Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Next generation sequencing unravels the biosynthetic ability of spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics.

BACKGROUND: Plant glandular trichomes are chemical factories with specialized metabolic capabilities to produce diverse compounds. Aromatic mint plants produce valuable essential oil in specialised glandular trichomes known as peltate glandular trichomes (PGT). Here, we performed next generation transcriptome sequencing of different tissues of Mentha spicata (spearmint) to identify differentially expressed transcripts specific to PGT. Our results provide a comprehensive overview of PGT's dynamic metabolic activities which will help towards pathway engineering. RESULTS: Spearmint RNAs from 3 different tissues: PGT, leaf and leaf stripped of PGTs (leaf-PGT) were sequenced by Illumina paired end sequencing. The sequences were assembled de novo into 40,587 non-redundant unigenes; spanning a total of 101 Mb. Functions could be assigned to 27,025 (67%) unigenes and among these 3,919 unigenes were differentially expressed in PGT relative to leaf - PGT. Lack of photosynthetic transcripts in PGT transcriptome indicated the high levels of purity of isolated PGT, as mint PGT are non-photosynthetic. A significant number of these unigenes remained unannotated or encoded hypothetical proteins. We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT. Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases. CONCLUSIONS: The extensive transcriptome data set renders a complete description of genes differentially expressed in spearmint PGT. This will facilitate the metabolic engineering of mint terpene pathway to increase yield and also enable the development of strategies for sustainable production of novel or altered valuable compounds in mint.

Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential.

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (-)-carvone and (-)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

Spearmint plantlet culture system as a means to study secondary metabolism.

Spearmint has one major monoterpene, (-)-carvone, that constitutes up to 90% of all the monoterpenes present. Likewise, the major phenylpropanoid-rosmarinic acid-in spearmint accounts for up to 70% of the phenylpropanoids produced from the plant. These two compounds are each produced by separate distinct biosynthetic pathways which provide an excellent opportunity to study the influence of a wide number of environmental and chemical conditions on secondary metabolism and plant growth. The techniques presented in this chapter employ 1 g of fresh weight material for each secondary metabolite analyses. Analysis of single compounds obtained from the two distinct metabolic pathways simplifies the interpretation of the metabolic results allowing for direct correlations of culture factors on secondary metabolism.

The combined and single effect of salinity and copper stress on growth and quality of Mentha spicata plants.

Copper is essential for plant growth, but in excess may cause adverse effects on plant physiology. Harmful effects are also caused by plant exposure to salinity (NaCl) due to the excessive use of fertilizers, soil degradation and/or the quality of the water used for irrigation. The impact of single and combined salinity (Sal) and copper (Cu) stress on spearmint metabolism were studied in hydroponics. Spearmint plants (Mentha spicata L.) were subjected to salinity stress (150 mM NaCl) and/or excessive Cu concentration (60 µM Cu) via the nutrient solution. Not only Sal and Cu, but also their combination suppressed plant growth by decreasing plant biomass, root fresh weight and plant height. Chlorophyll content decreased mainly for the combined stress treatment (Sal + Cu). Polyphenols and antioxidants (FRAP, DPPH, ABTS) increased in single stress treatments (Sal or Cu), but decreased in the combined stress (Sal + Cu). The application of Sal or Cu stress decreased Zn, N and K (leaves), K, Ca, P and Mg (roots) content. Copper application increased Ca and Mg in leaves. In conclusion, salinity stress and Cu exposure may change the primary metabolic pathways in favor of major volatile oil components biosynthesis, resulting in significant changes of essential oil yield and composition.

Physiological and biochemical attributes of Mentha spicata when subjected to saline conditions and cation foliar application.

Marginal water, including saline water, has been proposed as an alternative source of irrigation water for partially covering plant water requirements due to scarcity of adequate water supply in hot arid and semi-arid areas, such as those usually found in the Mediterranean basin. In the present study, spearmint plants (Mentha spicata L.) were grown in a deep flow hydroponic system under saline conditions, namely 0, 25, 50, and 100 mM NaCl. Moreover, foliar application of specific cations (K, Zn, Si) was tested as a means for alleviation of salinity stress under a plant physiological and biochemical approach. The results indicated that the highest salinity level of 100 mM NaCl severely affected plant growth, photosynthetic rates, leaf stomatal conductance, content of total phenolics and antioxidant status, while low to moderate salinity levels (25-50 mM NaCl) did not significantly affect plant growth and biochemical functions. In addition, leaf potassium and calcium accumulation decreased in saline-treated plants. Cations foliar application had small to no effect on plant growth, although it increased antioxidant activity and detoxified oxidative stress products/effects, through the increased enzymatic activities and proline accumulation. The present results have demonstrated that spearmint could be considered as a salinity tolerant species which is able to grow successfully under moderate salinity levels, while cation enrichment through foliar sprays was proved as a useful means to alleviate the stress effects caused by high salinity.

Treatment of chronic pain by designer cells controlled by spearmint aromatherapy.

Current treatment options for chronic pain are often associated with dose-limiting toxicities, or lead to drug tolerance or addiction. Here, we describe a pain management strategy, based on cell-engineering principles and inspired by synthetic biology, consisting of microencapsulated human designer cells that produce huwentoxin-IV (a safe and potent analgesic peptide that selectively inhibits the pain-triggering voltage-gated sodium channel NaV1.7) in response to volatile spearmint aroma and in a dose-dependent manner. Spearmint sensitivity was achieved by ectopic expression of the R-carvone-responsive olfactory receptor OR1A1 rewired via an artificial G-protein deflector to induce the expression of a secretion-engineered and stabilized huwentoxin-IV variant. In a model of chronic inflammatory and neuropathic pain, mice bearing the designer cells showed reduced pain-associated behaviour on oral intake or inhalation-based intake of spearmint essential oil, and absence of cardiovascular, immunogenic and behavioural side effects. Our proof-of-principle findings indicate that therapies based on engineered cells can achieve robust, tunable and on-demand analgesia for the long-term management of chronic pain.

Structural re-arrangement of depolymerized sodium alginate enriches peltate glandular trichomes and essential oil production of spearmint.

Over the past decade, radiation-degraded polysaccharides have been used as regulators of growth and development in several crop plants. In quest of the possible reasons of previously established growth-promotion activity of irradiated sodium alginate (ISA), structural parameters of irradiated and un-irradiated sodium alginate were analysed using Ultraviolet-visible spectroscopy (UV-vis) and Fourier Transform Infrared spectroscopic (FT-IR) studies to develop an understanding of structure-property relationship. Using foliar application, response to graded concentrations of ISA was tested in terms of yield and quality attributes of spearmint (Mentha spicata L.). Among different concentrations of ISA [0 (control), 40, 80, 120 and 160mgL-1], 80mgL-1 proved to be the optimum foliar-spray treatment for most of the parameters studied including peltate glandular-trichomes density, which was increased from 20 to 44mm-2. Measurements made at 150days after planting revealed that foliar application of ISA at 80mgL-1 increased the content and yield of spearmint essential oil (EO) by 36.0 and 122.6%, respectively, in comparison to the control. Compared to the control, gas chromatography mass spectrometry (GC-MS) analysis revealed an increase of 18.7% in the carvone content and a decrease of 15.7% in limonene content of the spearmint EO.

Metal absorption properties of Mentha spicata grown under tannery sludge amended soil-its effect on antioxidant system and oil quality.

Tannery sludge (TS) is hazardous to environment and its disposal in an ecofriendly manner is a major challenge. An experiment was conducted to investigate the metal absorption properties of Mentha spicata grown under different levels of TS amended soil (soil: sludge in 100:0, 75:25, 50:50, 25:75 and 0:100 ratio) and its effect on the antioxidant system and oil quality. At 75:25 ratio of sludge and soil, metal translocation factor was ≥0.5 for Cr, Cd, and Co and for Ni and for Pb ≥ 1. Carvone, limonene, dihydrocarvone and other oil constituents along with biomass were maximum in 75:25 ratio of sludge and soil. Superoxide dismutase (SOD), CAT (Catalases), POD (Peroxidases), MDA (Malondialdehyde) and proline play a major role in detoxification of reactive oxygen species generated due to TS (heavy metal stress). Antioxidant (SOD, CAT and POD), MDA and proline showed an increasing trend as the concentration of TS increased with the treatments. To test the relationship between 23 character principal component analysis (PCA) was performed. PC-I contributed 56% of total variance while PC-II contributed 37% of total variance. The results concluded that M. spicata performed well in terms of oil yield and multiple metal translocations in 75:25 sludge and soil ratio.

Generation and Functional Evaluation of Designer Monoterpene Synthases.

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes.

Ultrasound-assisted extraction coupled with under vacuum distillation of flavour compounds from spearmint (carvone-rich) plants: Comparison with conventional hydrodistillation.

Ultrasonically assisted extraction of flavour compounds from different varieties of Mentha spicata, using 70% ethanol, have been carried out for 5, 10 and 15min and coupled with under vacuum distillation. The ultrasound distilled extracts have been analysed by GC-MS and compared with essential oils obtained by hydrodistillation. The results have showed that ultrasonically assisted extraction in combination with under vacuum distillation have provided extracts with higher flavouring strength due to the increased concentration of desirable oxygenated compounds (from 5 to 8 times) compared with hydrodistillation. Extraction yields of flavour volatiles have been calculated giving a range 0.04-0.13% by ultrasound and 0.01-0.02% by hydrodistillation.

Characterization of the harmful effect of olive mill wastewater on spearmint.

In this study, changes in viability, biomass production, essential oil yield and essential oil composition of Mentha spicata L. (spearmint) exposed to olive mill wastewater (OMW) were investigated. Spearmint cuttings were sensitive to OMW and, after 6h of incubation in raw or diluted OMW, their viability was null. The short contact of raw OMW with mint cuttings caused an irreversible damage in rhizogenesis and shoots development. Roots were more sensitive to phytotoxicity than shoots. In a field essay, spearmint showed a good capability to recover when OMW was spread at 8 l m(-2) at the vegetative phase of growth (45 days after plantation). At this dose, a slight increase of mostly of the mint essential oil constituents was obtained. When the dose applied was 16 l m(-2), phytotoxicity was manifested by a high reduction of biomass and essential oil yield. The essential oil composition was also affected and a disappearance of many of mint essential oil constituents was observed with an increase of 59% for carvone, the major compound of spearmint essential oil. As far as we know, this is the first report on the effect of field application of OMW on an aromatic plant essential oil yield and composition.

Influence of autoclaved fungal materials on spearmint (Mentha spicata L.) growth, morphogenesis, and secondary metabolism.

The influence of autoclaved fungal materials such as culture filtrate, freeze-dried mycelium (FDM), mycelium suspension, and spore suspension (SS) on the growth, morphogenesis, and carvone production of spearmint (Mentha spicata L.) plants was studied. Fungal materials were either applied as a drench or spray on the plants. Spearmint plants (cv. "294099") drenched with SS (1 x 10(8) spores/ml) of Trichoderma reesei showed no significant differences in leaf numbers, root numbers, or shoot numbers compared with nontreated controls. However, significantly higher fresh weights and carvone levels were observed in plants drenched with T. reesei SS compared with the untreated controls. Fungal materials derived from Aspergillus sp., Fusarium graminearum, F. sporotrichoides, Penicillium sp., P. acculeatum, Rhizopus oryzae, and T. reesei were sprayed on spearmint foliage. F. graminearum, F. sporotrichoides, or R. oryzae elicited no enhanced growth, morphogenesis, or secondary metabolism responses. The best growth and morphogenesis responses were obtained employing Aspergillus sp., Penicillium sp., or T. reesei foliar sprays. For example, spearmint cv. "557807" plants sprayed with 100 mg/l FDM T. reesei isolate NRRL 11460 C30 stimulated higher fresh weights (75%), shoot numbers (39%), leaf numbers (57%), and root numbers (108%) compared with untreated plants. This effect was not dose-dependent because similar growth and morphogenesis responses were obtained by testing 10, 100, or 1000 mg/l FDM concentrations. Carvone levels in fungal-treated foliar-sprayed plants were comparable to nontreated controls. However, total carvone levels per plant were higher in fungal-treated plants because of their increased fresh weight.

Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco.

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.