Lohaus Gertrud
Molecular Plant Science/Plant Biochemistry, Bergische University Wuppertal, Wuppertal, Germany.
Methods Mol Biol. 2019;2014:235-251. doi: 10.1007/978-1-4939-9562-2_20.
The key step of carbon export from green leaves is the loading of sugars into the phloem. To fully understand and quantify this process, it is essential to know the concentration of sugars in the different compartments of the cells along the phloem loading pathway. However, determining subcellular metabolite concentrations has been technically challenging. This paper describes a technique to measure metabolite levels in the chloroplast, the cytosol, and the vacuole of mesophyll cells with high accuracy. The nonaqueous fractionation (NAF) technique is arguably the method of choice to analyze the subcellular metabolite distributions as it minimizes the risk of metabolite interconversions or redistribution during the process. The principle of NAF is the separation of small subcellular particles, which are obtained by homogenization, lyophilization, and sonication, in a nonaqueous density gradient. Due to the varying composition-dependent density of the fragments, their segregation reflects compartmental distributions throughout the gradient. By determining marker enzymes for chloroplast stroma, cytosol, and vacuole in gradient fractions the proportions of each subcellular compartment in each gradient fraction can be analyzed. The measured distribution of marker enzymes and of metabolites in each fraction of the gradient can be used to calculate the subcellular distribution of the metabolites.
绿叶中碳输出的关键步骤是将糖类装载到韧皮部。为了全面理解和量化这一过程，了解沿韧皮部装载途径的细胞不同区室中糖类的浓度至关重要。然而，确定亚细胞代谢物浓度在技术上具有挑战性。本文描述了一种能够高精度测量叶肉细胞叶绿体、细胞质和液泡中代谢物水平的技术。非水相分级分离（NAF）技术可以说是分析亚细胞代谢物分布的首选方法，因为它将过程中代谢物相互转化或重新分布的风险降至最低。NAF的原理是在非水密度梯度中分离通过匀浆、冻干和超声处理获得的小亚细胞颗粒。由于片段的密度因组成而异，它们的分离反映了整个梯度中的区室分布。通过测定梯度级分中叶绿体基质、细胞质和液泡的标记酶，可以分析每个梯度级分中每个亚细胞区室的比例。梯度各部分中标记酶和代谢物的测量分布可用于计算代谢物的亚细胞分布。
