A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms.

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.

Natural variation in wild tomato trichomes; selecting metabolites that contribute to insect resistance using a random forest approach.

BACKGROUND: Plant-produced specialised metabolites are a powerful part of a plant's first line of defence against herbivorous insects, bacteria and fungi. Wild ancestors of present-day cultivated tomato produce a plethora of acylsugars in their type-I/IV trichomes and volatiles in their type-VI trichomes that have a potential role in plant resistance against insects. However, metabolic profiles are often complex mixtures making identification of the functionally interesting metabolites challenging. Here, we aimed to identify specialised metabolites from a wide range of wild tomato genotypes that could explain resistance to vector insects whitefly (Bemisia tabaci) and Western flower thrips (Frankliniella occidentalis). We evaluated plant resistance, determined trichome density and obtained metabolite profiles of the glandular trichomes by LC-MS (acylsugars) and GC-MS (volatiles). Using a customised Random Forest learning algorithm, we determined the contribution of specific specialised metabolites to the resistance phenotypes observed. RESULTS: The selected wild tomato accessions showed different levels of resistance to both whiteflies and thrips. Accessions resistant to one insect can be susceptible to another. Glandular trichome density is not necessarily a good predictor for plant resistance although the density of type-I/IV trichomes, related to the production of acylsugars, appears to correlate with whitefly resistance. For type VI-trichomes, however, it seems resistance is determined by the specific content of the glands. There is a strong qualitative and quantitative variation in the metabolite profiles between different accessions, even when they are from the same species. Out of 76 acylsugars found, the random forest algorithm linked two acylsugars (S3:15 and S3:21) to whitefly resistance, but none to thrips resistance. Out of 86 volatiles detected, the sesquiterpene α-humulene was linked to whitefly susceptible accessions instead. The algorithm did not link any specific metabolite to resistance against thrips, but monoterpenes α-phellandrene, α-terpinene and ß-phellandrene/D-limonene were significantly associated with susceptible tomato accessions. CONCLUSIONS: Whiteflies and thrips are distinctly targeted by certain specialised metabolites found in wild tomatoes. The machine learning approach presented helped to identify features with efficacy toward the insect species studied. These acylsugar metabolites can be targets for breeding efforts towards the selection of insect-resistant cultivars.

Introgression of the sesquiterpene biosynthesis from Solanum habrochaites to cultivated tomato offers insights into trichome morphology and arthropod resistance.

MAIN CONCLUSION: Cultivated tomatoes harboring the plastid-derived sesquiterpenes from S. habrochaites have altered type-VI trichome morphology and unveil additional genetic components necessary for piercing-sucking pest resistance. Arthropod resistance in the tomato wild relative Solanum habrochaites LA1777 is linked to specific sesquiterpene biosynthesis. The Sesquiterpene synthase 2 (SsT2) gene cluster on LA1777 chromosome 8 controls plastid-derived sesquiterpene synthesis. The main genes at SsT2 are Z-prenyltransferase (zFPS) and Santalene and Bergamotene Synthase (SBS), which produce α-santalene, ß-bergamotene, and α-bergamotene in LA1777 round-shaped type-VI glandular trichomes. Cultivated tomatoes have mushroom-shaped type-VI trichomes with much smaller glands that contain low levels of monoterpenes and cytosolic-derived sesquiterpenes, not presenting the same pest resistance as in LA1777. We successfully transferred zFPS and SBS from LA1777 to cultivated tomato (cv. Micro-Tom, MT) by a backcrossing approach. The trichomes of the MT-Sst2 introgressed line produced high levels of the plastid-derived sesquiterpenes. The type-VI trichome internal storage-cavity size increased in MT-Sst2, probably as an effect of the increased amount of sesquiterpenes, although it was not enough to mimic the round-shaped LA1777 trichomes. The presence of high amounts of plastid-derived sesquiterpenes was also not sufficient to confer resistance to various tomato piercing-sucking pests, indicating that the effect of the sesquiterpenes found in the wild S. habrochaites can be insect specific. Our results provide for a better understanding of the morphology of S. habrochaites type-VI trichomes and paves the way to obtain insect-resistant tomatoes.

Plant terpenoid biosynthetic network and its multiple layers of regulation.

Terpenoids constitute one of the largest and most chemically diverse classes of primary and secondary metabolites in nature with an exceptional breadth of functional roles in plants. Biosynthesis of all terpenoids begins with the universal five­carbon building blocks, isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which in plants are derived from two compartmentally separated but metabolically crosstalking routes, the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. Here, we review the current knowledge on the terpenoid precursor pathways and highlight the critical hidden constraints as well as multiple regulatory mechanisms that coordinate and homeostatically govern carbon flux through the terpenoid biosynthetic network in plants.

Volatile communication in plants relies on a KAI2-mediated signaling pathway.

Plants are constantly exposed to volatile organic compounds (VOCs) that are released during plant-plant communication, within-plant self-signaling, and plant-microbe interactions. Therefore, understanding VOC perception and downstream signaling is vital for unraveling the mechanisms behind information exchange in plants, which remain largely unexplored. Using the hormone-like function of volatile terpenoids in reproductive organ development as a system with a visual marker for communication, we demonstrate that a petunia karrikin-insensitive receptor, PhKAI2ia, stereospecifically perceives the (-)-germacrene D signal, triggering a KAI2-mediated signaling cascade and affecting plant fitness. This study uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).

Genetic and physiological requirements for high-level sesquiterpene-production in tomato glandular trichomes.

Type-VI glandular trichomes of wild tomato Solanum habrochaites PI127826 produce high levels of the sesquiterpene 7-epizingiberene and its derivatives, making the plant repellent and toxic to several pest insects and pathogens. How wild tomato trichomes achieve such high terpene production is still largely unknown. Here we show that a cross (F1) with a cultivated tomato produced only minute levels of 7-epizingiberene. In the F2-progeny, selected for the presence of the 7-epizingiberene biosynthesis genes, only three percent produced comparable amounts the wild parent, indicating this trait is recessive and multigenic. Moreover, trichome density alone did not explain the total levels of terpene levels found on the leaves. We selected F2 plants with the "high-production active-trichome phenotype" of PI127826, having trichomes producing about 150 times higher levels of terpenes than F2 individuals that displayed a "low-production lazy-trichome phenotype". Terpene quantities in trichomes of these F2 plants correlated with the volume of the storage cavity and shape of the gland. We found that trichome morphology is not a predetermined characteristic, but cavity volume rather depended on gland-cell metabolic activity. Inhibitor assays showed that the plastidial-precursor pathway (MEP) is fundamental for high-level production of both cytosolic as well as plastid-derived terpenes in tomato trichomes. Additionally, gene expression profiles of isolated secretory cells showed that key enzymes in the MEP pathway were higher expressed in active trichomes. We conclude that the MEP pathway is the primary precursor-supply route in wild tomato type-VI trichomes and that the high-production phenotype of the wild tomato trichome is indeed a multigenic trait.