Domenico Lafiandra

Wheat glutenin subunits and dough elasticity: findings of the EUROWHEAT project

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

Increasing the amylose content of durum wheat through silencing of the SBEIIa genes

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.

Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress.

In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterized by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The nonprolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of nonprolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress versus control). The 2‐D patterns of nonprolamin proteins were monitored to identify polypeptides affected by heat stress during grain fill. This study shows that heat stress alters significantly the durum wheat seed proteome, although the changes range is only between 1.2‐ and 2.2‐fold. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI‐TOF and MALDI‐TOF‐TOF MS and included HSPs, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress‐related proteins. Many of the heat‐induced polypeptides are considered to be allergenic for sensitive individuals.

High resolution melting analysis for the detection of EMS induced mutations in wheat SbeIIa genes

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.

Characterisation and chromosomal localisation of C-type low-molecular-weight glutenin subunits in the bread wheat cultivar Chinese Spring

Abstract Low-molecular-weight glutenin subunits are classically divided into the B, C and D groups. Most attention has been paid to the characterisation of the B and D groups, whereas C subunits, although represented by a large number of protein components, have not been thoroughly characterised, mainly because they tend to separate with the gliadins in many fractionation procedures. Here we describe a procedure for obtaining a fraction strongly enriched in C subunits that has allowed us to determine the chromosomal location of these subunits in the bread wheat cultivar Chinese Spring. This analysis has shown that these subunits are coded on chromosome groups 1 and 6. Comparison between N-terminal amino acid sequencing of B and C subunits has shown that, whereas the former group includes mainly subunits with typical LMW-GS type sequences (76%), the C subunit group is made up almost completely of subunits with gliadin-like sequences (95%), including the α-type. These results indicate that the LMW-GSs are likely to be coded not only by the typical Glu-3 loci, but also by loci tightly linked to, and possibly included within, the Gli-1 and Gli-2 loci.

N-terminal amino acid sequences of chloroform/methanol-soluble proteins and albumins from endosperms of wheat, barley and related species: Homology with inhibitors of α-amylase and trypsin and with 2 S storage globulins

The N‐terminal amino acid sequences of two chloroform/methanol soluble globulins from barley and one form wheat are reported. They are homologous with N‐terminal sequences previously reported for α‐amylase and trypsin inhibitors from cereals and 2 S storage proteins from castor bean and rape. Three albumins were also purified from Aegilops squarrosa and Triticum monococcum. These had N‐terminal amino acid sequences most closely related to the α‐amylase and trypsin inhibitors. The relationships of this superfamily of seed proteins are discussed.

Production of novel allelic variation for genes involved in starch biosynthesis through mutagenesis

Given the important role that starch plays in food and non-food uses of many crops, particularly wheat, efforts are being made to manipulate its composition through modification of the amylose/amylopectin ratio. Approaches used to achieve this goal include the manipulation of the genes involved in the starch biosynthetic pathway using natural or induced mutations and transgenic methods. The use of mutagenesis to produce novel allelic variation represents a powerful tool to increase genetic diversity and this approach seems particularly appropriate for starch synthase genes for which limited variation exists. In this work, an EMS-mutagenised population of bread wheat cv. Cadenza has been screened by combining SDS–PAGE analysis of granule bound starch proteins with a TILLING (Targeting Induced Local Lesions IN Genomes) approach at the gene level. In particular we have focused on two groups of synthase genes, those encoding the starch synthase II (Sgp-1) and those corresponding to the waxy proteins (Wx). SDS–PAGE analysis of granule bound proteins allowed the identification of single null genotypes associated with each of the three homoeologous loci. Molecular characterization of induced mutants has been performed using genome specific primer pairs for Sgp-1 and Wx genes. Additional novel allelic variation has also been detected at the different Sgp-1 homoeoloci by using a reverse genetic approach (TILLING). In particular single nucleotide substitutions, introducing a premature stop codon and creating amino acid substitutions, have been identified.

PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus.

Genes encoding high-molecular-weight (HMW) glutenin subunits, present in bread-wheat lines and cultivars, were studied by RFLP (restriction fragment length polymorphism) and PCR (polymerase chain reaction) analyses. In particular, allelic subunits of the x-or y-type, encoded at the Glu-D1 locus present on the long arm of chromosome 1D, were investigated. The variation in size, observed in different allelic subunits, is mainly due to variation in the length of the central repetitive domain, typical of these proteins. Deletions or duplications, probably caused by unequal crossingover, have given rise to the size heterogeneity currently observed. The possibility of using the PCR technique for a detailed analysis of HMW glutenin genes in order to obtain a more accurate estimation of the molecular weight of their encoded subunits, and the detection of unexpressed genes, is also described.

Chromosomal assignment of genes coding for the wheat gliadin protein components of the cultivars ‘Cheyenne’ and ‘Chinese Spring’ by two-dimensional (two-pH) electrophoresis

SummaryThe gliadin proteins of the hexaploid bread wheat cultivar ‘Cheyenne’ (Triticum aestivum L. var. aestivum), which has good mixing and baking characteristics, were analyzed by 2-dimensional (2-pH) polyacrylamide gel electrophoresis (pH 3.2 in the 1st dimension, pH 9.2 in the 2nd). Genes for most of the 35 resolved components were assigned to the chromosomes of homoeologous groups 1 and 6 through the use of various aneuploids and substitution lines. A similar analysis was carried out for the cultivar ‘Chinese Spring’, which does not have good quality, and which had been analyzed by a different 2-dimensional procedure by Wrigley and Shepherd (Ann NY Acad Sci 209:154, 1973). A coordinate system was devised for description of the components in the 2-dimensional patterns.

A 1B-coded low-molecular-weight glutenin subunit associated with quality in durum wheats shows strong similarity to a subunit present in some bread wheat cultivars.

Good quality durum wheats usually present the LMW-2 type of SDS-PAGE pattern, whereas the LMW-I type of pattern is usually associated with poor quality durum wheats. The two patterns are distinguished mainly by the presence of a strongly expressed protein band with molecular weight around 42,000 (42 K subunit) in the LMW-2-type pattern; this subunit is absent in the LMW-1-type pattern. Here we show that this particular low-molecular-weight glutenin subunit has strong similarity to a subunit present in some bread wheat cultivars. This correspondence has been demonstrated through SDS-PAGE, PCR analysis of the corresponding genes, a comparison of the deduced amino acid sequences, and RP-HPLC. This last approach showed a slight difference in retention time between the 42 K protein of bread and durum wheats that might be attributed to the eight amino acid differences found between the deduced amino acid sequences of the two corresponding genes.