Metabolite profiling and assessment of metabolome compartmentation of soybean leaves using non-aqueous fractionation and GGMS analysis
Metabolite profiling and assessment of metabolome compartmentation of soybean leaves using non-aqueous fractionation and GGMS analysis
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DOI：
10.1007/s11306-007-0078-y
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发表时间：
2007-09-01
期刊：
影响因子：
3.6
通讯作者：
Osaki, Mitsuru
中科院分区：
文献类型：
--
作者：
Benkeblia, Noureddine;Shinano, Takuro;Osaki, Mitsuru
本研究利用非水相分离(NAQF)和气相色谱-质谱仪(GC-MS)对大豆成熟叶片的代谢进行了空间分辨研究。用CCl4-正庚烷和超速离心法对冷冻干燥的大豆叶片进行NAQF，得到由六个组分组成的梯度。以叶绿素含量为基质标记物，以磷酸烯醇式丙酮酸羧基酶(PEPC)和α-甘露糖苷酶(α-甘露糖苷酶)为标记酶，分别作为基质、细胞质和液泡标记。对每个组分的GC-MS分析结果表明，大约有100种不同的代谢物。这些化合物的分布从液泡到细胞质再到叶绿体基质的顺序是递减的。换句话说，与胞浆或基质相比，在液泡中发现了更多已鉴定的化合物。糖、有机酸和脂肪酸的水平在液泡中的相对丰度更高，分别占50%、55%和50%。胞质中氨基酸的相对丰度较高，占氨基酸总量的45%。液泡室中相对较大的糖和酚酸库意味着高水平的淀粉代谢和苯丙烷类生物合成。另一方面，低氨基酸库表明大豆叶片中的氮素积累较少。对最丰富的代谢物进行的系统聚类分析显示，有三个簇包含32个选定代谢物中的10个、20个和2个。根据大豆成熟叶片的NaQF和GC-MS分析，以及代谢产物在亚细胞水平上的分布和区划，对这些数据进行了讨论。
In the present study, non-aqueous fractionation (NAQF) and GC-MS were used to obtain a spatially resolved view of metabolism in mature leaves of soybean (Glycine max Merr.). NAQF of lyophilized soybean leaves was performed using CCl4-n-heptane and ultracentrifugation that yielded a gradient comprised of six fractions. Chlorophyll content, and marker enzyme activities, phosphoenolpyruvate carboxylase (PEPC) and alpha-mannosidase, were utilized as stroma, cytosol and vacuole markers, respectively. GC-MS analyses of each fraction resulted in the identification of around 100 different metabolites. The distribution of these identified compounds showed a decreasing order from the vacuole to cytosol to chloroplast stroma. In other words, a greater number of identified compounds were found in the vacuole when compared to the cytosol or stroma.Levels of sugars, organic acids and fatty acids showed greater relative abundances in the vacuole with 50, 55, and 50% of the respective pools. A greater relative abundance of amino acids was observed in the cytosol where 45% of the total of amino acids content was recorded. The relatively large pool of sugars and phenolic acids in the vacuole compartment implies high levels of starch metabolism and phenylpropanoid biosynthesis. The low amino acids pool, on the other hand, suggests low nitrogen accumulation in the leaves of soybean. Hierarchical cluster analysis on the most abundant metabolites revealed three clusters containing 10, 20, and 2 of the 32 selected metabolites. The data were discussed in term of NAQF and GC-MS analysis of soybean mature leaves, and also in term of distribution and compartmentation of metabolites at subcellular levels.