Domain loss has independently occurred multiple times in plant terpene synthase evolution

Abstract

The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these synthases have evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a approximately 220 amino acid 'internal sequence element' (now recognized as the γ domain) gave rise to the precursor of the modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function. This relationship demonstrates that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume that the 'internal/γ' domain loss represents a single evolutionary event. Here we provide evidence that such a loss was not a singular event, but rather has occurred multiple times. Specifically, we provide an example of a bi-domain diterpene synthase from Salvia miltiorrhiza, along with a sesquiterpene synthase from Triticum aestivum (wheat) that is not only closely related to diterpene synthases, but retains the ent-kaurene synthase activity relevant to the ancestral gibberellin metabolic function. Indeed, while the wheat sesquiterpene synthase clearly no longer contains the 'internal/γ' domain, it is closely related to rice diterpene synthase genes that retain the ancestral tri-domain structure. Thus, these findings provide examples of key evolutionary intermediates that underlie the bi-domain structure observed in the expansive plant TPS gene family, as well as indicating that 'internal/γ' domain loss has occurred independently multiple times, highlighting the complex evolutionary history of this important enzymatic family.

Figure 1. Diterpene synthase tri-domain structure

As exemplified by the crystal structure of taxadiene synthase from Taxus brevifolia (Köksal, et al. 2011b), with domain nomenclature as indicated.

Figure 2. KS(L) reactions

Reactions catalyzed by ent-kaurene synthases (e.g. from gibberellin phytohormone biosynthesis) and DsKSL.

Figure 3. DsKSL lacks “internal/γ” domain

Alignment of DsKSL with Solenaceae KSL homologs demonstrates homology only after the beginning of the β domain (indicated by *). Shown is alignment around N-terminal sequence of DsKSL.

Figure 4. TaKSL5 lacks “internal/γ” domain

Alignment of TaKSL5 with other cereal KSLs demonstrates homology only after the beginning of the β domain (indicated by *). Notably, TaKSL5 appears to have lost complete exons, as the point at which homology becomes readily evident is an exon boundary in its closest homolog, OsKSL3 (indicated by #; see Figure S5).

Figure 5. TaKSL5 diterpene synthase activity

GC-MS analysis of diterpene products from co-expression of TaKSL5d121 with ent-CPP synthase in E. coli. Shown are the chromatogram and the mass spectra of both products, with comparison to that of authentic standards for ent-kaurene and ent-beyerene (as indicated).

Figure 6. TaKSL5 is a sesquiterpene synthase

(a) GC-MS analysis of diterpene products from co-expression of TaKSL5d121 with cis-FPP synthase in E. coli. Shown is the chromatogram and the mass spectra, with comparison to that of an authentic standard (6E)-nerolidol. (b) Reaction catalyzed by TaKSL5 to produce (6E)-nerolidol from cis-FPP.

Figure 7. Phylogenetic relationships of diterpene synthases

Unrooted tree constructed via the neighbor-joining algorithm, including essentially only class I di-TPSs (along with a few closely related other TPSs defined in the text), and with previously defined TPS sub-family assignments indicated (Chen, et al. 2011).

Figure 8. Proposed process for the repeated evolution of bi-domain terpene synthases

Gene duplication and divergence of the ent-kaurene synthase (KS) required for gibberellin biosynthesis leads to KS-like (KSL) diterpene synthases, whose function can diverge. Subsequent loss of the “internal/γ” domain results in bi-domain KSLs such as DsKSL. Further functional divergence then enables reaction with other isoprenyl diphosphate substrates, presumably via intermediate states still able to catalyze the ancestral diterpene synthase reaction such as TaKSL5.