Mechanism On The Role Of HMW-GS Dy10 And Dx5subunits In The Formation Of Wheat Processing Quality

Wheat processing quality is mainly determined by the composition and content of wheat gluten.The gluten proteins,which are crucial components of the protein network,are composed of gliadins and glutenins.The HMW-GSs account for only 7%-12%of the glutenins in common wheat（Triticum aestivum）,but their composition and concentration have critical effects on gluten structure and determine the viscoelastic properties of dough through intermolecular disulfide bonds.Therefore,the identification of novel HMW-GS can provide excellent gene resources for wheat quality improvement.Previous research indicated that Dx5+Dy10 is the superior HMW-GS subunit pair for wheat processing quality.Here we investigate the mechanisms of Dy10 and Dx5 subunits on wheat processing quality by using three new genetic materials.The main results are as follows:1.After screening an EMS-induced‘Shumai 482’population,a mutant C144-10 line with an extra protein band in the HMW-GS zone was obtained.The extra protein band was derived from the Dy10 subunit by genetic analysis,immunoblot analysis,and mass spectrometry.Molecular cloning and sequence analysis revealed that the Dy10 allele,Dy10-m619SN,had a missense mutation（G to A）,resulting in an amino acid substitution（serine-to-asparagine）at the 619^thresidue within the C-terminal domain.C-terminal sequencing,prokaryotic expression and transient expression analysis showed that Dy10-m619SN produced two new peptides,due to a partial post-translational cleavage.Moreover,the post-translational cleavage of Dy10-m619SN subunit is synchronized with the synthesis of HMW-GS in wheat grain.2.In the C144-10 line,some members of Ta VPE family were significantly up-regulated by RNA-seq and q RT-PCR analysis.In vitro digestion experiments showed that vacuolar processing enzymes extracted from rice or wheat seeds can specifically cleave the Dy10-m619SN subunit.Phylogenetic analysis showed that multiple copies of seed-type Ta VPE were located on the chromosome 2 and originated from a common ancestor.RT-PCR analysis showed that all seed-type Ta VPE can be expressed in wheat endosperm,but they had different spatiotemporal gene expression patterns and expression levels.Subcellular localization indicated Ta VPEIa was located at the vacuole,suggesting that Ta VPE may be responsible for the post-translational cleavage.Purified Ta VPEIa can process the Dy10-m619SN subunit into mature forms.Similarly,other seed-type Ta VPEs were able to efficiently cleave the Dy10-m619SN subunit.3.The C144-10 mutant was backcrossed with the Shumai 482 to build near-isogenic lines.No signifcant differences in agronomic characteristics（i.e.,plant height,spikelet length,1000-kernel weight,kernel length and width）were detected between the Shumai 482 and C144-10.Dy10-m619SN expression in C144-10 decreased the size of glutenin polymers,gluten index,Zeleny sedimentation value,dough development and stability times and lower bread quality.However,the biscuits of C144-10 showed larger diameter,less thickness and higher spread ratio.These new peptides of Dy10-m619SN disrupted the interactions among gluten proteins because of the associated changes to the number of available cysteine residues for interchain disulfde bonds.Consequently,Dy10-m619SN expression decreased the size of glutenin polymers and weakened glutens,which resulted in wheat dough with improved cookie-making quality,with no change to the glutenin-to-gliadin ratio.4.A Dy10 subunit deletion mutant,C143-22,was screened from an EMS-induced‘Shumai 482’population by SDS-PAGE.Molecular cloning and sequence analysis showed that the Dy10-null allele had a nonsense mutation（C to T）,which resulted in a premature termination of translation at the 217^thamino acid residue.RT-PCR and western blotting revealed the Dy10-null allele was transcribed normally,but there was a lack of the Dy10subunit.C143-22 was backcrossed with the Shumai 482 to build near-isogenic lines.No significant difference in agronomic characteristics（i.e.,plant height,spikelet length,1000-kernel weight,kernel length and width）were detected between the Shumai 482 and C143-22.Our findings suggested that the Dy10-null allele decreased the glutenin-to-gliadin ratio and negatively affected dough strength（i.e.,Zeleny sedimentation value,gluten index,dough development time and stability time）and the bread-making quality;however,it positively affected the biscuit-making quality.The incorporation of various amounts of purified Dy10 into wheat flour gave rise to inferior biscuit-making quality.5.The morphology and distribution of protein bodies in developing and mature grains of transgenic wheat with high over-expression of HMW-GS 1Dx5 was observed by light microscopy,image analysis and immunofluorescence microscopy.The result confirmed that the number of the smallest PB was significantly higher in sub-aleurone cells.The segregation of the HMW-GS in ER-derived PB may affect grain processing quality as over-expressions of HMW-GS Dx5 is known to result in the formation of insoluble polymers and an over-strong dough phenotype.