Geraniol Synthase Research Articles

Identification and Characterization of a Fungal Monoterpene Synthase Responsible for the Biosynthesis of Geraniol.

Geraniol, an acyclic monoterpenoid of substantial value extracted from the essential oils of various aromatic plants, holds significant commercial and industrial importance in the realms of food, cosmetics, medicine, and bioenergy. Geraniol synthase, which is responsible for geraniol production, has been identified in only several plant species to date. Here, we present the first cloning and characterization of a geraniol synthase (PgfTPS) from Penicillium griseofulvum. This enzyme demonstrates pronounced specificity in catalyzing the conversion of geranyl diphosphate into geraniol. Moreover, through protein modeling and site-directed mutagenesis, we have identified key active-site residues crucial for the catalytic function of PgfTPS. Finally, we utilized engineered Saccharomyces cerevisiae as a host for PgfTPS expression to facilitate geraniol production. Our findings not only advance the development of efficient biocatalysts for geraniol generation but also establish a fundamental basis for further exploration into fungal monoterpene biosynthesis.

Identification and functional analysis of a deduced geraniol synthase from Camphora officinarum.

The online version contains supplementary material available at 10.1007/s12298-024-01463-4.

Identification of key genes controlling monoterpene biosynthesis of Citral-type Cinnamomum bodinieri Levl. Based on transcriptome and metabolite profiling

The citral-type is the most common chemotype in Cinnamomum bodinieri Levl (C. bodinieri), which has been widely used in the daily necessities, cosmetics, biomedicine, and aromatic areas due to their high citral content. Despite of this economic prospect, the possible gene-regulatory roles of citral biosynthesis in the same geographic environment remains unknown. In this study, the essential oils (EOs) of three citral type (B1, B2, B3) and one non-citral type (B0) varieties of C. bodinieri were identified by GC-MS after hydrodistillation extraction in July. 43 components more than 0.10% were identified in the EOs, mainly composed of monoterpenes (75.8–91.84%), and high content citral (80.63–86.33%) were identified in citral-type. Combined transcriptome and metabolite profiling analysis, plant-pathogen interaction(ko04626), MAPK signaling pathway-plant(ko04016), starch and sucrose metabolism(ko00500), plant hormone signal transduction(ko04075), terpenoid backbone biosynthesis (ko00900) and monoterpenoid biosynthesis (ko00902) pathways were enriched significantly. The gene expression of differential genes were linked to the monoterpene content, and the geraniol synthase (CbGES), alcohol dehydrogenase (CbADH), geraniol 8-hydroxylase-like (CbCYP76B6-like) and 8-hydroxygeraniol dehydrogenase (Cb10HGO) were upregulated in the citral-type, indicating that they were associated with high content of geraniol and citral. The activities of CbGES and CbADH in citral type were higher than in non-citral type, which was corroborated by enzyme-linked immunosorbent assay (ELISA). This study on the accumulation mechanism of citral provides a theoretical basis for the development of essential oil of C. bodinieri.

Silicon nanoparticles (SiNPs) stimulated secondary metabolism and mitigated toxicity of salinity stress in basil (Ocimum basilicum) by modulating gene expression: a sustainable approach for crop protection.

The underlying mechanisms through which silicon oxide nanoparticles (SiNPs) can confer salinity resistance to plants are poorly understood. This study explored the efficacy of supplementing nutrient solution with SiNPs (20-30nm; 10 mg kg-1 soil) to stimulate metabolism and alleviate the risks associated with salinity (0.73 g kg-1 soil) in basil seedlings. For this purpose, variations in photosynthetic indices, proline osmoprotectant, antioxidant markers, phenylpropanoid metabolism, and transcriptional behaviors of genes were investigated. SiNPs increased shoot fresh weight (38%) and mitigated the risk associated with the salinity stress by 14%. SiNPs alleviated the inhibitory effects of salinity on the total chlorophyll concentration by 15%. The highest increase (twofold) in proline content was recorded in the SiNP-treated seedlings grown under salinity. The nano-supplement enhanced the activity of enzymatic antioxidants, including peroxidase (2.5-fold) and catalase (4.7-fold). SiNPs induced the expression of gamma-cadinene synthase (CDS) and caffeic acid O-methyltransferase (COMT) genes by 6.5- and 18.3-fold, respectively. SiNPs upregulated the eugenol synthase (EGS1) and fenchol synthase (FES) genes by six- and nine-fold, respectively. Salinity transcriptionally downregulated the geraniol synthase (GES) gene, while this gene displayed an upward trend in response to SiNPs by eight-fold. The nano-supplement transcriptionally stimulated the R-linalool synthase (LIS) gene by 3.3-fold. The terpinolene synthase (TES) gene displayed a similar trend to that of the GES gene. The highest expression (25-fold) of the phenylalanine ammonia-lyase (PAL) gene was recorded in seedlings supplemented with SiNPs. The physiological and molecular assessments demonstrated that employing SiNPs is a sustainable strategy for improving plant primary/secondary metabolism and crop protection.

Transcriptome Analysis Provides Insights into Catalpol Biosynthesis in the Medicinal Plant Rehmannia glutinosa and the Functional Characterization of RgGES Genes.

Rehmannia glutinosa, a member of the Scrophulariaceae family, has been widely used in traditional Chinese medicine since ancient times. The main bioactive component of R. glutinosa is catalpol. However, the biogenesis of catalpol, especially its downstream pathway, remains unclear. To identify candidate genes involved in the biosynthesis of catalpol, transcriptomes were constructed from R. glutinosa using the young leaves of three cultivars, Beijing No. 3, Huaifeng, and Jin No. 9, as well as the tuberous roots and adventitious roots of the Jin No. 9 cultivar. As a result, 71,142 unigenes with functional annotations were generated. A comparative analysis of the R. glutinosa transcriptomes identified over 200 unigenes of 13 enzymes potentially involved in the downstream steps of catalpol formation, including 9 genes encoding UGTs, 13 for aldehyde dehydrogenases, 70 for oxidoreductases, 44 for CYP450s, 22 for dehydratases, 30 for decarboxylases, 19 for hydroxylases, and 10 for epoxidases. Moreover, two novel genes encoding geraniol synthase (RgGES), which is the first committed enzyme in catalpol production, were cloned from R. glutinosa. The purified recombinant proteins of RgGESs effectively converted GPP to geraniol. This study is the first to discover putative genes coding the tailoring enzymes mentioned above in catalpol biosynthesis, and functionally characterize the enzyme-coding gene in this pathway in R. glutinosa. The results enrich genetic resources for engineering the biosynthetic pathway of catalpol and iridoids.

Engineering Yarrowia lipolytica for the biosynthesis of geraniol

Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast.

A geraniol synthase regulates plant defense via alternative splicing in tea plants.

Geraniol is an important contributor to the pleasant floral scent of tea products and one of the most abundant aroma compounds in tea plants; however, its biosynthesis and physiological function in response to stress in tea plants remain unclear. The proteins encoded by the full-length terpene synthase (CsTPS1) and its alternative splicing isoform (CsTPS1-AS) could catalyze the formation of geraniol when GPP was used as a substrate in vitro, whereas the expression of CsTPS1-AS was only significantly induced by Colletotrichum gloeosporioides and Neopestalotiopsis sp. infection. Silencing of CsTPS1 and CsTPS1-AS resulted in a significant decrease of geraniol content in tea plants. The geraniol content and disease resistance of tea plants were compared when CsTPS1 and CsTPS1-AS were silenced. Down-regulation of the expression of CsTPS1-AS reduced the accumulation of geraniol, and the silenced tea plants exhibited greater susceptibility to pathogen infection than control plants. However, there was no significant difference observed in the geraniol content and pathogen resistance between CsTPS1-silenced plants and control plants in the tea plants infected with two pathogens. Further analysis showed that silencing of CsTPS1-AS led to a decrease in the expression of the defense-related genes PR1 and PR2 and SA pathway-related genes in tea plants, which increased the susceptibility of tea plants to pathogens infections. Both in vitro and in vivo results indicated that CsTPS1 is involved in the regulation of geraniol formation and plant defense via alternative splicing in tea plants. The results of this study provide new insights into geraniol biosynthesis and highlight the role of monoterpene synthases in modulating plant disease resistance via alternative splicing.

Volatile Composition and Classification of Paeonia lactiflora Flower Aroma Types and Identification of the Fragrance-Related Genes.

Flower scent is one of the main ornamental characteristics of herbaceous peony, and the improvement of flower fragrance is a vital objective of herbaceous peony breeding. In this study, 87 herbaceous peony cultivars were divided into three groups (no/light fragrance, medium fragrance, and strong fragrance) based on their sensory evaluation scores, and 16 strong fragrance cultivars and one no fragrance cultivar were selected for subsequent analysis. Sixty-eight volatile components were detected in these 17 cultivars based on solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS), and 26 types were identified as important scent components. They were composed of terpenoids, benzenoids/phenylpropanoids, and fatty acid derivatives. According to the content and odor threshold of these main aroma components, the characteristic aroma substances of herbaceous peony were identified, including linalool, geraniol, citronellol, and phenylethyl alcohol (2-PE). The cultivars of strong scented herbaceous peony were divided into three types: rose scent, lily scent, and mixed scent. We explored the possible key genes of characteristic aroma substances in herbaceous peony petals with different odors through the qRT-PCR. The key genes encoding monoterpene biosynthesis were found to be PlDXS2, PlDXR1, PlMDS1, PlHDR1, PlGPPS3, and PlGPPS4. In addition, the linalool synthase (LIS) gene and the geraniol synthase (GES) gene were also found. PlAADC1, PlPAR1, and PlMAO1, related to the biosynthesis of 2-PE were detected, and the synthetic pathway of 2-PE was speculated. In conclusion, these findings revealed that the difference in gene expression of monoterpene and 2-PE synthesis pathway was related to the difference in the fragrance of herbaceous peony. This study explored the releasing pathway of herbaceous peony characteristic aroma substances and provided key genetic resources for fragrance improvement.

Chemical ecology of Himalayan eggplant variety's antixenosis: identification of geraniol as an oviposition deterrent against the eggplant shoot and fruit borer.

Eggplant (Solanum melongena) suffers severe losses due to a multi-insecticide-resistant lepidopteran pest, shoot and fruit borer (SFB, Leucinodes orbonalis). Heavy and combinatorial application of pesticides for SFB control renders eggplant risky for human consumption. We observed that gravid SFB females do not oviposit on Himalayan eggplant variety RC-RL-22 (RL22). We hypothesized that RL22 contained an antixenosis factor. Females' behavior indicated that the RL22 cue they perceived was olfactory. To identify it, leaf volatile blends of seven eggplant varieties were profiled using solid phase microextraction and gas chromatography mass spectrometry. Seven RL22-specific compounds were detected in the plant headspace. In choice assays, oviposition deterrence efficacies of these candidate compounds were independently tested by their foliar application on SFB-susceptible varieties. Complementation of geraniol, which was exclusively found in RL22, reduced oviposition (> 90%). To validate geraniol's role in RL22's SFB-deterrence, we characterized RL22's geraniol synthase and silenced its gene in planta, using virus-induced gene silencing. Geraniol biosynthesis suppression rendered RL22 SFB-susceptible; foliar geraniol application on the geraniol synthase-silenced plants restored oviposition deterrence. We infer that geraniol is RL22's SFB oviposition deterrent. The use of natural compounds like geraniol, which influence the chemical ecology of oviposition, can reduce the load of hazardous synthetic larvicides.

Grapevine genome analysis demonstrates the role of gene copy number variation in the formation of monoterpenes.

Volatile organic compounds such as terpenes influence the quality parameters of grapevine through their contribution to the flavour and aroma profile of berries. Biosynthesis of volatile organic compounds in grapevine is relatively complex and controlled by multiple genes, the majority of which are unknown or uncharacterised. To identify the genomic regions that associate with modulation of these compounds in grapevine berries, volatile metabolic data generated via GC-MS from a grapevine mapping population was used to identify quantitative trait loci (QTLs). Several significant QTLs were associated with terpenes, and candidate genes were proposed for sesquiterpene and monoterpene biosynthesis. For monoterpenes, loci on chromosomes 12 and 13 were shown to be associated with geraniol and cyclic monoterpene accumulation, respectively. The locus on chromosome 12 was shown to contain a geraniol synthase gene (VvGer), while the locus on chromosome 13 contained an α-terpineol synthase gene (VvTer). Molecular and genomic investigation of VvGer and VvTer revealed that these genes were found in tandemly duplicated clusters, displaying high levels of hemizygosity. Gene copy number analysis further showed that not only did VvTer and VvGer copy numbers vary within the mapping population, but also across recently sequenced Vitis cultivars. Significantly, VvTer copy number correlated with both VvTer gene expression and cyclic monoterpene accumulation in the mapping population. A hypothesis for a hyper-functional VvTer allele linked to increased gene copy number in the mapping population is presented and can potentially lead to selection of cultivars with modulated terpene profiles. The study highlights the impact of VvTPS gene duplication and copy number variation on terpene accumulation in grapevine.

Creation of new germplasm resources, development of SSR markers, and screening of monoterpene synthases in thyme

BackgroundThyme derived essential oil and its components have numerous applications in pharmaceutical, food, and cosmetic industries, owing to their antibacterial, antifungal, and antiviral properties. To obtain thyme essential oil with different terpene composition, we developed new germplasm resources using the conventional hybridization approach.ResultsPhenotypic characteristics, including essential oil yield and composition, glandular trichome density, plant type, and fertility, of three wild Chinese and seven European thyme species were evaluated. Male-sterile and male-fertile thyme species were crossed in different combinations, and two F1 populations derived from Thymus longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq) crosses were selected, with essential oil yield and terpene content as the main breeding goals. Simultaneously, simple sequence repeat (SSR) primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F1 hybrids. A total of 300 primer pairs were selected, and polymerase chain reaction (PCR) was carried out on the parents of the two hybrid populations (Tl, Tvf, Tve, and Tq). Based on the chemotype of the parents and their F1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by quantitative real-time PCR (qRT-PCR).ConclusionWe used hybridization to create new germplasm resources of thyme, developed SSR markers based on the whole-genome sequence of T. quinquecostatus, and screened the expression of monoterpene synthase genes in thyme. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of quantitative trait loci (QTLs), and help gain insight into the mechanism of monoterpenoids biosynthesis in thyme.

Genetic and Biochemical Aspects of Floral Scents in Roses.

Floral scents possess high ornamental and economic values to rose production in the floricultural industry. In the past two decades, molecular bases of floral scent production have been studied in the rose as well as their genetic inheritance. Some significant achievements have been acquired, such as the comprehensive rose genome and the finding of a novel geraniol synthase in plants. In this review, we summarize the composition of floral scents in modern roses, focusing on the recent advances in the molecular mechanisms of floral scent production and emission, as well as the latest developments in molecular breeding and metabolic engineering of rose scents. It could provide useful information for both studying and improving the floral scent production in the rose.

Spatial and temporal patterns of secoiridoid and xanthone biosynthetic pathways during early development of Centaurium erythraea Rafn, as altered by ploidy level

Centaurium erythraea, used as medical plant from the earliest times, is an immense depot of quite rare bioactive compounds. Secoiridoids (sweroside, swertiamarin, and gentiopicrin) and xanthones (methylbellidifolin and decussatin) are predominant bioactive compounds in C. erythraea. The present study aims at providing new insights into how the content of these bioactive principles can be related with plant ploidy level by characterizing possible differences in their biosynthesis and accumulation between diploid and tetraploid genotypes from both spatial and temporal aspects. In general, shoots are determined as the major site of secoiridoids’ and xanthones’ accumulation, whose ratio vary during the development. Genes involved in iridoid and xanthone metabolic pathways were found to be coordinately regulated at the transcriptional level both during the development and among organs. Biosynthetic gene expression levels were found highly correlated with the content of major compounds from these two classes. Diversification in chemical profiles between tetraploid and diploid genotypes may result from the expression difference between homologous loci correspondent to several key biosynthetic genes, which trigger changes in the two metabolic routes. Thus, enhanced expression of genes coding for geraniol synthase (GES), 8-hydroxygeraniol oxidoreductase (8HGO), and 7-deoxyloganic acid hydrolase (7DLH2) is strongly associated with intensive production of iridoids. Interestingly, transcript levels of beta-glucosidase (CebGLU), a candidate to catalyze the first step in the secoiridoid catabolism, is significantly positively correlated with the content of major secoiridoids. Elevated expression of genes coding for benzophenone synthase (BS) and 3-hydroxybenzoate:CoA ligase (3HBL) appear to account for enhanced production of hexa-substituted xanthones. Regarding content of iridoids and xanthones, a diploid genotype appeared to be more productive than a tetraploid genotype under controlled in vitro conditions, therewithal displaying significantly higher biomass.

Cytosolic Nudix Hydrolase 1 Is Involved in Geranyl β-Primeveroside Production in Tea.

Geraniol is a potent tea odorant and exists mainly as geranyl glycoside in Camellia sinensis. Understanding the mechanisms of geraniol biosynthesis at molecular levels in tea plants is of great importance for practical improvement of tea aroma. In this study, geraniol and its glycosides from tea plants were examined using liquid chromatography coupled with mass spectrometry. Two candidate geraniol synthase (GES) genes (CsTPS) and two Nudix hydrolase genes (CsNUDX1-cyto and CsNUDX1-chlo) from the tea genome were functionally investigated through gene transcription manipulation and gene chemical product analyses. Our data showed that in tea leaves, levels of geranyl β-primeveroside were dramatically higher than those of geranyl β-glucoside, while free geraniol was undetectable in this study. A tempo-spatial variation of geranyl β-primeveroside abundance in tea plants existed, with high levels in young and green tissues and low levels in mature or non-green tissues. Cytosolic CsNUDX1-cyto showed higher hydrolysis activity of geranyl-pyrophosphate to geranyl-monophosphate (GP) in vitro than did chloroplastidial CsNUDX1-chlo. A transgenic study revealed that expression of CsNUDX1-cyto resulted in significantly more geranyl β-primeveroside in transgenic Nicotiana benthamiana compared with non-transgenic wild-type, whereas expression of CsNUDX1-chlo had no effect. An antisense oligo-deoxynucleotide study confirmed that suppression of CsNUDX1-cyto transcription in tea shoots led to a significant decrease in geranyl β-primeveroside abundance. Additionally, CsNUDX1-cyto transcript levels and geranyl β-primeveroside abundances shared the same tempo-spatial patterns in different organs in the tea cultivar “Shucha Zao,” indicating that CsNUDX1-cyto is important for geranyl β-primeveroside formation in tea plants. Results also suggested that neither of the two candidate GES genes in tea plants did not function as GES in transgenic N. benthamiana. All our data indicated that CsNUDX1-cyto is involved in geranyl β-primeveroside production in tea plants. Our speculation about possible conversion from the chemical product of CsNUDX1-cyto to geranyl β-primeveroside in plants was also discussed.

Unveiling Monoterpene Biosynthesis in Taiwania cryptomerioides via Functional Characterization.

Taiwania cryptomerioides is a monotypic species, and its terpenoid-rich property has been reported in recent years. To uncover monoterpene biosynthesis in T. cryptomerioides, this study used transcriptome mining to identify candidates with tentative monoterpene synthase activity. Along with the phylogenetic analysis and in vitro assay, two geraniol synthases (TcTPS13 and TcTPS14), a linalool synthase (TcTPS15), and a β-pinene synthase (TcTPS16), were functionally characterized. Via the comparison of catalytic residues, the Cys/Ser at region 1 might be crucial in determining the formation of α-pinene or β-pinene. In addition, the Cupressaceae monoterpene synthases were phylogenetically clustered together; they are unique and different from those of published conifer species. In summary, this study aimed to uncover the ambiguous monoterpenoid network in T. cryptomerioide, which would expand the landscape of monoterpene biosynthesis in Cupressaceae species.

Identification and Molecular Characterization of Geraniol and Linalool Synthase Genes Related to Monoterpene Biosynthesis in Damask Rose (Rosa damascena Mill.), Several Genes for Little Scent

Rosa damascena Mill. (Damask rose) is a famous traditional flower that is widely appreciated for its valuable essential oil and high-quality fragrance used in the perfume and cosmetics industries. Rose oil contains more than 300 volatile constituents, and the main constituents geraniol, linalool, nerol, and citronellol determine the essential oil quality. In the present study, we reported the cloning and characterization of three rose monoterpene synthase genes related to rose oil quality. The genes encoding geranyl diphosphate synthase (GDPS) designated as RdGDP (accession No. MN650820), geraniol synthase (GES) as RdGES (accession No. MN639696), and linalool synthase (LIS) as RdLIS (accession No. MN639697) and involved in the monoterpene synthases pathways were successfully cloned from R. damascena and their expression profiles in different flower developmental stages were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The deduced RdGDP, RdGES, and RdLIS proteins contain 383, 603, and 562 amino acids, and the corresponding size of their open-reading frames is 1152, 1812, and 1689 bp, respectively. Expression analysis revealed that these genes demonstrated persistent expressions from the budding to the full opening stages of R. damascena flower development, and the expression levels of RdLIS were lower than that of RdGDP and RdGES. These results suggest that the GDP and GES can regulate the biosynthesis of floral scents in Damask rose flowers. The cloning of rose genes, including RdGDP, RdGES, and RdLIS, could have far-reaching implications for improving the quality of R. damascena essential oils through metabolic engineering.

Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags

Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Basic helix‐loop‐helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in Catharanthus roseus. Two previously characterized subgroup‐IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated C. roseus genome sequence and discovered that BIS1 and BIS2 are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as BIS3. Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants Calotropis gigantea (giant milkweed) and Gelsemium sempervirens (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, BIS3 is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, geraniol synthase (GES), geraniol 10‐hydroxylase (G10H), 8‐hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), 7‐deoxyloganetic acid glucosyl transferase (7‐DLGT), and 7‐deoxyloganic acid hydroxylase (7DLH), but not iridoid oxidase (IO). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF‐associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the G10H promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G‐box (CACGTG) in the G10H promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of G10H suggests that the sequences adjacent to the G‐box are also involved in the regulation by BIS3. Overexpression of BIS3 in C. roseus flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. BIS2 expression was significantly induced by BIS3 although BIS3 did not directly activate the BIS2 promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.

Functional Characterization of a Dendrobium officinale Geraniol Synthase DoGES1 Involved in Floral Scent Formation.

Floral scent is a key ornamental trait that determines the quality and commercial value of orchids. Geraniol, an important volatile monoterpene in orchids that attracts pollinators, is also involved in responses to stresses but the geraniol synthase (GES) responsible for its synthesis in the medicinal orchid Dendrobium officinale has not yet been identified. In this study, three potential geraniol synthases were mined from the D. officinale genome. DoGES1, which was localized in chloroplasts, was characterized as a geraniol synthase. DoGES1 was highly expressed in flowers, especially in petals. DoGES1 transcript levels were high in the budding stage of D. officinale flowers at 11:00 a.m. DoGES1 catalyzed geraniol in vitro, and transient expression of DoGES1 in Nicotiana benthamiana leaves resulted in the accumulation of geraniol in vivo. These findings on DoGES1 advance our understanding of geraniol biosynthesis in orchids, and lay the basis for genetic modification of floral scent in D. officinale or in other ornamental orchids.

Metabolic Engineering of the Native Monoterpene Pathway in Spearmint for Production of Heterologous Monoterpenes Reveals Complex Metabolism and Pathway Interactions.

Spearmint produces and stores large amounts of monoterpenes, mainly limonene and carvone, in glandular trichomes and is the major natural source of these compounds. Towards producing heterologous monoterpenes in spearmint, we first reduced the flux into the native limonene pathway by knocking down the expression of limonene synthase (MsLS) by RNAi method. The MsLS RNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed that MsLS RNAi plants also showed an increase in sesquiterpene, phytosterols, fatty acids, flavonoids, and phenolic metabolites, suggesting an interaction between the MEP, MVA shikimate and fatty acid pathways in spearmint. Three different heterologous monoterpene synthases namely, linalool synthase and myrcene synthase from Picea abies and geraniol synthase from Cananga odorata were cloned and introduced independently into the MsLS RNAi mutant background. The expression of these heterologous terpene synthases resulted mainly in production of monoterpene derivatives. Of all the introduced monoterpenes geraniol showed the maximum number of derivatives. Our results provide new insights into MEP pathway interactions and regulation and reveals the existence of mechanisms for complex metabolism of monoterpenes in spearmint.