Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar.

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Alternative transcript splicing regulates UDP-glucosyltransferase-catalyzed detoxification of DIMBOA in the fall armyworm (Spodoptera frugiperda).

Herbivorous insects often possess the ability to detoxify chemical defenses from their host plants. The fall armyworm (Spodoptera frugiperda), which feeds principally on maize, detoxifies the maize benzoxazinoid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by stereoselective re-glucosylation using a UDP-glucosyltransferase, SfUGT33F28. SfUGT33F28 activity is induced by feeding on a DIMBOA-containing diet, but how this induction is regulated is unknown. In the present work, we describe the alternative splicing of the SfUGT33F28 transcript. Variant transcripts are differentially expressed in response to DIMBOA, and this transcriptional response is mediated by an insect aryl hydrocarbon receptor. These variants have large deletions leading to the production of truncated proteins that have no intrinsic UGT activity with DIMBOA but interact with the full-length enzyme to raise or lower its activity. Therefore, the formation of SfUGT33F28 splice variants induces DIMBOA-conjugating UGT activity when DIMBOA is present in the insect diet and represses activity in the absence of this plant defense compound.

Balancing nutrients in a toxic environment: the challenge of eating.

Insect herbivores can regulate their food intake by mixing food sources with different nutrient content, but face the resulting challenge of ingesting various plant secondary metabolites. How insects deal with toxins in a complex nutrient environment is unclear. Here we investigated the influence of a classic plant secondary metabolite, allyl glucosinolate (sinigrin), and its hydrolyzed product allyl isothiocyanate (AITC), on the development of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) when fed on diets with different protein-to-carbohydrate (p : c) ratios. We also examined the effects of these toxins on larval biochemistry, by chemically analyzing the frass produced by insects feeding on the different diets. As expected, AITC had a greater negative effect than sinigrin on H. armigera life-history traits. However, AITC at low concentration appeared to have a positive effect on some traits. Both sinigrin and AITC-induced detoxification activity in the gut, and the reaction was related to diet protein concentration. High-protein diets can provide the required free amino acid, especially cysteine, needed for the detoxification process. The nutrient content of the diet influences how plant secondary metabolites are handled, and the use of artificial diets in experiments investigating the metabolic fate of plant secondary compounds needs to be carefully evaluated.

So Much for Glucosinolates: A Generalist Does Survive and Develop on Brassicas, but at What Cost?

While plants produce complex cocktails of chemical defences with different targets and efficacies, the biochemical effects of phytotoxin ingestion are often poorly understood. Here, we examine the physiological and metabolic effects of the ingestion of glucosinolates (GSLs), the frontline chemical defenses of brassicas (crucifers), on the generalist herbivore Helicoverpa armigera. We focus on kale and cabbage, two crops with similar foliar GSL concentrations but strikingly different GSL compositions. We observed that larval growth and development were well correlated with the nutritional properties of the insect diets, with low protein contents appearing to exacerbate the negative effects of GSLs on growth, pupation and adult eclosion, parameters that were all delayed upon exposure to GSLs. The different GSLs were metabolized similarly by the insect, indicating that the costs of detoxification via conjugation to glutathione (GSH) were similar on the two plant diets. Nevertheless, larval GSH contents, as well as some major nutritional markers (larval protein, free amino acids, and fat), were differentially affected by the different GSL profiles in the two crops. Therefore, the interplay between GSL and the nitrogen/sulfur nutritional availability of different brassicas strongly influences the effectiveness of these chemical defenses against this generalist herbivore.

Gallocatechin biosynthesis via a flavonoid 3',5'-hydroxylase is a defense response in Norway spruce against infection by the bark beetle-associated sap-staining fungus Endoconidiophora polonica.

One of the best-studied defense responses to fungal infection in Norway spruce (Picea abies) is the biosynthesis of flavan-3-ols, which accumulate as monomers or polymers known as proanthocyanidins. The individual flavan-3-ol units consist of compounds with a 3',4'-dihydroxylated B ring [2,3-(trans)-(+)-catechin or 2,3-(cis)-(-)-epicatechin] and compounds with a 3',4',5'-trihydroxylated B ring [2,3 (trans)-(+)-gallocatechin or 2,3-(cis)-(-)-epigallocatechin]. While much is known about the biosynthesis and biological activity of catechin in Norway spruce, there is little comparable information about gallocatechin or epigallocatechin. We found that there was a significant increase in the gallocatechin content of Norway spruce bark and wood after inoculation with the bark beetle-associated sap-staining fungus Endoconidiophora polonica. Gallocatechins increased proportionally more than catechins as both monomers and units of polymers. A flavonoid 3',5'-hydroxylase gene identified in Norway spruce was shown by heterologous expression in Nicotiana benthamiana to be involved in the conversion of 2,3 (trans)-(+)-catechin to 2,3 (trans)-(+)-gallocatechin. The formation of the trihydroxylated B ring in Norway spruce occurs at the level of flavan-3-ols, rather than at the level of dihydroflavonols as in many angiosperms. The transcript abundance of the flavonoid 3',5'-hydroxylase gene also increased significantly during fungal infection underlining its importance in gallocatechin biosynthesis. Comparisons of the effect of 2,3 (trans)-(+)-catechin and 2,3 (trans)-(+)-gallocatechin on fungal growth revealed that 2,3 (trans)-(+)-catechin is a stronger inhibitor of fungal growth, while 2,3 (trans)-(+)-gallocatechin is a stronger inhibitor of melanin biosynthesis.

Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis.

The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.