Genome-wide identification and expression of BAHD acyltransferase gene family shed novel insights into the regulation of linalyl acetate and lavandulyl acetate in lavender.

The BAHD acyltransferase superfamily has a variety of biological functions, especially in catalyzing the synthesis of ester compounds and improving plant stress resistance. Linalyl acetate and lavandulyl acetate, the most important volatile esters in lavender, are generated by LaBAHDs. However, the systematic identification, expression characteristics of LaBAHD genes and their correlations with ester formation remain elusive. Here, 166 LaBAHD genes were identified from the lavender genome. Based on detailed phylogenetic analysis, the LaBAHD family genes were divided into five groups, among which the LaBAHDs involved in volatile ester biosynthesis belong to the IIIa and Va clades. Whole-genome duplications (WGDs) and tandem duplications (TDs) jointly drive the expansion of LaBAHD superfamily. The promoter regions of LaBAHDs contained a variety of stress- and hormone-related motifs, as well as binding sites with five types of transcription factors (TFs). Then, linalyl acetate- and lavandulyl acetate-regulated coexpression modules were established and some candidate TFs that may function in inducing ester formation were identified. Based on the correlation analysis between the ester contents and expression profiles of BAHD genes in different tissues, five candidate genes were screened for further examination. Drought, salt and MeJA treatments increased the accumulation of linalyl acetate and lavandulyl acetate, and induced the expression of LaBAHDs. Our results indicated that LaBAHD57, LaBAHD63, LaBAHD104, LaBAHD105 and LaBAHD119 are crucial candidate genes involved in linalyl acetate and lavandulyl acetate biosynthesis. Our findings offer a theoretical foundation for further studying the specific biological functions of LaBAHD family and improving the quality of lavender essential oil.

Chromosome-scale genome assembly of the 'Munstead' cultivar of Lavandula angustifolia.

OBJECTIVES: Lavandula angustifolia (English lavender) is commercially important not only as an ornamental species but also as a major source of fragrances. To better understand the genomic basis of chemical diversity in lavender, we sequenced, assembled, and annotated the 'Munstead' cultivar of L. angustifolia. DATA DESCRIPTION: A total of 80 Gb of Oxford Nanopore Technologies reads was used to assemble the 'Munstead' genome using the Canu genome assembler software. Following multiple rounds of error correction and scaffolding using Hi-C data, the final chromosome-scale assembly represents 795,075,733 bp across 25 chromosomes with an N50 scaffold length of 31,371,815 bp. Benchmarking Universal Single Copy Orthologs analysis revealed 98.0% complete orthologs, indicative of a high-quality assembly representative of genic space. Annotation of protein-coding sequences revealed 58,702 high-confidence genes encoding 88,528 gene models. Access to the 'Munstead' genome will permit comparative analyses within and among lavender accessions and provides a pivotal species for comparative analyses within Lamiaceae.

Terpene produced by coexpression of the TPS and P450 genes from Lavandula angustifolia protects plants from herbivore attacks during budding stages.

To deter herbivore attacks, plants employ a diverse array of volatile compounds, particularly during the early developmental stages. The highly expressed genes LaTPS7, LaTPS8, and LaCYP71D582 were identified during the budding phases of Lavandula angustifolia. In vitro studies revealed that LaTPS7 generated nine distinct compounds, including camphene, myrcene, and limonene. LaTPS8 enzymatically converted eight volatiles by utilizing geranyl diphosphate and nerolidyl diphosphate as substrates. Overexpression of plastid-localized LaTPS7 in Nicotiana benthamiana resulted in the production of limonene. Furthermore, the endoplasmic reticulum-associated enzyme LaCYP71D582 potentially converted limonene into carveol. In N. benthamiana, LaTPS8 is responsible for the synthesis of α-pinene and sylvestrene. Furthermore, leaves transfected with LaTPS7 and leaves cotransfected with LaTPS7 and LaCYP71D582 exhibited a repellent effect on aphids, with an approximate rate of 70%. In comparison, leaves with an empty vector displayed a repellent rate of approximately 20%. Conversely, tobacco leaves expressing LaTPS7 attracted ladybugs at a rate of 48.33%, while leaves coexpressing LaTPS7 and LaCYP71D582 attracted ladybugs at a slightly higher rate of 58.33%. Subsequent authentic standard tests confirmed that limonene and carveol repel Myzus persicae while attracting Harmonia axyridis. The promoter activity of LaTPS7 and LaCYP71D582 was evaluated in Arabidopsis thaliana using GUS staining, and it was observed that wounding stimulated the expression of LaTPS7. The volatile compounds produced by LaTPS7, LaTPS8, and LaCYP71D582 play a crucial role in plant defence mechanisms. In practical applications, employing biological control measures based on plant-based approaches can promote human and environmental health.

Decoupling subgenomes within hybrid lavandin provide new insights into speciation and monoterpenoid diversification of Lavandula.

Polyploidization and transposon elements contribute to shape plant genome diversity and secondary metabolic variation in some edible crops. However, the specific contribution of these variations to the chemo-diversity of Lamiaceae, particularly in economic shrubs, is still poorly documented. The rich essential oils (EOs) of Lavandula plants are distinguished by monoterpenoids among the main EO-producing species, L. angustifolia (LA), L. × intermedia (LX) and L. latifolia (LL). Herein, the first allele-aware chromosome-level genome was assembled using a lavandin cultivar 'Super' and its hybrid origin was verified by two complete subgenomes (LX-LA and LX-LL). Genome-wide phylogenetics confirmed that LL, like LA, underwent two lineage-specific WGDs after the γ triplication event, and their speciation occurred after the last WGD. Chloroplast phylogenetic analysis indicated LA was the maternal source of 'Super', which produced premium EO (higher linalyl/lavandulyl acetate and lower 1,8-cineole and camphor) close to LA. Gene expression, especially the monoterpenoid biosynthetic genes, showed bias to LX-LA alleles. Asymmetric transposon insertions in two decoupling 'Super' subgenomes were responsible for speciation and monoterpenoid divergence of the progenitors. Both hybrid and parental evolutionary analysis revealed that LTR (long terminal repeat) retrotransposon associated with AAT gene loss cause no linalyl/lavandulyl acetate production in LL, and multi-BDH copies retained by tandem duplication and DNA transposon resulted in higher camphor accumulation of LL. Advances in allelic variations of monoterpenoids have the potential to revolutionize future lavandin breeding and EO production.

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.

Cloning, functional characterization and evaluating potential in metabolic engineering for lavender ( +)-bornyl diphosphate synthase.

KEY MESSAGE: We isolated and functionally characterized a new ( +)-bornyl diphosphate synthase (( +)-LiBPPS) from Lavandula x intermedia. The in planta functions of ( +)-LiBPPS were evaluated in sense and antisense transgenic plants. The monoterpene ( +)-borneol contributes scent and medicinal properties to some plants. It also is the immediate precursor to camphor, another important determinant of aroma and medicinal properties in many plants. ( +)-Borneol is generated through the dephosphorylation of bornyl diphosphate (BPP), which is itself derived from geranyl diphosphate (GPP) by the enzyme ( +)-bornyl diphosphate synthase (( +)-BPPS). In this study we isolated and functionally characterized a novel ( +)-BPPS cDNA from Lavandula x intermedia. The cDNA excluding its transit peptide was expressed in E. coli, and the corresponding recombinant protein was purified with Ni-NTA agarose affinity chromatography. The recombinant ( +)-LiBPPS catalyzed the conversion of GPP to BPP as a major product, and a few minor products. We also investigated the in planta role of ( +)-LiBPPS in terpenoid metabolism through its overexpression in sense and antisense orientations in stably transformed Lavandula latifolia plants. The overexpression of ( +)-LiBPPS in antisense resulted in reduced production of ( +)-borneol and camphor without compromising plant growth and development. As anticipated, the overexpression of the gene led to enhanced production of borneol and camphor, although growth and development were severely impaired in most transgenic lines strongly and ectopically expressing the ( +)-LiBPPS transgene in sense. Our results demonstrate that LiBPPS would be useful in studies aimed at the production of recombinant borneol and camphor in vitro, and in metabolic engineering efforts aimed at lowering borneol and camphor production in plants. However, overexpression in sense may require a targeted expression of the gene in glandular trichomes using a trichome-specific promoter.

Exogenous application of methyl jasmonate affects the emissions of volatile compounds in lavender (Lavandula angustifolia).

The plant hormone, methyl jasmonate (MeJA), is an orthodox elicitor of secondary metabolites, including terpenoids. Lavandula angustifolia is an important aromatic plant generating, yet few studies have been performed to evaluate the function of MeJA on the biosynthesis of terpenoids in lavender. Five treatments (with concentrations of 0, 0.4, 4, 8, and 16 mM) were set, and the physiological indicators of each group were determined after 0, 6, 12, 24, 48, and 72 h. The results illustrate that (1) MeJA could affect the diurnal rhythm of the emission of volatiles and MeJA acted in a dose-dependent and time-dependent manner; (2) 8 mM MeJA treatment increased the total content of the volatiles, and the contents of monoterpenoids and sesquiterpenoids were up-regulated 0.46- and 0.74- fold than the control at 24 h and 12 h, respectively; (3) after MeJA treatment, all the genes expression analyzed changed to varying degrees, of which 3-carene synthase (La3CARS) gene changed most significantly (7.66- to 38.02- fold than the control); (4) MeJA application was associated with a rise in glandular trichome density. The positive effects of MeJA indicate that the exogenous application of MeJA could be a beneficial mean for studies on the biosynthesis of terpenoids in lavender.

The transcription factor LaMYC4 from lavender regulates volatile Terpenoid biosynthesis.

BACKGROUND: The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, are essential regulators of plant terpenoid biosynthesis and response to stresses. Lavender has more than 75 volatile terpenoids, yet few TFs have been identified to be involved in the terpenoid biosynthesis. RESULTS: Based on RNA-Seq, reverse transcription-quantitative polymerase chain reaction, and transgenic technology, this study characterized the stress-responsive transcription factor LaMYC4 regulates terpenoid biosynthesis. Methyl jasmonate (MeJA) treatment increased volatile terpenoid emission, and the differentially expressed gene LaMYC4 was isolated. LaMYC4 expression level was higher in leaf than in other tissues. The expression of LaMYC4 decreased during flower development. The promoter of LaMYC4 contained hormone and stress-responsive regulatory elements and was responsive to various treatments, including UV, MeJA treatment, drought, low temperature, Pseudomonas syringae infection, and NaCl treatment. LaMYC4 overexpression increased the levels of sesquiterpenoids, including caryophyllenes, in Arabidopsis and tobacco plants. Furthermore, the expression of crucial node genes involved in terpenoid biosynthesis and glandular trichome number and size increased in transgenic tobacco. CONCLUSIONS: We have shown that the stress-responsive MYC TF LaMYC4 from 'Jingxun 2' lavender regulates volatile terpenoid synthesis. This study is the first to describe the cloning of LaMYC4, and the results help understand the role of LaMYC4 in terpenoid biosynthesis.

Combining DNA Barcoding and Chemical fingerprints to authenticate Lavender raw material.

OBJECTIVE: This study was initiated and conducted by several laboratories, 3 of the main cosmetic ingredient suppliers and 4 brands of cosmetics in France. Its objective is to show the interest and robustness of coupling chemical and genetic analyses in the identification of plant species. In this study, the Lavandula genus was used. METHODS: In this study, we used two analytical methods. Chemical analysis from UHPLC (ultra-high-performance liquid chromatography) and genetic analysis from barcoding with genetic markers. RESULTS: Eleven lavender species were selected (botanically authenticated) and analysed. The results show that three chemical compounds (coumaric acid hexoside, ferulic acid hexoside and rosmarinic acid) and three genetic markers (RbcL, trnH-psbA and ITS) are of interest for the differentiation of species of the genus lavandula. CONCLUSION: The results show that the combination of complementary analytical methods is a relevant system to prove the botanical identification of lavender species. This first study, carried out on a plant of interest for cosmetics, demonstrates the need for authentication using a tool combining genetic and chemical analysis as an advance over traditional investigation methods used alone, in terms of identification and authentication reliability.

OBJECTIF: Cette étude a été lancée et menée par plusieurs laboratoires, trois des principaux fournisseurs d'ingrédients cosmétiques et quatre marques de cosmétiques en France. Son objectif est de montrer qu'associer les analyses chimiques et génétiques dans l'identification des espèces végétales présente un intérêt et est une approche solide. Dans cette étude, c'est le genre Lavandula qui a été utilisé. MÉTHODES: Dans cette étude, nous avons fait appel à deux méthodes analytiques. L'analyse chimique, à partir de la chromatographie en phase liquide à haute performance (ultra-high-performance liquid chromatography, UHPLC), et l'analyse génétique en procédant à un codage à barres avec des marqueurs génétiques. RÉSULTATS: Onze espèces de lavande ont été sélectionnées (authentifiées du point de vue botanique) et analysées. Les résultats montrent que trois composés chimiques (acide coumarique hexoside, acide ferulique hexoside et acide rosmarinique) et trois marqueurs génétiques (RbcL, trnH-psbA et ITS) présentent un intérêt pour la différenciation des espèces du genre lavandula. CONCLUSION: Les résultats montrent que la combinaison de méthodes analytiques complémentaires est un système pertinent pour prouver l'identification végétale des espèces de lavande. Cette première étude, réalisée sur une plante qui offre un intérêt pour les cosmétiques, démontre la nécessité de procéder à une authentification à l'aide d'un outil qui conjugue analyse génétique et chimique ; elle représente une avancée par rapport aux méthodes d'investigation traditionnelles utilisées seules, en termes d'identification et de fiabilité de l'authentification.

Genotyping Analysis by RAD-Seq Reads Is Useful to Assess the Genetic Identity and Relationships of Breeding Lines in Lavender Species Aimed at Managing Plant Variety Protection.

Lavender species are widely distributed in their wild forms around the Mediterranean Basin and they are also cultivated worldwide as improved and registered clonal varieties. The economic interest of the species belonging to the Lavandula genus is determined by their use as ornamental plants and important source of essential oils that are destinated to the production of cosmetics, pharmaceuticals and foodstuffs. Because of the increasing number of cases of illegal commercialization of selected varieties, the protection of plant breeders' rights has become of main relevance for the recognition of breeding companies' royalties. With this aim, genomic tools based on molecular markers have been demonstrated to be very reliable and transferable among laboratories, and also much more informative than morphological descriptors. With the rising of the next-generation sequencing (NGS) technologies, several genotyping-by-sequencing approaches are now available. This study deals with a deep characterization of 15 varietal clones, belonging to two distinct Lavandula species, by means of restriction-site associated DNA sequencing (RAD-Seq). We demonstrated that this technology screens single nucleotide variants that enable to assess the genetic identity of individual accessions, to reconstruct genetic relationships among related breeding lines, to group them into genetically distinguishable main subclusters, and to assign their molecular lineages to distinct ancestors. Moreover, a number of polymorphic sites were identified within genes putatively involved in biosynthetic pathways related to both tissue pigmentation and terpene production, useful for breeding and/or protecting newly registered varieties. Overall, the results highlighted the presence of pure ancestries and interspecific hybrids for the analyzed Lavandula species, and demonstrated that RAD-Seq analysis is very informative and highly reliable for characterizing Lavandula clones and managing plant variety protection.

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.

Lifetime genealogical divergence within plants leads to epigenetic mosaicism in the shrub Lavandula latifolia (Lamiaceae).

Epigenetic mosaicism is a possible source of within-plant phenotypic heterogeneity, yet its frequency and developmental origin remain unexplored. This study examines whether extant epigenetic heterogeneity within Lavandula latifolia (Lamiaceae) shrubs reflects recent epigenetic modifications experienced independently by different plant parts or, alternatively, it is the cumulative outcome of a steady lifetime process. Leaf samples from different architectural modules (branch tips) were collected from three L. latifolia plants and characterized epigenetically by global DNA cytosine methylation and methylation state of methylation-sensitive amplified fragment-length polymorphism (MS-AFLP) markers. Epigenetic characteristics of modules were then assembled with information on the branching history of plants. Methods borrowed from phylogenetic research were used to assess genealogical signal of extant epigenetic variation and reconstruct within-plant genealogical trajectory of epigenetic traits. Plants were epigenetically heterogeneous, as shown by differences among modules in global DNA methylation and variation in the methylation states of 6 to 8% of MS-AFLP markers. All epigenetic features exhibited significant genealogical signal within plants. Events of epigenetic divergence occurred throughout the lifespan of individuals and were subsequently propagated by branch divisions. Internal epigenetic diversification of L. latifolia individuals took place steadily during their development, a process which eventually led to persistent epigenetic mosaicism.

Quick and efficient approach to develop genomic resources in orphan species: Application in Lavandula angustifolia.

Next-Generation Sequencing (NGS) technologies, by reducing the cost and increasing the throughput of sequencing, have opened doors to generate genomic data in a range of previously poorly studied species. In this study, we propose a method for the rapid development of a large-scale molecular resources for orphan species. We studied as an example the true lavender (Lavandula angustifolia Mill.), a perennial sub-shrub plant native from the Mediterranean region and whose essential oil have numerous applications in cosmetics, pharmaceuticals, and alternative medicines. The heterozygous clone "Maillette" was used as a reference for DNA and RNA sequencing. We first built a reference Unigene, compound of coding sequences, thanks to de novo RNA-seq assembly. Then, we reconstructed the complete genes sequences (with introns and exons) using an Unigene-guided DNA-seq assembly approach. This aimed to maximize the possibilities of finding polymorphism between genetically close individuals despite the lack of a reference genome. Finally, we used these resources for SNP mining within a collection of 16 commercial lavender clones and tested the SNP within the scope of a genetic distance analysis. We obtained a cleaned reference of 8, 030 functionally in silico annotated genes. We found 359K polymorphic sites and observed a high SNP frequency (mean of 1 SNP per 90 bp) and a high level of heterozygosity (more than 60% of heterozygous SNP per genotype). On overall, we found similar genetic distances between pairs of clones, which is probably related to the out-crossing nature of the species and the restricted area of cultivation. The proposed method is transferable to other orphan species, requires little bioinformatics resources and can be realized within a year. This is also the first reported large-scale SNP development on Lavandula angustifolia. All the genomics resources developed herein are publicly available and provide a rich pool of molecular resources to explore and exploit lavender genetic diversity in breeding programs.

Transcriptome profiling of spike provides expression features of genes related to terpene biosynthesis in lavender.

Lavender (Lavandula angustifolia) is an important economic plant because of the value of its essential oil (EO). The Yili Valley in Xinjiang has become the largest lavender planting base in China. However, there is a lack of research on the gene expression regulation of EO biosynthesis and metabolism in local varieties. Here, de novo transcriptome analysis of inflorescence of three development stages from initial flower bud to flowering stage 50% from two lavender cultivars with contrasting EO production revealed the dynamics of 100,177 differentially expressed transcripts (DETs) in various stages of spike development within and across the cultivars. The lavender transcriptome contained 77 DETs with annotations related to terpenoid biosynthesis. The expression profiles of the 27 genes involved in the methylerythritol phosphate (MEP) pathway, 22 genes in the mevalonate (MVA) pathway, 28 genes related to monoterpene and sesquiterpene biosynthesis during inflorescence development were comprehensively characterized, and possible links between the expression changes of genes and contents of EO constituents were explored. The upregulated genes were mainly concentrated in the MEP pathway, while most genes in the MVA pathway were downregulated during flower development, and cultivars with a higher EO content presented higher expression of genes in the MEP pathway, indicating that EOs were chiefly produced through the MEP pathway. Additionally, MYB transcription factors constituted the largest number of transcripts in all samples, suggesting their potential roles in regulating EO biosynthesis. The sequences and transcriptional patterns of the transcripts will be helpful for understanding the molecular basis of lavender terpene biosynthesis.

Short-chain isoprenyl diphosphate synthases of lavender (Lavandula).

KEY MESSAGE: We reported the functional characterization of cDNAs encoding short-chain isoprenyl diphosphate synthases that control the partitioning of precursors for lavender terpenoids. Lavender essential oil is composed of regular and irregular monoterpenes, which are derived from linear precursors geranyl diphosphate (GPP) and lavandulyl diphosphate (LPP), respectively. Although this plant strongly expresses genes responsible for the biosynthesis of both monoterpene classes, it is unclear why regular monoterpenes dominate the oil. Here, we cloned and characterized Lavandula x intermedia cDNAs encoding geranyl diphosphate synthase (LiGPPS), geranylgeranyl diphosphate synthase (LiGGPPS) and farnesyl diphosphate synthase (LiFPPS). LiGPPS was heteromeric protein, consisting of a large subunit (LiGPPS.LSU) and a small subunit for which two different cDNAs (LiGPPS.SSU1 and LiGPPS.SSU2) were detected. Neither recombinant LiGPPS subunits was active by itself. However, when co-expressed in E. coli LiGPPS.LSU and LiGPPS.SSU1 formed an active heteromeric GPPS, while LiGPPS.LSU and LiGPPS.SSU2 did not form an active protein. Recombinant LiGGPPS, LiFPPS and LPP synthase (LPPS) proteins were active individually. Further, LiGPPS.SSU1 modified the activity of LiGGPPS (to produce GPP) in bacterial cells co-expressing both proteins. Given this, and previous evidence indicating that GPPS.SSU can modify the activity of GGPPS to GPPS in vitro and in plants, we hypothesized that LiGPPS.SSU1 modifies the activity of L. x intermedia LPP synthase (LiLPPS), thus accounting for the relatively low abundance of LPP-derived irregular monoterpenes in this plant. However, LiGPPS.SSU1 did not affect the activity of LiLPPS. These results, coupled to the observation that LiLPPS transcripts are more abundant than those of GPPS subunits in L. x intermedia flowers, suggest that regulatory mechanisms other than transcriptional control of LPPS regulate precursor partitioning in lavender flowers.

Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia.

MAIN CONCLUSION: Using RNA-Seq, we identified genes involved in floral development in lavenders and functionally characterized the floral repressor LaSVP. The molecular aspects of flower initiation and development have not been adequately investigated in lavender (Lavandula). In order to identify genes that control these processes, we employed RNA-Seq to obtain sequence information for transcripts originating from the vegetative shoot apical meristem (SAM) and developing inflorescence tissues of Lavandula angustifolia and Lavandula × intermedia plants, and assemble a comprehensive transcriptome of 105,294 contigs. Homology-based annotation provided gene ontology terms for the majority of transcripts, including over 100 genes homologous to those that control flower initiation and organ identity in Arabidopsis thaliana. Expression analysis revealed that most of these genes are differentially expressed during flower development. For example, LaSVP, a homolog of the floral repressor SHORT VEGETATIVE PHASE (SVP), was strongly expressed in vegetative SAM compared to developing flowers, implicating its potential involvement in flowering repression in lavender. To investigate LaSVP further, we constitutively expressed the gene in transformed A. thaliana plants, evaluating its effects on flower initiation and morphology. Expression of the LaSVP in A. thaliana delayed flowering and affected flower organ identity in a dosage-dependent manner. Two of the highest expressing lines produced sepals instead of petals and were sterile as they failed to develop proper seed pods. This study provides the foundation for future investigations aimed at elucidating flower initiation and development in lavender.

Diverse transcription factors control monoterpene synthase expression in lavender (Lavandula).

MAIN CONCLUSION: We cloned eight transcription factors that activate lavender monoterpene synthase promoters. In this study, we employed the Yeast One-Hybrid (Y1H) assay system to identify transcription factors that control promoters for two Lavandula × intermedia monoterpene synthase genes, linalool synthase (LiLINS) and 1,8-cineole synthase (LiCINS). The bait sequences used in the assay were either a 768-bp LiLINS, or a 1087-bp LiCINS promoter. The prey included proteins expressed in L. × intermedia floral tissue. The assay identified 96 sequences encoding proteins that interacted with one or both promoters. To explore the nature of this interaction, the LiLINS and LiCINS promoter fragments were each fused to the E. coli gusA (GUS) reporter gene. The constructs were separately transformed into tobacco (Nicotiana benthamiana) leaves co-expressing individually a subset of ten representative transcription factors (TFs) predicted to control these promoters. Six TFs induced expression from both promoters, two activated LiCINS promoter alone, and two did not induce expression from either promoter. The TFs identified in this study belong to various groups including those containing conserved domains typical of MYB, bZIP, NAC, GeBP and SBP-related proteins.

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.

A lavender ABC transporter confers resistance to monoterpene toxicity in yeast.

MAIN CONCLUSION: Functional expression of a multidrug resistance-type ABC transporter from Lavandulaangustifolia improved yeast resistance to geraniol, a monoterpene constituent of lavender essential oil. Plant ATP-binding cassette (ABC) transporters are a large family of membrane proteins involved in active and selective transport of structurally diverse compounds. In this study, we functionally evaluated LaABCB1, a multidrug resistance (MDR)-type ABC transporter strongly expressed in the secretory cells of lavender glandular trichomes, where monoterpene essential oil constituents are synthesized and secreted. We used LaABCB1 to complement a yeast knockout mutant in which 16 ABC transporters were deleted. Expression of LaABCB1 enhanced tolerance of yeast mutants to geraniol, a key constituent of essential oils in lavenders and numerous other plants. Our findings suggest a role for the MDR-type ABC transporters in the toxicity tolerance of at least certain essential oil constituents in lavender oil glands.