Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. Fusarium oxysporum) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in F. oxysporum, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in E. coli, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C18-polyunsaturated fatty acids to 13S-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives.

Plant glandular trichomes are specialized secretory structures located on the surface of the aerial parts of plants with large biosynthetic capacity, often with terpenoids as output molecules. The collection of plant trichomes requires a method to separate trichomes from leaf epidermal tissues. For metabolite profiling, trichome tissue needs to be rapidly quenched in order to maintain the indigenous state of intracellular intermediates. Appropriate extraction and chromatographic separation methods must be available, which address the wide-ranging polarity of metabolites. In this chapter, a protocol for trichome harvest using a frozen paint brush is presented. A work flow for broad-range metabolite profiling using LC-MS(2) analysis is described, which is applicable to assess very hydrophilic isoprenoid precursors as well as more hydrophobic metabolites from trichomes and other plant tissues.

Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

13Secretions from glandular trichomes potentially protect the plant against a variety of 14 aggressors. In the tomato genus, wild species constitute a rich source of chemical diversity 15 produced at the leaf surface by glandular trichomes. Previously, 7-epi-zingiberene produced 16 in several accessions of Solanum habrochaites was found to confer resistance to whiteflies 17 (Bemisia tabaci) and other insect pests. Here, we identify two derivatives of 7-epi-zingiberene 18 from S. habrochaites that had not been reported as yet. We identified them as 9-hydroxy-19 zingiberene and 9-hydroxy-10,11-epoxyzingiberene. Using a combination of genetics and 20 transcriptomics we identified a single cytochrome P450 oxygenase, ShCYP71D184 that 21 carries out two successive oxidations to generate the two sesquiterpenoids. Bioactivity assays 22 showed that only 9-hydroxy-10,11-epoxyzingiberene exhibits substantial toxicity against B. 23tabaci. In addition, both 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene display 24 substantial growth inhibitory activities against a range of microorganisms, including Bacillus 25 subtilis, Phytophtora infestans and Botrytis cinerea. Our work shows that trichome secretions 26 from wild tomato species can provide protection against a wide variety of organisms. In 27 addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-28 zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision 29 breeding. 30 sesquiterpene synthases able to convert Z,Z-FPP have been identified in S. habrochaites. The 59 santalene and bergamotene synthase (ShSBS) is a multiproduct cyclase which makes (+)-α-60