13 C-isotope labeling experiments to study metabolism in Catharanthus roseus

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, e.g. to protect the plant against herbivore or against microbial infections. 13 C-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- 13 C] Glucose was first applied to C. roseus seedlings grown in culture medium. In a time course the amount and position of 13 C incorporation into the metabolites were analysed by 1 H-NMR and 1 H- 13 C HSQC NMR. The results show that the fed 13 C-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 fluxes analysis.


Introduction
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 precursorin-vivo or in-vitro. Though now common in microorganisms, in plants, with their complex secondary metabolic pathways, MFA is so far mostly focused on primary metabolism. In fact, each metabolic flux reflects the function and performance of a specific pathway in a plant's development and its interaction with the environment, e.g. defense against herbivores or microorganisms (Ratcliffe and Shachar-Hill, 2006). Consequently metabolic fluxes represent the fourth dimension of a living organism. There are three dimensions of space, which form the phenotype, but the dynamics, the fluxes, represent life. Flux analysis based on 13 C-isotope labeling experiments ( 13 CLE) has been established as an effective method for determining the flux distribution through the compartmented pathways of primary metabolism in plant cells. The 13 C isotope is not radioactive, thus convenient to be used to label the metabolites in the pathways. Usually a specifically 13 C-isotope labeled substrate, e.g. [1-13 C] glucose, is used in a CLE study. After feeding, this labeled material is distributed over the various metabolic pathways. At various time points the distribution of the label over the various measurable metabolites, is measured by using different NMR or MS instruments (Szyperski, 1998;Mollney et al., 1999). By NMR the position of the label as well as the enrichment on every position in a molecule can be measured. By MS the overall enrichment of a molecule can be determined, whereas the position is can only to some extent be determined by analysis of the fragments.
There are two strategies for 13 C MFA: one is dynamic labeling strategy (time course experiments), 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 (Ratcliffe and Shachar-Hill, 2005). 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 is 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 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 levels of primary metabolites in plants is many fold 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 Catharanthus roseus, 13 C label have been applied for both pathway elucidation and systemwide flux quantification. By feeding [1-13 C] glucose to a cell culture of C. roseus with 13 C NMR spectroscopy (Contin et al, 1998) the biosynthetic pathway of secologanin was elucidated from which secologanin was found to originate from the triose-phosphate pathway. Salicylic acid biosynthesis was uncovered in C. roseus cell cultures by a retrobiosynthetic study based on 13 C feeding experiments . Flux quantification in central carbon metabolism of C. roseus hairy roots by 13 C labeling-based flux analysis, and quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in cell cultures of C. roseus were also reported (Sriram et al. 2007;Schuhr et al. 2003). Antonio et al. (2013) used plant cell suspension cultures of C. roseus to study the the changes in fluxes after elicitation with jasmonate. The incorporation of fully labeled pyruvate was measured by GC-MS and UPLC-MS. The elicitation was found to disturb various metabolic pathways, as could be concluded from the differences in incorporation of labels. Up to now 13 CLE-based MFA has not been implemented on intact C. roseus plants. The major reason is that intact plants are a more complex metabolic system than cell cultures or hairy root cultures which only have one a few different cell types. For example, previous research showed that some valuable TIAs (e.g. vindoline, vinblastine and vincristine) can only be produced in leaves of C. roseus, not in cell cultures and hairy roots, due to the tissue-and cell-specific organization of TIA biosynthesis. So a more detailed understanding of carbon flux distribution in the complex metabolic networks of intact C. roseus plants is a prerequisite for progress in metabolic engineering of TIA production in order to feed the rapidly growing market demands of these important TIAs.
In this study, the fate of [1-13 C] glucose fed to the intact C. roseus plants via the root system was analyzed in considerable detail. Labeling patterns of targeted metabolites were deduced from previous publications (Lundstrom et al., 2007;Contin et al., 1998) (Fig. 1), and confirmed by the current experiment. By tracing the label in some of the detected primary and secondary metabolites through a time course, we have information about the 13 C incorporation status of these compounds and thus in the metabolic fluxes in the C. roseus plant metabolism and the channeling of carbon into the MIA biosynthesis. Also the metabolic changes after elicitation were measured in this model.

Plant material and in-vitro culture
Catharanthus roseus seeds (Pacifica Cherry Red cultivar) were purchased from PanAmerican Seed Company (USA). The seeds were surface sterilized in 75 % of ethanol (v/v) and 10% of NaClO (v/v) for 1 min and 15 min (respectively), and subsequently washed three times with sterile distilled water. Sterilized seeds were germinated on solid MS medium (basal culture medium by Murashige and Skoog, 1962) with 1% non-labeled glucose. Finally, 54 seedlings were obtained, sub-cultured every 2 weeks in the same MS solid medium and used before flowering for the labeling experiments. After 10 weeks, 19 plants as control were transferred to glass tubes and reared (each) with 5 ml of 10 g/L non-labeled glucose solution; whereas the other 35 plants were placed in separate glass tubes containing 5 ml of 10 g/L [1-13 C] glucose solution. The plant cultures were grown in a climate chamber under a 16 h light and 8 h dark photoperiod at 25 ± 2 °C.

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 submerging the plant roots with 5 ml of [1-13 C] 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 h, 6 h, 24 h, and 72 h after treatment; young leaves, old leaves, stems and roots of C. roseus plants were harvested separately, immediately frozen and ground in liquid nitrogen into powder and freeze-dried for 72 h before NMR extraction (5 replicates per sample).

NMR analysis
1 H-NMR spectra were recorded in CH 3 OH-d 4 using a Bruker DMX 600 MHz spectrometer, whilst the coherence order selective gradient heteronuclear single quantum coherence (HSQC) spectra were recorded in CH 3 OH-d 4 by a Bruker AV 500 MHz spectrometer. HSQC spectra were recorded for a data matrix of 256 × 2048 points covering 30182.7 × 7812.5 Hz with 64 scans for each increment (Kim et al., 2010). INEPT transfer delays were optimized for a heteronuclear coupling of 145 Hz and a relaxation delay of 1.5 s was applied. Data was linear predicted in F 1 to 512 × 2048 using 32 coefficients and then zero-filled to 2048 × 2048 points prior to echo-anti echo type 2D Fourier transformation and a sine bell shaped window function shifted by π/2 in both dimensions was applied. 1D projection along the F 1 -axis was extracted using the build-in positive projection tool of Topspin (version 2.1, Bruker Biospin).
The signal intensity of carbons at certain positions of a given metabolite was obtained from peak height in the 13 C-dimension spectra abstracted from the 2D HSQC spectra. The signal height of CH 3 OH-d 4 was selected as standard and set as 1 in both labeled and non-labeled samples. The other signals were normalized and expressed relative to this signal. 13 C signal intensity ratio was calculated by comparison of normalized 13 C signal heights between 13 C labeled and non-labeled samples.

Comparison of growth and metabolism of C. roseus plants grown in the solid culture medium versus soil
Two batches of C. roseus seeds (each containing of 10 seeds) were germinated, one batch in soil and another one in solid MS medium with glucose. They were kept in the same condition of light and temperature. The height, the size and the leaf pairs of the plants from seedling until flowering were monitored and recorded regularly to determine the growth state of plants.
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 (Fig. 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 ( Fig. 2A and 2B). Moreover, the soil plantlets grew higher than those grown in MS medium (Fig. 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 1 H-NMR (Fig. 3). The 1 H-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.  Some groups of metabolites have a close correlation with plant growth and biomass, like the tricarboxylic acid cycle (TCA) cycle intermediates succinate, citrate or malate, as well as amino acids (Meyer et al., 2007). Both glutamine and asparagine are the major compounds for nitrogen fixing, transport and storage in plants (Lea et al., 2007). 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., (2007) 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 (Genix et al., 1990). In the present study, the plants cultured on solid MS medium require an aseptic jar with cap, which limits the space to grow, and also affects air exchange, CO 2 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 CO 2 in the sterile closed containers may thus be a reason for lower biomass production.
[1-13 C] glucose feeding experiment and JA elicitation on

Catharanthus 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 [1-13 C] glucose for five days, incorporation of 13 C label was found in some primary and secondary metabolites detected in all organs of the C. roseus plantlets. 13 C signals of some primary and secondary metabolites were assigned based on the "in-house" database and some references (Choi et al., 2004;, and confirmed in the chapter 4. Totally 14 amino acids, 9 organic acids, 2 carbohydrates, 6 phenylpropanoids, 5 TIAs, 2 terpenoids and 3 other compounds were identified. Among them, only the metabolites from which characteristic signals were clearly visible and non-overlapping in both 1 H and 13 C NMR spectra were quantified (Fig. 4). Those include some primary metabolites like amino acids (threonine, alanine, asparagine, aspartate, glutamine, glutamate and arginine) and malic acid (Fig. 4A), as well as some secondary metabolites like phenylpropanoids (chlorogenic acid, 4-O-caffeoylquinic acid), terpenoids (loganic acid and secologanin) and TIA (vindoline) (Fig. 4C). Figure 5 shows the 13 C-dimension HSQC spectra and 1 H-NMR spectra of the non-labeled sample and the 13 C-enriched sample determined in CH 3 OH-d 4 . As expected the superimposed 1 H-NMR spectra of leaves (Fig. 5) and stems (Data not shown), did not show any significant difference in proton signal intensity of the metabolites for the control and the 13 C-enriched sample. Production of these sugars caused a decrease in the levels of glucose and sucrose in roots but did not affect the metabolite levels in other organs. Except this, there was no significant change in metabolites levels of the plants fed with labeled-and non-labeled glucose solution (Table 2). This information is necessary to confirm that the 13 C signals of the spectra of enriched samples are due to incorporation of label, and not because of higher levels of production of the metabolites. Superimposed 13 C-dimension HSQC spectrum showed that the enriched sample had a much higher intensity of 13 C signals than the non-labeled one. The results indicate that the [1-13 C] glucose-fed C. roseus plants grew normally, and incorporated the labeled sugar into its metabolic network. Previous work with Arabidopsis support that 13 C feeding does not in itself distort the fluxes through the metabolic network in a plant (Kruger et al., 2007). H] HSQC spectrum of CH 3 OH-d 4 extract of Catharanthus roseus leaves. A, spectrum region displaying amino acid resonances; B, spectrum region displaying sugar resonances; C, spectrum region displaying aromatic resonances. 1, alanine; 2, threonine; 3, arginine; 4, glutamine; 5, glutamate; 6, malate; 7, aspartate; 8, asparagine; 9, vindoline; 10, loganic acid; 11, chlorogenic acid; 12, 4-O-Caffeoyl quinic acid.

C Incorporation into primary and secondary metabolites
The signals in the HSQC spectra of the enriched samples were identified (Fig. 4). The carbon position of 13 C incorporation into a metabolite was investigated by calculating 13 C signal intensity ratios between the same carbons of the metabolite in labeled and non-labeled samples (Table 3).
Among amino acids, the signals corresponding to C at δ 16.98, C-3 of alanine, exhibited a high 13 C relative enrichment ratio. Glycolysis introduces the C-1 or C-6 of glucose into alanine C-3 (Lundstrom et al., 2007). 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, 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 13 C 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 (Malloy et al., 1988). 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 implies that there might be a form of channeling that converts oxoglutarate C-4 to oxaloacetate C-2 or C-3 (Dieuaide-Noubhani et al., 1995).
In plant cells, the labeling of amino acids alanine, glutamate, and aspartate are found to reflect that of the corresponding α-oxoacids: pyruvate, α-oxoglutarate, and oxaloacetate, respectively (Salon et al., 1988). The organic acid malate showed a 6-fold increased intensity for the carbon signal at δ 43.40.
Besides primary metabolites, secondary metabolites also exhibited clear 13 C incorporation. Two phenylpropanoids, chlorogenic acid and its isomer 4-O-caffeoyl quinic acid, have an increased 13 C intensity of C-6. Incorporation of 13 C could be observed for C-3 and C-10 of loganic acid. These results are in agreement with the prediction shown in Fig. 1. The signal corresponding to C-18 of vindoline in the labeled sample was 2-fold higher than in the spectrum of the control.

C Incorporation in different organs
Based on 1 H-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 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.
The incorporation of 13 C in different organs (upper leaf, lower leaf, stem and root) were 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 13 C dimension of HSQC spectra of all organs, 13 C signals of labeled samples showed an apparently higher intensity in the amino acid and sugar areas than those of non-labeled ones (Fig. 6), which indicated that 13 C-isotope was efficiently incorporated into the primary metabolism of intact C. roseus plants via the roots. Glucose had higher 13 C intensity ratio in lower leaves and roots but relatively low in upper leaves and stems, thus showing a time dependent distribution through the plant. Glutamate and aspartate, directly derived from α-ketoglutarate and oxaloacetate of the TCA cycle, showed clear 13 C enrichment in all organs. So did malate, one of the bricks in the TCA cycle. Meanwhile, glutamate, aspartate and malate all displayed the highest 13 C intensity ratio in roots. These results indicate that 13 C was efficiently incorporated and recycled in the primary metabolism of intact plants. Upper leaves had higher levels and higher relative enrichment ratios of glutamate and malate compared with lower leaves, reflecting the faster rate of TCA cycle in the upper parts for plants growing. The glutamate-derived amino acids glutamine and arginine displayed a different pattern of 13 C incorporation. Glutamine showed 13 C incorporation in all organs with the highest intensity ratio in roots and the lowest in stems, whereas arginine showed low 13 C incorporation in all organs, implying a low flux in its biosynthetic pathway and low usage for other pathways. The 13 C incorporation of aspartate-derived amino acids asparagine and threonine was also different. Threonine had relatively high 13 C incorporation in upper leaves and roots, but relatively low in lower leaves and stems, indicating a high turnover in the upper leaves. Asparagine, except for roots, displayed low 13 C incorporation in the other organs. Pyruvate-derived alanine exhibited the lowest relative enrichment ratio in upper leaves, whilst the highest was found in stems and roots.  In stems and roots, no 13 C signals of vindoline, chlorogenic acid and 4-O-Caffeoyl quinic acid were detected with or without feeding [1-13 C] glucose. Vindoline is not found in roots due to its tissue-specific biosynthesis requiring chloroplasts for one of its biosynthetic steps (Zhou et al., 2011;Murata et al., 2008;Abbasi et al., 2007;Shukla et al., 2006;Murata and De Luca, 2005;De Luca and Cutler, 1987). The 13 C signal of loganic acid at δ 12.69 and 146.1 ppm was clearly present and showed a high relative enrichment ratio at the spectra of all organs while that of secologanin at δ 121.53 ppm was only found in the spectra of leaves. It was difficult to calculate the relative enrichment ratio of secologanin due to the signal overlapping. In roots and stems secologanin was too low for further analysis. The high levels of loganic acid in the roots are in line with a previous study that reported that LAMT activity, which converts loganic acid into loganin (the direct substrate of secologanin), was 4 to 8 times lower in hairy roots than that in the other organs of the plant (Murata et al., 2008).

Effect of JA elicitation on 13 C fluxes into metabolic pathways
JA was spiked into the labeled glucose solution at the 6th 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 (Fig. 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-O-caffeoylquinic acid showed an increase of the enrichments within 9 days. Previous study with C. roseus hairy roots grown in the light showed that the 13 C label was not diluted by CO 2 fixation (Schuhr et al., 2003). In tobacco plants grown on agar containing labeled glucose, the metabolism was studied on a quantitative basis showing that the labeled glucose was efficiently absorbed via the root system, metabolized and recycled (Ettenhuber et al., 2005). Our results indicate that the C. roseus plant system can reach a relatively steady isotopic state with plants growing in 13 CLE. 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 (Fig. 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 (Fig. 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 (Fig. 7). The level of loganic acid decreased with time (Fig. 8), leading to a dramatical decrease of its enrichment at both C-3 and C-10 from 6 h to 72 h. The levels of chlorogenic acid and 4-O-caffeoyl quinic acid in the time course did not change after JA elicitation (Fig. 8), but the enrichments were lower than those of the control labeled samples (Fig. 7). 13 C fluxes to various metabolic pathways, like glutamate and loganic acid, could be disturbed within 24 h after MeJA treatment (Antonio et al., 2013).

Conclusion
This study reports a comprehensive 13 C labeling-based metabolomics of a plant system. [1-13 C] glucose was efficiently absorbed via the root system and recycled in the whole plant of C. roseus. The plant system of C. roseus could reach a relatively steady isotopic state in 13 CLE, which appears to be well qualified to study flux contributions in the biosynthesis of sink metabolites for system biology. Combined with exogenous elicitation, 13 C MFA appears to be a good tool to study the crosslink among pathways in the complicated plant metabolic network