Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

Tomato plants colonised with the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum show systemic induced resistance to the foliar pathogen Alternaria alternata, as observed in interactions of other AM-colonised plants with a range of pathogens. The role of jasmonic (JA) and salicylic (SA) acid in expression of this mycorrhiza-induced resistance (MIR) against A. alternata was studied by measuring: (i) activity of enzymes reported to be involved in their biosynthesis, namely lipoxygenase (LOX) and phenylammonia lyase (PAL); and (ii) levels of methyl jasmonate (MeJA) and SA. Transcript abundance of some defence genes associated with JA and SA response pathways were also studied. Both LOX and PAL activity increased twofold in response to pathogen application to control plants. AM-colonised plants had three-fold higher LOX activity compared to control plants, but unlike controls, this did not increase further in response to pathogen application. Higher LOX activity in AM-colonised plants correlated with four-fold higher MeJA in leaves of AM-colonised plants compared to controls. Treatment of plants with the JA biosynthesis inhibitor salicylhydroxamic acid (SHAM) led to 50% lower MeJA in both control and AM-colonised plants and correlated with increased susceptibility to A. alternata, suggesting a causal role for JA in expression of MIR against the pathogen. Genes involved in JA biosynthesis (OPR3) and response (COI1) showed six- and 42-fold higher expression, respectively, in leaves of AM-colonised plants compared to controls. AM-colonised plants also showed increased expression of the SA response gene PR1 and that of the wound-inducible polypeptide prosystemin. Our results suggest that the systemic increase in JA in response to AM colonisation plays a key role in expression of MIR against A. alternata.

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Basic limonoids carrying a 4,4,8-trimethyl-17-furanylsteroid skeleton are a class of triterpenoids and wellknown for their insecticidal as well as a vast array of pharmacological activities. Rare and synthetically challenging 12β-and 17β-hydroxylation was achieved on the basic limonoid skeleton to produce a novel series of hydroxylated limonoids using fungi-mediated biocatalysis. The fungal system belonging to the genera of Mucor efficiently converted azadiradione, epoxyazadiradione, gedunin and their derivatives into corresponding 12β-and/or 17β-hydroxy derivatives. The position and stereochemistry of hydroxylation was determined by rigorous spectroscopic and crystallographic studies. This fungi-mediated stereoand regio-selective hydroxylation process was highly efficient and mild enough to sustain chemically sensitive functional groups around the basic limonoid skeleton. Modifications of specific functional groups and variation in biocatalyst were shown to bring selectivity among 12β-or 17β-hydroxylation.

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