Lohaus Gertrud, Pennewiss Kerstin, Sattelmacher Burkhard, Hussmann Melanie, Hermann Muehling Karl
Albrecht-von-Haller Institut für Pflanzenwissenschaften, Biochemie der Pflanze, Untere Karspüle 2, D-37073 Goettingen, Germany; Institute for Plant Nutrition and Soil Science, Christian-Albrechts University, Olshausenstrasse 40, D-24098 Kiel, Germany; Present address: University of California, Department of Land, Air and Water Resources, One Shields Avenue, Davis, California 95616-8627, USA.
Physiol Plant. 2001 Apr;111(4):457-465. doi: 10.1034/j.1399-3054.2001.1110405.x.
The suitability of the infiltration-centrifugation method for collection of apoplastic fluid from intact leaves was evaluated for different plant species. Large differences with respect to infiltrability of the leaves, which correlated inversely with stomatal and mesophyll resistance, became apparent. Osmolality of infiltration medium (deionised water, 0.2 mM CaCl2, 10 mM KCl, 180 mM 2-[N-morpholino]ethane-sulphonic acid) and incubation time, time passed between onset of infiltration and end of centrifugation, revealed relatively little influence on the composition of the apoplastic washing fluid (AWF). In contrast, the pH of the infiltrated solution highly influenced the concentration of sucrose and hexoses. With increasing centrifugation force, hexosephosphate isomerase (HPI) activity in the AWF, which was taken as an indication for cytoplasmic contamination, increased. At the same time, Ca2+ concentration in the AWF increased even more. Since Ca2+ cannot originate from the cytoplasm, the suitability of HPI as marker for cytoplasmic contamination is questioned. From the composition of the AWF, it is concluded that, if centrifugation force does not exceed 1 000 g, cytoplasmic contamination is negligible and that the infiltration-centrifugation technique reveals an easy and inexpensive way to study apoplastic solutes. The infiltration-centrifugation method was also suitable to determine apoplastic air volume (Vair) and apoplastic water volume (Vwater), which are necessary for the calculation of the ion concentration in the leaf apoplast. It could be shown that the leaves of different species and the apical and basal leaves of single plants differ in Vair and Vwater.
针对不同植物物种，评估了浸润-离心法从完整叶片收集质外体汁液的适用性。叶片的浸润性存在很大差异，且与气孔和叶肉阻力呈负相关。浸润介质（去离子水、0.2 mM氯化钙、10 mM氯化钾、180 mM 2-[N-吗啉代]乙磺酸）的渗透压和孵育时间（从开始浸润到离心结束所经过的时间）对质外体洗涤液（AWF）的成分影响相对较小。相比之下，浸润溶液的pH对蔗糖和己糖浓度影响很大。随着离心力增加，作为细胞质污染指标的AWF中的磷酸己糖异构酶（HPI）活性增加。同时，AWF中的Ca2+浓度增加得更多。由于Ca2+并非源自细胞质，因此HPI作为细胞质污染标志物的适用性受到质疑。从AWF的成分得出结论，若离心力不超过1000 g，细胞质污染可忽略不计，且浸润-离心技术为研究质外体溶质提供了一种简便且经济的方法。浸润-离心法也适用于测定质外体空气体积（Vair）和质外体水体积（Vwater），这对于计算叶片质外体中的离子浓度是必要的。结果表明，不同物种的叶片以及单株植物的顶部和基部叶片在Vair和Vwater方面存在差异。
