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Molecular structure and dynamics

Use of high-resolution NMR for solving the molecular structure of plant polymers comprising intact polymeric structures and their derivatives

The biopolyester cutin is ubiquitous in land plants, building the polymeric matrix of the plant's outermost defensive barrier, the cuticle. Cutin influences many biological processes in planta; however, due to its complexity and highly branched nature, the native structure remains partially unresolved. Our aim was to define an original workflow for the purification and systematic characterization of the molecular structure of cutin. To purify cutin we tested the ionic liquids cholinium hexanoate and 1-butyl-3-methyl-imidazolium acetate. The ensuing polymeric materials are highly esterified, amorphous, and have a typical monomeric composition as demonstrated by solid-state NMR, complemented by spectroscopic, thermal, and x-ray scattering analyses. We performed a systematic study by solution-state NMR of cryogenically milled cutins extracted from tomatoes

Wide-ranging NMR spectral characterization of Micro-Tom cutin isolated with cholinium hexanoate (2 h) and Micro-Tom untreated cuticle. The 1H NMR spectra of cutin (A) and cuticle (B) samples, indicate the relative abundance (percent) of linear aliphatic esters (LAE-α), total esters [α(C=O) esters], and free acids [α(C=O) acids]; HSQC regions correspond to cutin (C) and cuticle (D) aliphatics and CH (E) and CH2-X aliphatics (F). Some assignments (unlabeled) are uncertain or unidentified.


We resolved their molecular structures, relative distribution of ester aliphatics, free acid end-groups and free hydroxyl groups, differentiating between those derived from primary and secondary esters. Our data demonstrate the existence of free hydroxyl groups in cutin and provide insight into how the mutations affect the esterification arrangement of cutin. The usage of ionic liquids for studying plant polyesters has advantages over conventional approaches, since simple modifications can be applied to recover a biopolymer carrying distinct types/degrees of modifications (e.g. preservation of esters or cuticular polysaccharides), which in combination with the solution NMR methodologies developed here, constitutes essential tools to fingerprint the multifunctionality and the structure of cutin in planta.



Moreira CJS, Bento A, Pais J, Petit J, Escórcio R, Correia VG, Pinheiro A, Haliński ŁP, Mykhaylyk OO, Rothan C, Silva Pereira C (2020) An Ionic Liquid Extraction That Preserves the Molecular Structure of Cutin Shown by Nuclear Magnetic Resonance, Plant Physiology, 184: 592-606, doi: 10.1104/pp.20.01049