O'Leary Brendan M, Rico Arantza, McCraw Sarah, Fones Helen N, Preston Gail M
Department of Plant Sciences, University of Oxford.
School of Education of Vitoria-Gasteiz, University of the Basque Country (UPV/EHU).
J Vis Exp. 2014 Dec 19(94):52113. doi: 10.3791/52113.
The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.
质外体是植物组织中位于质膜之外的一个独特的细胞外区室，包括细胞壁。植物叶片的质外体区室是几个重要生物学过程的发生场所，包括细胞壁形成、细胞营养物质和水分的吸收与输出、植物与内生菌的相互作用以及对病原体的防御反应。浸润离心法是一种成熟的用于分析各种植物物种可溶性质外体成分的可靠技术。通过这种方法获得的液体通常称为质外体洗涤液（AWF）。以下方案描述了一种优化的真空浸润和离心方法，用于从菜豆（法国豆）品种嫩绿叶中提取AWF。讨论了该方法的局限性以及针对其他植物物种对方案的优化。回收的AWF可用于广泛的下游实验，这些实验旨在表征质外体的组成及其如何因植物物种和基因型、植物发育和环境条件而变化，或者确定微生物如何在质外体液体中生长并对其组成变化做出反应。
