Identification of traits underpinning good breadmaking performance of wheat grown with reduced nitrogen fertilisation.

BACKGROUND: Nitrogen fertiliser is the major input and cost for wheat production, being required to support the development of the canopy to maximise yield and for the synthesis of the gluten proteins that are necessary for breadmaking. Consequently, current high-yielding cultivars require the use of nitrogen fertilisation levels above the yield optimum to achieve the grain protein content needed for breadmaking. This study aimed to reduce this requirement by identifying traits that allow the use of lower levels of nitrogen fertiliser to produce wheat for breadmaking. RESULTS: A range of commercial wheat genotypes (cultivars) were grown in multiple field trials (six sites over 3 years) in the UK with optimal (200 kg Ha-1 ) and suboptimal (150 kg Ha-1 ) application of nitrogen. Bulked grain samples from four sites per year were milled and white flours were baked using three types of breadmaking process. This identified five cultivars that consistently exhibited good breadmaking quality when grown with the lower nitrogen application. Chemical and biochemical analyses showed that the five cultivars were characterised by exhibiting grain protein deviation (GPD) and high dough elasticity. CONCLUSIONS: It is possible to develop novel types of wheat that exhibit good breadmaking quality by selecting for GPD and high dough strength. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Allele mining in diverse accessions of tropical grasses to improve forage quality and reduce environmental impact.

BACKGROUND AND AIMS: The C4Urochloa species (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease the amount of land required per unit production, and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We therefore set out to look for such alleles in diverse accessions of Urochloa species and Megathyrsus maximus from the genebank collection held at the CIAT. METHODS: We studied allelic diversity of 20 target genes (11 for digestibility, nine for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNA sequencing and then bait capture DNA sequencing to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence. KEY RESULTS: We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified seven putative LOF alleles with high confidence, including those in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme. CONCLUSIONS: We demonstrated a novel, effective approach to allele discovery in diverse accessions using a draft reference genome from a single species. We used this to find gene variants in a collection of tropical grasses that could help reduce the environmental impact of cattle production.

High post-anthesis temperature effects on bread wheat (Triticum aestivum L.) grain transcriptome during early grain-filling.

BACKGROUND: High post-anthesis (p.a) temperatures reduce mature grain weights in wheat and other cereals. However, the causes of this reduction are not entirely known. Control of grain expansion by the maternally derived pericarp of the grain has previously been suggested, although this interaction has not been investigated under high p.a. temperatures. Down-regulation of pericarp localised genes that regulate cell wall expansion under high p.a. temperatures may limit expansion of the encapsulated endosperm due to a loss of plasticity in the pericarp, reducing mature grain weight. Here the effect of high p.a. temperatures on the transcriptome of the pericarp and endosperm of the wheat grain during early grain-filling was investigated via RNA-Seq and is discussed alongside grain moisture dynamics during early grain development and mature grain weight. RESULTS: High p.a. temperatures applied from 6-days after anthesis (daa) and until 18daa reduced the grain's ability to accumulate water, with total grain moisture and percentage grain moisture content being significantly reduced from 14daa onwards. Mature grain weight was also significantly reduced by the same high p.a. temperatures applied from 6daa for 4-days or more, in a separate experiment. Comparison of our RNA-Seq data from whole grains, with existing data sets from isolated pericarp and endosperm tissues enabled the identification of subsets of genes whose expression was significantly affected by high p.a. temperature and predominantly expressed in either tissue. Hierarchical clustering and gene ontology analysis resulted in the identification of a number of genes implicated in the regulation of cell wall expansion, predominantly expressed in the pericarp and significantly down-regulated under high p.a. temperatures, including endoglucanase, xyloglucan endotransglycosylases and a ß-expansin. An over-representation of genes involved in the 'cuticle development' functional pathway that were expressed in the pericarp and affected by high p.a. temperatures was also observed. CONCLUSIONS: High p.a. temperature induced down-regulation of genes involved in regulating pericarp cell wall expansion. This concomitant down-regulation with a reduction in total grain moisture content and grain weight following the same treatment period, adds support to the theory that high p.a. temperatures may cause a reduction in mature grain weight as result of decreased pericarp cell wall expansion.

Loss of TaIRX9b gene function in wheat decreases chain length and amount of arabinoxylan in grain but increases cross-linking.

Wheat contains abundant xylan in cell walls of all tissues, but in endosperm, there is an unusual form of xylan substituted only by arabinose (arabinoxylan; AX) that has long chains and low levels of feruloylation, a fraction of which is extractable in water (WE-AX). WE-AX acts as soluble dietary fibre but also gives rise to viscous extracts from grain, a detrimental trait for some non-food uses of wheat. Here, we show that a glycosyl transferase family 43 wheat gene abundantly expressed in endosperm complements the Arabidopsis irx9 mutant and so name the three homoeologous genes TaIRX9b. We generated wheat lines with a constitutive knockout of TaIRX9b by stacking loss-of-function alleles for these homeologues from a mutagenized hexaploid wheat population resulting in decreases in grain extract viscosity of 50%-80%. The amount and chain length of WE-AX molecules from grain of these triple-stack lines was decreased accounting for the changes in extract viscosity. Imaging of immature wheat grain sections of triple-stacks showed abolition of immunolabelling in endosperm with LM11 antibody that recognizes epitopes in AX, but also showed apparently normal cell size and shape in all cell types, including endosperm. We identified differentially expressed genes from endosperm of triple-stacks suggesting that compensatory changes occur to maintain this endosperm cell wall integrity. Consistent with this, we observed increased ferulate dimerization and increased cross-linking of WE-AX molecules in triple-stacks. These novel wheat lines lacking functional TaIRX9b therefore provide insight into control of wheat endosperm cell walls.

Genetic variation and heritability of grain protein deviation in European wheat genotypes.

There is a well-established negative relationship between the yield and the concentration of protein in the mature wheat grain. However, some wheat genotypes consistently deviate from this relationship, a phenomenon known as Grain Protein Deviation (GPD). Positive GPD is therefore of considerable interest in relation to reducing the requirement for nitrogen fertilization for producing wheat for breadmaking. We have carried out two sets of field experiments on multiple sites in South East England. The first set comprised 11 field trials of 6 cultivars grown over three years (2008-2011) and the second comprised 9 field trials of 40 genotypes grown over two years (2015-2017) and 5 field trials of 30 genotypes grown in a single year (2017-2018). All trials comprised three replicate randomized plots of each genotype and nutrient regime. These studies showed strong genetic variation in GPD, which also differed in stability between genotypes, with cultivars bred in the UK generally having higher GPD and higher stability than those bred in other European countries. The heritability of GPD was estimated as 0.44, based on data from the field trials of 30 and 40 genotypes. The largest component contributing to the genetic variance was genotype (0.30), with a smaller contribution of the interaction between genotype and year/site (0.11) and a small (but statistically significant) contribution of nitrogen level. These studies suggest that selection for GPD is a viable target for breeders.

Response of cell-wall composition and RNA-seq transcriptome to methyl-jasmonate in Brachypodium distachyon callus.

MAIN CONCLUSION: Methyl-jasmonate induces large increases in p-coumarate linked to arabinoxylan in Brachypodium and in abundance of GT61 and BAHD family transcripts consistent with a role in synthesis of this linkage. Jasmonic acid (JA) signalling is required for many stress responses in plants, inducing large changes in the transcriptome, including up-regulation of transcripts associated with lignification. However, less is known about the response to JA of grass cell walls and the monocot-specific features of arabinoxylan (AX) synthesis and acylation by ferulic acid (FA) and para-coumaric acid (pCA). Here, we show that methyl-jasmonate (MeJA) induces moderate increases in FA monomer, > 50% increases in FA dimers, and five-sixfold increases in pCA ester-linked to cell walls in Brachypodium callus. Direct measurement of arabinose acylated by pCA (Araf-pCA) indicated that most or all the increase in cell-wall pCA was due to pCA ester-linked to AX. Analysis of the RNA-seq transcriptome of the callus response showed that these cell-wall changes were accompanied by up-regulation of members of the GT61 and BAHD gene families implicated in AX decoration and acylation; two BAHD paralogues were among the most up-regulated cell-wall genes (seven and fivefold) after 24 h exposure to MeJA. Similar responses to JA of orthologous BAHD and GT61 transcripts are present in the RiceXPro public expression data set for rice seedlings, showing that they are not specific to Brachypodium or to callus. The large response of AX-pCA to MeJA may, therefore, indicate an important role for this linkage in response of primary cell walls of grasses to JA signalling.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses.

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)- and α-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases α-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for α-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content.

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.

The transcription factor ABI4 Is required for the ascorbic acid-dependent regulation of growth and regulation of jasmonate-dependent defense signaling pathways in Arabidopsis.

Cellular redox homeostasis is a hub for signal integration. Interactions between redox metabolism and the ABSCISIC ACID-INSENSITIVE-4 (ABI4) transcription factor were characterized in the Arabidopsis thaliana vitamin c defective1 (vtc1) and vtc2 mutants, which are defective in ascorbic acid synthesis and show a slow growth phenotype together with enhanced abscisic acid (ABA) levels relative to the wild type (Columbia-0). The 75% decrease in the leaf ascorbate pool in the vtc2 mutants was not sufficient to adversely affect GA metabolism. The transcriptome signatures of the abi4, vtc1, and vtc2 mutants showed significant overlap, with a large number of transcription factors or signaling components similarly repressed or induced. Moreover, lincomycin-dependent changes in LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN 1.1 expression were comparable in these mutants, suggesting overlapping participation in chloroplast to nucleus signaling. The slow growth phenotype of vtc2 was absent in the abi4 vtc2 double mutant, as was the sugar-insensitive phenotype of the abi4 mutant. Octadecanoid derivative-responsive AP2/ERF-domain transcription factor 47 (ORA47) and AP3 (an ABI5 binding factor) transcripts were enhanced in vtc2 but repressed in abi4 vtc2, suggesting that ABI4 and ascorbate modulate growth and defense gene expression through jasmonate signaling. We conclude that low ascorbate triggers ABA- and jasmonate-dependent signaling pathways that together regulate growth through ABI4. Moreover, cellular redox homeostasis exerts a strong influence on sugar-dependent growth regulation.

Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome.

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3;1,4)-ß-d-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Comparative Compositions of Grain of Bread Wheat, Emmer and Spelt Grown with Different Levels of Nitrogen Fertilisation.

Five cultivars of bread wheat and spelt and three of emmer were grown in replicate randomised field trials on two sites for two years with 100 and 200 kg nitrogen fertiliser per hectare, reflecting low input and intensive farming systems. Wholemeal flours were analysed for components that are suggested to contribute to a healthy diet. The ranges of all components overlapped between the three cereal types, reflecting the effects of both genotype and environment. Nevertheless, statistically significant differences in the contents of some components were observed. Notably, emmer and spelt had higher contents of protein, iron, zinc, magnesium, choline and glycine betaine, but also of asparagine (the precursor of acrylamide) and raffinose. By contrast, bread wheat had higher contents of the two major types of fibre, arabinoxylan (AX) and ß-glucan, than emmer and a higher AX content than spelt. Although such differences in composition may be suggested to result in effects on metabolic parameters and health when studied in isolation, the final effects will depend on the quantity consumed and the composition of the overall diet.

Recruitment of glutathione into the nucleus during cell proliferation adjusts whole-cell redox homeostasis in Arabidopsis thaliana and lowers the oxidative defence shield.

Cellular redox homeostasis and signalling are important in progression of the eukaryotic cell cycle. In animals, the low-molecular-weight thiol tripeptide glutathione (GSH) is recruited into the nucleus early in the cell proliferation cycle. To determine whether a similar process occurs in plants, we studied cell proliferation in Arabidopsis thaliana. We show that GSH co-localizes with nuclear DNA during the proliferation of A. thaliana cells in culture. Moreover, GSH localization in the nucleus was observed in dividing pericycle cells of the lateral root meristem. There was pronounced accumulation of GSH in the nucleus at points in the growth cycle at which a high percentage of the cells were in G(1) phase, as identified by flow cytometry and marker transcripts. Recruitment of GSH into the nucleus led to a high abundance of GSH in the nucleus (GSHn) and severe depletion of the cytoplasmic GSH pool (GSHc). Sequestration of GSH in the nucleus was accompanied by significant decreases in transcripts associated with oxidative signalling and stress tolerance, and an increase in the abundance of hydrogen peroxide, an effect that was enhanced when the dividing cells were treated with salicylic acid. Total cellular GSH and the abundance of GSH1 and GSH2 transcripts increased after the initial recruitment of GSH into the nucleus. We conclude that GSH recruitment into the nucleus during cell proliferation has a profound effect on the whole-cell redox state. High GSHn levels trigger redox adjustments in the cytoplasm, favouring decreased oxidative signalling and enhanced GSH synthesis.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Acclimation to high CO2 in maize is related to water status and dependent on leaf rank.

The responses of C(3) plants to rising atmospheric CO(2) levels are considered to be largely dependent on effects exerted through altered photosynthesis. In contrast, the nature of the responses of C(4) plants to high CO(2) remains controversial because of the absence of CO(2) -dependent effects on photosynthesis. In this study, the effects of atmospheric CO(2) availability on the transcriptome, proteome and metabolome profiles of two ranks of source leaves in maize (Zea mays L.) were studied in plants grown under ambient CO(2) conditions (350 +/- 20 µL L(-1) CO(2) ) or with CO(2) enrichment (700 +/- 20 µL L(-1) CO(2) ). Growth at high CO(2) had no effect on photosynthesis, photorespiration, leaf C/N ratios or anthocyanin contents. However, leaf transpiration rates, carbohydrate metabolism and protein carbonyl accumulation were altered at high CO(2) in a leaf-rank specific manner. Although no significant CO(2) -dependent changes in the leaf transcriptome were observed, qPCR analysis revealed that the abundance of transcripts encoding a Bowman-Birk protease inhibitor and a serpin were changed by the growth CO(2) level in a leaf rank specific manner. Moreover, CO(2) -dependent changes in the leaf proteome were most evident in the oldest source leaves. Small changes in water status may be responsible for the observed responses to high CO(2,) particularly in the older leaf ranks.

Accumulation and deposition of triacylglycerols in the starchy endosperm of wheat grain.

A combination of lipidomics, transcriptomics and bioimaging has been used to study triacylglycerol synthesis and deposition in the developing starchy endosperm of wheat. The content of TAG increased between 14 and 34 days after anthesis, from 50 to 115 mg/100 g dry wt and from about 35 to 175 mg/100 g dry wt in two experiments. The major fatty acids were C16 (palmitic C16:0 and palmitoleic C16:1) and C18 (stearic C18:0, oleic C18:1, linoleic C18:2 and linolenic C18:3), with unsaturated fatty acids accounting for about 75-80% of the total throughout development. Linoleic acid (C18:2) was the major component at all stages and the proportion increased during development. Transcript profiling indicated that predominant route to TAG synthesis and oil accumulation is via the Kennedy pathway and diacylglycerol acyltransferase (DGAT) activity. Confocal microscopy of stained tissue sections showed that TAG accumulated in droplets which are associated with protein and concentrated in the starchy endosperm cells below the sub-aleurone cells. Transcripts encoding 16kd oleosins were also expressed, indicating that the oil droplets are in part stabilised by oleosin proteins.

Flow Cytometry-Based Determination of Ploidy from Dried Leaf Specimens in Genomically Complex Collections of the Tropical Forage Grass Urochloa s. l.

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the 'brizantha' and 'humidicola' agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

Conditional modulation of NAD levels and metabolite profiles in Nicotiana sylvestris by mitochondrial electron transport and carbon/nitrogen supply.

Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO(2) and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO(2) and increased N supply both significantly enhanced NAD(+) and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD(+) and NADH compared to the WT, but the differences in NADH were smaller at high CO(2) than in air because high CO(2) increased WT NADH/NAD(+). The CMSII-linked increases in NAD(+) and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD(+) and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD(+) and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD(+) and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent signaling pathways.

Historical changes in the contents and compositions of fibre components and polar metabolites in white wheat flour.

Thirty-nine UK adapted wheat cultivars dating from between 1790 and 2012 were grown in replicated randomised field trials for three years, milled, and white flour analysed for the contents of dietary fibre components (arabinoxylan and ß-glucan) and polar metabolites (sugars, amino acids, organic acids, choline and betaine) to determine whether the composition had changed due to the effects of intensive breeding. The concentrations of components varied between study years, indicating strong effects of environment. Nevertheless, some trends were observed, with the concentrations of arabinoxylan fibre and soluble sugars (notably sucrose, maltose and fructose) increasing and most amino acids (including asparagine which is the precursor of acrylamide formed during processing) decreasing between the older and newer types. The concentration of betaine, which is beneficial for cardio-vascular health, also increased. The study therefore provided no evidence for adverse effects of intensive breeding on the contents of beneficial components in wheat flour.

Identification of a major QTL and associated molecular marker for high arabinoxylan fibre in white wheat flour.

Dietary fibre (DF) has multiple health benefits and wheat grains are major sources of DF for human health. However, DF is depleted in white wheat flour which is more widely consumed than wholegrain. The major DF component in white flour is the cell wall polysaccharide arabinoxylan (AX). We have identified the Chinese wheat cultivar Yumai 34 as having unusually high contents of AX in both water-soluble and insoluble forms. We have therefore used populations generated from crosses between Yumai 34 and four other wheat cultivars, three with average contents of AX (Ukrainka, Altigo and Claire) and one also having unusually high AX (Valoris), in order to map QTLs for soluble AX (determined as relative viscosity of aqueous extracts of wholemeal flours) and total AX (determined by enzyme fingerprinting of white flour). A number of QTL were mapped, but most were only detected in one or two crosses. However, all four crosses showed strong QTLs for high RV/total AX on chromosome 1B, with Yumai 34 being the increasing parent, and a KASP marker for the Yumai 34 high AX allele was validated by analysis of high AX lines derived from Yumai 34 but selected by biochemical analysis. A QTL for RV was also mapped on chromosome 6B in Yumai 34 x Valoris, with Valoris being the increasing allele, which is consistent with the observation of transgressive segregation for this population. Association studies in an independent germplasm panel identified marker trait associations for relative viscosity in these same locations while direct selection for fibre content in breeding resulted in high levels of enrichment for the Yumai 34 1B allele. The data therefore indicate that marker-assisted breeding can be used to develop wheat with high AX fibre in white flour.