Abstract
Subunit pair 1Dx5 + 1Dy10 was recognized as superior subunit combination in wheat and contained an extra repetitive-domain cysteine residue in 1Dx5 that was important for understanding the formation of dough viscoelasticity. In this research, one specific serine codon of the 1Ax1 gene corresponding to the extra cysteine residue of 1Dx5 was substituted by a cysteine codon through site-directed mutagenesis. Four homozygous transgenic lines (T4) expressing the mutant 1Ax1 gene (mut1Ax1) were produced. Their greater dough strength and stability were confirmed by mixograph and were associated with highly increased gluten index, larger amounts of gluten macropolymers, larger size distribution for glutenin macropolymer particles and varied sodium-dodecyl-sulfate sedimentation volumes, compared with those of the one line expressing wild 1Ax1 that had similar expression level of transgene. The contents of β-sheets in dough and disulfide groups in gluten of the mut1Ax1 transgenic lines were significantly increased. The microstructure of dough mixed to peak showed a more continuous gluten matrix in the mutant transgenic lines than the one line mentioned-above. It was concluded that the extra cysteine residue of mutant 1Ax1 subunit plays a positive role in contributing to dough strength and stability of wheat by cross-linking into gluten aggregates through inter-chain disulfide bonds.
Highlights
The unique viscoelasticity of wheat dough is conferred by decisive gluten proteins in wheat grains and produces different types of wheat end-use products, such as breads, cakes, and other flour-based foods[1, 2]
The sequencing results for the reverse transcription-polymerase chain reaction (PCR) (RT-PCR) products of target gene mutant 1Ax1 gene (mut1Ax1) showed that the introduced gene expressed in the transgenic wheat lines had been successfully site-mutated (Supplementary Fig. S2C and D)
Some researchers have attributed this mechanism to the presence of the extra cysteine residue in the repetitive domain[14], which is due to the “head-to-tail” HMW polymer formed by disulfide crosslinking among HMW-GSs34
Summary
The unique viscoelasticity of wheat dough is conferred by decisive gluten proteins in wheat grains and produces different types of wheat end-use products, such as breads, cakes, and other flour-based foods[1, 2]. The expression of the exogenous genes 1Dx5 and 1Ax1 had different impacts on flour functional properties. Over-expression of transgene 1Dx5 dramatically increased the amount of glutenin polymers and resulted in “over-strong” dough properties, which were not suitable for making bread[11]. Leόn et al.[16] reported that the expression of subunit genes 1Ax1 and 1Dx5 in the bread-making wheat cv. The extra cysteine in specific x-type HMW-GSs such as 1Bx7.1 may be not as important as previously reported or even interfere with glutenin polymerisation, which further has negative effect on the dough mixing properties[17, 18]. The effect of the extra cysteine residue of x-type HMW-GSs on the functional properties of flour remains unknown. Subunit 1Dx5 generally occurs with 1Dy1019, 20, and its effect might be influenced by the total amounts of both subunits, and the ratio of 1Dx5/1Dy10 and a balanced D-subunit composition[14, 21]
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