Petra M. Bleeker

The Tomato Terpene Synthase Gene Family

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.

The Role of Specific Tomato Volatiles in Tomato-Whitefly Interaction

Bemisia tabaci (whitefly) infestations and the subsequent transfer of viruses are the cause of severe losses in crop production and horticultural practice. To improve biological control of B. tabaci, we investigated repellent properties of plant-produced semiochemicals. The mix of headspace volatiles, collected from naturally repellent wild tomato accessions, influenced B. tabaci initial choice behavior, indicating a role for plant semiochemicals in locating host plants. A collection of wild tomato accessions and introgression lines (Solanum pennellii LA716 × Solanum lycopersicum ‘Moneyberg’) were extensively screened for attractiveness to B. tabaci, and their headspace profiles were determined by means of gas chromatography-mass spectrometry. Correlation analysis revealed that several terpenoids were putatively involved in tomato-whitefly interactions. Several of these candidate compounds conferred repellence to otherwise attractive tomato plants when applied to the plants branches on paper cards. The sesquiterpenes zingiberene and curcumene and the monoterpenes p-cymene, α-terpinene, and α-phellandrene had the strongest effects in free-choice bioassays. These terpenes also elicited a response of receptors on the insects antennae as determined by electroantennography. Conversely, the monoterpene β-myrcene showed no activity in both assays. B. tabaci apparently uses, besides visual cues, specific plant volatile cues for the initial selection of a host. Altering whitefly choice behavior by manipulation of the terpenoid composition of the host headspace may therefore be feasible.

Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative

Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.

Tomato-produced 7-epizingiberene and R-curcumene act as repellents to whiteflies

How whiteflies (Bemisia tabaci) make the choice for a host plant prior to landing, is not precisely known. Here we investigated whether they respond to specific volatiles of tomato. Zingiberene and curcumene were purified from Solanum habrochaites (PI127826), characterised by NMR and X-ray analysis and identified as 7-epizingiberene and R-curcumene. In contrast, oil from Zingiber officinalis contained the stereoisomers zingiberene and S-curcumene, respectively. Using a combination of free-choice bio-assays and electroantennography, 7-epizingiberene and its dehydrogenated derivative R-curcumene were shown to be active as semiochemicals to B. tabaci adults, whereas the stereoisomers from ginger were not. In addition, R-curcumene elicited the strongest electroantennographic response. Bio-assays showed that a cultivated tomato could be made less attractive to B. tabaci than its neighbouring siblings by the addition of the tomato stereoisomer 7-epizingiberene or its derivative R-curcumene. These sesquiterpenes apparently repel adult whiteflies prior to landing, presumably because it informs them that after landing they, or their offspring, may be exposed to higher and lethal concentrations of the same compounds.

RNA-seq discovery, functional characterization, and comparison of sesquiterpene synthases from Solanum lycopersicum and Solanum habrochaites trichomes

Solanum lycopersicum and Solanum habrochaites (f. typicum) accession PI127826 emit a variety of sesquiterpenes. To identify terpene synthases involved in the production of these volatile sesquiterpenes, we used massive parallel pyrosequencing (RNA-seq) to obtain the transcriptome of the stem trichomes from these plants. This approach resulted initially in the discovery of six sesquiterpene synthase cDNAs from S. lycopersicum and five from S. habrochaites. Searches of other databases and the S. lycopersicum genome resulted in the discovery of two additional sesquiterpene synthases expressed in trichomes. The sesquiterpene synthases from S. lycopersicum and S. habrochaites have high levels of protein identity. Several of them appeared to encode for non-functional proteins. Functional recombinant proteins produced germacrenes, β-caryophyllene/α-humulene, viridiflorene and valencene from (E,E)-farnesyl diphosphate. However, the activities of these enzymes do not completely explain the differences in sesquiterpene production between the two tomato plants. RT-qPCR confirmed high levels of expression of most of the S. lycopersicum sesquiterpene synthases in stem trichomes. In addition, one sesquiterpene synthase was induced by jasmonic acid, while another appeared to be slightly repressed by the treatment. Our data provide a foundation to study the evolution of terpene synthases in cultivated and wild tomato.

Plant Volatiles in Defence.

Abstract Plant odours are the most ubiquitous volatiles in nature. This chapter deals with the biochemistry and molecular biology of plant volatiles that are emitted from vegetative tissues during pathogen- or herbivore-induced stress and the contribution of these volatiles to plant defences. While mechanical tissue wounding causes the non-specific release of volatiles, herbivore-specific elicitors from their saliva distinctly alter the volatile signature. These volatiles acquired diverse roles in ecological interactions. Firstly, they can be toxic to, or repel conspecific or other herbivores from already infested plants. This function is referred to as ‘direct defence’. Secondly, volatiles function as prey-associated signals for foraging carnivorous arthropods. This is referred to as ‘indirect defence’. Finally, volatiles elicit metabolic changes in unattacked neighbouring plants resulting in priming and induction of defences already before herbivores have arrived. This is referred to as ‘plant–plant communication’. Although volatile production by native plants and crops is highly variable under different growth conditions and during development, its function in direct and indirect defences is remarkably robust. With the current state of knowledge, it is now possible to manipulate these direct and indirect defences through breeding or transgenic approaches. Taken together, plant volatiles play profound roles in plant–herbivore and plant–pathogen interactions and are promising targets for improved crop protection.

Engineering of Tomato Glandular Trichomes for the Production of Specialized Metabolites

Glandular trichomes are specialized tissues on the epidermis of many plant species. On tomato they synthesize, store, and emit a variety of metabolites such as terpenoids, which play a role in the interaction with insects. Glandular trichomes are excellent tissues for studying the biosynthesis of specialized plant metabolites and are especially suitable targets for metabolic engineering. Here we describe the strategy for engineering tomato glandular trichomes, first with a transient expression system to provide proof of trichome specificity of selected promoters. Using microparticle bombardment, the trichome specificity of a terpene-synthase promoter could be validated in a relatively fast way. Second, we describe a method for stable expression of genes of interest in trichomes. Trichome-specific expression of another terpene-synthase promoter driving the yellow-fluorescence protein-gene is presented. Finally, we describe a case of the overexpression of farnesyl diphosphate synthase (FPS), specifically in tomato glandular trichomes, providing an important precursor in the biosynthetic pathway of sesquiterpenoids. FPS was targeted to the plastid aiming to engineer sesquiterpenoid production, but interestingly leading to a loss of monoterpenoid production in the transgenic tomato trichomes. With this example we show that trichomes are amenable to engineering though, even with knowledge of a biochemical pathway, the result of such engineering can be unexpected.

Small RNAs from Bemisia tabaci Are Transferred to Solanum lycopersicum Phloem during Feeding

The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

Thrips advisor : Exploiting thrips-induced defences to combat pests on crops

Plants have developed diverse defence mechanisms to ward off herbivorous pests. However, agriculture still faces estimated crop yield losses ranging from 25% to 40% annually. These losses arise not only because of direct feeding damage, but also because many pests serve as vectors of plant viruses. Herbivorous thrips (Thysanoptera) are important pests of vegetable and ornamental crops worldwide, and encompass virtually all general problems of pests: they are highly polyphagous, hard to control because of their complex lifestyle, and they are vectors of destructive viruses. Currently, control management of thrips mainly relies on the use of chemical pesticides. However, thrips rapidly develop resistance to these pesticides. With the rising demand for more sustainable, safer, and healthier food production systems, we urgently need to pinpoint the gaps in knowledge of plant defences against thrips to enable the future development of novel control methods. In this review, we summarize the current, rather scarce, knowledge of thrips-induced plant responses and the role of phytohormonal signalling and chemical defences in these responses. We describe concrete opportunities for breeding resistance against pests such as thrips as a prototype approach for next-generation resistance breeding.

The Impact of Induced Plant Volatiles on Plant-Arthropod Interactions

Plants release volatile organic compounds from their vegetative tissues into their environment during most of their life cycle. The functions of these volatiles are diverse and not always known but some of these volatiles repel foraging herbivores while others, in turn, attract them and are feeding stimuli. Upon herbivory the amount of volatiles increases dramatically while, simultaneously, also the composition of the blend changes thereby enhancing the attractiveness of the plant to foraging natural enemies and in some cases increasing repellency to herbivores. Hence, herbivore-induced volatiles promote a natural form of biological pest control referred to as “indirect plant defense” and it has often been suggested that this phenomenon could be exploited to enhance crop protection. Here we will introduce the concept of indirect plant defense via volatiles and via other means and outline the current state of knowledge to the extent in which it contributes to protecting a plant to maximize its fitness under natural conditions in an evolutionary and ecological context. Moreover we will summarize the different approaches that have been undertaken to manipulate indirect defenses, either via application of synthetic volatiles or via transgenic manipulation of plant-volatile production, to control the movements of foraging arthropods to improve biological control. Finally, we will discuss to which extent IPM can be improved or even be disrupted via manipulation of plant volatiles.