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.

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.

BackgroundPrevious studies indicate that elevated amylose content in products from rice, corn, and barley induce lower postprandial glycaemic responses and higher levels of resistant starch (RS). Consumption of slowly digestible carbohydrates and RS has been associated with health benefits such as decreased risk of diabetes and cardiovascular disease.ObjectiveTo evaluate the postprandial glucose and insulin responses in vivo to bread products based on a novel wheat genotype with elevated amylose content (38%).DesignBread was baked from a unique wheat genotype with elevated amylose content, using baking conditions known to promote amylose retrogradation. Included test products were bread based on whole grain wheat with elevated amylose content (EAW), EAW with added lactic acid (EAW-la), and ordinary whole grain wheat bread (WGW). All test breads were baked at pumpernickel conditions (20 hours, 120°C). A conventionally baked white wheat bread (REF) was used as reference. Resistant starch (RS) content was measured in vitro and postprandial glucose and insulin responses were tested in 14 healthy subjects.ResultsThe results showed a significantly higher RS content (on total starch basis) in breads based on EAW than in WGW (p<0.001). Lactic acid further increased RS (p<0.001) compared with both WGW and EAW. Breads baked with EAW induced lower postprandial glucose response than REF during the first 120 min (p<0.05), but there were no significant differences in insulin responses. Increased RS content per test portion was correlated to a reduced glycaemic index (GI) (r=−0.571, p<0.001).ConclusionsThis study indicates that wheat with elevated amylose content may be preferable to other wheat genotypes considering RS formation. Further research is needed to test the hypothesis that bread with elevated amylose content can improve postprandial glycaemic response.

SummaryModifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end products. The starch synthesized in the grain of conventional wheats (Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes (GBSSI, SSIIa and SBEIIa) has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker‐assisted breeding exercise, has been used to generate three mutant lines that produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and nonstarch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross‐talk between the starch and carbohydrate synthesis pathways.

Plant-derived protein hydrolysates (PHs) have received increased attention in the last decade because of their potential to improve yield, nutritional quality as well as tolerance to abiotic stressors. The current study investigated the effects and the molecular mechanisms of a legume-derived PH under optimal and sub-optimal nitrogen (N) concentrations (112 and 7 mg L−1, respectively) in tomato (Solanum lycopersicum L.). Growth and mineral composition of tomato plants treated with PHs by foliar spray or substrate drench were compared to untreated plants. In addition, the expression was determined of genes encoding ammonium and nitrate transporters and seven enzymes involved in N metabolism: nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase 1 (GS1), glutamine synthetase 2 (GS2), ferredoxin-dependent glutamate synthase (GLT), NADH-dependent glutamate synthase (GLS), and glutamate dehydrogenase (GDH). The root and total plant dry weight, SPAD index and leaf nitrogen content were higher by 21, 17, 7, and 6%, respectively, in plants treated by a substrate drench in comparison to untreated tomato plants, whereas foliar application of PH gave intermediate values. PH-treated plants grown with lower N availability showed reduced expression of NR and NiR as well as of nitrate and ammonium transporter transcripts in both leaf and root tissues in comparison with untreated plants; this was especially pronounced after application of PH by substrate drench. Conversely, the transcript level of an amino acid transporter gene was up-regulated in comparison with untreated plants. At high N regime, the transcript levels of the ammonium and amino acid transporters and also NR, NiR, and GLT were significantly up-regulated in root after PH foliar and substrate drench applications compared with untreated plants. An up-regulation was also observed for GS1, GS2, and GDH transcripts in leaf after substrate drench. These results highlighted the potential benefits of using legume PH in vegetable production systems to increase growth and N-nutritional status of plants.

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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.

In recent years, awareness on sustainable land use has increased. Optimizing the practice of nitrogen fertilization has become crucially imperative in cropping management as a result of this current trend. The effort to improve the availability of organic nitrogen has incurred a bottleneck while seeking to achieve a high yield and quality performance for organic winter cereals. Field experiments were conducted, under rainfed Mediterranean conditions, over a period of two subsequent growing seasons. The objective was to investigate the effect of soil and foliar S application on the performance of three durum wheat cultivars fertilized with either organic or inorganic N. The hypothesis to be verified was if different S fertilization strategies could improve grain yield and quality when coupled with mineral or organic N fertilizer. There were three levels of treatment with mineral N fertilizer (120, 160 and 200 kg ha −1 ), two levels of organic N fertilizer (160 and 200 kg ha −1 ), two levels of S fertilizer applied to the soil (0 and 70 kg ha −1 ), and two levels of foliar S application at flag leaf stage (0 and 5 kg ha −1 ). Cultivars were Dylan, Iride and Saragolla. Analyzed traits were grain yield, yield components and quality features of grain. Overall, at the same N rate, grain yield and quality were markedly higher for mineral than organic N source. Cultivar × Year × N treatment interactions significantly affected grain yield and quality indices. Iride showed a high yield stability throughout the mineral N rates in the most favorable year (2011) and, in the same year, was the top performing cultivar in organic N treatments. Dylan was the top performing cultivar for protein content, while Saragolla for the SDS sedimentation test. Soil S fertilization had no effect on grain quality, whereas it significantly increased grain yield (+ 300 kg ha −1 ) when coupled with organic rather than a mineral N source. However, foliar S application at flag leaf stage did not affect grain yield, but it significantly enhanced quality indices such as test weight (81 vs. 79.9 kg hL −1 ), protein content (13.7% vs. 12.9 %) and SDS value (72.5 vs. 70.5 mm). A rate of 160 kg ha −1 of N (both mineral and organic) determined the optimal response for both grain yield and quality. Finally, soil and foliar application of S may help to contain the large yield and quality gap that still exists between mineral and organic fertilization of durum wheat.