Glenn W. Turner

Organization of Monoterpene Biosynthesis in Mentha. Immunocytochemical Localizations of Geranyl Diphosphate Synthase, Limonene-6-Hydroxylase, Isopiperitenol Dehydrogenase, and Pulegone Reductase

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) (−)-(4S)-limonene-6-hydroxylase, peppermint (−)-trans-isopiperitenol dehydrogenase, and peppermint (+)-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, (−)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (−)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-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.

A systems biology approach identifies the biochemical mechanisms regulating monoterpenoid essential oil composition in peppermint

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 (Ki = 300 μM). 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 μM). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways.

Terpenoid biosynthesis in trichomes—current status and future opportunities

Glandular 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.

Improving peppermint essential oil yield and composition by metabolic engineering

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.

Schizogenous Secretory Cavities of Citrus limon (L.) Burm. F. and A Reevaluation of the Lysigenous Gland Concept

The lysigenous appearance of Citrus limon (L) Burm. f. secretory cavities is the result of fixation artifacts. The glandular epithelial cells of lemon glands undergo rapid destructive swelling when immersed in commonly used fixatives. The swelling is most evident with mature glands, causing a false impression that the epithelial cells undergo autolysis as they complete their development. Epithelial cells of secretory cavities sliced open during tissue preparation show more extensive swelling than those left intact during fixation, indicating a possible cause for inconsistencies among reports of Citrus gland development. Aqueous primary fixation of intact secretory cavities results in sufficient epithelial cell swelling to give a false impression of precocious senescence; osmium vapor-fixed glandular cells show less swelling, contain intact organelles, and appear living at gland maturity. Although the concept of lysigeny is very old, recent evidence for lysigeny is based on investigations that used standard aqueous fixatives. Since fixation requirements for glands from other species of plants could be similar to those for Citrus glands, we suggest that lysigeny, in general, could be a false category of gland development, representing misinterpretation of artifacts.

Experimental sink removal induces stress responses, including shifts in amino acid and phenylpropanoid metabolism, in soybean leaves.

The repeated removal of flower, fruit, or vegetative buds is a common treatment to simulate sink limitation. These experiments usually lead to the accumulation of specific proteins, which are degraded during later stages of seed development, and have thus been designated as vegetative storage proteins. We used oligonucleotide microarrays to assess global effects of sink removal on gene expression patterns in soybean leaves and found an induction of the transcript levels of hundreds of genes with putative roles in the responses to biotic and abiotic stresses. In addition, these data sets indicated potential changes in amino acid and phenylpropanoid metabolism. As a response to sink removal we detected an induced accumulation of γ-aminobutyric acid, while proteinogenic amino acid levels decreased. We also observed a shift in phenylpropanoid metabolism with an increase in isoflavone levels, concomitant with a decrease in flavones and flavonols. Taken together, we provide evidence that sink removal leads to an up-regulation of stress responses in distant leaves, which needs to be considered as an unintended consequence of this experimental treatment.

Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.

Ultrastructure of Grapefruit Secretory Cavities and Immunocytochemical Localization of (+)-Limonene Synthase

Premise of research. The genus Citrus includes species that are among the most important tree fruit crops. Although fruit juice is the primary product, monoterpenoid essential oil obtained from the peel is an important value-added commodity. Peel monoterpenes are synthesized by subepidermal secretory cavities that consist of glandular cells surrounding an extracellular oil storage space. Several previous studies have focused on early secretory cavity development, ending with the initiation of secretion. In order to better understand the process of monoterpene formation, transport, and storage, it is important to obtain detailed information concerning plant oil glands during all phases of development. Methodology. TEM was performed on secretory cavities isolated from the exocarp of grapefruit (Citrus × paradisi Macfad. ‘Duncan’) preserved by microwave-assisted fixation or high-pressure freezing and freeze substitution. Immunocytochemistry was employed to localize (+)-limonene synthase. Tomography was used to examine leucoplast ultrastructure. Pivotal results. Glandular epithelial cells of secretory cavities persisted for several months after their formation. Lipid accumulation occurred within plastoglobule-like lipid bodies (PGs), vacuoles, and cytoplasmic lipid bodies of epithelial cells. (+)-Limonene synthase, the enzyme that catalyzes the primary flux-carrying reaction in Citrus monoterpene biosynthesis, was localized to tubules within oil gland leucoplasts and was not associated with PGs. No bulk movement of lipids from PGs to vacuoles or to the extracellular oil storage cavity was observed. Conclusions. Oil glands remain biosynthetically active throughout fruit development in grapefruit. The high degree of oil gland metabolic specialization is reflected in unique cellular features, such as leucoplasts, abundant plastid–endoplasmic reticulum membrane contact sites, and the accumulation of fibrillar material in vacuoles. The mechanism of the movement of monoterpenes from plastids to the central cavity is unknown, but our observations suggest that it does not involve vesicles or disruption of the large PGs.

Soybean vegetative lipoxygenases are not vacuolar storage proteins

The paraveinal mesophyll (PVM) of soybean is a distinctive uniseriate layer of branched cells situated between the spongy and palisade chlorenchyma of leaves that contains an abundance of putative vegetative storage proteins, Vspα and Vspβ, in its vacuoles. Soybean vegetative lipoxygenases (five isozymes designated as Vlx(A-E)) have been reported to co-localise with Vsp in PVM vacuoles; however, conflicting results regarding the tissue-level and subcellular localisations of specific Vlx isozymes have been reported. We employed immuno-cytochemistry with affinity-purified, isozyme-specific antibodies to reinvestigate the subcellular locations of soybean Vlx isozymes during a sink limitation experiment. VlxB and VlxC were localised to the cytoplasm and nucleoplasm of PVM cells, whereas VlxD was present in the cytoplasm and nucleoplasm of mesophyll chlorenchyma (MC) cells. Label was not associated with storage vacuoles or any evident protein bodies, so our results cast doubt on the hypothesis that Vlx isozymes function as vegetative storage proteins.

Utility of Aromatic Plants for the Biotechnological Production of Sustainable Chemical and Pharmaceutical Feedstocks

Currently, transportation fuels and many platform chemicals are derived primarily from petroleum. The use of this finite resource contributes substantially to the dangerous build-up of greenhouse gases in the earth’s atmosphere, which has led to an increasing demand for sustainably produced fuels and chemicals. Aromatic plants accumulate terpenoids and phenylpropanoids with physicochemical properties similar to those of petrochemical feedstocks. They are also a source of bioactive compounds used by the agrochemical and pharmaceutical industries. Recent successes with engineering the terpenoid pathway indicate that aromatic plants have the potential to play an important role in providing chemical and pharmaceutical feedstocks from renewable sources. In this review, we critically assess the current status of the field and discuss different biotechnological approaches to enhance the accumulation of commercially valuable target terpenoids.