Article pubs.acs.org/JAFC
Methylerythritol and Mevalonate Pathway Contributions to Biosynthesis of Mono‑, Sesqui‑, and Diterpenes in Glandular Trichomes and Leaves of Stevia rebaudiana Bertoni Ursula Wölwer-Rieck,† Bianca May,† Christa Lankes,‡ and Matthias Wüst*,† †
Institute of Nutrition and Food Sciences, Chair of Bioanalytics/Food Chemistry, Rheinische Friedrich-Wilhelms-Universität Bonn, Endenicher Allee 11-13, 53115 Bonn, Germany ‡ Institute of Crop Science and Resource Conservation, Chair of Horticultural Sciences, Rheinische Friedrich-Wilhelms-Universität Bonn, Auf dem Hügel 6, 53121 Bonn, Germany S Supporting Information *
ABSTRACT: The biosynthesis of the diterpenoid steviol glycosides rebaudioside A and stevioside in nonrooted cuttings of Stevia rebaudiana was investigated by feeding experiments using the labeled key precursors [5,5-2H2]-mevalonic acid lactone (d2MVL) and [5,5-2H2]-1-deoxy-D-xylulose (d2-DOX). Labeled glycosides were extracted from the leaves and stems and were directly analyzed by LC-(-ESI)-MS/MS and by GC-MS after hydrolysis and derivatization of the resulting isosteviol to the corresponding TMS-ester. Additionally, the incorporation of the proffered d2-MVL and d2-DOX into volatile monoterpenes, sesquiterpenes, and diterpenes in glandular trichomes on leaves and stems was investigated by headspace−solid phase microextraction−GC-MS (HS-SPME-GC-MS). Incorporation of the labeled precursors indicated that diterpenes in leaves and monoterpenes and diterpenes in glandular trichomes are predominately biosynthesized via the methylerythritol phosphate (MEP) pathway, whereas both the MEP and mevalonate (MVA) pathways contribute to the biosynthesis of sesquiterpenes at equal rates in glandular trichomes. These findings give evidence for a transport of MEP pathway derived farnesyl diphosphate precursors from plastids to the cytosol. Contrarily, the transport of MVA pathway derived geranyl diphosphate and geranylgeranyl diphosphate precursors from the cytosol to the plastid is limited. KEYWORDS: steviol glycosides, terpenes, biosynthesis, MVA pathway, MEP pathway, GC-MS, LC-(ESI)-MS/MS
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with D,L-[2-14C]sodium mevalonate, which demonstrates a limited participation of the MVA pathway, but Totté et al.5,6 demonstrated that the MEP pathway was exclusively used for steviol glycoside biosynthesis by using labeling experiments with [1-13C]-glucose. At the end point of the MEP or MVA pathway, the resulting dimethylallyl diphosphate (DMAPP) is converted by adding several isopentenyl diphosphate (IPP)/farnesyl diphosphate (FPP) units to monoterpenes (+1 IPP), sesquiterpenes (+2 IPP), diterpenes (+3 IPP), and triperpenes (+2 IPP +1 FFP).7 In diterpene biosynthesis, geranylgeranyl diphosphate (GGPP; Figure 1) is assembled via three successive condensation reactions. In a first cyclization reaction, copalyl diphosphate (CDP) is produced by protonation-initiated cyclization from GGPP and then converted to (−)-kaurene and oxidized in cytochrome P450 mediated steps located in the cytosol to kaurenoic acid. Steviol biosynthesis diverges from gibberellin biosynthesis with the hydroxylation of kaurenoic acid at C13 to steviol.2,7,8 Subsequent glycosylation of steviol results in steviolmonoside, steviolbioside, stevioside, and rebaudioside A. Further glucosylations to steviol glycosides containing up to seven glycosyl moieties9 as well as the incorporation of
INTRODUCTION Stevia rebaudiana Bertoni is a perennial herb of significant economic value due to its high content of natural sweeteners in its leaves.1 The sweet constituents are extracted from the plant and are approved as food additives with a purity of at least 95% in many countries throughout the world. Steviol glycosides are tetracyclic diterpenoids and differ mainly in the variable number of sugar units bound to the terpenoid skeleton. The best known steviol glycosides are stevioside (Stev) and rebaudioside A (RebA), which are found at the highest levels in the plant and which bear three or four glucose molecules attached to the steviol moiety, respectively. Two different biosynthetic pathways contribute in their early steps to the synthesis of plant-derived terpenes. Whereas the cytosolic acetate−mevalonic acid (MVA) pathway is involved in the biosynthesis of sesqui-, tri-, and polyterpenes, the plastidlocalized methylerythritol phosphate (MEP) pathway contributes to the synthesis of mono-, di-, and tetraterpenes.2 However, despite a significant amount of research on the activity of these pathways under different conditions, the relative contribution of each to the biosynthesis of diverse terpenes remains unclear.3 Steviol glycosides are tetracyclic diterpenoids of the ent-kaurene type and share four steps of their biosynthesis with the gibberellin pathway. Both are expected to be synthesized mainly via the MEP way. For steviol glycosides, Hanson and White4 showed an incorporation of 0.83% into (−)-kaurene after feeding the cut stems of the plant © 2014 American Chemical Society
Received: Revised: Accepted: Published: 2428
January 16, 2014 February 28, 2014 March 1, 2014 March 1, 2014 dx.doi.org/10.1021/jf500270s | J. Agric. Food Chem. 2014, 62, 2428−2435
Journal of Agricultural and Food Chemistry
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Figure 1. Proposed simplified scheme for the compartmentalization of terpene biosynthesis via MEP and MVA pathways in Stevia rebaudiana. Metabolic cross talk is mediated by a yet unidentified metabolite transporter, which is represented by a gray-shaded rectangle inserted into the plastid membrane. IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; MEP, methylerythritol phosphate; MVA, mevalonic acid; MVPP, 5-diphosphomevalonate.
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rhamnose and xylose in, for example, dulcoside A and rebaudiosides C and F cannot be explained until now10 due to lacking information about the participating enzymes. This study aims to reinvestigate the biosynthesis of steviol glycosides by feeding experiments using the labeled precursors of the MEP and MVA pathways, namely, [5,5-2H2]-1-deoxy-Dxylulose (d2-DOX) and [5,5-2H2]-mevalonic acid lactone (d2MVL), respectively. Two analytical methods were applied to detect the incorporation of the labeled intermediates: gas chromatography−mass spectrometry (GC-MS) and liquid chromatography−electrospray tandem mass spectrometry in negative ion mode (LC-(ESI)-MS/MS).11 This experimental design is fundamentally different from the one that was chosen by Totté et al. in the aforementioned study. We chose to feed labeled precursors of the isoprenoid pathways and used the more sensitive mass spectrometry to detect the incorporation of labeled precursors. Moreover, we investigated in parallel the incorporation of the precursors into essential oil isoprenoids that were generated in glandular trichomes of Stevia leaves. A comprehensive investigation of the chemical composition of leaf extracts of S. rebaudiana revealed that its essential oil contains numerous mono-, sesqui-, and diterpenes.12 An appropriate technique for the analysis of such volatile organic compounds (VOC) is headspace solid phase microextraction (HS-SPME) in combination with GC-MS.13 It has been recently employed for the study of the pathway utilization in the sesquiterpene biosynthesis of grape berries.14 Hence, analyses of the leaf and stem volatiles of S. rebaudiana, including mono-, sesqui-, and diterpenes, were performed by HS-SPME-GC-MS as well to gain further insight into the pathway utilization in glandular trichomes.
MATERIALS AND METHODS
Reagents. HPLC grade acetonitrile was obtained from VWR International (Leuven, Belgium). Purified water was purchased from TKA Genopure-UV system. [5,5-2H2]-Mevalonic acid lactone (d2MVL) was prepared according to the method of Simpson et al.,11 and [5,5-2H2]-1-deoxy-D-xylulose (d2-DOX) was prepared according to the method of Meyer et al.15 Spectral data of the labeled compounds were in all cases in good agreement with the data given in the references cited above. Stevioside, rebaudioside A, and isosteviol with purities of ≥99.0 and 98.0%, respectively, were obtained from Wako Chemicals (Neuss, Germany). Steviol with a purity of ≥98% (HPLC), αterpineol (≥96%), β-caryophyllene (≥80%), and pyridine (anhydrous ≥99.8%) were purchased from Sigma-Aldrich (Taufkirchen, Germany). N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) in 1 mL ampules was obtained from CS-Chromatographie Service (Langerwehe, Germany), diisopropyl ether, ACS (≥99%), and linalool were obtained from Roth (Karlsruhe, Germany), anhydrous sodium sulfate EMSURE ACS and sulfuric acid (90−91%) were obtained from Merck (Darmstadt, Germany), and sulfuric acid (1 mol/L) was obtained by diluting 90−91% sulfuric acid, respectively. Safe-lock tubes, 2.0 mL, were purchased from Eppendorf (Hamburg, Germany). Standard Solutions. To prepare stock solutions, the steviol glycosides were dried to constant weight at 103 °C, cooled, and stored in a desiccator until use. For LC-MS, 10 mg of dried steviol glycosides was dissolved in water (10 mL). Solutions of stevioside and rebaudioside A were prepared by diluting the respective steviol glycoside with acetonitrile/water (8:2 v/ v) to obtain final concentrations of 10 μg/mL and were used for tuning the mass spectrometer. For isosteviol identification by GC-MS, stock solutions of isosteviol (0.1 mg/mL in anhydrous pyridine) were prepared. Plant Material and Incubation. S. rebaudiana plants of the genotype Gawi were obtained from the Institute of Crop Science and 2429
dx.doi.org/10.1021/jf500270s | J. Agric. Food Chem. 2014, 62, 2428−2435
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GC-MS Analysis of Steviol Glycosides. The GC-MS system and basic settings were the same as mentioned above. The following settings were changed for steviol glycoside analysis: The injection port was set to 250 °C, and injections were made in the split mode with a ratio of 1:20. The column temperature program started at 180 °C and was kept at this temperature for 1 min. Then it was increased to 300 °C during 10 min at a rate of 8 °C/min. Mass spectra were recorded in a range of m/z 50−450 at a scan rate of 0.82 s/scan. Isosteviol was identified by comparison of its retention time and mass spectra with the standard substance. HPLC-(-ESI)-MS/MS Analysis. Liquid chromatography analysis was performed on a binary LC-20AD system (Shimadzu, Duisburg, Germany) equipped with an SIL-20A autosampler, a Spark Mistral column thermostat and a four-channel degasser. The HPLC system was coupled to an API2000 triple-quadrupole detector (AB Sciex, Darmstadt, Germany) equipped with an ESI source used in the tandem MS mode with multiple reaction monitoring (MRM). Analyses and data processing were performed using Analyst 1.6.1 software. The instrument operated in the negative ion mode with the following source adjustments: ion spray voltage, −4200 V; curtain gas, 20 psi; gas 1 and 2, 50 psi; capillary temperature, 400 °C. The compound-dependent parameters were for both stevioside and rebaudioside A a declustering potential of −151 V, a cell entrance potential of −42 V, a collision energy of −30 V, a focusing potential of −340 V for stevioside and of −260 V for rebaudioside A, an entrance potential of −12 V for stevioside and of −7.5 V for rebaudioside A, and a cell exit potential of −22 V for stevioside and of −26 V for rebaudioside A. The MRM method involved the precursor/product transitions m/z 803−811 to m/z 641−649 for stevioside/d8-stevioside and m/z 965− 973 to m/z 803−811 for rebaudioside A/d8-rebaudioside A. For HPLC separation, a YMC-Triart Diol-HILIC column (250 × 4.6 mm, particle size = 5 μm, YMC, Dinslaken, Germany) equipped with the corresponding guard column (4 × 3.0 mm) was used. The mobile phase consisted of acetonitrile/water mixtures. The column temperature was maintained at 40 °C, and the flow was set to 0.4 mL/ min. The sample injection volume was 10 μL. The solvent gradient started with 20% water for 20 min, increased linearly to 40% water over 5 min, and then decreased back to 20% water. Column reequilibration was set at 5 min. Incorporation Rates. In all cases, incorporation rates (%) were calculated by dividing the peak area of the completely labeled compound by the peak area of the genuine compound multiplied by 100.14
Resource Conservation (INRES), Chair of Horticultural Sciences of the University of Bonn. Shoot tip cuttings of plants with four leaves were placed in vials containing 11 mL of autoclaved half-strength MS16 nutrient medium (solution without solidifying agar) containing each 30 mg of d2-MVL or 30 mg of d2-DOX or only half-strength MS medium as control. Incubation was performed in a culture cabinet at 25 °C and 80% humidity under 16 h per day reduced light conditions of 150 μE·m−2·s−1 photosynthetic active radiation. After 5 days, leaves and stems were harvested. The leaves were grouped in “old” leaves (leaves that were almost fully expanded prior to incubation) and “new” leaves (leaves that were expanding during incubation). In 2012, orienting incubations were performed with d2-DOX, and in 2013 with d2-DOX and d2-MVL, respectively. In addition to the leaves, stems were sampled and analyzed separately. Leaves and stems were ground in a mortar in the presence of some milliliters of fluid nitrogen. After the evaporation of the nitrogen, the dried leaf/stem powder was stored immediately at −20 °C prior to analysis. Sample Extraction. Headspace−Solid-Phase Microextraction. The analysis of the leaf and stem volatiles was performed with the plant material of 2013. Approximately 1 cm2 of the freshly harvested leaves or stems was placed into 10 mL headspace vials without further preparation. The subsequent measurement of the leaf and stem volatiles was performed with a polyacrylate fiber, 85 μm (Supelco, Bellefonte, PA, USA). The equilibration time of the samples was 30 min at a temperature of 45 °C; afterward, the SPME fiber was exposed to the sample headspace for a further 10 min and was subsequently thermally desorbed in the injection port of the GC. Aqueous Extraction of Diterpene Glycosides. Freeze-dried Stevia leaves (25 mg) were weighed in a 2 mL Safe-lock tube and extracted three times with portions of 0.5 mL of water in a heating block set at 102 °C for 30 min. Each extract was cooled to room temperature and centrifuged (15 min, 6866g). The aqueous phases were transferred to a weighed tube and filled to 1.5 mL (weight correction) after the last extraction step. Recovery was 93% for rebaudioside A calculated by measuring a known sample in duplicate according to earlier investigations.17 Hydrolyis of Steviol Glycosides. To convert the extracted steviol glycosides to isosteviol, 100 μL of the Stevia extract was mixed with 4 mL of sulfuric acid (1 mol/L) and heated for 60 min at 102 °C. After cooling, the solution was extracted three times with 2 mL of diisopropyl ether each time. The combined ether phases were dried with sodium sulfate and evaporated to dryness after filtration. Silylation. Trimethylsilyl (TMS) ethers were prepared by adding 300 μL of anhydrous pyridine and 150 μL of MSTFA to the dried samples or to an aliquot of the isosteviol stock solution, for example, 25 μL. This mixture was heated for 15 min at 60 °C. After cooling, 1 μL was injected into the GC. Sample Preparation for LC-ESI-MS/MS Detection. Stevia extract was diluted with acetonitrile/water mixture (8:2 v/v) to obtain a final concentration of 0.01 mg/mL. Ten microliters was injected into the LC. GC-MS Analysis. HS-SPME-GC-MS Analysis. GC-MS analysis was performed on a Varian 450 GC coupled to a Varian 240 MS ion trap spectrometer (Varian, Darmstadt, Germany). The injection port was set to 220 °C. A splitless injection was used, and the split valve was opened after 3 min. Separation was achieved on a 30 m × 0.25 mm i.d. × 0.25 μm film thickness DB5 column (Varian, Darmstadt, Germany). The carrier gas (helium 5.0) was set to 1 mL/min as constant flow. The column temperature started at 35 °C for 3 min and was increased to 230 °C at a rate of 5 °C/min. The transfer line temperature was set to 260 °C and the ion source to 150 °C. The spectrometer operated in the electronic impact (EI) mode at 70 eV scanning the m/z 50−350 range at a rate of 0.64 s/scan. Acquisition was done using Varian MS Workstation 6.9.2. The analytes were identified by comparison of the retention times and mass spectra with standard substances or were identified by their mass spectra and Kovats retention indices using the Massfinder library available from Dr. Hochmuth (Hamburg, Germany).
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RESULTS AND DISCUSSION Stevia Plants. Cuttings of the S. rebaudiana genotype Gawi were tested together with other genotypes as plant material for the feeding experiments and proven to be the most robust with the best leaf production during the investigation period. The total sweetener content is about 10%, with stevioside being the most abundant (6−7%) and rebaudioside A with a content of about 3%.18 The feeding and incubation procedure was carried out according to the method of Totté et al.5 but without the chloramine T supplementation. Nevertheless, no microbial infestation was observed. For a better understanding of the distribution of labeled steviol glycosides, the leaves were harvested separately and divided into “old” and “new” leaves and stems (for a description, see Plant Material and Incubation). Analysis of Glandular Trichome Volatiles. Prior to the determination of the steviol glycosides, the plant material was analyzed by HS-SPME-GC-MS without further preparation. This method allows the selective detection of volatiles that are stored in the subcuticular space of glandular trichomes and released into the headspace.19,20 In the foliar, 10-celled lipophilic glandular trichomes of S. rebaudiana, the six secretory 2430
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Table 1. Analyzed Compounds with Their Ion Traces Used for Quantitation (GC-MS, Numbers 1−8) and Transitions Used for Quantitation (LC-MS, Numbers 9 and 10), Respectively no.
compound
molecular ion, genuine/labeled
ions and transitions for quantitation (m/z), genuine/labeled
max deuterium incorporation
1 2 3 4 5 6 7 8 9 10
linalool α-terpineol (E)-β-caryophyllene β-farnesenec germacrene D 13-epi-manoyloxidc ent-kaurenec isosteviol Stev RebA
154/158a 154/158a 204/210 204/210a 204/210 290/298a 272/280 390/397 803/811 965/973
93/96b 136/139b 161/(166b + 167) 161/(166b + 167) 161/166b 257/265 257/265 272/279 803→641/811→649 965→803/973→811
d4 d4 d6 d6 d6 d8 d8 d7 d8 d8
a
Molecular ions not observed due to intense fragmentation by EI ionization. bLoss of deuterium due to fragmentation.14 See Figure S4 in the Supporting Information for a detailed analysis of the fragmentation pattern and the cyclization process that gives rise to the observed labeling pattern. cTentatively identified by mass spectrum and retention index.
cells form three pairs of head cells. To form the secretory sheath, the cuticular membrane detaches from the outer walls of the apical secretory cells along a line that appears to be the pectin layer.21 For a typical HS-SPME-GC/MS chromatogram, see the Supporting Information (Figure S1). The trichome volatiles are dominated by mono-, sesqui-, and diterpenes. The identified volatiles are in agreement with those identified in previous studies on S. rebaudiana volatiles and also known as common headspace volatiles of other plant species.12,22−25 The analysis of the incubated cuttings provides a comprehensive insight into the pathway utilization of the different terpene glandular trichomes. After administration of the labeled precursors, genuine terpenoid volatiles were detectable in all samples. However, contents of volatiles in old leaves were 100 times lower than those in new leaves or stems (data not shown), which can be explained by a reduced biosynthetic activity in old leaves or a reduced density of trichomes per leaf area unit.26 Nevertheless, deuterium incorporation was detected in terpenoid volatiles in all investigated tissues. Labeled and genuine terpenes can be chromatographically separated due to the inverse isotopic effect in gas chromatography.27 Monoterpenes are formed by two units of IPP/DMAPP, sesquiterpenes by three units, and diterpenes by four units, respectively. Because 2H2-MVL and 2H2-DOX are metabolized to 2H2-IPP and/or 2H2-DMAPP, up to four deuterium atoms can be incorporated into monoterpenes, six into sesquiterpenes, and eight into diterpenes. Deuterium incorporation, molecular ions, and ion traces used for data evaluation are given in Table 1. Results obtained in the feeding experiments for both precursors in all plant tissues are given in Table 2. d2-DOX was incorporated into volatile mono-, sesqui-, and diterpenes and d2-MVL mainly into sesquiterpenes with similar incorporation rates for both precursors. However, incorporation of d2-MVL into mono- and diterpenes was barely detectable or 10−100 times lower than that obtained for d2-DOX. Analysis of Steviol Glycosides. Two independent analytical methods were used for determining the incorporation of deuterium into steviol glycosides. For GC-MS analysis, the steviol glycosides were hydrolyzed, yielding the aglycon steviol, which was converted to isosteviol and detected after silylation as a sum parameter. In the second method, the diterpenes were separated by LC and detected as individual steviol glycosides with (ESI)-MS/MS. GC-MS Analysis. The hydrolysis of steviol glycosides to steviol and its conversion to isosteviol were carried out with
Table 2. Incorporation Rates and Standard Deviations after Cut Stem Feeding Experiments of Stevia Cuttings with 2H2DOX and 2H2-MVL (n = 2, Plant Material of 2013)a 2 H2-DOX incorporation rate (%)
2 H2-MVL incorporation rate (%)
sample
compound
new leaves
linalool α-terpineol (E)-β-caryophyllene β-farnesene germacrene D 13-epi-manoyloxid ent-kaurene
10.9 10.7 5.3 37.1 25.2 7.1 94.5
± ± ± ± ± ± ±
4.3 0.9 2.8 0.8 8.4 3.1 32.2
nd 0.5 49.8 50.8 46.3 0.3 4.7
old leaves
linalool α-terpineol (E)-β-caryophyllene β-farnesene germacrene D 13-epi-manoyloxid ent-kaurene
37.8 19.5 3.0 27.4 10.9 2.5 257.7
± ± ± ± ± ± ±
3.2 0.7 0.5 3.0 0.1 0.5 16.7
nd nd 14.9 ± 2.6 nd 12.7 ± 0.4 nd 15.4 ± 9.8
stems
linalool α-terpineol (E)-β-caryophyllene β-farnesene germacrene D 13-epi-manoyloxid ent-kaurene
49.1 14.2 10.2 32.5 31.1 5.2 107.3
± ± ± ± ± ± ±
28.8 2.6 6.0 5.0 6.2 1.3 85.1
nd 0.4 25.2 31.7 27.0 0.3 1.4
± ± ± ± ± ±
± ± ± ± ± ±
0.6 41.0 16.5 37.3 0.4 6.6
0.5 0.5 1.8 2.3 0.2 2.0
a
HS-SPME-GC-MS analysis of volatile terpenoids according to Table 1. nd, not detectable: signal to noise ratio
