Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase      

Abstract

The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutindeficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

The glycerol-3-phosphate acyltransferase GPAT6 from tomato plays a central role in fruit cutin biosynthesis

“…In a previous study, we isolated from an EMSmutagenized tomato mutant population (cv Micro-Tom) a set of fruit brightness mutants (named glossy mutants) with abnormal fruit cutin amounts/compositions (Petit et al, 2014). In this study, we focused on one of these glossy mutants (line P23F12), which exhibits enhanced fruit brightness and a reduced cutin load.…”

“…Since the tomato fruit cuticle is so thick and easy to isolate, it has emerged as a model for studying cuticle formation and properties. In recent years, new insights into the regulation, synthesis, assembly, structure, and properties of the cuticle have been obtained using natural (Nadakuduti et al, 2012) or artificially induced genetic variability available in cultivated tomato (Adato et al, 2009;Isaacson et al, 2009;Shi et al, 2013;Petit et al, 2014) and in related wild species (Hovav et al, 2007;Yeats et al, 2012a). However, such studies also have raised many new questions, and many aspects of tomato fruit cuticle formation remain poorly characterized.…”

“…Using this strategy, we established that a point mutation in a GPAT gene (SlGPAT6) is responsible for a previously identified glossy mutation that affects fruit brightness (Petit et al, 2014). Assays using wild-type and mutant recombinant SlGPAT6 proteins produced in yeast (Saccharomyces cerevisiae), together with various chemically synthesized oxidized C16 fatty acyl-CoAs, demonstrated that GPAT activity is abolished in the DSlGPAT6 variant.…”

“…It is composed of cutin, a polyester of glycerol, hydroxy, and epoxy fatty acids, in addition to a range of waxes. In tomato (Solanum lycopersicum), as in many species, the main cutin monomers are C16-based fatty acids (C16 dihydroxy fatty acids, C16 v-hydroxy fatty acids, and C16 dicarboxylic acids; Mintz-Oron et al, 2008;Isaacson et al, 2009;Girard et al, 2012;Petit et al, 2014). Following their synthesis in the plastids, long-chain fatty acids are transported to the endoplasmic reticulum, where they undergo a series of modifications, including activation to CoA thioesters by long-chain acyl-CoA synthetases (LACS), oxidation by cytochrome P450-dependent fatty acid oxidases (CYP), and esterification to glycerol-based acceptor by glycerol-3-phosphate acyltransferase (GPAT) enzymes to produce acyl glycerols (Li-Beisson et al, 2013).…”

“…To better understand fruit cuticle formation, we recently isolated several cuticle mutants from an ethyl methanesulfonate (EMS)-mutagenized mutant collection in the miniature cv Micro-Tom genetic background and further identified by map-based cloning a mutation in a cutin synthase gene that is responsible for a glossy fruit phenotype (Petit et al, 2014). However, crossing the mutant line with a genetically distant genotype, as required for map-based cloning, introduces a large genetic diversity and, consequently, large phenotypic variability.…”