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Agro-industrial residues comprise a rich diversity of plant polymers and bioactive compounds, constituting promising sources for the development of materials, including bioplastics, and food supplements, among other applications. In particular, the polyester cutin is abundant in fruit peel, a plentiful constituent of pomace agro-industrial residues. The potential of diverse fruit pomaces as a source for the development of cutin-derived materials/products has been extensively sought out. This study expands the established knowledge: it sets proof of concept for the production of antimicrobial oligomers from cutin-rich materials isolated in a single step from tomato pomaces generated by two remote agro-industries. Specifically, it first analyzed how the chemical signature (nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS)) of a pomace (and of its major constituents) mirrors that of the corresponding cutin-rich material isolated using an ionic liquid extractant. The cutin-rich materials were then deconstructed (using mild hydrolyses), and the resultant mixtures were chemically characterized and screened for bactericidal activity against

Polyesters, as they exist in planta, are promising materials with which to begin the development of “green” replacements. Cutin and suberin, polyesters found ubiquitously in plants, are prime candidates. Samples enriched for plant polyesters, and in which their native backbones were largely preserved, were studied to identify “natural” structural features; features that influence critical physical properties. Quantitative nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and X-ray scattering methods were used to quantify structure–property relationships in these polymeric materials. The degree of esterification, namely, the presence of acylglycerol linkages in suberin and of secondary esters in cutin, and the existence of mid-chain epoxide groups defining the packing of the aliphatic chains were observed. This packing determines polymer crystallinity, the resulting crystal structure, and the melting temperature. To evaluate the strength of this rule, tomato cutin from the same genotype, studying wild-type plants and two well-characterized mutants, was analyzed. The results show that cutin’s material properties are influenced by the amount of unbound aliphatic hydroxyl groups and by the length of the aliphatic chain. Collectively, the acquired data can be used as a tool to guide the selection of plant polyesters with precise structural features, and hence physicochemical properties., A detailed insight into plant polyesters identifies correlations of structural features with physical properties that are translated into five essential rules for the development of novel green materials.

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 (Solanum lycopersicum 'Micro-Tom'; the wild type and the GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE [GPAT6] and CUTIN SYNTHASE [CUS1] mutants). 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.

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 processesin plantahowever 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 characterisation 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 polymers are highly esterified, amorphous and have the typical monomeric composition as demonstrated by solid state NMR, complemented by spectroscopic (GC-MS), thermal (DSC) and x-ray scattering (WAXS) analyses. A systematic study by solution-state NMR of cryogenically milled cutins extracted from Micro-Tom tomatoes (the wild type and thegpat6andcus1mutants) was undertaken. 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, were solved. The acquired data demonstrate the existence of free hydroxyl groups in cutin and reveal novel insights on how the mutations impact the esterification arrangement of cutin. Compared to conventional approaches, the usage of ionic liquids for the study of plant polyesters opens new avenues 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 now essential tools to fingerprint the multi-functionality and the structure of cutinin planta.

Understanding stress responses and signaling pathways in fungi became a fundamental need for the discovery of new specific antifungal targets for fighting emerging life-threatening pathogens and drug resistance. Ionic liquids constitute a unique class of chemicals, which structural diversity and tunable physical and chemical properties can provide a great diversity of stimuli. In this study, we propose the use of ionic liquids as tools to unravel signaling of stress responses in the filamentous fungus Aspergillus nidulans. We assessed how three ionic liquids with distinct effects over the cell wall and plasma membrane affect the biosynthesis of sphingolipids and accumulation of free sphingoid bases in this fungus. The stress imposed by each ionic liquid triggered the sphingolipid biosynthetic pathway and led to distinct profiles of sphingoid bases accumulation. Dodecyltributylphosphonium chloride and 1-decyl-3-methylimidazolium chloride induced the accumulation of sphingosine and of a yet unknown sphingoid base, respectively, while cholinium decanoate did not seem to accumulate any of these intermediates. This study brings further light to the roles of sphingoid bases in A. nidulans. In particular, sphingosine as a possible response mediator to cell wall damage induced by dodecyltributylphosphonium chloride, and involvement of an unknown sphingoid base in the response to plasma membrane permeabilization caused by 1-decyl-3-methylimidazolium chloride. In addition, we completed the genetic assignment of the glucosylceramide pathway in A. nidulans through the identification of the sphingolipid Δ4-desaturase gene (AN4405). The knowledge established reinforces the idea of targeting sphingolipids biosynthesis in the search of improved antifungal compounds.