The First Comprehensive Chemical Profiling of Vachellia gummifera (Willd.) Kyal. & Boatwr., a Plant with Medicinal Value.

Vachellia gummifera (Willd.) Kyal. & Boatwr. is a medicinal plant endemic to Morocco that has no documented studies on its chemical composition. In this study, the chemical composition of the water/methanol (4 : 1) extracts of air-dried leaf and stem samples of Moroccan V. gummifera was determined using UHPLC-MS and NMR. In total, over 100 metabolites were identified in our study. Pinitol was the major compound in both the leaf and stem extracts, being significantly more abundant in the former. Asparagine and 3-hydroxyheteroendrin were the second most abundant compounds in the stem and leaf extracts, respectively, though both compounds were present in each tissue. The other compounds included flavonoids based on quercetin, and phenolic derivatives. Eucomic acid, only identified in the stems and was the major aromatic compound distinguishing the leaf and stem profiles. Quercetin 3-O-(6''-O-malonyl)-ß-D-glucopyranoside was identified as the major flavonoid in the leaves but was also present in the stems. Other malonylated derivatives that were all flavonol glycosides based on myricetin, kaempferol, and isorhamnetin in addition to quercetin were also identified. This is the first report of eucomic acid and malonylated compounds in Vachellia species. This report provides valuable insights into the chemotaxonomic significance of the Vachellia genus.

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds.

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

A willow UDP-glycosyltransferase involved in salicinoid biosynthesis.

The salicinoids are phenolic glycosides that are characteristic secondary metabolites of the Salicaceae, particularly willows and poplars. Despite the well-known pharmacology of salicin, that led to the development of aspirin >100 years ago, the biosynthetic pathways leading to salicinoids have yet to be defined. Here, we describe the identification, cloning, and biochemical characterization of SpUGT71L2 and SpUGT71L3-isozymic glycosyltransferases from Salix purpurea-that function in the glucosylation of ortho-substituted phenols. The best substrate in vitro was salicyl-7-benzoate. Its product, salicyl-7-benzoate glucoside, was shown to be endogenous in poplar and willow. Together they are inferred to be early intermediates in the biosynthesis of salicortin and related metabolites in planta. The role of this UDP-glycosyltransferase was confirmed via the metabolomic analysis of transgenic plants produced by RNAi knockdown of the poplar orthologue (UGT71L1) in the hybrid clone Populus tremula×P. alba, INRA 717-1B4.

Oleuropein derivatives from olive fruit extracts reduce α-synuclein fibrillation and oligomer toxicity.

Aggregation of α-synuclein (αSN) is implicated in neuronal degeneration in Parkinson's disease and has prompted searches for natural compounds inhibiting αSN aggregation and reducing its tendency to form toxic oligomers. Oil from the olive tree (Olea europaea L.) represents the main source of fat in the Mediterranean diet and contains variable levels of phenolic compounds, many structurally related to the compound oleuropein. Here, using αSN aggregation, fibrillation, size-exclusion chromatography-multiangle light scattering (SEC-MALS)-based assays, and toxicity assays, we systematically screened the fruit extracts of 15 different olive varieties to identify compounds that can inhibit αSN aggregation and oligomer toxicity and also have antioxidant activity. Polyphenol composition differed markedly among varieties. The variety with the most effective antioxidant and aggregation activities, Koroneiki, combined strong inhibition of αSN fibril nucleation and elongation with strong disaggregation activity on preformed fibrils and prevented the formation of toxic αSN oligomers. Fractionation of the Koroneiki extract identified oleuropein aglycone, hydroxyl oleuropein aglycone, and oleuropein as key compounds responsible for the differences in inhibition across the extracts. These phenolic compounds inhibited αSN amyloidogenesis by directing αSN monomers into small αSN oligomers with lower toxicity, thereby suppressing the subsequent fibril growth phase. Our results highlight the molecular consequences of differences in the level of effective phenolic compounds in different olive varieties, insights that have implications for long-term human health.

Transitions in wheat endosperm metabolism upon transcriptional induction of oil accumulation by oat endosperm WRINKLED1.

BACKGROUND: Cereal grains, including wheat (Triticum aestivum L.), are major sources of food and feed, with wheat being dominant in temperate zones. These end uses exploit the storage reserves in the starchy endosperm of the grain, with starch being the major storage component in most cereal species. However, oats (Avena sativa L.) differs in that the starchy endosperm stores significant amounts of oil. Understanding the control of carbon allocation between groups of storage compounds, such as starch and oil, is therefore important for understanding the composition and hence end use quality of cereals. WRINKLED1 is a transcription factor known to induce triacylglycerol (TAG; oil) accumulation in several plant storage tissues. RESULTS: An oat endosperm homolog of WRI1 (AsWRI1) expressed from the endosperm-specific HMW1Dx5 promoter resulted in drastic changes in carbon allocation in wheat grains, with reduced seed weight and a wrinkled seed phenotype. The starch content of mature grain endosperms of AsWRI1-wheat was reduced compared to controls (from 62 to 22% by dry weight (dw)), TAG was increased by up to nine-fold (from 0.7 to 6.4% oil by dw) and sucrose from 1.5 to 10% by dw. Expression of AsWRI1 in wheat grains also resulted in multiple layers of elongated peripheral aleurone cells. RNA-sequencing, lipid analyses, and pulse-chase experiments using 14C-sucrose indicated that futile cycling of fatty acids could be a limitation for oil accumulation. CONCLUSIONS: Our data show that expression of oat endosperm WRI1 in the wheat endosperm results in changes in metabolism which could underpin the application of biotechnology to manipulate grain composition. In particular, the striking effect on starch synthesis in the wheat endosperm indicates that an important indirect role of WRI1 is to divert carbon allocation away from starch biosynthesis in plant storage tissues that accumulate oil.

The anaplerotic node is essential for the intracellular survival of Mycobacterium tuberculosis.

Enzymes at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.

Phenylalkanoid Glycosides (Non-Salicinoids) from Wood Chips of Salix triandra × dasyclados Hybrid Willow.

Salix triandra (almond leaved willow) is an established crop, grown in coppicing regimes for basket-making materials. It is known as a source of non-salicinoid phenolic glycosides, such as triandrin and salidroside. A spontaneous natural hybrid of S. triandra and S. dasyclados was subjected to metabolite profiling by high resolution LC-MS, and 22 phenolic glycosides, including 18 that are new to the Salicaceae, were identified. Structures were determined by HPLC isolation and NMR methods. The hybridisation process has introduced novel chemistry into the Salix phenolic glycoside palette, in particular, the ability to generate disaccharide conjugates where the glycosyl group is further extended by a range of sugars, including apiose, rhamnose, xylose, and arabinose. Also of note is the appearance of chavicol derivatives, also not previously seen in Salix spp. The work demonstrates the plasticity of the phenolic glycoside biosynthetic pathway, and the potential to improve established crops such as S. triandra and S. dasyclados, via high-value metabolites, for both basketry and bioenergy markets.

Feruloylation and structure of arabinoxylan in wheat endosperm cell walls from RNAi lines with suppression of genes responsible for backbone synthesis and decoration.

Arabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end-use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE-AX), then xylanase action (XE-AX) leaving an unextractable (XU-AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild-type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3-linked to mono-substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5-7 and 13-19 mg bound ferulate (g-1 AX), and their content of diferulates (diFA), <0.3, 1-1.7 and 4-5 mg (g-1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE-AX fraction than in the WE-AX fraction and largely unaffected in the XU-AX fraction. As the majority of diFA is associated with the XU-AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.

(1)H-NMR screening for the high-throughput determination of genotype and environmental effects on the content of asparagine in wheat grain.

Free asparagine in cereals is known to be the precursor of acrylamide, a neurotoxic and carcinogenic product formed during cooking processes. Thus, the development of crops with lower asparagine is of considerable interest to growers and the food industry. In this study, we describe the development and application of a rapid (1)H-NMR-based analysis of cereal flour, that is, suitable for quantifying asparagine levels, and hence acrylamide-forming potential, across large numbers of samples. The screen was applied to flour samples from 150 bread wheats grown at a single site in 2005, providing the largest sample set to date. Additionally, screening of 26 selected cultivars grown for two further years in the same location and in three additional European locations in the third year (2007) provided six widely different environments to allow estimation of the environmental (E) and G x E effects on asparagine levels. Asparagine concentrations in the 150 genotypes ranged from 0.32 to 1.56 mg/g dry matter in wholemeal wheat flours. Asparagine levels were correlated with plant height and therefore, due to recent breeding activities to produce semi-dwarf varieties, a negative relationship with the year of registration of the cultivar was also observed. The multisite study indicated that only 13% of the observed variation in asparagine levels was heritable, whilst the environmental contribution was 36% and the GxE component was 43%. Thus, compared to some other phenotypic traits, breeding for low asparagine wheats presents a difficult challenge.

Postprandial plasma betaine and other methyl donor-related responses after consumption of minimally processed wheat bran or wheat aleurone, or wheat aleurone incorporated into bread.

The bran and particularly the aleurone fraction of wheat are high in betaine and other physiological methyl donors, which may exert beneficial physiological effects. We conducted two randomised, controlled, cross-over postprandial studies to assess and compare plasma betaine and other methyl donor-related responses following the consumption of minimally processed bran and aleurone fractions (study A) and aleurone bread (study B). For both studies, standard pharmacokinetic parameters were derived for betaine, choline, folate, dimethylglycine (DMG), total homocysteine and methionine from plasma samples taken at 0, 0·5, 1, 2 and 3 h. In study A (n 14), plasma betaine concentrations were significantly and substantially elevated from 0·5 to 3 h following the consumption of both bran and aleurone compared with the control; however, aleurone gave significantly higher responses than bran. Small, but significant, increases were also observed in DMG measures; however, no significant responses were observed in other analytes. In study B (n 13), plasma betaine concentrations were significantly and substantially higher following consumption of the aleurone bread compared with the control bread; small, but significant, increases were also observed in DMG and folate measures in response to consumption of the aleurone bread; however, no significant responses were observed in other analytes. Peak plasma betaine concentrations, which were 1·7-1·8 times the baseline levels, were attained earlier following the consumption of minimally processed aleurone compared with the aleurone bread (time taken to reach peak concentration 1·2 v. 2·1 h). These results showed that the consumption of minimally processed wheat bran, and particularly the aleurone fraction, yielded substantial postprandial increases in plasma betaine concentrations. Furthermore, these effects appear to be maintained when aleurone was incorporated into bread.

Opposite variations in fumarate and malate dominate metabolic phenotypes of Arabidopsis salicylate mutants with abnormal biomass under chilling.

In chilling conditions (5°C), salicylic acid (SA)-deficient mutants (sid2, eds5 and NahG) of Arabidopsis thaliana produced more biomass than wild type (Col-0), whereas the SA overproducer cpr1 was extremely stunted. The hypothesis that these phenotypes were reflected in metabolism was explored using 600 MHz (1) H nuclear magnetic resonance (NMR) analysis of unfractionated polar shoot extracts. Biomass-related metabolic phenotypes were identified as multivariate data models of these NMR 'fingerprints'. These included principal components that correlated with biomass. Also, partial least squares-regression models were found to predict the relative size of plants in previously unseen experiments in different light intensities, or relative size of one genotype from the others. The dominant signal in these models was fumarate, which was high in SA-deficient mutants, intermediate in Col-0 and low in cpr1 at 5°C. Among signals negatively correlated with biomass, malate was prominent. Abundance of transcripts of the FUM2 cytosolic fumarase (At5g50950) showed strong positive correlation with fumarate levels and with biomass, whereas no significant differences were found for the FUM1 mitochondrial fumarase (At2g47510). It was confirmed that the morphological effects of SA under chilling find expression in the metabolome, with a role of fumarate highlighted.

Metabolomic analysis of Arabidopsis reveals hemiterpenoid glycosides as products of a nitrate ion-regulated, carbon flux overflow.

An understanding of the balance between carbon and nitrogen assimilation in plants is key to future bioengineering for a range of applications. Metabolomic analysis of the model plant, Arabidopsis thaliana, using combined NMR-MS revealed the presence of two hemiterpenoid glycosides that accumulated in leaf tissue, to ~1% dry weight under repeated nitrate-deficient conditions. The formation of these isoprenoids was correlated with leaf nitrate concentrations that could also be assayed in the metabolomic data using a unique flavonoid-nitrate mass spectral adduct. Analysis of leaf and root tissue from plants grown in hydroponics with a variety of root stressors identified the conditions under which the isoprenoid pathway in leaves was diverted to the hemiterpenoids. These compounds were strongly induced by root wounding or oxidative stress and weakly induced by potassium deficiency. Other stresses such as cold, saline, and osmotic stress did not induce the compounds. Replacement of nitrate with ammonia failed to suppress the formation of the hemiterpenoids, indicating that nitrate sensing was a key factor. Feeding of intermediates was used to study aspects of 2-C-methyl-d-erythritol-4-phosphate pathway regulation leading to hemiterpenoid formation. The formation of the hemiterpenoids in leaves was strongly correlated with the induction of the phenylpropanoids scopolin and coniferin in roots of the same plants. These shunts of photosynthetic carbon flow are discussed in terms of overflow mechanisms that have some parallels with isoprene production in tree species.

The mechanisms behind the contrasting responses to waterlogging in black-grass (Alopecurus myosuroides) and wheat (Triticum aestivum).

Black-grass (Alopecurus myosuroides ) is one of the most problematic agricultural weeds of Western Europe, causing significant yield losses in winter wheat (Triticum aestivum ) and other crops through competition for space and resources. Previous studies link black-grass patches to water-retaining soils, yet its specific adaptations to these conditions remain unclear. We designed pot-based waterlogging experiments to compare 13 biotypes of black-grass and six cultivars of wheat. These showed that wheat roots induced aerenchyma when waterlogged whereas aerenchyma-like structures were constitutively present in black-grass. Aerial biomass of waterlogged wheat was smaller, whereas waterlogged black-grass was similar or larger. Variability in waterlogging responses within and between these species was correlated with transcriptomic and metabolomic changes in leaves of control or waterlogged plants. In wheat, transcripts associated with regulation and utilisation of phosphate compounds were upregulated and sugars and amino acids concentrations were increased. Black-grass biotypes showed limited molecular responses to waterlogging. Some black-grass amino acids were decreased and one transcript commonly upregulated was previously identified in screens for genes underpinning metabolism-based resistance to herbicides. Our findings provide insights into the different waterlogging tolerances of these species and may help to explain the previously observed patchiness of this weed's distribution in wheat fields.

Breeding Novel Chemistry in Willow: New Hetero Diels-Alder Cyclodimers from Arbusculoidin and Salicortin Suggest Parallel Biosynthetic Pathways.

An investigation of phenolic glycosides extracted from Salix germplasm revealed that arbusculoidin (benzyl 1-O-ß-d-glucopyranosyl-1-hydroxy-6-oxo-2-cyclohexenyl carboxylate) and its enolic 6-glycoside isomer, isoarbusculoidin, are widespread across the Salix family. An analysis of natural hybrid species and progeny from a willow breeding programme demonstrated that the putative biosynthetic pathway leading to the salicinoid family of phenolic glycosides runs in parallel to a "benzyl"-based pathway to arbusculoidin. The introduction of a known Diels-Alder reaction trait from Salix dasyclados, as well as an acylation trait, into progeny containing both salicyl- and benzyl- pathways caused the formation of all possible hetero-cyclodimers from mixtures of reactive dienone (acyl)glycosides that participated in cross-over reactions. In addition to providing access to new analogues of the anti-cancer dimer miyabeacin, the analysis of the breeding progeny also indicated that these dienone (acyl)glycosides are stable in planta. Although the immediate biosynthetic precursors of these compounds remain to be defined, the results suggest that the (acyl)glycosylation reactions may occur later in the pathway than previously suggested by in vitro work on cloned UGT enzymes.

Aphid-induced accumulation of trehalose in Arabidopsis thaliana is systemic and dependent upon aphid density.

Trehalose is a disaccharide sugar that is now considered to be widely distributed among higher plants. Trehalose has been attributed a number of roles, including control of basic plant processes, such as photosynthesis, and conferring tolerance to abiotic stresses, such as desiccation and high salinity. Trehalose is also a common storage sugar used by insects. In this study, we used laboratory investigations to examine various aspects of trehalose dynamics in an aphid-host plant system (Arabidopsis and the peach potato aphid, Myzus persicae). Trehalose concentrations were measured by [1-H]-NMR. Myzus persicae reared on Arabidopsis, but not on black mustard or spring cabbage, contained considerable quantities of trehalose (5 % w/w dry matter). In Arabidopsis foliage, feeding by aphids induced a density-dependent accumulation of trehalose up to 5 mg g(-1) dry weight. Leaves that were not challenged directly by aphids also exhibited increased trehalose concentrations, indicating that this accumulation was systemic. Trehalose was measured at high concentrations in the phloem sap of plants challenged by aphids, suggesting that aphid feeding induced the plant to produce significant quantities of trehalose, which moved through the plant and into the aphids via the phloem sap. Trehalose was also excreted in the aphid honeydew. Further work is required to clarify whether this trehalose accumulation in Arabidopsis has a direct role or a signalling function in plant tolerance of, or resistance to, aphid feeding, and if a similar accumulation of this sugar occurs when other species or genotypes of aphids are reared on this host plant.

¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation.

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of 'misfire' products of Rubisco.

Comparative compositions of grain of tritordeum, durum wheat and bread wheat grown in multi-environment trials.

Three genotypes each of bread wheat, durum wheat and tritordeum were grown in randomized replicated field trials in Andalusia (Spain) for two years and wholemeal flours analysed for a range of components to identify differences in composition. The contents of all components that were determined varied widely between grain samples of the individual species and in most cases also overlapped between the three species. Nevertheless, statistically significant differences between the compositions of the three species were observed. Notably, tritordeum had significantly higher contents of protein, some minerals (magnesium and iron), total phenolics and methyl donors. Tritordeum also had higher levels of total amino acids (but not asparagine) and total sugars, including raffinose. By contrast, bread wheat and tritordeum had similar contents of the two major dietary fibre components in white flour, arabinoxylan and ß-glucan, with significantly lower contents in durum wheat.

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.