Menthol Biosynthesis Research Articles

Modulation of Phytochemical Pathways and Antioxidant Activity in Peppermint by Salicylic Acid and GR24: A Molecular Approach.

This study uncovers the potential of salicylic acid (SA) and synthetic Strigolactone (GR24) in enhancing menthol biosynthesis and antioxidant defense mechanisms in Mentha piperita L. Our comprehensive analysis, which included a series of controlled experiments and data analysis of the effects of these phytohormones on enzymatic antioxidants catalase (CAT) and ascorbate peroxidase (APX) and non-enzymatic antioxidants, including carotenoids and proline, revealed promising results. The study also examined their impact on lipid peroxidation, hydrogen peroxide levels, and the expression of genes critical to menthol and menthofuran synthesis. The results indicated that SA and GR24 significantly increased menthol production and reduced the levels of menthofuran and pulegone, suggesting upregulation in the plant's innate defense systems. Furthermore, the activities of CAT and APX were elevated, reflecting a strengthened antioxidant response. Interestingly, the menthofuran synthase (MFS) was higher in the control group. At the same time, pulegone reductase (PR) genes and menthol dehydrogenase (MDH) gene expression were upregulated, highlighting the protective effects of SA and GR24. These findings underscore the potential of SA and GR24 to serve as effective bio-stimulants, improving the quality and resilience of peppermint plants and thereby contributing to eco-friendly agricultural practices in pollution-stressed environments.

Discovery and Engineering of a Bacterial (+)-Pulegone Reductase for Efficient (-)-Menthol Biosynthesis.

The biosynthesis of valuable plant-derived monoterpene (-)-menthol from readily available feedstocks (e. g., (-)-limonene) is of great significance because of the high market demand for this product. However, biotransforming (+)-pulegone into (-)-menthone, the (-)-menthol precursor, through (+)-pulegone reductase (PGR) catalysis is inefficient because of the poor protein expression or catalytic efficiency (kcat/Km) of plant origin PGRs. In this study, a novel bacterial PGR from Pseudomonas resinovorans (PrPGR) was identified, and the most successful variant, PrPGRM2-1 (A50 V/G53 W), was obtained, showing respective 20-fold and 204-fold improvements in specific activity and catalytic efficiency. PrPGRM2-1 was employed to bioreduce (+)-pulegone, resulting in 4.4-fold and 35-fold enhancements in (-)-menthone titers compared with the bioreductions catalyzed by wild-type (WT) PrPGR and MpPGR, respectively. Furthermore, a whole-cell biocatalyst containing PrPGRM2-1, MpMMR, and BstFDH was constructed and achieved the highest (-)-menthol titer reported to date without externally supplemented NADPH/NADP+. Overall, this study details an efficient PGR with high catalytic efficiency that possesses great potential for (-)-menthol biosynthesis.

Studying the impact of titanium dioxide nanoparticles on the expression of pivotal genes related to menthol biosynthesis and certain biochemical parameters in peppermint plants (Mentha Piperita L.)

BackgroundThis study examines the impact of titanium dioxide nanoparticles (TiO2NPs) on gene expression associated with menthol biosynthesis and selected biochemical parameters in peppermint plants (Mentha piperita L.). Menthol, the active ingredient in peppermint, is synthesized through various pathways involving key genes like geranyl diphosphate synthase, menthone reductase, and menthofuran synthase. Seedlings were treated with different concentrations of TiO2NPs (50, 100, 200, and 300 ppm) via foliar spray. After three weeks of treatment, leaf samples were gathered and kept at -70 °C for analysis.ResultsAccording to our findings, there was a significant elevation (P ≤ 0.05) in proline content at concentrations of 200 and 300 ppm in comparison with the control. Specifically, the highest proline level was registered at 200 ppm, reaching 259.64 ± 33.33 µg/g FW. Additionally, hydrogen peroxide and malondialdehyde content exhibited a decreasing trend following nanoparticle treatments. Catalase activity was notably affected by varying TiO2NP concentrations, with a significant decrease observed at 200 and 300 ppm compared to the control (P ≤ 0.05). Conversely, at 100 ppm, catalase activity significantly increased (11.035 ± 1.12 units/mg of protein/min). Guaiacol peroxidase activity decreased across all nanoparticle concentrations. Furthermore, RT-qPCR analysis indicated increased expression of the studied genes at 300 ppm concentration.ConclusionsHence, it can be inferred that at the transcript level, this nanoparticle exhibited efficacy in influencing the biosynthetic pathway of menthol.Graphical

Effects of MhMYB1 and MhMYB2 transcription factors on the monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq.

Transcription factors MhMYB1 and MhMYB2 correlate with monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq, which could affect the contents of ( -)-menthol and ( -)-menthone. Mentha haplocalyx Briq., a plant with traditional medicinal and edible uses, is renowned for its rich essential oil content. The distinct functional activities and aromatic flavors of mint essential oils arise from various chemotypes. While the biosynthetic pathways of the main monoterpenes in mint are well understood, the regulatory mechanisms governing different chemotypes remain inadequately explored. In this investigation, we identified and cloned two transcription factor genes from the M. haplocalyx MYB family, namely MhMYB1 (PP236792) and MhMYB2 (PP236793), previously identified by our research group. Bioinformatics analysis revealed that MhMYB1 possesses two conserved MYB domains, while MhMYB2 contains a conserved SANT domain. Yeast one-hybrid (Y1H) analysis results demonstrated that both MhMYB1 and MhMYB2 interacted with the promoter regions of MhMD and MhPR, critical enzymes in the monoterpenoid biosynthesis pathway of M. haplocalyx. Subsequent virus-induced gene silencing (VIGS) of MhMYB1 and MhMYB2 led to a significant reduction (P < 0.01) in the relative expression levels of MhMD and MhPR genes in the VIGS groups of M. haplocalyx. In addition, there was a noteworthy decrease (P < 0.05) in the contents of ( -)-menthol and ( -)-menthone in the essential oil of M. haplocalyx. These findings suggest that MhMYB1 and MhMYB2 transcription factors play a positive regulatory role in ( -)-menthol biosynthesis, consequently influencing the essential oil composition in the l-menthol chemotype of M. haplocalyx. This study serves as a pivotal foundation for unraveling the regulatory mechanisms governing monoterpenoid biosynthesis in different chemotypes of M. haplocalyx.

A haplotype-resolved gap-free genome assembly provides novel insight into monoterpenoid diversification in Mentha suaveolens 'Variegata'.

Mentha is a commonly used spice worldwide, which possesses medicinal properties and fragrance. These characteristics are conferred, at least partially, by essential oils such as menthol. In this study, a gap-free assembly with a genome size of 414.3Mb and 31,251 coding genes was obtained for Mentha suaveolens 'Variegata'. Based on its high heterozygosity (1.5%), two complete haplotypic assemblies were resolved, with genome sizes of 401.9 and 405.7Mb, respectively. The telomeres and centromeres of each haplotype were almost fully annotated. In addition, we detected a total of 41,135 structural variations. Enrichment analysis demonstrated that genes involved in terpenoid biosynthesis were affected by these structural variations. Analysis of volatile metabolites showed that M. suaveolens mainly produces piperitenone oxide rather than menthol. We identified three genes in the M. suaveolens genome which encode isopiperitenone reductase (ISPR), a key rate-limiting enzyme in menthol biosynthesis. However, the transcription levels of ISPR were low. Given that other terpenoid biosynthesis genes were expressed, M. suaveolens ISPRs may account for the accumulation of piperitenone oxide in this species. The findings of this study may provide a valuable resource for improving the detection rate and accuracy of genetic variants, thereby enhancing our understanding of their impact on gene function and expression. Moreover, our haplotype-resolved gap-free genome assembly offers novel insights into molecular marker-assisted breeding of Mentha.

Potential application of light emitting diodes to improve molecular, physiological, and growth responses in peppermint plant

AbstractBackgroundClimate change and growing global population are driving forces that shift the agriculture from open fields to environmentally‐controlled confined systems. In such confined systems, plants are adapted to unique light spectra to achieve maximal qualitative and quantitative yields. In the present study, effects of five light spectra on morpho‐physiological and molecular attributes of peppermint were examined. Treatments included a traditional greenhouse considered as the control, a fluorescent light, and four types of light emitting diodes (LED): red, blue, red‐blue (70:30), and white. Leaf samples were collected from plants and analyzed for volatile constituents using a headspace GC–MS. Meanwhile, changes in the expression profile of genes involved in the biosynthesis of menthol were evaluated.ResultsOur results showed that maximal yield (wet and dry biomass) was achieved under greenhouse condition and the red‐blue LED. Application of the red‐blue LED resulted in maximal production of beneficial monoterpenes, which indicate superiority to greenhouse environment. A significant change in the gene expression profile was observed among plants grown under different light treatments. The red‐blue LED was associated with a significant up‐regulation of key genes in the menthol biosynthesis (menthol dehydrogenase and pulegone reductase). This phenomenon was consistent with a higher menthol production under the same condition.ConclusionThe application of red‐blue LED was associated with the highest level of yield and menthol production, whereas it resulted in lower concentrations of unfavorable by‐products (isomenthol, cis‐pulegone, and menthofuran). In conclusion, 70:30 red‐blue LED can be satisfactory applied to maximize growth yield and optimize favorable bioactive constituent in peppermint.

Glandular trichome specificity of menthol biosynthesis pathway gene promoters from Mentha × piperita.

Several cis-elements including Myb-binding motifs together confer glandular trichome specificity as revealed from heterologous expression and analysis of menthol biosynthesis pathway gene promoters. Glandular Trichomes (GTs) are result of division of epidermal cells that produce diverse metabolites. Species of mint family are important for their essential oil containing many high-value terpenoids, biosynthesized and stored in these GTs. Hence, GTs constitute attractive targets for metabolic engineering and GT-specific promoters are important. In this investigation, the upstream regions of the Mentha × piperita menthol biosynthetic pathway genes (-)-limonene synthase, (-)-P450 limonene-3- hydroxylase, (-)-trans-isopiperitenol dehydrogenase, (-)-Isopiperitenone reductase, ( +)-Pulegone reductase, (-)-Menthone reductase/ (-)-Menthol dehydrogenase and a branched pathway gene ( +)-menthofuran synthase were isolated and characterized. These fragments, fused to β-glucuronidase (GUS) reporter gene of pBI101 binary vector, are able to drive high level gene expression in transgenic tobacco trichomes with strong signals in GTs, except for (-)-Isopiperitenone reductase. The GT-enriched tissue from transformed plants were analysed for GUS enzyme activity and RNA expression which correlates the GUS staining. To characterize the cis-elements responsible for GT-specific expression, a series of 5' deletion constructs for MpPLS and MpPMFS were cloned and analysed in stable transgenic tobacco lines. The specificity of trichome expression was located to - 797 to- 598bp sequence for (-)-limonene synthase and- 629 to - 530bp for ( +)-menthofuran synthase promoters containing specific Myb-binding motifs in addition to other unique motifs described for developmental regulation without any defined pattern. All other pathway promoters also recruits specific but different Myb factors as indicated by this analysis.

Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis.

Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs like Mentha haplocalyx Briq., Nepeta tenuifolia Briq., Perilla frutescens (L.) Britt and Pogostemin cablin (Blanco) Benth. Pulegone reductase is the key enzyme in the biosynthesis of menthol and is required for the stereoselective reduction of the Δ2,8 double bond of pulegone to produce the major intermediate menthone, thus determining the stereochemistry of menthol. However, the structural basis and mechanism underlying the stereoselectivity of pulegone reductase remain poorly understood. In this study, we characterized a novel (−)-pulegone reductase from Nepeta tenuifolia (NtPR), which can catalyze (−)-pulegone to (+)-menthone and (−)-isomenthone through our RNA-seq, bioinformatic analysis in combination with in vitro enzyme activity assay, and determined the structure of (+)-pulegone reductase from M. piperita (MpPR) by using X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, molecular dynamics simulations, and biochemical analysis. We identified and validated the critical residues in the crystal structure of MpPR involved in the binding of the substrate pulegone. We also further identified that residues Leu56, Val282, and Val284 determine the stereoselectivity of the substrate pulegone, and mainly contributes to the product stereoselectivity. This work not only provides a starting point for the understanding of stereoselectivity of pulegone reductases, but also offers a basis for the engineering of menthone/menthol biosynthetic enzymes to achieve high-titer, industrial-scale production of enantiomerically pure products.

Removing the Obstacle to (-)-Menthol Biosynthesis by Building a Microbial Cell Factory of (+)-cis-Isopulegone from (-)-Limonene.

Microbial synthesis of plant-based (-)-menthol is of great interest because of its high demand (≈30 kiloton per year) as well as unique odor and cooling characteristics. However, this remains a great challenge due to the yet unfilled gap between (-)-limonene and (+)-cis-isopulegone. Herein, the first artificial and effective system was developed for (+)-cis-isopulegone biosynthesis from (-)-limonene by recruiting two bacterial enzymes to replace their inefficient counterparts from Mentha piperita, limonene-3-hydroxylase, and isopiperitenol dehydrogenase. A cofactor self-regenerative recombinant Escherichia coli strain was constructed by introducing a formate dehydrogenase for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and an engineered microbial isopiperitenol dehydrogenase. The production of (+)-cis-isopulegone (up to 281.2 mg L-1 ) was improved by 36 times compared with that of the initial strain. This work lays a reliable foundation for the microbial synthesis of (-)-menthol.

Discovery and Engineering of Bacterial (−)‐Isopiperitenol Dehydrogenases to Enhance (−)‐Menthol Precursor Biosynthesis

AbstractMicrobial synthesis of (−)‐menthol, a compound of plant origin, is of great importance because of the high demand for this product and related sustainability issues. However, the total biosynthesis of (−)‐menthol from easily available feedstocks like (−)‐limonene by engineered microbial hosts is stalled by the poor protein expression or activity of several enzymes from the native (−)‐menthol biosynthesis pathway of mint (Mentha piperita). Among these unsatisfied steps, (−)‐isopiperitenol dehydrogenase (IPDH) catalyzed oxidation reaction of (−)‐trans‐isopiperitenol was one of the bottlenecks that need to be optimized. In this work, two novel bacterial enzymes with IPDH activity were discovered to replace their inefficient counterpart from plant cells in microbial (−)‐menthol synthesis. Two key residues in PaIPDH from Pseudomonas aeruginosa were mutated to PaIPDHE95F/Y199V with 4.4‐fold improved specific activity than PaIPDH. The mechanism for the beneficial mutations was elucidated by molecular dynamics simulations. PaIPDHE95F/Y199V was used to synthesize (−)‐isopiperitenone from (−)‐limonene in vivo via a self‐sufficient cofactor cascade enzyme reaction, affording a 3.7‐fold enhanced titer of (−)‐isopiperitenone compared with that obtained using the original mint IPDH (MpIPDH). The bacterial enzyme PaIPDHE95F/Y199V can be applied in the future for constructing a more efficient artificial pathway to biosynthesize (−)‐menthol in a microbial whole‐cell system.magnified image

Application of Petri Net Theory for Modelling and Validation of Menthol Biosynthesis

An essential step in network modelling is to validate the network model. Petri net theory provides algorithms and methods, which can be applied directly to metabolic network modelling and analysis in order to validate the model. This paper describes the thriving application of Petri net theory for model validation of biosynthesis of menthol using the well-established Petri net analysis technique of place and transition invariants. Because of the complexity of metabolic networks and their regulation, formal modelling is a useful method to improve the understanding of these systems. A petri net representation, its validation and simulation of biosynthesis of menthol from geranyl diphosphate (GPP) has been performed with the objective of understating new insights of the structure of this pathway affecting the synthesis of menthol. The model has been validated for its P-invariant and T-invariant. T-invariant analysis suggest absence of any loop in the net which restore the initial state suggesting all reactions to be only forward. The net is covered by positive P-invariants and bounded. The net is utilized to simulate the time (pt) with concentrations of GPP, (−)-limonene, (+)-pulegone, (−)-menthone and (−)-menthol. Dimethylallyl diphosphate and isopentenyl diphosphate were the main precursors for this biosynthesis. Biological data needed for simulation where obtained from extensive survey of literature. The results were shown graphically and the nature of graphs represent the variation of concentrations with time.

Cyclodextrin-based Gas Chromatography and GC/MS methods for determination of chiral pair constituents in mint essential oils

ABSTRACT The genus Mentha has been known as culinary source of minty note. Besides, the essential oil imparts value to consumer products like confectionery, cosmetics, and pharmaceutical preparations. A simple and sensitive enantioselective gas chromatographic method is described for the quantitative analysis of terpenoid enantiomers in essential oils using different cyclodextrin coated stationary phases. Limonene, menthone, menthol, and menthyl acetate were identified as pure (-)-enantiomers whereas isomenthone, neomenthol, pulegone, and piperitone as pure (+)-enantiomers in menthol mint, and peppermint oils, respectively. The selectivity of each enantiomer was also demonstrated. A β-cyclodextrin phase with diethyl substitution provides baseline resolution for all target enantiomers with selectivity ranged from 1.004 to 1.050. Moreover, permethylated or diacetylated-β-cyclodextrin showed no selectivity for (+/-)-menthol enantiomers. The current study may facilitate the origin authentication studies of mint oils. Investigation of enantiomers involved in menthol biosynthesis revealed that only (-)-menthol is present in the essential oils of Mentha species.

Omeprazole alleviates water stress in peppermint and modulates the expression of menthol biosynthesis genes

Water stress is a worldwide agricultural challenge that limits crop growth and quality. Chemical compounds that promote tolerance to water stress, such as omeprazole showed recently promising results. The present study investigates the effect of weekly drenching applications of 0, 10, 50, 100, or 200 μM omeprazole on Mentha piperita (peppermint) subjected to water stress by watering at 100%, 70%, and 50% of container substrate capacity for 7 weeks in an experiment that spanned two seasons. Peppermint that received higher doses of omeprazole showed increased plant height, leaf number, leaf area, and dry weight under normal and water stress conditions. The amounts of chlorophyll and proline in the leaves as well as gas exchange increased in omeprazole-treated plants relative to the control plants. Omeprazole treatment also resulted in increased activity of the enzymes catalase and ascorbate peroxidase, reduced accumulation of the reactive oxygen species hydrogen peroxide, increase in the essential oil ratio, and improvement in essential oil composition. Omeprazole-treated plants showed higher ratios of menthol and menthone composition relative to the control plants. The changes in essential oil composition were associated with increased expression of genes associated with the menthol biosynthesis pathway. These findings indicate that omeprazole can ameliorate water stress in peppermint by increasing vegetative and root growth; increasing chlorophyll amount, photosynthetic rate, and gas exchange; reducing water loss by boosting leaf water potential and relative water content; increasing proline content; and modulating the gene expression of secondary metabolites.

Transcriptome Analysis of JA Signal Transduction, Transcription Factors, and Monoterpene Biosynthesis Pathway in Response to Methyl Jasmonate Elicitation in Mentha canadensis L.

Mentha canadensis L. has important economic value for its abundance in essential oils. Menthol is the main component of M. canadensis essential oils, which is certainly the best-known monoterpene for its simple structure and wide applications. However, the regulation of menthol biosynthesis remains elusive in M. canadensis. In this study, transcriptome sequencing of M. canadensis with MeJA treatment was applied to illustrate the transcriptional regulation of plant secondary metabolites, especially menthol biosynthesis. Six sequencing libraries were constructed including three replicates for both control check (CK) and methyl jasmonate (MeJA) treatment and at least 8 Gb clean bases was produced for each library. After assembly, a total of 81,843 unigenes were obtained with an average length of 724 bp. Functional annotation indicated that 64.55% of unigenes could be annotated in at least one database. Additionally, 4430 differentially expressed genes (DEGs) with 2383 up-regulated and 2047 down-regulated transcripts were identified under MeJA treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that “Monoterpenoid biosynthesis” was one of the most significantly enriched pathways in metabolism. Subsequently, DEGs involved in JA signal transduction, transcription factors, and monoterpene biosynthesis were analyzed. 9 orthologous genes involved in menthol biosynthesis were also identified. This is the first report of a transcriptome study of M. canadensis and will facilitate the studies of monoterpene biosynthesis in the genus Mentha.

Changes in the expression of key genes involved in the biosynthesis of menthol and menthofuran in Mentha piperita L. under drought stress

Peppermint (Mentha piperita) is known as an important medicinal plant throughout the world. In the present study, after exposing peppermint plants under drought stress, the qRT-PCR was use to analyze the expression of genes involved in menthol biosynthesis pathway and encoding: limonene synthase (lS), limon-3-hydroxylase (l3oh), trans-isopiperitenol dehydrogenase (ipd), isopiperitenone reductase (ipr), pulegone reductase (pr), menthol dehydrogenase (mdeh), and menthofuran synthase (mfs), which also evaluated the morphological and physiological traits. The results revealed that due to water stress, the gene expression levels of ipd, ipr, and mfs were increased, whereas the gene expression level of pr and mdeh was decreased under water stress conditions. The most of essential oil components (menthol, menthofuran, and plugene), which were analyzed by gas chromatography–mass spectrometry (GC–MS), was positively correlated with genes expression. Drought stress decreased morphological and induces increasing contents of pulegone and menthofuran and reduction in menthol percentages. Results from this study suggest that up-regulation of mfs might contribute to the altered of menthofuran as well as down-regulation of mdeh might cause the reduction of menthol. Furthermore, increasing ls gene expression levels might induce more essential oil yield, while reduction of mfs gene expression levels causes an improvement of essential oil quality.

Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome (GT) was carried out. In addition to the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway genes, about 210 and 196 different terpene synthases (TPSs) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (three from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data.

Relative expression of genes of menthol biosynthesis pathway in peppermint (Mentha piperita L.) after chitosan, gibberellic acid and methyl jasmonate treatments

Menthol as an important component of monoterpenes essential oil in peppermint (Mentha piperita L.) is widely applied for medical and industrial uses. In this study, the effect of exogenous applications of chitosan (200 mg/L), gibberellic acid (50 mg/L) and methyl jasmonate (300 μM) was investigated in the main genes of menthol biosynthesis pathways within a 72 h time period using qRT-PCR. Transcript levels of most genes were either unaffected or down-regulated following chitosan treatment relative to control plants. Decreasing of geranyl diphosphate synthase (GDS) and limonene synthase (LS) genes transcript in chitosan treatment could possibly be effective in reducing of limonene level. On the other hand, it seems that an increase in menthone-menthol reductase (MMR) transcription level at 72 h under these treatments had a positive role in increasing the amount of menthol in this plant. Since exogenous application of gibberellic acid (GA3) down-regulated transcript levels of several genes involved in menthol biosynthesis, there is this expectance that GA3 treatment might not have a prominent role in enhancing menthol yield via transcription regulation. Transcript level of the majority genes after methyl jasmonate treatment gradually increased and reached the highest level at 72 h, therefore, it is possible that methyl jasmonate improves medicinal properties of M. piperita.

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.

Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis

Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

(−)-Menthol biosynthesis and molecular genetics

(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint (Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general "allylic oxidation-conjugate reduction" scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1R, 3R, 4S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.