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Land plants produce a cuticle, an extracellular hydrophobic layer that covers aerial organs and is involved in many critical protective roles, most notably in preventing desiccation. The predominant component of the cuticle is the lipidic polyester, cutin, which is deposited in the epidermal primary cell wall. Most of cutin of tomato fruit, a model for cuticle research, is polymerized by the extracellular GDSL-hydrolase enzyme CUTIN SYNTHASE-LIKE 1 (CUS1). However, other enzymes involved in cutin assembly remain to be identified and characterized. In this current study, we investigated whether other GDSL-hydrolases that are highly expressed in fruit epidermis might also contribute to cutin polymerization and restructuring. Candidates include homologs of Arabidopsis thaliana CUTICLE DESTRUCTIVE FACTOR 1 (CDEF1), which has been reported to catalyze cutin hydrolysis, as well as other phylogenetically diverse and distantly related GDSL-hydrolases. We determined that members of the CUS and CDEF families can catalyze the transesterification of cutin precursors in vitro, and can modify tomato fruit cutin structure in semi-in vivo assays. Tomato mutant knockout lines of CUS and CDEF genes generated by CRISPR/ Cas9 and cross mutations with cus1 (previously cd1) were found to exhibit different fruit and flower phenotypes related to cutin assembly, including an effect on cutin monomer esterification, composition and content, cutin nanoridge formation in flowers, fruit cuticle permeability and permeance. Characterization of the mutant phenotypes, in combination with the enzyme analysis and bioassays, revealed distinct differences in the contribution of CUS and CDEF enzymes to cutin biosynthesis and remodeling. Our analysis also revealed unexpected spatiotemporal variation in cutin polymerization and structure coordinated by distinct GDSL-hydrolase enzymes over the fruit surface, which further suggests great complexity in cutin deposition and cuticle functions during organ development.
Competing Interest Statement
The authors have declared no competing interest.