Detailed studies of wheat glutenin subunits have provided novel details of their molecular structures and interactions which allow the development of a model to explain their role in determining the visco-elastic properties of gluten and dough. The construction and analysis of near-isogenic and transgenic lines expressing novel subunit combinations or increased amounts of specific subunits allows differences in gluten properties to be related to the structures and properties of individual subunits, with potential benefits for the production of cultivars with improved properties for food processing or novel end users #
Starch and cell wall polysaccharides (dietary fibre) of cereal grains contribute to the health benefits associated with the consumption of whole grain cereal products, including reduced risk of obesity, type 2 diabetes, cardiovascular disease and colorectal cancer. The physiological bases for these effects are reviewed in relation to the structures and physical properties of the polysaccharides and their behaviour (including digestion and fermentation) in the gastro-intestinal tract. Strategies for modifying the content and composition of grain polysaccharides to increase their health benefits are discussed, including exploiting natural variation and using mutagenesis and transgenesis to generate further variation. These studies will facilitate the development of new types of cereals and cereal products to face the major health challenges of the 21st century.
BackgroundHigh amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter.ResultsAmylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR).ConclusionWe have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.
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
BackgroundManipulation of the amylose-amylopectin ratio in cereal starch has been identified as a major target for the production of starches with novel functional properties. In wheat, silencing of starch branching enzyme genes by a transgenic approach reportedly caused an increase of amylose content up to 70% of total starch, exhibiting novel and interesting nutritional characteristics.In this work, the functionality of starch branching enzyme IIa (SBEIIa) has been targeted in bread wheat by TILLING. An EMS-mutagenised wheat population has been screened using High Resolution Melting of PCR products to identify functional SNPs in the three homoeologous genes encoding the target enzyme in the hexaploid genome.ResultsThis analysis resulted in the identification of 56, 14 and 53 new allelic variants respectively for SBEIIa-A, SBEIIa-B and SBEIIa-D. The effects of the mutations on protein structure and functionality were evaluated by a bioinformatic approach. Two putative null alleles containing non-sense or splice site mutations were identified for each of the three homoeologous SBEIIa genes; qRT-PCR analysis showed a significant decrease of their gene expression and resulted in increased amylose content. Pyramiding of different single null homoeologous allowed to isolate double null mutants showing an increase of amylose content up to 21% compared to the control.ConclusionTILLING has successfully been used to generate novel alleles for SBEIIa genes known to control amylose content in wheat. Single and double null SBEIIa genotypes have been found to show a significant increase in amylose content.
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