Figure 3. Immunoblot analysis of VER2 separated on 2-D electrophoresis and dephosphorylation of VER2 by phospha- tase in vernalized and devernalized wheat plants. (A) VER2 was separated into 3 main spots with pI = 5.6, 6.1 and 6.7, respectively, in vernalized wheat plants. (B) VER2 was detected as one spot with pI = 6.7 in devernalized wheat plants. (C) VER2 was immunoprecipitated and then treated with protein phosphatase for the time shown in vernalized and devernalized plants. (D) Immunoblot analysis showing the difference of migration distance of VER2 in vernalized and devernalized materials. The samples were separated by electrophoresis for enough time until the marker band with the molecular weight of 25 kDa shifted to the forefront of the 8 cm gel. doi:10.1371/journal.pone.0004854.g003 vernalization and explored the possibility of VER2 binding with O-GlcNAcylated proteins because of the carbohydrate specificity of VER2 to N-acetylglucosamine. We detected O-GlcNAc- modified proteins in nonvernalized, vernalized and devernalized wheat plants using the monoclonal antibody CTD110.6 originally produced to specifically recognize Ser- or Thr-O-GlcNAc- modified proteins in animal cells [34]. Western blot analysis VER2 and O-GlcNAc Signaling showed that vernalization increased the global level of O-GlcNAc- modified proteins as compared with nonvernalization; further- more, devernalization decreased the level of O-GlcNAc-modified total proteins, with only a few proteins detected (Figure 4A, B). We then immunoprecipitated VER2 from vernalized and devernalized extracts of wheat plumules using the anti-VER2 antibody. The antibody CTD110.6 was used to detect O-GlcNAc-modified proteins in immunoprecipitates. A protein band of approximately 35 kDa was detected in vernalized plants. In contrast, no signal was detected in devernalized materials (Figure 4C). Neither vernalized nor devernalized plants showed O-GlcNAc-modified signals corresponding to VER2. Figure 4D shows that VER2 immunoprecipitated with O-GlcNAc-modified proteins in vernal- ized but not devernalized plants. Subcellular location and intracellular motility of VER2- GFP in transgenic Arabidopsis We generated 35S::VER2-GFP and 35S::GFP transgenic Arabi- dopsis 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). In epidermal cells of young leaves of transgenic Arabidopsis overexpressing VER2-GFP, VER2-carrying nuclear and perinucle- ar structures were observed to change direction randomly and move within the cell (Figure 6A). The proportion of observed cells with nuclear motility is about 10%. In vein cells, nuclei showed axial migration with a velocity of approximately 30 mm/min (Figure 6B), which is much faster than nuclear movement in Arabidopsis root hairs (,10 mm/min) [35]. However, nuclear migration in 35S::GFP transgenic Arabidopsis plants could not be observed under the same conditions. Therefore, VER2-GFP could alter the motility and position of nuclei in transgenic plants. Overexpression of VER2 was suggested to facilitate nuclear motility. Punctate-targeted VER2- GFP was also observed with a mobile pattern in leaf epidermis and veins (Figure 6C,D), and mobile punctate signals could fuse together. Nuclei are reported to move along microtubules [36]. To examine whether perinuclear-distributed VER2-GFP is associated with cellular microtubules or moves passively with the nucleus, we treated Arabidopsis epidermis with the cytoskeleton-destabilizing agent propyzamid (5 mM). The propyzamid was dissolved in DMSO and diluted to working concentration to incubate Arabidopsis leaves. As a control, DMSO treatment alone did not alter the location pattern of VER2-GFP (Figure 7A), but treatment with propyzamid destroyed the perinuclear tubular extension of VER2-GFP (Figure 7B). Thus, VER2 overexpressed at the perinuclear region is associated with microtubules. VER2-GFP is targeted to the secretory pathway but not as a sorting receptor To identify organelles responsible for the mobile punctate distribution pattern of VER2-GFP in transgenic Arabidopsis, PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4854