Direct surface analysis mass spectrometry uncovers the vertical distribution of cuticle-associated metabolites in plants.

The plant cuticle covers the plant's entire aerial surface and acts as the outermost protective layer. Despite being crucial for the survival of plants, surprisingly little is known about its biosynthesis. Conventional analytical techniques are limited to the isolation and depolymerization of the polyester cutin, which forms the cuticular scaffold. Although this approach allows the elucidation of incorporated cutin monomers, it neglects unincorporated metabolites participating in cutin polymerization. The feasibility of a novel approach is tested for in situ analysis of unpolymerized cuticular metabolites to enhance the understanding of cuticle biology. Intact cotyledons of Brassica napus and Arabidopsis thaliana seedlings are immersed in organic solvents for 60 seconds. Extracts are analyzed using high-resolution direct infusion mass spectrometry. A variety of different diffusion routes of plant metabolites across the cuticle are discussed. The results reveal different feasibilities depending on the research question and cuticle permeabilities in combination with the analyte's polarity. Especially hydrophilic analytes are expected to be co-located in the cell wall beneath the cuticle causing systematic interferences when comparing plants with different cuticle permeabilities. These interferences limit data interpretation to qualitative rather than quantitative comparison. In contrast, quantitative data evaluation is facilitated when analyzing cuticle-specific metabolites or plants with similar cuticle permeabilities.