VER2 and O-GlcNAc Signaling 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 mm. doi:10.1371/journal.pone.0004854.g005 Discussion VER2 is a JRL responding to vernalization in wheat Plants are well known to contain lectins with carbohydrate binding and agglutination activity. JRL proteins are divided into galactose- and mannose-specific subgroups. We show here that VER2 is a JRL protein with N-acetylglucosamine and galactose specificity. Peumans et al. [1] reported that a mannose-specific jacalin, Calsepa, was localized in the cytoplasm, whereas a galactose-specific jacalin was shown to locate in small storage vacuoles. Nevertheless, another mannose-specific jacalin, Morni- gaM, with no transit peptides, showed considerable presence in the nucleus as well as cytoplasm. The nuclear location mechanism for MornigaM is not clear [37]. Nictaba has a typical nuclear localization signal corresponding to its nuclear location [5]. Nevertheless, the amino acid sequence of VER2 does not indicate any classical nuclear localization signals or signal peptides for subcellular targeting. However, VER2 was targeted to the nucleus, perinuclear region and cytoplasmic mobile punctate structures (Figures 2,5,6). These subcellular location patterns differed greatly from that of other reported lectins. Amino acid sequence analysis of VER2 showed a dirigent domain in the N terminus and a jacalin-like domain in the C terminus. We previously described a mannose-specific jacalin OsJAC1 with the same two domains as VER2 in rice [42], overexpressed OsJAC1-GFP showed the same location pattern as GFP alone in Arabidopsis (unpublished data). The N-terminal dirigent domain of plant lectins was suggested to play defensive or stress-related roles [43]. VER2 could act as a nonclassical lectin to respond to vernalization in wheat. Phosphorylation is necessary for VER2 functioning Phosphorylation and dephosphorylation can direct cytoplasmic or nuclear localization of proteins in response to environmental stress and the nutrient state [33,44,45]. Our data clearly indicate that phosphorylation of VER2 is induced by vernalization. The phosphorylation modification of VER2 corresponds to its nuclear/ perinuclear targeting and binding to O-GlcNAc-modified proteins. Removal of the phosphorylation state of VER2 eliminated the nuclear location and the binding of VER2 to O-GlcNAcylated protein. In transgenic Arabidopsis, site mutation of S33G disturbed phosphorylation modification of VER2 and changed its compart- mentalization. We suggest that phosphorylated VER2 is involved in the vernalization response and that phosphorylation at S33 is necessary for VER2 functioning. O-GlcNAc modification is an abundant and dynamic process, as is phosphorylation, for numerous nucleocytoplasmic proteins [46]. Cellular O-GlcNAc levels respond to environmental and nutrient stress in animal cells, and increasing global levels of O-GlcNAc- modified proteins increases stress tolerance and modulate the responsiveness of cells to extracellular signaling [47]. O-GlcNAc signaling and O-GlcNAcylated proteins in response to environmental cues are poorly understood in plants. Although SPY, the negative regulator of the gibberellic acid (GA) signaling pathway, has been identified in Arabidopsis as a homologue of O-GlcNAc transferase, O- GlcNAc-modified proteins involved in GA signal transduction have not been determined. In our study, vernalization as a special environmental stress increased protein O-GlcNAcylation at the global level in immunoblot assay with O-GlcNAc-specific antibody. Early studies determined the influence of carbohydrates on vernalization. Only exogenous sugar application could optimize vernalization of excised meristems. Glucose promotes and mannose inhibits vernalization [48]. Glucose may modulate vernalization through elevating cellular O-GlcNAc level. Our data indicate that vernalization needs the participation of O-GlcNAc-dependent intracellular signaling that may act as a novel regulatory signaling component. The relationship between O-GlcNAc modified proteins and plant lectins is largely undefined at present. Phosphorylated HSP-70, a nuclear/cytoplasmic GlcNAc-specific lectin from rat liver, was speculated to be involved in the shuttling of O- GlcNAcylation-modified proteins between cytoplasm and nucleus [16]. O-GlcNAc modification of HSP70 might mediate cellular protection by its binding to target proteins [49]. We speculate that phosphorylated VER2 may bind to O-GlcNAc modified protein(s) and regulate their stability and/or intracellular location during the vernalization process in winter wheat. The identification and functional analysis of O-GlcNAc-modified proteins induced by vernalization will be helpful to elucidate the mechanism of O- GlcNAc signaling in response to vernalization in plants, and additional studies of the relationship between VER2 regulation and O-GlcNAc modified proteins is of great importance. Overexpressed VER2 modulates nuclear movement in Arabidopsis Nuclear migration is critical for proper developmental processes in eukaryotes and depends on microtubules and actin filaments [36,50]. We have examined the nuclear movement and present evidence that overexpressed VER2 in transgenic Arabidopsis accelerated nuclear migration. Pre-mitotic nuclear migration is reported to occur in the G1 phase and the nuclear positioning indicates the potential division site [51]. During the post-mitotic process, nuclear migration includes the movement of nuclei toward daughter cells and the center position of interphase cells. Kinesin is involved in unidirectional nuclear motility and positioning [52,53]. The migration of interphase nuclei in fungi has been described to be intermittent and related with other cell cycle events [54]. Here, the nuclear motility we observed is possibly pre-mitotic or positioning in cells of post-mitotic stage. The previously published data showed that the heading and maturation time was delayed and tissue degeneration was observed at the top of spikes in antisense transgenic wheat plants [21]. In the present study, we compared the expression of VER2 protein in roots, stems, leaves, and panicles at different development stages by western blotting. Before heading, VER2 was detected in leaves and panicles of wheat but not roots and stems. The protein level in panicles was higher than that in leaves. After heading, VER2 was expressed only in panicles. The immunoblotting signal of tubulin was used as a loading control of total proteins (Figure S2). The specific accumulation of VER2 protein during heading indicates that VER2 is required for panicle development in wheat. The rate of nuclear migration functions as a influencing factor of cell cycle duration [55]. Our results provide some indication that VER2 possibly participates in spike development via regulating nuclear motility-associated mitotic process during heading stage. Rice lectin Orysata exhibits PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4854