Biocontrol Potential of Forest Tree Endophytes

Peer reviewe

Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis

Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis. A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum

Cytochrome P450s from Cynara cardunculus L. CYP71AV9 andCYP71BL5, catalyze distinct hydroxylations in the sesquiterpenelactone biosynthetic pathway

Cynara cardunculus (Asteraceae) is a cross pollinated perennial crop which includes the two cultivatedtaxa globe artichoke and cultivated cardoon. The leaves of these plants contain high concentrationsof sesquiterpene lactones (STLs) among which cynaropicrin is the most represented, and has recentlyattracted attention because of its therapeutic potential as anti-tumor and anti-photoaging agent. Costuno-lide is considered the common precursor of the STLs and three enzymes are involved in its biosyntheticpathway: i.e. the germacrene A synthase (GAS), the germacrene A oxidase (GAO) and the costunolidesynthase (COS). Here we report on the isolation of two P450 genes, (i.e. CYP71AV9 and CYP71BL5), ina set of ~19,000 C. cardunculus unigenes, and their functional characterization in yeast and in planta.The metabolite analyses revealed that the co-expression of CYP71AV9 together with GAS resulted in thebiosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid in yeast. The co-expression of CYP71BL5 andCYP71AV9 with GAS led to biosynthesis of the free costunolide in yeast and costunolide conjugates inNicotiana benthamiana, demonstrating their involvement in STL biosynthesis as GAO and COS enzymes.The substrate specificity of CYP71AV9 was investigated by testing its ability to convert amorpha-4,11-diene, (+)-germacrene D and cascarilladiene to their oxidized products when co-expressed in yeast withthe corresponding terpene synthases