Vernalization requirement duration in winter wheat is controlled by TaVRN-A1 at the protein level

Winter wheat requires a period of low temperatures to accelerate flowering (vernalization). This requirement could make winter wheat more vulnerable to elevated global temperature via insufficient vernalization. All known vernalization genes are cloned according to qualitative variation in vernalization requirement between spring and winter wheat, but the genes controlling quantitative variation for more or less vernalization requirement among winter wheat cultivars remain unknown. We report here that the gene for the vernalization requirement duration in winter wheat was cloned using a BC(1)F(2:3) population that segregated in a 3:1 ratio of early-flowering plants and late-flowering plants after vernalization for 3 weeks. The positional cloning of the gene for vernalization requirement duration demonstrated that this trait is controlled by TaVRN-A1 at the protein level. The Ala(180) in vrn-A1a, encoded by the dominant allele for 3-week vernalization, was mutated to Val(180) in vrn-A1b, encoded by the recessive allele for 6-week vernalization. Further studies with in vitro protein pull-down assays and immunoprecipitation analyses indicated that the mutated Val(180) in vrn-A1b protein decreased the ability to bind with TaHOX1 (the first homeobox protein in Triticum aestivum). The direct binding of TaVRN-A1 and TaHOX1 proteins was confirmed in the nucleus of living plant cells by bimolecular fluorescence complementation (BiFC) analyses. The TaHOX1 gene was found to be upregulated by low temperatures, and to have a significant genetic effect on heading date, suggesting that TaHOX1 functions in the flowering pathway in winter wheat.