To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism

Abstract

The diterpenoids are classically defined by their composition--four isoprenyl units (20 carbons)--and are generally derived from [E,E,E]-geranylgeranyl diphosphate (GGPP). Such metabolism seems to be ancient and has been extensively diversified, with ∼12,000 diterpenoid natural products known. Particularly notable are the gibberellin phytohormones, whose requisite biosynthesis has provided a genetic reservoir that gave rise to not only a large superfamily of ∼7,000 diterpenoids but also, to some degree, all plant terpenoid natural products. This review focuses on the diterpenoids, particularly the defining biosynthetic characteristics of the major superfamilies defined by the cyclization and/or rearrangement of GGPP catalyzed by diterpene synthases/cyclases, although it also includes some discussion of the important subsequent elaboration in the few cases where sufficient molecular genetic information is available. It additionally addresses the array of biological activity providing the selective pressures that drive the observed gene family expansion and diversification, along with biosynthetic gene clustering.

Figure 1

Schematic of the biological roles played by diterpenoid natural products, ranging from the growth promoting effects of the GA phytohormones to roles in ecological interactions – e.g., illustrated are the activities of momilactone B as a rice allelochemical, rhizathalene A as an herbivore (insect) antifeedant, and momilactone A as an antibiotic/phytoalexin against the rice fungal blast pathogen.

Figure 2

Sub-cellular localization of the initial stages of diterpenoid biosynthesis to plastids. Schematic of a plant cell with relevant chloroplast and leucoplast organelles shown, along with upstream MEP-dependent isoprenoid precursor pathway described elsewhere (104), as well as production of the general diterpenoid precursor GGPP catalyzed by isoprenyl diphosphate synthases also described elsewhere (17; 102).

Figure 3

Bicyclization of GGPP to labdane hydrocarbon backbone containing CPP that can proceed more prototypical allylic diphosphate ester ionization initiated reaction catalyzed by (class I) terpene synthases in diterpenoid biosynthesis.

Figure 4

Early steps in GA biosynthesis, from GGPP to first gibberellane intermediate, along with relevant enzymes (as defined in the text).

Figure 5

Staggered evolution of GA biosynthesis. While the production of ent-kaurenoic acid seems to have evolved early in land plants, as suggested by the presence of CPS/KS and KO homologs in the bryophyte P. patens, while GA biosynthesis more specifically seems to have arisen later, during the evolution of vascular plants, as suggested by the presence of homologs to the remaining genes in the early diverging, yet vascular, lycophyte S. moellendorffii (GA20ox, GA C20-oxidase; GA3ox, GA C3-oxidase). Shown is simplified plant phylogeny with dates of divergence (MYA, millions of years ago), along with components of the GA biosynthetic pathway alongside the approximate period in which they evolved.

Figure 6

Effect of GA phytohormone deficiency on stem elongation and flowering of A. thaliana.

Figure 7

Structure of the abietadiene synthase from A. grandis (AgAS), along with catalyzed reactions. AgAS exhibits a tri-domain structure (γβα, as indicated), wherein the class II active site sits between the N-terminal γβ domains, and the class I active site is within the C-terminal α domain. The structure is shown as a ribbon diagram, with the side-chains of the catalytic class II associated DxDD and class I associated DDxxD motifs shown in stick format.

Figure 8

Metabolic map of rice diterpenoid biosynthesis. Shown are the characterized diterpene synthases/cyclases along with the catalyzed reactions, and downstream natural products where known. Indicated in green, and by thicker arrows, are those enzymes and reactions required for GA biosynthesis in rice (83). Adapted from ref. , with permission.

Figure 9

Effect of single residue ‘switch’ on reaction catalyzed by ent-(iso)kaurene synthases. Shown is a scheme of the reaction catalyzed in the usual production of ent-kaurene from ent-CPP, which requires the presence of the conserved Ile residue indicated in the alignment of KSs from A. thaliana (AtKS), rice (OsKS), spruce (PgKS), and P. patens (PpCPSKS), which spans ~450 million years of evolution. If this residue is changed to Thr, the enzymes largely catalyze the abortive production of ent-pimaradiene instead. The potential role of the ionized pyrophosphate anion in driving carbocation migration also is indicated by its positioning within the scheme.

Figure 10

Reactions catalyzed by diterpene synthases from S. sclarea in production of sclareol (SsLPS, class II diterpene cyclase producing 8α-hydroxy-labdadienyl diphosphate, which is utilized by the class I diterpene synthase SsSS to produce sclareol). These reactions are noteworthy in their incorporation of water prior to concluding deprotonation.

Figure 11

Macrocyclization reactions catalyzed by casbene, neocembrene, and cembrenol synthases.

Figure 12

Loss of γ domain in prototypical (class I) plant terpene synthases. Illustrated by comparison of AgAS structure with that of the 5-epi-aristolochene (sesquiterpene) synthase (93). Domains are colored to assist visualization (γ, yellow; β, green; α, blue), with N-terminal helix that is retained during the ancient, but not more recent, γ domain loss event show in magenta. Note that the N-termini of AgAS (although not apparent in the crystal structure), as well epi-aristolochene synthase, fold back and form part of the class I active site in the C-terminal α domain.

Figure 13

Rizathalene cyclization reaction demonstrates ability of class I diterpene synthases to rearrange GGPP to geranyllinalyl diphosphate (shown) en route to 1,6-cyclization to the shown cyclohexenyl carbocation intermediate.

Figure 14

Hydroxylation reactions carried out by CYP in biosynthesis of more specialized rice labdane-related diterpenoids, along with resulting (both indirect and direct) natural products.

Figure 15

Illustrating the varied biological roles played by diterpenoid natural products. (a) The rhizathalenes produced by TPS08 act as antifeedants against herbivory by larva of the fungus gnat (Bradysia) in A. thaliana (103). Top row depicts undamaged roots (wild-type plants). Middle row depicts roots of wild-type plants after Bradysia feeding. Bottom row depicts roots from TPS08 knock-out (tps08-1) after Bradysia feeding. Reprinted with permission from ref. . (b) The momilactones, whose biosynthesis relies on OsCPS4, act as allelochemicals, suppressing the growth of other plants. Lettuce seedlings germinated in the presence of OsCPS4 knock-out (cps4) or its parental/wild-type (WT) rice seedlings. Reprinted with permission from ref. . This effect is most likely due to momilactone B (39). (c) The momilactones, most likely momilactone A, acts as an antibiotic phytoalexin against the rice fungal blast pathogen M. oryzae (101). Pictures of the lesions in the leaves of OsCPS knock-down (cps4-tos) or its parental/wild-type rice following infection with M. oryzae. Reprinted with permission from ref. .

Figure 16

Yin and yang of plant biosynthetic gene clusters, as illustrated by that for diterpenoid (momilactone) biosynthesis in rice. Schematic of gene cluster on chromosome 4 in the rice genome and known roles of the encoded enzymes in momilactone biosynthesis, along with effect of knocking-out OsKSL4 (ksl4) relative to its parental/wild-type (WT) line on allelopathy (increased growth of the endemic rice paddy weed barnyard grass with ksl4) and germination rate (decreased with ksl4). The combination of positive and negative selection pressure (here allelopathy versus decreased germination rate) is hypothesized to drive gene clustering.