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

MAIN CONCLUSION: 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 ß-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-  598 bp sequence for (-)-limonene synthase and-  629 to -   530 bp 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.

Application of insecticides on peppermint (Mentha × piperita L.) induces lignin accumulation in leaves by consuming phenolic acids and thus potentially deteriorates quality.

Irrational use of pesticides may lead to physiological and metabolic disorders in different crops. However, there are limited investigations on impacts of insecticides on physiology and biochemistry, secondary metabolic pathways, and associated quality of medicinal plants such as peppermint (Mentha × piperita L.). In this study, target metabolites in peppermint were monitored following foliar spraying of five insecticides: imidacloprid, pyriproxyfen, acetamiprid, chlorantraniliprole, and chlorfenapyr. Compared with the control, all insecticide treatments caused a significant loss of soluble protein (decreased by 22.3-38.7%) in peppermint leaves. Insecticides induced an increase in the levels of phytohormones jasmonic acid and abscisic acid in response to these chemical stresses. Among them, imidacloprid increased jasmonic acid by 388.3%, and pyriproxyfen increased abscisic acid by 98.8%. The contents of phenylpropanoid metabolites, including rutin, quercetin, apigenin, caffeic acid, 4-hydroxybenzoic acid, ferulic acid, syringic acid, and sinapic acid showed a decreasing trend, with pyriproxyfen decreasing the levels of quercetin and 4-hydroxybenzoic acid by 78.8% and 72.6%, respectively. Combined with correlation analysis, the content of lignin in leaves shows different degrees of negative correlations with several phenolic acids. It could be inferred that insecticides may trigger plant defense mechanisms that accumulate lignin (increased by 24.6-49.1%) in leaves by consuming phenolic acids to barricade absorption of insecticides. Through constructing networks between phytohormones and secondary metabolites, peppermint may regulate the contents of caffeic acid, 4-hydroxybenzoic acid, and sinapic acid by the antagonistic effect between salicylic acid and abscisic acid in response to insecticidal stresses. Principal component analysis and systemic cluster analysis revealed that the most pronounced changes in physiological indexes and metabolites were caused by the pyriproxyfen treatment. In conclusion, this study improves our understanding of the mechanism by which insecticides affect plant physiological and metabolic processes, thus potentially altering the quality and therapeutic value of peppermint as an example.

Peppermint essential oil: its phytochemistry, biological activity, pharmacological effect and application.

Mentha (also known as peppermint), a genus of plants in the taxonomic family Lamiaceae (mint family), is widely distributed throughout temperate regions of the world. Mentha contains various constituents that are classified as peppermint essential oil (PEO) and non-essential components. PEO, consisting mainly of menthol, menthone, neomenthol and iso-menthone, is a mixture of volatile metabolites with anti-inflammatory, antibacterial, antiviral, scolicidal, immunomodulatory, antitumor, neuroprotective, antifatigue and antioxidant activities. Mounting evidence indicates that PEO may pharmacologically protect gastrointestinal, liver, kidney, skin, respiratory, brain and nervous systems, and exert hypoglycemic and hypolipidemic effects. Clinically, PEO is used for gastrointestinal and dermatological diseases, postoperative adjuvant therapy and other fields. This review aims to address the advances in the extraction and isolation of PEO, its biological activities, pharmacological effects, toxicity and applications, with an emphasis on the efficacy of PEO on burn wounds and psoriasis, providing a comprehensive foundation for research, development and application of PEO in future.

Effects of Peppermint Essential Oil on Learning and Memory Ability in APP/PS1 Transgenic Mice.

OBJECTIVE: To explore the effect and mechanism of peppermint essential oil on learning and memory ability of APP/PS1 transgenic mice. METHODS: Morris water maze test and shuttle box test were used to explore the changes in learning and memory ability of APP/PS1 transgenic mice after sniffing essential oil. The cellular status of neurons in the hippocampal CA1 region of the right hemisphere, Aß deposition, oxidative stress level, and serum metabonomics were detected to explore its mechanism. RESULTS: Sniffing peppermint essential oil can improve the learning and memory ability of APP/PS1 transgenic mice. Compared with the model group, the state of neurons in the hippocampal CA1 region of the peppermint essential oil group returned to normal, and the deposition of Aß decreased. The MDA of brain tissue decreased significantly, and the activity of SOD and GSH-PX increased significantly to the normal level. According to the results of metabonomics, it is speculated that peppermint essential oil may improve cognitive function in AD by regulating arginine and proline metabolism, inositol phosphate metabolism, and cysteine and methionine metabolism.

Peppermint Essential Oil and Its Major Volatiles as Protective Agents against Soft Rot Caused by Fusarium sambucinum in Cera Pepper (Capsicum pubescens).

Cera pepper (Capsicum pubescens) is an exotic fruit considered as a rich source of nutraceuticals with known benefits for human health and also an economic resource for local producers in Mexico. The present investigation reports on the in vitro and in situ antifungal activity of the essential oil from Mentha piperita and its two major volatiles (menthol and menthone) against Fusarium sambucinum, which is a causal agent of soft rot in cera pepper. The application of these components in pepper fruits previously infected with F. sambucinum caused a significant delay (p<0.05) in the emergence of soft rot symptoms. This effect was reflected in the maintenance of pH and fruit firmness during a period of 10 days. The nutrimental content of the fruits (protein, fiber, fat and other proximate parameters) was conserved in the same period of time. The nutraceutical content of these fruits was estimated by the quantification of seven carotenoids (violaxanthin, cis-violaxanthin, luteoxanthin, antheraxanthin, lutein, zeaxanthin and ß-carotene), ascorbic acid and capsaicinoids (capsaicin and dihydrocapsaicin). According to our results, the essential oil from M. Piperita and its major volatiles exerted a preservative effect on these metabolites. Our findings demonstrated that the essential oil of M. Piperita and its major volatiles represent an ecological alternative for the control of fusariosis caused by F. sambucinum in cera peppers under postharvest conditions.

Does co-inoculation of mycorrhiza and Piriformospora indica fungi enhance the efficiency of chlorophyll fluorescence and essential oil composition in peppermint under irrigation with saline water from the Caspian Sea?

Symbiotic associations with endophytic fungi are ecologically important for medicinal and aromatic plants. Endophytic fungi highly affect the quantity and quality of herbal products. In this study, a pot experiment was carried out in the greenhouse to investigate the interactive effects of Piriformospora indica and arbuscular mycorrhizal (AMF) inoculation on the chlorophyll fluorescence, essential oil composition, and antioxidant enzymes of peppermint under saline condition. The results showed that Fo, YNPQ, YNO, and NPQ values were obviously increased under salinity conditions, while essential oil content, chlorophyll a and b, gs, Fm, Fv, ETR, ФPSII and Fv/Fm ratio decreased by increasing salinity. In addition, salt induced the excess Na+ uptake, whereas the opposite trend was observed for P and K+. The synergistic association of P. indica and AMF caused a considerable increase in the antioxidant ability, essential oil content, Fv/Fm ratio, ФPSII, and amount of P and K+ uptake in salt-stressed plants. The main peppermint oil constituents, menthol, menthone, and 1,8-cineole increased considerably in inoculated plants. Besides, the applied endophytic fungi positively enhanced the ability of peppermint to alleviate the negative effect of the salinity stress.

A prospective randomized controlled trial comparing simethicone, N-acetylcysteine, sodium bicarbonate and peppermint for visualization in upper gastrointestinal endoscopy.

OBJECTIVES: Early cancer detection is crucial in improving the patients' quality of life and upper gastrointestinal endoscopy (EGD) plays a key role in this detection. Many clearing mechanisms may be applied to create good endoscopic visualizations for the upper gastrointestinal tract using mucolytic agents, antifoaming agents, proteolytic enzymes and neutralizers. The aim of this study is to compare the effects of simethicone, N-acetylcysteine (NAC), sodium bicarbonate and peppermint as pre-medications for visualization of esophagogastroduodenoscopy (EGD). METHODS: This study was a single center prospective randomized controlled trial. The patients were randomly allocated to one of four treatment groups. Group A: water; Group B: water with simethicone; Group C: water with simethicone plus NAC 600 mg; Group D: water with simethicone, NAC, sodium bicarbonate and peppermint. RESULTS: A total of 128 patients were enrolled and evaluated in this study. Total visibility score (TVS) of Groups A, B, C, and D were 13.4 ± 1.86, 10.5 ± 1.45, 7.15 ± 0.98 and 6.4 ± 1.43, respectively. Group D showed lower TVS than other groups. The procedural durations of Groups C and D were significantly shorter than Group A. The volume of solution for mucosal cleansing of Groups C and D was significantly lower than Groups A and B. CONCLUSIONS: The application of simethicone plus NAC is safe, improves endoscopic visualization and requires a minimal amount of mucosal cleansing solution. The addition of sodium bicarbonate and peppermint further improved visualization for the upper and lower gastric body. Thai Clinical Trials Registry (TCTR) with a reference number; TCTR20190501002.

Peppermint extract inhibits protein aggregation.

The extracts of 7 herbs were screened and compared for their functional ability to inhibit the aggregation of trypsin as an appropriate model protein for in vitro fibrillation in aqueous ethanol at pH 7.0. Turbidity measurements, total phenolic content determination, aggregation kinetics, Congo red binding assay as well as transmission electron microscopy were used to analyse the inhibition of amyloid fibril formation. This correlated with the total phenolic content of the herb extracts. The peppermint extract proved to be the most potent anti-amyloidogenic agent. Results showed that the peppermint extract exerted dose-dependent inhibitory effect on trypsin fibril formation.

Antioxidant and enzyme-inhibitory activity of peppermint extracts and essential oils obtained by conventional and emerging extraction techniques.

Peppermint is widely used medicinal plant with distinguished bioactive potential, therefore, the aim of present work was to develop novel peppermint extracts with high activity by application of traditional and emerging separation techniques. Conventional hydrodistillation and microwave-assisted hydrodistillation (MWHD) were applied for recovery of essential oil (EO), while organic solvent extraction using Soxhlet apparatus, microwave-assisted and ultrasound-assisted process and supercritical fluid extraction (SFE) were applied for non-selective recovery of peppermint lipophilic extracts. Extracts were characterized in terms of terpenoids profile with special emphasis on content of major compounds (mentol, menthone, isomenthol and eucalyptol). Antioxidant activity (DPPH, ABTS, CUPRAC, FRAP, chelating and phosphomolybdenum assay) and enzyme-inhibitory assays (acetylcholinesterase, butyrylcholinesterase, tyrosinase, amylase and glucosidase inhibition) were used for screening of peppermint bioactivity. MWHD was recognized as alternative for traditional process in EO recovery, while SFE extracts were useful for green production of solvent-free peppermint extracts rich in terpenoids and other lipophilic bioactives.

A simple method to determine antioxidant status in aromatic plants subjected to drought stress.

Drought is a major environmental stress factor that affects the growth and development of plants. All plants have to maintain the reactive oxygen species within certain levels for normal cellular homeostasis by means of their antioxidant systems, which can be classified as enzymatic and non-enzymatic. Plants under drought stress generate an excess production of reactive oxygen species. At high concentrations, this can be detrimental by producing damage to the protein structures and inhibiting enzymes, as well as oxidizing macromolecules, which may eventually lead to cell death. There has been increasing attention paid to the antioxidant capacity of aromatic/medicinal plants, with a high antioxidant content having been reported in some plant extracts, such as in Mentha piperita (peppermint). Peppermint plants cultivated under drought stress also present high levels of phenolic compounds, peroxidase enzyme activity and lipid peroxidation of membranes. A simple and inexpensive laboratory class is proposed for teaching some mechanisms that plants have evolved to avoid reactive oxygen species damage. The series of lab experiments described is aimed at demonstrating the antioxidant status in aromatic plants subjected to drought stress, by measuring total phenolic compound content (non-enzymatic antioxidant compound), peroxidase activity (enzymatic antioxidant) and malondialdehyde, as convenient biomarkers for lipid peroxidation. The proposed class will be carried out by undergraduate students of the advanced biochemistry course, as part of our biology and agronomy studies. The experiment presented is intended to be used as a vehicle to emphasize the concepts that students have learned in their lectures. This lab exercise to be carried out by the students has dual goals: to apply a methodology only learned superficially on previous courses, and also to increase their understanding of how plants developed resistance mechanisms in order to tolerate drought stress.

Six Uridine-Diphosphate Glycosyltransferases Catalyze the Glycosylation of Bioactive C13-Apocarotenols.

C13-apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products that perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains poorly understood. Candidate uridine-diphosphate glycosyltransferase genes (UGTs) were selected based on their high transcript abundance in comparison with other UGTs in vegetative tissues of Nicotiana benthamiana and peppermint (Mentha × piperita), as these tissues are rich sources of apocarotenoid glucosides. Hydroxylated C13-apocarotenol substrates were produced by P450-catalyzed biotransformation and microbial/plant enzyme systems were established for the synthesis of glycosides. Natural substrates were identified by physiological aglycone libraries prepared from isolated plant glycosides. In total, we identified six UGTs that catalyze the glucosylation of C13-apocarotenols, where Glc is bound either to the cyclohexene ring or the butane side chain. MpUGT86C10 is a superior novel enzyme that catalyzes the glucosylation of allelopathic 3-hydroxy-α-damascone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionol (Blumenol C), and 3-hydroxy-7,8-dihydro-ß-ionol, whereas a germination test demonstrated the higher phytotoxic potential of a norisoprenoid glucoside in comparison to its aglycone. Glycosylation of C13-apocarotenoids has several functions in plants, including increased allelopathic activity of the aglycone, facilitating exudation by roots and allowing symbiosis with arbuscular mycorrhizal fungi. The results enable in-depth analysis of the roles of glycosylated norisoprenoid allelochemicals, the physiological functions of apocarotenoids during arbuscular mycorrhizal colonization, and the associated maintenance of carotenoid homeostasis.

Impact of Soil Rhizobacteria Inoculation and Leaf-Chewing Insect Herbivory on Mentha piperita Leaf Secondary Metabolites.

Secondary metabolites commonly play important physiological roles in plants and can be modified quantitatively and qualitatively by exposure to biotic and abiotic interactions. Plant growth promoting rhizobacteria (PGPR) and herbivory induce systemic resistance. In the present study, we analyzed the induction of secondary metabolites in peppermint plants in response to chewing insect herbivory on PGPR-inoculated Mentha piperita plants. The secondary metabolites of M. piperita plants were increased when plants were inoculated with PGPR and also exposed to caterpillar herbivory. It was found that the total essential oil yield in inoculated plants with insect damage was ~2.6-fold higher than in controls. The yield was similar to that of plants either damaged by insects or inoculated, indicating that there was no synergism. The same trend was observed for phenolic compounds. In contrast, VOC emissions were significantly higher in plants infested by insects, independent of whether they were inoculated. Insect damaged plants had 5.5 times higher monoterpene emissions than control plants, and ~ 2-fold higher emissions than on PGPR-inoculated plants without insects. To gain a better understanding of how herbivory on PGPR-inoculated plants can cause an increase in secondary metabolites of peppermint, we examined changes in plant defense hormones in inoculated plants after herbivory. We found that the combination of both treatments increased the endogenous jasmonic and salicylic acid levels to the same extent as in plants only inoculated or only insect-damaged. Because different interactions can alter the phytochemistry of plants such as M. piperita, this topic is both ecologically and economically relevant.

Production and purification of anti-tubercular and anticancer protein from Staphylococcus hominis under mild stress condition of Mentha piperita L.

The present study was investigated to purify and characterize anti-tubercular and anticancer protein from Staphylococcus hominis strain MANF2 under mild stress condition of Mentha piperita L. Initially, the in vitro anti-tubercular activity of strain MANF2 was determined against Mycobacterium tuberculosis H37Rv using luciferase reporter phage (LRP) assay which showed relative light unit reduction (RLU) of >90 %. Further, MTT test revealed promising in vitro anticancer trait of strain MANF2 against lung (A549) and colon (HT-29) cancer cell lines. Mild stress of M. piperita L. was provided to strain MANF2 at lag and log phase of its growth and the protein production was optimized statistically using central composite design of response surface methodology. Results showed enhanced protein production in the medium containing yeast extract (0.5 % w/v) and glycerol (1.5 % v/v), being supplemented with M. piperita L. (1.5 % v/v at log phase of strain MANF2. Protein was purified using standard purification techniques and showed single homogeneous band on SDS-PAGE with nominal molecular mass of 51293 Da, as confirmed by MALDI-TOF MS/MS. The N- amino acid sequencing showed homology with proline dehydrogenase (ProDH), thus, the protein was proposed to be new ProDH-like protein in S. hominis. Further, LRP test revealed concentration dependent (10-50 µg/mL) in vitro anti-tubercular properties of purified protein with significant RLU reductions of 36.8 ±â€¯0.3-78.5 ±â€¯0.4 %. The IC50 values of purified protein against A549 and HT-29 cancer cells were calculated as 42.2 and 48.4 µg/mL, respectively. In conclusion, protein purified from strain MANF2 under mild stress of M. piperita L can certainly be implied as efficacious anti-tubercular and anticancer agents in future.

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.

Changes in Ecophysiology, Osmolytes, and Secondary Metabolites of the Medicinal Plants of Mentha piperita and Catharanthus roseus Subjected to Drought and Heat Stress.

Global warming contributes to higher temperatures and reduces rainfall for most areas worldwide. The concurrent incidence of extreme temperature and water shortage lead to temperature stress damage in plants. Seeking to imitate a more natural field situation and to figure out responses of specific stresses with regard to their combination, we investigated physiological, biochemical, and metabolomic variations following drought and heat stress imposition (alone and combined) and recovery, using Mentha piperita and Catharanthus roseus plants. Plants were exposed to drought and/or heat stress (35 °C) for seven and fourteen days. Plant height and weight (both fresh and dry weight) were significantly decreased by stress, and the effects more pronounced with a combined heat and drought treatment. Drought and/or heat stress triggered the accumulation of osmolytes (proline, sugars, glycine betaine, and sugar alcohols including inositol and mannitol), with maximum accumulation in response to the combined stress. Total phenol, flavonoid, and saponin contents decreased in response to drought and/or heat stress at seven and fourteen days; however, levels of other secondary metabolites, including tannins, terpenoids, and alkaloids, increased under stress in both plants, with maximal accumulation under the combined heat/drought stress. Extracts from leaves of both species significantly inhibited the growth of pathogenic fungi and bacteria, as well as two human cancer cell lines. Drought and heat stress significantly reduced the antimicrobial and anticancer activities of plants. The increased accumulation of secondary metabolites observed in response to drought and/or heat stress suggests that imposition of abiotic stress may be a strategy for increasing the content of the therapeutic secondary metabolites associated with these plants.

Radiation-mediated molecular weight reduction and structural modification in carrageenan potentiates improved photosynthesis and secondary metabolism in peppermint (Mentha piperita L.).

In an attempt to gain insights into the possible relationship between the irradiation-mediated molecular weight reduction and structural modification and the growth-promotion activity, characterization of the polysaccharide before and after irradiation was carried out through Fourier Transform Infrared (FT-IR), Ultraviolet-visible (UV-vis) and Nuclear Magnetic Resonance (NMR) spectroscopic studies. Moreover, graded concentrations of irradiated carrageenan (IC) were applied through foliage to assess the performance of peppermint (Mentha piperita L.). Among the various concentrations of IC [0 (control), un-irradiated carrageenan (UC), 40, 80, 120, 160 and 200 mg L-1], the effect of 80 mg L-1 IC established to be most favorable for most of the parameters studied. Rubisco and phenylalanine ammonia lyase activities were maximally enhanced by 65.9% and 35.6% by the application of 80 mg L-1 IC, respectively; as compared to the control and UC. A maximum enrichment in the content (32.8%) and yield (88.3%) of essential oil was noted by the application of 80 mg L-1 IC, respectively. Results of the gas chromatography revealed that the contents of menthol and 1, 8-cineole were increased; however, menthone and menthyl-acetate contents were decreased by the application of IC over the control and UC.

Effect of Heat Stress on Yield, Monoterpene Content and Antibacterial Activity of Essential Oils of Mentha x piperita var. Mitcham and Mentha arvensis var. piperascens.

Heat stress affects the yield of medicinal plants and can reduce biomass and/or metabolite production. In order to evaluate the effect of heat-induced stress on the essential oil production in Mentha x piperita L. var. Mitcham (Mitcham mint) and Mentha arvensis var. piperascens Malinv. ex L. H. Bailey (Japanese mint), we studied the chemical composition of the oils of the two mint species under different heat shock stresses in growth chambers. The antibacterial activity of the essential oils was also evaluated; microscopic observation (fluorescence and electron transmission) was used to assess the effect of the tested samples on bacterial growth. The results obtained shed light on the mint essential oils composition and biological activity in relation to heat stress.

Elevated CO2 induces a global metabolic change in basil (Ocimum basilicum L.) and peppermint (Mentha piperita L.) and improves their biological activity.

Many studies have discussed the influence of elevated carbon dioxide (eCO2) on modeling and crop plants. However, much less effort has been dedicated to herbal plants. In this study, a robust monitoring for the levels of 94 primary and secondary metabolites and minerals in two medicinal herbs, basil (Ocimum basilicum L.) and peppermint (Mentha piperita L.), grwon under both ambient (aCO2, 360 ppm) and eCO2 (620 ppm) was performed. We also assessed how the changes in herbal tissue chemistry affected their biological activity. Elevated CO2 significantly increased herbal biomass, improved the rates of photosynthesis and dark respiration, and altered the tissue chemistry. Principal Component Analysis of the full data set revealed that eCO2 induced a global change in the metabolomes of the two plants. Moreover, Hierarchical Clustering Analyses showed quantitative differences in the metabolic profiles of the two plants and in their responsiveness to eCO2. Out of 94 metabolites, 38 and 31 significantly increased in basil and peppermint, respectively, as affected by eCO2. Regardless of the plant species, the levels of non-structural carbohydrates, fumarate, glutamine, glutathione, ascorbate, phylloquinone (vitamin K1), anthocyanins and a majority of flavonoids and minerals were significantly improved by eCO2. However, some metabolites tended to show species specificity. Interestingly, eCO2 caused enhancement in antioxidant, antiprotozoal, anti-bacterial and anticancer (against urinary bladder carcinoma; T24P) activities in both plants, which was consequent with improvement in the levels of antioxidant metabolites such as glutathione, ascorbate and flavonoids. Therefore, this study suggests that the metabolic changes triggered by eCO2 in the target herbal plants improved their biological activities.

Bioenergetics of Monoterpenoid Essential Oil Biosynthesis in Nonphotosynthetic Glandular Trichomes.

The commercially important essential oils of peppermint (Mentha × piperita) and its relatives in the mint family (Lamiaceae) are accumulated in specialized anatomical structures called glandular trichomes (GTs). A genome-scale stoichiometric model of secretory phase metabolism in peppermint GTs was constructed based on current biochemical and physiological knowledge. Fluxes through the network were predicted based on metabolomic and transcriptomic data. Using simulated reaction deletions, this model predicted that two processes, the regeneration of ATP and ferredoxin (in its reduced form), exert substantial control over flux toward monoterpenes. Follow-up biochemical assays with isolated GTs indicated that oxidative phosphorylation and ethanolic fermentation were active and that cooperation to provide ATP depended on the concentration of the carbon source. We also report that GTs with high flux toward monoterpenes express, at very high levels, genes coding for a unique pair of ferredoxin and ferredoxin-NADP+ reductase isoforms. This study provides, to our knowledge, the first evidence of how bioenergetic processes determine flux through monoterpene biosynthesis in GTs.

PEG-induced osmotic stress in Mentha x piperita L.: Structural features and metabolic responses.

The present study investigated whether osmotic stress induced by the exposure of peppermint (Mentha x piperita L.) to moderate and severe stress for short periods of time changes the plant's physiological parameters, leaf anatomy and ultrastructure and essential oil. Plants were exposed to two levels of polyethyleneglycol (50 g L(-1) and 100 g L(-1) of PEG) in a hydroponic experiment. The plants exposed to 50 g L(-1) maintained metabolic functions similar to those of the control group (0 g L(-1)) without changes in gas exchange or structural characteristics. The increase in antioxidant enzyme activity reduced the presence of free radicals and protected membranes, including chloroplasts and mitochondria. In contrast, the osmotic stress caused by 100 g L(-1) of PEG inhibited leaf gas exchange, reduced the essential oil content and changed the oil composition, including a decrease in menthone and an increase in menthofuran. These plants also showed an increase in peroxidase activity, but this increase was not sufficient to decrease the lipid peroxidation level responsible for damaging the membranes of organelles. Morphological changes were correlated with the evaluated physiological features: plants exposed to 100 g L(-1) of PEG showed areas with collapsed cells, increases in mesophyll thickness and the area of the intercellular space, cuticle shrinkage, morphological changes in plastids, and lysis of mitochondria. In summary, our results revealed that PEG-induced osmotic stress in M. x piperita depends on the intensity level of the osmotic stress applied; severe osmotic stress changed the structural characteristics, caused damage at the cellular level, and reduced the essential oil content and quality.