The Role of Glutamine Synthetase (GS) and Glutamate Synthase (GOGAT) in the Improvement of Nitrogen Use Efficiency in Cereals.

Cereals are the most broadly produced crops and represent the primary source of food worldwide. Nitrogen (N) is a critical mineral nutrient for plant growth and high yield, and the quality of cereal crops greatly depends on a suitable N supply. In the last decades, a massive use of N fertilizers has been achieved in the desire to have high yields of cereal crops, leading to damaging effects for the environment, ecosystems, and human health. To ensure agricultural sustainability and the required food source, many attempts have been made towards developing cereal crops with a more effective nitrogen use efficiency (NUE). NUE depends on N uptake, utilization, and lastly, combining the capability to assimilate N into carbon skeletons and remobilize the N assimilated. The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle represents a crucial metabolic step of N assimilation, regulating crop yield. In this review, the physiological and genetic studies on GS and GOGAT of the main cereal crops will be examined, giving emphasis on their implications in NUE.

Genetic Mapping of Flavonoid Grain Pigments in Durum Wheat.

Pigmented cereal grains with high levels of flavonoid compounds have attracted the attention of nutritional science backing the development of functional foods with claimed health benefits. In this study, we report results on the genetic factors controlling grain pigmentation in durum wheat using a segregant population of recombinant inbred lines (RILs) derived from a cross between an Ethiopian purple grain accession and an Italian amber grain cultivar. The RIL population was genotyped by the wheat 25K SNP array and phenotyped for total anthocyanin content (TAC), grain color, and the L*, a*, and b* color index of wholemeal flour, based on four field trials. The mapping population showed a wide variation for the five traits in the different environments, a significant genotype x environment interaction, and high heritability. A total of 5942 SNP markers were used for constructing the genetic linkage map, with an SNP density ranging from 1.4 to 2.9 markers/cM. Two quantitative trait loci (QTL) were identified for TAC mapping on chromosome arms 2AL and 7BS in the same genomic regions of two detected QTL for purple grain. The interaction between the two QTL was indicative of an inheritance pattern of two loci having complementary effects. Moreover, two QTL for red grain color were detected on chromosome arms 3AL and 3BL. The projection of the four QTL genomic regions on the durum wheat Svevo reference genome disclosed the occurrence of the candidate genes Pp-A3, Pp-B1, R-A1, and R-B1 involved in flavonoid biosynthetic pathways and encoding of transcription factors bHLH (Myc-1) and MYB (Mpc1, Myb10), previously reported in common wheat. The present study provides a set of molecular markers associated with grain pigments useful for the selection of essential alleles for flavonoid synthesis in durum wheat breeding programs and enhancement of the health-promoting quality of derived foods.

Fine Mapping and Candidate Gene Analysis of Pm36, a Wild Emmer-Derived Powdery Mildew Resistance Locus in Durum Wheat.

Powdery mildew (PM) is an economically important foliar disease of cultivated cereals worldwide. The cultivation of disease-resistant varieties is considered the most efficient, sustainable and economical strategy for disease management. The objectives of the current study were to fine map the chromosomal region harboring the wild emmer PM resistance locus Pm36 and to identify candidate genes by exploiting the improved tetraploid wheat genomic resources. A set of backcross inbred lines (BILs) of durum wheat were genotyped with the SNP 25K chip array and comparison of the PM-resistant and susceptible lines defined a 1.5 cM region (physical interval of 1.08 Mb) harboring Pm36. The genetic map constructed with F2:3 progenies derived by crossing the PM resistant line 5BIL-42 and the durum parent Latino, restricted to 0.3 cM the genetic distance between Pm36 and the SNP marker IWB22904 (physical distance 0.515 Mb). The distribution of the marker interval including Pm36 in a tetraploid wheat collection indicated that the positive allele was largely present in the domesticated and wild emmer Triticum turgidum spp. dicoccum and ssp. dicoccoides. Ten high-confidence protein coding genes were identified in the Pm36 region of the emmer, durum and bread wheat reference genomes, while three added genes showed no homologous in the emmer genome. The tightly linked markers can be used for marker-assisted selection in wheat breeding programs, and as starting point for the Pm36 map-based cloning.

Candidate Genes and Quantitative Trait Loci for Grain Yield and Seed Size in Durum Wheat.

Grain yield (YLD) is affected by thousand kernel weight (TKW) which reflects the combination of grain length (GL), grain width (GW) and grain area (AREA). Grain weight is also influenced by heading time (HT) and plant height (PH). To detect candidate genes and quantitative trait loci (QTL) of yield components, a durum wheat recombinant inbred line (RIL) population was evaluated in three field trials. The RIL was genotyped with a 90K single nucleotide polymorphism (SNP) array and a high-density genetic linkage map with 5134 markers was obtained. A total of 30 QTL were detected including 23 QTL grouped in clusters on 1B, 2A, 3A, 4B and 6B chromosomes. A QTL cluster on 2A chromosome included a major QTL for HT co-located with QTL for YLD, TKW, GL, GW and AREA, respectively. The photoperiod sensitivity (Ppd-A1) gene was found in the physical position of this cluster. Serine carboxypeptidase, Big grain 1 and ß-fructofuranosidase candidate genes were mapped in clusters containing QTL for seed size. This study showed that yield components and phenological traits had higher inheritances than grain yield, allowing an accurate QTL cluster detection. This was a requisite to physically map QTL on durum genome and to identify candidate genes affecting grain yield.

Functional Validation of Glutamine synthetase and Glutamate synthase Genes in Durum Wheat near Isogenic Lines with QTL for High GPC.

Durum wheat (Triticum turgidum L. ssp. durum) is a minor crop grown on about 17 million hectares of land worldwide. Several grain characteristics determine semolina's high end-use quality, such as grain protein content (GPC) which is directly related to the final products' nutritional and technological values. GPC improvement could be pursued by considering a candidate gene approach. The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle represents a bottleneck in the first step of nitrogen assimilation. QTL for GPC have been located on all chromosomes, and several major ones have been reported on 2A and 2B chromosomes, where GS2 and Fd-GOGAT genes have been mapped. A useful and efficient method to validate a putative QTL is the constitution of near-isogenic lines (NILs) by using the marker found to be associated to that QTL. Here, we present the development of two distinct sets of heterogeneous inbred family (HIF)- based NILs segregating for GS2 and Fd-GOGAT genes obtained from heterozygous lines at those loci, as well as their genotypic and phenotypic characterizations. The results allow the validation of the previously identified GPC QTL on 2A and 2B chromosomes, along with the role of these key genes in GPC control.

Mapping Powdery Mildew (Blumeria graminis f. sp. tritici) Resistance in Wild and Cultivated Tetraploid Wheats.

Wheat is the most widely grown crop and represents the staple food for one third of the world's population. Wheat is attacked by a large variety of pathogens and the use of resistant cultivars is an effective and environmentally safe strategy for controlling diseases and eliminating the use of fungicides. In this study, a collection of wild and cultivated tetraploid wheats (Triticum turgidum) were evaluated for seedling resistance (SR) and adult plant resistance (APR) to powdery mildew (Blumeria graminis) and genotyped with a 90K single nucleotide polymorphism (SNP) array to identify new sources of resistance genes. The genome-wide association mapping detected 18 quantitative trait loci (QTL) for APR and 8 QTL for SR, four of which were identical or at least closely linked to four QTL for APR. Thirteen candidate genes, containing nucleotide binding sites and leucine-rich repeats, were localized in the confidence intervals of the QTL-tagging SNPs. The marker IWB6155, associated to QPm.mgb-1AS, was located within the gene TRITD1Av1G004560 coding for a disease resistance protein. While most of the identified QTL were described previously, five QTL for APR (QPm.mgb-1AS, QPm.mgb-2BS, QPm.mgb-3BL.1, QPm.mgb-4BL, QPm.mgb-7BS.1) and three QTL for SR (QPm.mgb-3BL.3, QPm.mgb-5AL.2, QPm.mgb-7BS.2) were mapped on chromosome regions where no resistance gene was reported before. The novel QTL/genes can contribute to enriching the resistance sources available to breeders.

Fruit Development in Ficus carica L.: Morphological and Genetic Approaches to Fig Buds for an Evolution From Monoecy Toward Dioecy.

The mechanism behind the bud evolution towards breba or main crop in Ficus carica L. is uncertain. Anatomical and genetic studies may put a light on the possible similarities/differences between the two types of fruits. For this reason, we collected complimentary data from anatomical, X-ray imaging, and genetic techniques. The RNA seq together with structural genome annotation allowed the prediction of 34,629 known genes and 938 novel protein-coding genes. Transcriptome analysis of genes during bud differentiation revealed differentially expressed genes in two fig varieties (Dottato and Petrelli) and in breba and main crop. We chose Dottato and Petrelli because the first variety does not require pollination to set main crop and the latter does; moreover, Petrelli yields many brebas whereas Dottato few. Of the 1,615 and 1,904 loci expressed in Dottato and Petrelli, specifically in breba or main crop, respectively, only 256 genes appeared to be transcripts in both varieties. The buds of the two fig varieties were observed under optical microscope and using 3D X-ray tomography, highlighting differences mainly related to the stage of development. The X-ray images of buds showed a great structural similarity between breba and main crop during the initial stages of development. Analysis at the microscope indicated that inflorescence differentiation of breba was split in two seasons whereas that of main crop started at the end of winter of season 2 and was completed within 2 to 3 months. The higher expression of floral homeotic protein AGAMOUS in breba with respect to main crop, since this protein is required for normal development of stamens and carpels in the flower, may indicate an original role of these fruits for staminate flowers production for pollination of the main crop, as profichi in the caprifig. Several genes related to auxin (auxin efflux carrier, auxin response factor, auxin binding protein, auxin responsive protein) and to GA synthesis (GA20ox) were highly expressed in brebas with respect to main crop for the development of this parthenocarpic fruit.

A Comprehensive Peptidomic Approach to Characterize the Protein Profile of Selected Durum Wheat Genotypes: Implication for Coeliac Disease and Wheat Allergy.

The wheat varietal selection undertaken by breeders in recent decades has been tailored mainly to improve technological and productivity-related traits; however, the latter has resulted in a considerable impoverishment of the genetic diversity of wheat-based products available on the market. This pitfall has encouraged researchers to revalue the natural diversity of cultivated and non-cultivated wheat genotypes in light of their different toxic/immunogenic potential for celiac disease and wheat-allergic patients. In the present investigation, an advanced proteomic approach was designed for the global characterization of the protein profile of selected tetraploid wheat genotypes (Triticum turgidum). The approach combined proteins/peptides sequence information retrieved by specific enzymatic digestions (single and dual proteolytic enzymes) with protein digestibility information disclosed by means of in-vitro simulated human gastroduodenal digestion experiments. In both cases, the peptide pools were characterized by discovery analysis with liquid chromatography high-resolution tandem mass spectrometry, and specific amino acid sequences were identified via commercial software. The peptide list was screened for in silico toxicity/immunogenicity risk assessment, with the aid of various open-source bioinformatics tools for epitopes matching. Given the global information provided by the designed proteomic approach, the in silico risk assessment not only tackled toxicity implication for celiac disease patients, but also scouted for immunogenic sequences relevant for wheat allergic patients, achieving a comprehensive characterization of the protein profile of the selected genotypes. These latter were assessed to encrypt a variable number of toxic/immunogenic epitopes for celiac disease and wheat allergy, and as such they could represent convenient bases for breeding practices and for the development of new detoxification strategies.

Durum wheat genome highlights past domestication signatures and future improvement targets.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.

Scouting for Naturally Low-Toxicity Wheat Genotypes by a Multidisciplinary Approach.

Over the last years, great efforts have been devoted to develop effective gluten detoxification strategies with a consequent detrimental alteration of the technological properties as well. Obtaining low-gluten products without affecting the rheological properties of wheat could still be considered a new challenge to face. In this investigation, we presented a comprehensive characterization of durum wheat genotypes aimed at identifying low gluten ones, which combine the potential lower toxicity/immunogenicity with conserved yield and rheological properties to encompass the perspective usability for bread or pasta making. A preliminary profiling of gluten proteins was accomplished by immunoassay-based quantification and liquid chromatography coupled to UV detection, focusing on the gliadin fraction as main responsible for immunoreactivity in celiac disease patients. In addition, data on grain protein content, grain yield per spike, dry gluten and gluten index were collected in order to provide complementary information about productivity-related traits and quali-quantitative characteristics related to wheat nutritional value and its technological properties. The whole pool of data was statistically evaluated driving to the selection of a preferred list of candidate low-toxicity genotypes that were subjected to in-vitro simulated gastroduodenal digestion and untargeted HR-MS/MS peptide identification. Finally, an in-silico risk assessment of potential toxicity for celiac disease patients was performed according to the most recent guidance provided by EFSA.

The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments.

BACKGROUND: In plants carotenoids play an important role in the photosynthetic process and photo-oxidative protection, and are the substrate for the synthesis of abscisic acid and strigolactones. In addition to their protective role as antioxidants and precursors of vitamin A, in wheat carotenoids are important as they influence the colour (whiteness vs. yellowness) of the grain. Understanding the genetic basis of grain yellow pigments, and identifying associated markers provide the basis for improving wheat quality by molecular breeding. RESULTS: Twenty-four candidate genes involved in the biosynthesis and catabolism of carotenoid compounds have been identified in wheat by comparative genomics. Single nucleotide polymorphisms (SNPs) found in the coding sequences of 19 candidate genes allowed their chromosomal location and accurate map position on two reference consensus maps to be determined. The genome-wide association study based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs validated quantitative trait loci (QTLs) previously detected in biparental populations and discovered new QTLs for grain colour-related traits. Ten carotenoid genes mapped in chromosome regions underlying pigment content QTLs indicating possible functional relationships between candidate genes and the trait. CONCLUSIONS: The availability of linked, candidate gene-based markers can facilitate breeding wheat cultivars with desirable levels of carotenoids. Identifying QTLs linked to carotenoid pigmentation can contribute to understanding genes underlying carotenoid accumulation in the wheat kernels. Together these outputs can be combined to exploit the genetic variability of colour-related traits for the nutritional and commercial improvement of wheat products.

Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

Identification and Characterization of the Sucrose Synthase 2 Gene (Sus2) in Durum Wheat.

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase (SUS), which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for SUS in durum wheat (cultivars Ciccio and Svevo) is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur, and 5-BIL42). The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modeling approaches. The combined results of sucrose synthase 2 expression and activity levels were then considered in the light of their possible involvement in starch yield.

Characterization of ferredoxin-dependent glutamine-oxoglutarate amidotransferase (Fd-GOGAT) genes and their relationship with grain protein content QTL in wheat.

BACKGROUND: In higher plants, inorganic nitrogen is assimilated via the glutamate synthase cycle or GS-GOGAT pathway. GOGAT enzyme occurs in two distinct forms that use NADH (NADH-GOGAT) or Fd (Fd-GOGAT) as electron carriers. The goal of the present study was to characterize wheat Fd-GOGAT genes and to assess the linkage with grain protein content (GPC), an important quantitative trait controlled by multiple genes. RESULTS: We report the complete genomic sequences of the three homoeologous A, B and D Fd-GOGAT genes from hexaploid wheat (Triticum aestivum) and their localization and characterization. The gene is comprised of 33 exons and 32 introns for all the three homoeologues genes. The three genes show the same exon/intron number and size, with the only exception of a series of indels in intronic regions. The partial sequence of the Fd-GOGAT gene located on A genome was determined in two durum wheat (Triticum turgidum ssp. durum) cvs Ciccio and Svevo, characterized by different grain protein content. Genomic differences allowed the gene mapping in the centromeric region of chromosome 2A. QTL analysis was conducted in the Svevo×Ciccio RIL mapping population, previously evaluated in 5 different environments. The study co-localized the Fd-GOGAT-A gene with the marker GWM-339, identifying a significant major QTL for GPC. CONCLUSIONS: The wheat Fd-GOGAT genes are highly conserved; both among the three homoeologous hexaploid wheat genes and in comparison with other plants. In durum wheat, an association was shown between the Fd-GOGAT allele of cv Svevo with increasing GPC - potentially useful in breeding programs.

Structural analysis of the wheat genes encoding NADH-dependent glutamine-2-oxoglutarate amidotransferases and correlation with grain protein content.

BACKGROUND: Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutamine synthetase (GS), GOGAT maintains the flow of nitrogen from NH4 (+) into glutamine and glutamate, which are then used for several aminotransferase reactions during amino acid synthesis. RESULTS: The aim of the present work was to identify and analyse the structure of wheat NADH-GOGAT genomic sequences, and study the expression in two durum wheat cultivars characterized by low and high kernel protein content. The genomic sequences of the three homoeologous A, B and D NADH-GOGAT genes were obtained for hexaploid Triticum aestivum and the tetraploid A and B genes of Triticum turgidum ssp. durum. Analysis of the gene sequences indicates that all wheat NADH-GOGAT genes are composed of 22 exons and 21 introns. The three hexaploid wheat homoeologous genes have high conservation of sequence except intron 13 which shows differences in both length and sequence. A comparative analysis of sequences among di- and mono-cotyledonous plants shows both regions of high conservation and of divergence. qRT-PCR performed with the two durum wheat cvs Svevo and Ciccio (characterized by high and low protein content, respectively) indicates different expression levels of the two NADH-GOGAT-3A and NADH-GOGAT-3B genes. CONCLUSION: The three hexaploid wheat homoeologous NADH-GOGAT gene sequences are highly conserved - consistent with the key metabolic role of this gene. However, the dicot and monocot amino acid sequences show distinctive patterns, particularly in the transit peptide, the exon 16-17 junction, and the C-terminus. The lack of conservation in the transit peptide may indicate subcellular differences between the two plant divisions - while the sequence conservation within enzyme functional domains remains high. Higher expression levels of NADH-GOGAT are associated with higher grain protein content in two durum wheats.

The glutamine synthetase (GS2) genes in relation to grain protein content of durum wheat.

Glutamine synthetase (GS2) is a key enzyme in plant nitrogen metabolism responsible of the first step of ammonium assimilation and transformation into glutamine (an essential compound in the amino acid-biosynthetic pathway). The goal of the present study was to isolate and characterize GS2 genes and to assess the linkage with grain protein content (GPC), an important quantitative trait controlled by multiple genes. Here, we report the isolation of the complete glutamine synthetase gene sequences and their localization on the two homeologous chromosome 2A and 2B in durum wheat cvs. Ciccio and Svevo characterized by a different grain protein content. GS2-A2 located on 2A chromosome is comprised of 13 exons separated by 12 introns, and the allele sequence in the two cultivars were different for an insertion of 5 bp located in the third exon in the cv. Ciccio. The GS2-B2 has the same intron/exon structure, but the two cultivars differ for the insertion of a 33-bp sequence located in the second intron of the cv. Svevo. Specific primers were designed in the polymorphic region and amplified in a recombinant inbred line mapping population. The study localized GS genes (GS2-A2, GS2-B2 GSe, GSr) on chromosomes 2A, 2B, 4A, and 4B, where four significant QTLs for GPC were also located.