Mutations in starch biosynthesis genes affect chloroplast development in wheat pericarp.

Starch bioengineering in cereals has produced a plethora of genotypes with new nutritional and technological functionalities. Modulation of amylose content from 0 to 100% was inversely correlated with starch digestibility and promoted a lower glycemic index in food products. In wheat, starch mutants have been reported to exhibit various side effects, mainly related to the seed phenotype. However, little is known about the impact of altered amylose content and starch structure on plant metabolism. Here, three bread wheat starch mutant lines with extreme phenotypes in starch branching and amylose content were used to study plant responses to starch structural changes. Omics profiling of gene expression and metabolic patterns supported changes, confirmed by ultrastructural analysis in the chloroplast of the immature seeds. In detail, the identification of differentially expressed genes belonging to functional categories related to photosynthesis, chloroplast and thylakoid (e.g. CURT1), the alteration in the accumulation of photosynthesis-related compounds, and the chloroplast alterations (aberrant shape, grana stacking alteration, and increased number of plastoglobules) suggested that the modification of starch structure greatly affects starch turnover in the chloroplast, triggering oxidative stress (ROS accumulation) and premature tissue senescence. In conclusion, this study highlighted a correlation between starch structure and chloroplast functionality in the wheat kernel.

The functional implications of high-amylose wholegrain wheat flours: An in vitro digestion and fermentation approach combined with metabolomics.

Wheat flour is one of the most prevalent foodstuffs for human consumption, and novel strategies are underway to enhance its nutritional properties. This work evaluated wholegrain flours from bread wheat lines with different amylose/amylopectin ratios through in vitro starch digestion and large intestine fermentation. High-amylose flours presented a higher resistant starch content and lower starch hydrolysis index. Moreover, UHPLC-HRMS metabolomics was carried out to determine the profile of the resulting in vitro fermentates. The multivariate analysis highlighted distinctive profiles between the flours derived from the different lines compared to the wild type. Peptides, glycerophospholipids, polyphenols, and terpenoids were identified as the main markers of the discrimination. The high-amylose flour fermentates showed the richest bioactive profile, containing stilbenes, carotenoids, and saponins. Present findings pave the way toward applying high-amylose flours to design novel functional foods.

The suppression of TdMRP3 genes reduces the phytic acid and increases the nutrient accumulation in durum wheat grain.

Micronutrient malnutrition affects more than half of the world population. Reduced bioavailability of microelements in the raw materials is considered one of the main causes of mineral deficiency in populations whose diet is largely based on the consumption of staple crops. In this context, the production of low phytic acid (lpa) cereals is a main goal of the breeding programs, as phytic acid (PA) binds essential mineral cations such as iron (Fe), zinc (Zn), manganese (Mn), potassium (K), calcium (Ca) and magnesium (Mg) precipitating in the form of phytate salts poorly digested by monogastric animals, including humans, due to the lack of phytases in the digestive tract. Since PA limits the bioavailability of microelements, it is widely recognized as an anti-nutritional compound. A Targeting Induced Local Lesions IN Genomes (TILLING) approach has been undertaken to silence the genes encoding the TdABCC13 proteins, known as Multidrug-Resistance associated Proteins 3 (TdMRP3), transporters involved in the accumulation of PA inside the vacuole in durum wheat. The TdMRP3 complete null genotypes showed a significant reduction in the content of PA and were able to accumulate a higher amount of essential micronutrients (Fe, Zn, Mn) compared to the control. The number of spikelets and seeds per spike, traits associated with the agronomic performances, were reduced compared to the control, but the negative effect was in part balanced by the increased grain weight. The TdMRP3 mutant lines showed morphological differences in the root apparatus such as a significant decrease in the number of root tips, root length, volume and surface area and an increase in root average diameter compared to the control plants. These materials represent a promising basis for obtaining new commercial durum wheats with higher nutritional value.

Molecular insights into the role of amylose/amylopectin ratio on gluten protein organization.

Waxy (WX) and high-amylose (HA) wheat flours have interesting functional and/or nutritional characteristics, but low technological properties compared to regular wheat. Here a set of three wheat lines, having different amylose content but sharing the same varietal background, were compared to shed light on the role of the amylose/amylopectin ratio on the protein conformational changes that lead to gluten formation. Despite the absence of differences in their protein profile, as also confirmed by thiolomic approaches, both WX and HA lines developed a weaker gluten than the control sample. The altered amylose/amylopectin ratio exerts a matrix effect establishing a competition for water with proteins, leading to a different protein structure and three-dimensional organization of the gluten network. These results add a piece to the understanding of the molecular aspects that oversee matrix effects on gluten formation in wheat, which description can be helpful for a rational optimization of the transformation process.

The Triple Jags of Dietary Fibers in Cereals: How Biotechnology Is Longing for High Fiber Grains.

Cereals represent an important source of beneficial compounds for human health, such as macro- and micronutrients, vitamins, and bioactive molecules. Generally, the consumption of whole-grain products is associated with significant health benefits, due to the elevated amount of dietary fiber (DF). However, the consumption of whole-grain foods is still modest compared to more refined products. In this sense, it is worth focusing on the increase of DF fractions inside the inner compartment of the seed, the endosperm, which represents the main part of the derived flour. The main components of the grain fiber are arabinoxylan (AX), ß-glucan (ßG), and resistant starch (RS). These three components are differently distributed in grains, however, all of them are represented in the endosperm. AX and ßG, classified as non-starch polysaccharides (NSP), are in cell walls, whereas, RS is in the endosperm, being a starch fraction. As the chemical structure of DFs influences their digestibility, the identification of key actors involved in their metabolism can pave the way to improve their function in human health. Here, we reviewed the main achievements of plant biotechnologies in DFs manipulation in cereals, highlighting new genetic targets to be exploited, and main issues to face to increase the potential of cereals in fighting malnutrition.

Enrichment of provitamin A content in durum wheat grain by suppressing ß-carotene hydroxylase 1 genes with a TILLING approach.

KEY MESSAGE: The suppression of the HYD-1 gene by a TILLING approach increases the amount of ß-carotene in durum wheat kernel. Vitamin A deficiency is a major public health problem that affects numerous countries in the world. As humans are not able to synthesize vitamin A, it must be daily assimilated along with other micro- and macronutrients through the diet. Durum wheat is an important crop for Mediterranean countries and provides a discrete amount of nutrients, such as carbohydrates and proteins, but it is deficient in some essential micronutrients, including provitamin A. In the present work, a targeting induced local lesions in genomes strategy has been undertaken to obtain durum wheat genotypes biofortified in provitamin A. In detail, we focused on the suppression of the ß-carotene hydroxylase 1 (HYD1) genes, encoding enzymes involved in the redirection of ß-carotene toward the synthesis of the downstream xanthophylls (neoxanthin, violaxanthin and zeaxanthin). Expression analysis of genes involved in carotenoid biosynthesis revealed a reduction of the abundance of HYD1 transcripts greater than 50% in mutant grain compared to the control. The biochemical profiling of carotenoid in the wheat mutant genotypes highlighted a significant increase of more than 70% of ß-carotene compared to the wild-type sibling lines, with no change in lutein, α-carotene and zeaxanthin content. This study sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat and provides new genotypes that represent a good genetic resource for future breeding programs focused on the provitamin A biofortification through non-transgenic approaches.

A Green Nanostructured Pesticide to Control Tomato Bacterial Speck Disease.

Bacterial speck disease, caused by Pseudomonas syringae pv. tomato (Pst), is one of the most pervasive biological adversities in tomato cultivation, in both industrial and in table varieties. In this work synthesis, biochemical and antibacterial properties of a novel organic nanostructured pesticide composed of chitosan hydrochloride (CH) as active ingredient, cellulose nanocrystals (CNC) as nanocarriers and starch as excipient were evaluated. In order to study the possibility of delivering CH, the effects of two different types of starches, extracted from a high amylose bread wheat (high amylose starch-HA Starch) and from a control genotype (standard starch-St Starch), were investigated. Nanostructured microparticles (NMP) were obtained through the spray-drying technique, revealing a CH loading capacity proximal to 50%, with a CH release of 30% for CH-CNC-St Starch NMP and 50% for CH-CNC-HA Starch NMP after 24 h. Both NMP were able to inhibit bacterial growth in vitro when used at 1% w/v. Moreover, no negative effects on vegetative growth were recorded when NMP were foliar applied on tomato plants. Proposed nanostructured pesticides showed the capability of diminishing Pst epiphytical survival during time, decreasing disease incidence and severity (from 45% to 49%), with results comparable to one of the most used cupric salt (hydroxide), pointing out the potential use of CH-CNC-Starch NMP as a sustainable and innovative ally in Pst control strategies.

Biotechnological Resources to Increase Disease-Resistance by Improving Plant Immunity: A Sustainable Approach to Save Cereal Crop Production.

Plant diseases are globally causing substantial losses in staple crop production, undermining the urgent goal of a 60% increase needed to meet the food demand, a task made more challenging by the climate changes. Main consequences concern the reduction of food amount and quality. Crop diseases also compromise food safety due to the presence of pesticides and/or toxins. Nowadays, biotechnology represents our best resource both for protecting crop yield and for a science-based increased sustainability in agriculture. Over the last decades, agricultural biotechnologies have made important progress based on the diffusion of new, fast and efficient technologies, offering a broad spectrum of options for understanding plant molecular mechanisms and breeding. This knowledge is accelerating the identification of key resistance traits to be rapidly and efficiently transferred and applied in crop breeding programs. This review gathers examples of how disease resistance may be implemented in cereals by exploiting a combination of basic research derived knowledge with fast and precise genetic engineering techniques. Priming and/or boosting the immune system in crops represent a sustainable, rapid and effective way to save part of the global harvest currently lost to diseases and to prevent food contamination.

Can Manipulation of Durum Wheat Amylose Content Reduce the Glycaemic Index of Spaghetti?

Resistant starch (RS) in foods has positive benefits for potentially alleviating lifestyle diseases. RS is correlated positively with starch amylose content. This study aimed to see what level of amylose in durum wheat is needed to lower pasta GI. The silencing of starch synthases IIa (SSIIa) and starch branching enzymes IIa (SBEIIa), key genes involved in starch biosynthesis, in durum wheat cultivar Svevo was performed and spaghetti was prepared and evaluated. The SSIIa and SBEIIa mutants have a 28% and 74% increase in amylose and a 2.8- and 35-fold increase in RS, respectively. Cooked pasta was softer, with higher cooking loss but lower stickiness compared to Svevo spaghetti, and with acceptable appearance and colour. In vitro starch digestion extent (area under the digestion curve) was decreased in both mutants, but much more in SBEIIa, while in vivo GI was only significantly reduced from 50 to 38 in SBEIIa. This is the first study of the glycaemic response of spaghetti prepared from SBEIIa and SSIIa durum wheat mutants. Overall pasta quality was acceptable in both mutants but the SBEIIa mutation provides a clear glycaemic benefit and would be much more appealing than wholemeal spaghetti. We suggest a minimum RS content in spaghetti of ~7% is needed to lower GI which corresponded to an amylose content of ~58%.

Provitamin A Biofortification of Durum Wheat through a TILLING Approach.

Macro- and micronutrients, essential for the maintenance of human metabolism, are assimilated daily through the diet. Wheat and other major cereals are a good source of nutrients, such as carbohydrates and proteins, but cannot supply a sufficient amount of essential micronutrients, including provitamin A. As vitamin A deficiency (VAD) leads to several serious diseases throughout the world, the biofortification of a major staple crop, such as wheat, represents an effective way to preserve human health in developing countries. In the present work, a key enzyme involved in the branch of carotenoids pathway producing ß-carotene, lycopene epsilon cyclase, has been targeted by a Targeting Induced Local Lesions in Genomes (TILLING) approach in a "block strategy" perspective. The null mutant genotype showed a strong reduction in the expression of the lcyE gene and also interesting pleiotropic effects on an enzyme (ß-ring hydroxylase) acting downstream in the pathway. Biochemical profiling of carotenoids in the wheat mutant lines showed an increase of roughly 75% in ß-carotene in the grains of the complete mutant line compared with the control. In conclusion, we describe here the production and characterization of a new wheat line biofortified with provitamin A obtained through a nontransgenic approach, which also sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat.

Enhancing grain size in durum wheat using RNAi to knockdown GW2 genes.

KEY MESSAGE: Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Raising crop yield is a priority task in the light of the continuing growth of the world's population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains' starch content by 10-40%, their width by 4-13% and their surface area by 3-5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain.

Nanostructured starch combined with hydroxytyrosol in poly(vinyl alcohol) based ternary films as active packaging system.

Novel ternary films have been realized by using poly(vinyl alcohol) (PVA) as polymeric matrix, nanostructured starch as reinforcement phase and hydroxytyrosol (HTyr), a low-molecular phenolic compound present in olive oil, as antioxidant agent. Nanostructured starch, in the form of starch nanocrystals (NC) and nanoparticles (NP) obtained by acid hydrolysis and ultrasound irradiation of starch derived from the bread wheat variety Cadenza (WT, amylose content 33%) and a derived-high amylose line (HA, amylose content 75%), was considered. The developed multifunctional films were characterized in terms of morphological, thermal and optical properties, water absorption capacity, overall and specific migration into a food simulant and antioxidant properties. Experimental data showed a prolonged release of HTyr from all ternary films and the released HTyr retained a strong antioxidant activity. The data, compared to those of PVA/HTyr binary films, demonstrated the key role of nanostructured starch in the ternary formulations in promoting a gradual release of HTyr. Overall, PVA fillm combined with nanoparticles from low amylose starch and hydroxytyrosol resulted as the most promising ternary formulation for food packaging applications.

Combining mutations at genes encoding key enzymes involved in starch synthesis affects the amylose content, carbohydrate allocation and hardness in the wheat grain.

Modifications 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.

Metabolic response to amylose-rich wheat-based rusks in overweight individuals.

BACKGROUND/OBJECTIVES: The amylose-amylopectin ratio influences starch properties. A higher amylose content is associated with slower starch digestion thus reducing the postprandial plasma glucose response and improving the overall postprandial metabolism. So far, limited evidence is available on the metabolic effect of wheat-based foods rich in amylose. This randomised controlled study investigated the acute metabolic effects of amylose-rich wheat-based rusks in overweight subjects focusing on potential mechanisms. SUBJECTS/METHODS: Ten overweight subjects consumed in random order two test meals differing only in the carbohydrate source: rusks prepared with amylose-rich wheat flour (ARR) or conventional wheat flour (control). Blood samples were taken at fasting and over 4 h after the meal. Satiety and intestinal fermentation were evaluated by VAS and H2-breath test, respectively. RESULTS: ARR reduced plasma glucose response during the first two hours after the meal and the desire to eat, and increased breath hydrogen concentration at 4 h (p < 0.05 for all). Moreover, according to computational models, the ARR slightly reduced intestinal glucose absorption in the first hour after the meal and increased the overall postprandial insulin sensitivity. CONCLUSIONS: Rusks made with amylose-rich flour could be useful for improving postprandial glucose metabolism and reduce the desire to eat, thus possibly contributing to the prevention and treatment of overweight/obesity, impaired glucose tolerance or diabetes.

Molecular characterisation of two novel starch granule proteins 1 in wild and cultivated diploid A genome wheat species.

Starch synthase IIa, also known as starch granule protein 1 (SGP-1), plays a key role in amylopectin biosynthesis. The absence of SGP-1 in cereal grains is correlated to dramatic changes in the grains' starch content, structure, and composition. An extensive investigation of starch granule proteins in this study revealed a polymorphism in the electrophoretic mobility of SGP-1 between two species of wheat, Triticum urartu and T. monococcum; this protein was, however, conserved among all other Triticum species that share the A genome inherited from their progenitor T. urartu. Two different electrophoretic profiles were identified: SGP-A1 proteins of T. urartu accessions had a SDS-PAGE mobility similar to those of tetraploid and hexaploid wheat species; conversely, SGP-A1 proteins of T. monococcum ssp. monococcum and ssp. boeoticum accessions showed a different electrophoretic mobility. The entire coding region of the two genes was isolated and sequenced in an attempt to explain the polymorphism identified. Several single nucleotide polymorphisms (SNPs) responsible for amino acid changes were identified, but no indel polymorphism was observed to explain the difference in electrophoretic mobility. Amylose content did not differ significantly among T. urartu, T. monococcum ssp. boeoticum and T. monococcum ssp. monococcum, except in one accession of the ssp. boeoticum. Conversely, several interspecific differences were observed in viscosity properties (investigated as viscosity profiles using a rapid visco analyzer-RVA profiles) of these cereal grains. T. monococcum ssp. boeoticum accessions had the lowest RVA profiles, T. urartu accessions had an intermediate RVA profile, whereas T. monococcum ssp. monococcum showed the highest RVA profile. These differences could be associated with the numerous amino acid and structural changes evident among the SGP-1 proteins.

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 ß-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, ß-glucans and resistant starch. Furthermore the investigation of gene expression highlighted important differences in some genes involved in starch and ß-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 ß-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.

Analysis of Quality-Related Parameters in Mature Kernels of Polygalacturonase Inhibiting Protein (PGIP) Transgenic Bread Wheat Infected with Fusarium graminearum.

Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters.

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.