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      5-Azacytidine treatment and TaPBF-D over-expression increases glutenin accumulation within the wheat grain by hypomethylating the Glu-1 promoters.

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          Abstract

          5-azaC treatment and TaPBF - D over-expression decrease C-methylation status of three Glu - 1 gene promoters, and aid in enhancing the expression of the Glu - 1 genes. The wheat glutenins exert a strong influence over dough elasticity, but the regulation of their encoding genes has not been firmly established. Following treatment with 5-azacytidine (5-azaC), both the weight and glutenin content of the developing and mature grains were significantly increased. The abundance of transcript produced by the Glu-1 genes (encoding high-molecular-weight glutenin subunits), as well as those encoding demethylases and transcriptional factors associated with prolamin synthesis was higher than in grain of non-treated plants. These grains also contained an enhanced content of the prolamin box binding factor (PBF) protein. Bisulfite sequencing indicated that the Glu-1 promoters were less strongly C-methylated in the developing grain than in the flag leaf, while in the developing grain of 5-azaC treated plants, the C-methylation level was lower than in equivalent grains of non-treated plants. Both Glu-1 transcript abundance and glutenin content were higher in the grain set by three independent over-expressors of the D genome homoeolog of TaPBF than in the grain set by wild type plants. When assessed 10 days after flowering, the Glu-1 promoters' methylation level was lower in the developing grains set by the TaPBF-D over-expressor than in the wild type control. An electrophoretic mobility shift assay showed that PBF-D was able to bind in vitro to the P-box of Glu-1By8 and -1Dx2, while a ChIP-qPCR analysis revealed that a lower level of C-methylation in the Glu-1By8 and -1Dx2 promoters improved the TaPBF binding. We suggest that promoter DNA C-methylation is a key determinant of Glu-1 transcription.

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          Author and article information

          Journal
          Theor. Appl. Genet.
          TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik
          Springer Science and Business Media LLC
          1432-2242
          0040-5752
          Mar 2018
          : 131
          : 3
          Affiliations
          [1 ] The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Science, Shandong University, Jinan, 250100, China.
          [2 ] The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Science, Shandong University, Jinan, 250100, China. fanguo2002@sdu.edu.cn.
          Article
          10.1007/s00122-017-3032-z
          10.1007/s00122-017-3032-z
          29214328

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