Phosphorylation Modification of Wheat Lectin VER2 Is Associated with Vernalization-Induced O -GlcNAc Signaling and Intracellular Motility Lijing Xing1, Juan Li1, Yunyuan Xu 1 , Zhihong Xu 2 , Kang Chong1,2* 1 Research Center for Molecular Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing, China, 2 National Centre for Plant Gene Research, Beijing, China Abstract Background: O-linked b-N-acetylglucosamine (O-GlcNAc) modification of proteins mediates stress response and cellular motility in animal cells. The plant lectin concanavalin A can increase nuclear O-GlcNAc levels and decrease cytoplasmic O- GlcNAc levels in T lymphocytes. However, the functions of O-GlcNAc signaling in plants, as well as the relation between plant lectins and O-GlcNAc in response to environmental stimuli are largely undefined. Methodology/Principal Findings: We describe a jacalin-like lectin VER2 in wheat that shows N-acetylglucosamine and galactose specificity. Immunocytochemical localization showed VER2 expression induced predominantly at potential nuclear structures in shoot tips and young leaves and weakly in cytoplasm in response to vernalization. In contrast, under devernalization (continuous stimulation with a higher temperature after vernalization), VER2 signals appeared predominantly in cytoplasm. 2-D electrophoresis, together with western blot analysis, showed phosphorylation modification of VER2 under vernalization. Immunoblot assay with O-GlcNAc-specific antibody revealed that vernalization increased O-GlcNAc modification of proteins at the global level. An O-GlcNAc-modified protein co-immunoprecipitated with VER2 in vernalized wheat plants but not in devernalized materials. The dynamic of VER2 was observed in transgenic Arabidopsis overexpressing the VER2-GFP fusion protein. Overexpressed VER2 accelerated nuclear migration. Immunogold labeling and indirect immunofluoresence colocalization assay indicated that VER2-GFP was targeted to the secretory pathway. Conclusions/Significance: O-GlcNAc signaling is involved in the vernalization response in wheat, and phosphorylation is necessary for the lectin VER2 involving O-GlcNAc signaling during vernalization. Our findings open the way to studies of O- GlcNAc protein modification in response to environmental signals in plants. Citation: Xing L, Li J, Xu Y, Xu Z, Chong K (2009) Phosphorylation Modification of Wheat Lectin VER2 Is Associated with Vernalization-Induced O-GlcNAc Signaling and Intracellular Motility. PLoS ONE 4(3): e4854. doi:10.1371/journal.pone.0004854 Editor: Hany A. El-Shemy, Cairo University, Egypt Received September 22, 2008; Accepted February 9, 2009; Published March 16, 2009 Copyright: 2009 Xing et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (NSFC 30470167); the National Science Foundation for Distinguished Young Scholars of China (30525026); and the Major State Basic Research Program of the People’s Republic of China (2007CB108700). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chongk@ibcas.ac.cn Introduction Plant lectins are specific carbohydrate-binding proteins classi- fied into various families. Jacalin-related lectins (JRL) are further subdivided into the galactose- and mannose-specific groups [1]. On the basis of their carbohydrate-binding characteristics, plant lectins are also divided into ‘‘classical’’ and ‘‘non-classical’’ lectins [2]. Classical lectins are usually abundant proteins with protein storage and plant defense properties in some plant tissues. In contrast, non-classical lectins induced by exogenous or endoge- nous stimuli are involved in specific protein-carbohydrate interactions and are suggested to play specific endogenous roles in plant tissues or cells [2]. Jacalin-related mannose-specific lectins have been reported in monocotyledonous plants. Orysata, a mannose-specific jacalin in rice, is a potent mitogen of T lymphocytes and is involved in stress defense-related protein-carbohydrate interactions in plants [3]. Recently, horcolin, a new jacalin-related lectin specific to mannose, from Hordeum vulgare, was proposed to perceive and transfer environmental stress signaling [4]. Despite the well- characterized carbohydrate-binding activity of plant lectins, cellular signaling and regulation mediated by the specific interaction of plant lectins with glycosylated proteins and glycoconjugates remains to be clarified. Nictaba, a tobacco lectin, is localized in the cytoplasm and nucleus, and the nuclear distribution of the protein is directed by a nuclear localization signal [5]. The constitutively overexpressed EGFP-Nictaba fusion protein showed lectin activity. High affinity of Nictaba for N- glycans suggested that it interacts with N-glycosylated nuclear proteins by binding to the nuclear envelope [6]. Although N- glycosylated cytoplasm and nuclear proteins possibly play important roles by interacting with lectins, nucleocytoplasmic proteins modified by O-linked b-N-acetylglucosamine (O-GlcNAc) are ubiquitous and of great significance in plant and animal cells PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4854