Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer

Abstract

Both high-and low-molecular-weight glutenin subunits (LMW-GSThe glutenin fraction of the gluten proteins is primarily responsible for the viscoelastic properties of wheat (Triticum aestivum L.) flour doughs. It consists of various types of protein subunits that are linked together by intermolecular disulfide bonds. These form a polymeric mixture that has a broad molecular-weight distribution, with component polymers ranging from the dimeric forms with molecular weights as low as 60,000, to polymers containing many subunits with molecular weights in the millions (for review, see Kasarda, 1989; Wrigley, 1996). Variations in the types and amounts of subunits correlate with quality variations among wheat cultivars, probably by affecting the molecular-weight distribution of the glutenin polymers (Gupta et al., 1993(Gupta et al., , 1995. There are two main types of subunits, the HMW-GS and the LMW-GS, with the former having been much more extensively characterized than the latter.Difficulties in characterization of LMW-GS arose because they derive from many more genes than HMW-GS and because the subunits are somewhat insoluble after reduction of the intermolecular disulfide bonds (which is necessary for their purification, but which also breaks down intramolecular disulfide bonds to expose buried hydrophobic regions). Until recently, almost all attempts at cloning lmw-gs genes led to DNA sequences corresponding to similar protein products that are not representative of the major LMW-GS types; almost all had the apparent N-terminal sequence METSCIPGL-, relatively low molecular weights of about 35,000 or less, and a total of eight Cys residues, including the Cys at position 5 (for review, see Shewry and Tatham, 1997; Cassidy et al., 1998).In contrast to the apparently single type (with very minor variations) of the LMW-GS indicated by the DNA sequencing, two main types of LMW-GS have been defined on the basis of N-terminal amino acid sequences: the LMW-s and LMW-m types, with the former starting with the sequence SHIPGL-, and the latter represented by the METSHIPGL-, METSRIPGL-, or METSCIPGL-N-terminal sequences (Kasarda et al., 1988; Tao and Kasarda, 1989; Lew et al., 1992). The LMW-s types are predominant. They also tend to have higher molecular weights, in the approximate range of 35,000 to 45,000 relative to the LMW-m types, which seem to fall into the wider molecular-weight range of about 30,000 to 45,000 (Lew et al., 1992). In bread wheat cultivars, the LMW-m type with the METSHIPGLsequence was the next most abundant type of LMW-GS, followed by the METSRIPGL-type, whereas the METSCIPGL-N-terminal sequence, typical of the cloned sequences, appeared to be somewhat rare among the types defined by direct protein sequencing (Lew et al., 1992). Both LMW-s and LMW-m types are coded by genes present at the complex Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3 in hexaploid wheat). Only partial sequence information has been available for the LMW-s types because 1

Novel x-Type High-Molecular-Weight Glutenin Genes From Aegilops tauschii and Their Implications on the Wheat Origin and Evolution Mechanism of Glu-D1-1 Proteins

“…Similar phenomena were observed for LMW-GS gene recombination clones when using different E. coli strains (Altenbach 1998;Masci et al 1998). There, single deletions of 50-200 bp occurred within the repetitive domains of different recombinant clones.…”

“…Because illegitimate recombination requires only a few base pairs of sequence identity (Wicker et al 2003), it is likely to occur within Glu-D1-1 alleles because of a few long direct repeats generally present in the repetitive domain of x-type HMW genes as shown in 1Dx5 *t and 1Dx2.2 * (Figures 3 and 6). Although fragment deletions within the repetitive domains of glutenin genes in recombinant clones were found (Altenbach 1998;Masci et al 1998), their molecular origins are still unknown. Our results provide direct evidence for illegitimate recombination in 1Dx5 *t within the E. coli heterologous expression system and have led to a large fragment deletion and generation of a small protein.…”

Characterization of wheat gluten proteins by hplc and MALDI TOF mass spectrometry

“…However,©such©comparisons©of©intact©protein©masses must© be© viewed© with© caution© since© some© of© the© cloned LMW© subunits© genes© used© to© deduce© the© sequence© may have© contained© deletions© of© the© repetitive© domain© [21] resulting© in© underestimation© of© the© actual© MW.© Moreover,©several©of©the©subunits©have©intact©molecular weights©too©similar©to©be©resolved©by©linear©MALDI TOF.…”

An LMW-s Glutenin Gene of a Hard Red Winter Wheat is Similar to an LMW-s Gene of a Canadian Western Extra-strong Wheat

“…It is therefore inferred that a large number of wheat varieties carry LMW-GSs similar to KS2/KS3a and GL1/GL2. Maruyama-Funatsuki et al (2005) suggested that HA1 is highly similar to the 42-kDa subunit of 'Yecora Rojo' described by Masci et al (1998), since their mobilities in PAGE and their gene candidates are very similar. Masci et al (2000) showed that several bread wheat cultivars carry LMW-GSs similar to the 42-kDa subunit.…”