SUMMARYPlant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato (Solanum lycopersicum) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 (cd1), cd2 and cd3, the fruit cuticles of which have a dramatic (95-98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.

A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol (2-MHG). CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo, indicating that it is a cutin synthase.

Tomato (Solanum lycopersicum) is the primary model for the study of fleshy fruits, and research in this species has elucidated many aspects of fruit physiology, development, and metabolism. However, most of these studies have involved homogenization of the fruit pericarp, with its many constituent cell types. Here, we describe the coupling of pyrosequencing technology with laser capture microdissection to characterize the transcriptomes of the five principal tissues of the pericarp from tomato fruits (outer and inner epidermal layers, collenchyma, parenchyma, and vascular tissues) at their maximal growth phase. A total of 20,976 high-quality expressed unigenes were identified, of which more than half were ubiquitous in their expression, while others were cell type specific or showed distinct expression patterns in specific tissues. The data provide new insights into the spatial distribution of many classes of regulatory and structural genes, including those involved in energy metabolism, source-sink relationships, secondary metabolite production, cell wall biology, and cuticle biogenesis. Finally, patterns of similar gene expression between tissues led to the characterization of a cuticle on the inner surface of the pericarp, demonstrating the utility of this approach as a platform for biological discovery.

SUMMARYFull appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato (Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.

Summary The cuticle covers the aerial epidermis of land plants and plays a primary role in water regulation and protection from external stresses. Remarkable species diversity in the structure and composition of its components, cutin and wax, have been catalogued, but few functional or genetic correlations have emerged. Tomato (Solanum lycopersicum) is part of a complex of closely related wild species endemic to the northern Andes and the Galapagos Islands (Solanum Sect. Lycopersicon). Although sharing an ancestor less than seven million years ago, these species are found in diverse environments and are subject to unique selective pressures. Furthermore, they are genetically tractable, since they can be crossed with S. lycopersicum, which has a sequenced genome. With the aim of evaluating the relationships between evolution, structure and function of the cuticle, we characterized the morphological and chemical diversity of fruit cuticles of seven species from Solanum Sect. Lycopersicon. Striking differences in cuticular architecture and quantities of cutin and waxes were observed, with wild species wax coverage exceeding that of S. lycopersicum by up to seven fold. Wax composition varied in the occurrence of wax esters and triterpenoid isomers. Using a S. habrochaites introgression line population, we mapped triterpenoid differences to a genomic region that includes two S. lycopersicum triterpene synthases. Based on known metabolic pathways for acyl wax compounds, hypotheses are discussed to explain the appearance of wax esters with atypical chain lengths. These results establish a model system for understanding the ecological and evolutionary functional genomics of plant cuticles.