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First published online September 15, 2006; 10.1104/pp.106.086355 Plant Physiology 142:890-900 (2006) © 2006 American Society of Plant Biologists Up-Regulation of 1-Deoxy-D-Xylulose-5-Phosphate Synthase Enhances Production of Essential Oils in Transgenic Spike Lavender1Departamento de Biología Vegetal (Facultad de Farmacia), Universidad de Valencia, 46100 Burjasot, Valencia, Spain
Spike lavender (Lavandula latifolia) is an aromatic shrub cultivated worldwide for the production of essential oils. The major constituents of these oils are monoterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-Deoxy-D-xylulose-5-P synthase (DXS) catalyzes the first step of the MEP pathway. A cDNA coding for the Arabidopsis (Arabidopsis thaliana) DXS was constitutively expressed in spike lavender. Gas chromatography/mass spectrometry analyses revealed that transgenic plants accumulated significantly more essential oils compared to controls (from 101.5% to 359.0% and from 12.2% to 74.1% yield increase compared to controls in leaves and flowers, respectively). T0 transgenic plants were grown for 2 years, self-pollinated, and the T1 seeds obtained. The inheritance of the DXS transgene was studied in the T1 generation. The increased essential oil phenotype observed in the transgenic T0 plants was maintained in the progeny that inherited the DXS transgene. Total chlorophyll and carotenoid content in DXS progenies that inherited the transgene depended on the analyzed plant, showing either no variation or a significant decrease in respect to their counterparts without the transgene. Transgenic plants had a visual phenotype similar to untransformed plants (controls) in terms of morphology, growth habit, flowering, and seed germination. Our results demonstrate that the MEP pathway contributes to essential oil production in spike lavender. They also demonstrate that the DXS enzyme plays a crucial role in monoterpene precursor biosynthesis and, thus, in essential oil production in spike lavender. In addition, our results provide a strategy to increase the essential oil production in spike lavender by metabolic engineering of the MEP pathway without apparent detrimental effects on plant development and fitness.
Plant secondary metabolism is a source of both biologically and economically important compounds. Thus, constituents of essential oils are thought to be involved in plant-insect, plant-pathogen, and plant-plant interactions (Pichersky and Gershenzon, 2002  ). In addition, essential oils have an economic value as flavors, fragrances, and medicines (Verlet, 1993). Spike lavender (Lavandula latifolia Medicus), of the family Lamiaceae, is an aromatic shrub native to the Mediterranean region that is cultivated worldwide for the production of spike lavender oil. This oil is traditionally believed to be antibacterial, antifungal, carminative (smooth muscle relaxing), sedative, antidepressive, and effective for burns and insect bites (Font-Quer, 1978). Although the biological activities of this essential oil are still inconclusive, there are clinical data that support their traditional uses (Cavanagh and Wilkinson, 2002). The composition of spike lavender oil is determined mainly by the genetic make-up of each cultivar, although monoterpenes are always the major fraction, with three of them (linalool, cineol, and camphor) accounting for more than 80% of the total sample (Harborne and Williams, 2002). As in other Lamiaceae, these compounds seem to be synthesized and accumulated in the peltate glandular trichomes found on the aerial parts of the species (Hallahan, 2000).
Monoterpene biosynthesis can be divided into four phases (Mahmoud and Croteau, 2002
Although studies on metabolic engineering of monoterpenes in Lamiaceae are scarce, investigations in peppermint (Mentha x piperita) suggest that the MEP pathway may be the main source of precursors (IPP and DMAPP) for monoterpenes in this family (Wildung and Croteau, 2005 ). Also, there are clear indications that precursor supply is a limiting factor in the biosynthesis of monoterpenes from this pathway (Mahmoud and Croteau, 2002). The MEP pathway (Rohmer, 2003) starts by the transketolase-type condensation of two carbons from pyruvate with glyceraldehyde-3-P to form 1-deoxy-D-xylulose-5-P (DXP), catalyzed by DXP synthase (DXS). The subsequent synthesis of MEP, catalyzed by DXP reductoisomerase (DXR), is the previous step for the formation of the C5 units, following reactions in which the last enzyme of the MEP pathway (hydroxymehtylbutenyl diphosphate reductase) synthesizes IPP and DMAPP simultaneously (Fig. 1).
DXS has been postulated to catalyze a crucial step in the formation of plastid-derived isoprenoids (Lois et al., 2000 To investigate the effect of an increase of monoterpene precursors on the final production of essential oils, we targeted the first step in the MEP pathway by overexpressing the DXS cDNA from Arabidopsis in spike lavender. This approach also allowed us to study the contribution of the MEP pathway in the biosynthesis of essential oils in aromatic plants. To date, selection of high essential oil-yielding spike lavender has been exclusively based on conventional breeding programs. Progress in biotechnology offers an alternative and promising approach to improve spike lavender oil production by genetic engineering. Successful application of this approach will depend on the transgene being expressed and inherited in a stable and predictable way. Because of this, we also studied the expression and inheritance of the DXS gene in T1 progeny from transgenic lines that flowered 2 years after growing at the greenhouse.
Generation and Evaluation of Transgenic Spike Lavender
Using an Agrobacterium-based leaf culture protocol set up in our laboratory (Nebauer et al., 2000 All kanamycin-resistant plants were first screened by PCR for the presence of neomycin phosphotransferase II (nptII) and DXS genes (data not shown). All nptII+/DXS+ plants were cloned, acclimatized to ex vitro conditions, and transferred to the greenhouse for further analyses. Seven independent primary transformants (T0), designed as DXS1 to DXS7, were obtained. The transgenic plants did not show any obvious phenotypic differences compared to untransformed greenhouse-grown plants (Fig. 2, A and B ) in terms of morphology, growth habit, flowering, and seed germination.
The number of transgene inserts was determined by Southern blotting using both nptII and DXS probes. Five out of the seven independent transgenic lines (DXS1, DXS2, DXS3, DXS5, and DXS7) presented single-copy insertions, while transgenic lines DXS4 and DXS6 had two copies of the DXS gene (Fig. 3A ). Identical hybridization patterns were obtained for the nptII transgene (data not shown), corroborating the insertion of the complete T-DNA.
To correlate phenotypes of the DXS lines with gene expression, mRNA and protein levels of DXS were determined for transgenic T0 plants. Total RNA was isolated from leaves and flowers, and expression of the transgenic DXS mRNA was determined by northern-blot analyses. Figure 3, B and C, shows that there are large differences in expression levels of the DXS cDNA among the independent transgenic lines. In leaves (Fig. 3B), all lines but one (DXS3) showed detectable levels of DXS mRNA, with lines 1, 4, 5, 6, and 7 showing the highest levels. Again, transgene expression in flowers (Fig. 3C) was line dependent, with lines DXS1, 4, 6, and 7 showing the highest levels. Under the conditions used in our experiments, the DXS probe did not cross-react with the spike lavender DXS transcript because there was no detectable band in the control lane (Fig. 3B). Western blotting performed on extracts obtained from young leaves demonstrated that the processed DXS protein was detected in control and transgenic lines, but the latter showed a moderately higher level (Fig. 3D).
Essential oil analyses were accomplished by hydro-distillation of air-dried leaves or flowers followed by gas chromatography (GC) separation of components and quantification using internal standards. Identification of the oil components was corroborated by GC/mass spectrometry (MS). The most common components usually found in spike lavender oils were present in the oil samples analyzed (Harborne and Williams, 2002
Essential oil yield (milligram per gram dried tissue) in leaves and flowers of spike lavender was markedly increased in most of the transgenic plants that expressed the DXS cDNA (Table I
). In leaves, six out of seven transgenic lines accumulated significantly more essential oils than controls (from 101.5% to 359.0% yield increase in DXS7 and DXS1, respectively). In both transgenic and control plants, essential oil production in flowers was higher than in leaves. Moreover, flowers from all transgenic plants produced significantly more essential oils than the controls, but the yield increase (from 17.2% to 74.1% in DXS2 and DXS1, respectively) was lower than in leaves (Table I). Both hydrocarbon and oxygenated monoterpenes contributed to the increased oil yield of transgenic plants, but the latter showed the highest increases in relation to the controls (increase averages of 67.6% versus 171% and 25.1% versus 42.7% in leaves and flowers, respectively). Table II
summarizes the percentages of major essential oil constituents in transgenic and control spike lavender plants. In transgenic lines, these percentages were within the range already described in spike lavender essential oil and similar to the controls (Table II). Note that linalool, a major constituent in spike lavender oils from flowers, is a minor constituent in the leaf oils (
Inheritance of the DXS Gene in T1 Spike Lavender Plants Correlated with a Higher Essential Oil Yield To characterize integration of the transforming cDNA into the spike lavender genome and its inheritance, we analyzed T1 plants derived from self-pollinated seeds of the T0 lines that flowered after 2 years growing in the greenhouse. Inheritance of the DXS and nptII transgenes was determined by PCR (Table III ). Transgenic lines DXS1, 2, and 5, having only one DXS copy according to the Southern blot, showed the 3:1 segregation, whereas DXS6, having two copies, showed a 15:1 segregation (Fig. 3A). However, line DXS4, which according to the Southern blotting also had two copies (Fig. 3A), showed a 3:1 segregation, suggesting that in this plant both copies were integrated in the same chromosome. Segregation of the nptII gene was concordant with the DXS gene (data not shown). Thus, both transgenes behave as typical dominant, linked genes. T1 progenies from the highest essential oil-producing lines (DXS1, 4, and 6) were selected for further molecular and phenotypic analyses.
Southern blotting patterns of the DXS1-T1 plants that inherited the transgene were similar to that of the single-copy parent (Fig. 4A ). As expected, progeny of the two copy parent lines (DXS4 and 6) that inherited the transgene showed one or two copies in the corresponding Southern blot (Fig. 4, C and E). Note, however, that most of the DXS4-T1 positive plants inherited the two copies of the DXS transgene, corroborating the 3:1 segregation obtained from PCR analyses (Table III). Data from the Southern analysis is consistent with the northern analysis: plant material exhibiting transgene expression also exhibited the complete DNA repertoire, whereas material from nontransgene-expressing plants contained no detectable signal (Fig. 4).
Essential oil analyses of T1 plants showed that the enhanced monoterpene content observed in the first generation was stable in the subsequent generation (Tables I and IV ). Regardless of the T0 parental line, most of the T1 plants that inherited the DXS gene maintained their elevated essential oil phenotype; average oil yield increases in DXS1, DXS4, and DXS6 progenies that inherited the transgene were 77.3%, 38.4%, and 48.8% with respect to their T1 counterparts without the transgene (Table IV). Corroborating the results obtained in transgenic T0 plants, oxygenated monoterpenes constituted the main contribution to the increased oil yield (Tables I and IV). Note that northern-blot analyses indicated that increases in the expression of the DXS cDNA from Arabidopsis (Fig. 4) roughly paralleled increases in the monoterpene content of progeny that inherited the DXS gene (Table IV). The essential oil compositions of the T1 samples were similar to the T0 counterparts (Tables II and V ). All these results demonstrate that DXS transgene was stably transmitted to the progeny.
Overexpression of the DXS Gene Does Not Increase Chlorophyll and Carotenoid Content of the Spike Lavender Leaves
Experimental evidence has clearly demonstrated that plastid isoprenoids involved in photosynthesis, such as carotenoids and the phytol moiety of chlorophylls, are derived from the MEP pathway (Lichtenthaler, 1999
In higher plants, the common building C5 units for the synthesis of isoprenoid-derived primary and secondary metabolites are IPP and DMAPP. The two pathways leading to the biosynthesis of these C5 units are the cytosolic MVA and the plastidic MEP pathways (Rodriguez-Concepción and Boronat, 2002 ). Among the metabolites derived from the latter pathway are thought to be monoterpenes (Mahmoud and Croteau, 2002), the main chemical constituents of the essential oil from spike lavender.
Although DXR is the first committed step for terpenoid biosynthesis through the MEP pathway (Carretero-Paulet et al., 2002
Results reported here on DXS overexpression in spike lavender corroborate those obtained by up-regulation of another enzyme of the MEP pathway in peppermint (Mahmoud and Croteau, 2001
Despite the increased-monoterpene phenotype of the transgenic plants, essential oil profiles obtained from transgenic and control plants were quite similar to each other. It is well known that the variability in the spike lavender oil composition is primarily genotype dependent (Harborne and Williams, 2002
Gene expression variability and silencing are major problems in the production of stable transgenic plants (Kohli et al., 2003
Metabolic engineering of volatile terpenoids can impose a cost on plant growth and fitness, caused not only by the reduced supply of precursors to branching (primary metabolites) pathways but also by the toxicity of the resulting compounds to plant cells (Aharoni et al., 2005 In conclusion, this study demonstrates the potential of the first step of the MEP pathway as a site for metabolic engineering. To our knowledge, results herein represent one of the first examples of an aromatic crop in which end-product monoterpenes can be elevated by increasing the supply of precursors to specific branches of the isoprenoid pathway. Given the economic importance of spike lavender for fragrance, flavor, and pharmaceutical industries, results reported here can be of value to improve and shorten the breeding programs of the species. Besides the biotechnologically relevant enhancement in the yield of spike lavender oil, plants stably overexpressing DXS provide a valuable model for studying the monoterpene formation pathway and its regulatory mechanism.
Plant Material and Bacterial Strain
Bulked seeds of spike lavender (Lavandula latifolia Medicus) from Spanish natural populations (Intersemillas SA) were germinated under sterile conditions as described by Calvo and Segura (1988) Transgenic T0 lines refer to plants regenerated from explants originally infected with Agrobacterium tumefaciens. T1 plants (first generation) are seed-derived plants obtained from controlled self-pollination of T0 plants. Nontransgenic, wild-type spike lavender plants were grown under the same conditions as controls. Both flowers and either developing (first and second verticils) or fully expanded (fourth to 10th verticils) leaves were sampled for molecular and phenotypical analyses. Agrobacterium strain C58, containing the plasmid pLBI1DXSBS1, kindly provided by Dr. Boronat (University of Barcelona), was used for transformation experiments. This binary vector contains a nptII marker gene, driven by the nopaline synthase promoter and terminator, and the full-length open reading frame from the Arabidopsis (Arabidopsis thaliana) DXS cDNA (AT4G15560), under the control of the CaMV 35S promoter and nopaline synthase terminator.
Agrobacterium-mediated transformation and regeneration of spike lavender was carried out according to Nebauer et al. (2000)
After 2 years under greenhouse conditions, all transgenic T0 lines flowered. Progenies were obtained by selfing (July–August) in controlled conditions. T1 seeds were harvested in October, germinated in vitro as described by Calvo and Segura (1988)
DNA was isolated from leaves (50–100 mg fresh weight) of in vitro-grown transgenic and wild-type plants using the cetyl trimethyl ammonium bromide (CTAB) procedure described by Doyle and Doyle (1990) PCRs were performed in 50-µL reaction volumes containing 75 mM Tris-HCl, pH 9.0, 50 mM KCl, 2.5 mM MgCl2, 20 mM (NH4)2SO4, 0.1 mM dNTP, 0.25 mM each oligonucleotide primer, and 4 units of Taq polymerase (Biotools). Primer sets used were as follows: 5'-GTCGCTTGGTCGGTCATTTCG-3' and 5'-GTCATCTCACCTTGCTCCTGCC-3' for the nptII gene, and 5'-GTTCATTTCATTTGGAGAGGAC-3' and 5'-TGGGAATTGTTGTTGGGTTTC-3' for the DXS gene. The predicted sizes of the amplified DNA fragments were 526 bp and 320 bp for nptII and DXS, respectively. Amplification parameters for the nptII gene were 95°C for 5 min followed by 40 cycles of 95°C for 1 min and 65°C for 2 min. A 65°C incubation for 5 min as final step was included. Amplification parameters for the DXS gene were 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, with a final extension step of 72°C for 7 min. Amplified products were detected by UV light fluorescence after electrophoresis on 1% (w/v) agarose Tris-borate/EDTA gels with 1.27 µM ethidium bromide.
The Southern-blot analysis was performed with nonradioactive digoxigenin-11-dUTP labeled probes. DNA from each sample (20–30 µg) was individually digested with 240 units of EcoRI for 16 h at 37°C and subsequently separated on 1% (w/v) agarose Tris-borate/EDTA gel at 60 V for 6 to 8 h. The gel was incubated for 30 min in 0.25 N HCl, twice for 30 min in 0.5 N NaOH, 1.5 M NaCl, and twice for 30 min in 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl. DNA was transferred onto positively charged nylon membranes (Boehringer Mannheim) by capillary blotting (Sambrook et al., 1989
Total RNA was extracted from 0.8 g of fresh, fully expanded leaves and 0.4 g flowers of transgenic and wild-type plants by using the Tripure Isolation Reagent (Roche Applied Science) according to the supplier's protocol. RNA concentrations were determined by measuring A260. For RNA gel-blot analysis, 30 µg of total RNA was denatured for 15 min at 65°C in loading buffer (1.25x MAE, 55% [v/v] formamide, 7.4% [v/v] formaldehyde, 3.2% [v/v] glycerol, and 0.05% [w/v] bromphenol blue) prior to electrophoresis. RNA was separated at 60 V for 4 h on 1% (w/v) agarose gel in MAE containing 2.2% (v/v) formaldehyde and transferred by capillary action onto Hybond-N nylon membranes (Amershan Pharmacia). DNA probes, labeled with [
The polyclonal antibody Ab-AtDXS was generated in a rabbit injected with the synthetic peptide GPMHQLAAKVDV, corresponding to positions G296 to V307 of the Arabidopsis DXS protein. Both the peptide and the antibodies were ordered from Bio-Synthesis and produced in their facilities. For western blots, crude protein extracts from wild-type Arabidopsis var. Columbia and young spike lavender leaf tissues (0.5 g) were obtained by harvesting in liquid nitrogen and grinding in 1 mL of ice-cold homogenization buffer (50 mM Tris-HCl, pH 7.5, 15 mM MgCl2, 100 mM KCl, 0.25 mM Suc, 10% [v/v] glycerol, 1% [w/v] polyvinylpyrrolidone-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.4% [v/v] 2-mercaptoethanol) with 33.33 µL of the Protease Inhibitor Cocktail (Sigma, P-9599), followed by two successive centrifugations (25,000 rpm for 30 min at 4°C). Protein samples were quantified with Bradford reagent (Bio-Rad) using bovine serum albumin as a standard, and then 30 µg of total proteins was separated in 8% SDS-PAGE. To verify equal protein loading, a parallel gel was run and stained with Coomassie Brilliant Blue R-250 (Sigma) and destained with 40% (v/v) methanol and 10% (v/v) acetic acid. The proteins were electrotransferred onto Immune-Blot polyvinylidene difluoride membranes (Bio-Rad) using the Mini Tran-Blot Cell (Bio-Rad) for 16 h at 40 V with the transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, and 20% [v/v] methanol). Blots were blocked in PBS buffer and 0.1% (v/v) Tween 20 (PBS-T) with 5% (w/v) nonfat dry milk for 1 h. Incubation with the antiserum (1:2,000) in PBS-T was carried out for 1 h at room temperature. Blots were then washed three times for 5 min in PBS-T and incubated for 1 h with peroxidase-conjugated secondary antibody diluted 1:7,500 in PBS-T. After three washes in PBS-T, cross-reacting bands were detected using the ECL Western-Blotting Analysis System kit (Amersham Biosciences).
Leaves and inflorescences from each examined plant were treated separately for essential oil extraction, and the tissue was manually crushed and mixed to ensure sample uniformity. Air-dried (for 30 d), fully expanded leaves (1.5 g) or flowers (0.5 g of cymes with three to five open flowers) were distilled in 100 mL of water in a Clevenger-type apparatus for 1.5 h, containing n-tetradecane and naphthalene as internal standards. Oils obtained were recovered with hexane, dried over anhydrous sodium sulfate, filtered through 0.22 µm polyvinylidene difluoride Millipore membranes, and adjusted to a final volume of 10 or 50 mL with hexane to obtain 10 µg/mL n-tetradecane and 400 µg/mL naphthalene or 2 µg/mL n-tetradecane and 80 µg/mL naphthalene for leaves or flower distillates, respectively. Oils were kept in air-tight glass containers at 4°C until further use. Qualitative essential oil analysis was accomplished by GC and GC/MS. GC analyses were performed with a Focus GC (Thermo Finnigan) equipped with a flame ionization detector and fitted with a BP-20 capillary column (polyethylene glycol, 30-m x 0.25-mm x 0.25-µm film; SGE Europe), carrier gas He at 1 mL/min, and 1.5 µL was injected in splitless mode (0.8 min) with an AI 3000 Autosampler (Thermo Finnigan). The normal oven temperature was programmed initially at 40°C for 1 min, followed by a ramp of 4°C/min to 130°C, and finally held isothermal at 130°C for 25 min. Temperatures of injector and detector were 230°C and 260°C, respectively. To separate (–)-trans-pinocarveol and (1S)-cis-verbenol, the temperature was programmed at 40°C for 1 min, followed by a ramp of 2°C/min to 58°C, a second ramp of 1°C/min to 70°C, held isothermal at 70°C for 1 min, followed by a third ramp of 2°C/min to 130°C, and finally held isothermal at 130°C for 15 min. GC/MS analyses were carried out on an Agilent 6890N gas chromatograph with an HP-INNOWax Column (Agilent Technologies) similar to the previously used BP-20; the coupled mass spectrometer was an Agilent 5973N with a quadrupole mass selective detector. All mass spectra were acquired in the electron impact mode at 70 eV. The mass spectrometer scanned in the range of 30 to 550 mass-to-charge ratio at a rate of 5.36 scans/s. The other analytical conditions were the same as for GC analysis. Compounds were identified by comparison of retention indices and mass spectra to those of authentic standards, or by reference mass spectra in a computer library (Wiley7n). MS/GC was performed by the Central Service for the Support to Experimental Research (SCIE, University of Valencia). The products were quantified (milligram per gram tissue dried) by comparison of detector response with that of the internal standards, assuming equal response factors. Also, percentages of compounds were determined from their peak areas. The relative peak area for individual constituents was determined using the Chrom-Card S/W program (Thermo Finnigan). All analyses were performed at least four times.
Extraction and determination of total chlorophylls and carotenoids were conducted as described by Lichtenthaler (1987)
Significance of the variation in essential oil production and chlorophyll and carotenoid content between transgenic and control plants was determined using ANOVA (Statgraphics Plus for Windows version 2.1), and mean comparisons using Tukey's (1953) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number U27099. Received July 6, 2006; accepted September 13, 2006; published September 15, 2006.
1 This work was supported by Dirección General de Investigación Científica y Técnica, Madrid (project AGL2002–00977); by Generalitat Valenciana, Valencia, Spain (projects GV2001–020 and Grupos 03/102); and by a Formación de Profesorado Universitario Research Fellowship from the Spanish Ministerio de Educación y Cultura (to J.M.-B.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Juan Segura (juan.segura{at}uv.es). www.plantphysiol.org/cgi/doi/10.1104/pp.106.086355 * Corresponding author; e-mail juan.segura{at}uv.es; fax 34963544926.
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0.02%). Thus, a consistent increase of a specific essential oil constituent was not observed in leaves of transgenic plants as compared to controls. 
- and 
-pinene, cineole, camphor, linalool, 
guez-Concepción M, Ferrer A, Boronat A, Campos N (2002) Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Plant Physiol 129: 1581–1591
