Quantification of compartmented metabolic fluxes in developing soybean embryos by employing Biosynthetic ally directed fractional 13C labeling, [13C, 1H] two-dimensional nuclear magnetic resonance, and comprehensive isotopomer balancing
Quantification of compartmented metabolic fluxes in developing soybean embryos by employing Biosynthetic ally directed fractional 13C labeling, [13C, 1H] two-dimensional nuclear magnetic resonance, and comprehensive isotopomer balancing
复制标题
DOI：
10.1104/pp.104.050625
复制
发表时间：
2004-10-01
期刊：
影响因子：
7.4
通讯作者：
Shanks, JV
中科院分区：
文献类型：
--
作者：
Sriram, G;Fulton, DB;Shanks, JV
植物代谢通量定量有助于详细了解代谢，但很难在系统水平上进行。为了实现这一目标，我们报告了计算机辅助代谢通量分析工具的开发和应用，该分析工具能够同时评估几种主要代谢途径中的通量。通过将 U-C-13 Suc、天然丰富的 Suc 和 Gln 的混合物喂入发育中的大豆 (Glycine max) 胚胎来进行标记实验。获取了种子储存蛋白和淀粉水解物的二维 [C-13,H-1] NMR 谱，并产生了由 155 个 C-13 同位素异构体丰度组成的标记数据集。我们开发了一个计算机程序来自动计算这些数据的通量。该程序接受用户定义的代谢网络模型，并结合了最新的数学进展，以实现准确、高效的通量评估。计算通量并进行统计分析以获得 SD。通过氧化戊糖磷酸途径发现高通量（19.99 +/- 4.39 mumol d(-1) 子叶(-1)，或每 100 碳摩尔 Suc 吸收 104.2 碳摩尔 +/- 23.0 碳摩尔）。可以在质体和胞质溶胶中区分单独的转酮醇酶和转醛醇酶通量，并且发现质体中的通量至少高出 6 倍。还发现质体中从丙糖到磷酸己糖的回流是大量的（21.72 +/- 5.00 mumol d(-1) 子叶(-1)，或每 100 碳摩尔 Suc 吸收 113.2 碳摩尔 +/-26.0 碳摩尔）。可以区分回补通量的向前和向后方向。发现乙醛酸盐分流通量可以忽略不计。这种通用通量分析工具可以作为代谢研究和表型比较的定量工具，并且可以扩展到其他植物系统。
Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis toot that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-C-13 Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [C-13,H-1] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 C-13 isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain SDs. A high flux was found through the oxidative pentose phosphate pathway (19.99 +/- 4.39 mumol d(-1) cotyledon(-1), or 104.2 carbon mol +/- 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 +/- 5.00 mumol d(-1) cotyledon(-1), or 113.2 carbon mol +/-26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.