Identification of a Dolabellane Type Diterpene Synthase and other Root-Expressed Diterpene Synthases in Arabidopsis

Abstract

Arabidopsis thaliana maintains a complex metabolism for the production of secondary or specialized metabolites. Such metabolites include volatile and semivolatile terpenes, which have been associated with direct and indirect defensive activities in flowers and leaves. In comparison, the structural diversity and function of terpenes in Arabidopsis roots has remained largely unexplored despite a substantial number of root-expressed genes in the Arabidopsis terpene synthase (TPS) gene family. We show that five root-expressed TPSs of an expanded subfamily-a type clade in the Arabidopsis TPS family function as class I diterpene synthases that predominantly convert geranylgeranyl diphosphate (GGPP) to different semi-volatile diterpene products, which are in part detectable at low levels in the ecotypes Columbia (Col) and Cape Verde Island (Cvi). The enzyme TPS20 produces a macrocyclic dolabellane diterpene alcohol and a dolabellane-related diterpene olefin named dolathaliatriene with a so far unknown C6-C11 bicyclic scaffold besides several minor olefin products. The TPS20 compounds occur in all tissues of Cvi but are absent in the Col ecotype because of deletion and substitution mutations in the Col TPS20 sequence. The primary TPS20 diterpene products retard the growth of the root rot pathogen Pythium irregulare but only at concentrations exceeding those in planta. Together, our results demonstrate that divergence and pseudogenization in the Arabidopsis TPS gene family allow for structural plasticity in diterpene profiles of above- and belowground tissues.

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution

“…S2B). However, we did not observe the formation of sesquiterpenes or diterpenes, even for AtTPS22, -25, -29, and -30, which had previously been reported to synthesize these types of terpenes in vitro (31). This outcome may be because of the low activity of the encoding enzymes, inadequate precursor supply, or the loss of the volatile/semivolatile products in N. benthamiana, especially if these products were produced in very low abundance.…”

“…N-terminal transit peptide (N-tp)-truncated recombinant AtTPS18 and AtTPS19 proteins were recently coexpressed with N-tp AtGFPPS1 and AtGFPPS2 in E. coli and reported to synthesize (+)-arathanatriene and (−)-retigeranin B, respectively (22). Recombinant proteins encoded by N-tptruncated AtTPS22 and AtTPS25 were also recently expressed in E. coli but reported to have sesquiterpene synthase activity, making sesquiterpene mixtures when incubated with E,E-farnesyl diphosphate (E,E-FPP) as a substrate in vitro (31). Recombinant proteins encoded by N-tp-truncated AtTPS30 were found to have DTS activities when assayed with GGPP or ent-copalyl diphosphate (ent-CPP) as substrates in vitro, whereas AtTPS29 was not active in these assays (31).…”

“…Recombinant proteins encoded by N-tptruncated AtTPS22 and AtTPS25 were also recently expressed in E. coli but reported to have sesquiterpene synthase activity, making sesquiterpene mixtures when incubated with E,E-farnesyl diphosphate (E,E-FPP) as a substrate in vitro (31). Recombinant proteins encoded by N-tp-truncated AtTPS30 were found to have DTS activities when assayed with GGPP or ent-copalyl diphosphate (ent-CPP) as substrates in vitro, whereas AtTPS29 was not active in these assays (31). AtTPS17, Cr237, and Bo250 have not been investigated previously.…”

Nerylneryl diphosphate is the precursor of serrulatane, viscidane and cembrane-type diterpenoids in Eremophila species

“…The main pathway to diterpenoids in angiosperms involves the sequential activity of two TPSs (class II followed by class I) and leads to the formation of the labdane-related diterpenoids (characterised by a decalin core) [23]. Diterpenes can also be biosynthesised directly from geranylgeranyl diphosphate (GGPP) or nerylneryl diphosphate (NNPP, the all cis isomer of GGPP) by class I TPSs alone to generate linear [27,28], macrocyclic [29][30][31] and (poly) cyclic [32][33][34][35] products. These enzymes catalyse metal ion dependant ionization of the diphosphate bond of their prenyl diphosphate substrates to generate a reactive carbocation molecule.…”

In silico discovery of terpenoid metabolism in Cannabis sativa

“…It would be great if possible that the author could add this piece of information, which would be valuable for future characterization of these genes. An example study showing the expression pattern of terpene genes would be found in the paper by Wang, et al 2016 1 .…”