TY - JOUR
AU - Aharoni, Asaph
AB - Abstract The skin of fleshy fruit is typically covered by a thick cuticle. Some fruit species develop different forms of layers directly above their skin. Reticulation, for example, is a specialized suberin-based coating that ornaments some commercially important melon (Cucumis melo) fruit and is an important quality trait. Despite its importance, the structural, molecular, and biochemical features associated with reticulation are not fully understood. Here, we performed a multilevel investigation of structural attributes, chemical composition, and gene expression profiles on a set of reticulated and smooth skin melons. High-resolution microscopy, surface profiling, and histochemical staining assays show that reticulation comprises cells with heavily suberized walls accumulating large amounts of typical suberin monomers, as well as lignified cells localized underneath the specialized suberized cell layer. Reticulated skin was characterized by induced expression of biosynthetic genes acting in the core phenylpropanoid, suberin, lignin, and lignan pathways. Transcripts of genes associated with lipid polymer assembly, cell wall organization, and loosening were highly enriched in reticulated skin tissue. These signatures were exclusive to reticulated structures and absent in both the smooth surfaces observed in between reticulated regions and in the skin of smooth fruit. Our data provide important insights into the molecular and metabolic bases of reticulation and its tight association with skin ligno-suberization during melon fruit development. Moreover, these insights are likely to contribute to melon breeding programs aimed at improving postharvest qualities associated with fleshy fruit surface layers. The cuticle, a specialized barrier unique to plants, covers the outer surface of fleshy fruit, as for all other plant aerial organs (Martin and Rose, 2014). This structure hinders the loss of water from the fruit skin epidermis, providing a mechanical barrier against pathogen infection and herbivory. The absence or reduced number of stomata in most fruit augments the role of the cuticle as a barrier from the environment (Schreiber and Riederer, 1996). The cuticle is a unique lipophilic structure largely composed of the cutin polyester made of C16 and C18 ω-hydroxylated fatty acids combined with a complex mixture of very-long-chain epicuticular waxes (Pollard et al., 2008). In addition, the cuticle of different plant species may also contain metabolites belonging to various classes, including alkaloids, sterols, and phenylpropanoids (Adato et al., 2009). The structural characteristics of the cuticular layer are crucial for maintaining its function under the high-tension forces existing during fruit expansion. Yet, in cases of fruit skin damage, the cuticle might suffer from microcracking, reducing its capacity to act as a barrier. In response to cuticular malfunction, plants developed secondary surfaces called periderms, which typically contain different lipophilic material (other than cutin) such as lignin and/or suberin (Knoche and Lang, 2017). Lignin was shown in numerous studies to account for structural integrity and waterproofing capacities of native plant cells as well as wounded ones (Boerjan et al., 2003). In the case of fleshy fruit, suberin is considered the major specialized matrix deposited above the fruit skin to seal the tissue as part of a wound response. Compared with cutin, the suberin polyester is largely composed of longer C20 to C26 fatty acid derivatives, greater quantity of phenolics, together with glycerol-based alkyl ferulate waxes (Franke et al., 2005; Beisson et al., 2012). Certain varieties of some fruit crops such as apple (Malus × domestica) and pear (Pyrus communis) possess a network of suberized cells directly above the skin termed russeting, appearing as a brown and rough matrix deposition (Khanal et al., 2013; Lashbrooke et al., 2015). Suberized layers appear naturally in other specialized tissues and organs, including the root endodermis, tree bark, seed coat, and the phellem layer of potato (Solanum tuberosum) tuber skin (Pollard et al., 2008; Schreiber, 2010). They are easily recognized microscopically due to their typical appearance as a lamellar structure deposited in the inner side of the cell wall adjacent to the plasma membrane. Another known case of a specialized structure deposited above fruit skin during fruit development occurs in melon (Cucumis melo). While fruit of wild melon are small with almost no edible mesocarp and typically covered by a smooth thick skin (Monforte et al., 2014), domesticated melon fruit display a large diversity of fruit surface phenotypes. Some accessions possess a smooth skin appearance, while others are decorated by suberized regions forming patterns of reticulation or netting. The exclusive reticulated structure ornamenting melon fruit skin was reported in the early 1950s as a tissue originating from cracks appearing on the fruit surface (Meissner, 1952). Throughout fruit development and its enlargement, the length and width of these fissures increase, leading to their interconnection, eventually forming a complete reticulated structure once fruit are fully mature (Cutter, 1969; Webster and Craig, 1976). It appears that periderm cells below these fissures start to multiply rapidly, producing heavily suberized wall cells (Keren-Keiserman et al., 2004a). Furthermore, peroxidase activity was associated with processes of suberization and lignification occurring at the reticulated surface (Keren-Keiserman et al., 2004b). Differences in the structure of the skin surface, smooth or reticulated, impact fruit physiology and development, including firmness and elasticity (Rose et al., 1998; Dos-Santos et al., 2011; Puthmee et al., 2013), waterproofing capacity, and turgor pressure of hypodermal tissues (Saladié et al., 2007). They further confer tolerance against mechanical damage (Keren-Keiserman et al., 2004a). Thus far, several studies investigated the structure of reticulated regions merely through standard microscopical observations. Yet, despite the fundamental interest in suberization and the commercial importance of melon fruit, no data are currently available regarding the molecular and biochemical features associated with melon fruit skin reticulation. Here, we conducted a multilevel investigation of reticulated and smooth skin melon fruit to decipher structural attributes, chemical composition, and gene expression profiles associated with the reticulation phenomena. Several advanced microscopy techniques, histochemical staining procedures, and mass spectrometry approaches demonstrate that reticulation comprises specialized cells with heavily suberized walls with some portion of lignified cells beneath them. Reticulated regions accumulate large amounts of typical suberin monomers and display induced expression of genes functioning in the core phenylpropanoid, suberin, lignin, and lignan pathways. In addition, higher expression of genes involved in cell wall organization and loosening was detected in the reticulated fruit as compared with smooth skin varieties. The shift toward the metabolism of suberin and lignin at the onset of reticulation dramatically affects the biosynthesis of other skin compounds such as cutin, epicuticular waxes, and flavonoids. Altogether, this study provides important insights into fruit skin ligno-suberization hallmarks and is likely to assist in understanding the molecular mechanisms associated with the suberization process. RESULTS The Morphology and Fine Structure of Fruit Reticulation A collection of three smooth (cv Sakata’s Sweet, White Crenshaw, and Honeydew) and three reticulated (cv Sharlyn, Hearts of Gold, and Delicious 51) skin melon cultivars served as the primary genetic resource for this study (Fig. 1). Apart from morphological differences in skin appearance associated with reticulation, the six cultivars differ in flesh color and fruit maturity index (Fig. 1). Initially, skin surfaces from all fruit were manually dissected and examined for their fine morphology using light microscopy. While skin surfaces of reticulated cultivars displayed thick structures that protrude above the fruit surface, those of cv Hearts of Gold fruit appeared to rise higher and cover larger areas of the skin (Fig. 2A). Fruit surfaces were further analyzed using SEM, revealing that skin surfaces of both fruit types are covered by typical polygonal epidermal cells, with cv Sakata’s Sweet skin displaying very small cracks. Yet, in the three reticulated fruit, the polygonal cells are disrupted by large cracks in which dense cells with thick walls protract out vertically toward the surface plateau (Fig. 2, B and C). SEM was employed to examine epicuticular wax crystal deposition on smooth and reticulated skin surfaces. We used paraformaldehyde (PFA) and glutaraldehyde (GA) fixation buffers that have minimal effects on wax composition, as well as critical point dehydration procedures to avoid changes in skin morphology and preserve its structure. Closer examination of the polygonal cells’ surface in the three smooth skin fruit indicated considerable amounts of epicuticular wax crystals. Fewer crystals could be detected on the surface of all three reticulated skin fruit (Fig. 2D). Figure 1. Open in new tabDownload slide Smooth and reticulated skin melon fruit cultivars investigated in this study. Maturity index represents the number of days (d) from fertilization to full fruit maturity. Bar = 10 cm. Figure 1. Open in new tabDownload slide Smooth and reticulated skin melon fruit cultivars investigated in this study. Maturity index represents the number of days (d) from fertilization to full fruit maturity. Bar = 10 cm. Figure 2. Open in new tabDownload slide Light microscopy and scanning electron microscopy (SEM) of smooth and reticulated fruit surfaces. A, Light microscopy images of fruit surfaces from smooth and reticulated skin cultivars. B, SEM micrographs of fruit skin surfaces. White circles represent enlarged images that appear in C and D. C, Enlargements of skin surfaces (smooth fruit) and cracking regions (reticulated fruit) displayed in B. D, Enlargements of micrographs displayed in B and C showing the presence of epicuticular wax crystals (ewc). SEM observations were performed at least on two samples of skin surface from two different fruit of every cultivar. Bars = 1 cm (A), 300 µm (B), 10 µm (C), and 1 µm (D). Figure 2. Open in new tabDownload slide Light microscopy and scanning electron microscopy (SEM) of smooth and reticulated fruit surfaces. A, Light microscopy images of fruit surfaces from smooth and reticulated skin cultivars. B, SEM micrographs of fruit skin surfaces. White circles represent enlarged images that appear in C and D. C, Enlargements of skin surfaces (smooth fruit) and cracking regions (reticulated fruit) displayed in B. D, Enlargements of micrographs displayed in B and C showing the presence of epicuticular wax crystals (ewc). SEM observations were performed at least on two samples of skin surface from two different fruit of every cultivar. Bars = 1 cm (A), 300 µm (B), 10 µm (C), and 1 µm (D). Reticulation seemed to differ between the three reticulated cultivars. We therefore outlined the structure of their skin surfaces by an optical profiler microscope, allowing the construction of 3D images (see “Materials and Methods”; Fig. 3A). Using these models, we concluded that cv Sharlyn and Delicious 51 skin surfaces possess a concave structure of reticulation, as the top of reticulation was raised ∼75 and ∼147 µm above the bottom of reticulation, respectively (Fig. 3B). The reticulation in cv Hearts of Gold skin surface, however, exhibited a dome structure, as the surface of reticulation itself was 39 µm higher than its borders (Fig. 3B). Nonetheless, reticulation protruded differently above the skin surface of all three cultivars, ∼206, ∼312, and ∼436 µm in cv Sharlyn, Delicious 51, and Hearts of Gold, respectively (Fig. 3B). Finally, we calculated the percentage coverage of reticulation by generating reticulation maps (see “Materials and Methods”). These analyses demonstrated that 66% of the skin surface of cv Hearts of Gold was covered by reticulation, while 49% and 54% of those of cv Sharlyn and Delicious 51 were covered by reticulation, respectively (Fig. 3C). Altogether, these assays suggested that reticulation disrupts the surface of the skin epidermal layer. Yet, reticulated structures seem to differ in their morphology, patterning, and skin percentage coverage between all three reticulated cultivars. Figure 3. Open in new tabDownload slide Optical profiling microscopy (OPM) and characterization of reticulation patterns. A, Representative 3D images of reticulated skin surfaces derived from reticulated fruit cv Sharlyn, Delicious 51, and Hearts of Gold following OPM analyses. B, Reticulation models using 3D constructed OPM models with average height differences between the skin surface to the top of reticulation and between the top of reticulation to the reticulation surface itself. n = 12. C, Representative reticulation maps with calculated reticulation coverage in skin samples. n = 8. Figure 3. Open in new tabDownload slide Optical profiling microscopy (OPM) and characterization of reticulation patterns. A, Representative 3D images of reticulated skin surfaces derived from reticulated fruit cv Sharlyn, Delicious 51, and Hearts of Gold following OPM analyses. B, Reticulation models using 3D constructed OPM models with average height differences between the skin surface to the top of reticulation and between the top of reticulation to the reticulation surface itself. n = 12. C, Representative reticulation maps with calculated reticulation coverage in skin samples. n = 8. Reticulated Regions Comprise Cells with Heavily Suberized Walls To examine the impact of reticulation on the epidermis layer, we cross sectioned skin samples isolated from the six cultivars and observed them by light microscopy. Samples of the three smooth cultivars exhibited a typical cuticle built from well-organized conical cells covering the outermost epidermal cell layer (Fig. 4A). The skin of the reticulated cv Hearts of Gold showed severe disruption of the epidermis layer by a mass of unorganized cells bursting out of cracked regions (Fig. 4A). A similar phenomenon was observed in the skin of the reticulated cv Sharlyn and Delicious 51, in which unorganized cells exhibit a slightly more ordered structure as with those of cv Hearts of Gold (Fig. 4A). Following Toluidine Blue O staining, these specific cells within cracks were stained by faint blue, indicating that these areas are likely lignified and/or suberized and contain more aliphatic domains (Fig. 4A). Figure 4. Open in new tabDownload slide Reticulated regions comprise cells with heavily suberized walls. A, Light microcopy images of skin cross sections of smooth and reticulated skin cultivars. Sections were counterstained with Toluidine Blue O. White circles represent regions examined by transmission electron microscopy (TEM) that appear in B and C. B, TEM micrographs of cell walls located within cells of the outermost epidermal layer as indicated in A. C, TEM micrographs of cell walls located within cells of the inner collenchyma as indicated in A. pcw, Primary cell wall; sl, suberin lamellae. Bars = 200 µm (A), 50 nm (B), and 50 nm (C). Figure 4. Open in new tabDownload slide Reticulated regions comprise cells with heavily suberized walls. A, Light microcopy images of skin cross sections of smooth and reticulated skin cultivars. Sections were counterstained with Toluidine Blue O. White circles represent regions examined by transmission electron microscopy (TEM) that appear in B and C. B, TEM micrographs of cell walls located within cells of the outermost epidermal layer as indicated in A. C, TEM micrographs of cell walls located within cells of the inner collenchyma as indicated in A. pcw, Primary cell wall; sl, suberin lamellae. Bars = 200 µm (A), 50 nm (B), and 50 nm (C). As noted above, previous work suggested the existence of a network of suberized tissue within the reticulated regions. Yet, none of these studies examined the walls of cells building the reticulated structure or the cells adjacent to it. Here, we analyzed ultrathin skin sections of the six cultivars by means of TEM. In smooth skin samples, cells of the epidermis layer as well as those located in the inner collenchyma displayed typical thick cell walls (Fig. 4, B and C). However, suberized lamellae adjacent to the cell walls of the unorganized cells in the reticulated regions were clearly detected in skin of all three reticulated fruit (Fig. 4B). Markedly, these specialized suberized layers were entirely absent in the walls of the inner collenchyma cells of both smooth and reticulated cultivars (Fig. 4C). This indicated that cell wall suberization is physiologically and chemically restricted to the cells located at the upper layers of reticulated structures. Histochemical Assays Link Reticulation and Ligno-Suberization The results above suggested that reticulated structures are made from a mass of suberized and/or lignified specialized cells. To corroborate these findings, we stained skin cross sections with phloroglucinol-hydrochloric acid for the presence of lignin, producing a visible cherry-red color under bright field. We also used the fluorescent dye Fluorol Yellow 088 to detect the presence of suberin. No lignified cells could be detected in the skin samples of smooth fruit (Fig. 5A), in which the typical conical cells that build the cuticle were clearly observed by autofluorescence (Fig. 5B). Yet, in all three reticulated fruit, cells located beneath the upper epidermal cell layers showed a clear indication of lignin (Fig. 5C). The unorganized cells located at the outermost epidermal layer were subsequently shown to be suberized cells through Fluorol Yellow 088 staining (Fig. 5D). In cv Hearts of Gold fruit skin, where the epidermal cuticular layer seemed to be highly disrupted by reticulated structures, we could also detect lignin deposition within the upper suberized cells (Fig. 5C). Hence, histochemical assays supported the involvement of both suberin and lignin in fruit surface reticulation. Figure 5. Open in new tabDownload slide Linking reticulation to ligno-suberization. Light and fluorescence microscopy images of smooth and reticulated fruit skin cross sections following staining for lignin (phloroglucinol-hydrochloric acid staining) and suberin (Fluorol Yellow 088 staining). A, Light microscopy images of smooth skin fruit. B, Fluorescence microscopy 647-nm images of cuticle layer in smooth skin fruit. C, Light microscopy images of reticulated skin fruit. D, Fluorescence microscopy 488-nm images of suberized layer in reticulated skin fruit. Typical epidermis conical cells are clearly observed in the smooth skin samples with autofluorescence of cuticle layers observed under a Cy5 filter (647 nm). Lignified cells colored by faint purple are present in reticulated samples, together with fluorescence of suberized cells observed under a GFP filter (488 nm). No 488-nm images are presented for the three samples of smooth skin, as no suberin was detected; while no 647-nm images are presented for the three samples of reticulated skin, as the images focus solely on suberized areas where cuticle layer is absent. Bars = 50 µm. Figure 5. Open in new tabDownload slide Linking reticulation to ligno-suberization. Light and fluorescence microscopy images of smooth and reticulated fruit skin cross sections following staining for lignin (phloroglucinol-hydrochloric acid staining) and suberin (Fluorol Yellow 088 staining). A, Light microscopy images of smooth skin fruit. B, Fluorescence microscopy 647-nm images of cuticle layer in smooth skin fruit. C, Light microscopy images of reticulated skin fruit. D, Fluorescence microscopy 488-nm images of suberized layer in reticulated skin fruit. Typical epidermis conical cells are clearly observed in the smooth skin samples with autofluorescence of cuticle layers observed under a Cy5 filter (647 nm). Lignified cells colored by faint purple are present in reticulated samples, together with fluorescence of suberized cells observed under a GFP filter (488 nm). No 488-nm images are presented for the three samples of smooth skin, as no suberin was detected; while no 647-nm images are presented for the three samples of reticulated skin, as the images focus solely on suberized areas where cuticle layer is absent. Bars = 50 µm. Suberin Monomers Accumulate in Reticulated Structures But Not in Smooth Surfaces in between Reticulated Regions To examine if the suberin polymer accumulates within the reticulated area, we spatially localized ferulic acid, a major suberin building block, using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). Ferulic acid is a substantial component of suberin but minor in the chemically similar cutin polymer. Skin discs from all six cultivars were delipidated by extensive chloroform/methanol washings to ensure merely the existence of compounds associated with cell wall-bound cutin and suberin polyesters. Strikingly, MALDI-MSI revealed the presence of ferulic acid solely within reticulated regions in all three reticulated fruit but not in nearby smooth surfaces or in the surfaces of smooth fruit (Fig. 6, A and B). To provide additional quantitative evidence for the unique spatial distribution of suberin in reticulated regions but not in the smooth surfaces in between, these two types of tissues were manually isolated from the same fruit of reticulated cv Sharlyn and profiled for cutin and suberin monomers by gas chromatography-mass spectrometry (GC-MS; Fig. 6C). Total cutin-associated monomers in the isolated reticulated regions was 42% lower as compared with the adjacent smooth surfaces; however, it accumulated 7-fold higher total suberin-associated monomers (Fig. 6D). In agreement with MALDI-MSI observations, ferulic acid was one of the most abundant metabolites in reticulated regions, being 26-fold higher as compared with the traces found in the adjacent smooth surfaces. In contrast, coumarate and caffeate, normally present in the cutin polyester, and particularly C16-10,16-dihydroxyacid (the most abundant fruit cutin monomer) were all significantly lower in reticulated regions. Levels of almost all other characteristic suberin monomers, including long-chain fatty acids, alcohols, α-hydroxyacids, ω-hydroxyacids, and α,ω-diacids, were massively higher in the reticulated regions and virtually absent in the adjacent smooth surfaces (Fig. 6E). Figure 6. Open in new tabDownload slide MSI and cutin and suberin composition analyses in reticulated and in-between smooth regions of fruit skin tissues. A, Light microscopy analysis of fruit skin samples analyzed using MALDI-MSI. B, MALDI-MSI showing the spatial distribution of ferulic acid (195.065 ± 0.001 mass-to-charge ratio [m/z]) located in the reticulated areas of reticulated skin samples but absent in the smooth skin samples. Dashed white lines represent skin surface areas analyzed by MALDI-MSI. Bars in A and B = 1 mm. C, Manual dissection of reticulated regions and the smooth regions in-between reticulated ones from cv Sharlyn fruit skin. D, Total cutin and suberin contents in skin tissues dissected as shown in C. E, Levels of individual cutin and suberin monomers in skin tissues dissected as in C. Cutin and suberin profiles in D and E were analyzed by GC-MS; y axes represent relative peak areas following normalization to a C32-alkane internal standard. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit). Significance was calculated according to Student’s t test: *, P < 0.05; **, P < 0.001; and ***, P < 0.0001. Figure 6. Open in new tabDownload slide MSI and cutin and suberin composition analyses in reticulated and in-between smooth regions of fruit skin tissues. A, Light microscopy analysis of fruit skin samples analyzed using MALDI-MSI. B, MALDI-MSI showing the spatial distribution of ferulic acid (195.065 ± 0.001 mass-to-charge ratio [m/z]) located in the reticulated areas of reticulated skin samples but absent in the smooth skin samples. Dashed white lines represent skin surface areas analyzed by MALDI-MSI. Bars in A and B = 1 mm. C, Manual dissection of reticulated regions and the smooth regions in-between reticulated ones from cv Sharlyn fruit skin. D, Total cutin and suberin contents in skin tissues dissected as shown in C. E, Levels of individual cutin and suberin monomers in skin tissues dissected as in C. Cutin and suberin profiles in D and E were analyzed by GC-MS; y axes represent relative peak areas following normalization to a C32-alkane internal standard. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit). Significance was calculated according to Student’s t test: *, P < 0.05; **, P < 0.001; and ***, P < 0.0001. Suberin and Cuticular Lipid Profiling of Smooth and Reticulated Fruit Skin At this stage, we carried out a comparative profiling of suberin and cutin polyesters, as well as epicuticular waxes, in skin tissue dissected from the six investigated cultivars. A principle component analysis (PCA) plot (57 compounds in total) could clearly distinguish between smooth and reticulated skin samples (Supplemental Fig. S1). Twenty-two cutin and suberin monomers, including aromatics, fatty acids, alcohols, α-hydroxyacids, ω-hydroxyacids, and α,ω-diacids, were identified based on mass fragmentation patterns (Fig. 7A). The induction of suberin apparently affected the synthesis of cutin, as reticulated skin samples displayed a major decrease in its predominant aromatic domains coumarate and caffeate and lower levels of short-chain C16-C20 fatty acids, C16 and C18 ω-hydroxyacids, and C16-10,16-dihydroxyacid (Fig. 7A). In contrast, ferulate was significantly higher in all three reticulated skin samples compared with smooth skin (Fig. 7B). Apart from ferulate, reticulated skin samples accumulated higher levels of typical long-chain suberin monomers, including fatty acids (C22-C24), alcohols (C22-C28), ω-hydroxyacids (C20-C24), and α,ω-diacids (C16-C18; Fig. 7B). Figure 7. Open in new tabDownload slide Differential profiles of cutin, suberin, and epicuticular waxes in skin of smooth and reticulated fruit. Profiles are shown for cutin monomers (A), suberin monomers (B), and epicuticular waxes (C) in skin of smooth and reticulated fruit. All lipophilic compounds were analyzed by GC-MS; y axes in A and B represent the relative peak areas following normalization to a C32-alkane internal standard, while that in C represents relative peak areas following normalization to a C36-alkane internal standard. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit). Significance was calculated according to two-way ANOVA of P < 0.05 per metabolite class, where lowercase italic letters above bars represent statistical significance. Figure 7. Open in new tabDownload slide Differential profiles of cutin, suberin, and epicuticular waxes in skin of smooth and reticulated fruit. Profiles are shown for cutin monomers (A), suberin monomers (B), and epicuticular waxes (C) in skin of smooth and reticulated fruit. All lipophilic compounds were analyzed by GC-MS; y axes in A and B represent the relative peak areas following normalization to a C32-alkane internal standard, while that in C represents relative peak areas following normalization to a C36-alkane internal standard. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit). Significance was calculated according to two-way ANOVA of P < 0.05 per metabolite class, where lowercase italic letters above bars represent statistical significance. The metabolic rearrangements in reticulated skin cutin and suberin profiles were accompanied by substantial modification in epicuticular wax contents, including altered profiles of wax-derived fatty acids, n-alkanes, alcohols, and aldehydes (Fig. 7C). Major accumulation of short-chain fatty acids C16-C22 was detected in reticulated compared with smooth skin samples, which were accompanied by reductions in long-chain C24-C34 fatty acids (Fig. 7C). Reticulated skin samples contained significantly less C21-C33 n-alkanes as well as lowered levels of C22-C28 alcohols and C26-C30 aldehydes (Fig. 7C). Metabolomics of Skin Tissues Derived from Smooth and Reticulated Fruit We employed ultra performance-liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) for metabolomics analysis that detects mainly semipolar compounds. PCA displayed a clear separation between smooth and reticulated skin samples based on 870 raw mass signals that were detected in the subsets of both skin samples (Supplemental Fig. S2). Following filtering for signal thresholds and peak annotation (see “Materials and Methods”), we were able to positively annotate 17 semipolar phenylpropanoid pathway compounds showing differential accumulation between reticulated and smooth skin. The levels of nine out of 17 metabolites were higher in reticulated skin, including feruloyl and sinapoyl derivatives, vanillic acid, ferulic and benzoic acid hexoses, and two lariciresinol lignan isomers (Fig. 8A). Conversely, seven derivatives of five different flavonoids (i.e. luteolin, kaempferol, apigenin, isorhamnetin, and naringenin) as well as hydroxycinnamic acid hexose displayed lower abundance in reticulated skin samples (Fig. 8B). These findings imply that phenylpropanoid metabolism tightly associated with the suberin and lignin pathways is boosted in reticulated skin, while the activity of the branch leading to the biosynthesis of flavonoids is reduced. Figure 8. Open in new tabDownload slide Phenylpropanoid pathway metabolites are differentially accumulated in reticulated versus smooth fruit skin tissues. Compositional profiles are shown for phenylpropanoids (A) and flavonoids (B) in skin of smooth and reticulated fruit. Bar graphs represent the relative abundance of 17 annotated differential phenylpropanoid pathway metabolites and flavonoids in smooth and reticulated skin samples. MS data were obtained by UPLC-qTOF-MS in negative electrospray ionization mode. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit). Significance was calculated according to two-way ANOVA of P < 0.05 per metabolite, where lowercase italic letters above bars represent statistical significance. Figure 8. Open in new tabDownload slide Phenylpropanoid pathway metabolites are differentially accumulated in reticulated versus smooth fruit skin tissues. Compositional profiles are shown for phenylpropanoids (A) and flavonoids (B) in skin of smooth and reticulated fruit. Bar graphs represent the relative abundance of 17 annotated differential phenylpropanoid pathway metabolites and flavonoids in smooth and reticulated skin samples. MS data were obtained by UPLC-qTOF-MS in negative electrospray ionization mode. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit). Significance was calculated according to two-way ANOVA of P < 0.05 per metabolite, where lowercase italic letters above bars represent statistical significance. Cutin and Suberin Monomer Accumulation during Fruit Skin Development We next investigated reticulation by tracking the accumulation of cutin and suberin polyesters in skin tissues isolated from smooth (cv Sakata’s Sweet) and reticulated (cv Sharlyn) fruit at seven developmental stages. At 30 d after fertilization (DAF) we detected the first signs of reticulation near the fruit blossom scar of cv Sharlyn fruit. From this stage up to 90 DAF, reticulation gradually covered the skin surface, with the reticulated structures protruding high above the fruit skin surface (Fig. 9A). Examining the levels of individual cutin and suberin monomers during the development of the two fruit types indicated several suberin-associated monomers that accumulated mostly during the development of reticulated skin fruit while maintaining relatively stable low levels during development in smooth skin fruit. These included ferulate, C20, C22, and C24 fatty acids, C22, C24, and C28 fatty alcohols, and C20, C22, and C24 ω-hydroxyacids (Fig. 9B). In contrast, the two cutin-associated coumarate and caffeate aromatic compounds displayed higher levels in smooth compared with reticulated skin, particularly starting from mid to later stages of fruit development. A similar trend was also detected in the two most abundant monomers of the cutin polyester, C16 ω-hydroxyacid and C16-10,16-dihydroxyacid (Fig. 9B). Finally, C16 and C18 α,ω-diacids, which are associated with suberin, exhibited relatively continual increase along the development of the two skin types, starting from early stages up to 70 DAF, from which reticulated skin accumulated much higher levels toward full fruit maturity (Fig. 9B). Figure 9. Open in new tabDownload slide Patterns of cutin and suberin monomer accumulation during fruit skin development. A, Representative images of fruit at seven developmental stages (smooth cv Sakata’s Sweet and reticulated cv Sharlyn fruit). B, Cutin and suberin monomer profiles during fruit development. Cutin and suberin monomers were analyzed by GC-MS; y axes represent relative peak areas following normalization to a C32-alkane internal standard. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit) at each developmental stage. Figure 9. Open in new tabDownload slide Patterns of cutin and suberin monomer accumulation during fruit skin development. A, Representative images of fruit at seven developmental stages (smooth cv Sakata’s Sweet and reticulated cv Sharlyn fruit). B, Cutin and suberin monomer profiles during fruit development. Cutin and suberin monomers were analyzed by GC-MS; y axes represent relative peak areas following normalization to a C32-alkane internal standard. Data represent means ± se of three biological replicates each per sample (generated from a pool of skin tissues from four different fruit) at each developmental stage. Revealing Transcriptional Hallmarks Associated with Fruit Skin Reticulation To gain deeper insight into the molecular mechanisms underlying fruit skin reticulation, we performed comparative RNA sequencing analyses of flesh and skin tissues manually dissected from the six investigated cultivars 21 DAF. This provided us with information regarding gene expression programs enriched in the skin tissue and identified genes dedicated to reticulation. PCA of the transcriptome data demonstrated relatively minor differences between flesh samples of smooth and reticulated fruit. Yet, we observed clear separation between skin samples of both fruit types, implying major transcriptional changes (Supplemental Fig. S3). We used the flesh transcriptome to isolate genes expressed at least 10 times higher in skin compared with flesh tissues (comparison performed per skin type; Fig. 10A). The analysis revealed 421 smooth skin-enriched genes (Supplemental Table S1), 196 reticulated skin-enriched genes (Supplemental Table S2), and 173 genes that were shared between the two data sets (i.e. skin-enriched genes in both fruit types; Fig. 10A; Supplemental Table S3). To monitor transcriptional patterns among these 173 shared genes, they were represented by a heat map. Two distinct gene clusters were clearly observed: one of skin-enriched genes highly expressed in reticulated compared with smooth fruit (cluster I; Fig. 10B) and the second composed of genes displaying lower expression in reticulated skin (cluster II; Fig. 10B). Figure 10. Open in new tabDownload slide Comparative transcriptome analysis reveals transcriptional hallmarks associated with fruit skin reticulation. A, Scheme representing the steps and cutoffs employed in order to identify skin-enriched genes in smooth and reticulated fruit. The Venn diagram demonstrates 421 smooth skin-enriched genes, 196 reticulated skin-enriched genes, and 173 genes that were common between the two gene subsets. B, Heat map representing the relative normalized expression of the 173 common skin-enriched genes in smooth and reticulated fruit shown in the Venn diagram in A. The heat map was calculated according to normalized log10-transformed gene expression levels, mean centered, and scaled by the sd of each gene. Cluster I represents a subset of genes that were highly expressed in reticulated skin, while cluster II represents a subset of genes that were highly expressed in smooth skin. Figure 10. Open in new tabDownload slide Comparative transcriptome analysis reveals transcriptional hallmarks associated with fruit skin reticulation. A, Scheme representing the steps and cutoffs employed in order to identify skin-enriched genes in smooth and reticulated fruit. The Venn diagram demonstrates 421 smooth skin-enriched genes, 196 reticulated skin-enriched genes, and 173 genes that were common between the two gene subsets. B, Heat map representing the relative normalized expression of the 173 common skin-enriched genes in smooth and reticulated fruit shown in the Venn diagram in A. The heat map was calculated according to normalized log10-transformed gene expression levels, mean centered, and scaled by the sd of each gene. Cluster I represents a subset of genes that were highly expressed in reticulated skin, while cluster II represents a subset of genes that were highly expressed in smooth skin. Reticulation Entails Transcriptional Reprogramming Associated with Skin Ligno-Suberization and Cell Wall Organization The 173 genes included in the above two clusters can be considered as fundamental transcriptional programs associated with skin ligno-suberization. Table 1 summarizes 50 genes of particular interest that exhibited significantly altered expression in reticulated versus smooth skin tissue. These genes belong to the metabolism of phenylpropanoids, suberin, lignin, lignans, cutin, and wax, genes involved in lipid polymer assembly and transport, and genes that partake of cell wall loosening (Table 1). List of skin-enriched genes associated with skin ligno-suberization Table 1. List of skin-enriched genes associated with skin ligno-suberization The list includes 50 genes that exhibited significantly altered expression in reticulated versus smooth skin tissue according to transcriptome analyses. The genes listed represent part of the 173 common genes in the Venn diagram presented in Figure 10A (see full list of common genes in Supplemental Table S3). MELO Gene IDa . AGIb . Gene Descriptionb . Full Gene Nameb . Fold Change Reticulated/Smoothc . Phenylpropanoid metabolism  MELO3C014227 AT2G37040 PAL1 PHENYLALANINE AMONIA LYASE1 9.1  MELO3C025786 AT3G53260 PAL2 PHENYLALANINE AMONIA LYASE2 3.5  MELO3C019585 AT2G30490 C4H CINNAMATE 4-HYDROXYLASE 4.6  MELO3C024886 AT3G21240 4CL2 4-COUMARATE:COENZYME A LIGASE2 4.7  MELO3C018450 AT4G34050 CCOAOMT1 CAFFEOYL COENZYME A O-METHYLTRANSFERASE1 6.4  MELO3C025328 AT3G24503 ALDH1A ALDEHYDE DEHYDROGENASE1A 0.2  MELO3C002400 AT1G07250 UGT71C4 UDP-GLUCOSYL TRANSFERASE71C4 0.4  MELO3C017219 AT5G07990 TT7 TRANSPARENT TESTA7 0.5 Suberin metabolism  MELO3C026188 AT5G41040 ASFT ALIPHATIC SUBERIN FERULOYL-TRANSFERASE 8.9  MELO3C023876 AT3G11430 GPAT5 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE5 8.8  MELO3C020561 AT5G58860 CYP86A1 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE1 6.2  MELO3C023592 AT5G23190 CYP86B1 CYTOCHROME P450 FAMILY 86 SUBFAMILY B POLYPEPTIDE1 9.8  MELO3C021431 AT4G33790 FAR3 FATTY ACID REDUCTASE3 11.0  MELO3C025486 AT3G55090 ABCG16 ATP-BINDING CASSETTE G16 5.5 Lignin metabolism  MELO3C020517 AT2G30210 LAC3 LACCASE3 5.6  MELO3C019994 AT5G05340 PRX52 PEROXIDASE52 7.3  MELO3C007868 AT5G66390 PRX72 PEROXIDASE72 6.5 Lignan metabolism  MELO3C022763 AT1G32100 PRR1 PINORESINOL REDUCTASE1 3.7  MELO3C016325 AT1G64160 DIR5 DIRIGENT PROTEIN5 5.9  MELO3C020796 AT2G21100 DIR-like DIRIGENT-LIKE PROTEIN 6.5  MELO3C019288 AT4G38700 DIR-like DIRIGENT-LIKE PROTEIN 5.5  MELO3C014667 AT1G58170 DIR-like DIRIGENT-LIKE PROTEIN 3.2 Cutin and wax metabolism  MELO3C016930 AT2G26250 KCS10 3-KETOACYL-COA SYNTHASE10 0.2  MELO3C014475 AT1G06520 GPAT1 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE1 0.7  MELO3C021206 AT1G01610 GPAT4 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE4 0.6  MELO3C009182 AT2G38110 GPAT6 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE6 0.6  MELO3C004537 AT2G26910 ABCG32 ATP-BINDING CASSETTE G32 0.4  MELO3C010341 AT1G15360 SHN1 SHINE1 1.8  MELO3C010174 AT5G57800 CER3 ECERIFERUM3 0.7  MELO3C012928 AT2G47240 LACS1 LONG-CHAIN ACYL-COENZYME A SYNTHASE1 0.4  MELO3C021205 AT2G45970 LCR LACERATA 0.6  MELO3C009874 AT3G28910 MYB30 MYB DOMAIN PROTEIN30 0.3 Lipid polymer assembly  MELO3C005686 AT1G63710 CYP86A7 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE7 0.5  MELO3C017177 AT1G74460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 4.9  MELO3C014442 AT2G04570 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C002330 AT3G48460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C010132 AT5G33370 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.2 Lipid transport  MELO3C007279 AT2G34700 Pollen Ole e 1 POLLEN OLE E 1 ALLERGEN AND EXTENSIN FAMILY PROTEIN 13.3  MELO3C014397 AT2G48140 EDA4 EMBRYO SAC DEVELOPMENT ARREST4 7.4  MELO3C023497 AT2G18370 LTP LIPID TRANSFER PROTEIN 12.1  MELO3C012662 AT3G22620 LTP LIPID TRANSFER PROTEIN 8.0  MELO3C025219 AT5G01870 LTP LIPID TRANSFER PROTEIN 0.5  MELO3C014762 AT5G13900 LTP LIPID TRANSFER PROTEIN 8.6 Cell wall loosening  MELO3C010017 AT1G65570 RCPD ROOT CAP POLYGALACTURONASE 5.7  MELO3C002319 AT5G63180 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 2.6  MELO3C006092 AT3G61490 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 5.4  MELO3C005567 AT1G62770 PMEI9 PECTIN METHYLESTERASE INHIBITOR9 13.3  MELO3C012108 AT1G69530 EXPA1 EXPANSIN A1 4.7  MELO3C018743 AT4G28250 EXPB3 EXPANSIN B3 1.7  MELO3C004941 AT2G36870 XTH32 XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE32 9.5 MELO Gene IDa . AGIb . Gene Descriptionb . Full Gene Nameb . Fold Change Reticulated/Smoothc . Phenylpropanoid metabolism  MELO3C014227 AT2G37040 PAL1 PHENYLALANINE AMONIA LYASE1 9.1  MELO3C025786 AT3G53260 PAL2 PHENYLALANINE AMONIA LYASE2 3.5  MELO3C019585 AT2G30490 C4H CINNAMATE 4-HYDROXYLASE 4.6  MELO3C024886 AT3G21240 4CL2 4-COUMARATE:COENZYME A LIGASE2 4.7  MELO3C018450 AT4G34050 CCOAOMT1 CAFFEOYL COENZYME A O-METHYLTRANSFERASE1 6.4  MELO3C025328 AT3G24503 ALDH1A ALDEHYDE DEHYDROGENASE1A 0.2  MELO3C002400 AT1G07250 UGT71C4 UDP-GLUCOSYL TRANSFERASE71C4 0.4  MELO3C017219 AT5G07990 TT7 TRANSPARENT TESTA7 0.5 Suberin metabolism  MELO3C026188 AT5G41040 ASFT ALIPHATIC SUBERIN FERULOYL-TRANSFERASE 8.9  MELO3C023876 AT3G11430 GPAT5 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE5 8.8  MELO3C020561 AT5G58860 CYP86A1 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE1 6.2  MELO3C023592 AT5G23190 CYP86B1 CYTOCHROME P450 FAMILY 86 SUBFAMILY B POLYPEPTIDE1 9.8  MELO3C021431 AT4G33790 FAR3 FATTY ACID REDUCTASE3 11.0  MELO3C025486 AT3G55090 ABCG16 ATP-BINDING CASSETTE G16 5.5 Lignin metabolism  MELO3C020517 AT2G30210 LAC3 LACCASE3 5.6  MELO3C019994 AT5G05340 PRX52 PEROXIDASE52 7.3  MELO3C007868 AT5G66390 PRX72 PEROXIDASE72 6.5 Lignan metabolism  MELO3C022763 AT1G32100 PRR1 PINORESINOL REDUCTASE1 3.7  MELO3C016325 AT1G64160 DIR5 DIRIGENT PROTEIN5 5.9  MELO3C020796 AT2G21100 DIR-like DIRIGENT-LIKE PROTEIN 6.5  MELO3C019288 AT4G38700 DIR-like DIRIGENT-LIKE PROTEIN 5.5  MELO3C014667 AT1G58170 DIR-like DIRIGENT-LIKE PROTEIN 3.2 Cutin and wax metabolism  MELO3C016930 AT2G26250 KCS10 3-KETOACYL-COA SYNTHASE10 0.2  MELO3C014475 AT1G06520 GPAT1 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE1 0.7  MELO3C021206 AT1G01610 GPAT4 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE4 0.6  MELO3C009182 AT2G38110 GPAT6 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE6 0.6  MELO3C004537 AT2G26910 ABCG32 ATP-BINDING CASSETTE G32 0.4  MELO3C010341 AT1G15360 SHN1 SHINE1 1.8  MELO3C010174 AT5G57800 CER3 ECERIFERUM3 0.7  MELO3C012928 AT2G47240 LACS1 LONG-CHAIN ACYL-COENZYME A SYNTHASE1 0.4  MELO3C021205 AT2G45970 LCR LACERATA 0.6  MELO3C009874 AT3G28910 MYB30 MYB DOMAIN PROTEIN30 0.3 Lipid polymer assembly  MELO3C005686 AT1G63710 CYP86A7 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE7 0.5  MELO3C017177 AT1G74460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 4.9  MELO3C014442 AT2G04570 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C002330 AT3G48460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C010132 AT5G33370 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.2 Lipid transport  MELO3C007279 AT2G34700 Pollen Ole e 1 POLLEN OLE E 1 ALLERGEN AND EXTENSIN FAMILY PROTEIN 13.3  MELO3C014397 AT2G48140 EDA4 EMBRYO SAC DEVELOPMENT ARREST4 7.4  MELO3C023497 AT2G18370 LTP LIPID TRANSFER PROTEIN 12.1  MELO3C012662 AT3G22620 LTP LIPID TRANSFER PROTEIN 8.0  MELO3C025219 AT5G01870 LTP LIPID TRANSFER PROTEIN 0.5  MELO3C014762 AT5G13900 LTP LIPID TRANSFER PROTEIN 8.6 Cell wall loosening  MELO3C010017 AT1G65570 RCPD ROOT CAP POLYGALACTURONASE 5.7  MELO3C002319 AT5G63180 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 2.6  MELO3C006092 AT3G61490 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 5.4  MELO3C005567 AT1G62770 PMEI9 PECTIN METHYLESTERASE INHIBITOR9 13.3  MELO3C012108 AT1G69530 EXPA1 EXPANSIN A1 4.7  MELO3C018743 AT4G28250 EXPB3 EXPANSIN B3 1.7  MELO3C004941 AT2G36870 XTH32 XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE32 9.5 a MELO gene IDs correspond to the melon genome (Garcia-Mas et al., 2012) b Arabidopsis Genome Initiative (AGI) number, gene description, and full gene name are according to TAIR (https://www.arabidopsis.org/). c Statistically significant fold changes between the average gene expression in smooth versus reticulated skin samples according to Student’s t test at P < 0.05. Open in new tab Table 1. List of skin-enriched genes associated with skin ligno-suberization The list includes 50 genes that exhibited significantly altered expression in reticulated versus smooth skin tissue according to transcriptome analyses. The genes listed represent part of the 173 common genes in the Venn diagram presented in Figure 10A (see full list of common genes in Supplemental Table S3). MELO Gene IDa . AGIb . Gene Descriptionb . Full Gene Nameb . Fold Change Reticulated/Smoothc . Phenylpropanoid metabolism  MELO3C014227 AT2G37040 PAL1 PHENYLALANINE AMONIA LYASE1 9.1  MELO3C025786 AT3G53260 PAL2 PHENYLALANINE AMONIA LYASE2 3.5  MELO3C019585 AT2G30490 C4H CINNAMATE 4-HYDROXYLASE 4.6  MELO3C024886 AT3G21240 4CL2 4-COUMARATE:COENZYME A LIGASE2 4.7  MELO3C018450 AT4G34050 CCOAOMT1 CAFFEOYL COENZYME A O-METHYLTRANSFERASE1 6.4  MELO3C025328 AT3G24503 ALDH1A ALDEHYDE DEHYDROGENASE1A 0.2  MELO3C002400 AT1G07250 UGT71C4 UDP-GLUCOSYL TRANSFERASE71C4 0.4  MELO3C017219 AT5G07990 TT7 TRANSPARENT TESTA7 0.5 Suberin metabolism  MELO3C026188 AT5G41040 ASFT ALIPHATIC SUBERIN FERULOYL-TRANSFERASE 8.9  MELO3C023876 AT3G11430 GPAT5 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE5 8.8  MELO3C020561 AT5G58860 CYP86A1 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE1 6.2  MELO3C023592 AT5G23190 CYP86B1 CYTOCHROME P450 FAMILY 86 SUBFAMILY B POLYPEPTIDE1 9.8  MELO3C021431 AT4G33790 FAR3 FATTY ACID REDUCTASE3 11.0  MELO3C025486 AT3G55090 ABCG16 ATP-BINDING CASSETTE G16 5.5 Lignin metabolism  MELO3C020517 AT2G30210 LAC3 LACCASE3 5.6  MELO3C019994 AT5G05340 PRX52 PEROXIDASE52 7.3  MELO3C007868 AT5G66390 PRX72 PEROXIDASE72 6.5 Lignan metabolism  MELO3C022763 AT1G32100 PRR1 PINORESINOL REDUCTASE1 3.7  MELO3C016325 AT1G64160 DIR5 DIRIGENT PROTEIN5 5.9  MELO3C020796 AT2G21100 DIR-like DIRIGENT-LIKE PROTEIN 6.5  MELO3C019288 AT4G38700 DIR-like DIRIGENT-LIKE PROTEIN 5.5  MELO3C014667 AT1G58170 DIR-like DIRIGENT-LIKE PROTEIN 3.2 Cutin and wax metabolism  MELO3C016930 AT2G26250 KCS10 3-KETOACYL-COA SYNTHASE10 0.2  MELO3C014475 AT1G06520 GPAT1 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE1 0.7  MELO3C021206 AT1G01610 GPAT4 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE4 0.6  MELO3C009182 AT2G38110 GPAT6 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE6 0.6  MELO3C004537 AT2G26910 ABCG32 ATP-BINDING CASSETTE G32 0.4  MELO3C010341 AT1G15360 SHN1 SHINE1 1.8  MELO3C010174 AT5G57800 CER3 ECERIFERUM3 0.7  MELO3C012928 AT2G47240 LACS1 LONG-CHAIN ACYL-COENZYME A SYNTHASE1 0.4  MELO3C021205 AT2G45970 LCR LACERATA 0.6  MELO3C009874 AT3G28910 MYB30 MYB DOMAIN PROTEIN30 0.3 Lipid polymer assembly  MELO3C005686 AT1G63710 CYP86A7 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE7 0.5  MELO3C017177 AT1G74460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 4.9  MELO3C014442 AT2G04570 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C002330 AT3G48460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C010132 AT5G33370 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.2 Lipid transport  MELO3C007279 AT2G34700 Pollen Ole e 1 POLLEN OLE E 1 ALLERGEN AND EXTENSIN FAMILY PROTEIN 13.3  MELO3C014397 AT2G48140 EDA4 EMBRYO SAC DEVELOPMENT ARREST4 7.4  MELO3C023497 AT2G18370 LTP LIPID TRANSFER PROTEIN 12.1  MELO3C012662 AT3G22620 LTP LIPID TRANSFER PROTEIN 8.0  MELO3C025219 AT5G01870 LTP LIPID TRANSFER PROTEIN 0.5  MELO3C014762 AT5G13900 LTP LIPID TRANSFER PROTEIN 8.6 Cell wall loosening  MELO3C010017 AT1G65570 RCPD ROOT CAP POLYGALACTURONASE 5.7  MELO3C002319 AT5G63180 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 2.6  MELO3C006092 AT3G61490 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 5.4  MELO3C005567 AT1G62770 PMEI9 PECTIN METHYLESTERASE INHIBITOR9 13.3  MELO3C012108 AT1G69530 EXPA1 EXPANSIN A1 4.7  MELO3C018743 AT4G28250 EXPB3 EXPANSIN B3 1.7  MELO3C004941 AT2G36870 XTH32 XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE32 9.5 MELO Gene IDa . AGIb . Gene Descriptionb . Full Gene Nameb . Fold Change Reticulated/Smoothc . Phenylpropanoid metabolism  MELO3C014227 AT2G37040 PAL1 PHENYLALANINE AMONIA LYASE1 9.1  MELO3C025786 AT3G53260 PAL2 PHENYLALANINE AMONIA LYASE2 3.5  MELO3C019585 AT2G30490 C4H CINNAMATE 4-HYDROXYLASE 4.6  MELO3C024886 AT3G21240 4CL2 4-COUMARATE:COENZYME A LIGASE2 4.7  MELO3C018450 AT4G34050 CCOAOMT1 CAFFEOYL COENZYME A O-METHYLTRANSFERASE1 6.4  MELO3C025328 AT3G24503 ALDH1A ALDEHYDE DEHYDROGENASE1A 0.2  MELO3C002400 AT1G07250 UGT71C4 UDP-GLUCOSYL TRANSFERASE71C4 0.4  MELO3C017219 AT5G07990 TT7 TRANSPARENT TESTA7 0.5 Suberin metabolism  MELO3C026188 AT5G41040 ASFT ALIPHATIC SUBERIN FERULOYL-TRANSFERASE 8.9  MELO3C023876 AT3G11430 GPAT5 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE5 8.8  MELO3C020561 AT5G58860 CYP86A1 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE1 6.2  MELO3C023592 AT5G23190 CYP86B1 CYTOCHROME P450 FAMILY 86 SUBFAMILY B POLYPEPTIDE1 9.8  MELO3C021431 AT4G33790 FAR3 FATTY ACID REDUCTASE3 11.0  MELO3C025486 AT3G55090 ABCG16 ATP-BINDING CASSETTE G16 5.5 Lignin metabolism  MELO3C020517 AT2G30210 LAC3 LACCASE3 5.6  MELO3C019994 AT5G05340 PRX52 PEROXIDASE52 7.3  MELO3C007868 AT5G66390 PRX72 PEROXIDASE72 6.5 Lignan metabolism  MELO3C022763 AT1G32100 PRR1 PINORESINOL REDUCTASE1 3.7  MELO3C016325 AT1G64160 DIR5 DIRIGENT PROTEIN5 5.9  MELO3C020796 AT2G21100 DIR-like DIRIGENT-LIKE PROTEIN 6.5  MELO3C019288 AT4G38700 DIR-like DIRIGENT-LIKE PROTEIN 5.5  MELO3C014667 AT1G58170 DIR-like DIRIGENT-LIKE PROTEIN 3.2 Cutin and wax metabolism  MELO3C016930 AT2G26250 KCS10 3-KETOACYL-COA SYNTHASE10 0.2  MELO3C014475 AT1G06520 GPAT1 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE1 0.7  MELO3C021206 AT1G01610 GPAT4 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE4 0.6  MELO3C009182 AT2G38110 GPAT6 GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE6 0.6  MELO3C004537 AT2G26910 ABCG32 ATP-BINDING CASSETTE G32 0.4  MELO3C010341 AT1G15360 SHN1 SHINE1 1.8  MELO3C010174 AT5G57800 CER3 ECERIFERUM3 0.7  MELO3C012928 AT2G47240 LACS1 LONG-CHAIN ACYL-COENZYME A SYNTHASE1 0.4  MELO3C021205 AT2G45970 LCR LACERATA 0.6  MELO3C009874 AT3G28910 MYB30 MYB DOMAIN PROTEIN30 0.3 Lipid polymer assembly  MELO3C005686 AT1G63710 CYP86A7 CYTOCHROME P450 FAMILY 86 SUBFAMILY A POLYPEPTIDE7 0.5  MELO3C017177 AT1G74460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 4.9  MELO3C014442 AT2G04570 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C002330 AT3G48460 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.3  MELO3C010132 AT5G33370 GDSL-like GDSL-MOTIF ESTERASE/ACYLTRANSFERASE/LIPASE 0.2 Lipid transport  MELO3C007279 AT2G34700 Pollen Ole e 1 POLLEN OLE E 1 ALLERGEN AND EXTENSIN FAMILY PROTEIN 13.3  MELO3C014397 AT2G48140 EDA4 EMBRYO SAC DEVELOPMENT ARREST4 7.4  MELO3C023497 AT2G18370 LTP LIPID TRANSFER PROTEIN 12.1  MELO3C012662 AT3G22620 LTP LIPID TRANSFER PROTEIN 8.0  MELO3C025219 AT5G01870 LTP LIPID TRANSFER PROTEIN 0.5  MELO3C014762 AT5G13900 LTP LIPID TRANSFER PROTEIN 8.6 Cell wall loosening  MELO3C010017 AT1G65570 RCPD ROOT CAP POLYGALACTURONASE 5.7  MELO3C002319 AT5G63180 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 2.6  MELO3C006092 AT3G61490 Pectin lyase-like PECTIN LYASE SUPERFAMILY PROTEIN 5.4  MELO3C005567 AT1G62770 PMEI9 PECTIN METHYLESTERASE INHIBITOR9 13.3  MELO3C012108 AT1G69530 EXPA1 EXPANSIN A1 4.7  MELO3C018743 AT4G28250 EXPB3 EXPANSIN B3 1.7  MELO3C004941 AT2G36870 XTH32 XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE32 9.5 a MELO gene IDs correspond to the melon genome (Garcia-Mas et al., 2012) b Arabidopsis Genome Initiative (AGI) number, gene description, and full gene name are according to TAIR (https://www.arabidopsis.org/). c Statistically significant fold changes between the average gene expression in smooth versus reticulated skin samples according to Student’s t test at P < 0.05. Open in new tab The expression of five putative genes associated with the phenylpropanoid pathway was significantly higher in reticulated skin: PHENYLALANINE AMMONIA-LYASE1 (PAL1) and PAL2, CINNAMATE 4-HYDROXYLASE, 4-COUMARATE:COENZYME A LIGASE2, and CAFFEOYL COENZYME A O-METHYLTRANSFERASE1 (Table 1). On the other hand, ALDEHYDE DEHYDROGENASE1A, UDP-GLUCOSYL TRANSFERASE71C4, and TRANSPARENT TESTA7 all were reduced in reticulated skin (Table 1). As expected, the detection of distinct suberized cells within reticulated regions was corroborated by massive induction in the expression of putative suberin biosynthetic genes, including ALIPHATIC SUBERIN FERULOYL-TRANSFERASE, utilizing ferulate derivatives supplied from the phenylpropanoid pathway to form aromatic suberin metabolites (Li et al., 2007; Gou et al., 2009); GLYCEROL 3-PHOSPHATE sn-2-ACETYLTRANSFERASE5 (GPAT5), required for the synthesis of glycerol-based suberin monomers (Beisson et al., 2007); CYP86A1 and CYP86B1, two P450s encoding fatty acyl ω-hydroxylases involved in the formation of ω-hydroxy long-chain fatty acid suberin building blocks (Höfer et al., 2008; Compagnon et al., 2009; Molina et al., 2009); FATTY ACID REDUCTASE3, which generates long-chain fatty alcohols associated with suberin (Domergue et al., 2010; Vishwanath et al., 2013); and ATP-BINDING CASSETTE G16 (ABCG16), involved in the transport of suberin monomers from their biosynthetic sites toward their deposition sites at the plasma membrane (Yadav et al., 2014; Table 1). The list includes genes associated with the metabolism of lignin and lignans, two pathways that, as suberin tightly depends on precursor supply from the phenylpropanoid pathway, were also induced in reticulated skin. LACCASE3 and two peroxidases (PRX52 and PRX72) that oxidize monolignols in the last step of lignin biosynthesis, PINORESINOL REDUCTASE1, DIRIGENT PROTEIN5 (DIR5), and an additional three putative DIR-like genes all exhibited induced expression in reticulated skin (Table 1). In contrast, a subset of genes putatively associated with the metabolism of cutin and wax showed significantly lower expression in reticulated skin samples compared with smooth ones. This set of genes included the melon homologs of 3-KETOACYL-COENZYME A SYNTHASE10, GPAT1, GPAT4, and GPAT6, ABCG32, ECCERIFERUM3, LONG-CHAIN ACYL-COENZYME A SYNTHASE1, LACERATA, and the regulator MYB30 (Table 1). It appears that the significant changes observed in the expression of genes involved in the lipophilic barriers suberin, lignin, cutin, and wax were followed by altered expression of genes associated with lipid polymer assembly and transport. These include CYP86A7 and four putative GDSL ESTERASE/LIPASEs that were mostly reduced in reticulated skin. However, apart from one putative LIPID TRANSPORT PROTEIN (LTP), three additional LTPs as well as POLLEN OLE E1 ALLERGEN AND EXTENSIN FAMILY PROTEIN and EMBRYO SAC DEVELOPMENT ARREST4 all displayed substantial inductions in reticulated skin, suggesting possible roles in suberin polymer assembly (Table 1). Finally, it seems that skin suberization involves noteworthy modifications of the cell wall, as we detected the induction of several putative genes involved in cell wall-loosening processes, such as ROOT CAP POLYGALACTURONASE, two PECTIN-LYASE-like genes, and PECTIN METHYLESTERASE INHIBITOR9 that degrade cell wall pectin, as well as EXPANSIN A1, EXPANSIN B3, and XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE32 that putatively mediates splitting and reconnection of xyloglucan cross-links in the cell wall (Hara et al., 2014). DISCUSSION Reticulated Structures Comprise Specialized Suberized and Lignified Cells The skin of fruit constitutes the interface between the outer environment and the inner fruit fleshy tissues. As such, it serves as a mechanical protection layer and a physiological barrier controlling the exchange of water, solutes, and gases. Therefore, incomplete and/or severe disruptions of the skin result in negative outcomes to fruit development. Fruit skin failure can result from abrupt mechanical stress but also as part of a particular phase in fruit development, as occurs in many melon varieties. Early investigations suggested that during melon fruit enlargement, fissures of the outermost skin epidermal layer lead to continuous expansion of cracks (Meissner, 1952). These processes then induce rapid division of suberized and/or lignified cells as part of the developmental wounding response of the fruit skin. One hypothesis suggests that in cases when epidermal cells divide very rapidly and fruit enlarges, these cells are disrupted (Webster and Craig, 1976). Another premise proposes that the lower cell density of melon fruit epidermis weakens the epidermal cells, and this results in increased susceptibility to rupturing forces (Keren-Keiserman et al., 2004b). These reports provided the first link between melon fruit reticulation and skin ligno-suberization processes. Yet, they are merely based on primary histological techniques and lack in-depth examination of the reticulated structure and moreover its molecular basis. In this study, we carried out a multilevel analysis of skin reticulation in melon fruit and gained new insights into hallmarks of fruit skin ligno-suberization. Unique reticulated structures appeared roughly 30 DAF surrounding the blossom scar, gradually covering larger areas of skin surfaces throughout fruit development. At full maturity, fruit comprised a thick network of reticulation, yet to different degrees and shapes, in agreement with previous reports (Keren-Keiserman et al., 2004b; Li-Ying et al., 2011). Reticulation was visible already at earlier stages when fruit size was much smaller compared with its size at full maturity, implicating that fruit size and likely shape are not prime factors in the process of reticulation. In the same way, former studies in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) found no association between skin cracking density and fruit size (Johnson and Knavel, 1990; Ehret et al., 1993; Emmons and Scott, 1998). We show by several high-resolution microscopical approaches that both smooth and reticulated fruit possess characteristic thick cuticles covering their skin epidermal layer, although in reticulated ones these are severely disrupted by large cracks. Histochemical staining with specific dyes for suberin and lignin implies that these cracks are subsequently sealed by a mass of suberized cells localized explicitly at the upper layer of reticulation, while the lower cell layers underneath appear to be lignified. Suberized periderm confined to the outermost layer of fruit skin surface was also reported in potato tubers (Kolattukudy, 1981; Graça and Pereira, 2000), wounded tomato (Tao et al., 2016), and kiwifruit (Actinidia deliciosa; Han et al., 2017). In the same way, we assume that the initiation of ligno-suberization in melon is part of a developmental wound response occurring when the magnitude of tension forces applied on skin surfaces during fruit enlargement exceeds a certain threshold. Skin Reticulation in Developing Melon Fruit Is Associated with Key Metabolic Adjustments Several lines of evidence implied the involvement of suberin metabolism in reticulation, including the presence of suberin lamellae solely in the walls of cells that cover reticulated regions and the accumulation of suberin monomers in reticulated structures but not in smooth regions. These include typical aliphatic suberin monomers such as C22-C24 fatty acids, C22 alcohol, C20-C24 ω-hydroxyacids, and C16-C18 α,ω-diacids. Apparently, the accumulation of suberin monomers during the reticulation process has a substantial effect on cutin metabolism, leading to reduced levels of some cutin monomers, including C16 fatty acid, C16 and C18 ω-hydroxyacids, and particularly the most abundant fruit cutin monomer, C16-10,16-dihdroxyacid. We cannot rule out that the reduction in cutin-type monomers in reticulated skin might result from lower fractions of cutinized skin surface that is occupied by the reticulation structure. Still, these metabolic shifts suggest a tight association between damage to the fruit skin and the induction of suberization. These changes observed here in melon fruit accord with previous reports in other fruit species. For instance, in apple, russeting is considered as suberization of the skin layer that occurs naturally due to cuticular damage during fruit development (Khanal et al., 2013; Lashbrooke et al., 2015). Likewise, transgenic tomato with reduced expression of the cutin-associated protein DEFECTIVE IN CUTICULAR RIDGES displayed severe damage of the fruit skin that was covered by a rough suberized coating layer (Lashbrooke et al., 2016). Similar to reticulation in melon fruit, in both cases the induction of suberization was accompanied by intense changes in gene expression and metabolite profiles of cutin. Yet, the relationship between the biosynthetic pathways of cutin and suberin is likely far more complex, as the fruit skin of cutin deficient (cd) tomato mutants with perturbations in cutin biosynthesis (cd1 and cd3) or regulation (cd2) accumulated significantly lower levels of cutin metabolites but showed no signs of skin injury or suberization (Isaacson et al., 2009). The overaccumulation of suberin and lignin resulted in a significant shift in the profiles of aromatic components. Reticulated fruit skin accumulated suberin-associated ferulate at the expense of cutin-associated coumarate and caffeate. Suberin and lignin biosynthesis depends tightly on the supply of core phenylpropanoid pathway by-products utilized to produce suberin feruloyl monomers and lignin coumaryl, coniferyl, and sinapyl alcohols (Fraser and Chapple, 2011). Yet, an additional branch of the core phenylpropanoid pathway leads to flavonoid biosynthesis. Metabolic shifts in between branches of the phenylpropanoid pathway thus explain much lower levels of kaempferol, apigenin, naringenin, rhamnetin, and luteolin derivatives and larger amounts of feruloyl/sinapoyl derivatives and lignans in the reticulated skin. Interplay between epidermal suberin and cutin was also reported in russeted apple (Legay et al., 2017) and tomato (Lashbrooke et al., 2016) fruit. In a different study, Nicotiana benthamiana leaves agroinfiltrated with the apple MdMYB93 suberin pathway regulator displayed massive accumulation of suberin and its precursors, remobilization of phenylpropanoids, and increased levels of lignin precursors (Legay et al., 2016). This study, however, did not report changes in the levels of typical cutin monomers. Microscopy observations of skin surfaces suggested lower amounts of epicuticular wax crystals in reticulated compared with smooth skin, as later supported by major differences in wax composition. Increased levels of relatively short C16, C18, C20, and C22 fatty acids were detected in reticulated skin that were accompanied by substantial reductions in longer chain C26, C28, C30, and C32 fatty acids. Notably, we detected a dramatic reduction in almost all n-alkanes, alcohols, and aldehydes in reticulated skin wax. As in the case of melon fruit reticulated structures, a strong decrease in levels of waxes and triterpene contents was recently reported in russet regions of the semirusset apple variety Cox Orange Pippin (Legay et al., 2017). This suggests that developmental suberization of the fruit skin possesses substantial effects on its abilities to maintain epicuticular wax loads. As both fruit displayed lower amounts of very-long-chain fatty acid wax derivatives, we propose that the biosynthesis of these compounds is apparently delayed and/or decreased upon the formation of suberin, likely due to the rewiring of very-long-chain fatty acids toward the synthesis of suberin building blocks required for reticulation structures. The Transcriptional Hallmarks of Reticulation Transcriptional profiling showed that putative cutin and wax biosynthetic genes are repressed while phenylpropanoid biosynthetic genes and suberin deposition genes are highly active in the reticulated melon skin. This underlined the tight link between reticulation and suberization. We detected the induction of genes putatively involved in all steps of suberin metabolism, including biosynthesis of both aliphatic and aromatic monoacylglycerols, ω-hydroxylation, formation of alcohol monomers, and transport of suberin building blocks. At the same time, the melon homologs of GPAT4 and GPAT6, two well-characterized genes that are crucial for the synthesis of C16-based cutin monomers (Yang et al., 2010), showed lower expression values in reticulated skin, together with the ABCG11 homolog that is likely required for cutin transport to the extracellular matrix (Panikashvili et al., 2010). Repression of cutin-associated genes alongside increased expression of suberin genes were previously reported as distinct transcriptional signatures of suberin induction in fruit species possessing suberized skin phenotypes, including russet apple (Legay et al., 2015), russet pear (Wang et al., 2016), suberized bark tissues of poplar (Populus tremula × Populus alba; Rains et al., 2018), and severely wounded skin of tomato fruit (Lashbrooke et al., 2016). Our data also point at several GDSL genes that exhibited increased or decreased expression in either smooth or reticulated skin samples, implicating possible roles in the assembly of cutin and/or suberin polymers. This will necessitate further functional characterization. Genes involved in lignin also displayed higher expression in reticulated skin, in agreement with histochemical lignin staining marking the existence of lignified cells beneath the outer epidermal suberized cell layer. They included PRX52 and PRX72, two melon homolog peroxidases that are closely related to lignification processes (Herrero et al., 2013; Fernández-Pérez et al., 2015). Accordingly, high peroxidase activity was previously reported in reticulated regions of melon fruit skin (Keren-Keiserman et al., 2004b). As recently reported for suberized potato tuber skin (Vulavala et al., 2017), homologs of the Arabidopsis (Arabidopsis thaliana) CASP family members mediating lignification through the recruitment of its polymerization machinery (Roppolo et al., 2014) also displayed up-regulated expression in reticulated melon skin. The occurrence of fissures and microcracking of the fruit skin together with the formation of a ligno-suberized layer most likely requires activity of the enzymes involved in cell wall rearrangements. Indeed, several genes putatively encoding proteins associated with pectin and xyloglucan hydrolysis (Pauly and Keegstra, 2016) exhibited increased expression in the reticulated skin. Moreover, expression of genes putatively encoding EXPANSIN and EXTENSIN proteins, previously shown to play substantial roles in cell wall softening and breakdown (Li et al., 2003), was up-regulated in the reticulated skin. Thus, the transcriptome data suggested that reticulation in melon fruit is accompanied by dramatic structural rearrangements of the skin epidermal layer. Taken together, our findings provide insights into the molecular and metabolic foundations of skin reticulation and its tight association with the process of ligno-suberization. Moreover, we anticipate that the data obtained will serve as a valuable resource for discovering new genetic components associated with suberin metabolism and function. As fruit reticulation is one of several key features considered in melon breeding, the knowledge gained through this study could also be of value in fruit postharvest quality trait improvement. MATERIALS AND METHODS Plant Material and Growth Conditions Plants of smooth and reticulated skin cultivars of melon (Cucumis melo) were grown until fruit reached full maturity in a net house in Rehovot, Israel, and periodically fertilized with 20:20:20 N:P:K. Mature fruit were harvested from at least four independent plants of each cultivar, and flesh and skin tissues were manually dissected and stored at −80°C for further RNA extraction and LC-MS analyses. Alternatively, skin tissues were freshly extracted for cutin, suberin, and epicuticular wax analyses using GC-MS or immersed in fixation buffers for histology and microscopy. Microscopy For histological studies of skin morphology, skin samples from all six investigated fruit were immediately fixed in 4% (v/v) PFA and 3% (v/v) GA. The samples were next dehydrated in a graded ethanol series and infiltrated with paraffin. Sections of 6 µm were obtained using a Leica 2000 microtome and mounted on glass slides. The slides were then counterstained for 1 min with a freshly prepared solution of Toluidine Blue O (0.05% [w/v] in double distilled water) at room temperature and observed with an Olympus CLSM500 microscope. Histochemical observations of suberin and lignin in skin cross sections were achieved by staining with a freshly prepared solution of Fluorol Yellow 088 (0.01% [w/v] in lactic acid) for 30 min at 70°C and phloroglucinol-hydrochloric acid (2% [w/v] in 50% hydrochloric acid) for 30 min at room temperature, respectively. Stained sections were then observed with an Olympus CLSM500 microscope using bright-field and GFP filters. Autofluorescence of cuticle layers in smooth skin sections was observed with a Cy5 filter. For SEM and TEM imaging, skin samples were gently isolated from fresh fruits and immediately fixed in electron microscopy-grade 4% (v/v) PFA and 2% (v/v) GA in cacodylate 0.1 m buffer. PFA and GA were selected as fixation reagents to have minimal effects on skin epicuticular wax composition. Prior to SEM analyses, skin samples were carefully dehydrated according to the critical point dehydration method to avoid changes in skin morphology (Halbritter, 1998). Following dehydration, samples were mounted on SEM holders, coated with gold-palladium particles, and analyzed by an XL30 ESEM FEG microscope (FEI) at 5 to 10 kV. For TEM, following fixation, samples were stained with OsO4 and uranyl acetate, dehydrated in a graded ethanol series, and infiltrated with Epon, using an automated robot. Ultrathin sections were performed using a Leica 2000 microtome and mounted on TEM grids. Sections were observed with a Technai T12 TEM apparatus. 3D surface models of skin samples from the three reticulated fruit were constructed by a ZETA-20 optical profiler microscope. The instrument was set up to capture thin micrographs of small surface block squares sized 638 µm × 638 µm. Images were then compiled to generate a 3D image of 5.7 mm × 3.8 mm with changing values for the z axis, reflecting the actual structure of the fruit surface. Using the measurements tools embedded in the corresponding microscope software, height differences between reticulation peaks to the bottom of the smooth regions and between reticulation peaks to the bottom of the reticulated structure were calculated. Calculation of Reticulation Coverage To calculate the percentages of reticulation coverage for all reticulated fruit cultivars, representative high-resolution skin surface images were obtained from eight independent fruit for each reticulated skin cultivar. An in-house script was generated to primarily transform the images into black and white so natural differences in skin color would not affect accurate identification of the reticulated structure, and a reticulation map was generated for every image. The script was generated using the R software (https://www.r-project.org/), which was then implemented into the ImageJ software (https://imagej.net/). Transcriptome Analysis Total RNA was extracted from flesh and skin tissues of all six investigated fruit according to the common TRIzol method (Rio et al., 2010). Following quality control, RNA was used for preparation of strand-specific TranSeq libraries for Illumina high-throughput sequencing, as recently described (Tzfadia et al., 2018). Libraries were examined for purity and integrity before analysis using the Illumina Hi-Seq 2000 under default parameters, generating 50-bp single-end reads. Reads were mapped to the published melon genome (Garcia-Mas et al., 2012; http://www.melonomics.net/), generating normalized reads per kilobase of transcript per million mapped reads values for each gene. Profiling of Cutin, Suberin, and Epicuticular Wax Monomers For cutin and suberin monomer measurements, 1-cm skin disc samples were delipidated for 2 consecutive weeks in HPLC-grade methanol:chloroform buffer (v/v) and then dehydrated overnight in a hood before being transferred for 3 d for full dehydration in a desiccator containing activated silica-gel beads. Delipidated samples were then transesterified with 4 mL of boron trifluoride:methanol (Sigma-Aldrich; 99.8% or greater) and incubated 16 h at 70°C. n-Dotriacontane (C32) alkane internal standard was added into all transesterified samples followed by vigorous vortexing. The transesterification process was stopped by transferring the samples into 9-mL glass vials containing saturated NaHCO3/water. Two milliliters of chloroform was added three times into the samples to extract all cutin and suberin monomers available and then collected into new 9-mL vials. Phase separation was achieved by the addition of 1 mL of HPLC-grade water and vigorous vortexing. The upper polar phase was carefully discarded by a glass pipet before chloroform extracts were fully dried by the addition of anhydrous NaSO4. Dried extracts were transferred into 2-mL reaction vials and fully evaporated under a nitrogen stream. Epicuticular waxes were extracted from 1-cm skin disc samples immersed for 2 min in 4 mL of chloroform containing n-tetracosane (C24) alkane internal standard. Chloroform extracts were fully evaporated under a nitrogen stream. Prior to GC-MS running, both chloroform extracts containing cutin, suberin, or epicuticular waxes were resuspended in 100 µL of chloroform, derivatized with 20 µL of pyridine (Sigma-Aldrich; 99.8% or greater, anhydrous) and 20 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich; GC grade) at 70°C for 1 h, and transferred into GC-MS vials. A sample volume of 3 µL was injected in splitless mode on a GC-MS system (Agilent 7683 autosampler, 7890A gas chromatograph, and 5975C mass spectrometer). GC was performed (Zb-1 MS column; 30 m length, 0.25 mm i.d., and 0.5 mm film thickness; Zebron, Phenomenex) with injection temperature of 270°C, interface set to 250°C, and the ion source to 200°C. Helium was used as the carrier gas at a constant flow rate of 1.2 mL min−1. The temperature program was 0.5 min isothermal at 70°C, followed by a 30°C min−1 oven temperature ramp to 210°C and a 5°C min−1 ramp to 330°C, then kept constant during 21 min. Mass spectra were recorded with an m/z 40 to 850 scanning range. Chromatograms and mass spectra were evaluated using the MSD ChemStation software (Agilent). Integrated peaks of mass fragments were normalized for sample dry weight and the respective C24 or C32 alkane internal standard signal. For identification, the corresponding mass spectra and retention time indices were compared with in-house spectral libraries. Profiling of Semipolar Compounds Analysis of semipolar compounds was carried out with a UPLC-qTOF-MS instrument (Waters Premier; Waters Chromatography) equipped with a UPLC column connected online to a UV detector and then to the MS detector, as recently described (Sonawane et al., 2018). Separation of metabolites was performed by gradient elution (acetonitrile-water, containing 0.1% [v/v] formic acid) on a 100 × 2.1-mm i.d., 1.7-µm UPLC BEH C18 column (Waters Acquity). Masses of eluted compounds (m/z range from 50 to 1,500 D) were detected with a qTOF-MS device equipped with an electrospray ionization source performed in the negative mode. XCMS data processing (Smith et al., 2006) was carried out as previously described (Mintz-Oron et al., 2008). The raw data set was filtered for masses with signal greater than 6 and those that exhibited fold change greater than 2 or less than 0.5 between the two groups of skin samples. Seventeen differential metabolites were annotated in high confidence based on authentic standards and in-house spectral libraries. Retention times, peak masses identified according to negative electrospray ionization mode [M-H], error values, and molecular formulas of the 17 metabolites are listed in Supplemental Table S4. MSI Analyzing the spatial abundance of suberin-associated ferulic acid in delipidated melon fruit skin samples was performed by the use of MALDI-MSI. Fully dried delipidated skin discs from all six fruit were directly attached onto Superfrost Plus slides (Fisher Scientific) using double-sided tape. For fruit cross-section profiling, fresh skin cubes were embedded with M1 embedding matrix (Thermo Scientific) in Peel-A-Way disposable embedding molds (Peel-A-Way Scientific) and flash frozen in liquid nitrogen. The embedded tissues were transferred into a cryostat (Leica; CM3050) and allowed to thermally equilibrate at −18°C for 2 h. Cube samples were then cut into 25-µm-thick sections, mounted onto Superfrost Plus slides, and vacuum dried in a desiccator for 10 min. A TM sprayer (HTX Technologies) was used to coat the slides with 2,5-dihydroxybenzoic acid (DHB; Sigma-Aldrich; 99.8% or greater) matrix (40 mg mL−1 dissolved in 70% [w/v] methanol containing 0.2% [v/v] trifluoroacetic acid [Sigma-Aldrich; 9.8% or greater]). The nozzle temperature was set at 80°C, and the DHB matrix solution was sprayed for 16 passes over the tissue sections at a linear velocity of 120 cm min−1 with a flow rate of 50 μL min−1. MALDI imaging was performed using a 7T Solarix Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics). Ferulic acid standard was used to optimize the instrument parameters for maximum sensitivity, resolution, and mass accuracy. The data were collected in positive ion mode using lock mass calibration (DHB matrix peak, m/z 155.033886, [M-H]+) in a mass range of 100 to 2,000 m/z with a spatial resolution of 60 μm. Each mass spectrum was obtained from a single scan of 100 laser shots at a frequency of 1 kHz and a laser power of 18%. The acquired spectra were processed using the Flex-Imaging software 4.0 (Bruker Daltonics). The spectra were normalized to root-mean-square intensity, and MALDI images of ferulic acid were visualized at m/z 195.065 ± 0.001 with pixel interpolation on. Statistical Analyses Bar graphs were compiled using the GraphPad Prism 5.01 scientific software, and significance was calculated according to Student’s t test as follows: *, P < 0.05; **, P < 0.001; ***, P < 0.0001; or according to the two-way ANOVA of P < 0.05. The number of biological replicates is mentioned in the corresponding figure legend of each experiment. PCA of GC-MS, LC-MS, and TranSeq data was performed using MetaboAnalyst 4.0, a comprehensive tool suite for metabolomic data analysis (http://metaboanalyst.ca/; Xia et al., 2015), following data log10 transformation and pareto scaling (mean centered and divided by the square root of sd of each variable) manipulations. Supplemental Data The following supplemental materials are available. Supplemental Figure S1. PCA plot based on cutin, suberin, and epicuticular wax profiles in skin of smooth and reticulated fruit. Supplemental Figure S2. PCA plot based on semipolar metabolite profiles in skin of smooth and reticulated fruit. Supplemental Figure S3. PCA plot based on flesh and skin transcriptome profiles of smooth and reticulated fruit. Supplemental Table S1. List of 421 smooth skin-enriched genes identified by the Venn diagram in Figure 10A. Supplemental Table S2. List of 196 reticulated skin-enriched genes identified by the Venn diagram in Figure 10A. Supplemental Table S3. List of 173 skin-enriched genes shared between smooth and reticulated samples identified by the Venn diagram in Figure 10A. Supplemental Table S4. List of 17 semipolar compounds differentially accumulated in reticulated versus smooth skin tissues. LITERATURE CITED Adato A , Mandel T, Mintz-Oron S, Venger I, Levy D, Yativ M, Domínguez E, Wang Z, De Vos RC, Jetter R, et al. ( 2009 ) Fruit-surface flavonoid accumulation in tomato is controlled by a SlMYB12-regulated transcriptional network . 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Proc Natl Acad Sci USA 107 : 12040 – 12045 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Adelis Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, the Jeanne and Joseph Nissim Foundation for Life Sciences, and the Tom and Sondra Rykoff Family Foundation. 2 Author for contact: asaph.aharoni@weizmann.ac.il. 3 Senior author. 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). H.C. and A.A. planned and designed the research; H.C., Y.D., and J.L. performed the experiments; H.C., Y.D., J.S., S.M., E.A.-S., V.V.Z.-D., and L.S. analyzed the data; H.C. and A.A. wrote the article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.18.01158 © 2019 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - A Multilevel Study of Melon Fruit Reticulation Provides Insight into Skin Ligno-Suberization Hallmarks
JF - Plant Physiology
DO - 10.1104/pp.18.01158
DA - 2019-04-02
UR - https://www.deepdyve.com/lp/oxford-university-press/a-multilevel-study-of-melon-fruit-reticulation-provides-insight-into-opUhgDcjCP
SP - 1486
EP - 1501
VL - 179
IS - 4
DP - DeepDyve
ER -