Cuticle function is closely related to the structure of the cutin polymer. However, the structure and formation of this hydrophobic polyester of glycerol and hydroxy/epoxy fatty acids has not been fully resolved. An apoplastic GDSL-lipase known as CUTIN SYNTHASE1 (CUS1) is required for cutin deposition in tomato (Solanum lycopersicum) fruit exocarp. In vitro, CUS1 catalyzes the self-transesterification of 2-monoacylglycerol of 9(10),16-dihydroxyhexadecanoic acid, the major tomato cutin monomer. This reaction releases glycerol and leads to the formation of oligomers with the secondary hydroxyl group remaining nonesterified. To check this mechanism in planta, a benzyl etherification of nonesterified hydroxyl groups of glycerol and hydroxy fatty acids was performed within cutin. Remarkably, in addition to a significant decrease in cutin deposition, mid-chain hydroxyl esterification of the dihydroxyhexadecanoic acid was affected in tomato RNA interference and ethyl methanesulfonate-cus1 mutants. Furthermore, in these mutants, the esterification of both sn-1,3 and sn-2 positions of glycerol was impacted, and their cutin contained a higher molar glycerol-to-dihydroxyhexadecanoic acid ratio. Therefore, in planta, CUS1 can catalyze the esterification of both primary and secondary alcohol groups of cutin monomers, and another enzymatic or nonenzymatic mechanism of polymerization may coexist with CUS1-catalyzed polymerization. This mechanism is poorly efficient with secondary alcohol groups and produces polyesters with lower molecular size. Confocal Raman imaging of benzyl etherified cutins showed that the polymerization is heterogenous at the fruit surface. Finally, by comparing tomato mutants either affected or not in cutin polymerization, we concluded that the level of cutin cross-linking had no significant impact on water permeance.Cuticles are ubiquitous hydrophobic barriers at the surfaces of aerial plant organs. These complex hydrophobic assemblies consist of a biopolymer, cutin, coated and filled with waxes and can also comprise embedded cell wall polysaccharides. Waxes comprise solventsoluble aliphatic molecules with long hydrocarbon chains, terpenes, and steroids (Kunst and Samuels,

Summary The cuticle is an essential and ubiquitous biological polymer composite covering aerial plant organs, whose structural component is the cutin polyester entangled with cell wall polysaccharides. The nature of the cutin‐embedded polysaccharides (CEPs) and their association with cutin polyester are still unresolved Using tomato fruit as a model, chemical and enzymatic pretreatments combined with biochemical and biophysical methods were developed to compare the fine structure of CEPs with that of the noncutinized polysaccharides (NCPs). In addition, we used tomato fruits from cutin‐deficient transgenic lines cus1 (cutin synthase 1) to study the impact of cutin polymerization on the fine structure of CEPs. Cutin‐embedded polysaccharides exhibit specific structural features including a high degree of esterification (i.e. methylation and acetylation), a low ramification of rhamnogalacturonan (RGI), and a high crystallinity of cellulose. In addition to decreasing cutin deposition and polymerization, cus1 silencing induced a specific modification of CEPs, especially on pectin content, while NCPs were not affected. This new evidence of the structural specificities of CEPs and of the cross‐talk between cutin polymerization and polysaccharides provides new hypotheses concerning the formation of these complex lipopolysaccharide edifices.

With the aim of mimicking the plant cell wall, a layer by layer approach was used to build a thin film consisting of successive adsorption of pectin and extensin. Elaboration of the thin film was monitored by surface plasmon resonance, quartz crystal microbalance, and ellipsometry. All data indicate that formation of the film was successful and that growth occurred according to a nonuniform growth. It is likely that diffusion of the polymers occurred within the multilayer structure and that the final structure is not constituted by layered individual pectin and extensin films. Polymer rearrangements were also supported by the atomic force microscopy images that show a smoother surface after extensin adsorption than after pectin deposition.

The cuticle is a complex polymer matrix that protects all aerial organs of plants, fulfills multiple roles in plant-environment interactions, and is critical for plant development. These functions are associated with the structural features of cuticles, and the architectural modeling of cuticles during plant development is crucial for understanding their physical properties and biological functions. In this work, the in-depth architecture of the cutin polymer matrix during fruit development was investigated. Using cherry tomato fruit (Solanum lycopersicum) as a model from the beginning of the cell expansion phase to the red ripe stage, we designed an experimental scheme combining sample pre-treatment, Raman mapping, multivariate data analyses and biochemical analyses. These approaches revealed clear chemical areas with different contributions of cutin, polysaccharides and phenolics within the cutin polymer matrix. Besides, we demonstrated that these areas are finely tuned during fruit development, including compositional and macromolecular rearrangements. The specific spatio-temporal accumulation of phenolic compounds (p-coumaric acid and flavonoids) suggests that they fulfill distinct functions during fruit development. In addition, we highlighted an unexpected dynamic remodeling of the cutin-embedded polysaccharides pectin, cellulose and hemicellulose. Such structural tuning enables consistent adaption of the cutin-polysaccharide continuum and the functional performance of the fruit cuticle at the different developmental stages. This study provides insights into the plant cuticle architecture and in particular into the organization of the epidermal cell wall-cuticle.