Toward the apoplast metabolome: Establishing a leaf apoplast collection approach suitable for metabolomics analysis

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 (ddH₂O) 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 ddH₂O or 20% MeOH. This indicates that ddH₂O 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 ddH₂O 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.