Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells1

Abstract

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...

Developmental Regulation of Monoterpene Biosynthesis in the Glandular Trichomes of Peppermint

“…The rate of monoterpene biosynthesis determined by 14 CO 2 pulse labeling (Gershenzon et al, 1999) has been shown to rise rapidly in newly emerging leaves, reaching a sharp peak between 12 and 20 d, and then rapidly declining as full leaf expansion is reached (Fig. 2A).…”

“…This pattern of development in monoterpene biosynthetic capability and accumulation is entirely consistent with the population dynamics of the peltate oil glands, which pass through the one-, two-, four-, and eight-celled stages and complete oil secretion prior to full leaf expansion (G. Turner, J. Gershenzon, and R. Croteau, unpublished data). Since neither monoterpene catabolism (Mihaliak et al, 1991) nor monoterpene volatilization contribute significantly to alterations in the level of stored product (Gershenzon et al, 1999), the pattern of monoterpene accumulation in peppermint glandular trichomes appears to chiefly reflect the rate of biosynthesis.…”

“…The relative levels of the different enzyme activities readily rationalize the mature oil composition observed (Gershenzon et al, 1999). Thus, relatively low levels of the early pathway steps (geranyl diphosphate synthase, (Ϫ)-limonene synthase, Cyt P450, (Ϫ)-limonene-3-hydroxylase), coupled to much higher levels of the central pathway steps [(Ϫ)-trans-isopiperitenol dehydrogenase, (Ϫ)-isopiperitenone reductase, (ϩ)-cis-isopulegone isomerase] and somewhat lower levels of the final steps (pulegone reductase, menthone reductase) combine to yield only very low levels Developmental Regulation of Monoterpene Biosynthesisof pathway intermediates [a few percent each of (Ϫ)-limonene and (ϩ)-pulegone, with all other intermediates at trace levels] and result in the accumulation of (Ϫ)-menthone and (Ϫ)-menthol as the principal products.…”

“…Peppermint has been developed as a model system for the study of monoterpene metabolism because of the commercial value of the essential oil, the fact that the plant is clonal and easily propagated vegetatively, and because the oil is chemically complex and the biosynthetic pathway involves essentially all of the representative reaction types of terpenoid metabolism (Croteau and Gershenzon, 1994). Monoterpene biosynthesis and accumulation in mint is specifically localized to the glandular trichomes (Gershenzon et al, 1989;McCaskill et al, 1992), and the pathway originates in the plastids (leucoplasts) of the secretory cells of these highly specialized, nonphotosynthetic glandular structures (Turner et al, 1999). The monoterpene family of natural products therefore is derived from the plastidial, mevalonate-independent pathway for isoprenoid metabolism (Eisenreich et al, 1997;Sagner et al, 1998), which provides isopentenyl diphosphate (and, by isomerization, dimethylallyl diphosphate) as the universal precursors of the terpenoids (Lichtenthaler et al, 1997;Eisenreich et al, 1998;McCaskill and Croteau, 1999).…”

Morphology and histochemistry of glandular trichomes in Hyptis villosa Pohl ex Benth. (Lamiaceae) and differential labeling of cytoskeletal elements

“…Total lipids and terpenes were abundantly stained by the histochemical tests in the morphotype I, mainly in the head cells. In fact, the observation of several plastids lacking thylakoids in the head cells is associated with the production of oils (Fahn 1979;Rodrigues & Machado 2012); in addition, the presence of tubular inclusions in these plastids is common reported in cells secreting monoterpenes (Turner et al 1999). In general way, lipids are released from the secretory cells via ecrine (Evert 2006), cross the cell wall driven by the mechanical pressure exercised by the protoplast (Paiva 2016) and accumulate in the subcuticular space; oil material can cross the cuticle and reach the outer side of the cells without requiring rupture of pore formation in the cuticle.…”