Validation of carbon isotopologue distribution measurements by GC-MS and application to 13C-metabolic flux analysis of the tricarboxylic acid cycle in Brassica napus leaves

Abstract

The estimation of metabolic fluxes in photosynthetic organisms represents an important challenge that has gained interest over the last decade with the development of ¹³C-Metabolic Flux Analysis at isotopically non-stationary steady-state. This approach requires a high level of accuracy for the measurement of Carbon Isotopologue Distribution in plant metabolites. But this accuracy has still not been evaluated at the isotopologue level for GC-MS, leading to uncertainties for the metabolic fluxes calculated based on these fragments. Here, we developed a workflow to validate the measurements of CIDs from plant metabolites with GC-MS by producing tailor-made E. coli standard extracts harboring a predictable binomial CID for some organic and amino acids. Overall, most of our TMS-derivatives mass fragments were validated with these standards and at natural isotope abundance in plant matrices. Then, we applied this validated MS method to investigate the light/dark regulation of plant TCA cycle by incorporating U-¹³C-pyruvate to Brassica napus leaf discs. We took advantage of pathway-specific isotopologues/isotopomers observed between two and six hours of labeling to show that the TCA cycle can operate in a cyclic manner under both light and dark conditions. Interestingly, this forward cyclic flux mode has a nearly four-fold higher contribution for pyruvate-to-citrate and pyruvate-to-malate fluxes than the phosphoenolpyruvate carboxylase (PEPc) flux reassimilating carbon derived from some mitochondrial enzymes. The contribution of stored citrate to the mitochondrial TCA cycle activity was also questioned based on dynamics of ¹³C-enrichment in citrate, glutamate and succinate and variations of citrate total amounts under light and dark conditions. Interestingly, there was a light-dependent ¹³C-incorporation into glycine and serine showing that decarboxylations from pyruvate dehydrogenase complex and TCA cycle enzymes were actively reassimilated and could represent up to 5% to net photosynthesis.

Keywords: Krebs cycle; TCA cycle; accuracy; isotope C distribution 13; phosphoenol pyruvate carboxylase; plant; pyruvate dehydrogenase complex (PDC); trimethylsilyl derivatives (TMS).

Figure 1

Workflow used in this study. A GC-MS method was built with abundant molecular ions deriving from TMS-derivatives of organic and amino acids that contained a large number of informative carbons. Then this method was evaluated by using: i) chemical standards and tailor-made ¹³C-standards that had a predictable CID using E. coli as a metabolic factory (¹³C-PT); ii) unlabeled and ¹³C-labeled plant samples.

Figure 2

Evaluation of Carbon Isotopologue Distribution (CID) measurements for organic and amino acids by GC-MS using ¹³C-PT reference samples. (A), Evaluation of amino acids using ¹³C-PT protein hydrolysates. (B), Evaluation of organic acids using ¹³C-PT full metabolites extracts. Black bars correspond to expected CIDs and blue bars to the measured CIDs. The results are presented as the mean ± SD of four independent biological replicates. Statistical differences between predicted and measured CID for each isotopologue of each fragment are denoted with asterisks (*) and were established by considering the 95% confidence intervals of the measured CID. GC-MS fragments are denoted according to the considered metabolite, its MEOX/TMS-derivatives analyzed and the metabolite carbon backbone [for example, Glutamate_3TMS_C2C5 means that the fragment comes from glutamate(3TMS) and contains the carbon C2-C3-C4-C5 ( Table 1 )].

Figure 3

Evaluation of the measurement accuracy for CID and mean ¹³C enrichment at ¹³C-PT and ¹³C natural abundance in plants. (A), CID accuracy and (B), Mean ¹³C enrichment for the selected TMS-derivatives of organic and amino acids using ¹³C-PT samples. (C), Residual mean ¹³C enrichment measured in chemical standards, Arabidopsis seedlings and (B) napus leaves after correction for isotope natural abundance. (D), Isotopologue-specific biases identified at ¹³C natural abundance for proline, valine and threonine fragments. The results are presented as the mean ± SD of four independent biological replicates or independent preparations for commercial chemical standards.

Figure 4

Dynamics of U-¹³C-pyruvate incorporation in Brassica napus leaf discs exposed to light or dark conditions. (A), Mean fractional ¹³C enrichment and (B), Metabolite pool sizes. Mean fractional ¹³C enrichment were monitored with GC-MS, while absolute quantification of organic and amino acids was performed respectively with GC-FID and HPLC-UV. The results are presented as the mean ± SD of four independent biological replicates. Statistical differences between dark and light conditions are denoted with asterisks according to a Student test (*, p-value<0.05; **, p-value<0.01; ***, p-value<0.001). The complete dataset is available in Tables S2 , S3 . Supplementary statistics (ANOVA and post-hoc Tukey-HSD tests) are available in Tables S4 , S5 .

Figure 5

Fractional ¹³C enrichment at the isotopologue level for TCA-cycle derived metabolites. (A), Metabolic network considered based on the ¹³C-labeling strategy. (B), Light conditions and (C), Dark conditions. The M0 isotopologue was not represented in the graphs to facilitate the visualization of low-enriched isotopologues. The results are presented as the mean ± SD of four independent biological replicates. The complete dataset is available in Table S2 . In the proposed network, the second “turn” reflected the commitment of a molecule that has already experienced all reactions of the TCA cycle to another round of reactions (Orange and blue labels to compare acetyl-CoA-derived labeling from 1^st and 2^nd “turn”). Note that M0 istopologues of all metabolites will still continue to produce M0 istopologues at an important rate after a “turn” of the TCA cycle, given the ¹³C-enrichment for acetyl-CoA active pool. PEPc, Phosphoenolpyruvate carboxylase; PDC, Pyruvate dehydrogenase complex, CS, Citrate synthase; ACO, Aconitase; ICDH, Isocitrate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase; SDH, Succinate dehydrogenase complex; FUM, Fumarase; MDH, Malate dehydrogenase.

Figure 6

Fractional contribution of TCA cycle and PEPc-dependent reassimilation of mitochondrial CO₂ for pyruvate to malate and pyruvate to citrate in light and dark conditions. (A), Pathway-specific mass isotopologues considered for the calculations. (B), Fractional contribution of PEPc and TCA cycle to malate and citrate based on pathway-specific mass isotopologues/isotopomers. For these calculations, only the timepoint T4 and T6 hours were used and pooled (metabolic steady-state and isotopic steady-state for isotopologue ratios). The results are presented as the mean ± SD of four independent biological replicates. Statistical comparison of acetyl-CoA enrichment for light and dark conditions was performed with a Student-test, while fractional contributions were compared with an ANOVA followed by a post-hoc Tukey-HSD test (different letters indicate groups that were separated with a p-value < 0.05).

Figure 7

Fractional contribution of PDC and TCA-cycle derived decarboxylations to photosynthesis. (A), Fractional ¹³C enrichment at the isotopologue level for glycine and serine. (B), Working hypotheses to explain the photorespiratory-dependent labeling pattern of glycine and serine. (C), Estimation of the fractional contribution of PDC (pyruvate dehydrogenase complex) and TCA-cycle CO₂ to photosynthesis with Serine (expressed in %). For these calculations, only the timepoint T4 and T6 hours were used and pooled (metabolic and isotopic steady-state, assumption with ¹³C/¹²C of CO₂ released). The results are presented as the mean ± SD of four independent biological replicates. Statistical comparison of the two serine fragments was performed with a Student-test.

Figure 8

Proposed flux modes for TCA cycle in B. napus leaves under light conditions. The contribution of stored citrate is unlikely to interact with mitochondrial TCA cycle. The mitochondrial TCA cycle operate in both cyclic and non-cyclic flux modes. To which extent 2-oxoglutarate molecules escape the cycle and imbalance this cyclic/non-cyclic ratio remain to be determined.