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1.
Planta ; 254(3): 54, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34410495

RESUMO

MAIN CONCLUSION: The expression of full-length cDNAs encoding lavender AGAMOUS-like (LaAG-like) and SEPALLATA3-like (LaSEP3-like) transcription factors induces early flowering and impacts the leaf morphology at a strong expression level in Arabidopsis. Lavandula angustifolia is widely cultivated as an ornamental plant due to its attractive flower structure, and as a source of valuable essential oils for use in cosmetics, alternative medicines, and culinary products. We recently employed RNA-Seq and transcript profiling to describe a number of transcription factors (TFs) that potentially control flower development in this plant. In this study, we investigated the roles of two TFs, LaAGAMOUS-like (LaAG-like) and LaSEPALLATA3-like (LaSEP3-like), that exhibited substantial homology to Arabidopsis thaliana floral development genes, AGAMOUS and SEPALLATA3, respectively, in flowering initiation in Arabidopsis. We stably and constitutively expressed LaAG-like and LaSEP3-like cDNAs in separate Arabidopsis plants. All transgenic plants flowered earlier than the wild-type controls. However, plants that modestly overexpressed the gene were phenotypically normal, while those that strongly expressed the transgene developed curly leaves. We also assessed the expression of five endogenous flowering time regulating genes, from which high expression of Flowering Locus T (AtFT) mRNA in both LaAG-like (type-I and -II) and LaSEP3-like (type-I), and Leafy (AtLFY) mRNAs in LaSEP3-like (type-I) transgenic plants were detected, compared to wild-type controls. Our results suggest that with controlled expression, lavender AG-like and SEP3-like genes are potentially useful for the regulation of flowering time in commercial lavender species, and could be used for plant improvement studies through molecular genetics and targeted breeding programs.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Lavandula , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Lavandula/metabolismo , Proteínas de Domínio MADS/genética , Melhoramento Vegetal , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Repressoras
2.
Front Plant Sci ; 12: 699157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34349773

RESUMO

The isoprenoids play important ecological and physiological roles in plants. They also have a tremendous impact on human lives as food additives, medicines, and industrial raw materials, among others. Though some isoprenoids are highly abundant in nature, plants produce many at extremely low levels. Glandular trichomes (GT), which cover the aerial parts of more than 25% of vascular plants, have been considered as natural biofactories for the mass production of rare industrially important isoprenoids. In several plant genera (e.g., Lavandula and Mentha), GTs produce and store large quantities of the low molecular weight isoprenoids, in particular mono- and sesquiterpenes, as essential oil constituents. Within each trichome, a group of secretory cells is specialized to strongly and specifically express isoprenoid biosynthetic genes, and to synthesize and deposit copious amounts of terpenoids into the trichome's storage reservoir. Despite the abundance of certain metabolites in essential oils and defensive resins, plants, particularly those lacking glandular trichomes, accumulate small quantities of many of the biologically active and industrially important isoprenoids. Therefore, there is a pressing need for technologies to enable the mass production of such metabolites, and to help meet the ever-increasing demand for plant-based bioproducts, including medicines and renewable materials. Considerable contemporary research has focused on engineering isoprenoid metabolism in GTs, with the goal of utilizing them as natural biofactories for the production of valuable phytochemicals. In this review, we summarize recent advances related to the engineering of isoprenoid biosynthetic pathways in glandular trichomes.

3.
Planta ; 253(1): 17, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33392743

RESUMO

MAIN CONCLUSION: Using RNA profiling, we identified several silver thiosulfate-induced genes that potentially control the masculinization of female Cannabis sativa plants. Genetically female Cannabis sativa plants normally bear female flowers, but can develop male flowers in response to environmental and developmental cues. In an attempt to elucidate the molecular elements responsible for sex expression in C. sativa plants, we developed genetically female lines producing both female and chemically-induced male flowers. Furthermore, we carried out RNA-Seq assays aimed at identifying differentially expressed genes responsible for male flower development in female plants. The results revealed over 10,500 differentially expressed genes, of which around 200 potentially control masculinization of female cannabis plants. These genes include transcription factors and other genes involved in male organ (i.e., anther and pollen) development, as well as genes involved in phytohormone signalling and male-biased phenotypes. The expressions of 15 of these genes were further validated by qPCR assay confirming similar expression patterns to that of RNA-Seq data. These genes would be useful for understanding predisposed plants producing flowers of both sex types in the same plant, and help breeders to regulate the masculinization of female plants through targeted breeding and plant biotechnology.


Assuntos
Cannabis , Proteínas de Plantas , RNA-Seq , Cannabis/genética , Cannabis/crescimento & desenvolvimento , Cannabis/metabolismo , Flores/genética , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Proteínas de Plantas/metabolismo
4.
Plant Mol Biol ; 102(4-5): 517-535, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927660

RESUMO

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.


Assuntos
Alquil e Aril Transferases/metabolismo , Dimetilaliltranstransferase/metabolismo , Flores/enzimologia , Lavandula/enzimologia , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/genética , Catálise , Dimetilaliltranstransferase/genética , Difosfatos , Diterpenos , Escherichia coli/genética , Escherichia coli/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Lavandula/genética , Monoterpenos/metabolismo , Fases de Leitura Aberta , Fenótipo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Terpenos/metabolismo , Tabaco/metabolismo
5.
Planta ; 251(2): 41, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31907678

RESUMO

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.


Assuntos
Flores/metabolismo , Lavandula/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Lavandula/genética , Meristema/genética , Meristema/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
6.
Planta ; 251(1): 5, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776766

RESUMO

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.


Assuntos
Lavandula/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Lavandula/enzimologia , Lavandula/genética , Regiões Promotoras Genéticas/genética , Tabaco/genética , Tabaco/metabolismo , Fatores de Transcrição/genética
7.
Planta ; 249(1): 251-256, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30269192

RESUMO

MAIN CONCLUSION: The first draft genome for a member of the genus Lavandula is described. This 870 Mbp genome assembly is composed of over 688 Mbp of non-gap sequences comprising 62,141 protein-coding genes. Lavenders (Lavandula: Lamiaceae) are economically important plants widely grown around the world for their essential oils (EOs), which contribute to the cosmetic, personal hygiene, and pharmaceutical industries. To better understand the genetic mechanisms involved in EO production, identify genes involved in important biological processes, and find genetic markers for plant breeding, we generated the first de novo draft genome assembly for L. angustifolia (Maillette). This high-quality draft reveals a moderately repeated (> 48% repeated elements) 870 Mbp genome, composed of over 688 Mbp of non-gap sequences in 84,291 scaffolds with an N50 value of 96,735 bp. The genome contains 62,141 protein-coding genes and 2003 RNA-coding genes, with a large proportion of genes showing duplications, possibly reflecting past genome polyploidization. The draft genome contains full-length coding sequences for all genes involved in both cytosolic and plastidial pathways of isoprenoid metabolism, and all terpene synthase genes previously described from lavenders. Of particular interest is the observation that the genome contains a high copy number (14 and 7, respectively) of DXS (1-deoxyxylulose-5-phosphate synthase) and HDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase) genes, encoding the two known regulatory steps in the plastidial isoprenoid biosynthetic pathway. The latter generates precursors for the production of monoterpenes, the most abundant essential oil constituents in lavender. Furthermore, the draft genome contains a variety of monoterpene synthase genes, underlining the production of several monoterpene essential oil constituents in lavender. Taken together, these findings indicate that the genome of L. angustifolia is highly duplicated and optimized for essential oil production.


Assuntos
Lavandula/metabolismo , Óleos Voláteis/metabolismo , Terpenos/metabolismo , Lavandula/genética
8.
Planta ; 249(1): 139-144, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30535718

RESUMO

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.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Lavandula/genética , Monoterpenos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/genética , Saccharomyces cerevisiae/genética
9.
Planta ; 249(1): 271-290, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29948128

RESUMO

MAIN CONCLUSION: Using RNA-Seq, we cloned and characterized a unique monoterpene synthase responsible for the formation of a scent-determining S-linalool constituent of lavender oils from Lavandula × intermedia. Several species of Lavandula produce essential oils (EOs) consisting mainly of monoterpenes including linalool, one of the most abundant and scent-determining oil constituents. Although R-linalool dominates the EOs of lavenders, varying amounts (depending on the species) of the S-linalool enantiomer can also be found in these plants. Despite its relatively low abundance, S-linalool contributes a sweet, pleasant scent and is an important constituent of lavender EOs. While several terpene synthase genes including R-linalool synthase have been cloned from lavenders many important terpene synthases including S-linalool synthase have not been described from these plants. In this study, we employed RNA-Seq and other complementary sequencing data to clone and functionally characterize the sparsely expressed S-linalool synthase cDNA (LiS-LINS) from Lavandula × intermedia. Recombinant LiS-LINS catalyzed the conversion of the universal monoterpene precursor geranyl diphosphate to S-linalool as the sole product. Intriguingly, LiS-LINS exhibited very low (~ 30%) sequence similarity to other Lavandula terpene synthases, including R-linalool synthase. However, the predicted 3D structure of this protein, including the composition and arrangement of amino acids at the active site, is highly homologous to known terpene synthase proteins. LiS-LINS transcripts were detected in flowers, but were much less abundant than those corresponding to LiR-LINS, paralleling enantiomeric composition of linalool in L. × intermedia oils. These data indicate that production of S-linalool is at least partially controlled at the level of transcription from LiS-LINS. The cloned LiS-LINS cDNA may be used to enhance oil composition in lavenders and other plants through metabolic engineering.


Assuntos
Alquil e Aril Transferases/metabolismo , Hidroliases/metabolismo , Lavandula/metabolismo , Odorantes , Óleos Voláteis/metabolismo
10.
Plant Mol Biol ; 93(6): 641-657, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28258552

RESUMO

KEY MESSAGE: A methyl jasmonate responsive 3-carene synthase (Li3CARS) gene was isolated from Lavandula x intermedia and functionally characterized in vitro. Lavenders produce essential oils consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene 3-carene. In this study we isolated and functionally characterized a leaf-specific, methyl jasmonate (MeJA)-responsive monoterpene synthase (Li3CARS) from Lavandula x intermedia. The ORF excluding transit peptides encoded a 64.9 kDa protein that was expressed in E. coli, and purified with Ni-NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as the major product, with K m and k cat of 3.69 ± 1.17 µM and 2.01 s-1 respectively. Li3CARS also accepted NPP as a substrate to produce multiple products including a small amount of 3-carene. The catalytic efficiency of Li3CARS to produce 3-carene was over ten fold higher for GPP (k cat /K m = 0.56 µM-1s-1) than NPP (k cat /K m = 0.044 µM-1s-1). Production of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves (16-fold) as compared to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene production, and was up-regulated (1.18- to 3.8-fold) with MeJA 8-72 h post-treatment. The results suggest that Li3CARS may have a defensive role in Lavandula.


Assuntos
Acetatos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Ciclopentanos/metabolismo , Lavandula/genética , Oxilipinas/metabolismo , Proteínas de Plantas/genética , Alquil e Aril Transferases/química , Domínio Catalítico , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Lavandula/enzimologia , Monoterpenos/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Homologia Estrutural de Proteína
11.
Nat Prod Commun ; 11(4): 523-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27396210

RESUMO

A growing body of literature indicates that many synthetic pesticides have adverse effects on human, animal, and environmental health. As a result, plant-derived natural products are quickly gaining momentum as safer and less ecologically damaging alternatives due to their low toxicity, high biodegradability, and good specificity. Essential oils of Lavandula angustifolia, Lavandula x intermedia cv Grosso, and Lavandida x intermedia cv Provence as well as various mono- and sesquiterpene essential oil constituents were tested in order to assess their antifungal potential on three important agricultural pathogens: Botrytis cinerea, Mucor piriformis, and Penicillium expansum. Fungal susceptibility testing was performed using disk diffusion assays. The majority of essential oil constituents tested did not have a significant effect; however, 3-carene, carvacrol, geraniol, nerol and perillyl alcohol demonstrated significant inhibition at concentrations as low as 1 µ/mL. In vivo testing using strawberry fruit as a model system supported in vitro results and revealed that perillyl alcohol, carvacrol and 3-carene were effective in limiting infection by postharvest pathogens.


Assuntos
Antifúngicos/análise , Lavandula/química , Óleos Voláteis , Óleos Vegetais , Botrytis , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Mucor , Penicillium
12.
Planta ; 242(3): 709-19, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25998527

RESUMO

MAIN CONCLUSION: Two alcohol acetyltransferases, LiAAT-3 and LiAAT-4, from L. x intermedia were cloned, expressed in bacteria, and functionally characterized. Two monoterpene acetyltransferase cDNA clones (LiAAT-3 and LiAAT-4) were isolated from L. x intermedia glandular trichomes, expressed in bacteria to produce, and functionally characterize the encoded proteins in vitro. The recombinant LiAAT-3 and LiAAT-4 proteins had molecular weights of ca. 47 and 49 kDa, respectively, as evidenced by SDS-PAGE. The K m (mM) values for the recombinant LiAAT-3 and LiAAT-4 were 1.046 and 0.354 for lavandulol, 1.31 and 0.279 for geraniol, and 0.87 and 0.113 for nerol, respectively. The V max (pkat/mg) values for LiAAT-3 and LiAAT-4 were 92.13 and 105.1 for lavandulol, 81.07 and 52.17 for geraniol, and 15.02 and 15.8 for nerol, correspondingly. Catalytic efficiencies (mM(-1) min(-1)) for LiAAT-3 and LiAAT-4 were 0.27 and 0.85 for lavandulol, 0.19 and 0.54 for geraniol, and 0.052 and 0.4 for nerol, respectively. These kinetic properties are in the range of those reported for other plant acetyltransferases, and indicate that LiAAT-4 has a better catalytic efficiency than LiAAT-3, with lavandulol serving as the preferred substrate for both enzymes. Transcripts for both genes were abundant in L. angustifolia and L. x intermedia flowers, where monoterpene acetates are produced, and were undetectable (or present in trace quantities) in L. latifolia flowers, which do not accumulate significant amounts of these metabolites.


Assuntos
Acetiltransferases/metabolismo , Lavandula/enzimologia , Proteínas de Plantas/metabolismo , Tricomas/enzimologia , Acetiltransferases/genética , Regulação da Expressão Gênica de Plantas , Lavandula/genética , Monoterpenos/metabolismo , Proteínas de Plantas/genética , Tricomas/genética
13.
Planta ; 241(4): 987-1004, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25534945

RESUMO

MAIN CONCLUSION: We identified and characterized EST-SSRs with strong discrimination power against Lavandula angustifolia and Lavandula x intermedia . The markers also showed considerable cross-species transferability rate into six related Lavandula species. Lavenders (Lavandula) are important economical crops grown around the globe for essential oil production. In an attempt to develop genetic markers for these plants, we analyzed over 13,000 unigenes developed from L. angustifolia and L. x intermedia EST databases, and identified 3,459 simple sequence repeats (SSR), which were dominated by trinucleotides (41.2 %) and dinucleotides (31.45 %). Approximately, 19 % of the unigenes contained at least one SSR marker, over 60 % of which were localized in the UTRs. Only 252 EST-SSRs were 18 bp or longer from which 31 loci were validated, and 24 amplified discrete fragments with 85 % polymorphism in L. x intermedia and L. angustifolia. The average number of alleles in L. x intermedia and L. angustifolia were 3.42 and 3.71 per marker with average PIC values of 0.47 and 0.52, respectively. These values suggest a moderate to strong level of informativeness for the markers, with some loci producing unique fingerprints. The cross-species transferability rate of the markers ranges 50-100 % across eight species. The utility of these markers was assessed in eight Lavandula species and 15 L. angustifolia and L. x intermedia cultivars, and the dendrogram deduced from their similarity indexes successfully delineated the species into their respective sections and the cultivars into their respective species. These markers have potential for application in fingerprinting, diversity studies and marker-assisted breeding of Lavandula.


Assuntos
Lavandula/genética , Repetições de Microssatélites/genética , Alelos , DNA de Plantas/genética , Etiquetas de Sequências Expressas , Marcadores Genéticos/genética , Filogenia , Especificidade da Espécie
14.
Phytochemistry ; 102: 64-73, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24636455

RESUMO

Terpenoids are a large and diverse class of specialized metabolites that are essential for the growth and development of plants, and have tremendous industrial applications. The mericarps of Coriandrum sativum L. (coriander) produce an essential oil (EO) rich in monoterpenes, volatile C10 terpenoids. To investigate EO metabolism, the transcriptome of coriander mericarps, at three developmental stages (early, mid, late) was sequenced via Illumina technology and a transcript library was produced. To validate the usability of the transcriptome sequences, two terpene synthase candidate genes, CsγTRPS and CsLINS, encoding 558 and 562 amino acid proteins were expressed in bacteria, and the recombinant proteins purified by Ni-NTA affinity chromatography. The 65.16 (CsγTRPS) and 65.91 (CsLINS)kDa recombinant proteins catalyzed the conversion of geranyl diphosphate, the precursor to monoterpenes, to γ-terpinene and (S)-linalool, respectively, with apparent Vmax and Km values of 2.24±0.16 (CsγTRPS); 19.63±1.05 (CsLINS)pkat/mg and 66.25±13 (CsγTRPS); 2.5±0.6 (CsLINS)µM, respectively. Together, CsγTRPS and CsLINS account for the majority of EO constituents in coriander mericarps. Investigation of the coriander transcriptome, and knowledge gained from these experiments will facilitate future studies concerning essential and fatty acid oil production in coriander. They also enable efforts to improve the coriander oils through metabolic engineering or plant breeding.


Assuntos
Coriandrum/enzimologia , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Transcriptoma/genética , Clonagem Molecular , Biologia Computacional , Coriandrum/química , Coriandrum/ultraestrutura , Microscopia Eletrônica de Varredura , Estrutura Molecular , Terpenos/química , Terpenos/metabolismo
15.
Planta ; 238(5): 983-9, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23918183

RESUMO

The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.


Assuntos
Lavandula/enzimologia , Lavandula/genética , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo , Tricomas/enzimologia , Tricomas/genética , Sequência de Aminoácidos , Clonagem Molecular , Cruzamentos Genéticos , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
16.
J Biol Chem ; 288(9): 6333-41, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23306202

RESUMO

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 µm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.


Assuntos
Acetatos , Alquil e Aril Transferases , Genes de Plantas , Lavandula , Monoterpenos , Proteínas de Plantas , Acetatos/química , Acetatos/metabolismo , Monoterpenos Acíclicos , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , DNA Complementar/genética , Escherichia coli/genética , Lavandula/enzimologia , Lavandula/genética , Monoterpenos/química , Monoterpenos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Arch Biochem Biophys ; 528(2): 163-70, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23058847

RESUMO

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.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Lavandula/enzimologia , Lavandula/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oxirredutases do Álcool/química , Sequência de Aminoácidos , Sequência de Bases , Canfanos/metabolismo , Cânfora/metabolismo , Clonagem Molecular , DNA de Plantas/genética , Flores/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Cinética , Dados de Sequência Molecular , Peso Molecular , Óleos Voláteis/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
18.
Plant Mol Biol ; 79(4-5): 393-411, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22592779

RESUMO

Several members of the genus Lavandula produce valuable essential oils (EOs) that are primarily constituted of the low molecular weight isoprenoids, particularly monoterpenes. We isolated over 8,000 ESTs from the glandular trichomes of L. x intermedia flowers (where bulk of the EO is synthesized) to facilitate the discovery of genes that control the biosynthesis of EO constituents. The expression profile of these ESTs in L. x intermedia and its parents L. angustifolia and L. latifolia was established using microarrays. The resulting data highlighted a differentially expressed, previously uncharacterized cDNA with strong homology to known 1,8-cineole synthase (CINS) genes. The ORF, excluding the transit peptide, of this cDNA was expressed in E. coli, purified by Ni-NTA agarose affinity chromatography and functionally characterized in vitro. The ca. 63 kDa bacterially produced recombinant protein, designated L. x intermedia CINS (LiCINS), converted geranyl diphosphate (the linear monoterpene precursor) primarily to 1,8-cineole with K ( m ) and k ( cat ) values of 5.75 µM and 8.8 × 10(-3) s(-1), respectively. The genomic DNA of CINS in the studied Lavandula species had identical exon-intron architecture and coding sequences, except for a single polymorphic nucleotide in the L. angustifolia ortholog which did not alter protein function. Additional nucleotide variations restricted to L. angustifolia introns were also observed, suggesting that LiCINS was most likely inherited from L. latifolia. The LiCINS mRNA levels paralleled the 1,8-cineole content in mature flowers of the three lavender species, and in developmental stages of L. x intermedia inflorescence indicating that the production of 1,8 cineole in Lavandula is most likely controlled through transcriptional regulation of LiCINS.


Assuntos
Carbono-Carbono Liases/genética , Lavandula/enzimologia , Lavandula/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Carbono-Carbono Liases/química , Carbono-Carbono Liases/metabolismo , Clonagem Molecular , Cicloexanóis/metabolismo , DNA de Plantas/genética , Eucaliptol , Etiquetas de Sequências Expressas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genoma de Planta , Lavandula/crescimento & desenvolvimento , Redes e Vias Metabólicas , Dados de Sequência Molecular , Monoterpenos/metabolismo , Óleos Voláteis/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
19.
Planta ; 233(4): 685-96, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21165645

RESUMO

En route to building genomics resources for Lavandula, we have obtained over 14,000 ESTs for leaves and flowers of L. angustifolia, a major essential oil crop, and identified a number of previously uncharacterized terpene synthase (TPS) genes. Here we report the cloning, expression in E. coli, and functional characterization of ß-phellandrene synthase, LaßPHLS. The ORF--excluding the transit peptide--for this gene encoded a 62.3 kDa protein that contained all conserved motifs present in plant TPSs. Expression in bacteria resulted in the production of a soluble protein that was purified by Ni-NTA agarose affinity chromatography. While the recombinant LaßPHLS did not utilize FPP as a substrate, it converted GPP (the preferred substrate) and NPP into ß-phellandrene as the major product, with K (m) and k (cat) of 6.55 µM and 1.75 × 10(-2) s(-1), respectively, for GPP. The LaßPHLS transcripts were highly abundant in young leaves where ß-phellandrene is produced, but were barely detectable in flowers and older leaves, where ß-phellandrene is not synthesized in significant quantities. This data indicate that ß-phellandrene biosynthesis is transcriptionally and developmentally regulated. We also cloned and expressed in E. coli a second TPS-like protein, LaTPS-I, that lacks an internal stretch of 73 amino acids, including the signature DDxxD divalent metal binding motif, compared to other plant TPSs. The recombinant LaTPS-I did not produce detectable products in vitro when assayed with GPP, NPP or FPP as substrates. The lack of activity is most likely due to the absence of catalytically important amino acid residues within the missing region.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Cicloexenos/metabolismo , Lavandula/enzimologia , Lavandula/genética , Monoterpenos/metabolismo , Alquil e Aril Transferases/química , Sequência de Aminoácidos , Clonagem Molecular , Monoterpenos Cicloexânicos , Cicloexenos/química , Ensaios Enzimáticos , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Monoterpenos/química , Óleos Voláteis/análise , Filogenia , Alinhamento de Sequência , Análise de Sequência de Proteína , Transcrição Genética
20.
Planta ; 231(4): 835-45, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20043174

RESUMO

We are developing Lavandula angustifolia (lavender) as a model system for investigating molecular regulation of essential oil (a mixture of mono- and sesquiterpenes) production in plants. As an initial step toward building the necessary 'genomics toolbox' for this species, we constructed two cDNA libraries from lavender leaves and flowers, and obtained sequence information for 14,213 high-quality expressed sequence tags (ESTs). Based on homology to sequences present in GenBank, our EST collection contains orthologs for genes involved in the 1-deoxy-D: -xylulose-5-phosphate (DXP) and the mevalonic acid (MVA) pathways of terpenoid biosynthesis, and for known terpene synthases and prenyl transferases. To gain insight into the regulation of terpene metabolism in lavender flowers, we evaluated the transcriptional activity of the genes encoding for 1-deoxy-D: -xylulose-5-phosphate synthase (DXS) and HMG-CoA reductase (HMGR), which represent regulatory steps of the DXP and MVA pathways, respectively, in glandular trichomes (oil glands) by real-time PCR. While HMGR transcripts were barely detectable, DXS was heavily expressed in this tissue, indicating that essential oil constituents are predominantly produced through the DXP pathway in lavender glandular trichomes. As anticipated, the linalool synthase (LinS)-the gene responsible for the production of linalool, a major constituent of lavender essential oil-was also strongly expressed in glands. Surprisingly, the most abundant transcript in floral glandular trichomes corresponded to a sesquiterpene synthase (cadinene synthase, CadS), although sesquiterpenes are minor constituents of lavender essential oils. This result, coupled to the weak activity of the MVA pathway (the main route for sesquiterpene production) in trichomes, indicates that precursor supply may represent a bottleneck in the biosynthesis of sesquiterpenes in lavender flowers.


Assuntos
Genômica/métodos , Lavandula/genética , Lavandula/metabolismo , Óleos Voláteis/metabolismo , Monoterpenos Acíclicos , Biologia Computacional , Etiquetas de Sequências Expressas , Flores/genética , Flores/metabolismo , Ácido Mevalônico/metabolismo , Modelos Biológicos , Monoterpenos/metabolismo , Pentosefosfatos/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Terpenos/metabolismo
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