Expression Of Terpene Synthase Genes Research Articles

Identification of TPS-d subfamily genes and functional characterization of three monoterpene synthases in slash pine

Oleoresin terpenoids have been widely used in the food, pharmaceutical, and chemical industries. A thorough understanding of oleoresin terpenoid biosynthesis is important for the metabolic engineering of high-valued terpenoid products in plants and microbes and the improvement of oleoresin traits using molecular breeding strategies. TPS-d subfamily members, specific to conifers, determine the oleoresin terpenoid compound diversity. Here, we aimed to identify the TPS-d genes in slash pine (Pinus elliottii) and characterize their function and expression patterns in population. Our study reveals two main results: (1) A total of 52 terpene synthase (TPS) candidate genes were identified based on long-read transcripts of slash pine, 12 of which were successfully cloned from the secondary developing xylem RNA. Two levopimaradiene/abietadiene synthases (LAS), one monofunctional isopimaradiene synthase (mISO1)-like, and two α-pinene synthases were identified by comparing with previously known TPS. Next, the two α-pinene synthases and one β-pinene synthase producing multiple products were validated through heterologous expression and in vitro enzyme activity assays, which showed different product profiles from previously identified TPS. (2) The expression status of the cloned TPS genes in 240 slash pine individuals were analyzed. Four exhibited very low expression levels, including those identical to copalyl diphosphate synthases, hemiterpene synthases, diterpene synthases without activity, and one obtained without soluble protein in the heterologous expression. The expression levels of eight TPS genes participating in slash pine oleoresin biosynthesis varied in different individuals and were highly correlated with multiple transcriptional regulators, namely, MYB and MYC. The expression of two α-pinene synthase genes was significantly higher than that of the other TPS genes. Some TPS gene expression levels were positively correlated with the corresponding oleoresin component content. Our results provide an essential reference for exploring the mechanisms of terpene biosynthesis and regulation.

Identification and characterization of three nearly identical linalool/nerolidol synthase from Acorus calamus

Acorus calamus is a perennial aromatic medicinal plant from the Acorusaceae family, known for its pharmaceutical and medicinal value. A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of volatile organic compounds (VOCs) in rhizomes and leaves of A. calamus essential oil. Here, we performed VOC profiling and transcriptome-based identification and functional characterization of terpene synthase (TPS) genes. A total of 110 VOCs were detected from the rhizomes and leaves of A. calamus, and some VOCs showed significant differences between them. The further transcriptome-based analysis led to the identification of six putative TPSs genes. In phylogenetic analysis, three TPSs belonged to the TPS-g clade, one to each of the TPS-a, TPS-c, and TPS-e clades. The heterologous E. coli-based expression of recombinant TPSs identified three genes (AcTPS3, AcTPS4, and AcTPS5) as bifunctional linalool/nerolidol synthase. The correlation of TPS gene expression and VOC metabolite profiles supported the function of these genes in A. calamus. Our findings provide a roadmap for future efforts to enhance the molecular mechanisms of terpene biosynthesis and our understanding of Acorus-insect interactions.

Dynamic regulation of volatile terpenoid production and emission from Chrysanthemum morifolium capitula

Flower-associated communities consist of both mutualistic and antagonistic organisms. We have limited knowledge on how flowers regulate volatiles to balance their defense against antagonists and the attraction of beneficial organisms necessary for reproductive success. Asteraceae is the largest family among flowering plants. Its representatives are characterized by unique inflorescence called capitulum, which has been reduced to a reproduction unit resembling a single flower. Here, we chose Chrysanthemum morifolium, a model species of Asteraceae, to investigate how the capitulum balances the accumulation and emission of floral terpenoid volatiles that are implicated in defense and pollinator attraction, respectively. Our results showed that the capitula of C. morifolium produce and emit complex mixtures of monoterpenoids and sesquiterpenoids. The highest concentrations of terpenoids were detected in the bud stage of the capitula. In contrast, the capitulum reached the highest emission level prior to full blooming. The disc florets were the dominant organs of terpenoid accumulation and emission in the full-openness stage. To understand the molecular basis of volatile terpenoid biosynthesis in C. morifolium, experiments were designed to study terpene synthase (TPS) genes, which are pivotal for terpene biosynthesis. Eight CmCJTPS genes were identified in the transcriptomes of C. morifolium, and the proteins encoded by five genes were found to be biochemically functional. CmCJTPS5 and CmCJTPS8 were the multi-product enzymes catalyzing the monoterpenoid and sesquiterpenoid formation, which closely matched the major terpenoids produced in the flower heads. The five functional terpene synthase genes exhibited similar temporal expression patterns but diverse spatial expression levels, suggesting tissue-specific functions. Altogether, our results illustrate the dynamic patterns of accumulation and emission of floral volatile terpenoids implicated in defense and attracting pollinators in C. morifolium, for which both the regulation of TPS gene expression and the regulation of release may play critical roles.

Genome-wide identification and characterization of terpene synthase genes in Gossypium hirsutum

Terpenoids are widely distributed in plants and play important roles in the regulation of plant growth and development and in the interactions between plants and both the environment and other organisms. However, terpene synthase (TPS) genes have not been systematically investigated in the tetraploid Gossypium hirsutum. In this study, whole genome identification and characterization of the TPS family from G. hirsutum were carried out. Eighty-five TPS genes, including 47 previously unidentified genes, were identified in the G. hirsutum genome and classified into 5 subfamilies according to protein sequence similarities, as follows: 43 GhTPS-a, 29 GhTPS-b, 4 GhTPS-c, 7 GhTPS-e/f, and 2 GhTPS-g members. These 85 TPS genes were mapped onto 19 chromosomes of the G. hirsutum genome. Segmental duplications and tandem duplications contributed greatly to the expansion of TPS genes in G. hirsutum and were followed by intense purifying selection during evolution. Indentification of cis-acting regulatory elements suggest that the expression of TPS genes is regulated by a variety of hormones. RNA sequencing (RNA-seq) expression profile analysis revealed that the TPS genes had distinct spatiotemporal expression patterns, and several genes were highly and preferentially expressed in the leaves of cotton with gossypol glands (glanded cotton) versus a glandless strain. Virus-induced gene silencing (VIGS) of three TPS genes yielded plants characterized by fewer, smaller, and lighter gossypol glands, which indicated that these three genes were responsible for gland activity. Taken together, our results provide a solid basis for further elucidation of the biological functions of TPS genes in relation to gland activity and gossypol biosynthesis to develop cotton cultivars with low cottonseed gossypol contents.

Terpene synthase gene expression evaluation through RT-qPCR in Thymus caespititius

Thymus caespititius Brot. is an interesting aromatic plant due to the valuable monoterpenes it produces considering their properties and biological activities. Several chemotypes have been identified in this species, however, essential oil composition can vary throughout the year in the terpenes’ relative percentage. The first objective was to evaluate chemical stability, based on a 20-month survey in essential oil (EO) composition through GC and GC-MS, in two accessions belonging to the carvacrol chemotype, Tc2 and Tc4. Together with this evaluation, a quantitative study of gene expression was performed through reverse transcription quantitative PCR (RT-qPCR), for the most important genes involved in the biosynthesis of γ-terpinene (precursor of thymol and carvacrol), TcTPS2 and TcTPS4, and α-terpineol, TcTPS5. This second objective addressed the correlation between seasonal EO variation and terpene synthase gene expression patterns and compared the trends of seasonal variation between both plant accessions. The results show 22, 48 and 90-fold variation in gene expression for TcTPS2, TcTPS4 and TcTPS5, respectively. Expression of TcTPS5 was much more variable than those coding for γ-terpinene synthase. The two accessions evaluated showed Tc2 had a more stable gene expression compared to Tc4. Although gene expression and terpene accumulation were in accordance for most timepoints, no significant overall correlation could be found. Our results suggest a transcriptional regulation for TcTPS2 and TcTPS4. For TcTPS5 regulation at post transcriptional level must be present, accounting for differences in the α-terpineol relative amount and adding new layers of complexity in specialized metabolism. Further studies should be pursued to identify all the players involved in controlling the final bouquet of essential oil composition.

Genome-wide identification and expression analysis of terpene synthase gene family in Aquilaria sinensis

Agarwood is the resinous portion of Aquilaria trees, and has been widely used as medicine and incense. Sesquiterpenes are the main chemical characteristic constituents of agarwood. Terpene synthase (TPS) is a critical enzyme responsible for biosynthesis of sesquiterpene compounds. However, limited information is available on genome-wide identification and characterization of the TPS family in Aquilaria trees. In this study, TPS gene family was identified and characterized in Aquilaria sinensis by bioinformatics methods. The expression of those genes was analyzed by RNA-seq and quantitative real-time PCR. Transcription factors regulating TPS gene expression were identified by yeast one-hybrid and dual-luciferase assay. In total, 26 AsTPS genes (AsTPS1–AsTPS26) were identified, which were classified into five subgroups. Many putative cis-elements putatively involved in stresses and phytohormones (especially jasmonic acid) were identified in the promoter regions of AsTPSs, suggesting that AsTPSs genes may be regulated by stresses and jasmonic acid. Expression analysis revealed seven TPS genes encoding sesquiterpene synthetases were induced by wounding and methyl jasmonic acid (MeJA), which may be related to sesquiterpene biosynthesis. By yeast one-hybrid screening, a ERF transcription factor AsERF1 was identified to interact with the AsTPS1 promoter. Subcellular localization analysis indicated AsERF1 was a nucleus-localized protein. Transient transfection of AsERF1 in leaves of Nicotiana benthamiana significantly enhanced the promoter activation of AsTPS1, suggesting AsERF1 may participate in sesquiterpene biosynthesis by regulating AsTPS1 expression. These data generated in this study provide a foundation for future studies on functional roles and regulation mechanisms of AsTPS in sesquiterpene biosynthesis and agarwood formation.

Genome-wide identification and expression pattern of SnRK gene family under several hormone treatments and its role in floral scent emission in Hedychium coronarium.

The SnRK (Snf1-Related protein Kinase) gene family plays crucial roles in various plant signaling pathways and stress-adaptive responses including biotic and abiotic stresses via activating protein phosphorylation pathways. However, there is no information available on the role of the SnRK gene family in Hedychium coronarium. H. coronarium is an important crop widely cultivated as an ornamental plant, herb, spice, or condiment. In this study, 60 HcSnRK genes were identified from the H. coronarium genomic and transcriptome data. Phylogenetic and gene structure analysis showed that the HcSnRK genes were divided into three groups (HcSnRK1, HcSnRK2 and HcSnRK3) and among them HcSnRK3 subfamily was further subdivided into two clades according to the number of introns. Chromosome localization analysis showed that HcSnRK genes were unevenly mapped onto all chromosomes, and the Ka/Ks ratio of 24 paralogues includes four tandems and 20 segmental duplications indicated that the HcSnRK gene family underwent a purifying selection. Cis-regulatory elements analysis suggested that the HcSnRK genes respond to multiple hormones and other stresses. The responsiveness of HcSnRK genes to several hormones was analyzed by quantitative real-time PCR. Based on the different transcriptome data, two candidates HcSnRK genes (HcSnRK2.2 and HcSnRK2.9) were screened out for further characterization . The subcellular localization experiment revealed that both genes were located in the nucleus and cytoplasm. Moreover, virus-induced gene silencing (VIGS) of HcSnRK2.2 and HcSnRK2.9 significantly reduced the floral volatile contents by suppressing the expression of terpene synthase genes (HcTPS1, HcTPS3, and HcTPS5), indicating that HcSnRK2.2 and HcSnRK2.9 genes play an important role in the regulatory mechanism of floral aroma. These results will provide novel insights into the functional dissection of H. coronarium SnRK gene family.

Transcriptomics Integrated with Free and Bound Terpenoid Aroma Profiling during "Shine Muscat" (Vitis labrusca × V. vinifera) Grape Berry Development Reveals Coordinate Regulation of MEP Pathway and Terpene Synthase Gene Expression.

Terpenes and their derivatives are important biomarkers of grape quality as they contribute to the flavor and aroma of grapes. However, the molecular basis of terpene biosynthesis throughout the grapevine phenological developmental cycle remains elusive. Our current study investigates the free and bound terpene biosynthesis of berries at different phenological stages from preveraison to harvest. Detailed gene expression (transcriptomics) analysis, terpenoid volatile production by gas chromatography-mass spectrometry (GC-MS), and in planta transient expression were employed. Our results show that concentrations of most individual terpenes at different stages are distinctive and increase from preveraison to the veraison stage followed by a decrease from veraison to maturity. The combined transcriptomic analysis and terpene profiling revealed that 22 genes belonging to the MEP pathway and multiple classes of transcription factor family members including bHLH and several hormone biosynthesis- or signaling-related genes likely participate in the regulation of terpenoid biosynthesis according to their specific expression patterns in berries. Quantitative real-time polymerase chain expression analysis of 8 key differentially expressed genes in MEP pathways and further 12 randomly selected genes was performed during 8 sampling stages and validated the RNA-seq-derived expression profiles. To further confirm the function of a subset of the differentially expressed genes, we investigated the effects of combined overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (VvDXS1-LOC100249323), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (VvDXR-LOC100248516), and terpene synthase (VvTPS56-LOC100266449) on the production of terpenes by transient overexpression in Nicotiana benthamiana leaves. The overall developmental patterns of total terpenes and gene expression profiles will help guide the functional analyses of further candidate genes important for terpene biosynthesis of grape as well as identifying the master transcriptional and hormonal regulators of this pathway in the future.

Monoterpene synthases responsible for the terpene profile of anther glands in Eucalyptus polybractea R.T. Baker (Myrtaceae).

Research on terpene biosynthesis in the genus Eucalyptus (Myrtaceae) is poorly developed, but recently large numbers of terpene synthase (TPS) genes have been identified. Few of these have been characterized or their expression localized to specific tissues. A prime candidate for detailed examination of TPS gene expression is the bisexual eucalypt flower-composed of male and female reproductive organs, and vegetative tissues that may express different TPS genes. We aimed to characterize and compare the terpene profile and TPS genes expressed in anthers and gynoecia in the high oil-yielding Eucalyptus polybractea R.T. Baker. We hypothesized that gynoecia will produce greater amounts of defensive terpenes, whereas anthers will have a terpene profile that is biased towards a role in pollination. Microscopy of isolated anthers showed them to possess a single, prominent oil gland. Chemical analysis of whole floral structures at different stages of development showed total oil per unit dry mass increased as flower buds expanded, with highest concentrations in mature flower buds just prior to flower opening. The oil profile of gynoecia was dominated by the monoterpene 1,8-cineole, whereas that of isolated anthers were enriched with the monoterpene α-pinene. Through transcriptomic analysis and recombinant protein expression, we were able to identify monoterpene synthases responsible for the different profiles. Synthases for α-pinene and 1,8-cineole were expressed in each tissue type, but the relative expression of the former was higher in anthers. Sequence comparison and site-directed mutagenesis of the α-pinene synthase allowed us to identify amino acids that influence the α-pinene to β-pinene ratio of the product profile. We suggest the terpene constituents of anthers may have multiple roles including attracting pollinators through emission of volatile α-pinene, deterrence of palynivores through emission of volatile 1,8-cineole and adhesion of pollen to pollinators via the release of sticky α-pinene onto the anther surface.

Diversity and function of terpene synthases in the production of carrot aroma and flavor compounds

Carrot (Daucus carota L.) is an important root vegetable crop with high nutritional value, characteristic flavor, and benefits to human health. D. carota tissues produce an essential oil that is rich in volatile terpenes and plays a major role in carrot aroma and flavor. Although terpene composition represents a critical quality attribute of carrots, little is known about the biosynthesis of terpenes in this crop. Here, we functionally characterized 19 terpene synthase (TPS) genes in an orange carrot (genotype DH1) and compared tissue-specific expression profiles and in vitro products of their recombinant proteins with volatile terpene profiles from DH1 and four other colored carrot genotypes. In addition to the previously reported (E)-β-caryophyllene synthase (DcTPS01), we biochemically characterized several TPS proteins with direct correlations to major compounds of carrot flavor and aroma including germacrene D (DcTPS7/11), γ-terpinene (DcTPS30) and α-terpinolene (DcTPS03). Random forest analysis of volatiles from colored carrot cultivars identified nine terpenes that were clearly distinct among the cultivars and likely contribute to differences in sensory quality. Correlation of TPS gene expression and terpene metabolite profiles supported the function of DcTPS01 and DcTPS03 in these cultivars. Our findings provide a roadmap for future breeding efforts to enhance carrot flavor and aroma.

MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana.

Previously, linalool was found to be the most abundant component among the cocktail of volatiles released from flowers of Freesia hybrida. Linalool formation is catalysed by monoterpene synthase TPS1. However, the regulatory network developmentally modulating the expression of the TPS1 gene in Freesia hybrida remains unexplored. In this study, three regulatory genes, FhMYB21L1, FhMYB21L2, and FhMYC2, were screened from 52 candidates. Two MYB transcription factor genes were synchronously expressed with FhTPS1 and could activate its expression significantly when overexpressed, and the binding of FhMYB21L2 to the MYBCORE sites in the FhTPS1 promoter was further confirmed, indicating a direct role in activation. FhMYC2 showed an inverse expression pattern compared with FhTPS1; its expression led to a decreased binding of FhMYB21 to the FhTPS1 promoter to reduce its activation capacity when co-expressed, suggesting a role for an MYB-bHLH complex in the regulation of the FhTPS1 gene. In Arabidopsis, both MYB21 and MYC2 regulators were shown to activate the expression of sesquiterpene synthase genes, and the regulatory roles of AtMYB21 and AtMYC2 in the expression of the linalool synthase gene were also confirmed, implying conserved functions of the MYB-bHLH complex in these two evolutionarily divergent plants. Moreover, the expression ratio between MYB21 and MYC2 orthologues might be a determinant factor in floral linalool emission.

Oleoresin defenses in conifers: chemical diversity, terpene synthases and limitations of oleoresin defense under climate change.

Conifers have evolved complex oleoresin terpene defenses against herbivores and pathogens. In co-evolved bark beetles, conifer terpenes also serve chemo-ecological functions as pheromone precursors, chemical barcodes for host identification, or nutrients for insect-associated microbiomes. We highlight the genomic, molecular and biochemical underpinnings of the large chemical space of conifer oleoresin terpenes and volatiles. Conifer terpenes are predominantly the products of the conifer terpene synthase (TPS) gene family. Terpene diversity is increased by cytochromes P450 of the CYP720B class. Many conifer TPS are multiproduct enzymes. Multisubstrate CYP720B enzymes catalyse multistep oxidations. We summarise known terpenoid gene functions in various different conifer species with reference to the annotated terpenoid gene space in a spruce genome. Overall, biosynthesis of terpene diversity in conifers is achieved through a system of biochemical radiation and metabolic grids. Expression of TPS and CYP720B genes can be specific to individual cell types of constitutive or traumatic resin duct systems. Induced terpenoid transcriptomes in resin duct cells lead to dynamic changes of terpene composition and quantity to fend off herbivores and pathogens. While terpenoid defenses have contributed much to the evolutionary success of conifers, under new conditions of climate change, these defences may become inconsequential against range-expanding forest pests.

Terpene Synthase Genes in Quercus robur - Gene Characterization, Expression and Resulting Terpenes Due to Cockchafer Feeding.

Root herbivory caused by larvae of the forest cockchafer (Melolontha hippocastani) enhances the impact of drought on trees, particularly in oak forest rejuvenations. In Germany, geographically distant oak stands show differences in infestation strength by the forest cockchafer. While in Southwestern Germany this insect causes severe damage, oak forests in northern Germany are rarely infested. It is known that root-released volatile organic compounds (VOCs) are perceived by soil herbivores, thus guiding the larvae toward the host roots. In this work, we exposed seedlings of two distant oak provenances to forest cockchafer larvae and studied their population genetic properties, their root-based VOC chemotypes, their attraction for larvae and terpene synthase gene expression. Based on nuclear and chloroplast marker analysis, we found both oak populations to be genetically highly variable while showing typical patterns of migration from different refugial regions. However, no clear association between genetic constitution of the different provenances and the abundance of cockchafer populations on site was observed. In contrast to observations in the field, bioassays revealed a preference of the larvae for the northeastern oak provenance. The behavior of larvae was most likely related to root-released volatile terpenes and benzenoids since their composition and quantity differed between oak populations. We assume repellent effects of these compounds because the populations attractive to insects showed low abundance of these compounds. Five different oak terpene synthase (TPS) genes were identified at the genomic level which can be responsible for biosynthesis of the released terpenes. TPS gene expression patterns in response to larval feeding revealed geographic variation rather than genotypic variation. Our results support the assumption that root-released VOC are influencing the perception of roots by herbivores.

Temporal regulation of terpene synthase gene expression in Eucalyptus globulus leaves upon ozone and wounding stresses: relationships with stomatal ozone uptake and emission responses

Ozone and wounding are key abiotic factors but, their interactive effects on temporal changes in terpene synthase gene expression and emission responses are poorly understood. Here, we applied combined acute ozone and wounding stresses to the constitutive isoprenoid-emitter Eucalyptus globulus and studied how isoprene, 1,8-cineole, and isoledene synthase genes were regulated, and how the gene expression was associated with temporal changes in photosynthetic characteristics, product emission rates, and stomatal ozone uptake through recovery phase. Photosynthetic characteristics and emission rate of isoprene, 1,8-cineole, and isoledene were synergistically altered, while three TPS gene expressions were antagonistically altered by combined stress applications. A time-delay analysis indicated that the best correspondences between gene expression and product emission rates were observed for 0 h time-shift for wounding and 0–2 h time-shifts for separate ozone, and combined ozone and wounding treatments. The best correspondence between ozone uptake and gene expression was observed for 0–4 h time-shifts for separate ozone and combined ozone and wounding treatments. Overall, this study demonstrated that expression profiles of isoprene, the monoterpene 1,8-cineole, and the sesquiterpene isoledene synthase genes differentially influenced their corresponding product emissions for separate and combined ozone and wounding treatments through recovery.

Temporal and spatial expression of terpene synthase genes associated with agarwood formation in Aquilaria malaccensis Lam

The diseased wood, agarwood, from the tropical tree taxa Aquilaria, is famed for its unique fragrance and medicinal values, mainly due to its richness in secondary metabolites such as the sesquiterpenes. The presence of sesquiterpenes in high numbers and amounts correlates with agarwood of high quality. To understand the synthesis of this important compound, we cloned two candidate genes in the terpenoid synthesis pathway from Aquilaria malaccensis Lam., a major agarwood tree species. The genes encoding sesquiterpene synthase (AmSesTPS1) and δ-guaiene synthase (AmGuaiS1) were successfully cloned from callus RNA, using specific primers derived from transcriptomic data, in a reverse transcription PCR reaction. The full-length complementary DNA (cDNA) sequence of AmSesTPS1 was 1632 bp encoding for 544 amino acids, and AmGuaiS1 was 1644 bp encoding for 547 amino acids. Sequence alignment analysis showed that AmSesTPS1 shared between 99 to 100 % identity with sesquiterpene synthase from Aquilaria sinensis (Lour.) Spreng. while AmGuaiS1 shared between 95 to 99 % identity with δ-guaiene synthases from Aquilaria crassna Pierre ex Lecomte and A. sinensis. The genes were functionally characterised in a time course wounding experiment using 3-year-old living trees. Two types of wood samples were collected: (1) from wounded area (S1) and (2) from 5 cm below the wounded area (S2). AmSesTPS1 was highly expressed after 6 h post wounding for both S1 and S2, at a level three- to six-fold higher than that of the unwounded control (0 h), while AmGuaiS1 was induced after just 2 h of wounding (18- and 5.5-fold at S1 and S2, respectively), after which the expression of both genes was lowered. The average normalised expression of both genes at S1 and S2 indicates the genes were also upregulated in the distal area from the wounding site. It can be deduced that wounding triggers these two genes in the sesquiterpene synthesis pathway, which ultimately leads to agarwood formation.

The Eucalyptus terpene synthase gene family.

BackgroundTerpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts.ResultsThe genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus.ConclusionsOur data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1598-x) contains supplementary material, which is available to authorized users.

Expression of terpene synthase genes associated with the formation of volatiles in different organs of Vitis vinifera

Plants produce a plethora of volatile organic compounds (VOCs) which are important in determining the quality and nutraceutical properties of horticultural food products, including the taste and aroma of wine. Given that some of the most prevalent grape aroma constituents are terpenoids, we investigated the possible variations in the relative expression of terpene synthase (TPS) genes that depend on the organ. We thus analysed mature leaves, young leaves, stems, young stems, roots, rachis, tendrils, peduncles, bud flowers, flowers and berries of cv Moscato bianco in terms of their VOC content and the expression of 23 TPS genes.In terms of the volatile characterization of the organs by SPME/GC–MS analysis, flower buds and open flowers appeared to be clearly distinct from all the other organs analysed in terms of their high VOC concentration. Qualitatively detected VOCs clearly separated all the vegetative organs from flowers and berries, then the roots and rachis from other vegetative organs and flowers from berries, which confirms the specialization in volatile production among different organs.Our real-time RT-PCR results revealed that the majority of TPS genes analysed exhibited detectable transcripts in all the organs investigated, while only some were found to be expressed specifically in one or just a few organs. In most cases, we found that the known products of the in vitro assay of VvTPS enzymes corresponded well to the terpenes found in the organs in which the encoding gene was expressed, as in the case of (E)-β-caryophyllene synthases, α-terpineol synthase and α-farnesene synthase. In addition, we found groups of homologous TPS genes, such as (E)-β-caryophyllene and β-ocimene synthases, expressed distinctively in the various tissues. This thus confirmed the subfunctionalization events and a specialization on the basis of the organs in which they are mostly expressed.

Influence of culture media and fungal extracts on essential oils composition and on terpene synthase gene expression in Thymus caespititius

Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high diversity and some of them could be used in pharmaceutical and food industries. The effect of culture media composition and fungal extracts on essential oils composition and on the expression of terpene synthase (TPS) genes was evaluated in T. caespititius shoots cultures. Nutrient composition influenced quantitatively the essential oils content. A decrease in the acetate compounds in the essential oils was observed in SH medium as compared with MS. The TPS transcripts accumulation was higher in all genotypes grown in SH, except in sabinene/carvacrol (SC) genotype. B. cinerea extracts highly induced TPS gene expression in the first hours after treatment in SC genotype, while in carvacrol/thymol (CT) only a slight increase was observed. TPS down-regulation observed during the next time points could result from reallocation of energy resources to plants’ vital functions. Fungal extracts did not affect qualitatively and quantitatively the CT essential oils, however in SC, the sabinene relative content decreased and this was inversely related to the increase of two antifungal compounds, carvacrol and thymol, in the oil. T. caespititius in vitro cultures revealed to be a good system for terpene metabolism study.

Differential expression of limonene synthase gene affects production and composition of essential oils in leaf and floret of transgenic lavandin (Lavandula × intermedia Emeric ex Loisel.)

Transgenic lavandin (Lavandula × intermedia) expressing the limonene synthase gene (LIMS) cDNA of true lavender (L. angustifolia) driven by a constitutive 35S promoter was generated from leaf-derived callus inoculated with disarmed Agrobacteriumtumefaciens. Only three LIMS regenerants could be acclimatized and their transgenes confirmed. Of them, LM-3, showed closed internodes and short stalks with small spikes, similar to the morphological characters of dwarfism. These results might be attributable to depleted geranyl diphosphate, the monoterpene precursor, which is required for the production of gibberellin. LIMS was differentially expressed in leaves and florets of the transgenic plant, which affected the production of several monoterpenes and therefore essential oil production. In vegetative leaves, overexpressed LIMS increased not only limonene but also total essential oil production, although no alteration in fragrance was observed. Conversely, suppression of LIMS expression in florets of the reproductive stage reduced their total essential oil production, including the dramatic decrease of limonene, linalool, and linalyl acetate. These results suggest that the constitutive promoter acts as a suppressor in tissues in which endogenous targeted gene expression is strong. Consequently, a slight change in fragrance at weak intensity in florets of the transgenic plants, producing a camphoraceous odor, was apparent when compared with non-transgenic plants, because the relative proportions of camphoraceous compounds such as 1,8-cineole, camphor, and borneol were increased in the transgenic plants. These results suggest that the suppression of terpene synthase gene expression is an effective way to alter fragrance, despite a dramatic reduction in total essential oil production.

Analysis of the expression of terpene synthase genes in relation to aroma content in two aromatic Vitis vinifera varieties.

Grape (Vitis vinifera L.) flavour management in the vineyard requires knowledge of the derivation of individual flavour and aroma characteristics. Some of the most prevalent wine grape aroma constituents are terpenoids and this study represents a wide report about grape terpene synthase (TPS) gene transcript profiling in different tissues of two aromatic grapevine varieties, particularly flowers and developing berries, correlated with the accumulation patterns of free aroma compounds. All investigated genes belonging to the TPS-a and TPS-b subfamilies reached the highest expression in accordance with the peak of accumulation of the respective compounds. In the TPS-g subfamily, only one of the genes characterised for linalool synthases showed major transcript abundance in ripening berries, whereas the only geraniol synthase had a peak of expression in green berries and at the beginning of ripening, when geraniol concentration started to increase and overcome the linalool concentration. The genes identified in this study as being mainly responsible for linalool and geraniol synthesis during berry development, and the phenological phases in which they are mostly expressed, should be of interest to viticulturists and wine makers to improve decision making along the chain of production.