Analysis of sugar accumulation in a single parenchyma cell of the fruit

本研究对果实中完整的单个薄壁细胞的分析方法进行了研究。所开发的方法首先是收集果实单个细胞的液泡汁液，然后在皮升水平上进行分析，以研究果实单个薄壁细胞中糖的积累。显微操作系统用于收集液泡汁液。将玻璃微吸管插入目标细胞，通过降低压力从细胞中取出空泡汁液。然后，收集液泡汁液，排入5μL油滴中，储存在-20℃下进行糖分分析。葡萄糖、果糖、蔗糖或山梨醇的含量由NADPH的强度确定，NADPH是由15pl的液泡液通过酶促反应产生的，使用配备有光度计的荧光显微镜。转化酶活性测定也采用相同的方法。对单细胞液泡液渗透压和钾含量的测定方法进行了试验。用皮升渗透压计测量汁液的冰点降低值来测定渗透压，用扫描电子显微镜和能谱仪进行X射线微区分析来测定钾含量。利用上述方法，对柿、葡萄、桃、日本梨和苹果果实的糖积累进行了单个薄壁细胞的分析。结果表明，各种果实的单细胞糖积累规律与果实整体组织的糖积累规律一致，渗透压的增加是细胞内糖积累的结果。此外，个别细胞在某些果实中表现出不同的糖组成，但在其他果实中具有相同的组成。

The analysis methods for an intact single parenchyma cell of the fruit was examined in this research. The method developed is firstly due to collect vacuolar sap from a single cell of the fruit, and then to analyze at picolitre levels for studying sugar accumulation in a single parenchyma cell of the fruit.1. The maicromanipulator system were used for collecting vacuolar sap. The glass micropipette was inserted into a targeted cell, and vacuolar sap was withdrawn from the cell by lowering the pressure. Then, the collected vacuolar sap was expelled into 5 μL of an oil drop and stored at -20℃ for sugar analysis. The content of glucose, fructose, sucrose, or sorbitol were determined by the intensity of NADPH, which was produced with enzymatic reaction of 15 pl vacuolar sap, using a fluorescence microscope equipped with a photometer produced. The invertase activities were also determined by the same method.2. The method for determining the osmotic pressure and potassium content of the vacuolar sap from a single cell were tested. Osmotic pressure was determined by measuring freezing point depression of the sap using a picolitre osmometer, and potassium content was determined by X-ray microanalysis using a scanning electron microscopy (SEM) with energy dispersive spectrometers.3. Using above methods mentioned, analysis of sugar accumulation was conducted to the fruit of persimmon, grape, peach, Japanese pear, and apple on a single parenchyma cell basis. It was revealed that sugar accumulation pattern of a single cell is coincident with that of bulk tissue of the fruit in every kind of fruit, and that increase in osmotic pressure is due to accumulation of the sugars in the cells. Furthermore, individual cells showed different sugar compositions in some fruit, but it had the same compositions in other fruit.