Stefania Masci

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.

Comparative proteomic and transcriptional profiling of a bread wheat cultivar and its derived transgenic line overexpressing a low molecular weight glutenin subunit gene in the endosperm

We carried out a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that overexpresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding nontransformed genotype. Proteomic analyses showed that, during seed development, several classes of endosperm proteins were differentially accumulated in the transformed endosperm. As a result of the strong increase in the amount of the transgenic protein, the endogenous glutenin subunit, all subclasses of gliadins, and metabolic as well as chloroform/methanol soluble proteins were diminished in the transgenic genotype. The differential accumulation detected by proteomic analyses, both in mature and developing seeds, was paralleled by the corresponding changes in transcript levels detected by microarray experiments. Our results suggest that the most evident effect of the strong overexpression of the transgenic glutenin gene consists in a global compensatory response involving a significant decrease in the amounts of polypeptides belonging to the prolamin superfamily. It is likely that such compensation is a consequence of the diversion of amino acid reserves and translation machinery to the synthesis of the transgenic glutenin subunit.

Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene

The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer.

Comparison of the B and D subunits of glutenin encoded at the Glu-D3 locus in two biotypes of the common wheat cultivar Newton with different technological characteristics

The common wheat cultivar Newton occurs as two biotypes with different electrophoretic patterns of the Gli-D1-encoded ω-gliadins, which resemble the analogous patterns found in the cultivars Chinese Spring and Cheyenne. The α-, β-and γ-gliadin and high molecular weight glutenin subunit patterns are identical, however. The two biotypes, which have different technological properties, have been used to study the B and D groups of low molecular weight glutenin subunits encoded at the Glu-D3 locus, which is tightly linked to Gli-D1, in order to detect possible relations between these subunits and breadmaking properties. Whereas Newton (Chinese Spring type) possessed D subunits of glutenin, Newton (Cheyenne type) had only ω-gliadins in the corresponding field of the two-dimensional electrophoresis gel. D subunits appear to originate from mutated ω-gliadin coding genes. A comparable situation was also found in the cultivars Chinese Spring and Cheyenne. Analysis of the B group of low molecular weight glutenin subunits indicated that each biotype possessed one specific component that differed between the two. The 1D-encoded basic low molecular weight glutenin subunits of the cultivars Chinese Spring and Cheyenne were similarly differentiated. The role of these different low molecular weight subunits of glutenin in the two Newton biotypes in determining breadmaking quality characteristics is now under investigation.

Constitutive expression of the xylanase inhibitor TAXI-III delays Fusarium head blight symptoms in durum wheat transgenic plants.

Cereals contain xylanase inhibitor (XI) proteins which inhibit microbial xylanases and are considered part of the defense mechanisms to counteract microbial pathogens. Nevertheless, in planta evidence for this role has not been reported yet. Therefore, we produced a number of transgenic plants constitutively overexpressing TAXI-III, a member of the TAXI type XI that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We also showed that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium head blight disease symptoms caused by Fusarium graminearum but do not significantly influence leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide, for the first time, clear evidence in planta that XI are involved in plant defense against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum.

Sequence analysis of the 5' non-coding regions of active and inactive 1Ay HMW glutenin genes from wild and cultivated wheats

Abstract The genes encoding the HMW glutenin subunits belong to a small gene family where not all members are expressed in cultivated wheat varieties. In particular, the Ay HMW glutenin subunit is always absent. On the basis of nucleotide sequence comparisons of the putative promoter regions of the 1Ay gene of six different active HMW glutenin genes, it was previously hypothesized that the silencing of the 1Ay gene was caused by a few specific nucleotide substitutions in the 5′ flanking region. The availability of wild wheat progenitors expressing the Ay glutenin subunit allowed us to compare the 5′ flanking region of active and inactive genes. We have found a nearly perfect nucleotide sequence identity in this region between active and inactive 1Ay genes, and thus demonstrating that the inactivation of the gene encoding this subunit in cultivated wheats is not due to the 10 substitutions occurring in this region.

Differential Expression of Durum Wheat Gluten Proteome under Water Stress during Grain Filling.

Environmental stress during grain filling may affect wheat protein composition, thus influencing its final quality. A proteomic approach was used to evaluate changes in storage protein composition under water stress of two Italian durum wheat (Triticum turgidum ssp. durum) cultivars, Ciccio and Svevo. The high-molecular-weight glutenin region increased progressively in both cultivars and under two water regimens. The L48-35 region, corresponding to low-molecular-weight (LMW) glutenin subunits, increased slightly during grain development and decreased under water stress in both cultivars. In particular, an s-type LMW related to superior technological quality was down-expressed in the early-mid period in Svevo and in the mid-late period in Ciccio. Finally, the L<35 region, corresponding to gliadin-like proteins, decreased slightly during grain development and increased under stress in both cultivars. Several α-gliadins, associated with immunological potential, increased their expression under water stress, especially in Svevo in the early-mid stage of grain filling.

A Relaxed Specificity in Interchain Disulfide Bond Formation Characterizes the Assembly of a Low-Molecular-Weight Glutenin Subunit in the Endoplasmic Reticulum

Wheat (Triticum spp.) grains contain large protein polymers constituted by two main classes of polypeptides: the high-molecular-weight glutenin subunits and the low-molecular-weight glutenin subunits (LMW-GS). These polymers are among the largest protein molecules known in nature and are the main determinants of the superior technological properties of wheat flours. However, little is known about the mechanisms controlling the assembly of the different subunits and the way they are arranged in the final polymer. Here, we have addressed these issues by analyzing the formation of interchain disulfide bonds between identical and different LMW-GS and by studying the assembly of mutants lacking individual intrachain disulfides. Our results indicate that individual cysteine residues that remain available for disulfide bond formation in the folded monomer can form interchain disulfide bonds with a variety of different cysteine residues present in a companion subunit. These results imply that the coordinated expression of many different LMW-GS in wheat endosperm cells can potentially lead to the formation of a large set of distinct polymeric structures, in which subunits can be arranged in different configurations. In addition, we show that not all intrachain disulfide bonds are necessary for the generation of an assembly-competent structure and that the retention of a LMW-GS in the early secretory pathway is not dependent on polymer formation.

An asparagine residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm

BackgroundWheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase.ResultsWe performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing.ConclusionsOur results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.