Synthesis of Dihydrohelminthosporic Acids and their Plant Growth-promoting Activity

In the reduction of (?)-isomenthone with sodium in aqueous ammonia, it was found that the reduction product gives a mixture consisting of 75.5% of (?)-isomenthol (a,e,e), 9.5% of (+)-menthol (e,e,e) and 8.6% of (?)-neoisomenthol (a,a,e). From this fact, it might be concluded that this reduction is stereospecific for isomenthone and is mechanistically different from reduction with sodium and alcohol.     

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.     

Metabolism of monoterpenes: demonstration that (+)-cis-isopulegone, not piperitenone, is the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint (Mentha piperita)

Piperitenone is commonly considered to be the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint; however, [3H]piperitenone gave rise only to the inert metabolite (+)-piperitone when incubated with peppermint leaf discs. Under identical conditions, (-)-[3H]isopiperitenone was efficiently incorporated into (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and yielded an additional metabolite identified as (+)-cis-isopulegone; piperitenone was poorly labeled. Moreover, (+)-cis-[3H]isopulegone was rapidly converted to (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and the reduction of (+)-[3H]pulegone to (-)-menthone and (+)-isomenthone was similarly documented. Each step of the pathway was demonstrated in a crude soluble preparation from peppermint leaf epidermis and each of the relevant enzymes was partially purified in order to compare relative rates of catalysis. The results of these studies indicate that the endocyclic double bond of (-)-isopiperitenone is reduced to yield (+)-cis-isopulegone, which is isomerized to (+)-pulegone as the immediate precursor of (-)-menthone and (+)-isomenthone, and they rule out piperitenone as an intermediate of the pathway.     

Chemogenetic evidence supporting multiple allele control of the biosynthesis of (−)-menthone and (+)-isomenthone stereoisomers in Mentha species

The essential oils of certain Mentha species and chemotypes have proportions of (?)-menthone and (+)-isomenthone which differ but show a high degree of heritability in clonal propagation. Oil from an F₂ individual (69–296), selected from numerous 4n M. longifolia (4n = 48) × M. crispa (2n = 48) hybrids for high isomenthone content, had 41.3% isomenthone; the associated but seldom observed alcohols, 1.6% isomenthol, 10.3% neoiso-menthol; and 13% of their esters; in contrast to 8% menthone with 0.1% menthol, 5.0% neo-menthol, and 1.7% esters. Self-pollination of strain 69–296 gave a 3:1 ratio of high isomenthone: high menthone. Crosses with a true breeding high menthone plant having 80% menthone and 3.2% isomenthone gave a 1:1 ratio of the parental phenotypes by GLC analyses and herbage odor. This and data from high isomenthone and high menthone crosses with tester strains lead us to postulate the involvement of a single locus having multiple alleles with true breeding menthone having the genotype P^s P^s, true breeding isomenthone P^r P^r, 69–296 P^r P^s, and high pulegone pp. The P^r allele is not completely dominant over the P^s allele in 69–296 as about 18% of the total ketone derived from pulegone is menthone. Both are dominant over the recessive allele p that largely prevents menthone development. The quantitative amounts of the two isomers are believed to be controlled by the six combinations of the three alleles in a diploid species with graded effects obtained in the more complex genotypes possible in double diploid and octoploid species. 69–296 has (?)-piperitone even though (+)-piperitone is believed to be the common isomer in Mentha.     

Metabolism of Monoterpenes: Demonstration of (+)-Neomenthyl-beta-d-Glucoside as a Major Metabolite of (-)-Menthone in Peppermint (Mentha Piperita)

(-)-Menthone, the major monoterpene component of the essential oil of maturing peppermint (Mentha piperita L.) leaves (6 micromoles per leaf) is rapidly metabolized at the onset of flowering with a concomitant rise in the level of (-)-menthol (to about 2 micromoles per leaf). Exogenous (-)-[G-(3)H]menthone is converted into (-)-[(3)H]menthol as the major steam-volatile product in leaf discs in flowering peppermint (10% of incorporated tracer); however, the major portion of the incorporated tracer (86%) resided in the nonvolatile metabolites of (-)-[G-(3)H]menthone. Acid hydrolysis of the nonvolatile material released over half of the radioactivity to the steamvolatile fraction, and the major component of this fraction was identified as (+)-neomenthol by radiochromatographic analysis and by synthesis of crystalline derivatives, thus suggesting the presence of a neomenthyl glycoside. Thin layer chromatography, ion exchange chromatography, and gel permeation chromatography on Bio-Gel P-2 allowed the purification of the putative neomenthyl glycoside, and these results suggested that the glycoside contained a single, neutral sugar residue. Hydrolysis of the purified glycoside, followed by reduction of the resulting sugar moiety with NaB(3)H(4), generated a single labeled product that was subsequently identified as glucitol by radio gas-liquid chromatography of both the hexatrimethylsilyl ether and hexaacetate derivative, and by crystallization to constant specific radioactivity of both the alditol and the corresponding hexabenzoate. These results, along with studies on the hydrolysis of the glycoside by specific glycosidases, strongly suggest that (+)-neomenthyl-beta-d-glucoside is a major metabolite of (-)-menthone in flowering peppermint. This is the first report on the occurrence of a neomenthyl glycoside, and the first evidence implicating glycosylation as an early step in monoterpene catabolism.     

Effect of (+)-pulegone and other oil components of Mentha x Piperita on cucumber respiration

Peppermint (Mentha x piperita L.) essential oil and main components were assessed for their ability to interfere with plant respiratory functions. Tests were conducted on both root segments and mitochondria isolated by etiolated seedlings of cucumber (Cucumis sativus L.). Total essential oil inhibited 50% of root and mitochondrial respiration (IC50) when used at 324 and 593 ppm, respectively. (+)-Pulegone was the most toxic compound, with a 0.08 and 0.12 mM IC50 for root and mitochondrial respiration, respectively. (-)-Menthone. followed (+)-pulegone in its inhibitory action (IC50 values of 1.11 and 2.30 mM for root and mitochondrial respiration respectively), whereas (-)-menthol was the less inhibitory compound (IC50 values of 1.85 and 3.80 mM respectively). A positive correlation was found for (+)-pulegone, (-)-menthone and (-)-menthol between water solubility and respiratory inhibition. The uncoupling agent. carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP), lowered (-)-menthol and (-)menthone inhibition and annulled (+)-pulegone inhibition of mitochondrial respiration, whereas salicyl-hydroxamic acid (SHAM) 2-hydroxybenzohydroxamic acid, the alternative oxidase (AO) inhibitor, increased (-)-menthone inhibition and annulled both (+)-pulegone and (-)-menthol inhibitory activity. The possible interaction of (-)-pulegone and (-)-menthol with AO and the mechanism of action of(+)-pulegone, (-)-menthone and (-)-menthol on mitochondrial respiration are discussed.     

Studies on the Reduction of Terpenes with Sodium in Aqueous-ammonia

On reduction of (?)-menthone by various methods, generally, a mixture of (?)-menthol and (+)-neomenthol has been obtained. In the present work, it is found that (?)-menthol can be prepared almost quantitatively from (?)-menthone by treatment with sodium in aqueous-ammonia.     

Demonstration of the Intercellular Compartmentation of l-Menthone Metabolism in Peppermint (Mentha piperita) Leaves

The metabolism of l-menthone, which is synthesized in the epidermal oil glands of peppermint (Mentha piperita L. cv. Black Mitcham) leaves, is compartmented; on leaf maturity, this ketone is converted to l-menthol and l-menthyl acetate in one compartment, and to d-neomenthol and d-neomenthyl glucoside in a separate compartment. All of the enzymes involved in these reactions are soluble when prepared from whole-leaf homogenates. Mechanical separation of epidermal fragments from the mesophyll, followed by preparation of the soluble enzyme fraction from each tissue, revealed that the neomenthol dehydrogenase and the glucosyl transferase resided specifically in the mesophyll layer, whereas the menthol dehydrogenase and substantial amounts of the acetyl transferase were located in the epidermis, presumably within the epidermal oil glands. These results suggest that the compartmentation of menthone metabolism in peppermint leaves is intercellular, not intracellular.     

满天香精油化学成分的研究

采用Finngan-4510型毛细管气相色谱/质谱/电子计算机联用(GC/MS/DS)技术,辅以~1H NMR,~18C NMR和化学的手段,对广西龙州产满天香精油进存定性定量分析,分离出37个组分,鉴定了其中26个,占精油总量的97.40％。其主要成分为薄荷酮(51.09％)、异满天酮(7.95％)、(+)-新薄荷醇(19.42％)、枞油烯(5.71％)、薄荷醇,β-水芹烯等。薄荷香是一种富含薄荷酮、薄荷醇的新资源植物。     

Effect of Mentha x piperita essential oil and monoterpenes on cucumber root membrane potential.

Peppermint (Mentha piperita L.) essential oil and its main components were assessed for their ability to interfere with plant plasma membrane potentials. Tests were conducted on root segments isolated from etiolated seedlings of cucumber (Cucumis sativus L.). Increasing the concentration of peppermint essential oil from 5 to 50 ppm caused a decrease in membrane potential (Vm) hyperpolarization of 10-3 mV, whereas concentrations from 100 up to 900 ppm caused an increasing depolarization of Vm (from 5 to 110 mV). When tested at 300 ppm, (+)-menthyl acetate, (-)-limonene and 1,8-cineole did not exert any significant effect on V(m), whereas (+)-menthofuran (73 mV), (+)-pulegone (85 mV), (+)-neomenthol (96 mV), (-)-menthol (105 mV) and (-)-menthone (111 mV) showed increased ability to depolarize V(m). A plot of log of octanol-water partition coefficient (K(ow)) against their depolarizing effect showed a significant negative correlation, suggesting that among all monoterpenoids increased membrane depolarization depends on lower K(ow). However, among monoterpene ketones, alcohols and furans, increased membrane depolarization is associated with a decline in water solubility. The possible effect of monoterpenoids on membrane ion fluxes is also discussed, since changes in the bioelectric potential of cells imply changes in the flux of ions across the plasma membrane     

Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.     

Biotransformations. XIX. Reduction of some terpenic ketones by means of immobilized cells of rhodotorula mucilaginosa

Reduction of (?)-menthone ((?)- 1 ), (+)-(R)-methyl-α-campholenone ((+)- 2 ), (+)-carvone ((+)- 3 ), and eucarvone ( 4 ) was carried out by means of cells of the Rhodotorula mucilaginosa species immobilized in polyacrylamide gel. Alcohols with the (S)-configuration, (+)-neomenthol ((+)- 1a ), (+)-(R)-methyl-α-campholenol ((+)- 2a ), (?)-neoisodihydrocarveol ((?)- 3a ), dihydroeucarveol ((?)- 4a ), and small amounts of (?)-dihydroeucarvone ((?)- 5 ), were obtained. The cells of R. mucilaginosa maintained after this reaction ability to reduce standard acetophenone to (?)- 1 -phenyl- 1 -ethanol.     

Biotransformation of (-)-menthone by human liver microsomes

The aim of the current study was to investigate the metabolism of (-)-menthone by liver microsomes of humans. (-)-Menthone (1) was metabolized to (+)-neomenthol (2) (3-reduction) and 7-hydroxymenthone (3) by human liver microsomes. The metabolites formed were analyzed on GC and GC-MS. Kinetic analysis showed that K(m) and V(max) values for the metabolized (-)-menthone to respective (+)-neomenthol and 7-hydroxymenthone by liver microsomes of human sample HG70 were 0.37 mM and 4.91 nmol/min/mg protein and 0.07 mM and 0.71 nmol/min/mg protein.     

Metabolism of Monoterpenes : Early Steps in the Metabolism of d-Neomenthyl-beta-d-Glucoside in Peppermint (Mentha piperita) Rhizomes

Previous studies have shown that the monoterpene ketone l-[G-³H] menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaves of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-β-d-glucoside which is then transported to the rhizome (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). Analysis of the disposition of l-[G-³H]menthone applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis and transport of the monoterpenyl glucoside to be determined, and gave strong indication that the glucoside was subsequently metabolized in the rhizome. Studies with d-[G-³H]neomenthyl-β-d-glucoside as substrate, using excised rhizomes or rhizome segments, confirmed the hydrolysis of the glucoside as an early step in metabolism at this site, and revealed that the terpenoid moiety was further converted to a series of ether-soluble, methanol-soluble, and water-soluble products. Studies with d-[G-³H]neomenthol as the substrate, using excised rhizomes, showed the subsequent metabolic steps to involve oxidation of the alcohol back to menthone, followed by an unusual lactonization reaction in which oxygen is inserted between the carbonyl carbon and the carbon bearing the isopropyl group, to afford 3,4-menthone lactone. The conversion of menthone to the lactone, and of the lactone to more polar products, were confirmed in vivo using l-[G-³H]menthone and l-[G-³H]-3,4-menthone lactone as substrates. Additional oxidation products were formed in vivo via the desaturation of labeled neomenthol and/or menthone, but none of these transformations appeared to lead to ring opening of the p-menthane skeleton. Each step in the main reaction sequence, from hydrolysis of neomenthyl glucoside to lactonization of menthone, was demonstrated in cell-free extracts from the rhizomes of flowering mint plants. The lactonization step is of particular significance in providing a means of cleaving the p-menthane ring to afford an acyclic carbon skeleton that can be further degraded by modifications of the well-known β-oxidation sequence.     

Volatile Terpenoids of Endophyte-free and Infected Peppermint (<Emphasis Type="Italic">Mentha piperita</Emphasis> L.): Chemical Partitioning of a Symbiosis

The study reports the effects on volatiles of an endophytic fungus inhabiting asymptomatically the leaves of peppermint. By means of headspace solid-phase microextraction (HS-SPME) and gaschromatography-mass spectrometry (GC-MS) terpenoids were sampled in time course from the head space of peppermint leaves and roots. After removal of the mycelium from peppermint tissues, fungal volatiles were analyzed and compared with those of pure fungal cultures. In the presence of the endophyte, the relative amount of all main compounds increased in leaves. Starting from the first 14 d of culture, (−)-menthone and (+)-neomenthol were consistently higher than in control plants. On the contrary, (+)-menthofuran increased only by 28 d of culture. Root volatiles were also dramatically altered by the presence of the fungus, with (+)-pulegone accounting for at least 44% of the total volatile emission. (+)-Pulegone was also the main compound of PGP-HSF mycelium isolated from peppermint roots. The sesquiterpenoid cuparene was found as a novel compound of peppermint leaf headspace and was a main volatile of ex planta and pure culture mycelia. The chemical spectrum of terpenoids and their distribution among peppermint roots, leaves, and mycelia are likely to account for a fine regulation of the mutualism in planta and for the acquisition by the fungus of novel metabolic competences. This work is dedicated to the memory of Prof. Silvano Scannerini.     

(+)-Menthol and its hydroxy derivatives,novel fungal monoterpenols from the fusicoccin-producing fungi,Phomopsis amygdali F6a and Niigata 2

In our search for new fusicoccins of unique diterpene glucosides from Phomopsis amygdali, we found that a fragrant substance was formed in the early stage of fusicoccin fermentation. This fragrant constituent was isolated and identified as (+)-menthol, which is a novel fungal metabolite as the enantiomer of well-known peppermint (-)-menthol. (+)-7-Hydroxymenthol and new (+)-(6S)-hydroxymenthol were also isolated and identified as fungal metabolites. In addition, p-menthanetriol, which has been reported as the first fungal monoterpene from the fungus, was also isolated. The possible biosynthetic relationship of these metabolites is discussed.     

Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.     

Mathematical Modeling-Guided Evaluation of Biochemical,Developmental, Environmental,and Genotypic Determinants of Essential Oil Composition and Yield in Peppermint Leaves

We have previously reported the use of a combination of computational simulations and targeted experiments to build a first generation mathematical model of peppermint (Mentha × piperita) essential oil biosynthesis. Here, we report on the expansion of this approach to identify the key factors controlling monoterpenoid essential oil biosynthesis under adverse environmental conditions. We also investigated determinants of essential oil biosynthesis in transgenic peppermint lines with modulated essential oil profiles. A computational perturbation analysis, which was implemented to identify the variables that exert prominent control over the outputs of the model, indicated that the essential oil composition should be highly dependent on certain biosynthetic enzyme concentrations [(+)-pulegone reductase and (+)-menthofuran synthase], whereas oil yield should be particularly sensitive to the density and/or distribution of leaf glandular trichomes, the specialized anatomical structures responsible for the synthesis and storage of essential oils. A microscopic evaluation of leaf surfaces demonstrated that the final mature size of glandular trichomes was the same across all experiments. However, as predicted by the perturbation analysis, differences in the size distribution and the total number of glandular trichomes strongly correlated with differences in monoterpenoid essential oil yield. Building on various experimental data sets, appropriate mathematical functions were selected to approximate the dynamics of glandular trichome distribution/density and enzyme concentrations in our kinetic model. Based on a χ² statistical analysis, simulated and measured essential oil profiles were in very good agreement, indicating that modeling is a valuable tool for guiding metabolic engineering efforts aimed at improving essential oil quality and quantity.The essential oil distilled from peppermint (Mentha × piperita) leaves is used in numerous consumer products (e.g. chewing gum, toothpaste, and mouthwash), as a flavor in the confectionary and pharmaceutical industries, and as a source of active ingredients for aromatherapy. Peppermint oil consists primarily of p-menthane-type monoterpenes, with smaller amounts of other monoterpenes and very minor quantities of sesquiterpenes (Rohloff, 1999). The essential oil is synthesized and accumulated in specialized anatomical structures called peltate glandular trichomes (Gershenzon et al., 1989; McCaskill et al., 1992). These trichomes contain secretory cells, arranged in an eight-celled disc, which are responsible for the synthesis of the oil. Nascent essential oil is secreted into an emerging cavity formed by the separation of a preformed layer of cuticular material (Amelunxen, 1965). Over the last two decades, the entire complement of genes and enzymes involved in the peppermint monoterpenoid essential oil biosynthetic pathway has been characterized (for review, see Croteau et al., 2005).Transgenic peppermint plants have been generated in efforts aimed at modulating essential oil yield and composition. Mahmoud and Croteau (2001) reported that, by overexpressing the gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), oil yield increases (compared with wild-type plants) of up to 50% were observed. Antisense suppression of the (+)-menthofuran synthase (MFS) gene led to a dramatic decrease in the amounts of the undesirable side product (+)-menthofuran (elite transgenic line designated MFS7a; Mahmoud and Croteau, 2001). A slight increase in overall monoterpene yields was reported for transgenic plants with increased expression levels of the gene encoding (−)-limonene synthase (LS; Diemer et al., 2001), whereas only negligible effects on yield were detected in an independent study (Krasnyansky et al., 1999). Transgenic plants overexpressing the gene coding for (−)-limonene 3-hydroxylase (L3H) did not accumulate increased levels of the recombinant protein, and the composition and yield of the essential oils were the same as in wild-type controls; however, cosuppression of the L3H gene resulted in a vastly increased accumulation of the intermediate (−)-limonene, without notable effects on oil yield (elite transgenic line designed L3H20; Mahmoud et al., 2004).Mathematical modeling can be a powerful tool to support metabolic engineering efforts, including those performed with peppermint. Stoichiometric modeling only requires knowledge of the topology of reactions in the pathway and inputs/outputs. This is a particularly useful approach to determine flux distributions and the systemic characteristics of metabolic networks (for review, see Llaneras and Picó, 2008). When experimental designs supporting metabolic and isotopic steady state are employed, isotope labeling data can be utilized for the development of quantitative flux maps of metabolic pathways (for review, see Libourel and Shachar-Hill, 2008). For dynamic systems, kinetic modeling is regarded as the generally most suitable method (McNeil et al., 2000; Poolman et al., 2004; Bruggeman and Westerhoff, 2006; Rios-Estepa and Lange, 2007; Mendes et al., 2009). Building on the rich body of published data on the enzymology and physiology of the peppermint monoterpene pathway (for review, see Croteau et al., 2005), we recently developed a first generation kinetic model to simulate the dynamics of peppermint monoterpene composition (Rios-Estepa et al., 2008). Modeling indicated that the monoterpene profiles observed in leaves of plants grown under low-light conditions could be explained if one assumed that (+)-menthofuran, a dead-end side product, acted as a heretofore unknown competitive inhibitor against (+)-pulegone, the primary substrate of the branch point enzyme (+)-pulegone reductase (PR; Fig. 1). Follow-up biochemical studies established that this prediction was correct (Rios-Estepa et al., 2008), thus illustrating the utility of an approach that integrates mathematical modeling with experimental testing.Open in a separate windowFigure 1.Outline of p-menthane monoterpene biosynthesis in peppermint glandular trichomes. The following enzymes are involved in this pathway: 1, 1-deoxy-d-xylulose 5-phosphate synthase; 2, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; 3, 2C-methyl-d-erythritol 4-phosphate cytidyltransferase; 4, 4-(cytidine 5′-diphospho)-2C-methyl-d-erythritol kinase; 5, 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; 6, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate synthase; 7, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase; 8, isopentenyl diphosphate isomerase; 9, geranyl diphosphate synthase; 10, (−)-limonene synthase; 11, (−)-limonene 3-hydroxylase; 12, (−)-trans-isopiperitenol dehydrogenase; 13, (−)-trans-isopiperitenone reductase; 14, (+)-cis-isopulegone isomerase; 15, (+)-menthofuran synthase; 16a, (+)-pulegone reductase [(−)-menthone-forming activity]; 16b, (+)-pulegone reductase [(+)-isomenthone-forming activity]; 17a, (−)-menthone:(−)-menthol reductase [(−)-menthol-forming activity]; 17b, (−)-menthone:(−)-menthol reductase [(+)-neoisomenthol-forming activity]; 18a, (−)-menthone:(+)-neomenthol reductase [(+)-neomenthol-forming activity]; 18b, (−)-menthone:(+)-neomenthol reductase [(+)-isomenthol-forming activity]. The subcellular compartmentation of p-menthane metabolic enzymes is color coded as follows: Cyt (blue), cytosol; ER (orange), endoplasmic reticulum; Lpl (green), leucoplasts; Mit (red), mitochondria. The inhibitory effects of (+)-menthofuran on (+)-pulegone reductase and geranyl diphosphate on isopentenyl diphosphate isomerase are indicated by red arcs with orthogonal red lines. Names of selected metabolites are shown in the colors that are used to indicate the corresponding profiles in Figures 2 to 5​5​​.As part of this study, a computational perturbation analysis was used to predict factors with the potentially greatest impacts on peppermint essential oil yield and composition (specific biosynthetic enzymes and the density of oil-synthesizing trichomes). To test these modeling predictions experimentally, we first acquired biometric data with peppermint plants grown under several environmental conditions known to adversely affect oil accumulation (Burbott and Loomis, 1967; Clark and Menary, 1980) and the transgenic line MFS7a, for which an altered essential oil profile had been reported earlier (Mahmoud and Croteau, 2001). Building on these experimental data sets, we then developed a second generation model that accounts for biochemical, developmental, environmental, and genotypic factors of essential oil formation. This updated model was then used to simulate monoterpenoid essential oil profiles for the transgenic line MFS7a grown under low-light environmental stress conditions and the transgenic line L3H20, which had previously been shown to have vastly reduced expression levels of the gene encoding L3H. In both cases, simulated and measured monoterpene patterns were very similar, indicating that mathematical modeling has great potential for guiding efforts aimed at developing peppermint lines with high oil yields and favorable composition, even under adverse environmental conditions.     

A role for a menthone reductase in resistance against microbial pathogens in plants

Plants elaborate a vast array of enzymes that synthesize defensive secondary metabolites in response to pathogen attack. Here, we isolated the pathogen-responsive CaMNR1 [menthone: (+)-(3S)-neomenthol reductase] gene, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, from pepper (Capsicum annuum) plants. Gas chromatography-mass spectrometry analysis revealed that purified CaMNR1 and its ortholog AtSDR1 from Arabidopsis (Arabidopsis thaliana) catalyze a menthone reduction with reduced nicotinamide adenine dinucleotide phosphate as a cofactor to produce neomenthol with antimicrobial activity. CaMNR1 and AtSDR1 also possess a significant catalytic activity for neomenthol oxidation. We examined the cellular function of the CaMNR1 gene by virus-induced gene silencing and ectopic overexpression in pepper and Arabidopsis plants, respectively. CaMNR1-silenced pepper plants were significantly more susceptible to Xanthomonas campestris pv vesicatoria and Colletotrichum coccodes infection and expressed lower levels of salicylic acid-responsive CaBPR1 and CaPR10 and jasmonic acid-responsive CaDEF1. CaMNR1-overexpressing Arabidopsis plants exhibited enhanced resistance to the hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 and the biotrophic pathogen Hyaloperonospora parasitica isolate Noco2, accompanied by the induction of AtPR1 and AtPDF1.2. In contrast, mutation in the CaMNR1 ortholog AtSDR1 significantly enhanced susceptibility to both pathogens. Together, these results indicate that the novel menthone reductase gene CaMNR1 and its ortholog AtSDR1 positively regulate plant defenses against a broad spectrum of pathogens.