Allison M. Heskes

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

The first two steps of the biosynthesis of forskolin are active in Coleus forskohlii root cork cells harboring hydrophobic intracellular compartments used for terpenoid storage. Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.

Cytochromes P450 for Terpene Functionalisation and Metabolic Engineering

Plants have evolved the capacity to produce a striking array of specialised metabolites. Terpenoids are the oldest and most diverse class of such compounds and have attracted interest for industrial and pharmaceutical applications. The development of biotechnological alternatives for their production is the focus of intense research. Photosynthetic systems provide new strategies for autotrophic metabolic engineering. Focusing on cytochromes P450, involved in the functionalisation of the core terpene molecules, this review highlights the latest approaches in this field and looks towards recent discoveries that have the potential to shape the future of terpenoid bioengineering.

Contrasting ontogenetic trajectories for phenolic and terpenoid defences in Eucalyptus froggattii

BACKGROUND AND AIMS Plant defence metabolites are considered costly due to diversion of energy and nutrients away from growth. These costs combined with changes in resource availability and herbivory throughout plant ontogeny are likely to promote changes in defence metabolites. A comprehensive understanding of plant defence strategy requires measurement of lifetime ontogenetic trajectories--a dynamic component largely overlooked in plant defence theories. This study aimed to compare ontogenetic trajectories of foliar phenolics and terpenoids. Phenolics are predicted to be inexpensive to biosynthesize, whereas expensive terpenoids also require specialized, non-photosynthetic secretory structures to avoid autotoxicity. Based on these predicted costs, it is hypothesized that phenolics would be maximally deployed early in ontogeny, whereas terpenoids would be maximally deployed later, once the costs of biosynthesis and foregone photosynthesis could be overcome by enhanced resource acquisition. METHODS Leaves were harvested from a family of glasshouse-grown Eucalyptus froggattii seedlings, field-grown saplings and the maternal parent tree, and analysed for total terpenoids and phenolics. KEY RESULTS Foliar phenolics were highest in young seedlings and lowest in the adult tree. Indeed the ratio of total phenolics to total terpenoids decreased in a significantly exponential manner with plant ontogeny. Most individual terpene constituents increased with plant ontogeny, but some mono- and sesquiterpenes remained relatively constant or even decreased in concentration as plants aged. CONCLUSIONS Plant ontogeny can influence different foliar defence metabolites in directionally opposite ways, and the contrasting trajectories support our hypothesis that phenolics would be maximally deployed earlier than terpenoids. The results highlight the importance of examining ontogenetic trajectories of defence traits when developing and testing theories of plant defence, and illustrate an advantage of concurrently studying multiple defences.

Isolation of intact sub-dermal secretory cavities from Eucalyptus

BackgroundThe biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.ResultsLeaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories.ConclusionsThe protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.

Synthesis of the monoterpenoid esters cypellocarpin C and cuniloside B and evidence for their widespread occurrence in Eucalyptus

Short syntheses of cuniloside B and cypellocarpin C, (+)-(R)-oleuropeic acid-containing carbohydrates, are reported. Also disclosed are syntheses of the noreugenin glycosides, undulatoside A and corymbosins K(1) and K(2). Leaf extracts of 28 diverse eucalypts revealed cuniloside B to be present in all, and cypellocarpin C to be present in most, of the species examined. The widespread occurrence of these carbohydrate monoterpenoid esters supports their roles in essential oil biosynthesis or mobilization from sites of synthesis to secretory cavity lumena.

Micropropagation of Eucalyptus polybractea selected for key essential oil traits

A protocol for the micropropagation of Eucalyptus polybractea R.T. Baker (blue mallee) using axillary bud proliferation from lignotuber-derived explants is described. Three different ages of plants were used as explant sources: glasshouse-grown seedlings, field-grown saplings, and coppice of field-grown mature lignotubers. Explants from each source initiated successfully and no significant difference was observed for shoot proliferation, rooting success or hardening success between explant sources. Leaf oil quantity and quality for hardened clones transplanted to a field plantation were assessed after 3 months of growth. Ramets of all clones contained high quality oil with over 80% 1,8-cineole. For seedling-derived clones, foliar oil concentrations of ramets were higher than those of the ortets from which they were derived. For sapling and mature lignotuber derived clones the opposite was the case. This suggests that ontogenetic and physiological constraints may be influencing yield in the young ramets. The age of the explant source did not appear to influence the success of micropropagation, and as a result older plants (for which key oil traits are known) can be selected as elite plants for multiplying selected genotypes via micropropagation.

Multiphoton fluorescence lifetime imaging shows spatial segregation of secondary metabolites in Eucalyptus secretory cavities

Multiphoton fluorescence lifetime imaging provides an excellent tool for imaging deep within plant tissues while providing a means to distinguish between fluorophores with high spatial and temporal resolution. Ideal candidates for the application of multiphoton fluorescence lifetime imaging to plants are the embedded secretory cavities found in numerous species because they house complex mixtures of secondary metabolites within extracellular lumina. Previous investigations of this type of structure have been restricted by the use of sectioned material resulting in the loss of lumen contents and often disorganization of the delicate secretory cells; thus it is not known if there is spatial segregation of secondary metabolites within these structures. In this paper, we apply multiphoton fluorescence lifetime imaging to investigate the spatial arrangement of metabolites within intact secretory cavities isolated from Eucalyptus polybractea R.T. Baker leaves. The secretory cavities of this species are abundant (up to 10 000 per leaf), large (up to 6 nL) and importantly house volatile essential oil rich in the monoterpene 1,8‐cineole, together with an immiscible, non‐volatile component comprised largely of autofluorescent oleuropeic acid glucose esters. We have been able to optically section into the lumina of secretory cavities to a depth of ∼80 μm, revealing a unique spatial organization of cavity metabolites whereby the non‐volatile component forms a layer between the secretory cells lining the lumen and the essential oil. This finding could be indicative of a functional role of the non‐volatile component in providing a protective region of low diffusivity between the secretory cells and potentially autotoxic essential oil.

Localization of Oleuropeyl Glucose Esters and a Flavanone to Secretory Cavities of Myrtaceae

We report the widespread occurrence of structurally diverse oleuropeyl glucose esters, including the new diester eucaglobulin B, localized specifically to the essential oil secretory cavities of myrtaceous species. Clear taxonomic patterns in the composition of cavity extracts within the genus Eucalyptus are shown with species from subgenus Symphyomyrtus dominated by oleuropeyl glucose esters and species from subgenus Eucalyptus dominated instead by the flavanone, pinocembrin. We also examined the intra-species occurrence of oleuropeyl glucose esters by quantifying the abundant constituents cuniloside B and froggattiside A in trees from two populations of Eucalyptus polybractea R.T. Baker. All trees contained both compounds, which were positively correlated with total essential oil concentration. This apparent ubiquity of oleuropeyl glucose esters at both intra- and inter-specific levels in Eucalyptus is indicative of important physiological or ecological functions. The significance of their prevalence and the sequestration of these esters and also pinocembrin to the extracellular domain of secretory cavities is discussed in light of their potential biological activities and our findings that they are spatially segregated to the exterior of cavity lumina. The localization of oleuropeyl glucose esters to a specific and isolatable tissue type has the potential to aid in future elucidation of function and biosynthesis.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L. DOI: http://dx.doi.org/10.7554/eLife.23001.001

High-resolution PTP1B inhibition profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy: Proof-of-concept and antidiabetic constituents in crude extract of Eremophila lucida

Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC-HRMS-SPE-NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10 min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0 ± 5.9 μM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.