Cryofixation and conventional chemical fixation methods were employed to examine the ultrastructure of developing peltate glandular trichomes of peppermint (Mentha ϫ piperita). Our results are discussed in relation to monoterpene production and the mechanism of essential oil secretion. Peltate glands arise as epidermal protuberances (initials) that divide asymmetrically to produce a vacuolate basal cell, a stalk cell, and a cytoplasmically dense apical cell. Further divisions of the apical cell produce a peltate trichome with one basal cell, one stalk cell, and eight glandular (secretory) disc cells. Presecretory gland cells resemble meristematic cells because they contain proplastids, small vacuoles, and large nuclei. The secretory phase coincides with the separation and filling of the sub-cuticular oil storage space, the maturation of glandular disc cell leucoplasts in which monoterpene biosynthesis is known to be initiated, and the formation of extensive smooth endoplasmic reticulum at which hydroxylation steps of the monoterpene biosynthetic pathway occur. The smooth endoplasmic reticulum of the secretory cells appears to form associations with both the leucoplasts and the plasma membrane bordering the sub-cuticular oil storage cavity, often contains densely staining material, and may be involved with the transport of the monoterpene-rich secretion product. Associated changes in the ultrastructure of the secretory stage stalk cell are also described, as is the ultrastructure of the fragile post-secretory gland for which cryofixation methods are particularly well suited for the preservation of organizational integrity.

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (2)-(4S)-limonene-6-hydroxylase, peppermint (2)-trans-isopiperitenol dehydrogenase, and peppermint (1)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (2)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (2)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (1)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.Monoterpenes are a large and diverse class of volatile C 10 isoprenoids that are the major constituents of many plant essential oils and resins. These natural products play important chemoecological roles in the interactions of plants with their environments. Some monoterpenes have been implicated as allelopathic agents, and they often directly, or indirectly, protect plants from herbivores and pathogens (Pickett, 1991;Harborne, 1991;Langenheim, 1994;Wise and Croteau, 1999;Hallahan, 2000). As important constituents of floral scents, monoterpenes also function to attract pollinators (Dudareva and Pichersky, 2000). Some plants release volatile monoterpenes and sesquiterpenes in response to herbivore damage that function to attract predatory insects that in turn feed on, or parasitize, the herbivorous insects (Langenheim, 1994;Degenhardt et al., 2003). In conifer species, mechanical wounding, insect attack, or applications of methyl jasmonate can induce resin secretion and differentiation of traumatic resin ducts within wounded tissues, producing a protective barrier of resin at the site of wounding (Steele et al., 1995;Trapp and Croteau, 2001;Franceschi et al., 2002;Martin et al., 2002). Many plant species constitutively produce large quantities of terpenoid-rich resins and essential oils within specialized glandular tissues, such as glandular trichomes, secretory cavities, and secretory ducts (Fahn, 1979(Fahn, , 2000. These natural stores of plant terpenoids probably serve as deterrents to herbivorous insects, but they also provide commercially important sources of flavorings, fragrances, resins, and pharmaceuticals (Langenheim, 1994;Wise and Croteau, 1999). The glandular cells of these secretory tissues are of interest for their remarkable ability to rapidly generate substantial amounts of specific terpenoid products.Peppermint (Mentha x piperita) has been employed as a model system for the study of monoterpene biosynthesis (Wise an...

The pattern of peltate glandular trichome initiation and ontogeny on expanding peppermint (Mentha ϫ piperita) leaves was defined by surveying the populations of peltate glands in each of seven developmental stages within sampling areas of leaf apical, mid-, and basal zones for both abaxial and adaxial surfaces. It was shown that new peltate glands continue to form until leaf expansion ceases and that regions of active gland initiation are unevenly distributed. The distribution of gland initiation reflects the basipetal pattern of leaf maturation, with relatively immature regions at the leaf base continuing to produce oil glands long after gland production has stopped at the leaf apex. The proportion of glands in the secretory stage as a function of leaf development and the direct observations of living glands over a period of 33 h indicate that a period of only 20 to 30 h of secretory activity is required for filling of the gland storage compartment with essential oil. These findings are discussed in relation to earlier literature describing age-related changes in glandular essential oil content.

Two Acanthamoeba species, fed at three temperatures, expelled vesicles containing living Legionella pneumophilacells. Vesicles ranged from 2.1 to 6.4 μm in diameter and theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.

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.Peppermint (Mentha ϫ piperita), spearmint (Mentha spicata), and other essential oil plants of the Lamiaceae produce and accumulate monoterpenes in anatomically specialized glandular trichomes (Fahn, 1979). The mints bear two types of nonphotosynthetic glandular trichomes, a small capitate type with a limited capacity to store secreted material, and a peltate type containing a basal cell, a stalk cell, and eight secretory cells arranged in a disc ( Fig. 1) (Amelunxen, 1965; Amelunxen et al., 1969; Fahn, 1979). The latter type develops a large oil-storage space at the apex of the glandular trichome, where the thick cuticle separates from the secretory cells to produce a subcuticular pocket and is therefore thought to be responsible for the production of the bulk of the monoterpenoid essential oil (Amelunxen et al., 1969).Biochemical studies with isolated peppermint peltate glandular trichomes have revealed that the secretory cells are not only responsible for the secretion of monoterpenes into the oil-storage space, but also serve as the actual site of monoterpene biosynthesis (Gershenzon et al., 1992; McCaskill et al., 1992). Plant essential oil-and resin-secreting glands often share a syndrome of specialized ultrastructural features, including numerous amoeboid leucoplasts and abundant smooth ER (Schnepf, 1974; Dell and McComb, 1978; Fahn, 1988). There have been relatively few ultrastructural studies of Lamiaceae oil glands, and most of those that have been done have stressed the possible role of the extensive ER or the densely staining cytoplasm in monoterpene biosynthesis (Amelunxen, 1965; Schnepf, 1974; Bosaba...

Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.

The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint (Mentha ؋ piperita). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (؉)-pulegone and the side product (؉)-menthofuran. Our simulations indicated that the environmentally regulated changes in monoterpene profiles could only be explained when, in addition to effects on biosynthetic enzyme activities, as yet unidentified inhibitory effects of (؉)-menthofuran on the branchpoint enzyme pulegone reductase (PR) were assumed. Subsequent in vitro analyses with recombinant protein confirmed that (؉)-menthofuran acts as a weak competitive inhibitor of PR (K i ‫؍‬ 300 M). To evaluate whether the intracellular concentration of (؉)-menthofuran was high enough for PR inhibition in vivo, we isolated essential oil-synthesizing secretory cells from peppermint leaves and subjected them to steam distillations. When peppermint plants were grown under low-light conditions, (؉)-menthofuran was selectively retained in secretory cells and accumulated to very high levels (up to 20 mM), whereas under regular growth conditions, (؉)-menthofuran levels remained very low (<400 M). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways.kinetic modeling ͉ monoterpene biosynthesis ͉ isoprenoid ͉ menthofuran ͉ pulegone reductase

SummaryGlandular trichomes are anatomical structures specialized for the synthesis of secreted natural products. In this review we focus on the description of glands that accumulate terpenoid essential oils and oleoresins. We also provide an in-depth account of the current knowledge about the biosynthesis of terpenoids and secretion mechanisms in the highly specialized secretory cells of glandular trichomes, and highlight the implications for metabolic engineering efforts.