Ermelinda Botticella

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.

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.

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.

Approaches for modification of starch composition in durum wheat.

ABSTRACT Manipulation of starch composition in cereals and particularly in wheat is receiving increasing attention due to recognition of its important role in food and nonfood applications. The amylose/ amylopectin ratio influences the physicochemical properties of starches and nutritional value of derived end products. Identification of the key enzymes involved in the starch biosynthetic pathway has opened new avenues for altering the amylose and amylopectin content in durum and bread wheat. The granule bound starch synthases (GBSSI), or waxy proteins, are the enzymes responsible for amylose synthesis in storage tissues; amylopectin is produced by the concerted action of different enzymes, including starch synthases (SS), branching (SBE), and debranching enzymes (DBE). By altering the level of key enzymes involved in the regulation of starch synthesis, it is possible to generate novel starches with unique functional properties. In this respect, both low and high amylose starches are particularly interest...

TILLING mutants of durum wheat result in a high amylose phenotype and provide information on alternative splicing mechanisms.

The amylose/amylopectin ratio has a major influence over the properties of starch and determines its optimal end use. Here, high amylose durum wheat has been bred by combining knock down alleles at the two homoelogous genes encoding starch branching enzyme IIa (SBEIIa-A and SBEIIa-B). The complete silencing of these genes had a number of pleiotropic effects on starch synthesis: it affected the transcriptional activity of SBEIIb, ISA1 (starch debranching enzyme) and all of the genes encoding starch synthases (SSI, SSIIa, SSIII and GBSSI). The starch produced by grain of the double SBEIIa mutants was high in amylose (up to ∼1.95 fold that of the wild type) and contained up to about eight fold more resistant starch. A single nucleotide polymorphism adjacent to the splice site at the end of exon 10 of the G364E mutant copies of both SBEIIa-A and SBEIIa-B resulted in the loss of a conserved exonic splicing silencer element. Its starch was similar to that of the SBEIIa double mutant. G364E SBEIIa pre-mRNA was incorrectly processed, resulting in the formation of alternative, but non-functional splicing products.

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.

Development of a TILLING resource in durum wheat for reverse- and forward-genetic analyses

Abstract. A durum wheat TILLING (targeting induced local lesions in genomes) population of 2601 M3 families was developed from cv. Svevo using ethyl methanesulfonate as a chemical mutagen. The entire M3 population was field-grown for phenotypic evaluations. Despite the polyploid nature of the wheat genome, a preliminarily phenotypic screening showed a high frequency of morphological alterations (∼22%); specific phenotyping for seed morphology was undertaken. Furthermore, a reverse-genetics experiment was performed on DNA collected from M2 leaves for the homoeologous genes SBEIIa-A and SBEIIa-B involved in starch metabolism. One non-sense mutation for both genes was identified; specific crosses are planned in order to pyramid the two mutations.

The impact of the SSIIa null mutations on grain traits and composition in durum wheat

Starch represents a major nutrient in the human diet providing essentially a source of energy. More recently the modification of its composition has been associated with new functionalities both at the nutritional and technological level. Targeting the major starch biosynthetic enzymes has been shown to be a valuable strategy to manipulate the amylose-amylopectin ratio in reserve starch. In the present work a breeding strategy aiming to produce a set of SSIIa (starch synthases IIa) null durum wheat is described. We have characterized major traits such as seed weight, total starch, amylose, protein and β-glucan content in a set of mutant families derived from the introgression of the SSIIa null trait into Svevo, an elite Italian durum wheat cultivar. A large degree of variability was detected and used to select wheat lines with either improved quality traits or agronomic performances. Semolina of a set of two SSIIa null lines showed new rheological behavior and an increased content of all major dietary fiber components, namely arabinoxylans, β-glucans and resistant starch. Furthermore the investigation of gene expression highlighted important differences in some genes involved in starch and β-glucans biosynthesis.

The down-regulation of the genes encoding Isoamylase 1 alters the starch composition of the durum wheat grain

In rice, maize and barley, the lack of Isoamylase 1 activity materially affects the composition of endosperm starch. Here, the effect of this deficiency in durum wheat has been characterized, using transgenic lines in which Isa1 was knocked down via RNAi. Transcriptional profiling confirmed the partial down-regulation of Isa1 and revealed a pleiotropic effect on the level of transcription of genes encoding other isoamylases, pullulanase and sucrose synthase. The polysaccharide content of the transgenic endosperms was different from that of the wild type in a number of ways, including a reduction in the content of starch and a moderate enhancement of both phytoglycogen and β-glucan. Some alterations were also induced in the distribution of amylopectin chain length and amylopectin fine structure. The amylopectin present in the transgenic endosperms was more readily hydrolyzable after a treatment with hydrochloric acid, which disrupted its semi-crystalline structure. The conclusion was that in durum wheat, Isoamylase 1 is important for both the synthesis of amylopectin and for determining its internal structure.

TILLING for Improved Starch Composition in Wheat

Starch, the main component of wheat flour, is known to significantly influence the quality of wheat-based food products. In the last 20 years, research efforts have enabled the development of a number of wheat lines differing in starch composition and characterized by new chemical physical properties potentially able to confer new added value to food products. Scientists have focused on the opportunity to modify starch composition by targeting the main actors of its biosynthetic pathway: switching off the various starch synthetic enzymes has allowed for the production of a set of wheat starches with an amylose content ranging from 0 up to 75 %. Actually, amylose/amylopectin (AM/AP) ratio is considered the main factor affecting starch properties. Low amylose wheat is currently being investigated for its potential to improve the shelf life of baked products, frozen quality and the texture of noodles. High amylose wheat is of great interest for its healthy and nutritional properties comparable to those of a functional dietary fiber. In this context, reverse-genetics approaches based on the discovery and investigation of new allelic variants are becoming increasingly important. In particular, TILLING (Targeting Induced Local Lesions IN Genomes) has been widely adopted in wheat as well as in other important crops. Herewith, we review how TILLING is being exploited to improve starch composition in wheat.