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

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.

Over-Expressing TaSPA-B Reduces Prolamin and Starch Accumulation in Wheat (Triticum aestivum L.) Grains

“…TaSPA-B and TaPBF-D have been identified to regulate wheat glutenin gene, and their homologs in maize (O2 and PBF) show stimulatory and additive effects on maize zein and wheat glutenin genes [14,34,38]. In our results, ω-1,2 gliadin gene and TaPBF showed a little reduction in TaSPA-B OE-3 lines compared with WT ( Figure 5, Table 2).…”

“…Consistent with these results, TaPBF was reduced in TaSPA-B OE-3 lines (Table 2). TaPBF activated glutenin genes by regulating methylation of the promoter [34]. Our DLR assay revealed that TaSPA-B and TaPBF-D could activate the expression of ω-1,2 gliadin gene, and TaPBF-D showed a higher activation than TaSPA-B ( Figure 7B).…”

“…Other TF genes related to SSRGs, such as the negative regulatory factor genes TabZIP151, TabZIP194.3 and TabZIP229.1 (up-regulated), and the positive regulatory factor gene TabZIP167.2 (down-regulated), were also differentially expressed (Table 2) [13]. A series of TFs, including TaSPA, TaGAMYB, TaFUSCA3, TaSHP, and TaPBF-D, regulate the glutenin genes in wheat [17,19,33,34,36,38]. In the TaSPA-B OE-3 lines, TaSPA-A and TaSPA-D were down-regulated ( Figure 5); TaSHP, encoding a transcriptional repressor of glutenin genes, was up-regulated; TaPBF, encoding a transcriptional activator of glutenin genes was down-regulated ( Table 2).…”

“…Previous researches focused on the modification of the prolamin genes and starch synthesis genes to obtain ideal phenotypes [31,32]. With advances in genomics, the regulatory mechanism underlying the TFs and gene networks becomes a hot topic in wheat grain research [13,17,19,27,33,34]. Maize O2 is a typical TF with a broad influence, especially on starch and protein synthesis [28,35].…”

EpiRILs

“…One alternative to the generation of epigenetic mutants is the use of drugs interfering with epigenetic pathways. Inhibitors of DNA methylases, such as 5-Azacytidine and Zebularine, have been successfully used to induce DNA demethylation in plants (Griffin, Niederhuth, & Schmitz, 2016;Pecinka & Liu, 2014), including crops (Sano, Kamada, Youssefian, Katsumi, & Wabiko, 1990;Santos et al, 2002;Zhu et al, 2018). Although most of hypomethylation and transcriptional changes induced by these drugs are only transient (Baubec, Pecinka, Rozhon, & Mittelsten Scheid, 2009), transgenerational effects have been observed in rice treated with 5-Azacytidine (Sano et al, 1990).…”