Deconstructing a Plant Macromolecular Assembly: Chemical Architecture, Molecular Flexibility, And Mechanical Performance of Natural and Engineered Potato Suberins

Abstract

Periderms present in plant barks are essential protective barriers to water diffusion, mechanical breakdown, and pathogenic invasion. They consist of densely packed layers of dead cells with cell walls that are embedded with suberin. Understanding the interplay of molecular structure, dynamics, and biomechanics in these cell wall-associated insoluble amorphous polymeric assemblies presents substantial investigative challenges. We report solid-state NMR coordinated with FT-IR and tensile strength measurements for periderms from native and wound-healing potatoes and from potatoes with genetically modified suberins. The analyses include the intact suberin aromatic–aliphatic polymer and cell-wall polysaccharides, previously reported soluble depolymerized transmethylation products, and undegraded residues including suberan. Wound-healing suberized potato cell walls, which are 2 orders of magnitude more permeable to water than native periderms, display a strikingly enhanced hydrophilic–hydrophobic balance, a degradation-resistant aromatic domain, and flexibility suggestive of an altered supramolecular organization in the periderm. Suppression of ferulate ester formation in suberin and associated wax remodels the periderm with more flexible aliphatic chains and abundant aromatic constituents that can resist transesterification, attenuates cooperative hydroxyfatty acid motions, and produces a mechanically compromised and highly water-permeable periderm.

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

“…High power heteronuclear 1 H decoupling (175-185 kHz) was achieved using the small phase incremental alternation pulse sequence (33), and acquisition was carried out with a 3-s recycle delay. Typical experimental parameters (16,20,24) on the Varian spectrometer included 46 kHz sweep width, ϳ25-ms acquisition time, and 128 -1024 transients for 13 C-enriched pigments. 13 C multiple CP experiments (34) were validated against traditional direct polarization measurements and used to obtain high throughput quantification of the pigment composition.…”

“…Furthermore, we can selectively isolate for investigation those cellular constituents that are closely associated with the pigment and thereby protected from both environmental effects and chemical degradation. Finally, high resolution solid-state nuclear magnetic resonance (NMR) approaches can yield direct insights into the atomic level structure, dynamics, and action mechanisms of noncrystalline bioassemblies, including plant and microbial complexes that have polysaccharide or lipid constituents (11)(12)(13)(14)(15)(16). Thus, it is feasible to circumvent the difficulties of solubilization or crystallization to access macromolecular structure for pigments derived from known small molecule isotopically enriched precursors by using the C. neoformans fungal melanin and solid-state NMR.…”

Partial depolymerization of genetically modified potato tuber periderm reveals intermolecular linkages in suberin polyester

“…The intact suberin was studied by solid-state (CPMAS) NMR, in potato periderm, either natural or wound-induced (Bernards et al, 1995;Serra et al, 2014;Stark and Garbow, 1992), as well as in other suberized plant materials, like in Quercus suber cork (Gil et al, 1997;Neto et al, 1995). These NMR results showed that suberin included methylene chains with different dynamic behavior, the shorter with the highest mobility and the longer with the lowest, together with the spatial separation of the aliphatic and aromatic domains (Gil et al, 1997;Yan and Stark, 1998).…”

“…A further approach to the comprehension of the suberin macromolecule can be given by the identification and manipulation of genes and proteins presumably involved in the biosynthesis of its monomers and their ester assembling (Serra et al, 2014). Lists of candidate genes for suberin biosynthesis were drawn from the analysis of cork differentiating tissue (Soler et al, 2007) and Arabidopsis mutants (Franke et al, 2012).…”

“…Results of solid state NMR analyses combined with data from complete depolymerization of genetically engineered periderms allowed several observations. The analysis of StKCS6-RNAi potato periderms shows that a modest reduction in chain elongation capacity beyond C28 produces a periderm similar to wild type in suberin composition, in mechanical performance and in water permeability (Serra et al, 2014(Serra et al, , 2009a. On the other hand, CYP86A33-RNAi periderms showing a reduced capacity of terminal hydroxylation and crosslinking, have a greater fraction of mobile alkyl chains (Serra et al, 2014), lose the typical lamellar structure and have enhanced water permeability (Serra et al, 2009b).…”