Formation mechanism of glandular trichomes involved in the synthesis and storage of terpenoids in lavender.

BACKGROUND: Lavender (genus Lavandula, family Lamiaceae) is an aromatic plant widely grown as an ornamental plant. The chemical composition of lavender is characterized by monoterpenoids, sesquiterpenoids, and other compounds, which are primarily synthesized and stored in epidermal secretory structures called glandular trichomes (GTs). Volatile organic compounds (VOCs) are responsible for the aroma characteristics of plant oil that drive consumer preference. Aroma is usually regarded as a characteristic trait for the classification of aromatic plants. Interestingly, VOCs are synthesized and stored in GTs. Lamiaceae species such as purple perilla, peppermint, basil, thyme, and oregano usually possess two types of GTs: peltate glandular trichomes (PGTs) and capitate glandular trichomes (CGTs). But the development process of PGTs in lavender has been reported in only a few studies to date. RESULTS: In this study, we identified and quantified the VOCs in four lavender cultivars by headspace-solid phase micro extraction-gas chromatography mass spectrometry (HS-SPME-GC-MS). A total of 66 VOCs were identified in these four cultivars, the most prominent of which were linalyl acetate and linalool, and flowers were the main site of accumulation of these VOCs. Here, we examined the developmental process of PGTs, including the formation of their base, body, and apex. The apex cells contained secretory cavities, which produced VOCs. Based on the reference genome sequence of the lavender cultivar 'Jingxun 2', several R2R3-MYB subfamily genes related to GT formation were identified. These results will guide the engineering of GTs and molecular breeding of lavender for improving the VOC content. CONCLUSIONS: In this study, we identified the VOCs in four lavender cultivars. We analyzed the formation of GTs, and compared the number and diameter size of PGTs among four lavender cultivars. Additionally, we identified four candidate genes belonging to the R2R3-MYB family.

Lavandula Species, Their Bioactive Phytochemicals, and Their Biosynthetic Regulation.

Lavandula species are one of the most useful aromatic and medicinal plants and have great economic potential. The phytopharmaceutical contribution of the secondary metabolites of the species is unquestionable. Most recent studies have been focusing on the elucidation of the genetic background of secondary metabolite production in lavender species. Therefore, knowledge of not only genetic but especially epigenetic mechanisms for the regulation of secondary metabolites is necessary for the modification of those biosynthesis processes and the understanding of genotypic differences in the content and compositional variability of these products. The review discusses the genetic diversity of Lavandula species in relation to the geographic area, occurrence, and morphogenetic factors. The role of microRNAs in secondary-metabolites biosynthesis is described.

Physiological response and secondary metabolites of three lavender genotypes under water deficit.

Lavandula genus is a considerable medicinal plant in pharmaceutical and cosmetics industries. Considering increasing threat of drought in the world, it is important to identify genotypes which can tolerate drought. It is also important to characterize quantity and quality of essential oils, and tolerance indicators of these genotypes against drought stress. Therefore, an experiment was conducted in Gorgan University of Agricultural Sciences and Natural Resources, Iran, during 2017 and 2018, to investigate these factors. It was a factorial experiment based on randomized complete block design with two treatments, three genotypes (Lavandula angustifolia cv. Hidcote, Lavandula angustifolia cv. Munstead, and Lavandula stricta), and four levels of drought stress (irrigation regimes) (I1: 100-90% (control), I2: 80-70%, I3: 60-50% and I4: 30-40% of field capacity) which was done with three repetitions. Drought increased amount of proline in leaves, antioxidant activity, activity of catalase, peroxidase, ascorbate peroxidase, and superoxide enzymes, malondialdehyde content, total flavonoids, total phenol, total sugar and essential oil percentage. The PCA analysis of different irrigation regimes showed that in the first component, the best traits are antioxidant enzymes CAT, SOD, APX, while in the second component, only the trait Catalase is the best trait. The results of PCA analysis in lavender genotypes showed that L. stricta exhibits the most affected physiological changes while trying to adjust to changes in the water status of the environment, under the imposed conditions and shows the highest resistance. But it reduced dry weight of aerial parts, relative water content of leaves, and efficacy of essential oil. Lavandula stricta genotype had the highest amount of essential oil, but the highest dry weight of the aerial parts and essential oil yield were related to L. angustifolia cv. Hidcote and L. angustifolia cv. Munstead genotypes. In all evaluated genotypes, with increasing drought stress, monoterpene compounds were decreased and sesquiterpene compounds were increased. Totally it was shown that drought effect on evaluated traits depends on genotype and nature of traits; this indicates that by choosing drought-tolerant genotypes in breeding programs, high quantity and quality of essential oil, as well as tolerance to drought stress can be achieved.

Time-series transcriptome provides insights into the gene regulation network involved in the volatile terpenoid metabolism during the flower development of lavender.

BACKGROUND: Essential oils (EOs) of Lavandula angustifolia, mainly consist of monoterpenoids and sesquiterpenoids, are of great commercial value. The multi-flower spiciform thyrse of lavender not only determines the output of EOs but also reflects an environmental adaption strategy. With the flower development and blossom in turn, the fluctuation of the volatile terpenoids displayed a regular change at each axis. However, the molecular mechanism underlying the regulation of volatile terpenoids during the process of flowering is poorly understood in lavender. Here, we combine metabolite and RNA-Seq analyses of flowers of five developmental stages at first- and second-axis (FFDSFSA) and initial flower bud (FB0) to discover the active terpenoid biosynthesis as well as flowering-related genes. RESULTS: A total of 56 mono- and sesquiterpenoids were identified in the EOs of L. angustifolia 'JX-2'. FB0' EO consists of 55 compounds and the two highest compounds, ß-trans-ocimene (20.57%) and (+)-R-limonene (17.00%), can get rid of 74.71 and 78.41% aphids in Y-tube olfactometer experiments, respectively. With sequential and successive blossoms, temporally regulated volatiles were linked to pollinator attraction in field and olfaction bioassays. In three characteristic compounds of FFDSFSA' EOs, linalyl acetate (72.73%) and lavandulyl acetate (72.09%) attracted more bees than linalool (45.35%). Many transcripts related to flowering time and volatile terpenoid metabolism expressed differently during the flower development. Similar metabolic and transcriptomic profiles were observed when florets from the two axes were maintained at the same maturity grade. Besides both compounds and differentially expressed genes were rich in FB0, most volatile compounds were significantly correlated with FB0-specific gene module. Most key regulators related to flowering and terpenoid metabolism were interconnected in the subnetwork of FB0-specific module, suggesting the cross-talk between the two biological processes to some degree. CONCLUSIONS: Characteristic compounds and gene expression profile of FB0 exhibit ecological value in pest control. The precise control of each-axis flowering and regular emissions at transcriptional and metabolic level are important to pollinators attraction for lavender. Our study sheds new light on lavender maximizes its fitness from "gene-volatile terpenoid-insect" three layers.

Cyanobacterial production of plant essential oils.

MAIN CONCLUSION: Synechocystis (a cyanobacterium) was employed as an alternative host for the production of plant essential oil constituents. ß-Phellandrene synthase (PHLS) genes from different plants, when expressed in Synechocystis, enabled synthesis of variable monoterpene hydrocarbon blends, converting Synechocystis into a cell factory that photosynthesized and released useful products. Monoterpene synthases are secondary metabolism enzymes that catalyze the generation of essential oil constituents in terrestrial plants. Essential oils, including monoterpene hydrocarbons, are of interest for their commercial application and value. Therefore, heterologous expression of monoterpene synthases for high-capacity essential oil production in photosynthetic microorganism transformants is of current interest. In the present work, the cyanobacterium Synechocystsis PCC 6803 was employed as an alternative host for the production of plant essential oil constituents. As a case study, ß-phellandrene synthase (PHLS) genes from different plants were heterologously expressed in Synechocystis. Genomic integration of individual PHLS-encoding sequences endowed Synechocystis with constitutive monoterpene hydrocarbons generation, occurring concomitant with photosynthesis and cell growth. Specifically, the ß-phellandrene synthase from Lavandula angustifolia (lavender), Solanum lycopersicum (tomato), Pinus banksiana (pine), Picea sitchensis (Sitka spruce) and Abies grandis (grand fir) were active in Synechocystis transformants but, instead of a single product, they generated a blend of terpene hydrocarbons comprising ß-phellandrene, α-phellandrene, ß-myrcene, ß-pinene, and δ-carene with variable percentage ratios ranging from < 10 to > 90% in different product combinations and proportions. Our results suggested that PHLS enzyme conformation and function depends on the cytosolic environment in which they reside, with the biochemical properties of the latter causing catalytic deviations from the products naturally observed in the corresponding gene-encoding plants, giving rise to the terpene hydrocarbon blends described in this work. These findings may have commercial application in the generation of designer essential oil blends and will further assist the development of heterologous cyanobacterial platforms for the generation of desired monoterpene hydrocarbon products.

Functional characterization of terpene synthases and chemotypic variation in three lavender species of section Stoechas.

Lavandula pedunculata (Mill.) Cav. subsp. lusitanica, Lavandula stoechas L. subsp. stoechas and Lavandula viridis l'Hér. are three lavender taxa that belong to the botanical section Stoechas and are widely used as aromatherapy, culinary herb or folk medicine in many Mediterranean regions. The analysis of their bioactive volatile constituents revealed the presence of 124 substances, the most abundant being the bicyclic monoterpenes fenchone, camphor and 1,8-cineole that give these three species their respective chemotypes. Most noteworthy was fenchone which, with its reduced form fenchol, made 48% of the total volatile constituents of L. pedunculata while present at 2.9% in L. stoechas and undetectable in L. viridis. In order to provide a molecular explanation to the differences in volatile compounds of these three species, two monoterpene synthases (monoTPS) and one sesquiterpene synthase (sesquiTPS) were cloned in L. pedunculata and functionally characterized as fenchol synthase (LpFENS), α-pinene synthase (LpPINS) and germacrene A synthase (LpGEAS). The two other lavender species contained a single orthologous gene for each of these three classes of TPS with similar enzyme product specificities. Expression profiles of FENS and PINS genes matched the accumulation profile of the enzyme products unlike GEAS. This study provides one of the rare documented cases of chemotype modification during plant speciation via changes in the level of plant TPS gene expression, and not functionality.

Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.

Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 µM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering.

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development.

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub-groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.

Suppression of linalool acetate production in Lavandula x intermedia.

Linalool acetate, one of the major constituent of several essential oils, is heat-labile and decomposes upon exposure to the high injector temperature during gas chromatography. Here we report the development of an improved method for detection of this compound by gas chromatography mass spectrometry (GCMS) using cold on-column injection of the sample. By using this sensitive method, it has been demonstrated that a lavandin (L. x intermedia) mutant accumulates trace quantities of linalool acetate and camphor and higher amounts of cineole and borneol compared to its parent. This plant, which very likely carries a point mutation in one or more of the genes involved in essential oil production, provides a unique tool for investigating regulation of essential oil biogenesis in plants.