Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii

Abstract

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.

Figure 1.

Localization of oil bodies within the root cork of C. forskohlii. A, Cross section of entire root with thick-fissured cork. Bottom right inset, the location of cork cells. B, Rows of cork cells each with one prominent oil body. C to F, Confocal imaging of Nile Red-labeled oil bodies. C, Transmitted light image of a cork cell with two oil bodies. Fluorescence images of the same oil bodies showing discrimination between neutral lipids (green fluorescence, D) and polar lipids (magenta fluorescence, E). F, Overlay of the two fluorescence images. Bars = 200 µm (A) and 10 µm (B–F).

Figure 2.

Forskolin content (mg g^–1 dry weight [DW]) as determined by HPLC-ELSD analysis from different tissues of C. forskohlii. Data are the mean ± se of three independent biological replicates. Ck, Root cork; CS, root cortex and stele; Fl, flowers; St, stems; Lv, leaves.

Figure 3.

Detection of forskolin (a) and (13R) manoyl oxide (b) in C. forskohlii root cork and isolated oil bodies. A, LC-MS EIC of m/z 433.2197 [forskolin + Na]⁺ in isolated oil bodies and in root cork tissue from C. forskohlii. B, GC-MS EIC of m/z 275 corresponding to manoyl oxide in isolated oil bodies and in root cork tissue from C. forskohlii. C, Bright-field microscope image of isolated C. forskohlii oil bodies. Bar = 5 μm. D, Molecular structure of (13R) manoyl oxide. E, Mass spectrum obtained from manoyl oxide identified in root cork tissue (top) and reference spectrum (bottom) from Wiley mass spectrum database.

Figure 4.

GC-MS analysis of hexane extracts from C. forskohlii tissues. The letter “b” indicates (13R) manoyl oxide, “h” indicates dehydroabietadiene, “f” indicates miltiradiene, and “i” indicates abietadiene. IS, Internal standard (1 mg L⁻¹ 1-eicosene).

Figure 5.

Phylogenetic classification of C. forskohlii diterpene synthases with known class II (A) and class I (B) sequences. The phylograms are rooted with the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase from the moss P. patens. Asterisks indicate nodes supported by greater than 80% bootstrap confidence, and the scale bar indicates 0.1 amino acid changes. The numbers indicated at each enzyme refer to their respective enzymatic products, the structures of which are given on the right. Species and GenBank accession are given in Supplemental Table S2.

Figure 6.

Relative expression of CfTPS genes in C. forskohlii tissues. Transcript abundance of CfTPS genes expressed in arbitrary units was measured by quantitative PCR using the translation initiation factor (TIF4a) for normalization. Each value represents the average of three biological replicates, each of which was performed in at least three technical replicates. Ck, Root cork; CS, root cortex and stele; Fl, flowers; St, stems; Lv, leaves.

Figure 7.

GC-MS analysis of in vitro assays with C. forskohlii diTPS. A, In vitro assays with CfTPS1 alone and coupled assays with CfTPS1 and CfTPS3 and CfTPS4. Extracts of CfTPS1 assays were treated with calf intestinal alkaline phosphatase (CIP). B, In vitro assays with CfTPS2 and coupled with CfTPS3 and CfTPS4. Extracts of CfTPS2 were treated with calf intestinal alkaline phosphatase. The letter “b” indicates (13R) manoyl oxide, “c” indicates (13S) manoyl oxide, “d” indicates labd-13-en-8,15-diol, “e” indicates labden-8-ol, “f” indicates miltiradiene, and “g” indicates copal-15-ol. IS, Internal standard (1 mg L⁻¹ 1-eicosene). C, Mass spectra of compounds identified from assays. Structures tentatively identified as described in “Materials and Methods.”

Figure 8.

GC-MS analysis of hexane extracts from N. benthamiana transiently expressing C. forskohlii diTPSs. A, EIC of m/z 275. B, EIC of m/z 272. The letter “b” indicates (13R) manoyl oxide, “c” indicates (13S) manoyl oxide, “h” indicates dehydroabietadiene, “f” indicates miltiradiene, and “i” indicates trace amount of abietadiene.

Figure 9.

Scheme of the biosynthetic routes from GGPP to specialized and general diterpenoids of the abietane, labdane, and ent-kaurene class. Dashed arrows indicate reactions without experimental evidence in C. forskohlii. ¹Detection of (+)-ferruginol in C. forskohlii was reported earlier (Hurley, 1998); ²CYP76AH1 from the close relative S. miltiorrhiza was proposed to convert miltiradiene to ferruginol (Guo et al., 2013).