alexa Profiling Of Terpene Metabolism in 13CO2-Labelled Thymus Transcaucasicus | Open Access Journals
ISSN: 2153-0769
Metabolomics:Open Access
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Profiling Of Terpene Metabolism in 13CO2-Labelled Thymus Transcaucasicus

Kutzner E1#, Manukyan A2# and Eisenreich W1*

1Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany

2Center of Life and Food Sciences Weihenstephan, Technische Universität München, Dürnast 4, 85350, Freising-Weihenstephan, Germany

#Both authors contributed equally to the paper

*Corresponding Author:
Eisenreich W
Lehrstuhl für Biochemie
Technische Universität München
Lichtenbergstraβe 4, 85748 Garching, Germany
Tel: +49 89 28913336
Fax: +49 89 28913363
E-mail: [email protected]

Received date: November 18, 2013; Accepted date: January 24, 2014; Published date: January 31, 2014

Citation: Kutzner E, Manukyan A, Eisenreich W (2014) Profiling of Terpene Metabolism in 13CO2-Labelled Thymus transcaucasicus. Metabolomics 4:129. doi: 10.4172/2153-0769.1000129

Copyright: © 2014 Kutzner E, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Metabolomics:Open Access


Metabolism is characterized by the functional pathways and fluxes connecting substrates, intermediates and products of a given organism. Pathways in carbon metabolism can be identified by incorporating 13C-labelled tracers. In the present model study, we have evaluated potential benefits from a single 13CO2 pulse-chase experiment with the Caucasian endemic plant Thymus transcaucasicus for the analysis of terpene composition and biosynthesis. The study design was conducive of low 13C-enrichments (< 1%) in terpenes that were detected at enhanced NMRsensitivities without hampering GC-MS-based methods for terpene profiling. From the specific 13C-labelling patterns, pathways of terpene biosynthesis could be gleaned as exemplified for the mono-terpenethymol which is made via the non-mevalonate route under the physiological in vivo conditions of the 13CO2 experiment.


Transcaucasian thyme; Thymus transcaucasicus; Lamiaceae; Biosynthesis; Pathway; 13CO2; Terpene


Non-targeted or semi-targeted metabolite profiling has become a powerful method to determine the composition of small molecules in plant extracts using apolar or polar solvents. In principle, metabolite profiles are specific snapshots (fingerprints) of the metabolic processes that have occurred in the plant under study. Using the tools of GCMS, LC-MS or 1H-NMR spectroscopy, crude mixtures can be analyzed to provide information about metabolic profiles. Depending on the specific method, dozens to hundreds (by 1H-NMR) or hundreds to thousands (by GC-MS or LC-MS) of metabolites can be detected and assigned in a single run [1,2]. Whereas some of these methods (e.g. GC-MS) typically need chemical transformation of the compounds under study, others (e.g. 1H-NMR) are qualified to deal with the crude mixtures without any prior chemical treatment. On the other hand, the 1H-NMR approach is hampered by low sensitivity in comparison to MS-based methods, as well as by limited resolution due to the narrow chemical shift range of 1H-frequencies (i.e. approximately 10 ppm). Due to the even lower sensitivity of 13C-NMR as compared to 1H-NMR, 13C-profiling is not established in metabolomics, although the 13C-NMR chemical shift range is much larger (i.e. about 200 ppm) than in 1H-NMR and therefore less compromised by signal overlap. Only recently, 13C-NMR has been introduced as a tool for metabolomics, particularly for the analysis of 13CO2-grown photosynthetic bacteria [3]. Using special growth chambers, 13CO2-labelling experiments with intact plants revealed considerable details about metabolic pathways under physiological conditions [4-10]. However, all of these studies have not addressed the possibility to combine benefits of 13C-labelling for pathway analysis and 13C-based metabolite profiling from a single experiment. Therefore, we have now performed a model study with 13CO2-labelled Transcaucasian thyme (Thymus transcaucasicus Ronn.) (Lamiaceae) to assess both metabolite profiles (e.g. isoprenoids) as well as metabolic pathways from the same experiment. This Caucasian endemic species was selected since it is known as a rich producer of essential oils including terpenes [11-13]. DMAPP can be synthesized by the well-known mevalonate pathway in the cytosolic compartment of the cell or by the more recently discovered MEP pathway that is operative in the plastids (Figure 1A). Depending on the specific compartment where the final terpene is made, the respective pathway leads to the product. Typically, monoand diterpenes originate from the plastids and are therefore derived from the MEP pathway (for reviews, see [14-17]). However, mixed biosynthetic patterns were also reported for certain plant terpenes [17-20].


Figure 1: Monoterpene biosynthesis. A. The terpenoid precursors isopentenyl pyrophosphate (IPP, 10) and dimethylallyl pyrophosphate (DMAPP, 11) are synthesized in plants via two different pathways, the mevalonate and the methylerythritol phosphate (MEP) pathway. DMAPP (11) and IPP (10) are further condensed to geranyl pyrophosphate (GPP, 12) which can be converted into monoterpenes such as thymol (15). The isoprene unit derived from IPP (10) is highlighted in red, the isoprene unit derived from DMAPP (11) in green. B. Biosynthesis of thymol (15) by aromatization of γ-terpinene (13) to p-cymene (14) followed by hydroxylation of p-cymene (14).

Until now, there is no information available on monoterpene biosynthesis in endemic Thymus species, like T. transcaucasicus, while it is established knowledge that thymol from common thyme (T. vulgaris) is biosynthesized by aromatization of γ-terpinene to p-cymene followed by hydroxylation of p-cymene [21] (Figure 1B). More recently, it was also reported for cut shoots of T. vulgaris that the two isoprene units of thymol are made via the MEP pathway [22]. However, the experimental design of the former study [22] does not rule out effects on the metabolic network due to wounding or the usage of artificial carbon substrates which were used as 13C-labelled tracers in the earlier experiment. In sharp contrast, these intrinsic drawbacks can be avoided by the experimental design of the current study using intact plants and the natural carbon substrate CO2 as tracer.


Plant material and study design

A one year old full blooming plant of T. transcaucasicus from controlled greenhouse soilless culture was placed into a gas incubation chamber (Advance Optima Biobox AO2000, GWS, Berlin, Germany). The plant was illuminated with white light. The temperature was adjusted to 25°C. Prior to the labelling period with 13CO2 (pulse phase), the chamber was flushed with synthetic air (Westfalen AG, Münster, Germany) containing only oxygen (20.5 vol. %) and nitrogen (79.5 vol. %) until the atmospheric unlabelled CO2 was almost completely removed from the chamber. The plant was then labelled using a mixture of synthetic air and 13CO2. The concentration of 13CO2 was held constant at 700 ppm by permanently adding the gas from the reservoir (Sigma-Aldrich, Steinheim, Germany) for 4 hours. During this labelling period, the plant consumed about 120 ml of 13CO2. Subsequently, the plant was allowed to grow at room temperature under standard greenhouse conditions and under a natural atmosphere (i.e. containing 12CO2) for 12 days.

Metabolite profiling: Extraction of terpenes

Terpenes were extracted from the leaves as well as from the flowers of T. transcaucasicus with cold chloroform-D (CDCl3) (Sigma-Aldrich, Steinheim, Germany). In more detail, 400 mg of the plant material (fresh weight) were placed into a 10 ml test tube. 2 ml of CDCl3 were added and mixed in an attempt to cover all leaves with the solvent. The mixture was left at room temperature for several minutes. The solvent was then transferred into the next test tube containing another set of 400 mg of plant material from the same plant. These leaves were treated by the same way as the ones in the first test tube. The procedure was repeated again with another portion of 400 mg of fresh material. The solvent was collected and 100 mg of dry MgSO4 were added to remove water. The mixture was held for 30-90 minutes under periodically shaking. Flowers were in general subjected to the same procedure as the leaves. Without further preparation, 200 μl of the extract were used for GC-MS measurements (see below). For NMR analysis, 600 μl of the extract were filled into a 5 mm NMR tube and measured.

Metabolite and isotopologue profiling: Gas chromatography – mass spectrometry

The gas chromatography – mass spectrometry (GC-MS) measurements were performed on a GC 2010 Gas Chromatograph and a GCMS-QP 2010 Plus mass spectrometer coupled to a QP-5000 mass selective detector (Shimadzu, Duisburg, Germany) working with electron impact (EI) ionisation at 70 eV. A Silica capillary column Equity TM-5 (30 m x 0.25 mm x 0.25 μm film thickness) from Supelco Inc. (Bellefonte, PA, USA) was used. An aliquot of the chloroform extract was injected in 1:10 split mode at 230°C and a helium inlet pressure of 82.8 kPa. The interface temperature was 260°C and the helium column flow was 1.17 ml/min. The column was developed at 90°C for 2 minutes and then with a temperature gradient of 5°C/min to a temperature of 150°C followed by a gradient of 50°C/min to a final temperature of 250°C that was held for 2 minutes. Each sample was analyzed three times in order to acquire selected ion monitoring (SIM) data. The identification of the essential oil components was carried out by comparing the retention times and mass data of pure reference compounds, and by comparing data from the NIST05 and NIST05s mass spectral reference library. The relative intensities of the standards and the samples obtained from GC-MS analysis (peak integration) were processed with an in-house Excel-based software package according to [23-26]. This evaluation resulted in the molar 13C-excess of the carbon isotopologues in thymol.

Metabolite and isotopologue profiling: NMR spectroscopy

For the measurement of 13C NMR and INADEQUATE spectra an Avance III 500 system (Bruker, Rheinstetten, Germany) with a cryo probe head (5 mm CPQNP, 1H/13C/31P/ 19F/29Si; Z-gradient) was used. 1H and ADEQUATE spectra were measured with an Avance I 500 system (Bruker) and an inverse 1H-13C probe head. The resonance frequencies of 1H and 13C were 500.13 MHz and 125.82 MHz, respectively. The temperature was 300 K. The data analysis was done with the MestReNova Software Version 7.0.0 (Mestrelab Research, Santiago de Compostela, Spain). The one-dimensional 13C NMR spectra as well as the one- and two-dimensional spectra 1H NMR spectra were measured with standard Bruker parameter sets.

Results and Discussion

A GC chromatogram of a leave extract from 13CO2-labelled T. transcaucasicus is shown in Figure 2A. About 20 - 30 peaks (> 2% in relation to the highest peak) were detected in typical runs. The identification of the essential oil components was performed by comparison with reference data from the NIST05 and NIST05s libraries. It turned out that the weak 13C-enrichments (< 1%) did not disturb the identification of most compounds in the mixture. As a result, the apolar fraction obtained from the leaves as well as from the flowers of T. transcaucasicus was predominantly (more than 90% in the overall intensity of all detected peaks) composed of monoterpenes including thymol, γ-terpinene, α-pinene and 1,8-cineol (Figure 3). Sesquiterpenes were only detected at minor amounts, e.g. caryophyllene, germacrene D, α-bisabolene and β-ocimene. In addition to these compounds, the flowers produced additional terpenes, e.g. borneol. These results are in good accordance to previous investigations with T. transcaucasicus and other thyme species [12,13,27].


Figure 2: GC-MS analysis of chloroform extract of T. transcaucasicus flowers harvested 9 days after the labelling with 13CO2 for 4 hours. A. Gas chromatogram performed as described under Experimental. B. Corresponding mass spectrum and fragmentation pattern of thymol (molar mass 150.22 g/mol) from the labelled sample. C. Corresponding mass spectrum of unlabelled thymol from the NIST05s library.


Figure 3: Chemical structures and numbering of the major monoterpenes detected in chloroform extracts of T. transcaucasicus. A, thymol. B, α-pinene. C, 1,8-cineole. D, γ-terpinene.

The same chloroform extracts were now also analysed by NMR spectroscopy. A typical 1H NMR spectrum is shown in Figure 4. Not unexpectedly, the high-field NMR region was crowded due to the presence of multiple compounds in the crude extract. On the other hand, signals in the down-field region were well separated and immediately allowed the assignments of some intense signals to thymol, γ-terpinene, and α-pinene (Figure 4) by comparison with 1H NMR reference data (own library of 1H NMR spectra of terpenes (see also Table 1) or data provided by the Spectral Database for Organic Compounds, SDBS). Moreover, the assignments of the signals were confirmed by two-dimensional NMR experiments with the crude mixture (e.g. COSY, HMQC, HMBC) (Table 1 and Figure 5). Notably, the weak 13C-enrichments (< 1%) did not disturb the assignments of the 1H-NMR signals since the 13C-induced 1H-satellite signals were small (< 1% in the overall signal intensity of a given 1H-atom). However, the 13C-enrichments significantly increased the sensitivity of HSQC and HMBC experiments, thus benefitting the quality of these spectra. More than 100 well defined correlation peaks were observed in the full HMBC spectrum of the extract from the 13CO2 labelled plant (Figure 6A), whereas ca. 50 peaks were detected in the spectrum from the unlabelled plant (Figure 6B) (using the same amount of plant material and the same NMR parameters). In accordance to the GC-MS data, thymol was again identified by NMR as the most abundant metabolite in the crude extracts of leaves and flowers. The minor terpenes 1,8-cineole, α-pinene and γ-terpinene could also be clearly assigned in the 13C-enriched sample (Table 1 and Figure 4,5), but hardly in the unlabelled isolate. Nevertheless, it should be noted that the NMR sensitivity with the 13C-enriched sample was still lower than the sensitivity of the GCMS method. As a consequence, we could hardly detect by NMR some minor terpene compounds which were clearly observed by GC-MS. However, a major benefit of 13C-NMR analysis became obvious when analyzing the metabolic processes leading to terpenes.

Compound 13C atom Chem. Shift δ (ppm) 1H atom Chem. Shift δ (ppm) Coupling constant (Hz) COSY NOESYa HMBCa
Thymol C-8/9   22.7 A 1.28 (6 H, d) 6.9   C B, C, G, D(w)  
  C-10   20.9 B 2.31 (3 H, s)     - A, D, E, F G(w), C, B(w), A
  C-7   26.7 C 3.20 (1 H, m) 6.9   A A, D, G G(w), F, E
   -OH     - D 4.65 (1 H, s)     - E, C, B, A(w) G, E(w), A
  C-6 116.0 E 6.61 (1 H, s)     - B, D, F, G -
  C-4 121.7 F 6.77 (1 H, d) 7.9   G B, E, G G(w), F, D, B
  C-3 126.2 G 7.12 (1 H, d) 7.7   F A, C, E, F G(w), E, B
  C-1 152.5               F(w), D(w), C, B(w)
  C-5 136.7               G, F(w), E(w), D, C, B(w)
  C-2 131.3               G, F(w), E(w), B
                    G(w), F, E, D, C, B, A
(+)-α-Pinene C-9   20.8                
  C-7   31.5 A 0.86 (3 H, s)     - C, D, E(w), G/H, I, J  
  C-8   26.4 B 1.18 (1 H, d) 8.5   I C, E(w), F(w), G/H, I, J(w) B, C
  C-10   23.0 C 1.29 (3 H, s)     - A, B, D, E, F, G, H, I, J due to [28]
  C-1   47.0 D 1.68 (3 H, dd) 2.1   - A, C, E, F, G, H, I, J A, B, E
  C-5   40.7 E 1.96 (1 H, t) 11.2 1.5   F, I A(w), B(w), C, D, F(w) E
  C-4   31.3 F 2.10 (1 H, m)     E, I B(w), C, D, E(w), G, I(w), J(w) A, B, C, D, F, I, H(w)
  C-4   31.3 G 2.18 (1 H, m) 17.3 (dd) H A, B, C, D, F, H, I, J A, B, C, E, I, H(w)
  C-7   31.5 H 2.26 (1 H, m) 17.3 (dd) G A, B, C, D, G, I, J due to [28]
  C-3 116.0 I 2.36 (1 H, m) 8.5 11.2   B, E, F A, B, C, D, F, G, H, J due to [28]
  C-6   38.0 J 5.21 (1 H, m)     - A, B, C, D, E, F, G, H, I due to [28]
  C-2 144.5               -
                    A, B, C, E, I, F(w), G(w)
                    B, D, E, I, C(w), G(w), H(w)
1,8-Cineole C-7   27.6                
  C-9/10   28.9 A 1.07 (3 H, s)     - B, E(w)  
  C-4   32.9 B 1.26 (6 H, s)     - A, C(w), D(w), F D(w), E
  C-2/3/5/6   22.8 / 31.5 C 1.42 (1 H, s)     - B(w), D, E(w), F B, D(w)
  C-2/6   31.5 D (endo)b 1.51 (4 H, m)     E, F B(w), C, E, F B, D, E(w), F
  C-3/5   22.8 E (exo)b 1.68 (2 H, m)     D, F D, C(w), F(w)  
  C-1   69.8 F (exo)b 2.03 (2 H, m)     D, E B, C, D, E(w) A, C, D, E, F
  C-8   73.7               A(w), C(w), D, E, F
                    A, D(w), E, F
                    B, D, E(w), F(w)
γ-Terpinenec C-8/9     1.0                
  C-10   23.0 A 1.01            
  C-7   34.6 B 1.66            
  C-1/4   31.7 C 2.20            
  C-3/6 118.9 D 2.60            
  C-2 140.6 E 5.43            
  C-5 131.3                

Table 1: NMR signal assignment of main compounds in essential oils of Thymus transcaucasicus. The solvent was CDCl3.


Figure 4: 1H NMR spectrum of crude leaves extract of T. transcaucasicus. Leaves were extracted (see section Experimental) after a chase period of 12 days. The number of scans in the 1H experiment accounted for 64 and the solvent was CDCl3. The FID was multiplied with a Gaussian function (lb = -1.00; gb = 0.8 Hz).


Figure 5: Expanded view of HSQC (A) and HMBC (B) spectra of crude leaves extract of T. transcaucasicus. Leaves were extracted (see section Experimental) after a chase period of 12 days. The number of scans accounted for 8 and 16, respectively. The solvent was CDCl3.


Figure 6: Two-dimensional HMBC spectra of crude leaves extracts of T. transcaucasicus. A. Leaves (1.7 g) of the 13CO2-labelled plant (4 hour pulse period) harvested after a chase period of 12 days. B. Leaves (1.7 g) of an unlabelled T. transcaucasicus plant (same developmental stage as the labelled plant). Both spectra were measured with the same standard parameter sets and 16 scans per increment.

As a model compound for pathway analysis, we have selected thymol as the most prominent terpene in the chloroform extract of T. transcaucasicus. We have started with a concise analysis of the isotope pattern detected in the MS spectrum of the 13CO2-labelled compound. The mass spectrum is shown in Figure 2B with a peak at m/z = 150 reflecting the molar mass of thymol. The peaks at m/z = 151, 152, 153, etc. are due to thymol molecules carrying one, two, three, etc. 13C-atoms, respectively. By comparison of the mass intensities with the corresponding ones of a thymol sample at natural 13C-abundance, the 13C-excess values of each isotopologue due to incorporation of 13CO2 were calculated. The normalized ratios are displayed in Figure 7. It turned out that thymol was a complex mixture of isotopologues with M+1, M+2, M+3, and M+4 as the most prominent specimens. Isotopologues with more than four 13C-atoms were less abundant (< 20% in all 13C-isotopologues).


Figure 7: Relative isotopologue distribution (excess) of thymol analyzed by GC-MS. Thymol was extracted with chloroform from T. transcaucasicus leaves and flowers at different points of time after the labelling with 13CO2 for 4 hours (see Experimental). Excess values were obtained by subtracting the natural 13C abundance of 1.11%. The M+1 bar in the diagram represents molecules carrying only one 13C atom (position unknown), the M+2 bar stands for molecules carrying two 13C atoms, and so on.

Tentatively, isotopologues in thymol comprising three 13C atoms (M+3) can be explained by the MEP pathway which contributes three 13C atoms to the terpene precursors, IPP and DMAPP, via 13C- 3 labelled GAP (Figure 8). In contrast, the mevalonate pathway can only contribute two 13C atoms to a given isoprene precursor via the two-carbon moiety in acetyl-CoA. Nevertheless, one has to take into consideration that M+2 isotopologues not only originate from the MVA pathway (starting from acetyl-CoA) but are also transferred to IPP/DMAPP via the pyruvate building block in the MEP pathway (one carbon of pyruvate is lost as CO2 during the biosynthetic pathway). One also has to take into account that the GAP precursor in the MEP pathway is probably not only 13C3 labelled, but also carries a substantial amount of 13C-2 labelled isotopologues that subsequently result in additional M+2 isotopologues of thymol via the MEP pathway. Therefore, it is not possible to assess the absolute rates of thymol formation via the MEP and/or the mevalonate pathway on the basis of these MS data only. Another weakness of the GC-MS thymol data is the lack of positional label information. Unfortunately, the smaller fragments detected in the MS spectrum of the monoterpene are not useful to further elaborate the positional 13C-distribution in the molecule. Nevertheless, the significant amounts of (M+3) isotopologues in the labelled thymol at least suggest its predominant formation via the MEP pathway, as shown earlier for thymol from cut shoots of T. vulgaris [22].


Figure 8: Incorporation of 13C atoms derived from labelling experiments with 13CO2 into various metabolites during the plant metabolism. The possible positions of 13C atoms are highlighted as colored bars and small boxes, respectively; the color depends on the metabolic pathway which leads to a certain metabolite. 3-Phosphoglycerate (PGA) and glyceraldehyde 3-phosphate (GAP) are built up in the Calvin cycle which generate either molecules with two 13C atoms (pink bar) or molecules carrying three 13C atoms (red bars). A label at position 1 exclusively is also possible. The synthesis of monoterpenes is possible via the MEP pathway (left side) based on pyruvate and GAP (see Figure 1) which leads to a labelled C2 block originating from pyruvate (dark red bar and dark green bar, respectively) and/or a C2 or C3 block originating from GAP carrying either two 13C atoms (pink and light green bar, respectively) or three 13C atoms (red/green bar and red/green box, respectively), respectively. Red color marks carbon atoms coming from IPP and green color marks carbon atoms coming from DMAPP. On the right side the predicted labelling pattern of thymol arising from the mevalonate pathway based on acetyl-CoA is shown. This path does not generate monoterpenes carrying three 13C atoms. Boxed thymol is displayed with the labelling pattern observed by NMR spectroscopy.

In order to further support this hypothesis, the monoterpene was subjected to a detailed NMR analysis. 1H- and 13C-NMR signals of thymol (detected in the chloroform extract, Fig. 9B) could be unequivocally assigned on the basis of literature data and on the basis of two-dimensional experiments (COSY, HSQC, HMBC, ADEQUATE) (Tables 1 and 2) (Figure 9B). For comparison, a 13C-NMR spectrum of an authentic thymol reference is shown in Figure 9A. A closer inspection of the signals of the labelled thymol sample revealed satellite signals due to 13C-13C couplings (Figure 10). To improve the spectral quality, the central and the satellite signals were fitted using the GSD algorithm implemented in the MestReNova software. Mathematically, this module applies a de-convolution of complex spectra transforming them into individual lines. The shape of the lines can further be approximated by using combinations of Lorentzian and Gaussian functions. Whereas for most of the signals sharp satellites were observed due to coupling with one neighboured 13C-atom, the satellite signals of C-1 and C-3 were more complex with an additional fine splitting (8.3 Hz and 2.5 Hz, respectively) indicating the presence of 13C-3-motifs in the molecule. Interestingly, corresponding coupling constants were observed for satellites close to the central signals for C-4 and C-8/9, respectively (see also Table 2 with all detected coupling constants). By comparing these coupling constants, pairs or triples of 13C-atoms in thymol can be assigned as indicated in Table 2. More direct experimental evidence could be provided by ADEQUATE experiments that are based on magnetization transfer via one-bond 13C-13C couplings and a subsequent transfer to the attached proton of the 13C-pair. Due to the specific labelling pattern in the 13CO2 labelled thymol samples, only a few (but highly significant) signals were detected in these experiments. As shown in Figure 11B, the 1,1-ADEQUATE experiment displayed signals due to the following transfer paths: 13C-8 → 13C-7 → H-7, 13C- 7 → 13C-8 → H-8, 13C-2 → 13C-3 → H-3, 13C-1 → 13C-6 → H-6, 13C-5 → 13C-10 → H-10. This clearly indicates that the biosynthetic process afforded four pairs of directly adjacent 13C-atoms, i.e. 8-7, 1-6, 5-10 and 2-3 (shown in Figure 11B as bars connecting 13C-atoms). Carbon atoms C-4 and C-9 were not incorporated in form of a directly-bound 13C-pair, but only showed long-range 13C-13C couplings (see Figure 10). Indeed, the expected long-range couplings between C-4 and C-1, and C-9 and C-3, respectively, could be confirmed by 1,n-ADEQUATE experiments which are based on long-range 13C-13C transfer (i.e. via two, or three bonds) followed by direct 13C- 1H coupling (i.e. via one bond). As shown in Figure 11C, peaks due to 13C-1 → 13C-4 → H-4 and 13C-3 → 13C-9 → H-9 were observed. These correlations reflected that the carbon atoms 1, 6, 4 and 3, 2, 9 were incorporated as 13C-3-moieties (indicated in Figure 11C as arrows connecting the outlier 13C with a pair of 13C-atoms) during the biosynthetic process. Indeed, these 13C-3 species can be predicted for thymol from 13C-3-labelled GAP via the MEP pathway.

  Chemical shift Coupling constanta 13C enrichment
Position 13C δ (ppm) JCC (Hz) % 13Crel.b % 13C13C c
1 152.50 66.9  (6) 1.10 28.57
    8.3  (4)    
2 131.34 59.9  (3) 1.24 27.01
3 126.23 59.8  (2) 1.12 22.48
    2.5  (9)    
4 121.67 8.4  (1) 1.24 16.67
5 136.66 44.4  (10) 1.17 19.35
6 116.00 66.6  (1) 1.34 21.26
7 26.70 34.6  (8) 1.24 23.66
 8/9 22.71 34.5  (7) 1.47 13.79
    2.6  (3)    
10 20.91 44.2  (5) 1.16 22.48

Table 2: 13C-NMR data of thymol obtained from T. transcaucasicus flowers labelled with 13CO2 for four hours and extracted with chloroform 12 days after the labelling pulse. The solvent was CDCl3.


Figure 9: One-dimensional 13C NMR spectra of thymol. A. Standard sample (with natural 13C abundance of 1.1%) of thymol solved in CDCl3. Signals were assigned to the corresponding carbon atoms of thymol. B. Sample of T. transcaucasicus flowers harvested and extracted with chloroform 12 days after the labelling with 13CO2 (4 hours). The solvent was CDCl3.


Figure 10: Thymol signals from the one-dimensional 13C NMR spectrum of T. transcaucasicus flowers. Flowers were harvested and extracted with chloroform 12 days after the labelling with 13CO2 (4 hours). The solvent was CDCl3. The spectrum was processed with MestReNova Software (zero filling 256k). The original spectrum was multiplied by a Gaussian window function of 0.7 Hz except for signals C-8/9 and C-5. For signal C-8/9 and signal C-5 a function of 0.5 Hz and of 1.2 was applied. The numbering of the carbon atoms is displayed in Figure 3.


Figure 11: 1,1- and 1,n-ADEQUATE spectra of crude leaves extract of Thymus transcaucasicus. A. 1,1-ADEQUATE (65 Hz) of thymol standard solution in CDCl3 (44 mM). B. 1,1-ADEQUATE (65 Hz) of crude leaves extract. Leaves were extracted (see section Experimental) after a chase period of 12 days. The solvent was CDCl3. C. 1,n-ADEQUATE (8 Hz) of crude leaves extract. Leaves were extracted (see section Experimental) after a chase period of 12 days. The solvent was CDCl3.

In summary, the NMR-based pathway analysis confirmed that the monoterpene thymol from T. transcaucasicus was made predominantly or exclusively via the MEP route of isoprenoid biosynthesis under the physiological conditions of the CO2 experiment. It should also be emphasized that thymol from the flowers showed the same labelling patterns as that from the leaves. Obviously, thymol was either made in both plant organs via the same pathway or was made in one organ and then rapidly distributed over the whole plant. The labelling pattern also reflected that the linear precursor geranyl diphosphate was converted into the cyclic compound via the known mechanisms of thymol biosynthesis.


The 13CO2 pulse-chase experiment was useful for pathway analysis in T. transcaucasicus. It should be noted that the experimental method is not restricted to the analysis of terpene metabolism but can also be applied to virtually any metabolic (end)-product found in 13CO2-labelled plants as shown by many earlier studies [6-10]. The model study with T. transcaucasicus shows that a single 13CO2 pulse-chase experiment can not only provide valuable data for pathway analysis, but also can enable 13C-based metabolite profiling at significantly enhanced NMR sensitivity without hampering GC-MS based compound assignment. However, the NMR sensitivity is still lower than in GC-MS analysis, mainly due to the low 13C-enrichments afforded by 13CO2 pulsechase experiments. On this basis, the study design combines benefits of isotope labelling for pathway analysis as well as for metabolite detection. Thus, the experimental settings appear of general value in metabolomics studies with intact plants.


We thank the Hans-Fischer-Gesellschaft (München) and the Deutsche Forschungsgemeinschaft (EI 384/8-1) for generous sponsoring of this research work. We also thank the DAAD for supporting A.M.


Select your language of interest to view the total content in your interested language
Post your comment

Share This Article

Article Usage

  • Total views: 11790
  • [From(publication date):
    August-2014 - Oct 21, 2017]
  • Breakdown by view type
  • HTML page views : 8006
  • PDF downloads :3784

Post your comment

captcha   Reload  Can't read the image? click here to refresh

Peer Reviewed Journals
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

Agri, Food, Aqua and Veterinary Science Journals

Dr. Krish

[email protected]

1-702-714-7001 Extn: 9040

Clinical and Biochemistry Journals

Datta A

[email protected]

1-702-714-7001Extn: 9037

Business & Management Journals


[email protected]

1-702-714-7001Extn: 9042

Chemical Engineering and Chemistry Journals

Gabriel Shaw

[email protected]

1-702-714-7001 Extn: 9040

Earth & Environmental Sciences

Katie Wilson

[email protected]

1-702-714-7001Extn: 9042

Engineering Journals

James Franklin

[email protected]

1-702-714-7001Extn: 9042

General Science and Health care Journals

Andrea Jason

[email protected]

1-702-714-7001Extn: 9043

Genetics and Molecular Biology Journals

Anna Melissa

[email protected]

1-702-714-7001 Extn: 9006

Immunology & Microbiology Journals

David Gorantl

[email protected]

1-702-714-7001Extn: 9014

Informatics Journals

Stephanie Skinner

[email protected]

1-702-714-7001Extn: 9039

Material Sciences Journals

Rachle Green

[email protected]

1-702-714-7001Extn: 9039

Mathematics and Physics Journals

Jim Willison

[email protected]

1-702-714-7001 Extn: 9042

Medical Journals

Nimmi Anna

[email protected]

1-702-714-7001 Extn: 9038

Neuroscience & Psychology Journals

Nathan T

[email protected]

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

John Behannon

[email protected]

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

[email protected]

1-702-714-7001 Extn: 9042

© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version