Abstract
Key message
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.
Abstract
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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Abbreviations
- HMW-GS:
-
High-molecular-weight glutenin subunit
- LMW-GS:
-
Low-molecular-weight glutenin subunit
- PBF:
-
Prolamin-box binding factor
- SPA:
-
Storage protein activator
- GAMYB:
-
GA-dependent MYB transcription factor
- MET:
-
Methyltransferase
- CMT:
-
Chromomethylase
- DRM:
-
Domains-rearranged methyltransferase
- DNMT:
-
DNA methyltransferase homologue
- DME:
-
Demethyltransferase
- DML:
-
DME-like genes
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Acknowledgements
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.
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Communicated by Alan H. Schulman.
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122_2017_3032_MOESM1_ESM.pdf
Fig. S1 PCR assay used to validate the TaPBF-D over-expressor line. Lanes 1-7, line PBF-5-1 (T2 generation); lanes 9-22, line PBF-10-3 (T2 generation); lanes 24-26, line PBF-23-2 (T2 generation); Lanes 8, 23, and 26, positive control; Marker, λ/EcoRI + HindIII size marker. (PDF 86 kb)
122_2017_3032_MOESM2_ESM.pdf
Fig. S2 Induction of expression (a) and enriched (b) of recombinant TaPBF-D-GST. Crude bacterial extracts and the eluted protein were resolved by SDS-PAGE. Molecular mass markers are indicated on the left in kDa. (PDF 73 kb)
122_2017_3032_MOESM3_ESM.pdf
Fig. S3 The response of PBs to 5-azaC treatment. (a) Transmission electron micrographs of endosperm tissue sampled at 10, 15, and 20 DAF. (b)-(e) The mean number and size distribution of PBs. PB: protein body, N: nucleus, S: starch. Whiskers indicate the confidence interval based on a Student’s t test (P < 0.05, n = 3); asterisks indicate values which differ significantly between grain set by non-treated and treated plants. (PDF 198 kb)
122_2017_3032_MOESM4_ESM.pdf
Fig. S4 C-methylation in the Glu-1 promoters in DNA recovered from the developing grain set by non-treated (CS) and treated (5-azaC-CS) plants. (a) -1Bx7 (-561 to -90), (b) -1By8 (-445 to -5), (c) -1By8 (-551 to -105). The two types of cytosine residues, CG (red circles), CHG (blue square) were shown in the map. Filled and empty circles or squares denote methylated and unmethylated cytosines, respectively. The arrows indicate the location of different methylation types: CHG (empty arrows); CG (filled arrows). (PDF 389 kb)
122_2017_3032_MOESM5_ESM.pdf
Fig. S5 The transcription of TaSPA and genes encoding methylases and demethylases in wild type and the TaPBF-D over-expressor line. Whiskers indicate the confidence interval based on a Student’s t test (P < 0.05, n = 3); asterisks indicate values which differ significantly between grain set by wild type and the TaPBF-D over-expressor lines. (PDF 103 kb)
122_2017_3032_MOESM6_ESM.pdf
Fig. S6 Global C-methylation levels in developing grain set by wild type and the TaPBF-D over-expressor lines. (PDF 111 kb)
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Zhu, J., Fang, L., Yu, J. et al. 5-Azacytidine treatment and TaPBF-D over-expression increases glutenin accumulation within the wheat grain by hypomethylating the Glu-1 promoters. Theor Appl Genet 131, 735–746 (2018). https://doi.org/10.1007/s00122-017-3032-z
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DOI: https://doi.org/10.1007/s00122-017-3032-z