Dellero Younès, Berardocco Solenne, Berges Cécilia, Filangi Olivier, Bouchereau Alain
Institute for Genetics, Environment and Plant Protection (IGEPP), National Research Institute for Agriculture, Food and Environment (INRAE), Université Rennes, Institut Agro, Le Rheu, France.
Metabolic Profiling and Metabolomics platform (P2M2), Institute for Genetics, Environment and Plant Protection (IGEPP), Biopolymers Interactions Assemblies (BIA), Le Rheu, France.
Front Plant Sci. 2023 Jan 10;13:885051. doi: 10.3389/fpls.2022.885051. eCollection 2022.
The estimation of metabolic fluxes in photosynthetic organisms represents an important challenge that has gained interest over the last decade with the development of C-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 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-C-pyruvate to 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 C-enrichment in citrate, glutamate and succinate and variations of citrate total amounts under light and dark conditions. Interestingly, there was a light-dependent C-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.
光合生物中代谢通量的估计是一项重要挑战，在过去十年中随着同位素非稳态稳态下的C代谢通量分析的发展而受到关注。这种方法需要高精度测量植物代谢物中的碳同位素异构体分布。但对于气相色谱 - 质谱法（GC-MS）在同位素异构体水平上的这种准确性仍未进行评估，这导致基于这些片段计算的代谢通量存在不确定性。在这里，我们开发了一种工作流程，通过制备含有某些有机和氨基酸可预测二项式碳同位素异构体分布的特制标准提取物，来验证用GC-MS对植物代谢物中碳同位素异构体分布（CIDs）的测量。总体而言，我们的大多数三甲基硅烷（TMS）衍生物质量片段都通过这些标准以及植物基质中的天然同位素丰度得到了验证。然后，我们应用这种经过验证的质谱方法，通过将13C-丙酮酸掺入叶盘来研究植物三羧酸循环（TCA循环）的光/暗调节。我们利用在标记两到六小时之间观察到的途径特异性同位素异构体/同位素体，表明TCA循环在光照和黑暗条件下都可以循环运行。有趣的是，这种正向循环通量模式对丙酮酸到柠檬酸和丙酮酸到苹果酸通量的贡献比磷酸烯醇式丙酮酸羧化酶（PEPc）通量重新同化源自某些线粒体酶的碳的贡献高出近四倍。基于柠檬酸、谷氨酸和琥珀酸中碳富集的动态以及光照和黑暗条件下柠檬酸总量的变化，储存的柠檬酸对线粒体TCA循环活性的贡献也受到了质疑。有趣的是，存在光依赖的碳掺入甘氨酸和丝氨酸的现象，表明丙酮酸脱氢酶复合物和TCA循环酶的脱羧反应被积极重新同化，并且可能占净光合作用的5%。
