Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis

Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.     

Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants

Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and –pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods. Received: 22 November 1998 / Accepted: 4 Januar 1999     

Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells

Circumstantial evidence based on ultrastructural correlation, specific labeling, and subcellular fractionation studies indicates that at least the early steps of monoterpene biosynthesis occur in plastids. (4S)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised to the native enzyme demonstrated the specific localization of limonene synthase to the leucoplasts of peppermint (Mentha × piperita) oil gland secretory cells during the period of essential oil production. Labeling was shown to be absent from all other plastid types examined, including the basal and stalk cell plastids of the secretory phase glandular trichomes. Furthermore, in vitro translation of the preprotein and import experiments with isolated pea chloroplasts were consistent in demonstrating import of the nascent protein to the plastid stroma and proteolytic processing to the mature enzyme at this site. These experiments confirm that the leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps of monoterpene biosynthesis, in peppermint leaves. However, succeeding steps of monoterpene metabolism in mint appear to occur outside the leucoplasts of oil gland cells.     

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.     

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.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

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.     

A candidate cDNA clone for (−)-limonene-7-hydroxylase from Perilla frutescens

Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (−)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (−)-limonene. The perilla cDNA was a partial clone of 1550 bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively.     

Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation

Arabidopsis thaliana is frequently used as a model for the study of oilseed biology and metabolism. However, the very small seeds of Arabidopsis can complicate analysis of their oil content and influence the application of results to larger-seeded plants. Here, we describe how seed anatomy, light, and plant-to-plant variation influence the content and measurement of oil in Arabidopsis seeds. The anatomy of Arabidopsis and Brassica napus seeds were compared and the distribution of mass, oil and the fatty acid composition of different seed parts were determined. In Brassica, 90% of the seed oil resides in the cotyledons that contribute 74% of seed mass. By contrast, the values for Arabidopsis are 60% and 45%, respectively, with a higher fraction of the oil deposited in the radicle, hypocotyl, endosperm and seed coat. Growth of Arabidopsis plants with 600 micromol m(-2) s(-1) light resulted in a two-fold higher seed yield, a 40% increase in mass per seed and a 60% increase in oil per seed compared to growth at 100 micromol m(-2) s(-1). Factors that influence the analysis of oil content were evaluated. Intact-seed transmethylation followed by gas chromatography (GC) analysis provided reproducible analysis of Arabidopsis seed oil. However, plant-to-plant variation in oil content is large and we analyzed how this influences the ability to detect statistically valid changes in oil between different genotypes. These observations establish a reference data set on the fatty acid composition and distribution of mass and oil between tissues of Arabidopsis seeds that should help to predict the applicability of results obtained with Arabidopsis to other oilseeds.     

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-delta-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.     

Plant growth promoting rhizobacteria improve growth and essential oil yield in Origanum majorana L.

Effects of root colonization by plant growth promoting rhizobacteria (PGPR) on biomass, and qualitative and quantitative composition of essential oils, were determined in the aromatic crop Origanum majorana L. (sweet marjoram). PGPR strains evaluated were Pseudomonas fluorescens, Bacillus subtilis, Sinorhizobium meliloti, and Bradyrhizobium sp. Only P. fluorescens and Bradyrhizobium sp. showed significant increases in shoot length, shoot weight, number of leaf, number of node, and root dry weight, in comparison to control plants or plants treated with other PGPR. Essential oil yield was also significantly increased relative to non-inoculated plants, without alteration of oil composition. P. fluorescens has clear commercial potential for economic cultivation of O. majorana.     

Plant growth‐promoting effects of native Pseudomonas strains on Mentha piperita (peppermint): an in vitro study

Plant growth‐promoting rhizobacteria (PGPR) affect growth of host plants through various direct and indirect mechanisms. Three native PGPR (Pseudomonas putida) strains isolated from rhizospheric soil of a Mentha piperita (peppermint) crop field near Córdoba, Argentina, were characterised and screened in vitro for plant growth‐promoting characteristics, such as indole‐3‐acetic acid (IAA) production, phosphate solubilisation and siderophore production, effects of direct inoculation on plant growth parameters (shoot fresh weight, root dry weight, leaf number, node number) and accumulation and composition of essential oils. Each of the three native strains was capable of phosphate solubilisation and IAA production. Only strain SJ04 produced siderophores. Plants directly inoculated with the native PGPR strains showed increased shoot fresh weight, glandular trichome number, ramification number and root dry weight in comparison with controls. The inoculated plants had increased essential oil yield (without alteration of essential oil composition) and biosynthesis of major essential oil components. Native strains of P. putida and other PGPR have clear potential as bio‐inoculants for improving productivity of aromatic crop plants. There have been no comparative studies on the role of inoculation with native strains on plant growth and secondary metabolite production (specially monoterpenes). Native bacterial isolates are generally preferable for inoculation of crop plants because they are already adapted to the environment and have a competitive advantage over non‐native strains.     

Essential oil composition of nine Apiaceae species from western United States that attract the female Indra Swallowtail butterfly (Papilio indra)

Aletes acaulis, Cymopterus hendersonii, Cymopterus panamintensis var. acutifolius, Lomatium rigidum, Lomatium scabrum var. tripinnatum, Musineon tenuifolium, Sphenosciadium capitellatum, Tauschia arguta and Tauschia parishii are among the twenty-two species of the Apiaceae family to which female Indra Swallowtail butterflies (Papilio indra: Lepidoptera) are attracted for oviposition. Because plant volatile oils are known to be attractants for female butterflies, the percent composition of the essential oils of each species was studied. Amongst the nine host plants 168 essential oil components were identified representing between 84% and 99% of the oils. Principal Components Analysis and hierarchical cluster analysis on the essential oil compositions of the larval host plants against four non-larval host plants separated the hosts from the non-hosts into distinct clusters. Volatile components of the oils common to the nine species of Apiaceae are correlated with the expression of physiological attraction behavior by the butterfly.     

Chromosome doubling influences the morphological,physiological, biochemical and genetic traits related to essential oil biosynthesis of peppermint (Mentha piperita) under salinity stress

Peppermint (Mentha piperita L.) is an important medicinal aromatic plant. In this study, the morphology, physiology, biochemistry and gene expression of chromosomes doubling peppermint (D1 lines) were analyzed. The analysis showed that D1 lines had larger, thicker and darker leaves, and stronger roots when planted in the pots, but delayed growth in the field condition. Under NaCl stress, the D1 lines increased cell oxidative defense through more active antioxidant enzymes and decreased the oxidative damages of cell membrane, leading to a significantly greater survival rate and photosynthesis intensity than WT lines. The size and density of glandular trichomes of D1 lines was larger, which contributed to its higher essential oil yield. In addition, chromosome doubling reduced the inhibition of NaCl stress on essential oil yield and quality, through changing the expression of genes in the oil biosynthesis pathway. The traits of chromosome doubling peppermint provide new technical and theoretical evidence for peppermint germplasm improvement.

     

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.