The vernalization-induced long non-coding RNA VAS functions with the transcription factor TaRF2b to promote TaVRN1 expression for flowering in hexaploid wheat.

Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.

The Protein Modifications of O-GlcNAcylation and Phosphorylation Mediate Vernalization Response for Flowering in Winter Wheat.

O-GlcNAcylation and phosphorylation are two posttranslational modifications that antagonistically regulate protein function. However, the regulation of and the cross talk between these two protein modifications are poorly understood in plants. Here we investigated the role of O-GlcNAcylation during vernalization, a process whereby prolonged cold exposure promotes flowering in winter wheat (Triticum aestivum), and analyzed the dynamic profile of O-GlcNAcylated and phosphorylated proteins in response to vernalization. Altering O-GlcNAc signaling by chemical inhibitors affected the vernalization response, modifying the expression of VRN genes and subsequently affecting flowering transition. Over a vernalization time-course, O-GlcNAcylated and phosphorylated peptides were enriched from winter wheat plumules by Lectin weak affinity chromatography and iTRAQ-TiO2, respectively. Subsequent mass spectrometry and gene ontology term enrichment analysis identified 168 O-GlcNAcylated proteins that are mainly involved in responses to abiotic stimulus and hormones, metabolic processing, and gene expression; and 124 differentially expressed phosphorylated proteins that participate in translation, transcription, and metabolic processing. Of note, 31 vernalization-associated proteins were identified that carried both phosphorylation and O-GlcNAcylation modifications, of which the majority (97%) exhibited the coexisting module and the remainder exhibited the potential competitive module. Among these, TaGRP2 was decorated with dynamic O-GlcNAcylation (S87) and phosphorylation (S152) modifications, and the mutation of S87 and S152 affected the binding of TaGRP2 to the RIP3 motif of TaVRN1 in vitro. Our data suggest that a dynamic network of O-GlcNAcylation and phosphorylation at key pathway nodes regulate the vernalization response and mediate flowering in wheat.

Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering.

Post-translational modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) is catalyzed by O-GlcNAc transferases (OGTs). O-GlcNAc modification of proteins regulates multiple important biological processes in metazoans. However, whether protein O-GlcNAcylation is involved in epigenetic processes during plant development is largely unknown. Here, we show that loss of function of SECRET AGENT (SEC), an OGT in Arabidopsis, leads to an early flowering phenotype. This results from reduced histone H3 lysine 4 trimethylation (H3K4me3) of FLOWERING LOCUS C (FLC) locus, which encodes a key negative regulator of flowering. SEC activates ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), a histone lysine methyltransferase (HKMT), through O-GlcNAc modification to augment ATX1-mediated H3K4me3 histone modification at FLC locus. SEC transfers an O-GlcNAc group on Ser947 of ATX1, which resides in the SET domain, thereby activating ATX1. Taken together, these results uncover a novel post-translational O-GlcNAc modification-mediated mechanism for regulation of HKMT activity and establish the function of O-GlcNAc signaling in epigenetic processes in plants.

JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis.

Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1's function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1's regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis.

Smoking was associated with poor response to intravenous steroids therapy in Graves' ophthalmopathy.

BACKGROUND: Previous studies have shown that smoking is closely related to the occurrence, severity and response to orbital radiation in Graves' ophthalmopathy (GO). The aim of this study was to investigate whether smoking impacts the response to intravenous 4.5 g methylprednisolone therapy in patients with active moderate-to-severe GO. METHODS: Ninety-two individuals with active moderate-to-severe GO who were treated with cumulative doses of 4.5 g intravenous methylprednisolone within 3 months were recruited. The patients were grouped as never smokers, active smokers (including smokers and quit smokers) and passive smokers. RESULTS: We observed significantly greater response rate in never smokers compared with active smokers (73.9% vs 29.0%, p=0.001). After adjusting the confounding factors such as age, sex, body mass index, clinical activity score, thyroid-stimulating hormone receptor antibody and the duration of GO, smoking was independently associated with poor intravenous glucocorticoid (GC) response (OR 12.40, 95% CI 1.20 to 128.14, p=0.035). We also found the response rate was significantly higher in never smokers than in quit smokers (73.9% vs 16.7%, p=0.001), while no statistical significance between current smokers and quit smokers (36.8% vs 16.7%, p=0.228). There was a trend of poor response for passive smokers compared with never smokers (64.7% vs 72.2%, p=0.583). CONCLUSIONS: Smoking, even past smoking, was an independent risk factor associated with impaired response to intravenous corticosteroids in patients with GO. Smokers with GO should be given optimised treatment strategy such as higher dose of GC or combined radiation therapy.

O-GlcNAc-mediated interaction between VER2 and TaGRP2 elicits TaVRN1 mRNA accumulation during vernalization in winter wheat.

Vernalization, sensing of prolonged cold, is important for seasonal flowering in eudicots and monocots. While vernalization silences a repressor (FLC, MADS-box transcription factor) in eudicots, it induces an activator (TaVRN1, an AP1 clade MADS-box transcription factor) in monocots. The mechanism for TaVRN1 induction during vernalization is not well understood. Here we reveal a novel mechanism for controlling TaVRN1 mRNA accumulation in response to prolonged cold sensing in wheat. The carbohydrate-binding protein VER2, a jacalin lectin, promotes TaVRN1 upregulation by physically interacting with the RNA-binding protein TaGRP2. TaGRP2 binds to TaVRN1 pre-mRNA and inhibits TaVRN1 mRNA accumulation. The physical interaction between VER2 and TaGRP2 is controlled by TaGRP2 O-GlcNAc modification, which gradually increases during vernalization. The interaction between VER2 and O-GlcNAc-TaGRP2 reduces TaGRP2 protein accumulation in the nucleus and/or promotes TaGRP2 dissociation from TaVRN1, leading to TaVRN1 mRNA accumulation. Our data reveal a new mechanism for sensing prolonged cold in temperate cereals.

Requirement of histone acetyltransferases HAM1 and HAM2 for epigenetic modification of FLC in regulating flowering in Arabidopsis.

Histone acetylation is an important posttranslational modification associated with gene activation. In Arabidopsis, two MYST histone acetyltransferases HAM1 and HAM2 work redundantly to acetylate histone H4 lysine 5 (H4K5ace) in vitro. The double mutant ham1/ham2 is lethal, which suggests the critical role of HAM1 and HAM2 in development. Here, we used an artificial microRNA (amiRNA) strategy in Arabidopsis to uncover a novel function of HAM1 and HAM2. The amiRNA-HAM1/2 transgenic plants showed early flowering and reduced fertility. In addition, they responded normally to photoperiod, gibberellic acid treatment, and vernalization. The expression of flowering-repressor FLOWERING LOCUS C (FLC) and its homologues, MADS-box Affecting Flowering genes 3/4 (MAF3/4), were decreased in amiRNA-HAM1/2 lines. HAM1 overexpression caused late flowering and elevated expression of FLC and MAF3/4. Mutation of FLC almost rescued the late flowering with HAM1 overexpression, which suggests that HAM1 regulation of flowering time depended on FLC. Global H4 acetylation was decreased in amiRNA-HAM1/2 lines, but increased in HAM1-OE lines, which further confirmed the acetyltransferase activity of HAM1 in vivo. Chromatin immunoprecipitation revealed that H4 hyperacetylation and H4K5ace at FLC and MAF3/4 were less abundant in amiRNA-HAM1/2 lines than the wild type, but were enriched in HAM1-OE lines. Thus, HAM1 and HAM2 may affect flowering time by epigenetic modification of FLC and MAF3/4 chromatins at H4K5 acetylation.

Phosphorylation modification of wheat lectin VER2 is associated with vernalization-induced O-GlcNAc signaling and intracellular motility.

BACKGROUND: O-linked beta-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.

Cloning and expression of a novel cDNA encoding a mannose-specific jacalin-related lectin from Oryza sativa.

Lectin plays an important role in defense signaling in plants. A few genes of this family have been cloned. Here we report on a mannose-specific jacalin-related lectin in rice. Using sequence information of wheat gene VER2, which we had previously cloned, we were able to amplify a cDNA of OsJAC1 from Oryza sativa by RT-PCR. The cDNA of OsJAC1 was 1172 bp and contained a 921-bp open reading frame (ORF) encoding dirigent (amino acids 26-139) and jacalin (amino acids 175-305) domains of 306 amino acids. Comparison of the OsJAC1 sequence with those of other lectins (jacalin) from rice, wheat and other species revealed that OsJAC1 had the 12 amino acid positions conserved in all mannose-binding lectins. Semi-quantitative RT-PCR revealed that OsJAC1 expression was present in stems, leaves and young spikes but not young roots; the expression was high in leaves and low in stems and spikes. And methyl jasmonate could induce the expression of OsJAC1. To test the activity of OsJAC1, the jacalin domain at the C-terminal was expressed in E. coli. BL21 (DE3). The purified recombinant protein could agglutinate red blood cells of rabbit, and the agglutination activity was strongly inhibited by mannose compared with other carbohydrates. These results indicate that lectin with dirigent and jacalin domains exists in rice as well as wheat. This is the first report of cDNA cloning of mannose-binding jacalin-related lectin with a dirigent domain in N-terminal region from O. sativa.