Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization

Abstract

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.

Figure 1.

Characterization of tomato cutin in cus1 mutants and Pro-35S:SlCUS1^RNAi lines. A, Immunoblot analysis of the SlCUS1 protein in 20-DPA fruits. B, Nonnormalized areas of methylene (CH₂; 2,978–2,838 cm⁻¹) and carbonyl (CO; 1,750–1,690 cm⁻¹) bands, and esterification index, the ratio of CH₂ to CO areas in red ripe fruits. Left, cus1 mutants; right, Pro-35S:SlCUS1^RNAi lines. WT, Wild type. Mean values ± sd were calculated from 32 independent measurements (four biological samples and eight technical replicates each). Significant differences from the wild type are indicated by asterisks (Student’s t test; **, P < 0.01).

Figure 2.

Esterification levels of glycerol OH groups of tomato fruit cutin isolated from the cus1-a mutant and Pro-35S:SlCUS1^RNAi lines. A and C, Fruits at 20 DPA. B and D, Red ripe fruits. Left, cus1-a; right, Pro-35S:SlCUS1^RNAi lines. WT, Wild type. Mean values ± sd were calculated from nine independent measurements (three fruits and three technical replicates each). Significant differences from the corresponding wild type are indicated by asterisks (Student’s t test; *, P < 0.05 and **, P < 0.01).

Figure 3.

Esterification levels of 9(10),16-dihydroxyhexadecanoic acid [9(10),16-diOHC16 acid] OH groups of tomato fruit cutin from the cus1-a mutant and Pro-35S:SlCUS1^RNAi lines. A and C, Fruits at 20 DPA. B and D, Red ripe fruits. Left, cus1-a; right, Pro-35S:SlCUS1^RNAi lines. WT, Wild type. Mean values ± sd were calculated from nine independent measurements (three fruits and three technical replicates each). Significant differences from the corresponding wild type are indicated by asterisks (Student’s t test; *, P < 0.05; **, P < 0.01; and ***, P < 0.001).

Figure 4.

Raman mapping of nonesterified OH groups within cutin polyester from tomato. Cutin from wild-type (WT; A and B) and Pro-35S:SlCUS1^RNAi line L17 (D and D) red ripe fruit were benzyl etherified and analyzed by Raman microspectroscopy. Each image was obtained by mapping the characteristic band of aromatic groups at 1,001 cm⁻¹. Images B and D correspond to higher magnification views (dashed squares) of the Raman images (A and C, respectively).

Figure 5.

The cutin-deficient mutant cud1 is not affected in the polymerization of 9(10),16-dihydroxyhexadecanoic acid. A, Quantification of major cutin monomers. 16-OHC16 acid, 16-Hydroxyhexadecanoic acid; 9(10),16-diOHC16 acid, 9(10),16-dihydroxyhexadecanoic acid; 9-OH-1,16-C16 diacid, 9-hydroxy-1,16-hexadecanedioic acid. B, Esterification level of 9(10),16-dihydroxyhexadecanoic acid OH groups from cud1 cutin compared with wild-type (WT) and cus1-a cutins. Data are expressed as mean values, and error bars represent sd (n = 3). Significant differences from the wild type are indicated by asterisks (Student’s t test; *, P < 0.05 and **, P < 0.01).

Figure 6.

Comparison of the water permeance of tomato cutin in red ripe fruits from wild-type and tomato EMS mutant plants. Data are shown as median values ± minimum/maximum (n = 8–10). Differences between the wild type (WT), cus1 mutants (cus1-a, cus1-b, and cus1-c), and cud1 were tested with the Mann-Whitney U test. Asterisks indicate significant differences from the wild type (*, P < 0.05).