Mass Spectrometry Characterization of the SDS-PAGE Protein Profile of Legumins and Vicilins from Chickpea Seed.

Chickpea (Cicer arietinum L.) seed proteins show a lot of functional properties leading this legume to be an interesting component for the development of protein-enriched foods. However, both the in-depth proteomic investigation and structural characterization of chickpea seed proteins are still lacking. In this paper a detailed characterization of chickpea seed protein fraction by means of SDS-PAGE, in-gel protein digestion, high-resolution mass spectrometry, and database searching is reported. Through this approach, twenty SDS gel bands were cut and analyzed. While the majority of the bands and the identified peptides were related to vicilin and legumin storage proteins, metabolic functional proteins were also detected. Legumins, as expected, were revealed at 45-65 kDa, as whole subunits with the α- and ß-chains linked together by a disulphide bond, but also at lower mass ranges (α- and ß-chains migrating alone). Similarly, but not expected, the vicilins were also spread along the mass region between 65 and 23 kDa, with some of them being identified in several bands. An MS structural characterization allowed to determine that, although chickpea vicilins were always described as proteins lacking cysteine residues, they contain this amino acid residue. Moreover, similar to legumins, these storage proteins are firstly synthesized as pre-propolypeptides (Mr 50-80 kDa) that may undergo proteolytic steps that not only cut the signal peptides but also produce different subunits with lower molecular masses. Overall, about 360 different proteins specific of the Cicer arietinum L. species were identified and characterized, a result that, up to the current date, represents the most detailed description of the seed proteome of this legume.

Influence of Drought and Salt Stress on Durum Wheat Grain Quality and Composition: A Review.

Durum wheat is a staple crop for the Mediterranean diet because of its adaptability to environmental pressure and for its large use in cereal-based food products, such as pasta and bread, as a source of calories and proteins. Durum wheat whole grains are also highly valued for their peculiar amount of dietary fiber and minerals, as well as bioactive compounds of particular interest for their putative health-beneficial properties, including polyphenols, carotenoids, tocopherols, tocotrienols, and phytosterols. In Mediterranean environments, durum wheat is mostly grown under rainfed conditions, where the crop often experiences environmental stresses, especially water deficit and soil salinity that may induce a hyperosmotic stress. In particular, changes in C and N accumulation due to these abiotic conditions, during grain filling, can influence starch and storage protein amount and composition in durum wheat caryopsis, thus influencing yield and quality traits. Recent advancements regarding the influence of water deficit and salinity stress on durum wheat are critically discussed. In particular, a focus on stress-induced changes in (a) grain protein content and composition in relation to technological and health quality; (b) starch and dietary fiber accumulation and composition; (c) phytochemical composition; (d) health-related grain micronutrient accumulation, such as Fe and Zn.

Changes in Antioxidant Defence System in Durum Wheat under Hyperosmotic Stress: A Concise Overview.

Durum wheat is one of the most commonly cultivated species in the world and represents a key commodity for many areas worldwide, as its grain is used for production of many foods, such as pasta, bread, couscous, and bourghul. Durum wheat grain has a relevant role in the human diet, providing carbohydrates, proteins, lipids, fibres, vitamins, and minerals, as well as highly valued bioactive compounds contributing to a healthy diet. Durum wheat is largely cultivated in the Mediterranean basin, where it is mainly grown under rain-fed conditions, thus currently undergoing drought stress, as well as soil salinity, which can hamper yield potential and influence the qualitative characteristics of grain. When plants suffer drought and/or salinity stress, a condition known as hyperosmotic stress is established at cellular level. This leads to the accumulation of ROS thus generating in turn an oxidative stress condition, which can ultimately result in the impairment of cellular integrity and functionality. To counteract oxidative damage due to excessive ROS production under stress, plants have evolved a complex array of both enzymatic and non-enzymatic antioxidant mechanisms, working jointly and synergically for maintenance of ROS homeostasis. Enhancement of antioxidant defence system has been demonstrated as an adaptive mechanism associated to an increased tolerance to hyperosmotic stress. In the light of these considerations, this review provides a concise overview on recent advancements regarding the role of the ascorbate-glutathione cycle and the main antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) in durum wheat response to drought and salt stresses that are expected to become more and more frequent due to the ongoing climate changes.

Isolation, Screening, and Characterization of Plant-Growth-Promoting Bacteria from Durum Wheat Rhizosphere to Improve N and P Nutrient Use Efficiency.

The main goal of this paper was to select promising microorganisms which could potentially act as plant-growth-promoting bacteria (PGPB) for durum wheat of Foggia County. At this scope, a new statistical framework, based on multivariate analyses and the evaluation of the statistical distribution of each trait, was used. Four hundred and seventy-four isolates were isolated from the rhizosphere of durum wheat in Foggia County and preliminarily screened as a function of four target indices (ammonium production, siderophores production, P-solubilization, and nitrification). After this step, the number of strains was reduced and the remaining isolates were tested through a quantitative approach, to assess the production of IAA (indole acetic acid), P-mineralization, and nitrification. In this second step, the cut-off was based on the whole population trend by evaluating for each trait the medians and quartiles. As a result, 16 promising isolates were selected and identified by 16S rDNA sequencing (Bacillus, Pseudomonas, Stenotrophomonas, and Lysinibacillus). The last step of this research was a preliminary validation in a growth chamber on eight strains. As screening and simple indices, two quantitative measures were chosen. The main result was the selection of at least three isolates (6P, 20P, and 25A) for a future field validation. They increased biomass and height by respectively 50% and 25%.

Assessment of Antioxidant Capacity and Putative Healthy Effects of Natural Plant Products Using Soybean Lipoxygenase-Based Methods. An Overview.

In the last decades, increasing demand of antioxidant-rich foods and growing interest in their putative role in prevention of degenerative diseases have promoted development of methods for measuring Antioxidant Capacity (AC). Nevertheless, most of these assays use radicals and experimental conditions far from the physiological ones, and are able to estimate only one or a few antioxidant mechanisms. On the other hand, the novel LOX/RNO and LOX⁻FL methods, based on secondary reactions between the soybean lipoxygenase (LOX)-1 isoenzyme and either 4-nitroso-N,N-dimethylaniline (RNO) or fluorescein (FL), may provide a more comprehensive AC evaluation. In fact, they are able to detect simultaneously many antioxidant functions (scavenging of some physiological radical species, iron ion reducing and chelating activities, inhibition of the pro-oxidant apoenzyme) and to highlight synergism among phytochemicals. They are applied to dissect antioxidant properties of several natural plant products: food-grade antioxidants, cereal and pseudocereal grains, grain-derived products, fruits. Recently, LOX⁻FL has been used for ex vivo AC measurements of human blood samples after short- and long-term intakes of some of these foods, and the effectiveness in improving serum antioxidant status was evaluated using the novel Antioxidant/Oxidant Balance (AOB) parameter, calculated as an AC/Peroxide Level ratio. An overview of data is presented.

Data set of enzyme fingerprinting of dietary fibre components (arabinoxylan and ß-glucan) in old and modern Italian durum wheat genotypes.

The data presented are related to the research article entitled "Comparison of the dietary fibre composition of old and modern durum wheat (Triticum turgidum spp. durum) genotypes" (De Santis et al., 2018) [1]. This article provides details of the structures of the major dietary fibre components, arabinoxylan and ß-glucan, in semolina and wholemeal flour of old and modern Italian durum wheat genotypes grown in two seasons, determined by enzyme digestion followed by high-performance anion-exchange chromatography (enzyme fingerprinting).

Comparison of the dietary fibre composition of old and modern durum wheat (Triticum turgidum spp. durum) genotypes.

It has been suggested that intensive breeding has led to decreased contents of health-promoting components in modern wheats. We therefore compared the contents and compositions of the major dietary fibre components, arabinoxylan and ß-glucan, in semolina and wholemeal flour of old and modern Italian durum wheats. No differences in total arabinoxylan content were observed but the modern varieties had higher proportions of soluble arabinoxylan in wholemeals and of ß-glucan in semolina. The study therefore provides no evidence that intensive breeding has had negative effects on the contents of dietary fibre components in durum wheat. However, comparison of material grown over two years indicated that the content and composition of arabinoxylan and ß-glucan were more stable in the older than in the modern genotypes. The identification of modern cultivars with high viscosity associated with a high content of ß-glucan suggests that they are good sources of fibre for human health.

Differences in gluten protein composition between old and modern durum wheat genotypes in relation to 20th century breeding in Italy.

The impact of breeding on grain yields of wheat varieties released during the 20th century has been extensively studied, whereas less information is available on the changes in gluten quality associated with effects on the amount and composition of glutenins and gliadins. In order to explore the effects of breeding during the 20th century on gluten quality of durum wheat for processing and health we have compared a set of old and modern Italian genotypes grown under Mediterranean conditions. The better technological performance observed for the modern varieties was found to be due not only to the introgression of superior alleles of high (HMW-GS) and low molecular weight (LMW-GS) glutenin subunits encoded at Glu-B1 and Glu-B3 loci, but also to differential expression of specific storage proteins. In particular, the higher gluten index observed in modern genotypes was correlated with an increased glutenin/gliadin ratio and the expression of B-type LMW-GS which was, on average, two times higher in the modern than in the old group of durum wheat genotypes. By contrast, no significant differences were found between old and modern durum wheat genotypes in relation to the expression of α-type and γ-type gliadins which are major fractions that trigger coeliac disease (CD) in susceptible individuals. Furthermore, a drastic decrease was observed in the expression of ω-type gliadins in the modern genotypes, mainly ω-5 gliadin (also known as Tri a 19) which is a major allergen in wheat dependent exercise induced anaphylaxis (WDEIA). Immunological and 2DE SDS-PAGE analyses indicated that these differences could be related either to a general down-regulation or to differences in numbers of isoforms. Lower rainfall during grain filling period was related to overall higher expression of HMW-GS and ω-gliadins. In conclusion, breeding activity carried out in Italy during the 20th century appears to have improved durum wheat gluten quality, both in relation to technological performance and allergenic potential.

The role of Plant Growth Promoting Bacteria in improving nitrogen use efficiency for sustainable crop production: a focus on wheat.

Due to the increase in both human population growth and environmental pressure, it is necessary to raise agricultural productivity without enhancing environmental footprint. Within this context, soil inoculation with PGPB (Plant Growth Promoting Bacteria) may be considered a promising tool of integrated management systems. In particular, PGPB may improve plant growth either directly, by facilitating resource use or modulating plant hormone levels, or indirectly by decreasing the inhibitory effects of various pathogenic agents. PGPB comprise different functional and taxonomic groups of bacteria like Pseudomonas, Bacillus, Rhizobium and others. Their ability to either mobilize mineral or organic bound nutrients from the pedosphere or to fix atmospheric N2 and make it available to the plants, is a crucial feature in their application. In literature some data are available on the use of commercial PGPB, while less efforts have been made on the study of the effect of autochthonous PGPB isolated from soils on sustainability of cropping systems; thus a literature survey on these aspects was carried out with special focus on wheat, a staple food for a large part of world population. In particular, the main topic of this review is the potential of PGPB to enhance use efficiency of agro-environmental resources focusing on the interaction PGPB-wheat for improving nitrogen use efficiency.

Functional, textural and sensory properties of dry pasta supplemented with lyophilized tomato matrix or with durum wheat bran extracts produced by supercritical carbon dioxide or ultrasound.

A study was carried out to produce functional pasta by adding bran aqueous extract (BW) and bran oleoresin (BO) obtained using ultrasound and supercritical CO2, respectively, or a powdery lyophilized tomato matrix (LT). The bioactive compounds, hydrophilic and lipophilic antioxidant activity (HAA and LAA) in vitro, were evaluated. BW supplementation did not improve antioxidant activity, whilst LT pasta showed unconventional taste and odor. BO pasta had good levels of tocochromanols (2551µg/100g pasta f.w.) and carotenoids (40.2µg/100g pasta f.w.), and the highest HAA and LAA. The oleoresin altered starch swelling and gluten network, as evidenced by scanning electron microscopy, therefore BO pasta had structural characteristics poor compared with the control (4.8% vs. 3.2% cooking loss), although this difference did not affect significantly overall sensory judgment (74 vs. 79 for BO and control, respectively). BO supplementation was most effective for increasing antioxidant activity without jeopardizing pasta quality.

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.

Evaluation of Phenolic Antioxidant Capacity in Grains of Modern and Old Durum Wheat Genotypes by the Novel QUENCHERABTS Approach.

The QUENCHERABTS (QUick, Easy, New, CHEap and Reproducible) approach for antioxidant capacity (AC) determination is based on the direct reaction of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation with fine solid food particles. So, it may resemble the antioxidant action in foods or in human gastrointestinal trait. Here, the QUENCHER approach was used to study AC of durum wheat (Triticum durum Desf.) grains. Firstly, it was assessed which kind of antioxidants determines QUENCHER response. This has been performed by comparing AC measured by QUENCHERABTS and that measured by classical TEACABTS (Trolox equivalent antioxidant capacity) in four different extracts from whole flour of 10 durum wheat varieties containing: lipophilic, hydrophilic, insoluble-bound phenolic (IBP) and free-soluble phenolic (FSP) compounds. QUENCHERABTS data were unrelated to AC of water-extractable antioxidants and weakly correlated (r = 0.405, P < 0.05) to AC of the lipophilic ones; on the contrary, QUENCHERABTS response was mainly related to AC of IBP (r = 0.907, P < 0.001) and to a lesser extent of FSP extracts (r = 0.747, P < 0.001). Consistently, correlation was also found with the phenolic content of IBP and FSP (r = 0.760, P < 0.001 and r = 0.522, P < 0.01, respectively), thus confirming that QUENCHERABTS assay mainly assesses AC due to IBP. So, this assay was used in a first screening study to compare AC of bioactive IBP of thirty-six genotypes/landraces covering a century of cultivation in Italy. Interestingly, no relevant AC difference between modern and old genotypes was found, thus suggesting that a century of plant breeding did not decrease phenol-dependent health potential in durum wheat.

Comparative analysis of gluten proteins in three durum wheat cultivars by a proteomic approach.

The gluten protein composition and expression level influence dough properties and are cultivar and environment dependent. To broaden the knowledge of the durum wheat gluten proteome, three cultivars were compared in two different growing seasons by a proteomic approach. Cultivar-specific and differentially expressed spots in the two years were identified by mass spectrometry. Significant differences were observed among the cultivars: Ofanto showed the lowest protein spot volumes in the high molecular weight (HMW) and low molecular weight (LMW) <35,000 regions and the highest in the LMW 48,000-35,000 region, Latino the lowest in the LMW 48,000-35,000 region, and Simeto an intermediate expression level in both LMW regions. In the warmer year the up-regulation of HMW glutenins, α-gliadins, and a globulin 3 protein and the down-expression of LMW glutenins and γ-gliadins were observed. Among the cultivars, Simeto showed the highest stability across the environments.

Antioxidant activity of free and bound compounds in quinoa (Chenopodium quinoa Willd.) seeds in comparison with durum wheat and emmer.

UNLABELLED: Antioxidant activity (AA) of quinoa (Chenopodium quinoa Willd.) seeds, as well as of durum wheat (Triticum turgidum L. ssp. durum Desf.) and of emmer (T. turgidum L. ssp. dicoccum Schübler) grains, was evaluated by studying hydrophilic (H), lipophilic (L), free-soluble (FSP) and insoluble-bound (IBP) phenolic extracts using the new lipoxygenase/4-nitroso-N,N-dimethylaniline (LOX/RNO) method, able to simultaneously detect different antioxidant mechanisms, as well as using the Oxygen Radical Absorbance Capacity (ORAC) and the Trolox Equivalent Antioxidant Capacity (TEAC) assays, which measure the scavenging activity against peroxyl and ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)] radicals, respectively. The species under study were compared with respect to the sum of AA values of H, L and FSP extracts (AA(H+L+FSP)), containing freely solvent-soluble antioxidants, and AA values of IBP extracts (AA(IBP)), representing the phenolic fraction ester-linked to insoluble cell wall polymers. The LOX/RNO and ORAC methods measured in quinoa flour a remarkable AA(H+L+FSP) higher than durum wheat, although lower than emmer; according to the same assays, the IBP component of quinoa resulted less active than the durum wheat and emmer ones. The TEAC protocol also revealed a high AA(H+L+FSP) for quinoa. Interestingly, the ratio AA(H+L+FSP)/AA(H+L+FSP+IBP), as evaluated by the LOX/RNO and ORAC assays, resulted in quinoa higher than that of both durum wheat and emmer, and much higher than durum wheat, according to the TEAC protocol. This may suggest that antioxidants from quinoa seeds may be more readily accessible with respect to that of both the examined wheat species. PRACTICAL APPLICATIONS: Quinoa seeds may represent an excellent source of natural antioxidant compounds and, in particular, of the free-soluble antioxidant fraction. These compounds may improve nutritive and health-beneficial properties of quinoa-based gluten-free products, thus expanding interest for quinoa utilization from celiac patients to the general population.

New tool to evaluate a comprehensive antioxidant activity in food extracts: bleaching of 4-nitroso-N,N-dimethylaniline catalyzed by soybean lipoxygenase-1.

In this study the 4-nitroso-N,N-dimethylaniline (RNO) bleaching associated with linoleic acid hydroperoxidation catalyzed by the soybean lipoxygenase (LOX)-1 isoenzyme (LOX/RNO reaction) was used to determine the antioxidant activity (AA) of hydrophilic and lipophilic pure antioxidant compounds and of mixtures of antioxidants extracted from durum wheat whole flour (DWWF). By means of a simple and rapid experimental protocol (about 3 min/assay), the LOX/RNO reaction may simultaneously detect many antioxidant functions (scavenging of some physiological radical species, iron ion reducing and chelating activities, inhibition of the pro-oxidant apoenzyme), thus providing a comprehensive AA evaluation. Consistently, the LOX/RNO assay was very sensitive to hydrophilic, lipophilic, and phenolic antioxidant extracts from DWWF, providing AA values at least 35 and 30 times higher than those by TEAC and ORAC methods, respectively. Moreover, the new method was able to highlight synergism (among extracts) 3 times more than the ORAC method, whereas TEAC did not measure synergism under our experimental conditions.

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria.

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.

Seawater stress applied at germination affects mitochondrial function in durum wheat (Triticum durum) early seedlings.

Seawater stress effects on mitochondrial ATP synthesis and membrane potential (ΔΨ) were investigated in germinating durum wheat seedlings under moderate (22% seawater osmolarity, -0.62 MPa) and severe (37% seawater osmolarity, -1.04 MPa) stress. To estimate the osmotic component of salt stress, mannitol solutions (0.25 and 0.42 m) iso-osmotic with the saline ones were used. Moderate stress intensity only delayed mean germination time (MGT), whereas higher seawater osmolarity reduced germination percentage as well. In contrast, Na+ and Cl- accumulation showed a sharp increase under moderate stress and only a small further increase under severe stress, which was more pronounced for Cl-. Only severe stress significantly damaged succinate-dependent oxidative phosphorylation, which may be related to the stress-induced alteration in inner mitochondrial membrane permeability, as indicated by changes in ΔΨ profiles. Proline-dependent oxidative phosphorylation, however, was inhibited under moderate stress. This suggests the occurrence of an adaptation mechanism leading to proline accumulation as an osmoprotectant. Moreover, both the osmotic and the toxic components of seawater stress were detrimental to oxidative phosphorylation. Damage to germination and MGT, in contrast, were mainly caused by osmotic stress. Therefore, mitochondrial function appears to be a more sensitive target of toxic stress than growth. In conclusion, the effects of seawater stress on mitochondrial ATP synthesis vary in relation to the substrate oxidised and stress level, inducing both adaptive responses and damage.