David McCaskill

Isolation of Secretory Cells from Plant Glandular Trichomes and Their Use in Biosynthetic Studies of Monoterpenes and Other Gland Products

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.

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

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 [3H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [3H]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 [14C]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.

Monoterpene and sesquiterpene biosynthesis in glandular trichomes of peppermint (Mentha x piperita) rely exclusively on plastid-derived isopentenyl diphosphate

The subcellular compartmentation of isopentenyl diphosphate (IPP) synthesis was examined in secretory cells isolated from glandular trichomes of peppermint (Mentha x piperita L. cv. Black Mitcham). As a consequence of their anatomy and the conditions of their isolation, the isolated secretory cells are non-specifically permeable to low-molecular-weight water-soluble metabolites. Thus, the cytoplasm is readily accessible to the exogenous buffer whereas the selective permeability of subcellular organelles is maintained. With the appropriate choice of exogenous substrates, this feature allows the assessment of cytoplasmic and organellar (e.g. plastidic) metabolism in situ. Glycolytic substrates such as [14C]glucose-6-phosphate and [14C]pyruvic acid are incorporated into both monoterpenes and sesquiterpenes with a monoterpene:sesquiterpene ratio that closely mimics that observed in vivo, indicating that the correct subcellular partitioning of these substrates is maintained in this model system. Additionally, exogenous [14C]mevalonic acid and [14C]IPP, which are both intitially metabolized in the cytoplasm, produce an abnormally high proportion of sesquiterpenes. In contrast, incubation with either [14C]citrate or [14C]acetyl-CoA results in the accumulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) with no detectable isoprenoids formed. Taken together, these results indicate that the cytoplasmic mevalonic acid pathway is blocked at HMG-CoA reductase and that the IPP utilized for both monoterpene and sesquiterpene biosynthesis is synthesized exclusively in the plastids.

Some caveats for bioengineering terpenoid metabolism in plants

The engineering of terpenoid formation in plants, although highly appealing from a biotechnological viewpoint, is particularly challenging because of the myriad of terpenoids produced from a single intermediate (isopentenyl diphosphate) and the complex organization and subtle regulatory features of the biosynthetic pathways. This article surveys many of the biochemical issues that must be appreciated before attempting to develop rational strategies for the bioengineering of terpenoid biosynthesis.

Strategies for bioengineering the development and metabolism of glandular tissues in plants

Glandular tissues in plants produce a wide variety of commercially important chemicals. We review specific model systems that can be exploited for bioengineering the development and metabolism of these specialized structures, and the economic considerations that must be satisfied to permit commercially viable bioengineering approaches to specific chemicals and that constrain the choice of production systems.

ISOPENTENYL DIPHOSPHATE IS THE TERMINAL PRODUCT OF THE DEOXYXYLULOSE-5-PHOSPHATE PATHWAY FOR TERPENOID BIOSYNTHESIS IN PLANTS

Abstract Secretory cells specialized for monoterpene biosynthesis via the deoxyxylulose-5-phosphate pathway for terpenoid biosynthesis were isolated from peppermint ( Mentha piperita ) leaves and used to identify the terminal metabolite of this novel pathway. Incorporation of both [ 13 C]- and [ 14 C]pyruvic acid into monoterpenes in the presence of 2-(dimethylamino)ethyl diphosphate, an inhibitor of isopentenyl diphosphate isomerase, results in accumulation of isopentenyl diphosphate, and not dimethylallyl diphosphate.

A mass spectrometry method for measuring 15N incorporation into pheophytin.

The 15N content of pheophytin, the magnesium-free derivative of chlorophyll, can be measured with great accuracy and precision using positive-ion atmospheric pressure ionization electrospray mass spectroscopy following a simple solvent extraction of small amounts of plant tissue. The molecular weight of pheophytin prepared from Chlamydomonas reinhardtii grown in different ratios of 14N/15N showed linear regression with the isotopic input, with a precision of 0.5-1%. Using an isotope dilution strategy, we have shown that nitrogen fixation can contribute substantial 14N to pheophytin isolated from Medicago truncatula plants grown in symbiosis with Sinorhizobium meliloti. The assay is sensitive, precise, rapid, simple, and robust. These features suggest that it could become an important tool for measuring the contribution of symbiotic and associative nitrogen fixation to plant metabolism.

Biosynthetic methods for plant natural products: New procedures for the study of glandular trichome consistuents

Many types of plant natural products, including terpenoids, phenolics and sucrose esters, accumulate in modified epidermal hairs known as glandular trichomes.1–4 These substances have long been of interest to phytochemists because of their structural complexity and their importance as flavoring, perfumery and pharmaceutical agents. In addition, a number of glandular trichome constituents are thought to have a role in plant defense because of their toxicity and deterrency to herbivorous insects, and their anti-fungal and antibacterial activity.2–5