Terpenoids from Salvia sclarea

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.

http://www.plantphysiol.org/content/plantphysiol/133/3/1272.full.pdf

Pathogen-Responsive Expression of a Putative ATP-Binding Cassette Transporter Gene Conferring Resistance to the Diterpenoid Sclareol Is Regulated by Multiple Defense Signaling Pathways in Arabidopsis

Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

/pdf/discovery-and-functional-characterization-of-two-diterpene-4zauelgqn0.pdf

Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea(L.) and their relevance for perfume manufacture

The potato pathogenesis-related gene PR-10a is transcriptionally activated in response to pathogen infection or elicitor treatment. Characterization of the cis-acting elements of the PR-10a promoter revealed the presence of a silencing element between residues −52 and −27 that contributes to transcriptional regulation. In this study, we have isolated a silencing element binding factor (SEBF) from potato tuber nuclei that binds to the coding strand of the silencing element in a sequence-specific manner. The consensus binding site of SEBF, PyTGTCNC, is present in a number of PR genes and shows striking similarity to the auxin response element. Mutational analysis of the PR-10a promoter revealed an inverse correlation between the in vitro binding of SEBF and the expression of PR-10a. SEBF was purified to homogeneity from potato tubers, and sequencing of the N terminus of the protein led to the isolation of a cDNA clone. Sequence analysis revealed that SEBF is homologous with chloroplast RNA binding proteins that possess consensus sequence–type RNA binding domains characteristic of heterogenous nuclear ribonucleoproteins (hnRNPs). Overexpression of SEBF in protoplasts repressed the activity of a PR-10a reporter construct in a silencing element–dependent manner, confirming the role of SEBF as a transcriptional repressor.

/pdf/a-diterpene-as-an-endogenous-signal-for-the-activation-of-39ina6oaov.pdf

A Diterpene as an Endogenous Signal for the Activation of Defense Responses to Infection with Tobacco mosaic virus and Wounding in Tobacco

Abstract The prenylated isoflavone luteone has been isolated from healthy leaves of Lupinus albus and 11 other lupin species. Evidence is presented that this compound occurs as a leaf surface constituent. In vitro tests indicate that luteone and a second unidentified isoflavone frorn L. albus possess antifungal activity sufficient to support their proposed role as pre-infectional resistance factors. No evidence was obtained to suggest that phytoalexins were produced by the fungus-infected leaves of L. albus .

The isopentenyl isoflavone luteone as a pre-infectional antifungal agent in the genus

SUMMARY: The diterpenes sclareol and 13-epi-sclareol were isolated as a eutectic mixture from leaves of Nicotiana glutinosa. These compounds were probably present in liquid droplets which occurred on the leaf surface. These diterpenes did not prevent germination of fungal spores but markedly inhibited the radial extension of colonies growing on agar. This inhibition is shown to be due to an effect on the morphology of the fungi in which the degree of hyphal branching is increased. The possibility that sclareol/13-epi-sclareol influence the growth-regulatory processes of fungal hyphae and the potential of these compounds to affect growth of fungi on the surface of leaves of N. glutinosa is discussed.

R. S. Burden

Papers

Diterpenes from Nicotiana glutinosa and their Effect on Fungal Growth