Dragišić Maksimović Jelena J, Zivanović Branka D, Maksimović Vuk M, Mojović Miloš D, Nikolic Miroslav T, Vučinić Zeljko B
Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11030 Belgrade, Serbia.
Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia.
Plant Sci. 2014 Jun;223:49-58. doi: 10.1016/j.plantsci.2014.03.009. Epub 2014 Mar 13.
Apoplastic fluid was extracted from maize (Zea mays L.) roots using two procedures: collection from the surface of intact plant roots by filter paper strips (AF) or vacuum infiltration and/or centrifugation from excised root segments (AWF). The content of cytoplasmic marker (glucose-6-phosphate, G-6-P) and antioxidative components (enzymes, organic acids, phenolics, sugars, ROS) were compared in the extracts. The results obtained demonstrate that AF was completely free of G-6-P, as opposed to AWF where the cytoplasmic constituent was detected even at mildest centrifugation (200×g). Isoelectric focusing of POD and SOD shows the presence of cytoplasmic isoforms in AWF, and HPLC of sugars and phenolics a much more complex composition of AWF, due to cytoplasmic contamination. Organic acid composition differed in the two extracts, much higher concentrations of malic acid being registered in AF, while oxalic acid due to intracellular contamination being present only in AWF. EPR spectroscopy of DEPMPO spin trap in the extracts showed persistent generation of hydroxyl radical adduct in AF. The results obtained argue in favor of the filter strip method for the root apoplastic fluid extraction, avoiding the problems of cytoplasmic contamination and dilution and enabling concentration measurements in minute regions of the root.
采用两种方法从玉米（Zea mays L.）根中提取质外体汁液：用滤纸从完整植株根表面收集（AF），或对离体根段进行真空渗透和/或离心（AWF）。比较提取物中细胞质标记物（葡萄糖 - 6 - 磷酸，G - 6 - P）和抗氧化成分（酶、有机酸、酚类、糖类、活性氧）的含量。结果表明，AF完全不含G - 6 - P，而AWF即使在最温和的离心条件（200×g）下也能检测到细胞质成分。过氧化物酶（POD）和超氧化物歧化酶（SOD）的等电聚焦显示AWF中存在细胞质同工型，由于细胞质污染，糖类和酚类的高效液相色谱（HPLC）显示AWF的组成更为复杂。两种提取物中的有机酸组成不同，AF中苹果酸浓度高得多，而由于细胞内污染，草酸仅存在于AWF中。提取物中DEPMPO自旋捕集剂的电子顺磁共振（EPR）光谱显示AF中持续产生羟基自由基加合物。所得结果支持采用滤纸条法提取根质外体汁液，避免了细胞质污染和稀释问题，并能在根的微小区域进行浓度测量。
