells [7,67]. In neutrophils, O-GlcNAcylation induced by GlcNH2 can increase cellular motility [12] and regulate activities of intermediates of Rac and PI3K involved in cellular migration [68]. In plant cells, the relation between O-GlcNAc and intracellular motility is largely unknown. We propose that VER2 binds with specific O-GlcNAc-modified proteins in the secretory pathway and mediates intracellular motility modulated by O- GlcNAc signals. Our findings provide novel insights into lectin function in the vernalization response and motility-associated signaling in plants. Further studies are required to define more precisely the role of VER2 in O-GlcNAc signaling induced by vernalization. Materials and Methods Plant materials, growth conditions, and devernalization treatment Seeds of winter wheat (Triticum aestivum L.) were surface sterilized in 2% NaClO for 20 min, then rinsed overnight with flowing water. For all treatments, seeds were cultured under darkness. For vernalization treatment, seeds were sown on moist filter paper and grown at 4uC for 21 days. Nonvernalized control seeds were grown at 25uC for 3 days. Seedlings vernalized for 21 days were subsequently transferred to an incubator at 35uC for 5 days, defined as devernalization. Purification of GST-VER2 recombinant protein Two primers were designed (59-TAAGAATTCATGGC- CAAATTCCAGATTAC-39 and 59-AATCTCGAGGACCGTG- TAAACACCAAATG-39) to amplify VER2 from cDNA. The PCR product was excised with EcoRI and XhoI, then purified and cloned into the corresponding sites of the pGEX-4T-3 vector. The construct was verified by DNA sequencing. The GST-VER2 fusion protein was purified according to the manufacturer’s instructions (Amersham Biosciences). Removal of GST tag from the recombinant protein and purification of VER2 To cleave the GST tag, the fusion protein was cleaved with thrombin at room temperature for 16 h according to the manufacturer’s recommendations (Amersham Biosciences). GST was removed by column purification on glutathione sepharose 4B beads. Purified VER2 in the flow-through was collected. For preparation of anti-VER2 antibody, purified VER2 was injected into rabbits to produce polyclonal antibody. The anti- VER2 antibody was used for western blot analysis. Hemagglutination and Hapten carbohydrate-binding specificity of VER2 Agglutination assays were carried out in 96-U-well plates in a final volume of 40 mL containing 20 mL of purified VER2 serially diluted in two-fold increments and 20 ml of a 2% suspension of rabbit erythrocytes, according to a two-fold serial dilution procedure [69]. Agglutination was assessed visually after 1 h at room temperature. Hapten inhibition of the agglutination was determined as follows. A total of 20 mL of sugars or glycoprotein, including galactose, glucose, N-acetyl-D-glucosamine, mannose, sucrose, PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4854