LabPlant, Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, 60451-970, Brazil.
LabPlant, Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, 60451-970, Brazil.
Plant Physiol Biochem. 2024 Oct;215:109080. doi: 10.1016/j.plaphy.2024.109080. Epub 2024 Sep 1.
The leaf apoplast contains several compounds that play important roles in the regulation of different physiological processes in plants. However, this compartment has been neglected in several experimental and modelling studies, which is mostly associated to the difficulty to collect apoplast washing fluid (AWF) in sufficient amount for metabolomics analysis and as free as possible from symplastic contamination. Here, we established an approach based in an infiltration-centrifugation technique that use little leaf material but allows sufficient AWF collection for gas chromatography mass spectrometry (GC-MS)-based metabolomics analysis in both tobacco and Arabidopsis. Up to 54 metabolites were annotated in leaf and apoplast samples from both species using either 20% (v/v) methanol (20% MeOH) or distilled deionized water (ddHO) as infiltration fluids. The use of 20% MeOH increased the yield of the AWF collected but also the level of symplastic contamination, especially in Arabidopsis. We propose a correction factor and recommend the use of multiple markers such as MDH activity, protein content and conductivity measurements to verify the level of symplastic contamination in MeOH-based protocols. Neither the concentration of sugars nor the level of primary metabolites differed between apoplast samples extracted with ddHO or 20% MeOH. This indicates that ddHO can be preferentially used, given that it is a non-toxic and highly accessible infiltration fluid. The infiltration-centrifugation-based approach established here uses little leaf material and ddHO as infiltration fluid, being suitable for GC-MS-based metabolomics analysis in tobacco and Arabidopsis, with great possibility to be extended for other plant species and tissues.
质外体包含几种化合物，它们在植物的不同生理过程的调控中发挥重要作用。然而，这个隔室在许多实验和建模研究中被忽视了，这主要与收集足够数量的质外体洗脱液（AWF）用于代谢组学分析以及尽可能避免与胞质污染有关的困难有关。在这里，我们建立了一种基于渗透离心技术的方法，该方法使用少量叶片材料，但可以收集足够的 AWF，用于在烟草和拟南芥中进行基于气相色谱质谱（GC-MS）的代谢组学分析。使用 20%（v/v）甲醇（20% MeOH）或去离子蒸馏水（ddHO）作为渗透液，在两种物种的叶片和质外体样本中注释了多达 54 种代谢物。使用 20% MeOH 增加了收集的 AWF 的产量，但也增加了胞质污染的水平，尤其是在拟南芥中。我们提出了一个校正因子，并建议使用多个标记物，如 MDH 活性、蛋白质含量和电导率测量，以验证基于 MeOH 的方案中的胞质污染水平。ddHO 或 20% MeOH 提取的质外体样本中的糖浓度或初级代谢物水平没有差异。这表明 ddHO 可以优先使用，因为它是一种无毒且易于获得的渗透液。这里建立的基于渗透离心的方法使用少量叶片材料和 ddHO 作为渗透液，适用于烟草和拟南芥的 GC-MS 代谢组学分析，并有很大可能扩展到其他植物物种和组织。
