VER2 and O-GlcNAc Signaling Figure 2. Immunocytochemical localization of VER2, showing labeling signals in shoot apex and young leaves. Sections were probed with anti-VER2 antibody followed by a goat anti-rabbit alkaline phosphatase (AP)-conjugated secondary antibody. (A) Plants were vernalized for 21 days. VER2 is predominantly targeted to potential nuclear structures. Weaker labeling was detected in cytoplasm. (B) An enlarged image showing labeling signals in shoot apical meristem. (C) An enlarged image showing labeling signals in young leaves. (D) Showing hematoxylin stained nuclei of young leaves. (E) Plants were first vernalized for 21 days, then devernalized. Signals were dispersed in the cytoplasm. (F) No immunocytochemical signal detected in nonvernalized plants. (G) Negative control performed by omitting the first antibody. Bars, 20 mm. doi:10.1371/journal.pone.0004854.g002 and temporal expression patterns of VER2 in response to vernalization and devernalization. Devernalization treatment is a valuable control system of vernalization at the morphological or physiological level. The specificity of the antibody prepared with purified VER2 was detected by western blot analysis before immunocytochemical labelling. The anti-VER2 antibody specif- ically recognized a protein band at the expected molecular weight for VER2 (Figure S1) in wheat plants vernalized for 3 weeks. Furthermore, the labelling signal from the anti-VER2 antibody was consistent with the labelling results for antibody prepared with a synthesized polypeptide of VER2 [32]. Vernalized, devernalized and non-vernalized wheat plants were used for immunolabeling. Labeling signals were detected in shoot apical meristem and young leaves of plumules vernalized for 3 weeks (Figure 2A to C). Compared with nuclei indicated by hematoxylin staining in young leaves (Figure 2D), VER2 protein was shown to target predom- inantly to potential nuclear structures. In devernalized plants, immunolabeling signals were detected only in cytoplasm (Figure 2E), with no signal detected in nonvernalized plants (Figure 2F) or in the negative control (Figure 2G) under the same conditions. As well, immunolabeling signals were not detected in plants vernalized for 1 and 2 weeks (data not shown). Vernalization induces phosphorylation modification of VER2 in wheat The nucleocytoplasmic exchange of lectin plays a role in response to osmotic stress in yeast cells [33]. Amino acid sequence analysis indicated that VER2 does not contain a signal sequence for subcellular targeting. The different subcellular localization patterns of VER2 in response to vernalization and devernalization indicated that post-translational modification of VER2 might be involved in regulating its intracellular targeting. To address this possibility, total proteins from vernalized and devernalized materials were analyzed by 2-D gel electrophoresis and immunoblotted with anti-VER2 antibody. Continuous signal spots were apparent in wheat samples vernalized for 3 weeks (Figure 3A). Nevertheless, only a single spot was detected in devernalized plants (Figure 3B). Compared with the predicted isoelectric point (pI) of 6.6 (http://us.expasy.org/tools/ pi_tool.html) of VER2, the signal detected from vernalized plants (Figure 3A) showed an acidic shift. In the devernalized sample, the immunoblotting signal was at the approximate expected pI of VER2, which suggested that vernalization could induce post- translational phosphorylation modification of VER2. To further confirm the phosphorylation modification of VER2 in vernalized wheat plants, VER2 in vernalized and devernalized materials was immunoprecipitated and treated with protein phosphatase for 1, 2 and 3 h, then analyzed by immunoblotting. VER2 in vernalized plants was detected as two bands. Such mobility shift indicated that the fast migrating band in vernalized samples is the dephosphorylation form of VER2. The level of the dephosphorylated form of VER2 increased with prolonged treatment time. No band shift of VER2 occurred in the devernalized sample after 3 h of phosphatase treatment (Figure 3C). Because of lack of difference in shift distance of VER2 before phosphatase treatment in vernalized and deverna- lized materials due to the shorter migration distance, samples for SDS-PAGE were separated for more time before immunoblotting analysis. The following immunoblot result showed that VER2 in devernalized material migrated faster than that in vernalized material when the marker band of 25 kDa was allowed to move to the forefront of the 8-cm gel (Figure 3D). Thus, the difference in migration distance of VER2 between vernalized and devernalized wheat plants resulted from the post-translational phosphorylation modification of VER2 induced by vernalization. Vernalization induces an increase of O-GlcNAc modification of proteins at the global level and the binding of VER2 to O-GlcNAc-modified protein(s) in wheat To test whether the O-GlcNAc modification of proteins is affected by vernalization, which could be regarded as one kind of special environmental stress, we detected the variation of O- GlcNAc modified proteins at the global level in response to PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4854