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 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 the gpat6 and cus1 mutants) 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 support 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 cutin in planta.

中文翻译：

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基于离子液体的新型萃取方法，可保留角质素的分子结构

生物聚酯角质素在陆地植物中无处不在，建立了植物最外层防御屏障-角质层的聚合物基质。角质影响植物中的许多生物过程，但是由于其复杂性和高度分支的性质，其天然结构仍然部分未被解析。我们的目的是为角质素分子结构的纯化和系统表征定义一个原始的工作流程。为了纯化角质，我们测试了离子液体己酸胆碱和乙酸1-丁基-3-甲基咪唑鎓盐。随后的聚合物是高度酯化的，无定形的，具有典型的单体组成，如固态NMR所示，并辅以光谱（GC-MS），热（DSC）和X射线散射（WAXS）分析。通过溶液状态NMR对从Micro-Tom番茄（野生型以及gpat6和cus1突变体）提取的低温研磨的角质进行了系统研究。解决了它们的分子结构，脂族酯的相对分布，游离酸端基和游离羟基的区别，区别于伯酯和仲酯。获得的数据支持角质中游离羟基的存在，并揭示了有关突变如何影响角质酯化安排的新颖见解。与传统方法相比，将离子液体用于植物聚酯的研究开辟了新的途径，因为可以进行简单的修饰来回收带有不同类型/程度修饰的生物聚合物（例如保存酯或表皮多糖），