Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes

Leaves of tobacco (Nicotiana tabacum) are covered with glandular trichomes that produce sucrose esters and diterpenoids in varying quantities, depending on cultivar type. The bicyclic diterpene Z‐abienol is the major labdanoid present in some oriental tobacco cultivars, where it constitutes a precursor of important flavours and aromas. We describe here the identification and characterization of two genes governing the biosynthesis of Z‐abienol in N. tabacum. As for other angiosperm labdanoid diterpenes, the biosynthesis of Z‐abienol proceeds in two steps. NtCPS2 encodes a class‐II terpene synthase that synthesizes 8‐hydroxy‐copalyl diphosphate, and NtABS encodes a kaurene synthase‐like (KSL) protein that uses 8‐hydroxy‐copalyl diphosphate to produce Z‐abienol. Phylogenetic analysis indicates that NtABS belongs to a distinct clade of KSL proteins that comprises the recently identified tomato (Solanum habrochaites) santalene and bergamotene synthase. RT‐PCR results show that both genes are preferentially expressed in trichomes. Moreover, microscopy of NtCPS2 promoter‐GUS fusion transgenics demonstrated a high specificity of expression to trichome glandular cells. Ectopic expression of both genes, but not of either one alone, driven by a trichome‐specific promoter in transgenic Nicotiana sylvestris conferred Z‐abienol formation to this species, which does not normally produce it. Furthermore, sequence analysis of over 100 tobacco cultivars revealed polymorphisms in NtCPS2 that lead to a prematurely truncated protein in cultivars lacking Z‐abienol, thus establishing NtCPS2 as a major gene controlling Z‐abienol biosynthesis in tobacco. These results offer new perspectives for tobacco breeding and the metabolic engineering of labdanoid diterpenes, as well as for structure–function relationship studies of terpene synthases.     

Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa

After herbivore damage, many plants increase their emission of volatile compounds, with terpenes usually comprising the major group of induced volatiles. Populus trichocarpa is the first woody species with a fully sequenced genome, enabling rapid molecular approaches towards characterization of volatile terpene biosynthesis in this and other poplar species. We identified and characterized four terpene synthases (PtTPS1-4) from P. trichocarpa which form major terpene compounds of the volatile blend induced by gypsy moth (Lymantria dispar) feeding. The enzymes were heterologously expressed and assayed with potential prenyl diphosphate substrates. PtTPS1 and PtTPS2 accepted only farnesyl diphosphate and produced (−)-germacrene D and (E,E)-α-farnesene as their major products, respectively. In contrast, PtTPS3 and PtTPS4 showed both mono- and sesquiterpene synthase activity. They produce the acyclic terpene alcohols linalool and nerolidol but exhibited opposite stereospecificity. qRT-PCR analysis revealed that the expression of the respective terpene synthase genes was induced after feeding of gypsy moth caterpillars. The TPS enzyme products may play important roles in indirect defense of poplar to herbivores and in mediating intra- and inter-plant signaling.     

Behavioural responses of the Colorado potato beetle to trichomes and leaf surface chemicals of Solanum tarijense

Wild Solanum species constitute a source of resistance to several pests and diseases of potato. Several species of wild tuber‐bearing potato have been identified as resistant to the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), including Solanum tarijense Hawkes (Solanaceae). Our objective was to determine the mechanism of resistance of S. tarijense to the Colorado potato beetle and, because the resistance is limited to the adult stage of the insect, to study the host selection behaviour on resistant plants. In the field, Colorado potato beetles demonstrated a unique behaviour when in contact with S. tarijense, abandoning the plant by falling to the ground after a few minutes. The abundant trichomes on the leaves of S. tarijense induced the falling behaviour. However, on S. tarijense feeding remained low even after the trichomes were mechanically removed. Observations demonstrated that the normal sequence of behaviour leading to feeding was interrupted before adult beetles fed on S. tarijense leaves. Feeding experiments using volatile and non‐volatile fractions of leaf surface extracts identified a phagodeterrent effect of the volatile fraction. Our results contrast with a similar evaluation of the mode of resistance of Solanum berthaultii Hawkes, a close relative of S. tarijense, on which some feeding occurred and adults did not show falling behaviour. This study presents information on S. tarijense as a new source of resistance to the Colorado potato beetle that can be used for potato breeding.     

A Novel Pathway for Sesquiterpene Biosynthesis from Z,Z-Farnesyl Pyrophosphate in the Wild Tomato Solanum habrochaites

In the wild tomato Solanum habrochaites, the Sst2 locus on chromosome 8 is responsible for the biosynthesis of several class II sesquiterpene olefins by glandular trichomes. Analysis of a trichome-specific EST collection from S. habrochaites revealed two candidate genes for the synthesis of Sst2-associated sesquiterpenes. zFPS encodes a protein with homology to Z-isoprenyl pyrophosphate synthases and SBS (for Santalene and Bergamotene Synthase) encodes a terpene synthase with homology to kaurene synthases. Both genes were found to cosegregate with the Sst2 locus. Recombinant zFPS protein catalyzed the synthesis of Z,Z-FPP from isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP), while coincubation of zFPS and SBS with the same substrates yielded a mixture of olefins identical to the Sst2-associated sesquiterpenes, including (+)-α-santalene, (+)-endo-β-bergamotene, and (−)-endo-α-bergamotene. In addition, headspace analysis of tobacco (Nicotiana sylvestris) plants expressing zFPS and SBS in glandular trichomes afforded the same mix of sesquiterpenes. Each of these proteins contains a putative plastid targeting sequence that mediates transport of a fused green fluorescent protein to the chloroplasts, suggesting that the biosynthesis of these sesquiterpenes uses IPP and DMAPP from the plastidic DXP pathway. These results provide novel insights into sesquiterpene biosynthesis and have general implications concerning sesquiterpene engineering in plants.     

Biosynthesis of the Diterpenoid Lycosantalonol via Nerylneryl Diphosphate in Solanum lycopersicum

We recently reported that three genes involved in the biosynthesis of monoterpenes in trichomes, a cis-prenyltransferase named neryl diphosphate synthase 1 (NDPS1) and two terpene synthases (TPS19 and TPS20), are present in close proximity to each other at the tip of chromosome 8 in the genome of the cultivated tomato (Solanum lycopersicum). This terpene gene “cluster” also contains a second cis-prenyltransferase gene (CPT2), three other TPS genes, including TPS21, and the cytochrome P450-oxidoreductase gene CYP71BN1. CPT2 encodes a neryneryl diphosphate synthase. Co-expression in E. coli of CPT2 and TPS21 led to the formation of the diterpene lycosantalene, and co-expression in E. coli of CPT2, TPS21 and CYP71BN1 led to the formation of lycosantalonol, an oxidation product of lycosantalene. Here we show that maximal expression of all three genes occurs in the petiolule part of the leaf, but little expression of these genes occurs in the trichomes present on the petiolules. While lycosantalene or lycosantalonol cannot be detected in the petiolules of wild-type plants (or anywhere else in the plant), lycosantalene and lycosantalonol are detected in petiolules of transgenic tomato plants expressing CPT2 under the control of the 35S CaMV promoter. These results suggest that lycosantalene and lycosantalonol are produced in the petiolules and perhaps in other tissues of wild-type plants, but that low rate of synthesis, controlled by the rate-limiting enzyme CPT2, results in product levels that are too low for detection under our current methodology. It is also possible that these compounds are further modified in the plant. The involvement of CPT2, TPS21 and CYP71BN1 in a diterpenoid biosynthetic pathway outside the trichomes, together with the involvement of other genes in the cluster in the synthesis of monoterpenes in trichomes, indicates that this cluster is further evolving into “sub-clusters” with unique biochemical, and likely physiological, roles.     

Expression of lima bean terpene synthases in rice enhances recruitment of a beneficial enemy of a major rice pest

Volatile terpenoids play a key role in plant defence against herbivory by attracting parasitic wasps. We identified seven terpene synthase genes from lima bean, Phaseolus lunatus L. following treatment with either the elicitor alamethicin or spider mites, Tetranychus cinnabarinus. Four of the genes (Pltps2, Pltps3, Pltps4 and Pltps5) were up‐regulated with their derived proteins phylogenetically clustered in the TPS‐g subfamily and PlTPS3 positioned at the base of this cluster. Recombinant PlTPS3 was able to convert geranyl diphosphate and farnesyl diphosphate to linalool and (E)‐nerolidol, the latter being precursor of the homoterpene (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT). Recombinant PlTPS4 showed a different substrate specificity and produced linalool and (E)‐nerolidol, as well as (E,E)‐geranyllinalool from geranylgeranyl diphosphate. Transgenic rice expressing Pltps3 emitted significantly more (S)‐linalool and DMNT than wild‐type plants, whereas transgenic rice expressing Pltps4 produced (S)‐linalool, DMNT and (E,E)‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT). In laboratory bioassays, female Cotesia chilonis, the natural enemy of the striped rice stemborer, Chilo suppressalis, were significantly attracted to the transgenic plants and their volatiles. We further confirmed this with synthetic blends mimicking natural rice volatile composition. Our study demonstrates that the transformation of rice to produce volatile terpenoids has the potential to enhance plant indirect defence through natural enemy recruitment.     

Spearmint R2R3‐MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU)

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT‐specific R2R3‐MYB gene, MsMYB, from comparative RNA‐Seq data of spearmint and functionally characterized it. Analysis of MsMYB‐RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB‐overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene‐ and diterpene‐derived metabolite production. In addition, we found that MsMYB binds to cis‐elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3‐MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.     

Functionally Divergent Alleles and Duplicated Loci Encoding an Acyltransferase Contribute to Acylsugar Metabolite Diversity in Solanum Trichomes

Glandular trichomes from tomato (Solanum lycopersicum) and other species in the Solanaceae produce and secrete a mixture of O-acylsugars (aliphatic esters of sucrose and glucose) that contribute to insect defense. Despite their phylogenetic distribution and diversity, relatively little is known about how these specialized metabolites are synthesized. Mass spectrometric profiling of acylsugars in the S. lycopersicum x Solanum pennellii introgression lines identified a chromosome 11 locus containing a cluster of BAHD acyltransferases with one gene (named Sl-ASAT3) expressed in tip cells of type I trichomes where acylsugars are made. Sl-ASAT3 was shown to encode an acyl-CoA-dependent acyltransferase that catalyzes the transfer of short (four to five carbons) branched acyl chains to the furanose ring of di-acylsucrose acceptors to produce tri-acylsucroses, which can be further acetylated by Sl-ASAT4 (previously Sl-AT2). Among the wild tomatoes, diversity in furanose ring acyl chains on acylsucroses was most striking in Solanum habrochaites. S. habrochaites accessions from Ecuador and northern Peru produced acylsucroses with short (≤C5) or no acyl chains on the furanose ring. Accessions from central and southern Peru had the ability to add short or long (up to C12) acyl chains to the furanose ring. Multiple ASAT3-like sequences were found in most accessions, and their in vitro activities correlated with observed geographical diversity in acylsugar profiles.     

Location of resistance factors in the leaves of potato and wild tuber-bearing Solanum species to the aphid Myzus persicae

Analysis of electrically recorded feeding behaviour of aphids was combined with colony‐development tests to search for sources of resistance to Myzus persicae (Sulzer) (Homoptera: Aphididae) in tuber‐bearing Solanum species (Solanaceae), aiming at a reduction of potato leaf roll virus (PLRV) transmission. Twenty genotypes, originating from 14 gene bank accessions, representing 13 wild tuber‐bearing Solanum spp., three Solanum tuberosum L. (potato) cultivars, and one S. tuberosum breeding line, were selected. Colony‐development tests were carried out in no‐choice experiments by placing adult aphids on plants of each genotype and counting numbers of nymphs and adults on young plants after 8 and 15 days, and on flowering plants after 14 and 30 days. Large differences were observed among genotypes: some developed small colonies and others developed large ones. Also, in a few genotypes, resistance in mature plants was different for leaves of different ages; young leaves were resistant to aphids whereas old senescent leaves were susceptible. The electrical penetration graph (DC‐EPG system) technique was used to study aphid feeding behaviour on each Solanum genotype for 6 h. Electrical penetration graph (EPG) results also showed large differences among the genotypes, indicating resistance at the leaf surface and at three different levels of plant tissue (epidermis, mesophyll, and phloem). Therefore, it was concluded that different mechanisms of resistance to M. persicae exist among the genotypes analysed. EPGs recorded from aphids on Solanum berthaultii Hawkes and Solanum tarijense Hawkes with and without glandular trichomes showed that strong surface resistance can bias EPG parameters associated with resistance located in deeper tissues. Experimental evidence is presented that the resistance to aphids in the genotypes with glandular trichomes strongly depends on these morphological structures.     

Tomato linalool synthase is induced in trichomes by jasmonic acid

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato.     

Trichome‐based host plant resistance of Lycopersicon species and the biocontrol agent Mallada signata: are they compatible?

Trichome‐based host plant resistance of Lycopersicon (Solanaceae) species offers the potential to reduce pesticide use in tomato production, but its compatibility with biocontrol agents is largely unknown. The effect of trichome‐based host plant resistance on the lacewing biocontrol agent, Mallada signata, was assessed for five accessions of L. cheesmanii, four accessions of Lycopersicon hirsutum, two accessions of Lycopersicon pennellii, and one Lycopersicon esculentum cultivar. An intact leaf was isolated from the whole plant using Tangletrap to coat the petiole and 20 green peach aphids [Myzus persicae (Sulzer) (Homoptera: Aphididae)] were placed on the leaf surface. After 24 h, 10 lacewings were placed on the leaf. The numbers of dead, trapped by exudates, untrapped and predated lacewings and aphids, and the numbers that had left the leaf were recorded a further 24 h later. Differences in insect designations between accessions were analysed using ANOVA. A General Linear Model (GLM), consisting of the densities of each trichome type and leaf area, was fitted to the data to determine the role of trichomes on the observed effects on lacewings and aphids. Lacewing mortality was greater on one accession of L. pennellii and one accession of L. hirsutum than on L. esculentum. The GLM indicated that type IV trichomes decreased the numbers of aphids predated, and increased cannibalism and, along with type III trichomes, increased entrapment‐related predator mortality. Although there were no differences in the numbers of predated aphids, with the majority predated for all accessions, the compatibility of trichome‐based host plant resistance of Lycopersicon species and the biocontrol of aphids by lacewings is questionable.