Response of exogenous salicylic acid on cadmium induced photosynthetic damage,antioxidant metabolism and essential oil production in peppermint

The present inquest was undertaken in view of the alarming increase in the concentration of cadmium, nickel etc., in the arable soils of Uttar Pradesh, India and the little attention paid to the effect of these heavy metals on the performance of pharmaceutically imperative essential oil (EO) producing crops like peppermint (Mentha piperita L.). We devised a pot experiment to study the influence of exogenously sourced salicylic acid (SA) (10^?4 M) in the amelioration of growth, protection of photosynthesis and essential oil production against 30, 60 and 120 mg kg^?1 soil of cadmium (Cd)-accrued stress in peppermint. Plants grown with Cd showed remarkably deleterious effects on growth, photosynthesis, carbon and nitrate assimilating enzymes and yield and active constituents of EO in addition to the marked elevation in the oxidative stress. SA successfully alleviated the Cd induced toxicity in peppermint, improved photosynthesis by enhancing activity of RuBisCo and carbonic anhydrase and minimized the oxidative stress by mitigating the production of free radicals by the maintenance of free radical scavenging enzymes and reduced glutathione (GSH) pool. Furthermore, the decrease in the concentration of EO and menthol due to Cd stress was successfully alleviated by SA application which was evident from the gas chromatograms of EO of Cd stressed SA treated plants.     

Influence of phosfon D and cycocel on growth and essential oil content of sage and peppermint

Foliar application of Phosfon D at 50–100 ppm stimulates the growth of Salvia officinalis (sage) and moderately retards the growth of Mentha piperita (peppermint), while increasing the essential oil yield of both species by 50–70 % Phosfon D increases the proportions of (−)-3-isothujone and (+)-3-thujone in sage oil and decreases the level of (−)-β-pinene and (+)-camphor, whereas this growth retardant increases the proportions of (+)-isomenthone and (+)-neoisomenthol in peppermint oil and decreases the level of(−)-menthone and (−)-menthoL Foliar application of Cycocel at 250–500 ppm slightly stimulates growth and essential oil formation in peppermint, and retards growth of sage with little effect on oil yield. The influence of Cycocel on sage oil composition was the opposite of that of Phosfon, with a tendency to increase the level of (−)-β-pinene and decrease the level of (−)-3-isothujone under severe stunting. The effect of Cycocel on the composition of peppermint varied with concentration. The influence of growth retardants on essential oil composition and yield are most readily explained by alterations in the levels or activities of the relevant enzymes.     

Response of growth,essential oil composition,endogenous hormones and microbial activity of Mentha piperita to some organic and biofertilizers agents

The effect of organic (poultry and cattle manures) and biological (effective microorganisms, EM) fertilizers on growth, essential oil yield and its compositions, endogenous phytohormones content and antibacterial activity of peppermint plants grown in pot over 12 weeks was studied. Application of organo- and bio-fertilizers greatly affected on growth, essential oil production and other estimated parameters of peppermint plants. Slight stimulation effect was happened due to soil application of organic manures. Soil application of EM alone or in combination with organic fertilizers significantly increased growth, yield and components of essential oils, endogenous hormones of peppermint as compared to other treatments. Using disc diffusion method, the extracted oil of peppermint plants amended with organic and biofertilizers recorded the highest antibacterial activity against tested pathogenic bacteria like Klebsiella pneuumoniae and Staphylococcus aureus.     

Antibacterial properties and major bioactive components of Mentha piperita essential oils against bacterial fruit blotch of watermelon

Plant-derived essential oil is an alternative to antibiotics, eliminating the concern of developing antibiotic-resistant bacterial strains. In this study, using the half-divided Petri plate assays, 32 volatile essential oils were screened for their antibacterial activity against Acidovorax citrulli (Acc). Sweet basil and peppermint oils were the most effective against Acc, with subsequent trials showing that peppermint oil to be the most active. Using gas chromatography–mass spectrometry, the major compositions of peppermint oil were analysed. Among the various compositions of peppermint oil, menthol, neomenthol, isopulegone and 1,8-cineole were significantly active against Acc and each component at 0.2% concentration inhibited all bacterial growth. This study demonstrated in vitro and in vivo antibacterial activities of peppermint oil and its active components against Acc. These results suggest the use of peppermint oil as a potential antibacterial agent to treat seed with Acc.     

Plant growth regulators ameliorate the ill effect of salt stress through improved growth,photosynthesis, antioxidant system,yield and quality attributes in <Emphasis Type="Italic">Mentha piperita</Emphasis> L.

The roles of plant growth regulators (PGRs) in plants are well documented. However, there is a little information regarding their roles in alleviating salt stress in plants, particularly peppermint. This necessitated the evaluation of the efficiency of three selected PGRs in counteracting the ill effect of salt stress by conducting a pot experiment on peppermint (Lamiaceae). Three uniform size suckers were transplanted in each pot containing proper nutrients. Thirty day old plants were subjected to 4 levels of salinity, viz. 0, 50, 100 or 150 mM NaCl. Salt stress was given at 30 days after their transplantation (DAT). Plants were sprayed twice, i.e., at 60 and 75 DAT with 10^?6 M each of gibberellic acid (GA₃), salicylic acid (SA) or triacontanol (Tria). The sampling was made at 100 DAT and harvesting at 120 DAT. The graded levels of salinity decreased growth, photosynthesis, carbonic anhydrase (CA) activity, NPK content, peltate glandular trichome (PGT) density, essential oil (EO) and menthol content and herb, EO and menthol yield, but increased catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD) activities and proline content linearly. Spray of PGRs particularly SA improved all parameters under both salt and salt free conditions. The maximum values for yields of herb, EO and menthol were noted with 0 mM NaCl?×?SA. However, antioxidants, proline content, PGT density and EO content were found to be maximum with 150 mM NaCl?×?SA.     

Influence of ethephon and daminozide on growth and essential oil content of peppermint and sage

Foliar application of Daminozide at 1000 ppm reduces the growth of Salvia officinalis (sage) and decreases essential oil yield, but increases both growth and essential oil yield of Mentha piperita at the same concentration. Ethephon at 250 ppm reduces the growth and essential oil yield of peppermint and slightly increases growth and essential oil content of sage. Both growth regulators markedly reduce the level of menthone and menthol in peppermint oil and increase the level of isomenthone and neoisomenthol. Both growth regulators decrease the level of camphor and increase the level of β-pinene in sage oil. Changes in essential oil composition induced by these growth regulators are most readily explained by alterations in the levels or activities of the relevant biosynthetic enzymes.     

DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma

Abstract

Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B’, p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.     

Disruption of Autophagy Results in Constitutive Oxidative Stress in Arabidopsis

Plant cells frequently encounter oxidative stress, leading to oxidative damage and inactivation of proteins. We have recently demonstrated that oxidative stress induces autophagy in Arabidopsis seedlings in an AtATG18a-dependent manner and that RNAi-AtATG18a transgenic lines, which are defective in autophagosome formation, are hypersensitive to reactive oxygen species. Analysis of protein oxidation indicated that oxidized proteins are degraded in the vacuole after uptake by autophagy, and this degradation is impaired in RNAi-AtATG18a lines. Our results also suggest that in the absence of a functional autophagy pathway, plants are under increased oxidative stress, even under normal growth conditions.

Addendum to:

Degradation of Oxidized Proteins by Autophagy during Oxidative Stress in Arabidopsis

Y. Xiong, A.L. Contento, N.Q. Phan and D.C. Bassham

Plant Physiol 2007; 143:291-9     

Strigolactone and Methyl Jasmonate-Induced Antioxidant Defense and the Composition Alterations of Different Active Compounds in Dracocephalum kotschyi Boiss Under Drought Stress

Strigolactone (SL) and methyl jasmonate (MeJA) are one of the most important plant hormones that exert biological activity in plant responses to environmental stresses. Considering the undetected role of SL in drought tolerance and essential oil yield of medicinal plants as well as conceivable interaction among MeJA and SL, a factorial experiment was performed as a complete randomized design with three replications. Experimental factors including two irrigation regimes such as irrigation to 80% field capacity (control) and 40% field capacity (drought stress) and spraying treatments include MeJA (0 and 0.5 mM) and SL (0 and 10 μM) were applied. Treatment of plants with SL and MeJA resulted in higher tolerance to drought stress due to higher fresh and dry weights as well as lower electrolyte leakage, malondialdehyde, H₂O₂, total phenol content, total antioxidant activity and antioxidant power assay. The most important essential oil constituents of D. kotschyi included geranyl acetate (41.1–48.6%), α-pinene (16.2–18.9%), geranial (7.9–10.1%), limonene (5.5–7.0%), neral (3.5–4.1%), methyl geranate (2.3–3.3%) and geraniol (1–2.2%), the least of which was found in non-MeJA- and SL-treated plants under drought and the highest in MeJA- and SL-treated plants under drought stress. Drought tolerance of D. kotschyi became more intense and the amount of essential oil components of water stressed plants was the highest (99.2%) when these plant hormones were used together. These results suggest a cross-link between MeJA and SL in improving drought resistance and optimizing the production of essential oil of D. kotschyi.

     

Genetic engineering of peppermint for improved essential oil composition and yield

The biochemistry, organization, and regulation of essential oil metabolism in the epidermal oil glands of peppermint have been defined, and most of the genes encoding enzymes of the eight-step pathway to the principal monoterpene component (−)-menthol have been isolated. Using these tools for pathway engineering, two genes and two expression strategies have been employed to create transgenic peppermint plants with improved oil composition and yield. These experiments, along with related studies on other pathway genes, have led to a systematic, stepwise approach for the creation of a ‘super’ peppermint.     

Salinity-induced changes in essential oil,pigments and salts accumulation in sweet basil (Ocimum basilicum) in relation to alterations of morphological development

The objective of the project was to study salinity-induced effects on essential oil, pigments and salts accumulation in sweet basil (Ocimum basilicum, the cultivar Perrie) in relation to the alteration of plant morphological development and yield production. Hydroponically grown plants were exposed to one of six NaCl concentrations (1, 25, 50, 75, 100 and 130 mM NaCl). Inhibitory effects of salinity on biomass production of the shoot and the root, and area of individual leaves were apparent already under cultivation with 25 mM NaCl. Elevation of salinity from 1 to 100 mM NaCl induced 63% and 61% reductions in fresh and dry herb biomass production, respectively. The stress-induced reduction of foliage biomass sourced mainly from inhibition of leaf area development rather than reduction of internode and leaf number. Cl and Na concentrations in the leaves, stems and roots increased with elevation of NaCl concentration in the cultivation solution. While the extent of Cl accumulation was leaves>stems>roots, Na was largely excluded from the leaves and was preferentially accumulated in roots and the stems, potentially accounting for the moderate sensitivity of the leaf tissue to salinity. Salt stress increased the contents of essential oil and carotenoids in the leaves that may further account for the moderate sensitivity of sweet basil to salinity and suggest a potential for agro-industrial production. A twofold increase in both carotenoid concentration and the percent of essential oil in the fresh tissue was observed by elevation of the salinity from 1 to 130 mM NaCl. Overall, the stress induced increase of the percent of essential oil in the tissue in the salinity range 1–75 mM NaCl was about 50%, and thereby compensated for the similar reduction of biomass production in this salinity range, so that oil production on per plant basis was not reduced by salinity.     

Absence of MGST1 mRNA and protein expression in human neuroblastoma cell lines and primary tissue

A recent study identified a haplotype on a small region of chromosome 12, between markers D12S1725 and D12S1596, shared by all patients with familial neuroblastoma (NB). We previously localized the human MGST1 gene, whose gene product protects against oxidative stress, to this very same chromosomal region (12p112.1–p13.33). Owing to the chromosomal location of MGST1; its roles in tumorigenesis, drug resistance, and oxidative stress; and the known sensitivity of NB cell lines to oxidative stress, we considered a role for MGST1 in NB development. Surprisingly there was no detectable MGST1 mRNA or protein in either NB cell lines or NB primary tumor tissue, although all other human tissues, cell lines, and primary tumor tissue examined to date express MGST1 at high levels. The mechanism behind the failure of NB cells and tissue to express MGST1 mRNA is unknown and involves the failure of MGST1 pre-mRNA expression, but does not involve chromosomal rearrangement or nucleotide variation in the promoter, exons, or 3' untranslated region of MGST1. MGST1 provides significant protection against oxidative stress and constitutes 4 to 6% of all protein in the outer membrane of the mitochondria. As NB cells are extremely sensitive to oxidative stress, and often used as a model system to investigate mitochondrial response to endogenous and exogenous stress, these findings may be due to the lack of expression MGST1 protein in NB. The significance of this finding to the development of neuroblastoma (familial or otherwise), however, is unknown and may even be incidental. Although our studies provide a molecular basis for previous work on the sensitivity of NB cells to oxidative stress, and possibly marked variations in NB mitochondrial homeostasis, they also imply that the results of these earlier studies using NB cells are not transferable to other tumor and cell types that express MGST1 at high concentrations.     

Somatic hybridization in mint: identification and characterization of Mentha piperita (+) M. spicata hybrid plants

 Twenty eight somatic hybrid plants were identified following protoplast fusions between peppermint (Mentha piperita L. cv Black Mitcham), producing high-quality oil, and spearmint (Mentha spicata L. cv Native Spearmint), likewise producing high-quality oil and also possessing resistance to verticillium wilt. Prior to fusion, peppermint protoplasts were subjected to iodoacetic acid to inhibit cell division. Protoplasts of peppermint and spearmint were fused using polyethylene glycol plus DMSO. Fusion products were cultured according to an efficient protoplast-to-plant-cycle protocol developed for peppermint. Using this protocol, iodoacetic acid-treated peppermint protoplasts were not able to divide, whereas untreated spearmint protoplasts had the ability to produce callus but not shoots. Therefore, selection of somatic hybrid calli was based on the presumed capability of hybrid cells to form calli and shoots. Shoots in vitro were initially identified as hybrids using RAPD profiles. Subsequently, observations on morphology, chromosome counts, and Southern-hybridization patterns confirmed their hybrid status. The results of verticillium tests revealed that 18 somatic hybrids were more susceptible than Native Spearmint, while hybrid II-14 had a level of susceptibility intermediate between that of the fusion parents. Oil-analysis of hybrid plants indicated that they all have a GC-profile typical of spearmint oil. Received: 8 February 1997 / Accepted: 9 April 1997     

A mechanism coordinating root elongation,endodermal differentiation,redox homeostasis and stress response

Root growth relies on both cell division and cell elongation, which occur in the meristem and elongation zones, respectively. SCARECROW (SCR) and SHORT-ROOT (SHR) are GRAS family genes essential for root growth and radial patterning in the Arabidopsis root. Previous studies showed that SCR and SHR promote root growth by suppressing cytokinin response in the meristem, but there is evidence that SCR expressed beyond the meristem is also required for root growth. Here we report a previously unknown role for SCR in promoting cell elongation. Consistent with this, we found that the scr mutant accumulated a higher level of reactive oxygen species (ROS) in the elongation zone, which is probably due to decreased expression of peroxidase gene 3, which consumes hydrogen peroxide in a reaction leading to Casparian strip formation. When the oxidative stress response was blocked in the scr mutant by mutation in ABSCISIC ACID 2 (ABA2) or when the redox status was ameliorated by the upbeat 1 (upb1) mutant, the root became significantly longer, with longer cells and a larger and more mitotically active meristem. Remarkably, however, the stem cell and radial patterning defects in the double mutants still persisted. Since ROS and peroxidases are essential for endodermal differentiation, these results suggest that SCR plays a role in coordinating cell elongation, endodermal differentiation, redox homeostasis and oxidative stress response in the root. We also provide evidence that this role of SCR is independent of SHR, even though they function similarly in other aspects of root growth and development.     

Screening for inhibitory activities of essential oils on the growth of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., the causal agent of leaf spot disease of Murraya koenigii L.

Seven essential oils namely clove, cedar wood, lemongrass, peppermint, eucalyptus, citronella and neem oils were tested for their inhibitory effect on spore germination, growth of germ tube and mycelial growth of Colletotrichum gloeosporioides isolated from diseased Murraya koenigii. All essential oils inhibited the germination and growth of germ tube at different concentrations. However, significant reduction in colony growth was observed with citrus, lemongrass and peppermint oils at 1000, 1500 and 2000 ppm concentrations, respectively. Citrus oil at 1360 ppm inhibited the maximum growth of the fungus followed by lemongrass oil at 1720 ppm and peppermint at 2260 ppm, respectively. The effect of essential oils on mycelial dry weight also showed antifungal activity on the growth of Colletotrichum gloeosporioides. The study revealed the possible utilisation of these essential oils for foliar spray for the management of leaf spot disease of Murraya koenigii.     

Leaf dehydroascorbate reductase and catalase activity is associated with soil drought tolerance in bread wheat

A number of morphological, physiological and phenological traits have been suggested as significant markers of adaptation to drought in bread wheat (Triticum aestivum L.). This study was aimed at the identification of a relationship between dehydroascorbate reductase (DHAR, EC 1.8.5.1) and catalase (CAT, EC 1.11.1.6) activities in leaves of wheat plants and stability of yield components under water deficit. The single chromosome substitution lines of cv. Chinese Spring carrying separate chromosomes from the donor Synthetic 6x, an artificial hexaploid combining the genomes of the two wild species, Triticum dicoccoides (AABB) and Aegilops tauschii (DD), were the objects of the investigations. The activities of the DHAR and CAT were correlated with flag leaf relative water content and two indexes of stability of grain yield components under drought across the set substitution lines. The lines carrying a synthetic hexaploid homologous pair of chromosomes 1B, 1D, 2D, 3D or 4D all expressed a low constitutive level of DHAR and the lines carrying chromosomes 3B, 1D, 2D and 3D a low constitutive level of CAT. All were able to increase this level (by fourfold for DHAR and by 1.5-fold for CAT) in response to stress caused by water deficit. When challenged by drought stress, these lines tended to be the most effective in retaining the water status of the leaves and preventing the grain yield components from being compromised. The discovered genetic variability for enzymes activity in leaves of wheat might be a useful selection criterion for drought tolerance.     

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.