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.

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.

A growth- and bioluminescence-based bioreporter for the in vivo detection of novel biocatalysts.

The use of bioreporters in high-throughput screening for small molecules is generally laborious and/or expensive. The technology can be simplified by coupling the generation of a desired compound to cell survival, causing only positive cells to stay in the pool of generated variants. Here, a dual selection/screening system was developed for the in vivo detection of novel biocatalysts. The sensor part of the system is based on the transcriptional regulator AraC, which controls expression of both a selection reporter (LeuB or KmR; enabling growth) for rapid reduction of the initially large library size and a screening reporter (LuxCDABE; causing bioluminescence) for further quantification of the positive variants. Of four developed systems, the best system was the medium copy system with KmR as selection reporter. As a proof of principle, the system was tested for the selection of cells expressing an l-arabinose isomerase derived from mesophilic Escherichia coli or thermophilic Geobacillus thermodenitrificans. A more than a millionfold enrichment of cells with l-arabinose isomerase activity was demonstrated by selection and exclusion of false positives by screening. This dual selection/screening system is an important step towards an improved detection method for small molecules, and thereby for finding novel biocatalysts.

Reduced seed germination in Arabidopsis over-expressing SWI/SNF2 ATPase genes.

In the life of flowering plants, seed germination is a critical step to ensure survival into the next generation. Generally the seed prior to germination has been in a dormant state with a low rate of metabolism. In the transition from a dormant seed to a germinating seed, various epigenetic mechanisms play a regulatory role. Here, we demonstrate that the over-expression of chromatin remodeling ATPase genes (AtCHR12 or AtCHR23) reduced the frequency of seed germination in Arabidopsis thaliana up to 30% relative to the wild-type seeds. On the other hand, single loss-of-function mutations of the two genes did not affect seed germination. The reduction of germination in over-expressing mutants was more pronounced in stress conditions (salt or high temperature), showing the impact of the environment. Reduced germinations upon over-expression coincided with increased transcript levels of seed maturation genes and with reduced degradation of their mRNAs stored in dry seeds. Our results indicate that repression of AtCHR12/23 gene expression in germinating wild-type Arabidopsis seeds is required for full germination. This establishes a functional link between chromatin modifiers and regulatory networks towards seed maturation and germination.