Comparison of metabolite levels in
Plant metabolism is a complex network. Pathways are correlated and affect each other. Secondary metabolic pathways in plant cells are regulated strictly, and upon an intra- or extra-stimuli (e.g. stress), the metabolic fluxes will change as a response on the stimuli, for example, to protect the plant against herbivores or against microbial infections. 13C-isotope-labeling experiment has been performed on cell cultures and hairy roots of Catharanthus roseus to measure fluxes through some pathways. However, due to the complexity of the total metabolic network in an intact plant, no experiments have yet been carried on C. roseus plants. In this study, [1-13C] glucose was first applied to C. roseus seedlings grown in Murashige and Skoog’s (MS) medium. In a time course, the amount and position of 13C incorporation into the metabolites were analyzed by proton nuclear magnetic resonance (1H NMR) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR. The results show that the fed 13C-isotope was efficiently incorporated into and recycled in metabolism of the intact C. roseus plant. The C. roseus plants seem to be a good system for metabolic flux analysis.
- 13C-isotope labeling
- Catharanthus roseus
- intact plant
- metabolic fluxes
Metabolic flux analysis (MFA) aims at the quantitation of the carbon flow through a metabolic network by measuring the enrichment and position of labels in the various measurable metabolites after feeding a labeled precursor
There are two strategies for 13C MFA: one is dynamic-labeling strategy (time course experiments), and the other is steady-state-labeling strategy. The dynamic-labeling strategy has an advantage in studying small partial networks and it is particularly effective for the analysis of secondary metabolism . In this approach, a specific labeled advanced precursor of a pathway is pulse fed, and after a given time the incorporation is measured in the products of the pathway involved. In a steady-state-labeling strategy, the organisms are permanently growing in a medium containing a very early substrate for primary metabolism (e.g., a labeled sugar of pyruvate) and the diffusion of the label through all pathways is monitored by measuring the incorporation and position of the label in all measurable metabolites. This approach is usually utilized in studies of central carbon metabolism. In fact, the limiting factor in flux analyses in plants is the detection limits for the various metabolites, as the level of primary metabolites in plants is many folds higher than of secondary metabolites, the dynamic range analytical tools hamper the analysis of minor compounds. Therefore, often specific and selective extraction methods are used for the dynamic approach, whereas for the steady-state approach one uses the more general metabolomics analytical protocols.
In this study, the fate of [1-13C] glucose fed to the intact
2. Materials and methods
2.1. Plant material and in vitro culture
Murashige & Skoog medium (including vitamins) and gelrite (strength: 550–850 g/cm2) were purchased from Duchefa Biochemie. D (+)-Glucose (>99.0%) was obtained from Fluka Chemie (Buchs, Germany), whereas [1-13C]-D-glucose (>99.0%, with > 99% atom 1-13C) was from Campro Scientific (Veenendaal, The Netherlands). Jasmonic acid (JA) was from Sigma-Aldrich Chemie (Steinheim, Germany).
2.3. Jasmonic acid elicitation
A stock solution of 10 mg/ml of JA 40% EtOH was prepared, filter sterilized, and used for elicitation. After 5 days of submerging the plant roots with 5 ml of [1-13C] glucose solution (1%, w/v), 11 μl of the JA stock solution was aseptically spiked into each tube. The control samples received only the same volume of 40% EtOH. The plants were harvested at 0, 6, 24, and 72 h after treatment; young leaves, old leaves, stems, and roots of
2.4. NMR analysis
1H NMR spectra were recorded in CH3OH-
The signal intensity of carbons at certain positions of a given metabolite was obtained from peak height in the 13C-dimension spectra abstracted from the 2D HSQC spectra. The signal height of CH3OH-
3. Results and discussion
3.1. Comparison of growth and metabolism of
C. roseus plants grown in the solid MS medium versus soil
Two batches of
After 10–12 days, seeds in both batches germinated and produced their first pair of leaves. In the first 3 weeks after germination, there were no significant differences of height, leaf pairs, and leaf size between plantlets grown in MS medium and in the soil (Figure 2). However, in the following days, the plantlets in MS medium provided one more pair of leaves than those in soil did, but the leaf size was much smaller than that of plantlets grown in the soil (Figure 2A and B). Moreover, the soil plantlets grew higher than those grown in MS medium (Figure 2C). Plantlets in MS medium entered flowering time around 100 days after sowing, whereas those in soil flowered at 75 days. The plantlets grown in soil had a higher growth rate and a larger biomass than those grown in MS medium.
Metabolic differences between the plants grown in soil and MS medium were observed by 1H NMR (Figure 3). The 1H NMR spectra showed that qualitatively metabolites of plants grown in soil or MS medium were similar, but the levels varied (Table 1). Plants grown in soil produced higher levels of organic acids and sugars (malate, fumaric acid, glucose, and sucrose) than those grown in MS medium, indicating a low function/reduced level of carbon fixation in the leaves of the MS-grown plants. Also, secondary metabolites (such as secologanin, vindoline, quercetin, and kaempherol) were found in higher levels in soil-grown plants than the plants grown in MS medium. On the other hand, plants cultured in MS medium displayed significantly higher levels of arginine, glutamine, and asparagine but relatively low level of glucose and sucrose. The levels of threonine, glutamate, quinic acid, and lactic acid were also higher in plants grown in MS medium than those in soil.
|Compounds||Signal intensity||Signal intensity ratio (S/M)|
|Soil (S)||Medium (M)|
Some groups of metabolites have a close correlation with plant growth and biomass, such as the tricarboxylic acid cycle (TCA) intermediates succinate, citrate, or malate, as well as amino acids . Both glutamine and asparagine are the major compounds for nitrogen fixing, transport, and storage in plants . With the much more abundant nitrogen source in the medium than in the soil, the high levels of the amino acids in the medium grown plants could be explained. Meyer et al.  reported a negative correlation to the plant biomass with glutamine, which is in line with our findings. Sucrose starvation may lead to the presence of a large excess of asparagine in plant cells . In the present study, the plants cultured on solid MS medium require an aseptic jar with a cap, which limits the space to grow, and also affects air exchange, CO2 availability, and accumulation of volatiles in the head space if compared with plants grown in soil. Despite the uptake of carbohydrates from the medium through the roots, the growth was less than the plants grown in soil which are dependent of carbon fixation by leaves. The limited availability of CO2 in the sterile closed containers may thus be a reason for lower biomass production.
3.2. [1-13C] glucose feeding experiment and JA elicitation on
C. roseus plantlets
Samples from different organs (upper and lower leaves, stems, and roots) were measured by proton and carbon NMR. After feeding the plants with [1-13C] glucose for 5 days, the incorporation of 13C label was found in some primary and secondary metabolites detected in all organs of the
Figure 5 shows the 13C-dimension HSQC spectra and 1H NMR spectra of the non-labeled sample and the 13C-enriched sample determined in CH3OH-
|Compounds||Ratio of metabolite levels in labeled and non-labeled samples, (L0/C0), based on 1H NMR|
|Upper leaf||Lower leaf||Stem||Root|
3.3. 13C incorporation into primary and secondary metabolites
The signals in the HSQC spectra of the enriched samples were identified (Figure 4). The carbon position of 13C incorporation into a metabolite was investigated by calculating 13C signal intensity ratios between the same carbons of the metabolite in labeled and non-labeled samples (Table 3).
|Compound||Chemical Shift||Peak height||Relative intensity to CH3OH-
||Relative enrichment ratio (
Among amino acids, the signals corresponding to C at δ 16.98, C-3 of alanine, exhibited a high 13C relative enrichment ratio. Glycolysis introduces the C-1 or C-6 of glucose into alanine C-3 . Carbon signals at δ 20.47 of threonine and at δ 37.21 of aspartate also showed a relatively high labeling. The carbons of arginine and asparagine were apparently less labeled.
Glutamate (C-3 at δ 27.74, C-4 at δ 34.44, and C-5 at δ 55.67) and glutamine (C-3 at δ 27.11, C-4 at δ 31.83, and C-5 at δ 55.02) showed clear high 13C incorporation. The relative enrichment ratios of C-3 and C-2 of glutamine were lower than that of C-4, which indicate the entry of a diluting flux of C4 compounds into the TCA cycle . For glutamate, however, C-4 had a lower relative enrichment ratio than C-3 and C-2. Non-symmetrical enrichment ratios of C-2 and C-3 imply that there might be a form of channeling that converts oxoglutarate C-4 to oxaloacetate C-2 or C-3 .
In plant cells, the labeling of amino acids alanine, glutamate, and aspartate is found to reflect that of the corresponding α-oxoacids: pyruvate, α-oxoglutarate, and oxaloacetate, respectively . The organic acid malate showed a sixfold increased intensity for the carbon signal at δ 43.40.
Besides primary metabolites, secondary metabolites also exhibited clear 13C incorporation. Two phenylpropanoids, chlorogenic acid and its isomer 4-
3.4. 13C incorporation in different organs
Based on 1H NMR spectra, relative levels of primary and secondary metabolites in different organs were calculated by normalizing the integral of signal peaks to the internal standard (TSP). Table 4 showed that leaves, especially upper leaves, contained higher levels of amino acids, phenylpropanoids, iridoids, and vindoline, than stems and roots. In roots, phenylpropanoids and vindoline, which are biosynthesis dependent on chloroplasts, were not detected, whereas iridoids displayed a much lower level in roots while glucose and sucrose had relatively higher levels than in other organs.
|Compounds||Relative levels of metabolites|
|Upper leaf||Lower leaf||Stem||Root|
The incorporation of 13C in different organs (upper leaf, lower leaf, stem, and root) was also investigated by comparison of relative enrichment ratios in order to have a clue about the accumulation of label in different organs and its connection with transport and compartmentation of the pathways in the plants (Table 5). From the 13C dimension of HSQC spectra of all organs, 13C signals of labeled samples showed an apparently higher intensity in the amino acid and sugar areas than those of non-labeled ones (Figure 6), which indicated that 13C-isotope was efficiently incorporated into the primary metabolism of intact
|Compounds||13C chemical shift||Relative enrichment ratio (labeled:control)|
|Upper leaf||Lower leaf||stem||Root|
Based on the 13C dimension of HSQC spectra, leaves had more 13C signals in the area of >δ 100 ppm than stems and roots (Figure 6), even after feeding [1-13C] glucose. Upper leaves had relatively high 13C incorporation for vindoline, chlorogenic acid, and 4-
In stems and roots, no 13C signals of vindoline, chlorogenic acid, and 4-
3.5. Effect of JA elicitation on 13C fluxes into metabolic pathways
JA was spiked into the labeled glucose solution at the sixth day after submerging the plant roots in the solution. The control plants were also reared in labeled glucose solution but without JA elicitation. Leaves were harvested at 0, 6, 24, and 72 h (6, 7, and 9 d of incubation with the labeled glucose solution) after elicitation and measured by 1H NMR and HSQC.
For control plants, NMR spectra showed that the enrichments of malic acid and of the amino acids alanine, arginine, glutamate, glutamine, aspartate, and asparagine in the leaves were nearly identical at 6 and 9 d of incubation with the labeled glucose solution (Figure 7), suggesting the establishment of steady state at 6 d. However, the incorporation of label in glucose and threonine increased continuously within the measured period of 9 days. Besides, loganic acid and chlorogenic acid kept the same enrichments while vindoline and 4-
JA elicitation had little effect on the level of most metabolites, except glutamate, glutamine, vindoline, and loganic acid. Although JA induced an increase of glutamate and glutamine levels (Figure 8), their relative enrichment ratio remained unchanged compared with the controls. At the same time, the enrichment of alanine at C-3 showed an increase without levels changing compared to the controls. Vindoline levels showed an increase and reached the highest level at 72 h (23% higher than the controls) after JA treatment (Figure 8). However, the relative enrichment ratio of the C-18 signal of vindoline was lower in JA-elicited samples than in the controls, especially at 6 h (Figure 7). The level of loganic acid decreased with time (Figure 8), leading to a dramatical decrease of its enrichment at both C-3 and C-10 from 6 to 72 h. The levels of chlorogenic acid and 4-
4. Future prospects
Metabolic flux analysis is the quantification of all intracellular fluxes in an organism, which is thus an important cornerstone of metabolic engineering and systems biology. This study reports a comprehensive 13C-labeling-based metabolomics of a plant system. [1-13C] glucose was efficiently absorbed via the root system and recycled in the whole plant of
Ratcliffe RG, Shachar-Hill Y. Measuring multiple fluxes through plant metabolic networks. Plant J. 2006;45: 490–511.
Szyperski T. C-NMR, MS and metabolic flux balancing in biotechnology research. Q Rev Biophys. 1998;31: 41–31106.
Möllney M, Wiechert W, Kownatzki D, de Graaf AA. Bidirectional reaction steps in metabolic networks: IV. Optimal design of isotopomer labeling experiments. Biotechnol Bioeng. 1999;66: 86–103.
Contin A, van der Heijden R, Lefeber AWM, Verpoorte R. The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a Catharanthus roseuscell culture. FEBS Lett. 1998;434: 413–416.
Mustafa NR, Kim HK, Choi YH, Erkelens C, Lefeber AW, et al. Biosynthesis of salicylic acid in fungus elicited Catharanthus roseuscells. Phytochemistry. 2009;70: 532–539.
Sriram G, Fulton DB, Shanks JV. Flux quantification in central carbon metabolism of Catharanthus roseushairy roots by 13C labeling and comprehensive bondomer balancing. Phytochemistry. 2007;68: 2243–2257.
Schuhr CA, Radykewicz T, Sagner S, Latzel C, Zenk MH, et al. Quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in plants by NMR spectroscopy. Phytochem Rev. 2003;2: 3–16.
Lundström P, Teilum K, Carstensen T, Bezsonova I, Wiesner S, et al. Fractional 13C enrichment of isolated carbons using [1-13C]-or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Cα and side-chain methyl positions in proteins. J Biomol NMR. 2007;38: 199–212.
Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum. 1962;15: 473–497.
Kim HK, Khan S, Wilson EG, Kricun SDP, Meissner A, et al. Metabolic classification of South American Ilex species by NMR-based metabolomics. Phytochemistry. 2010;71: 773–784.
Meyer RC, Steinfath M, Lisec J, Becher M, Witucka-Wall H, et al. The metabolic signature related to high plant growth rate in Arabidopsis thaliana. Proc Nat Acad Sci. 2007;104: 4759–4764.
Lea PJ, Sodek L, Parry MA, Shewry PR, Halford NG. Asparagine in plants. Ann App Biol. 2007;150: 1–26.
Genix P, Bligny R, Martin J-B, Douce R. Transient accumulation of asparagine in sycamore cells after a long period of sucrose starvation. Plant Physiol. 1990;94: 717–722.
Choi YH, Tapias EC, Kim HK, Lefeber AWM, Erkelens C, et al. Metabolic discrimination of Catharanthus roseusleaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis. Plant Physiol. 2004;135: 2398–2410.
Mustafa NR, Kim HK, Choi YH, Verpoorte R. Metabolic changes of salicylic acid-elicited Catharanthus roseuscell suspension cultures monitored by NMR-based metabolomics. Biotechnol Let. 2009;31: 1967–1974.
Kruger NJ, Huddleston JE, Le Lay P, Brown ND, Ratcliffe RG. Network flux analysis: Impact of 13 C-substrates on metabolism in Arabidopsis thalianacell suspension cultures. Phytochemistry. 2007;68: 2176–2188.
Malloy CR, Sherry AD, Jeffrey F. Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy. J Biol Chem. 1988;263: 6964–6971.
Dieuaide-Noubhani M, Raffard G, Canioni P, Pradet A, Raymond P. Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C-or 14C-labeled glucose. J Biol Chem. 1995;270: 13147–13159.
Salon C, Raymond P, Pradet A. Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos. J Biol Chem. 1988;263: 12278–12287.
Shukla AK, Shasany AK, Gupta MM, Khanuja SP. Transcriptome analysis in Catharanthus roseusleaves and roots for comparative terpenoid indole alkaloid profiles. J Exp Bot. 2006;57: 3921–3932.
Zhou ML, Zhu XM, Shao JR, Tang YX, Wu YM. Production and metabolic engineering of bioactive substances in plant hairy root culture. Appl Microbiol Biotechnol. 2011;90: 1229–1239.
Murata J, Roepke J, Gordon H, De-Luca V. The leaf epidermome of Catharanthus roseusreveals its biochemical specialization. Plant Cell. 2008;20: 524–542.
Abbasi BH, Tian CL, Murch SJ, Saxena PK, Liu CZ. Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of Echinacea purpurea. Plant Cell Rep. 2007;26: 1367–1372.
Murata J, De-Luca V. Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus. Plant J. 2005;44: 581–594.
De Luca V, Cutler AJ. Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol. 1987;85: 1099–1102.
Ettenhuber C, Radykewicz T, Kofer W, Koop H-U, Bacher A, et al. Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants. Phytochemistry. 2005;66: 323–335.
Antonio C, Mustafa NR, Osorio S, Tohge T, Giavalisco P, Willmitzer L, Rischer H, Oksman-Caldentey KM, Verpoorte R, Fernie AR. Analysis of the interface between primary and secondary metabolism in Catharanthus roseuscell cultures using 13C-stable isotope feeding and coupled mass spectrometry. Mol Plant. 2013;6: 581–584.