A comprehensive approach to phytochemical analysis of macromolecular composites that protect tubers: case studies in suberized potato periderm tissues

Terrestrial plants rely on protection conferred by their outer coverings to defend against desiccation, bruising, and microbial invasion. For food staples such as potato tubers, the periderm contains the phellem (tuber skin) which creates a hydrophobic barrier by depositing macromolecular composites comprised of waxes, soluble phenolics, and a complex aliphatic suberin polyester (or suberin aliphatic domain) and lignin-like biopolymer (or suberin polyphenolic domain) within the previously formed polysaccharide cell wall. The antibacterial activity of both the soluble chemical constituents and their solid polymeric assemblies provides essential plant defense; their antioxidant and waterproofing properties also offer practical potential for sustainable food preservation and packaging applications. To characterize these phytochemical composites comprehensively and in molecular detail, we developed an approach that coordinates ‘bottom-up’ analysis of extracted metabolites that include suberin precursors, solid-state NMR spectroscopy of the polymers in intact skins or solid suspensions, and ‘top-down’ analysis of chemical breakdown products of suberin. The usefulness of analytical methods that include LC–MS, GC–MS, multivariate analysis, solid-state NMR, SEM, and TEM is illustrated for studies of molecular and supramolecular structures that underlie protective function in three potato periderm systems: (1) native tuber periderms versus suberized wound-healing tissues including closing layer and wound periderm; (2) metabolites unleashed in rapid response to wounding prior to formation of suberized tissues; (3) wild type versus genetically modified potato varieties with altered suberin deposition. We also demonstrate how enrichment with stable ¹³C and ¹⁵N isotopes can improve our understanding of how the suberin biopolymer molecular structure develops, increasing the reach of MS, 2D solid-state NMR, and dynamic nuclear polarization spectroscopic methods and revealing phenolic amide constituents that could represent an underappreciated part of the plant’s defensive arsenal.