TY - JOUR
AU - Aharoni, Asaph
AB - Abstract The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning. The epidermal layer of plants is a multifunctional and diverse tissue. Epidermal cells interact directly with the surrounding environment and as such, are involved in a number of processes, including osmotic regulation, defense, and pollinator attraction (Glover, 2000; Martin and Glover, 2007; Whitney et al., 2009). To optimally perform these functions, cells of the epidermis are differentiated into a variety of cell types. For the aboveground parts of plants, this typically includes pavement cells, trichomes, and stomata guard cells. Each of these cell types may be further differentiated, resulting in a range of more subtle variations and morphological shapes. An excellent example of this subtle variation can be seen in the conical cell shape of petal epidermal cells (Whitney et al., 2009, 2011). The widespread occurrence of these conical petal epidermal cells in nature has been associated with petal color, petal reflection, scent production, petal wettability, and pollinator grip on the flower surface (Neinhuis and Barthlott, 1997; Riederer and Muller, 2006; Whitney et al., 2009). One of the major evolutionary developments of land plants was the formation of the cuticular membrane (or cuticle) found to the exterior of the source of its building blocks, the epidermis layer. The cuticle provides a waterproof barrier between epidermal cells and the comparatively dry external environment (Riederer and Muller, 2006). It also plays an important role in the protection of plants against biotic and abiotic stresses, such as pathogen attack and damaging UV light (Bargel et al., 2006). Recent research has highlighted the cuticle as an important element in the plant’s detection and transmission of osmotic stress signals (Wang et al., 2011). The cuticle is comprised of a polymerized cutin matrix embedded with waxes (Kolattukudy, 2001). In many fleshy fruit, including tomato (Solanum lycopersicum), the major monomer of the cutin matrix is the C16-9/10, 16-dihydroxy fatty acid (DiHFA; Mintz-Oron et al., 2008). A number of the enzymatic steps for the biosynthesis, extracellular transport, and polymerization of the cutin matrix have been described in Arabidopsis (Arabidopsis thaliana) and to some extent, tomato. In short, free fatty acids are synthesized in the chloroplast, transported to the endoplasmic reticulum, and subsequently modified. Modification includes mid- and terminal-chain hydroxylation and CoA activation followed by transfer to a glycerol moiety. Enzymes responsible for these steps include long-chain acyl-CoA synthetases (LACSs; Schnurr et al., 2004; Jessen et al., 2011), oxidases (CYTOCHROME P450 86A [CYP86A] and CYP77A; Li-Beisson et al., 2009; Pinot and Beisson, 2011; Shi et al., 2013), and acyltransferases (GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASEs [GPATs] and likely, DEFECTIVE IN CUTICULAR RIDGES [DCR]; Panikashvili et al., 2009; Rani et al., 2010; Yang et al., 2010, 2012). The resultant monoacylglycerols undergo extracellular transport and finally, polymerization to form the cutin matrix. Enzymes responsible for these steps include ATP-binding cassette transporters (Bird et al., 2007; Bessire et al., 2011; Panikashvili et al., 2011) and cutin synthases (GDSL-motif lipase/hydrolase [GDSL]/CUTIN SYNTHASE and BODYGUARD [BDG]; Kurdyukov et al., 2006a; Girard et al., 2012; Yeats et al., 2012, 2014). Interestingly, the characterization of mutants and transgenic plants altered in cuticle assembly revealed a major effect on the patterning of epidermis cells. Arabidopsis mutants for cyp77a6 and gpat6 both exhibit a lack of cuticular nanoridges as well as petals with flatter epidermal cells (Li-Beisson et al., 2009). The lack of cuticular nanoridges was also observed in dcr Arabidopsis mutants (Panikashvili et al., 2009), whereas Arabidopsis mutants for genes possibly responsible for cutin synthesis, bdg and gdsl, show changes to epidermal cell shape (Kurdyukov et al., 2006a, 2006b). Study of transcriptional regulators further revealed a tight relationship between the cuticle and epidermal cell. It seems that some of the reported regulators of epidermal cell differentiation and cuticle biosynthesis are closely related proteins belonging to the same gene families (Hen-Avivi et al., 2014). Of particular interest in this regard are transcription factors from the SHINE1 (SHN1)/WAX INDUCER1 (WIN1) clade of the APETELA2 (AP2) domain superfamily, and the class IV homeodomain-leucine zipper (HD-ZIP IV) and R2-R3 MYB DNA-binding domain (MYB) factor families. Members of the HD-ZIP IV family were shown to regulate cuticle metabolism in tomato and maize (Zea mays), including CUTIN DEFICIENT2 (CD2; Isaacson et al., 2009; Nadakuduti et al., 2012) and OUTER CELL LAYER1 (Javelle et al., 2010; Depège-Fargeix et al., 2011), respectively. Other HD-ZIP IV proteins, including the Arabidopsis GLABRA2 (AtGL2; Rerie et al., 1994; Ishida et al., 2007; Tominaga-Wada et al., 2009, 2013), PROTODERMAL FACTOR2 (AtPDF2; Abe et al., 2003), Arabidopsis HOMEODOMAIN GLABROUS11 (Nakamura et al., 2006), MERISTEM LAYER1 (AtML1; Abe et al., 2003; Takada et al., 2013), and cotton (Gossypium hirsutum) HOMEODOMAIN1 (Walford et al., 2012), were shown to be involved in the regulation of epidermal cell differentiation. Two members of the SHN1/WIN1 clade of transcription factors from Arabidopsis and tomato (AtSHN1 and SlSHN3, respectively) have been shown to be required for both cutin biosynthesis as well as epidermal cell patterning (Aharoni et al., 2004; Broun et al., 2004; Shi et al., 2011, 2013). Finally, members of the R2R3 MYB transcription factor superfamily, AtMYB41 and AtMYB96, have also been shown to regulate wax deposition during abiotic stress (Cominelli et al., 2008; Seo et al., 2011), although more recent results suggest that AtMYB41 likely promotes suberin biosynthesis and deposition (Kosma et al., 2014). Members of the MYB protein family termed MIXTA or MIXTA-like were shown to be key regulators of epidermal cell differentiation across multiple plants species (Martin and Glover, 2007; Brockington et al., 2013). Orthologs of the MIXTA/MIXTA-like clade have been reported specifically to act as positive regulators of conical epidermal cell formation (Noda et al., 1994; Baumann et al., 2007; Di Stilio et al., 2009), trichome development (Perez-Rodriguez et al., 2005; Gilding and Marks, 2010; Plett et al., 2010), and cotton fiber development (Machado et al., 2009; Walford et al., 2011). Recently, Arabidopsis MIXTA-like orthologs, AtMYB16 and AtMYB106, were shown to regulate cuticle development (Oshima et al., 2013). To date, however, MIXTA/MIXTA-like clade genes were not characterized with relation to fleshy fruit surface. In a recent work characterizing SlSHN3 activity in tomato, it was suggested that this transcription factor may exert its influence on epidermal cell patterning through HD-ZIP IV and/or MIXTA transcription factors (Shi et al., 2013). Although expression of a tomato MIXTA-like gene (i.e. SlMIXTA-like) was shown earlier to be enriched in the epidermal tissue throughout tomato fruit development (Mintz-Oron et al., 2008), its functional role in tomato fruit surface was not examined. In this work, we functionally characterize the tomato SlMIXTA-like gene through the detailed investigation of lines with transgenically silenced SlMIXTA-like expression. Our findings show that SlMIXTA-like not only promotes conical epidermal cell development in tomato fruit but is a major positive regulator of cuticular lipids, more specifically cutin monomer biosynthesis as well as cuticle assembly. This is apparent from the wide range of down-regulated cutin biosynthetic genes in tomato lines silenced for SlMIXTA-like expression as well as the notable correlation between SlMIXTA-like expression and the deposition of cuticle. Thus, this study further advances the understanding of coregulation between epidermal cell patterning and cuticle biosynthesis during organ formation. RESULTS The Tomato Genome Contains a Single Expressed Member of the MIXTA/MIXTA-Like Gene Family That Is Predominantly Expressed in the Fruit Epidermal Layer Analysis of the tomato genome through BLAST searches using well-characterized MIXTA and MIXTA-like proteins from snapdragon (Antirrhinum majus) and Petunia hybrida (AmMIXTA [Noda et al., 1994] and PhMYB1 [Baumann et al., 2007], respectively) as input identified a number of potential tomato orthologs of this clade. Molecular phylogenetic analysis of the best hits revealed that the tomato genome retains seven potential orthologs to the clade containing MIXTA and MIXTA-like proteins (R2R3-MYB group 9; Supplemental Fig. S1; Stracke et al., 2001; Brockington et al., 2013). This group of MYB factors also includes MYB17 proteins, with which two of the tomato candidates (Solyc01g094360 and Solyc05g048830) group. These candidates can therefore be ruled out as potential MIXTA/MIXTA-like orthologs (Supplemental Fig. S1). Of the remaining five candidates, four group separately from both the MYB17 and the MIXTA/MIXTA-like branch (Solyc01g010910, Solyc04g005600, Solyc05g007690, and Solyc05g007710) and may be considered a unique group (Supplemental Fig. S1). These four genes show no expression in previously generated large-scale multiorgan expression data (Itkin et al., 2013) and thus, were not investigated further. The final identified candidate (Solyc02g088190) groups in the MIXTA-like branch of the phylogenetic tree (Supplemental Fig. S1). This protein, termed SlMIXTA-like, shares 75% and 80% amino acid identity with the well-characterized MIXTA-like epidermal cell differentiation factors snapdragon MYB MIXTA-like2 and PhMYB1, respectively (Baumann et al., 2007). A molecular phylogenetic tree for all previously characterized MIXTA and MIXTA-like proteins showed that SlMIXTA-like groups with MIXTA-like orthologs (Fig. 1A). Previous results have shown that SlMIXTA-like has enriched transcript levels in the surface layers of fruit (Mintz-Oron et al., 2008; Shi et al., 2013). Real-time quantitative reverse transcription (qRT)-PCR analysis of SlMIXTA-like expression in five stages of tomato fruit development in skin (epidermal enriched) and flesh tissues revealed that, although expression levels were highest in the skin at the immature green stage of fruit development, expression remained high in this tissue throughout development (Mintz-Oron et al., 2008). SlMIXTA-like transcripts in the skin were found to be at least 16-fold higher than in flesh at all measured stages of development. Examination of publically available large-scale gene expression data for various fruit cell types (Matas et al., 2011) revealed that SlMIXTA-like expression is predominantly detected in the outer and inner epidermis of the fruit, albeit to a lesser extent in the latter (Fig. 1B). The inner epidermal layer of tomato fruit separates the locular region from the pericarp. A very low level of SlMIXTA-like expression was detected in the collenchyma tissue, and none was detected in the vasculature and parenchyma. Additionally, the expression profile of SlMIXTA-like across 20 tomato tissues (Itkin et al., 2013) showed that, although fruit transcripts were enriched in the skin (at all five stages of fruit development examined), significant expression could be detected in petals, buds, and pollen (Fig. 1C). Figure 1. Open in new tabDownload slide The tomato MIXTA-like gene is predominantly expressed in fruit epidermal layers. A, Molecular phylogenetic analysis of the functionally characterized members of the MIXTA/MIXTA-like protein clade are shown together with the assigned functional roles. ClustalW and the MEGA6 Software were used to align the proteins and compute the neighbor-joining tree with significance percentages (bootstrap values out of 1,000). Bar represents the relative amino acid difference. Alignments can be viewed in Supplemental Figure S3. B, An epidermal-specific expression pattern is observed for the SlMIXTA-like gene. Normalized expression of SlMIXTA-like in immature green fruit tissues showed outer and inner epidermal expression (data were extracted from previously published results; Matas et al., 2011). collen, Collenchyma; inepi, inner epidermis; outepi, outer epidermis; paren, parenchyma; RPKM; vascu, vascular tissue. C, Normalized expression of SlMIXTA-like across 20 tomato tissues highlights skin-enriched expression in developing tomato fruit as well as significant expression in the petals and pollen (data extracted from previously published work; Itkin et al., 2013). Br, Breaker; IG, immature green; MG, mature green; Or, orange; R, red; RPKM, reads per kilobase per million; Y, young. Figure 1. Open in new tabDownload slide The tomato MIXTA-like gene is predominantly expressed in fruit epidermal layers. A, Molecular phylogenetic analysis of the functionally characterized members of the MIXTA/MIXTA-like protein clade are shown together with the assigned functional roles. ClustalW and the MEGA6 Software were used to align the proteins and compute the neighbor-joining tree with significance percentages (bootstrap values out of 1,000). Bar represents the relative amino acid difference. Alignments can be viewed in Supplemental Figure S3. B, An epidermal-specific expression pattern is observed for the SlMIXTA-like gene. Normalized expression of SlMIXTA-like in immature green fruit tissues showed outer and inner epidermal expression (data were extracted from previously published results; Matas et al., 2011). collen, Collenchyma; inepi, inner epidermis; outepi, outer epidermis; paren, parenchyma; RPKM; vascu, vascular tissue. C, Normalized expression of SlMIXTA-like across 20 tomato tissues highlights skin-enriched expression in developing tomato fruit as well as significant expression in the petals and pollen (data extracted from previously published work; Itkin et al., 2013). Br, Breaker; IG, immature green; MG, mature green; Or, orange; R, red; RPKM, reads per kilobase per million; Y, young. Silencing of SlMIXTA-Like Results in the Flattening of Epidermal Cells and a Thinner Cuticle in Tomato Fruit To investigate the function of SlMIXTA-like in tomato fruit surface development, silenced (SlMIXTA-RNA interference [RNAi]) lines were generated (Fig. 2). SlMIXTA-RNAi tomato lines exhibited a variety of phenotypes at the vegetative stage, including retarded growth and smaller, crinkled, and curled leaves (Fig. 2, B and C). Fruit of the SlMIXTA-RNAi plants appeared glossier than wild-type fruits and were subsequently subject for detailed investigation. Figure 2. Open in new tabDownload slide Growth and developmental phenotypes of tomato plants with altered SlMIXTA-like expression. A, Confirmation of SlMIXTA-like gene silencing confirmed by real-time qRT-PCR analysis of transgenic tomato ‘MT’ plants. Data show means and se for three individually transformed lines (n = 3). **, P < 0.01. B, Silencing of the SlMIXTA-like gene resulted in smaller, crinkled, and curled leaves. C, Altered expression of the SlMIXTA-like gene in tomato results in changes to plant architecture. Lines silenced for SlMIXTA-like gene expression were smaller and displayed stunted growth. WT, Wild type. Figure 2. Open in new tabDownload slide Growth and developmental phenotypes of tomato plants with altered SlMIXTA-like expression. A, Confirmation of SlMIXTA-like gene silencing confirmed by real-time qRT-PCR analysis of transgenic tomato ‘MT’ plants. Data show means and se for three individually transformed lines (n = 3). **, P < 0.01. B, Silencing of the SlMIXTA-like gene resulted in smaller, crinkled, and curled leaves. C, Altered expression of the SlMIXTA-like gene in tomato results in changes to plant architecture. Lines silenced for SlMIXTA-like gene expression were smaller and displayed stunted growth. WT, Wild type. A variety of microscopy techniques was used to investigate the changes in the surface morphology of the tomato lines with reduced SlMIXTA-like expression. Initial observations by light microscopy and lipid staining (using Sudan IV stain) revealed significantly less cuticle deposition in the fruit of SlMIXTA-RNAi lines throughout development and what appeared to be flatter epidermal cells (Fig. 3). Although in wild-type tomato, the cuticle frequently surrounds the entire epidermal cell (Mintz-Oron et al., 2008; Matas et al., 2011), SlMIXTA-RNAi fruit displayed almost no subepidermal staining (Fig. 3, A and B). Cryo-scanning electron microscopy (SEM) and atomic force microscopy (AFM) confirmed the change in cell shape and a concomitant change in the surface morphology of the tomato fruit (Fig. 3, C and D). Analysis by AFM revealed a disrupted and rough microsurface (Fig. 3D; Supplemental Fig. S2). Transmission electron microscopy (TEM) illustrated the typical conical shape of tomato fruit epidermal cells in the wild type and confirmed that lines silenced for SlMIXTA-like expression possessed flatter cells (Fig. 3E). Figure 3. Open in new tabDownload slide Epidermal cell patterning and cuticle deposition in fruit surface of SlMIXTA-like silenced tomato plants. A, Light microscopy observations of Sudan IV-stained tomato skin sections showing reduced cuticle deposition and flatter epidermal cells at the mature green fruit stage. B, Same as in A but in the red stage of development. ec, Epidermal cell. C, Cryo-SEM of the surface of tomato fruit at the mature green stage illustrates the effect of cell shape on surface morphology. Note the flat cells in the SlMIXTA-like silenced fruit. D, AFM images of red, ripe tomato fruit surface displaying a flatter, more irregular, and rough surface in the SlMIXTA-RNAi sample. Images are mixed light shaded to contrast the small features on the overall large topography. Bars = 20 µm (x and y axes), and 2 µm (z axis). E, TEM observations of tomato fruit epidermal cells (red fruit stage). A thinner cuticle as well as a distinct cell shape can be observed in the SlMIXTA-RNAi sample. cl, Cuticular layer; pcw, primary cell wall. Figure 3. Open in new tabDownload slide Epidermal cell patterning and cuticle deposition in fruit surface of SlMIXTA-like silenced tomato plants. A, Light microscopy observations of Sudan IV-stained tomato skin sections showing reduced cuticle deposition and flatter epidermal cells at the mature green fruit stage. B, Same as in A but in the red stage of development. ec, Epidermal cell. C, Cryo-SEM of the surface of tomato fruit at the mature green stage illustrates the effect of cell shape on surface morphology. Note the flat cells in the SlMIXTA-like silenced fruit. D, AFM images of red, ripe tomato fruit surface displaying a flatter, more irregular, and rough surface in the SlMIXTA-RNAi sample. Images are mixed light shaded to contrast the small features on the overall large topography. Bars = 20 µm (x and y axes), and 2 µm (z axis). E, TEM observations of tomato fruit epidermal cells (red fruit stage). A thinner cuticle as well as a distinct cell shape can be observed in the SlMIXTA-RNAi sample. cl, Cuticular layer; pcw, primary cell wall. To more deeply investigate cell shape, focused ion beam (FIB)-SEM was used. This technique is able to produce a three-dimensional (3D) image with the resolution typical of electron microscopy. Briefly, a biological sample is repeatedly milled (by the FIB) and then imaged (by the SEM), producing hundreds of SEM images from a few micrometers of tissue. These images can then be stacked (in silico), and the 3D structure of the observed specimen can be elucidated. In the case of the analysis of epidermal cells from ripe tomato fruit, wild-type fruit clearly possessed conical epidermal cells, whereas the epidermal cells from SlMIXTA-RNAi lines were considerably flatter (Fig. 4; Supplemental Movies S1 and S2). Figure 4. Open in new tabDownload slide 3D reconstruction from FIB-SEM acquisition shows that fruit of tomato lines silenced for SlMIXTA-like expression possesses flatter epidermal cells. A to D, Images acquired from the fruit skin of wild-type tomato display conical epidermal cell shape. E to H, Images of an epidermal cell from SlMIXTA-RNAi tomato fruit display a considerably flattened shape. I and J, The observations above were corroborated by in silico construction of 3D models of epidermal cells for both the wild type (I) and SlMIXTA-RNAi (J). For 3D model construction, see Supplemental Movies S1 and S2. Bars = 20 µm. Figure 4. Open in new tabDownload slide 3D reconstruction from FIB-SEM acquisition shows that fruit of tomato lines silenced for SlMIXTA-like expression possesses flatter epidermal cells. A to D, Images acquired from the fruit skin of wild-type tomato display conical epidermal cell shape. E to H, Images of an epidermal cell from SlMIXTA-RNAi tomato fruit display a considerably flattened shape. I and J, The observations above were corroborated by in silico construction of 3D models of epidermal cells for both the wild type (I) and SlMIXTA-RNAi (J). For 3D model construction, see Supplemental Movies S1 and S2. Bars = 20 µm. SlMIXTA-Like Expression Is Positively Correlated with the Accumulation of Cutin Monomers in Tomato Fruit Cuticle Analysis of enzymatically isolated cuticles from fruit altered in SlMIXTA-like expression revealed a positive correlation between SlMIXTA-like expression and cuticle mass (Table I; Supplemental Data Set S1). Cuticles isolated from SlMIXTA-RNAi fruit displayed a lower mass compared with wild-type cuticles. Deeper chemical analysis by gas chromatography (GC)-mass spectrometry (MS) was performed to determine which elements of the cuticle were contributing to the observed change in mass. The overall cutin monomer abundance was significantly altered in the SlMIXTA-RNAi lines (down 27%; Table I). Quantification of the total cuticular waxes from the tomato lines revealed no significant change (Supplemental Table S1). The change in cutin monomers was underlined by the significant reduction of aromatics, dicarboxylic fatty acids (DFAs), and midchain and terminal hydroxylated fatty acids (ω-HFAs) in the SlMIXTA-RNAi lines (Fig. 5; Table I). No significant change was observed in the accumulation of the saturated fatty acids in the different genotypes examined (Table I). Quantification of cutin monomer composition in fruit of SlMIXTA-like silenced lines and wild-type lines Table I. Quantification of cutin monomer composition in fruit of SlMIXTA-like silenced lines and wild-type lines Cutin monomers quantified after boron trifluoride depolymerization of enzymatically isolated, dewaxed tomato fruit cuticles (red-stage fruit). Concentrations (micrograms per centimeter−2) are shown for lines silenced for SlMIXTA-like (SlMIXTA-RNAi) and the corresponding wild type. Extractions were performed on three independently transformed lines. Monomers that show significant changes (Student’s t test) from the wild type are indicated (*, P < 0.01; **, P < 0.05). Cutin Monomers . Wild Type . SlMIXTA-RNAi . Average . se . Average . se . Aromatic  cis-Coumaric acid 0.92 0.23 0.55 0.17  trans-Coumaric acid 50.52 1.57 11.26* 3.01  Subtotal 51.44 1.44 11.81* 2.84 Saturated fatty acids  C16:0 fatty acids 2.84 0.14 2.45 0.21  C18:0 fatty acids 5.51 0.26 4.89 0.2  Subtotal 8.35 0.4 7.33 0.41 DFA  C16:0 DFA 24.92 1.34 11.22* 1.14  C16-9,10 hydroxy-DFA 67.53 4.73 40.57* 4.3  Subtotal 92.46 4.78 51.79* 5.37 Midchain hydroxylated fatty acids  C16-9/10,16 DiHFA 797.66 43.11 640.48* 35.58  C18-9,18 DiHFA 2.97 0.33 2.77 0.2  C18:1-9,10,18-trihydroxylated fatty acid 1.0 0.27 0.32** 0.03  C18-9,10 DiHFA 43.69 3.72 30.18* 2.32  Subtotal 845.31 47.32 673.75* 37.7 ω-HFA  C16:1-ω-HFA 7.37 0.6 4.13* 0.93  C16-ω-HFA 142.55 1.82 77.46* 6.75  Subtotal 149.91 2.28 81.59* 7.58 Epoxy fatty acids  C18:1-9,10-epoxy-18-ω-HFA 2.34 0.24 2.38 0.07  C18-9,10-epoxy-18-ω-HFA 3.44 0.31 3.69 0.23  Subtotal 5.78 0.55 6.08 0.27 Other  Naringenin 22.58 4.89 27.66 13.45  Naringenin dimer 23.88 0.49 13.86** 4.37  Subtotal 46.46 4.66 41.52 12.8 Unknown  Subtotal 187.38 6.46 134.1* 19.26 Total 1,387.09 53.86 1,012.65* 70.94 Cutin Monomers . Wild Type . SlMIXTA-RNAi . Average . se . Average . se . Aromatic  cis-Coumaric acid 0.92 0.23 0.55 0.17  trans-Coumaric acid 50.52 1.57 11.26* 3.01  Subtotal 51.44 1.44 11.81* 2.84 Saturated fatty acids  C16:0 fatty acids 2.84 0.14 2.45 0.21  C18:0 fatty acids 5.51 0.26 4.89 0.2  Subtotal 8.35 0.4 7.33 0.41 DFA  C16:0 DFA 24.92 1.34 11.22* 1.14  C16-9,10 hydroxy-DFA 67.53 4.73 40.57* 4.3  Subtotal 92.46 4.78 51.79* 5.37 Midchain hydroxylated fatty acids  C16-9/10,16 DiHFA 797.66 43.11 640.48* 35.58  C18-9,18 DiHFA 2.97 0.33 2.77 0.2  C18:1-9,10,18-trihydroxylated fatty acid 1.0 0.27 0.32** 0.03  C18-9,10 DiHFA 43.69 3.72 30.18* 2.32  Subtotal 845.31 47.32 673.75* 37.7 ω-HFA  C16:1-ω-HFA 7.37 0.6 4.13* 0.93  C16-ω-HFA 142.55 1.82 77.46* 6.75  Subtotal 149.91 2.28 81.59* 7.58 Epoxy fatty acids  C18:1-9,10-epoxy-18-ω-HFA 2.34 0.24 2.38 0.07  C18-9,10-epoxy-18-ω-HFA 3.44 0.31 3.69 0.23  Subtotal 5.78 0.55 6.08 0.27 Other  Naringenin 22.58 4.89 27.66 13.45  Naringenin dimer 23.88 0.49 13.86** 4.37  Subtotal 46.46 4.66 41.52 12.8 Unknown  Subtotal 187.38 6.46 134.1* 19.26 Total 1,387.09 53.86 1,012.65* 70.94 Open in new tab Table I. Quantification of cutin monomer composition in fruit of SlMIXTA-like silenced lines and wild-type lines Cutin monomers quantified after boron trifluoride depolymerization of enzymatically isolated, dewaxed tomato fruit cuticles (red-stage fruit). Concentrations (micrograms per centimeter−2) are shown for lines silenced for SlMIXTA-like (SlMIXTA-RNAi) and the corresponding wild type. Extractions were performed on three independently transformed lines. Monomers that show significant changes (Student’s t test) from the wild type are indicated (*, P < 0.01; **, P < 0.05). Cutin Monomers . Wild Type . SlMIXTA-RNAi . Average . se . Average . se . Aromatic  cis-Coumaric acid 0.92 0.23 0.55 0.17  trans-Coumaric acid 50.52 1.57 11.26* 3.01  Subtotal 51.44 1.44 11.81* 2.84 Saturated fatty acids  C16:0 fatty acids 2.84 0.14 2.45 0.21  C18:0 fatty acids 5.51 0.26 4.89 0.2  Subtotal 8.35 0.4 7.33 0.41 DFA  C16:0 DFA 24.92 1.34 11.22* 1.14  C16-9,10 hydroxy-DFA 67.53 4.73 40.57* 4.3  Subtotal 92.46 4.78 51.79* 5.37 Midchain hydroxylated fatty acids  C16-9/10,16 DiHFA 797.66 43.11 640.48* 35.58  C18-9,18 DiHFA 2.97 0.33 2.77 0.2  C18:1-9,10,18-trihydroxylated fatty acid 1.0 0.27 0.32** 0.03  C18-9,10 DiHFA 43.69 3.72 30.18* 2.32  Subtotal 845.31 47.32 673.75* 37.7 ω-HFA  C16:1-ω-HFA 7.37 0.6 4.13* 0.93  C16-ω-HFA 142.55 1.82 77.46* 6.75  Subtotal 149.91 2.28 81.59* 7.58 Epoxy fatty acids  C18:1-9,10-epoxy-18-ω-HFA 2.34 0.24 2.38 0.07  C18-9,10-epoxy-18-ω-HFA 3.44 0.31 3.69 0.23  Subtotal 5.78 0.55 6.08 0.27 Other  Naringenin 22.58 4.89 27.66 13.45  Naringenin dimer 23.88 0.49 13.86** 4.37  Subtotal 46.46 4.66 41.52 12.8 Unknown  Subtotal 187.38 6.46 134.1* 19.26 Total 1,387.09 53.86 1,012.65* 70.94 Cutin Monomers . Wild Type . SlMIXTA-RNAi . Average . se . Average . se . Aromatic  cis-Coumaric acid 0.92 0.23 0.55 0.17  trans-Coumaric acid 50.52 1.57 11.26* 3.01  Subtotal 51.44 1.44 11.81* 2.84 Saturated fatty acids  C16:0 fatty acids 2.84 0.14 2.45 0.21  C18:0 fatty acids 5.51 0.26 4.89 0.2  Subtotal 8.35 0.4 7.33 0.41 DFA  C16:0 DFA 24.92 1.34 11.22* 1.14  C16-9,10 hydroxy-DFA 67.53 4.73 40.57* 4.3  Subtotal 92.46 4.78 51.79* 5.37 Midchain hydroxylated fatty acids  C16-9/10,16 DiHFA 797.66 43.11 640.48* 35.58  C18-9,18 DiHFA 2.97 0.33 2.77 0.2  C18:1-9,10,18-trihydroxylated fatty acid 1.0 0.27 0.32** 0.03  C18-9,10 DiHFA 43.69 3.72 30.18* 2.32  Subtotal 845.31 47.32 673.75* 37.7 ω-HFA  C16:1-ω-HFA 7.37 0.6 4.13* 0.93  C16-ω-HFA 142.55 1.82 77.46* 6.75  Subtotal 149.91 2.28 81.59* 7.58 Epoxy fatty acids  C18:1-9,10-epoxy-18-ω-HFA 2.34 0.24 2.38 0.07  C18-9,10-epoxy-18-ω-HFA 3.44 0.31 3.69 0.23  Subtotal 5.78 0.55 6.08 0.27 Other  Naringenin 22.58 4.89 27.66 13.45  Naringenin dimer 23.88 0.49 13.86** 4.37  Subtotal 46.46 4.66 41.52 12.8 Unknown  Subtotal 187.38 6.46 134.1* 19.26 Total 1,387.09 53.86 1,012.65* 70.94 Open in new tab Figure 5. Open in new tabDownload slide SlMIXTA-like is required for cutin biosynthesis and deposition of the cuticle. A generic cutin biosynthetic pathway using the most abundant C16 fatty acid moiety as a substrate. Transcriptomic analysis pertaining to the pathway is displayed in the form of graphs indicating relative gene expression. Values are means of three independently transformed lines ± se (n = 3). For specific fold change values, see Table II. Relative changes to major cutin monomers are also shown (green arrows; n = 3). FA, Fatty acid. Figure 5. Open in new tabDownload slide SlMIXTA-like is required for cutin biosynthesis and deposition of the cuticle. A generic cutin biosynthetic pathway using the most abundant C16 fatty acid moiety as a substrate. Transcriptomic analysis pertaining to the pathway is displayed in the form of graphs indicating relative gene expression. Values are means of three independently transformed lines ± se (n = 3). For specific fold change values, see Table II. Relative changes to major cutin monomers are also shown (green arrows; n = 3). FA, Fatty acid. Silencing of SlMIXTA-Like Results in the Down-Regulation of a Wide Spectrum of Cutin Biosynthesis and Assembly Genes Further investigation of the SlMIXTA-like-RNAi lines identified a pertinent array of gene expression changes in the fruit skin. Global expression analysis by microarrays and real-time qRT-PCR assays for validation revealed a considerable down-regulation of cutin biosynthesis genes in the skin of SlMIXTA-RNAi fruit at the mature green stage (Table II; Supplemental Data Set S2; Supplemental Fig. S4). The mature green stage of fruit development was selected for gene expression analysis, because at this stage, the skin and flesh are more easily separated compared with the earlier stages of development, and cuticle biosynthesis genes are still strongly enriched (Mintz-Oron et al., 2008). Mining the microarray data to identify genes with at least 2-fold change (P ≤ 0.05) generated a relatively concise list of 93 down-regulated and 153 up-regulated genes in the SlMIXTA-RNAi fruit skin compared with the wild type (Supplemental Data Set S2). Gene ontology enrichment analysis of the 93 down-regulated genes revealed significant enrichment for lipid metabolism, specifically fatty acid and lignin biosynthesis (P < 0.01). Among the 93 down-regulated genes, 13 orthologs of genes previously functionally characterized and associated with cuticle assembly were annotated (Fig. 5; Table II). Because of the fact that SlSHN3 has been shown by promotor binding assays to act upstream of SlMIXTA-like (Shi et al., 2013), the initial list of down-regulated genes was further extended by qRT-PCR analysis of candidates previously shown to be down-regulated in SlSHN3-RNAi lines (Shi et al., 2013) but not detected in the SlMIXTA-RNAi microarray experiment because of either not being present on the array or expression levels below the background of detection. In total, 17 genes putatively involved in cuticle biosynthesis and assembly were identified to be down-regulated in SlMIXTA-RNAi lines (Table II). It is also important to note that qRT-PCR confirmed no significant change in expression of SlSHN3, SlCD2, SlPDF2d, SlDCR, and SlCD1 (Supplemental Fig. S4). Genes putatively involved in cuticle assembly showing down-regulation in the SlMIXTA-RNAi lines Table II. Genes putatively involved in cuticle assembly showing down-regulation in the SlMIXTA-RNAi lines A 2-fold change cutoff and P ≤ 0.05 were used. The majority of values are derived from microarray experiments; however, values in parentheses are derived from real-time qRT-PCR analysis. LB, Contains the L1-box promoter motif (Abe et al., 2001); L1, L1 layer-specific expression (Filippis et al., 2013); PDR4, PLEIOTROPIC DRUG RESISTANCE4. Gene Identification . Gene Name . Putative Function . Fold Change . P . Cluster . Characterized Ortholog . Reference . Solyc05g054330 α/β-Hydrolase Cutin polymerization −7.8 0.001 IV.iib AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc03g117800L1 CER3 Alkane biosynthesis −7.7 0.014 IV.iia AtCER3 (At5g57800) Bernard et al. (2012) Solyc08g080190L1 HTH Dicarboxylic acid biosynthesis −6.8 0.001 IV.iia AtHTH (At1g72970) Kurdyukov et al. (2006b) Solyc03g121180L1 GDSL esterase/lipase Cutin polymerization −4.3 0.040 IV.i SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g109180LB,L1 LACS2 Acyl activation −3.7 (−8.3) 0.001 (0.033) IV.iia AtLACS2 (At1g49430) Schnurr et al. (2004); Jessen et al. (2011) Solyc09g075140LB α/β-Hydrolase Cutin polymerization −3.7 0.001 IV.iia AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc01g095750LB,L1 LACS4 Acyl activation −3.6 (−4.3) 0.003 (0.011) IV.iia AtLACS4 (At4g23850) Schnurr et al. (2004); Jessen et al. (2011) Solyc03g019760LB,L1 ABCG11 Cuticular lipids transport −3.1 0.009 IV.iia AtABCG11 (At1g17840) Bird et al. (2007); Panikashvili et al. (2007) Solyc11g072990LB,L1 KCS3 Fatty acid elongation −3.0 0.003 IV.iia AtKCS3 (At1g07720) Millar et al. (1999) Solyc05g055400LB,L1 CYP77A2 Midchain fatty acid oxidationa −2.5 (−20.7) 0.038 (0.022) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc06g065670LB,L1 PDR4/ABCG32 Cuticular lipids transport −2.1 0.043 IV.iib AtABGC32 (At2g26910) Bessire et al. (2011) Solyc04g081770LB,L1 GDSL esterase/lipase Cutin polymerization −2.0 0.025 IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g094700LB,L1 GPAT4 Acyl transfer (−38.1) (0.013) IV.iia AtGPAT4 (At1g01610) Li et al. (2007) Solyc11g007540LB,L1 CYP77A1 Midchain fatty acid oxidationa (−13.3) (0.045) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc01g094750LB CYP86A68 Terminal fatty acid oxidationa (−4.8) (0.039) IV.iia SlCYP86A69 (Solyc08g081220) Shi et al. (2013) Solyc07g049440LB,L1 GDSLa Cutin polymerization (−4.2) (0.003) IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc03g120620LB GL2 Cuticle regulation (−3.6) (0.037) IV.iia SlCD2 (Solyc01g091630) Isaacson et al. (2009); Nadakuduti et al. (2012) Gene Identification . Gene Name . Putative Function . Fold Change . P . Cluster . Characterized Ortholog . Reference . Solyc05g054330 α/β-Hydrolase Cutin polymerization −7.8 0.001 IV.iib AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc03g117800L1 CER3 Alkane biosynthesis −7.7 0.014 IV.iia AtCER3 (At5g57800) Bernard et al. (2012) Solyc08g080190L1 HTH Dicarboxylic acid biosynthesis −6.8 0.001 IV.iia AtHTH (At1g72970) Kurdyukov et al. (2006b) Solyc03g121180L1 GDSL esterase/lipase Cutin polymerization −4.3 0.040 IV.i SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g109180LB,L1 LACS2 Acyl activation −3.7 (−8.3) 0.001 (0.033) IV.iia AtLACS2 (At1g49430) Schnurr et al. (2004); Jessen et al. (2011) Solyc09g075140LB α/β-Hydrolase Cutin polymerization −3.7 0.001 IV.iia AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc01g095750LB,L1 LACS4 Acyl activation −3.6 (−4.3) 0.003 (0.011) IV.iia AtLACS4 (At4g23850) Schnurr et al. (2004); Jessen et al. (2011) Solyc03g019760LB,L1 ABCG11 Cuticular lipids transport −3.1 0.009 IV.iia AtABCG11 (At1g17840) Bird et al. (2007); Panikashvili et al. (2007) Solyc11g072990LB,L1 KCS3 Fatty acid elongation −3.0 0.003 IV.iia AtKCS3 (At1g07720) Millar et al. (1999) Solyc05g055400LB,L1 CYP77A2 Midchain fatty acid oxidationa −2.5 (−20.7) 0.038 (0.022) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc06g065670LB,L1 PDR4/ABCG32 Cuticular lipids transport −2.1 0.043 IV.iib AtABGC32 (At2g26910) Bessire et al. (2011) Solyc04g081770LB,L1 GDSL esterase/lipase Cutin polymerization −2.0 0.025 IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g094700LB,L1 GPAT4 Acyl transfer (−38.1) (0.013) IV.iia AtGPAT4 (At1g01610) Li et al. (2007) Solyc11g007540LB,L1 CYP77A1 Midchain fatty acid oxidationa (−13.3) (0.045) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc01g094750LB CYP86A68 Terminal fatty acid oxidationa (−4.8) (0.039) IV.iia SlCYP86A69 (Solyc08g081220) Shi et al. (2013) Solyc07g049440LB,L1 GDSLa Cutin polymerization (−4.2) (0.003) IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc03g120620LB GL2 Cuticle regulation (−3.6) (0.037) IV.iia SlCD2 (Solyc01g091630) Isaacson et al. (2009); Nadakuduti et al. (2012) a Putative function confirmed in this article. Open in new tab Table II. Genes putatively involved in cuticle assembly showing down-regulation in the SlMIXTA-RNAi lines A 2-fold change cutoff and P ≤ 0.05 were used. The majority of values are derived from microarray experiments; however, values in parentheses are derived from real-time qRT-PCR analysis. LB, Contains the L1-box promoter motif (Abe et al., 2001); L1, L1 layer-specific expression (Filippis et al., 2013); PDR4, PLEIOTROPIC DRUG RESISTANCE4. Gene Identification . Gene Name . Putative Function . Fold Change . P . Cluster . Characterized Ortholog . Reference . Solyc05g054330 α/β-Hydrolase Cutin polymerization −7.8 0.001 IV.iib AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc03g117800L1 CER3 Alkane biosynthesis −7.7 0.014 IV.iia AtCER3 (At5g57800) Bernard et al. (2012) Solyc08g080190L1 HTH Dicarboxylic acid biosynthesis −6.8 0.001 IV.iia AtHTH (At1g72970) Kurdyukov et al. (2006b) Solyc03g121180L1 GDSL esterase/lipase Cutin polymerization −4.3 0.040 IV.i SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g109180LB,L1 LACS2 Acyl activation −3.7 (−8.3) 0.001 (0.033) IV.iia AtLACS2 (At1g49430) Schnurr et al. (2004); Jessen et al. (2011) Solyc09g075140LB α/β-Hydrolase Cutin polymerization −3.7 0.001 IV.iia AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc01g095750LB,L1 LACS4 Acyl activation −3.6 (−4.3) 0.003 (0.011) IV.iia AtLACS4 (At4g23850) Schnurr et al. (2004); Jessen et al. (2011) Solyc03g019760LB,L1 ABCG11 Cuticular lipids transport −3.1 0.009 IV.iia AtABCG11 (At1g17840) Bird et al. (2007); Panikashvili et al. (2007) Solyc11g072990LB,L1 KCS3 Fatty acid elongation −3.0 0.003 IV.iia AtKCS3 (At1g07720) Millar et al. (1999) Solyc05g055400LB,L1 CYP77A2 Midchain fatty acid oxidationa −2.5 (−20.7) 0.038 (0.022) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc06g065670LB,L1 PDR4/ABCG32 Cuticular lipids transport −2.1 0.043 IV.iib AtABGC32 (At2g26910) Bessire et al. (2011) Solyc04g081770LB,L1 GDSL esterase/lipase Cutin polymerization −2.0 0.025 IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g094700LB,L1 GPAT4 Acyl transfer (−38.1) (0.013) IV.iia AtGPAT4 (At1g01610) Li et al. (2007) Solyc11g007540LB,L1 CYP77A1 Midchain fatty acid oxidationa (−13.3) (0.045) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc01g094750LB CYP86A68 Terminal fatty acid oxidationa (−4.8) (0.039) IV.iia SlCYP86A69 (Solyc08g081220) Shi et al. (2013) Solyc07g049440LB,L1 GDSLa Cutin polymerization (−4.2) (0.003) IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc03g120620LB GL2 Cuticle regulation (−3.6) (0.037) IV.iia SlCD2 (Solyc01g091630) Isaacson et al. (2009); Nadakuduti et al. (2012) Gene Identification . Gene Name . Putative Function . Fold Change . P . Cluster . Characterized Ortholog . Reference . Solyc05g054330 α/β-Hydrolase Cutin polymerization −7.8 0.001 IV.iib AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc03g117800L1 CER3 Alkane biosynthesis −7.7 0.014 IV.iia AtCER3 (At5g57800) Bernard et al. (2012) Solyc08g080190L1 HTH Dicarboxylic acid biosynthesis −6.8 0.001 IV.iia AtHTH (At1g72970) Kurdyukov et al. (2006b) Solyc03g121180L1 GDSL esterase/lipase Cutin polymerization −4.3 0.040 IV.i SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g109180LB,L1 LACS2 Acyl activation −3.7 (−8.3) 0.001 (0.033) IV.iia AtLACS2 (At1g49430) Schnurr et al. (2004); Jessen et al. (2011) Solyc09g075140LB α/β-Hydrolase Cutin polymerization −3.7 0.001 IV.iia AtBDG (At1g64670) Kurdyukov et al. (2006a) Solyc01g095750LB,L1 LACS4 Acyl activation −3.6 (−4.3) 0.003 (0.011) IV.iia AtLACS4 (At4g23850) Schnurr et al. (2004); Jessen et al. (2011) Solyc03g019760LB,L1 ABCG11 Cuticular lipids transport −3.1 0.009 IV.iia AtABCG11 (At1g17840) Bird et al. (2007); Panikashvili et al. (2007) Solyc11g072990LB,L1 KCS3 Fatty acid elongation −3.0 0.003 IV.iia AtKCS3 (At1g07720) Millar et al. (1999) Solyc05g055400LB,L1 CYP77A2 Midchain fatty acid oxidationa −2.5 (−20.7) 0.038 (0.022) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc06g065670LB,L1 PDR4/ABCG32 Cuticular lipids transport −2.1 0.043 IV.iib AtABGC32 (At2g26910) Bessire et al. (2011) Solyc04g081770LB,L1 GDSL esterase/lipase Cutin polymerization −2.0 0.025 IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc01g094700LB,L1 GPAT4 Acyl transfer (−38.1) (0.013) IV.iia AtGPAT4 (At1g01610) Li et al. (2007) Solyc11g007540LB,L1 CYP77A1 Midchain fatty acid oxidationa (−13.3) (0.045) IV.iia AtCYP77A4 (At5g04660) Li-Beisson et al. (2009) Solyc01g094750LB CYP86A68 Terminal fatty acid oxidationa (−4.8) (0.039) IV.iia SlCYP86A69 (Solyc08g081220) Shi et al. (2013) Solyc07g049440LB,L1 GDSLa Cutin polymerization (−4.2) (0.003) IV.iia SlCD1 (Solyc11g006250) Girard et al. (2012); Yeats et al. (2012) Solyc03g120620LB GL2 Cuticle regulation (−3.6) (0.037) IV.iia SlCD2 (Solyc01g091630) Isaacson et al. (2009); Nadakuduti et al. (2012) a Putative function confirmed in this article. Open in new tab Of the 17 cuticle-associated down-regulated genes, 14 could be linked to various steps of cutin biosynthesis and assembly (Fig. 5). These consisted of two LACS genes (SlLACS2 and SlLACS4) putatively involved in the acyl activation of fatty acids (Fig. 5). Orthologs to these genes have been shown to be required for normal cuticle development in Arabidopsis: AtLACS1, AtLACS2, and AtLACS4 (Schnurr et al., 2004; Jessen et al., 2011). Three CYP450s were identified: SlCYP77A1, SlCYP77A2, and SlCYP86A68 (Fig. 5; Table II). Orthologs of CYP77A have been shown to be responsible for the midchain hydroxylation of C16 fatty acids during cutin biosynthesis (Li-Beisson et al., 2009), whereas CYP86A orthologs have been shown to catalyze the terminal hydroxylation of free fatty acids (Han et al., 2010; Shi et al., 2013). Another candidate for the modification of fatty acids is the down-regulated tomato HOTHEAD (SlHTH) gene. The Arabidopsis ortholog, AtHTH, has been shown to be crucial for the formation of DFAs (Kurdyukov et al., 2006b). The final step in the biosynthesis of cutin monomers is acyl transfer of the activated fatty acid to a glycerol moiety. This reaction is catalyzed by AtGPAT4 and Brassica napus GPAT4 in Arabidopsis (Li et al., 2007) and B. napus (Chen et al., 2011), respectively. The tomato ortholog to these genes (SlGPAT4) was down-regulated in SlMIXTA-RNAi lines (Fig. 5; Table II). The monoacylglycerols formed through the action of the enzymes described above are subsequently transported to the exterior of the cell before polymerization occurs (Pollard et al., 2008). SlMIXTA-RNAi lines showed down-regulation of two extracellular transporter genes: tomato ATP-BINDING CASSETTE11 (SlABCG11) and SlABCG32. SlABCG11 is orthologous to the characterized cutin and wax monomer transporter in Arabidopsis, AtABCG11 (Bird et al., 2007; Panikashvili et al., 2007), whereas SlABCG32 is the tomato ortholog to AtABCG32, an extracellular transporter of cuticle components (Bessire et al., 2011). Finally, five genes potentially involved in the extracellular polymerization of the cutin matrix were found to be down-regulated in the SlMIXTA-RNAi lines (Fig. 5; Table II). Three orthologs to the tomato CD1 gene, recently reported to be an extracellular cutin polymerase (Girard et al., 2012; Yeats et al., 2012), were also down-regulated in the SlMIXTA-RNAi lines (Solyc07g049440, Solyc03g121180, and Solyc04g081770) as well as two α/β-hydrolases (Solyc09g075140 and Solyc05g054330) orthologous to the Arabidopsis AtBDG that was suggested to be an extracellular synthase required for cutin formation (Kurdyukov et al., 2006a). Only 2 of the 17 cuticle-associated genes down-regulated in the SlMIXTA-RNAi lines could putatively be linked to wax biosynthesis (Solyc03g117800 and Solyc11g072990). The Arabidopsis ortholog of Solyc03g117800, ECERIFERUM3 (AtCER3), has been reported to catalyze the conversion of very long-chain acyl-CoAs to very long-chain alkanes (Bernard et al., 2012), whereas the ortholog of Solyc11g072990, Arabidopsis 3-KETOACYL-COA SYNTHASE3 (AtKCS3), catalyzes the first step of fatty acid elongation (Millar et al., 1999). Putative SlMIXTA-Like Downstream Targets Display an L1 Layer-Specific Enrichment In silico promoter analysis of the genes identified to be down-regulated in SlMIXTA-RNAi lines revealed a significant enrichment (P < 0.01) for the L1-box motif -TAAATGYA- (Table II; Supplemental Data Set S2). This motif has previously been shown to be the binding site for two L1-specific HD-ZIP transcriptional regulators in Arabidopsis: AtPDF2 and AtML1 (Abe et al., 2003; Takada et al., 2013). In the SlMIXTA-RNAi lines, an ortholog to these genes, SlGL2 (Solyc03g120620), was shown to be down-regulated (Table II). The 93 down-regulated genes were also analyzed for their L1-layer specificity by comparison with a recently generated data set (Filippis et al., 2013). In this work, 255 of the 13,277 genes examined were identified to exhibit L1 layer-specific tomato expression. Of the reported 255 L1 layer-specific gene set, 18 genes were found to be down-regulated in the SlMIXTA-RNAi lines (Table II; Supplemental Data Set S2). Hypergeometric analysis shows that this is an extremely strong enrichment (P < 0.01) and further suggests a role for SlMIXTA-like in L1 layer-specific gene control. By comparison, of the 153 up-regulated genes in the SlMIXTA-RNAi lines, only 3 are reportedly L1-layer specific, showing no enrichment (P = 0.57). The previous observation that SlMIXTA-like possessed a fruit skin-associated expression profile (Mintz-Oron et al., 2008; Shi et al., 2013) was further confirmed by the data extracted from recently generated large-scale transcriptomic analysis of multiple tomato tissues (Itkin et al., 2013; Fig. 1C). The hypothesis that putative downstream targets of SlMIXTA-like will likely exhibit a similar L1 layer-specific expression pattern was tested by extracting the expression patterns of the SlMIXTA-RNAi down-regulated genes in skin and flesh tissues from the data set and performing hierarchical clustering (Fig. 6). Data for 91 genes were collected, and four major clusters were identified, with the largest cluster (cluster IV) containing SlMIXTA-like and 40 other genes (Fig. 6; Table II; Supplemental Data Set S2). Cluster IV had a high homogeneity (r value = 0.81) and displayed skin-enriched (or L1 layer-specific) expression pattern, particularly in the early stages of fruit development (Fig. 6). All of the 16 genes described as potential SlMIXTA-like targets involved in cuticle biosynthesis were found to be part of cluster IV. The expanded expression profiles (including seed, leaf, flower, and root expression data) of genes described in this report can be seen in Supplemental Figure S5. Figure 6. Open in new tabDownload slide Hierarchical clustering of genes down-regulated in SlMIXTA-RNAi lines. The fruit development expression profiles of 91 genes identified as down-regulated in SlMIXTA-RNAi lines were extracted from a previously published large-scale expression data set (Itkin et al., 2013), and hierarchical clustering was performed. Four major clusters were identified, and their mean representative expression profiles are shown adjacent to the heat map (marked I–IV). Pearson correlation coefficients illustrating the homogeneity of the clusters are displayed below the cluster number. The expression profile of SlMIXTA-like located in cluster IV is highlighted by a yellow outline. Additional genes potentially involved in cuticle biosynthesis are labeled and detailed in Table II. For remaining genes, see Supplemental Data Set S2. Red and green colors represent pairwise distances for transcript expression from the mean (black). IG, Immature green; MG, mature green; Br, breaker; Or, orange; R, red. Figure 6. Open in new tabDownload slide Hierarchical clustering of genes down-regulated in SlMIXTA-RNAi lines. The fruit development expression profiles of 91 genes identified as down-regulated in SlMIXTA-RNAi lines were extracted from a previously published large-scale expression data set (Itkin et al., 2013), and hierarchical clustering was performed. Four major clusters were identified, and their mean representative expression profiles are shown adjacent to the heat map (marked I–IV). Pearson correlation coefficients illustrating the homogeneity of the clusters are displayed below the cluster number. The expression profile of SlMIXTA-like located in cluster IV is highlighted by a yellow outline. Additional genes potentially involved in cuticle biosynthesis are labeled and detailed in Table II. For remaining genes, see Supplemental Data Set S2. Red and green colors represent pairwise distances for transcript expression from the mean (black). IG, Immature green; MG, mature green; Br, breaker; Or, orange; R, red. Finally, of the 143 genes found to be up-regulated in the microarray analysis, 1 gene was of particular interest and displayed a dramatic up-regulation in SlMIXTA-RNAi lines. An HD-ZIP IV transcription factor, SlANL2b (Solyc06g035940), was up-regulated 43-fold (Supplemental Data Set S2). Tomato ANTHOCYANINLESS 2b (SlANL2b) is a close ortholog in tomato to the cuticle regulating SlCD2 gene (Isaacson et al., 2009; Nadakuduti et al., 2012) and significantly, also an ortholog to the epidermis- and L1 layer-related transcription factors AtPDF2 and AtML1 (Supplemental Fig. S6). Putative SlMIXTA-Like Downstream Target Genes Encode Members of the Cytochrome P450 CYP77A and CYP86A Subfamilies, Catalyzing the Formation of Major Cutin Monomers in Tomato Members of the cytochrome P450 CYP77A and CYP86A subfamilies have been reported previously to be involved in cutin monomers biosynthesis (largely in Arabidopsis), performing midchain and terminal oxidation of fatty acids, respectively (Li-Beisson et al., 2009). Because three members of these subfamilies were identified here as putative SlMIXTA-like downstream targets (Fig. 5; Table II; Supplemental Fig. S7), we explored the activity of the corresponding recombinant enzymes through expression in yeast (Saccharomyces cerevisiae) cells. Initial incubation of a variety of fatty acids with microsomes of yeast expressing the three CYPs (SlCYP77A1, SlCYP77A2, and SlCYP86A68) confirmed that they were all active enzymes able to catalyze hydroxylation of fatty acids (Fig. 7; Supplemental Fig. S8). More extensive analysis of SlCYP77A1 showed that the enzyme was able to hydroxylate C16:0 fatty acids (Fig. 7). To determine the position of the hydroxylation, we performed MS analysis on the recombinant SlCYP77A1 assay products and revealed a mixture of 11,16-; 10,16-; 9,16-; and 8,16-dihydroxyhexadecanoic acids (Fig. 7C). This result corresponds strongly with the reduced levels of 9/10,16-dihydroxyhexadecanoic acid in fruit of the SlMIXTA-RNAi lines. Figure 7. Open in new tabDownload slide Recombinant SlCYP77A1 catalyzes the formation of dihydroxyhexadecanoic acids. A and B, Microsomes of yeast cells expressing SlCYP77A1 were incubated with 100 μm hexadecanoic acid in the absence (A) or presence (B) of NADPH. Reactions products were extracted with diethyl ether, derivatized, and subjected to GC-MS analysis. C, Fragmentation pattern of the dihydroxyhexadecanoic acid peak is presented and identified to be a mixture of 8,16-; 9,16-; 10,16-; and 11,16-dihydroxyhexadecanoic acids. The molecular structure of the most abundant tomato cuticle fatty acid, 9,16-dihydroxyhexadecanoic acid, is displayed. Figure 7. Open in new tabDownload slide Recombinant SlCYP77A1 catalyzes the formation of dihydroxyhexadecanoic acids. A and B, Microsomes of yeast cells expressing SlCYP77A1 were incubated with 100 μm hexadecanoic acid in the absence (A) or presence (B) of NADPH. Reactions products were extracted with diethyl ether, derivatized, and subjected to GC-MS analysis. C, Fragmentation pattern of the dihydroxyhexadecanoic acid peak is presented and identified to be a mixture of 8,16-; 9,16-; 10,16-; and 11,16-dihydroxyhexadecanoic acids. The molecular structure of the most abundant tomato cuticle fatty acid, 9,16-dihydroxyhexadecanoic acid, is displayed. Silencing of SlMIXTA-Like Increased Postharvest Fruit Water Loss and Susceptibility to Postharvest Fungal Infection Postharvest analysis of SlMIXTA-RNAi fruit revealed that reduced expression of SlMIXTA-like resulted in an increase in postharvest water loss as well as increased susceptibility to fungal infection (Fig. 8). Fruit of SlMIXTA-RNAi lines lost 80% more water than those from wild-type lines over 30 d after harvest (Fig. 8A). Similarly, the susceptibility to postharvest fungal infection was significantly higher in SlMIXTA-RNAi lines. Fruits at the breaker stage were inoculated with the tomato fruit pathogen Colletotrichum coccodes, which causes black dot anthracnose disease (Dillard, 1989). Despite no significant difference in the rate of conidia germination and appressoria formation on the surface of wild-type and SlMIXTA-RNAi fruit (Supplemental Fig. S9), a 5-fold increase in the decay diameter after 1 week of inoculation was observed on SlMIXTA-RNAi fruit (Fig. 8B). Figure 8. Open in new tabDownload slide Reduced SlMIXTA-like expression impacts postharvest water loss and susceptibility to fungal infection. A, Water loss progression in fruit of wild-type (WT) and SlMIXTA-RNAi lines was measured over 40 d of postharvest incubation at room temperature. B, Decay area rates of C. coccodes colonization were measured after inoculation of breaker-stage tomato fruit cuticle for 7 d. Error bars represent se (n = 30). Figure 8. Open in new tabDownload slide Reduced SlMIXTA-like expression impacts postharvest water loss and susceptibility to fungal infection. A, Water loss progression in fruit of wild-type (WT) and SlMIXTA-RNAi lines was measured over 40 d of postharvest incubation at room temperature. B, Decay area rates of C. coccodes colonization were measured after inoculation of breaker-stage tomato fruit cuticle for 7 d. Error bars represent se (n = 30). DISCUSSION SlMIXTA-Like Forms Part of a Transcriptional Network Regulating the Development of Conical Epidermal Cells in Tomato Fruit To date, MIXTA orthologs across a variety of plant species have been reported to be involved in the regulation of epidermal cell differentiation (Brockington et al., 2013). The earliest characterized MIXTA ortholog (AmMIXTA) was shown to promote conical epidermal cell development in the petals of snapdragon (Noda et al., 1994; Brockington et al., 2013). Subsequently, members of the MIXTA transcription factor clade from Arabidopsis, Thalictrum thalictroides, and P. hybrida were also reported to regulate conical epidermal cell development (Baumann et al., 2007; Di Stilio et al., 2009), whereas orthologs from Arabidopsis, Populus trichopoda, Mimulus guttatus, snapdragon, Medicago trunculata, and Dendrobium crumenatum have been shown to be positive regulators of trichome development (Perez-Rodriguez et al., 2005; Gilding and Marks, 2010; Plett et al., 2010; Scoville et al., 2011). Phylogenetic analysis showed that the SlMIXTA-like protein characterized here is most closely related to the P. hybrida PhMYB1 ortholog, which has been reported to regulate conical epidermal cell differentiation in petals (Baumann et al., 2007; Fig. 1A). Four additional tomato proteins were identified through genome analysis and seemed related to the MIXTA/MIXTA-like clade; however, phylogenetic analysis found these proteins to be distinct from MIXTA/MIXTA-like orthologs (Supplemental Fig. S1). This relationship was not investigated in more detail, because no expression was detected for these genes in previously published large-scale expression studies (Itkin et al., 2013). It is possible that these genes are nonexpressed pseudogenes that are no longer under selective pressure to maintain activity. The prevalence of conical epidermal cells in flower petals suggests an important role for this particular morphology, likely the attraction of pollinators; however, the mode of action for this attraction is not obvious. Research into this question has shown that the reflection of light by the epidermal cells of petals is significantly altered by cell morphology (Gorton and Vogelmann, 1996), and this likely plays a role in pollinator attraction; however, more recent research has shown that pollinators are able to discriminate between petal surfaces by tactile sensation (Whitney et al., 2009). The conical epidermal cells of petals likely facilitate the physical interaction between pollinators and flowers (Whitney et al., 2009). In contrast to petals, the abundance of this morphology in fleshy fruit surface has received less attention. As in the case of petals, the conical cells in fruit likely play a role in the reflection of light and therefore, may be involved in the attraction of seed dispersal agents. Recent studies confirm earlier reports that change in epidermal cell morphology can dramatically alter the human perceived glossiness of tomato fruit (Isaacson et al., 2009; Mahjoub et al., 2009; Nadakuduti et al., 2012; Shi et al., 2013; Petit et al., 2014), although how these fruits are viewed by seed dispersers is still not known. Although no epidermal-specific petal phenotype was observed, leaves of SlMIXTA-RNAi lines were small and folded, whereas fruits were glossier than corresponding wild-type fruit, despite the reduction in cuticle deposition seen in these lines. Morphological examination of tomato fruit silenced for SlMIXTA-like expression revealed a flattening of the epidermal cells, which likely leads to the glossier surface, because light reflects from the surface with less scattering. The observed enrichment of the L1-box promoter motif (Abe et al., 2001) in the promoters of the genes down-regulated in the SlMIXTA-RNAi lines together with the enrichment of L1 layer-specific genes implicate SlMIXTA-like as a possible central regulator of L1-layer cell identity. This might occur through coregulation with the HD-ZIP IV transcription factor SlGl2, which is significantly down-regulated in lines silenced for SlMIXTA-like expression and therefore, may occur downstream of SlMIXTA-like in this regulatory pathway. The ortholog of SlGL2 in Arabidopsis, AtGL2, is a well-characterized regulator of epidermal cell differentiation (Tominaga-Wada et al., 2009). The L1-box motif has been found to be an important binding site for HD-ZIP IV transcription factors regulating epidermal cell differentiation in both Arabidopsis and cotton (Abe et al., 2003; Ohashi et al., 2003; Zhang et al., 2010). Significantly, an HD-ZIP IV ortholog from cotton GbML1 was shown to bind to not only the L1-box motif but also, a cotton MIXTA family protein, GbMYB25 (Zhang et al., 2010). In SlMIXTA-RNAi lines, the HD-ZIP IV transcription factor SlANL2b (Supplemental Fig. S6) was massively up-regulated (40-fold; Supplemental Data Set S2). If a similar mechanism found in cotton exists in tomato and SlANL2b can be regarded as having the orthologous function to GbML1 (i.e. binding both the L1-box motif and SlMIXTA-like), then the massive increase in SlANL2b expression in SlMIXTA-RNAi lines may represent a compensating mechanism for the loss of SlMIXTA-like expression. These results suggest that the influence of SlMIXTA-like (and possibly, MIXTA orthologs in general) on epidermal cell differentiation may be mediated through a regulatory network with L1-box motif binding HD-ZIP IV transcription factors (such as SlGL2 and SlANL2b). This view of such a network between MIXTA and HD-ZIP IV transcription factors in relation to epidermal cell differentiation identity can be expanded when a recent work on the AP2 domain SlSHN3 transcription factor is considered (Shi et al., 2013). The results of this earlier study suggested that SlSHN3 was able to regulate epidermal cell patterning in tomato by SlGL2 and/or SlMIXTA-like activities. SlGL2 and SlMIXTA-like were both down-regulated in SlSHN3-RNAi tomato lines, and luciferase reporter assays showed that SlSHN3 acted on the promoters of both genes (Shi et al., 2013). This together with the fact that SlSHN3 displayed no change in expression in SlMIXTA-RNAi lines suggest that, in tomato fruit, SlSHN3 seems to act above SlMIXTA-like in the regulatory pathway controlling epidermal cell patterning (Fig. 9). Figure 9. Open in new tabDownload slide The SlMIXTA-like regulatory network controls cuticle assembly and cell patterning. The SlSHN and SlMIXTA-like proteins directly or indirectly regulate cuticle deposition in tomato fruit skin as part of a larger program of epidermal cell patterning. This network likely involves interaction with additional regulators, such as members of the HD-ZIP IV family of transcription factors. Figure 9. Open in new tabDownload slide The SlMIXTA-like regulatory network controls cuticle assembly and cell patterning. The SlSHN and SlMIXTA-like proteins directly or indirectly regulate cuticle deposition in tomato fruit skin as part of a larger program of epidermal cell patterning. This network likely involves interaction with additional regulators, such as members of the HD-ZIP IV family of transcription factors. Finally, it should be noted that, although strong evidence is presented for the involvement of L1 layer-specific genes as the mediators of SlMIXTA-like activity in relation to epidermal cell shape, it may be that this morphological alteration is in fact a secondary effect caused by the observed reduction in cuticle deposition. Specifically, tomato fruit epidermal cells are either totally or partially enveloped by the cuticle, which likely contributes to the cell structure. Recent results have indeed found that the fruit surface structure is influenced by cutin deposition; however, in this work, a change in cutin deposition was not necessarily shown to result in a change in epidermal cell shape (Petit et al., 2014). Furthermore, silencing of CHALCONE SYNTHASE expression (a gene responsible for the inclusion of naringenin chalcone in the tomato cuticle) results in a stiffer, less elastic cuticle surrounding flatter epidermal cells (Schijlen et al., 2007; España et al., 2014). These results hint at a potential dependence on the surrounding cuticle with regard to epidermal cell structure. Further investigations into the relationship between cuticular lipid composition and epidermal cell development will prove invaluable to our understanding of plant surface formation. SlMIXTA-Like Is a Positive Regulator of Tomato Fruit Cutin Biosynthesis and Assembly Expression analysis of SlMIXTA-like in developing tomato fruit revealed a skin-enriched expression profile (predominantly at the early green stages of fruit development), whereas transcriptome analysis across multiple tissues and developmental stages revealed that SlMIXTA-like was also significantly expressed in the petals and pollen. This expression profile is typical of genes involved in cuticle biosynthesis (Mintz-Oron et al., 2008). Moreover, laser microdissection coupled to transcriptomic analysis of tomato fruit cell types showed that SlMIXTA-like is expressed in the inner as well as outer epidermal layers but not collenchyma, parenchyma, or vascular tissues (Matas et al., 2011; Hen-Avivi et al., 2014; Fig. 1B). The inner epidermal layer of tomato fruit produces a waxy cuticle separating the fleshy endocarp from the locular region of the fruit (Mintz-Oron et al., 2008; Matas et al., 2011), and thus, it seems likely that SlMIXTA-like plays a role in the regulation of this inner cuticle as well as the outer fruit cuticle. It is interesting to note that the previously characterized tomato cuticle regulators, SlSHN3 and SlCD2, are also expressed in both the inner and outer epidermis (Hen-Avivi et al., 2014). Analysis of the tomato lines altered in SlMIXTA-like expression showed a significant correlation between cuticle deposition and SlMIXTA-like expression. This was evident in the microscopic analysis, which displayed a significantly thinner cuticle in the SlMIXTA-like-RNAi lines, and the chemical analysis, which highlighted that this change can be predominantly attributed to a modification in cutin monomer biosynthesis. In fact, the change in cutin levels in the transgenics correlated strongly with the change in the overall weight of the isolated cuticle. Significant differences in the cutin composition were particularly evident in the highly abundant modified C16 fatty acid, more specifically C16 9/10,16 DiHFA, the dominant cutin monomer of tomato fruit, as well as DFAs and ω-HFAs. The saturated fatty acids, however, showed few significant changes in plants exhibiting modified SlMIXTA-like expression, suggesting that SlMIXTA-like exerts its activity at the downstream modification steps of the cutin biosynthetic pathway. This hypothesis was confirmed through the transcriptional analysis of the transgenic plants. Numerous genes that are directly responsible for the biosynthesis of specific modified fatty acids were down-regulated in plants with reduced SlMIXTA-like expression. In addition, genes involved in the extracellular transport and the polymerization of the cutin matrix were also found to display similar, SlMIXTA-like-dependent expression patterns. In most cases, the enzymatic activity of the proteins coded by these various genes was inferred through the homology with characterized orthologs, but for a number of the candidate enzymes, assays were performed to confirm these activities. Thus, for a few of the steps in the cutin metabolic pathway, namely the terminal hydroxylation of activated fatty acids by SlCYP86A68 and the midchain hydroxylation of fatty acids by both SlCYP77A1 and SlCYP77A2, we are able to present cohesive data for SlMIXTA-like regulated gene expression, enzyme activity, and the resultant metabolite change in the fruit skin (Figs. 5 and 7; Table I). Previously, SlCYP86A69 was shown to be directly regulated by SlSHN3 and the resultant activity responsible for cutin biosynthesis (Shi et al., 2013). Although SlCYP86A69 does not seem to be regulated in an SlMIXTA-like-dependent manner, we show that SlCYP77A1 action is responsible for the formation of the major cutin monomer found in tomato skin, namely C16-9/10, 16-DiHFA, and that SlCYP77A1 is regulated in an SlMIXTA-like-dependent manner. The broad spectrum of influence exerted through SlMIXTA-like activity strongly suggests a central role for SlMIXTA-like in the positive regulation of cuticle deposition (particularly cutin). Significantly, recent work has shown that the Arabidopsis orthologs of SlMIXTA-like, AtMYB16, and AtMYB106 also play a role in the regulatory network of cuticle assembly (Oshima et al., 2013), although their effect is less specific, because they regulate both the wax and cutin biosynthetic pathways. Interestingly, one of the orthologs, AtMYB106, is able to regulate the expression of AtSHN1, which seems to be an inversion of the relationship seen between these two orthologs in tomato (Fig. 9), pointing to a complex evolutionary relationship between these proteins. SlMIXTA-Like Regulates Postharvest Water Loss Prevention and Resistance to Fungal Infection Previous studies showed the important role of the cuticle in protecting plants from biotic and abiotic stresses and that abnormal cuticle formation can lead to plants with altered susceptibility to dehydration stress and fungal infection (Bargel et al., 2006; Riederer and Muller, 2006; Isaacson et al., 2009). In the case of SlMIXTA-RNAi lines, an increase in susceptibility to both fungal infection and postharvest water loss was observed. It seems likely that the significantly thinner cuticle allows easier penetration of the epidermal cells by C. coccodes and thus, faster proliferation of the fungus. This conclusion was supported by the facts that C. coccodes fungus had similar rates of germination and appressoria formation but that the decay area was significantly larger. However, the increased postharvest water loss is more difficult to explain. Previous research has suggested that cuticular waxes rather than the cutin matrix are responsible for the waterproofing of the plant (Vogg et al., 2004; Isaacson et al., 2009). However, in the case of tomato lines reduced in SlMIXTA-like expression, there seems to be limited changes to the total (epi- and intracuticular) cuticular wax composition. It may be that severity of reduction in the cutin polymer provided a reduced matrix to which the intracuticular waxes may be attached and embedded. Therefore, it is possible that altered cuticular structure and arrangement are the main factors responsible for the increase in water loss. Recent work showed that the cuticle is involved in the transmission of osmotic stress signals by abscisic acid (ABA) biosynthesis (Wang et al., 2011). The fact that SlMIXTA-RNAi lines displayed down-regulation of the ABA biosynthesis gene NINE-CIS-EPOXYCAROTENOID DIOXYGENASE1 (Sun et al., 2012; Ji et al., 2014) suggests that the reason for the increased susceptibility to dehydration may be caused by the impairment of a signal originating from the cuticle (Supplemental Fig. S4; Supplemental Data Set S2). Furthermore, increased cuticle permeability in ABA-deficient mutants has been reported (Curvers et al., 2010), suggesting a feedback loop connecting ABA biosynthesis and cuticle formation that might be regulated through the action of SlMIXTA-like. Furthermore, ABA biosynthesis was recently shown to be up-regulated in tomato fruit’s resistance response to C. coccodes (Alkan et al., 2015). This, therefore, may further contribute to the enhanced fungal colonization of SlMIXTA-RNAi fruit surface. CONCLUSION The evolution of land plants resulted in a number of important changes to the epidermal layer of aerial organs, including of fleshy fruit. Over time, epidermal cells began to differentiate to form a variety of cell types, while concurrently developing the specialized cuticular membrane necessary for overcoming the challenges of life on land. It is thus anticipated that the coevolution of the epidermis cell layer and the cuticle coating should intersect, particularly at the regulatory level. Here, we provide evidence that SlMIXTA-like transcriptionally modulates epidermal cell patterning as well as cuticle assembly and forms part of a transcriptional regulatory network that mediates the patterning of fruit surface. Hence, this work highlights the strong evolutionary and regulatory relationship between plant epidermal cell development and cuticle deposition and points to SlMIXTA-like as a major player in this relationship. MATERIALS AND METHODS Plant Materials and Transformation Silencing of tomato (Solanum lycopersicum) MIXTA-like (Solyc02g088190) was performed in cv MicroTom (MT). The construct for the posttranscriptional silencing (SlMIXTA-RNAi) was generated by PCR, isolating a 273-bp fragment of SlMIXTA from cv MT complementary DNA and cloning into pENTR/D-TOPO (Invitrogen). LR Clonase (Invitrogen) was used to recombine this fragment into the pH7GWIWG2(II) binary vector (Karimi et al., 2002). The primers used in the creation of the constructs in this study are listed in Supplemental Table S2. Cotyledon transformation of ‘MT’ tomato was performed as previously described (Dan et al., 2006). Subsequent gene expression analysis, chemical characterization, and fungal infection and water loss assays were performed using at least three independently transformed lines. In Silico Analysis Nucleotide and protein sequence retrieval from the SOL Genomics Network database was performed by BLAST (Fernandez-Pozo et al., 2015). Protein alignments were performed with ClustalW (Larkin et al., 2007), and the resultant molecular phylogenetic trees were visualized using MEGA6 (Tamura et al., 2013). Neighbor-joining trees were constructed with bootstrapping calculated from 1,000 instances. Promoter regions consisting of 1 kb of upstream sequence from the start codons were analyzed for binding motifs using the PLACE database (Higo et al., 1999). Determination of enrichment of promoter motifs and L1 layer-specific genes was performed using the hypergeometric distribution analysis provided by GeneProf (Halbritter et al., 2014). Promoter motif presence was compared to 6,000 kb of randomly extracted genomic DNA spanning all 12 tomato chromosomes (Fernandez-Pozo et al., 2015). Hierarchical clustering of genes based on available large-scale expression data (Itkin et al., 2013) was performed using EXPANDER (Ulitsky et al., 2010) with the default parameters. Gene Expression Analysis Total RNA extractions were performed with Trizol Reagent (Invitrogen) from manually dissected skin and flesh tissues pooled from five to six tomato fruits. Skin tissue can be thought of as enriched for epidermal cells. RNA was extracted from mature green peel tissue from three independently transformed tomato and wild-type lines. The complementary DNA was synthesized using Invitrogen’s SuperScript II Reverse Transcriptase Kit. Real-time qRT-PCR analysis was performed using gene-specific oligonucleotides on an ABI 7300 Instrument (Applied Biosystems) with the Platinum SYBR SuperMix (Invitrogen) under default parameters. Mean expression values were calculated for three independent transformation events. Applied Biosystems software was used to generate expression data. Sequences of gene-specific oligonucleotides are provided in Supplemental Table S2. Microarray analysis was performed using the 34,000-gene EUTOM3 exon array (http://www.ebi.ac.uk/arrayexpress/arrays/A-MEXP-2227) as described previously (Powell et al., 2012). Light Microscopy, Electron Microscopy, and AFM Samples for electron microscopy were prepared and analyzed as described previously (Panikashvili et al., 2009). Samples prepared for TEM analysis were also used for FIB-SEM analysis. The FIB-SEM tomography of the region of interest, containing a number of epidermal cells, was performed with the FEI Slice and View Software. Milling was done at an acceleration voltage of 30 kV and current of 2.7 nA. SEM images of the milled surface were acquired with the through-the-lens detector (secondary electrons mode) in immersion mode and an electron beam of 2 kV, 340 pA, and 30 μs of dwell time. Subsequent 3D reconstruction was performed by AVIZO Software application. For light microscopy, skin tissue samples were fixed and embedded in wax as described previously (Mintz-Oron et al., 2008). Sections were cut to 5 to 10 µm on a Leica 2000 Microtome and placed on glass slides. The slides were stained with Sudan IV (Buda et al., 2009) or toluidine blue (Tanaka et al., 2004) and then observed with an Olympus CLSM500 Microscope. Freshly harvested skin samples were used for AFM imaging. Samples were mounted on a stub and transferred to the AFM for imaging. All imaging was performed on an NT-MDT NTEGRA Instrument using the SMENA scanning head. Imaging was performed in either contact mode using Bruker DNP-S Probes or Olympus ORC-PS-W Probes with a nominal spring constant of 0.1 N m−1 or semicontact mode using Olympus AC240TS Probes with nominal frequency of 70 kHz and spring constant of 2 Nm−1. There was no difference in images obtained by the two modes, with the exception of one sample with some contamination that was swept aside in contact mode. The raw data were processed only by two-dimensional leveling. Cuticular Lipid Analyses Fruit skin discs were prepared for cuticular lipid extraction as previously described (Shi et al., 2013). Cuticular waxes were then extracted and analyzed as previously described (Kurdyukov et al., 2006a, 2006b) followed by cutin extraction and analysis as described previously (Franke et al., 2005). Enzyme Activity Assays Enzyme activity assays were performed using heterologously expressed SlCYP77A1, SlCYP77A2, and SlCYP68A68 according to methods previously described (Grausem et al., 2014). Briefly, the genes under investigation were cloned and expressed in a yeast (Saccharomyces cerevisiae) expression system specifically developed for the expression of P450 enzymes (Pompon et al., 1996; Supplemental Table S2). Expression of the tomato P450s was induced, microsomes were extracted, microsomal proteins were incubated with radiolabeled substrate, and the enzyme assay was initiated by the addition of NADPH. Only three cytochromes P450 are encoded by the yeast genome and are not expressed or expressed at a negligible level during the applied growth conditions. Furthermore, none of them are able to metabolize fatty acids (Kandel et al., 2005; Sauveplane et al., 2009), ensuring that the metabolism described results from enzymatic reactions catalyzed by the heterologously expressed cytochrome P450s. Completed assays were directly spotted on thin-layer chromatography plates for the separation of possible products formed. Plates were scanned with a radioactivity detector, and the areas corresponding to the metabolites were identified for subsequent GC-MS analysis. GC-MS analysis was carried out and analyzed as described previously (Eglinton et al., 1968; Grausem et al., 2014). Metabolites generated from 16-hydroxypalmitic acid were identified as described previously (Li-Beisson et al., 2009). Because no standards are available, position of hydroxyl group is given by fragmentation on both sides of the derivatized hydroxyl. This results in ions that allow one to distinguish between products. Fungal Infection and Dehydration Assays Colletotrichum coccodes isolate 138 (Alkan et al., 2008) was used to inoculate fruit of freshly harvested breaker-stage tomato fruits. The fruits were surface sterilized in 0.3% (v/v) hypochlorite for 3 min, rinsed thoroughly with sterile water, and dried. Drop inoculation was performed directly on the fruit cuticle by applying 7 µL of a conidia suspension (106 conidia mL−1). Each line had 30 biological inoculation repeats. After inoculation, fruits were incubated in humid chambers (22°C and approximately 95% relative humidity). At 19 h postinoculation, percentages of C. coccodes conidia germination and appressorium formation were evaluated under a light microscope. Disease evaluation of inoculated tomato fruits was performed by measuring the decay diameter over 7 d postinoculation. The experiment was repeated three times. For water loss measurement assays, red-stage fruit was harvested and stored at room temperature for 40 d. Fruit mass was periodically recorded, and water loss percentage was calculated. Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Identification of potential tomato MIXTA/MIXTA-like orthologs. Supplemental Figure S2. Protein alignments used to construct molecular phylogenetic trees. Supplemental Figure S3. AFM images of immature green tomato surface of wild-type and SlMIXTA-like silenced plants. Supplemental Figure S4. Validation of microarray-based differential gene expression results by means of qRT-PCR analysis. Supplemental Figure S5. Normalized expression across 20 tomato tissues of selected genes described in this study to be down-regulated in the SlMIXTA-RNAi tomato lines. Supplemental Figure S6. Molecular phylogeny of the HD-ZIP IV family proteins associated with epidermal cell patterning and ones identified in this study. Supplemental Figure S7. Molecular phylogeny of the HD-ZIP IV family proteins associated with epidermal cell patterning and ones identified in this study. Supplemental Figure S8. Radiochromatographic resolution by thin-layer chromatography of metabolites generated in incubations of dodecanoic acid with microsomes from yeast expressing SlCYP86A68 or SlCYP77A2. Supplemental Figure S9. Germination rate and appressoria formation of C. coccodes are unchanged on SlMIXTA-like silenced fruit surface. Supplemental Table S1. Quantification of cuticular waxes in fruit of SlMIXTA-like silenced lines. Supplemental Table S2. Oligonucleotides used in this study. Supplemental Data Set S1. Generation and analysis of tomato lines overexpressing SlMIXTA-like. Supplemental Data Set S2. Significantly down- and up-regulated genes in skin tissue of SlMIXTA-RNAi lines identified by microarray analysis. Supplemental Data Set S3. Microarray gene expression data of SlMIXTA-RNAi and wild-type lines. Supplemental Movie S1. 3D reconstruction from FIB-SEM acquisition of an epidermal cell from wild-type tomato fruit. Supplemental Movie S2. 3D reconstruction from FIB-SEM acquisition of an epidermal cell from tomato fruit silenced for SlMIXTA-like expression. ACKNOWLEDGMENTS We thank Sidney Cohen for assistance in performing the AFM analysis and Gilgi Friedlander for assistance in the analysis of the microarray data. Glossary ABA abscisic acid AFM atomic force microscopy 3D three-dimensional DFA dicarboxylic fatty acid DiHFA 16-dihydroxy fatty acid FIB focused ion beam GC gas chromatography ω-HFA terminal hydroxylated fatty acid MS mass spectrometry MT MicroTom qRT quantitative reverse transcription SEM scanning electron microscopy TEM transmission electron microscopy LITERATURE CITED Abe M , Katsumata H, Komeda Y, Takahashi T ( 2003 ) Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis . Development 130 : 635 – 643 Google Scholar Crossref Search ADS PubMed WorldCat Abe M , Takahashi T, Komeda Y ( 2001 ) Identification of a cis-regulatory element for L1 layer-specific gene expression, which is targeted by an L1-specific homeodomain protein . 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J Exp Bot 61 : 3599 – 3613 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Israel Science Foundation (personal grant no. 646/11 to A.Ah.), the Adelis Foundation (to A.Ah.), the Leona M. and Harry B. Helmsley Charitable Trust (to A.Ah.), the Jeanne and Joseph Nissim Foundation for Life Sciences (to A.Ah.), the Tom and Sondra Rykoff Family Foundation Research (to A.Ah.), the Raymond Burton Plant Genome Research Fund (to A.Ah.), and the Peter J. Cohn Professorial Chair (to A.Ah.). * Address correspondence to asaph.aharoni@weizmann.ac.il. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Asaph Aharoni (asaph.aharoni@weizmann.ac.il). J.L. designed and performed the research, analyzed the data, and cowrote the article; A.Ad. designed and performed the research and analyzed the data; O.L., N.A., T.T., K.R., J.-P.F.-M., E.W., B.G., F.P., and A.G. performed the research and analyzed the data; F.C. analyzed the data and cowrote the article; A.Ah. designed the research, analyzed the data, and cowrote the article; all authors read, made relevant suggestions, and approved the final article. www.plantphysiol.org/cgi/doi/10.1104/pp.15.01145 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly
JF - Plant Physiology
DO - 10.1104/pp.15.01145
DA - 2015-12-09
UR - https://www.deepdyve.com/lp/oxford-university-press/the-tomato-mixta-like-transcription-factor-coordinates-fruit-epidermis-5Gm50JV2fA
SP - 2553
EP - 2571
VL - 169
IS - 4
DP - DeepDyve
ER -