Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells

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 x 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.

Figure 1

Schematic diagram of a peppermint leaf peltate glandular trichome illustrating the placement of these epidermal structures and the relationship of the disc of secretory cells to the stalk and basal cells and to the subcuticular storage space.

Figure 2

Pathway for the conversion of C₅ isoprenoid units via geranyl diphosphate and limonene to the principal essential oil components (−)-menthol (peppermint) and (−)-carvone (spearmint). The responsible enzymes are: isopentenyl diphosphate isomerase (1); geranyl diphosphate synthase (2); 4S-limonene synthase (3); 4S-limonene-3-hydroxylase (4); and 4S-limonene-6-hydroxylase (5). The broken arrow indicates five enzymatic steps. OPP, Diphosphate moiety.

Figure 3

A, Immunogold labeling against limonene synthase. Colloidal gold particles strongly label leucoplasts (P) of a secretory stage glandular cap cell, while cytoplasm and other organelles are essentially unlabeled. Bar = 2 μm. B, High magnification of a leucoplast from a secretory stage glandular cap cell showing immunolabeling against limonene synthase. Bar = 1 μm. C, Preimmune serum control. Leucoplasts (P) of this secretory stage glandular cap cell are unlabeled after treatment with preimmune serum and colloidal gold labeled secondary antibodies. Bar = 1 μm. D, High magnification of a leucoplast from a secretory stage glandular cap cell showing no labeling when treated with preimmune serum and colloidal gold labeled secondary antibodies. Bar = 500 nm. E, High magnification of a plastid (P) from a stalk cell of a secretory stage peltate gland immunolabeled against limonene synthase showing very little labeling. Bar = 1 μm. F, Leucoplasts from a presecretory stage glandular cap cell immunolabeled against limonene synthase showing relatively little labeling. Bar = 1 μm. G, Leucoplasts (P) from a microwave-fixed, postsecretory-stage glandular cap cell immunolabeled against limonene synthase showing relatively little labeling. (Generally, microwave-fixed specimens showed less labeling than specimens prepared by other methods.) Bar = 1 μm. H, Plastid from a capitate glandular trichome immunolabeled against limonene synthase showing relatively little labeling. Bar = 1 μm.

Figure 4

Analysis of import and localization of limonene synthase. A, Time course of import of ³⁵S-labeled limonene synthase into pea chloroplasts. Following confirmation of binding at 0°C to 4°C in the dark in the presence of 0.1 mm Mg-ATP (data not shown), import assays were performed at room temperature in the presence of 4 mm Mg-ATP. Aliquots of the chloroplast preparation were removed at 0, 5, 10, and 20 min, sedimented through a 40% Percoll cushion, lysed, and then separated into a crude pellet (P) and a supernatant (S) fraction. All samples were analyzed by SDS-PAGE and fluorography. Tr, Unmodified limonene synthase translation product at about 72 kD. The protein band at about 65 kD is diffuse, likely due to imprecise proteolytic processing to mature forms of limonene synthase (Williams et al., 1998). B, Protease treatment and fractionation of imported limonene synthase. Limonene synthase was imported into pea chloroplasts for 20 min at room temperature as above and the intact chloroplasts, recovered by sedimentation through a 40% Percoll cushion, were incubated in the absence (−) or presence (+) of thermolysin for 30 min on ice in the dark. Proteolysis was terminated by the addition of EDTA, and the intact chloroplasts were recovered by sedimentation through a 40% Percoll cushion containing 5 mm EDTA. Chloroplasts were lysed and separated into a crude membrane fraction (P) and a soluble fraction (S). MW, Molecular-mass standards (in kilodaltons); Tr, limonene synthase translation product; SSU, positive control experiment conducted with the precursor to the SSU.