Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.)

Abstract

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ∼ 1800). Intact secretory cell clusters very efficiently incorporated [(3)H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [(3)H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD(+), ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [(14)C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.

Developmental Regulation of Monoterpene Biosynthesis in the Glandular Trichomes of Peppermint

“…This lack of information on metabolic controls is a serious impediment to the development of strategies for bioengineering of essential oil composition and yield (McCaskill and Croteau, 1997;Haudenschild and Croteau, 1998;Lange and Croteau, 1999b). The development of methods for the selective isolation from mint species of oil gland cells, in which the biosynthesis of essential oil terpenes specifically occurs (Gershenzon et al, 1989;McCaskill et al, 1992), has provided the means for verifying the target biosynthetic pathways, for isolating and purifying the corresponding enzymes, and for constructing highly enriched cDNA libraries from this very specialized tissue. This advance has led to the cloning of several monoterpene biosynthetic genes and has provided, for the first time to our knowledge, DNA probes and polyclonal antibodies as tools for examining the regulation of monoterpene biosynthesis at the molecular level.…”

“…Since monoterpene biosynthesis in peppermint is confined to the secretory cells of glandular trichomes (Gershenzon et al, 1989;McCaskill et al, 1992), to obtain extracts enriched in monoterpene biosynthetic enzymes, purified preparations of secretory cells were isolated by modification of a previously described surface abrasion protocol . Freshly harvested leaves were soaked in deionized water for 1 h at 4°C, and their surfaces abraded using a cell disrupter (Bead-Beater, Biospec Products, Bartlesville, OK) filled with 20 g of plant material, 120 g of 0.5-mm-diameter glass beads, 20 g of XAD-4 polystyrene resin beads, and a buffer comprised of 25 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, containing 200 mm sorbitol, 2 mm Suc, 10 mm KCl, 5 mm MgCl 2 , 5 mm dithiothreitol, 0.5 mm KH 2 PO 4 , 0.1 mm Na 2 P 2 O 7 , 1% (w/v) polyvinylpyrrolidone (M r ϭ 40,000), and 0.6% (w/v) methyl cellulose.…”

“…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).…”

Mathematical Modeling-Guided Evaluation of Biochemical, Developmental, Environmental, and Genotypic Determinants of Essential Oil Composition and Yield in Peppermint Leaves

“…Peppermint oil consists primarily of p-menthane-type monoterpenes, with smaller amounts of other monoterpenes and very minor quantities of sesquiterpenes (Rohloff, 1999). The essential oil is synthesized and accumulated in specialized anatomical structures called peltate glandular trichomes (Gershenzon et al, 1989;McCaskill et al, 1992). These trichomes contain secretory cells, arranged in an eight-celled disc, which are responsible for the synthesis of the oil.…”

Avaliação sazonal do rendimento de óleo essencial em espécies de menta

“…Em Mentha, foi demonstrado que a biossíntese de óleos essenciais ocorre especificamente nos tricomas glandulares peltados, distribuídos ao longo da superfície foliar (GERSHENZON et al, 1989;MCCASKILL et al, 1992). A produção e a distribuição dos tricomas na folha não é uniforme (TURNER et al, 2000) e, embora ocorra em ambas as superfícies, apresenta geralmente maior densidade na superfície abaxial (MARTINS & MAJER, 1998).…”