Quantitative resistance is generally controlled by several genes. More than 100 resistance quantitative trait loci (QTLs) have been identified in wheat and barley against Fusarium head blight (FHB), caused by Gibberella zeae (anamorph: Fusarium graminearum), implying the possible occurrence of several resistance mechanisms. The objective of this study was to apply metabolomics to identify the metabolites in barley that are related to resistance against FHB. Barley genotypes, Chevron and Stander, were inoculated with mock or pathogen during the anthesis stage. The disease severity was assessed as the proportion of spikelets diseased. The genotype Chevron (0.33) was found to have a higher level of quantitative resistance than Stander (0.88). Spikelet samples were harvested at 48 h post-inoculation; metabolites were extracted and analysed using an LC-ESI-LTQ-Orbitrap (Thermo Fisher, Waltham, MA, USA). The output was imported to an XCMS 1.12.1 platform, the peaks were deconvoluted and the adducts were sieved. Of the 1826 peaks retained, a t-test identified 496 metabolites with significant treatment effects. Among these, 194 were resistance-related (RR) constitutive metabolites, whose abundance was higher in resistant mock-inoculated than in susceptible mock-inoculated genotypes. Fifty metabolites were assigned putative names on the basis of accurate mass, fragmentation pattern and number of carbons in the formula. The RR metabolites mainly belonged to phenylpropanoid, flavonoid, fatty acid and terpenoid metabolic pathways. Selected RR metabolites were assayed in vitro for antifungal activity on the basis of fungal biomass production. The application of these RR metabolites as potential biomarkers for screening and the potential of mass spectrometry-based metabolomics for the identification of gene functions are discussed.

The mechanisms of resistance in barley to fusarium head blight (FHB), caused by Gibberella zeae are complex. Metabolomics technology was explored to phenotype resistance. Spikelets of barley genotypes with contrasting levels of resistance to FHB, mock inoculated or with the pathogen, were extracted with aqueous methanol and the metabolites were analyzed using liquid chromatography and hybrid mass spectrometry. Peaks were de-convoluted using XCMS and annotated using CAMERA and IntelliXtract bioinformatics tools. A t-test, of a total of 1608 purified peaks, selected 626 metabolites with significant treatment effects, of which 161 were identified as resistance related (RR) metabolites. A total of 53 metabolites, that are RR or pathogenicity related (PR), were assigned with putative compound names. These mainly belonged to three metabolic pathways: fatty acid (jasmonic acid, methyl jasmonate, 9,10-dihydroisojasmonate, linolenic acid, linoleic acid, traumatic acid), phenylpropanoid (p-coumaric acid, caffeyl alcohol, dimethoxy-4-phenylcoumarin, rosmarinic acid, diphyllin, 5-methoxypodophyllotoxin) and flavonoid (naringenin, catechin, quercetin, and alpinumisoflavone). A few PR/RR metabolites significantly reduced mycelial growth of G. zeae in vitro.

Resistance of barley to Fusarium graminearum was studied using a pair each of resistant and susceptible black and yellow barley lines. The spikelets were inoculated with a trichothecene-producing isolate, a trichothecene-nonproducing isolate (tri5 ) ), or a mock solution. Spikelets were collected 72 h after inoculation and metabolites were analysed using a LC-hybrid MS system. Metabolite abundances were used to identify the constitutive (RRC) and induced resistance-related metabolites (RRI). The pathogen virulence factor, DON, and its plant detoxification product, DON-3-O-glucoside (D3G), were also identified and designated as resistance-indicator (RI) metabolites. The RRC, RRI and RI metabolites were putatively identified. Jasmonic acid was significantly induced in barley following inoculation with a trichothecene-producing isolate, but not with a tri5) isolate. The former isolate reduced the induction of both the number and amount of RR metabolites. The metabolites cinnamic acid, sinapoyl alcohol, coniferin, catechin and naringin were identified only in response to the inoculation with a tri5 ) mutant. The abundances of p-coumaric acid, coniferaldehyde and sinapaldehyde increased more in response to the tri5 ) mutant than to the trichothecene-producing isolate. The total amount of DON synthesized and its conversion to D3G varied greatly between the resistant and susceptible black barley, but not in yellow barley. Interestingly, an increase in the amount of total DON produced was associated with a decrease in the conversion of DON to D3G. The roles of RRC, RRI and RI metabolites in plant defence and their further use as potential biomarkers in screening are discussed.

SUMMARYUpon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water ? CO 2 ) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl-CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50- to 100-fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30- to 50-fold. More important, these cells had a 2-fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl-CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.

We have constructed and experimentally tested a comprehensive genome-scale model of photoautotrophic growth, denoted iSyp821, for the cyanobacterium Synechococcus sp. PCC 7002. iSyp821 incorporates a variable biomass objective function (vBOF), in which stoichiometries of the major biomass components vary according to light intensity. The vBOF was constrained to fit the measured cellular carbohydrate/protein content under different light intensities. iSyp821 provides rigorous agreement with experimentally measured cell growth rates and inorganic carbon uptake rates as a function of light intensity. iSyp821 predicts two observed metabolic transitions that occur as light intensity increases: 1) from PSI-cyclic to linear electron flow (greater redox energy), and 2) from carbon allocation as proteins (growth) to carbohydrates (energy storage) mode. iSyp821 predicts photoautotrophic carbon flux into 1) a hybrid gluconeogenesis-pentose phosphate (PP) pathway that produces glycogen by an alternative pathway than conventional gluconeogenesis, and 2) the photorespiration pathway to synthesize the essential amino acid, glycine. Quantitative fluxes through both pathways were verified experimentally by following the kinetics of formation of C metabolites fromCO fixation. iSyp821 was modified to include changes in gene products (enzymes) from experimentally measured transcriptomic data and applied to estimate changes in concentrations of metabolites arising from nutrient stress. Using this strategy, we found that iSyp821 correctly predicts the observed redistribution pattern of carbon products under nitrogen depletion, including decreased rates of CO uptake, amino acid synthesis, and increased rates of glycogen and lipid synthesis.

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