Deletion of high-molecular-weight glutenin subunits in wheat significantly reduced dough strength and bread-baking quality

Abstract

Background: High-molecular-weight glutenin subunits (HMW-GS) play important roles in the elasticity of dough made from wheat. The HMW-GS null line is useful for studying the contribution of HMW-GS to the end-use quality of wheat.

Methods: In a previous work, we cloned the Glu-1E^bx gene from Thinopyrum bessarabicum and introduced it into the wheat cultivar, Bobwhite. In addition to lines expressing the Glu-1E^bx gene, we also obtained a transgenic line (LH-11) with all the HMW-GS genes silenced. The HMW-GS deletion was stably inherited as a dominant and conformed to Mendel's laws. Expression levels of HMW-GS were determined by RT-PCR and epigenetic changes in methylation patterns and small RNAs were analyzed. Glutenins and gliadins were separated and quantitated by reversed-phase ultra-performance liquid chromatography. Measurement of glutenin macropolymer, and analysis of agronomic traits and end-use quality were also performed.

Results: DNA methylation and the presence of small double-stranded RNA may be the causes of post-transcriptional gene silencing in LH-11. The accumulation rate and final content of glutenin macropolymer (GMP) in LH-11 were significantly lower than in wild-type (WT) Bobwhite. The total protein content was not significantly affected as the total gliadin content increased in LH-11 compared to WT. Deletion of HMW-GS also changed the content of different gliadin fractions. The ratio of ω-gliadin increased, whereas α/β- and γ-gliadins declined in LH-11. The wet gluten content, sedimentation value, development time and stability time of LH-11 were remarkably lower than that of Bobwhite. Bread cannot be made using the flour of LH-11.

Conclusions: Post-transcriptional gene silencing through epigenetic changes and RNA inhibition appear to be the causes for the gene expression deficiency in the transgenic line LH-11. The silencing of HMW-GW in LH-11 significantly reduced the dough properties, GMP content, wet gluten content, sedimentation value, development time and stability time of flour made from this wheat cultivar. The HMW-GS null line may provide a potential material for biscuit-making because of its low dough strength.

Keywords: Common wheat; Dough quality; Gliadin content; Glutenin macropolymer; High-molecular-weight glutenin subunits; Post-transcriptional gene silencing.

Fig. 1

Comparison of glutenin subunits and gliadins in wild-type and transgenic line LH-11 detected by SDS-PAGE and RP-UPLC. a SDS-PAGE analysis of LH-11. WT, wild-type Bobwhite; 1–4, transgenic line LH-11 in T₁ generation; 5–8, transgenic line LH-11 in T₂ generation; (b) RP-UPLC analysis of glutenin subunits; (c) RP-UPLC analysis of gliadins

Fig. 2

Expression analysis of HMW-GS genes (Glu-1) and Glu-1E^bx gene using RT-PCR. Almost all the HMW-GS genes were silenced in transgenic line LH-11. a Glu-1By9 and Glu-1Dy10 genes. b Glu-1Ax2* gene. c Glu-1Bx7 gene. d Glu-1Dx5 gene. e Glu-1By9, Glu-1By9, and Glu-1E^bx genes. f β-tubulin. M, marker; 1–6 cDNA from the seeds of transgenic line LH-11 at 6, 9, 12, 15, 18 and 21 days after flowering (DAF); 7–10 cDNA from the seeds of wild-type Bobwhite at 9, 12, 15 and 18 DAF. The numbers on the left side of the figure indicate the sizes (kb) of the PCR bands. The characters on the right side of the figure are the gene names

Fig. 3

DNA methylation analysis of transgenic line LH-11. DNA methylations were detected in Glu-1Bx7, Glu-1Dx5, Glu-1Dy10 and Glu-1E^bx genes of LH-11. a Glu-1Bx7 gene. H, HpaII/HindIII digestion; M, MspI/HindIII digestion. b Glu-1Dx5 gene. H, HpaII/HindIII digestion; M, MspI/HindIII digestion. c Glu-1Dy10 gene. H, HpaII/NaeI digestion; M, MspI/NaeI digestion. d Glu-1E^bx gene. H, HpaII/EcoRI digestion; M, MspI/EcoRI digestion

Fig. 4

Small RNA analysis of LH-11. Two hybridization signals of small RNA (about 20–25 nt in size) were detected in LH-11, whereas no signal in wild-type Bobwhite. Arrows point to the fragments of small RNA

Fig. 5

Accumulations of GMP during seed development in wild-type Bobwhite and transgenic line LH-11. a year 2008; b year 2009. WT, wild-type Bobwhite. Statistical significance was determined by a Student’s t-test at P < 0.01