5-Azacytidine treatment and TaPBF-D over-expression increases glutenin accumulation within the wheat grain by hypomethylating the Glu-1 promoters
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.
High-molecular-weight glutenin subunit
Low-molecular-weight glutenin subunit
Prolamin-box binding factor
Storage protein activator
GA-dependent MYB transcription factor
DNA methyltransferase homologue
This research was supported by Natural Science Foundation of China (31271706, 31471486) and Agricultural industrialization development project of high-quality seed from Shandong Province (2013). Authors thank former Prof. Robert Koebner in John Innes Centre of UK for critical comments and language improvement.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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