MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Grapevine fatty acid hydroperoxide lyase generates actin-disrupting volatiles and promotes defence-related cell death.

Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity.

The Aphid-Transmitted Turnip yellows virus Differentially Affects Volatiles Emission and Subsequent Vector Behavior in Two Brassicaceae Plants.

Aphids are important pests which cause direct damage by feeding or indirect prejudice by transmitting plant viruses. Viruses are known to induce modifications of plant cues in ways that can alter vector behavior and virus transmission. In this work, we addressed whether the modifications induced by the aphid-transmitted Turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana also apply to the cultivated plant Camelina sativa, both belonging to the Brassicaceae family. In most experiments, we observed a significant increase in the relative emission of volatiles from TuYV-infected plants. Moreover, due to plant size, the global amounts of volatiles emitted by C. sativa were higher than those released by A. thaliana. In addition, the volatiles released by TuYV-infected C. sativa attracted the TuYV vector Myzus persicae more efficiently than those emitted by non-infected plants. In contrast, no such preference was observed for A. thaliana. We propose that high amounts of volatiles rather than specific metabolites are responsible for aphid attraction to infected C. sativa. This study points out that the data obtained from the model pathosystem A. thaliana/TuYV cannot be straightforwardly extrapolated to a related plant species infected with the same virus.

Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.

Genetic analysis of the biosynthesis of 2-methoxy-3-isobutylpyrazine, a major grape-derived aroma compound impacting wine quality.

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.

Genetic analysis of geraniol metabolism during fermentation.

Geraniol produced by grape is the main precursor of terpenols which play a key role in the floral aroma of white wines. We investigated the fate of geraniol during wine fermentation by Saccharomyces cerevisiae. The volatile compounds produced during fermentation of a medium enriched with geraniol were extracted by Stir-bar sorptive extraction and analysed by GC-MS. We were able to detect and quantify geranyl acetate but also citronellyl- and neryl-acetate. The presence of these compounds partly explains the disparition of geraniol. The amounts of terpenyl esters are strain dependant. We demonstrated both by gene overexpression and gene-deletion the involvement of ATF1 enzyme but not ATF2 in the acetylation of terpenols. The affinity of ATF1 enzyme for several terpenols and for isoamyl alcohol was compared. We also demonstrated that OYE2 is the enzyme involved in geraniol to citronellol reduction. Fermenting strain deleted from OYE2 gene produces far less citronellol than wild type strain. Moreover lab strain over-expressing OYE2 allows 87% geraniol to citronellol reduction in bioconversion experiment compared to about 50% conversion with control strain.

Metabolic consequences of various fruit-based diets in a generalist insect species.

Most phytophagous insect species exhibit a limited diet breadth and specialize on a few or a single host plant. In contrast, some species display a remarkably large diet breadth, with host plants spanning several families and many species. It is unclear, however, whether this phylogenetic generalism is supported by a generic metabolic use of common host chemical compounds ('metabolic generalism') or alternatively by distinct uses of diet-specific compounds ('multi-host metabolic specialism')? Here, we simultaneously investigated the metabolomes of fruit diets and of individuals of a generalist phytophagous species, Drosophila suzukii, that developed on them. The direct comparison of metabolomes of diets and consumers enabled us to disentangle the metabolic fate of common and rarer dietary compounds. We showed that the consumption of biochemically dissimilar diets resulted in a canalized, generic response from generalist individuals, consistent with the metabolic generalism hypothesis. We also showed that many diet-specific metabolites, such as those related to the particular color, odor, or taste of diets, were not metabolized, and rather accumulated in consumer individuals, even when probably detrimental to fitness. As a result, while individuals were mostly similar across diets, the detection of their particular diet was straightforward. Our study thus supports the view that dietary generalism may emerge from a passive, opportunistic use of various resources, contrary to more widespread views of an active role of adaptation in this process. Such a passive stance towards dietary chemicals, probably costly in the short term, might favor the later evolution of new diet specializations.

Most insects that feed on green plants are specialists, meaning that they feed on just a narrow range of plant species. This reduces competition, especially if the host plant contains chemical deterrents that are toxic to other insects. But specialists cannot easily switch to feed on other plants, making them vulnerable to changes in the availability of the particular food type that they eat. Generalist insects, on the other hand, are able to consume a wide range of diets. This makes them more robust to changes in food availability, but it is unclear how these insects deal with the wider range of chemical compositions of their food. Do they convert food into energy using the same chemical process, or metabolism, for all the different things they eat? Or do generalists have a specific metabolic pathway for each food type? To answer this question, Olazcuaga, Baltenweck et al. studied the metabolism of a generalist fruit fly species. The team compared four types of fruit (blackcurrant, cherry, cranberry and strawberry) and isolated separate groups of flies so that they each ate only one type of fruit. By comparing the chemical composition of the flies with that of the fruit they ate, they were able to work out how each fruit type was metabolised. They found that the flies converted food into energy using the same process regardless of the type of fruit they ate. This lack of a specialist metabolic pathway for each fruit type meant that some chemicals were not metabolised and accumulated in the fly's body instead. This build-up of unprocessed chemicals is likely to be harmful to the fly. The results of Olazcuaga, Baltenweck et al. suggest that generalist insects do not actively adapt their metabolism to new food types. It's more likely that they try different types of food as the opportunity arises, regardless of the fact that some of the food will not be converted into energy and may harm them long term. These findings are important because they give us an insight into how the chemistry of a plant can shape the physiology of the organisms that consume it, and vice-versa. These insights are a crucial step in developing sustainable agriculture practices that must consider tackle how plants are pollinated, how plant seeds are dispersed and what type of pest control to use.

Co-Localization of Resistance and Metabolic Quantitative Trait Loci on Carrot Genome Reveals Fungitoxic Terpenes and Related Candidate Genes Associated with the Resistance to Alternaria dauci.

Alternaria leaf blight, caused by the fungus Alternaria dauci, is the most damaging foliar disease of carrot. Some carrot genotypes exhibit partial resistance to this pathogen and resistance Quantitative Trait Loci (rQTL) have been identified. Co-localization of metabolic QTL and rQTL identified camphene, α-pinene, α-bisabolene, ß-cubebene, caryophyllene, germacrene D and α-humulene as terpenes potentially involved in carrot resistance against ALB. By combining genomic and transcriptomic analyses, we identified, under the co-localization regions, terpene-related genes which are differentially expressed between a resistant and a susceptible carrot genotype. These genes include five terpene synthases and twenty transcription factors. In addition, significant mycelial growth inhibition was observed in the presence of α-humulene and caryophyllene.

QTL mapping of the production of wine aroma compounds by yeast.

BACKGROUND: Wine aroma results from the combination of numerous volatile compounds, some produced by yeast and others produced in the grapes and further metabolized by yeast. However, little is known about the consequences of the genetic variation of yeast on the production of these volatile metabolites, or on the metabolic pathways involved in the metabolism of grape compounds. As a tool to decipher how wine aroma develops, we analyzed, under two experimental conditions, the production of 44 compounds by a population of 30 segregants from a cross between a laboratory strain and an industrial strain genotyped at high density. RESULTS: We detected eight genomic regions explaining the diversity concerning 15 compounds, some produced de novo by yeast, such as nerolidol, ethyl esters and phenyl ethanol, and others derived from grape compounds such as citronellol, and cis-rose oxide. In three of these eight regions, we identified genes involved in the phenotype. Hemizygote comparison allowed the attribution of differences in the production of nerolidol and 2-phenyl ethanol to the PDR8 and ABZ1 genes, respectively. Deletion of a PLB2 gene confirmed its involvement in the production of ethyl esters. A comparison of allelic variants of PDR8 and ABZ1 in a set of available sequences revealed that both genes present a higher than expected number of non-synonymous mutations indicating possible balancing selection. CONCLUSIONS: This study illustrates the value of QTL analysis for the analysis of metabolic traits, and in particular the production of wine aromas. It also identifies the particular role of the PDR8 gene in the production of farnesyldiphosphate derivatives, of ABZ1 in the production of numerous compounds and of PLB2 in ethyl ester synthesis. This work also provides a basis for elucidating the metabolism of various grape compounds, such as citronellol and cis-rose oxide.

Metabolic engineering of monoterpene synthesis in yeast.

Terpenoids are one of the largest and most diverse families of natural compounds. They are heavily used in industry, and the trend is toward engineering modified microorganisms that produce high levels of specific terpenoids. Most studies have focused on creating specific heterologous pathways for sesquiterpenes in Escherichia coli or yeast. We subjected the Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) to a set of amino acid mutations in the catalytic site at position K197. Mutated strains have been shown to exhibit various growth rate, sterol amount, and monoterpenol-producing capacities. These results are discussed in the context of the potential use of these mutated strains for heterologous expression of monoterpenoid synthases, which was investigated using Ocimum basilicum geraniol synthase. The results obtained with up to 5 mg/L geraniol suggest a major improvement compared with previous available expression systems like Escherichia coli or yeast strains with an unmodified ERG20 gene that respectively delivered amounts in the 10 and 500 µg/L range or even a previously characterized K197E mutation that delivered amounts in the 1 mg/L range.

Link between carrot leaf secondary metabolites and resistance to Alternaria dauci.

Alternaria Leaf Blight (ALB), caused by the fungus Alternaria dauci, is the most damaging foliar disease affecting carrots (Daucus carota). In order to identify compounds potentially linked to the resistance to A. dauci, we have used a combination of targeted and non-targeted metabolomics to compare the leaf metabolome of four carrot genotypes with different resistance levels. Targeted analyses were focused on terpene volatiles, while total leaf methanolic extracts were subjected to non-targeted analyses using liquid chromatography couple to high-resolution mass spectrometry. Differences in the accumulation of major metabolites were highlighted among genotypes and some of these metabolites were identified as potentially involved in resistance or susceptibility. A bulk segregant analysis on F3 progenies obtained from a cross between one of the resistant genotypes and a susceptible one, confirmed or refuted the hypothesis that the metabolites differentially accumulated by these two parents could be linked to resistance.

PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses.

The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta.

Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems.

Numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (GPP) as a precursor. Sweet basil (Ocimum basilicum) geraniol synthase (GES) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. Grapevine (Vitis vinifera) calli were transformed using Agrobacterium tumefasciens and the plants were regenerated. Thale cress (Arabidopsis thaliana) was transformed using the floral dip method. Tobacco (Nicotiana benthamiana) leaves were agro-infiltrated for transient expression. Although, as expected, geraniol was the main product detected in the leaves, different minor products were observed in these plants (V. vinifera: citronellol and nerol; N. benthamiana: linalool and nerol; A. thaliana: none). O. basilicum GES expression was also carried out with microbial system yeasts (Saccharomyces cerevisiae) and Escherichia coli. These results suggest that the functional properties of a monoterpenol synthase depend not only on the enzyme's amino-acidic sequence, but also on the cellular background. They also suggest that some plant species or microbial expression systems could induce the simultaneous formation of several carbocations, and could thus have a natural tendency to produce a wider spectrum of monoterpenols.

Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma.

Madagascar periwinkle (Catharanthus roseus [L.] G. Don, Apocynaceae) produces monoterpene indole alkaloids (MIAs), secondary metabolites of high interest due to their therapeutic value. A key step in the biosynthesis is the generation of geraniol from geranyl diphosphate (GPP) in the monoterpenoid branch of the MIA pathway. Here we report on the cloning and functional characterization of C. roseus geraniol synthase (CrGES). The full-length CrGES was over-expressed in Escherichia coli and the purified recombinant protein catalyzed the conversion of GPP into geraniol with a K(m) value of 58.5 µM for GPP. In vivo CrGES activity was evaluated by heterologous expression in a Saccharomyces cerevisiae strain mutated in the farnesyl diphosphate synthase gene. Analysis of culture extracts by gas chromatography-mass spectrometry confirmed the excretion of geraniol into the growth medium. Transient transformation of C. roseus cells with a Yellow Fluorescent Protein-fusion construct revealed that CrGES is localized in plastid stroma and stromules. In aerial plant organs, RNA in situ hybridization showed specific labeling of CrGES transcripts in the internal phloem associated parenchyma as observed for other characterized genes involved in the early steps of MIA biosynthesis. Finally, when cultures of Catharanthus cells were treated with the alkaloid-inducing hormone methyl jasmonate, an increase in CrGES transcript levels was observed. This observation coupled with the tissue-specific expression and the subcellular compartmentalization support the idea that CrGES initiates the monoterpenoid branch of the MIA biosynthetic pathway.

Identification of a lysine residue important for the catalytic activity of yeast farnesyl diphosphate synthase.

The Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) has been subjected to a set of mutations at the catalytic site, at position K254 to determine the in vivo impact. The mutated strains have been shown to exhibit various growth rates, sterol profiles and monoterpenol producing capacities. The results obtained suggest that K at position 254 helps to stabilize one of the three Mg(2+) forming a bridge between the enzyme and DMAPP, and demonstrate that destabilizing two of the three Mg(2+) ions, by introducing a double mutation at positions K197 and K254, results in a loss of FPPS activity and a lethal phenotype.

A grapevine (Vitis vinifera L.) deoxy-D: -xylulose synthase gene colocates with a major quantitative trait loci for terpenol content.

Linalool, geraniol, nerol, citronellol and alpha-terpineol are isoprenoid molecules responsible for specific aromas found in grapes and wines. Total concentrations (free and bound forms) of these compounds were measured in the skins of mature berries during 2 successive years in two progenies obtained from Muscat Ottonel and Gewurztraminer selfings. Partial genetic maps based on microsatellite markers were constructed and several quantitative trait loci (QTLs) related to terpenol content were detected. A major QTL on linkage group (LG) 5 colocated with a deoxy-D: -xylulose synthase gene, coding for the first enzyme of the plastidial isoprenoid biosynthesis pathway. The number of favourable alleles at this locus determined the level of terpenol synthesis. A second QTL, on LG 10, was found to determine the balance linalool versus geraniol and nerol in the Muscat self-progeny plants.

A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine.

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl(3) treatment.