Apple is among the most consumed fruits worldwide with approximately 81 million tons produced in 2013 (FAOSTAT). The Malus × domestica species have a long history of breeding and cross hybridization which started by the import of the first Malus sieversii cultivars in Europe, through the Silk Route (Cornille et al., 2012, 2014). Later, breeding efforts aimed at improving yield, tolerance to pathogens and fruit quality traits, such as flesh firmness, juiciness, sugar, and polyphenol contents, to name a few (Laurens, 1998; Morgan et al., 2002; Juniper and Mabberley, 2006). Since the last decades, consumers and breeders have been progressively more attentive to the skin phenotype, favoring the development of glossy and colored skin varieties also called “waxy” varieties. However, the production of fruits with no damage is extremely dependent on environmental pressures and compels farmers to use increasing quantities of pesticides and fertilizers (Faust and Shear, 1972). Despite their efforts, some defects on the fruit surface can still occur. Among these, russeting is one of the most important causes of economic loss in the apple production sector. Fruits exhibiting this rough brown phenotype on the surface are usually redirected to the transformed product sector with a concomitant price downgrading, compared to the consumer's market.

The common apple skin surface is constituted by a thick cuticle linked to the epidermal cell walls of external tissues acting as protective barrier against environment. The cuticle composition varies across plant species and its chemical composition is characterized by the presence of multiple classes of hydrophobic hydrocarbons. Indeed, the cuticle is composed of a covalently linked macromolecular scaffold of cutin constituted by C16 and C18 ω-hydroxyacids and α,ω-dicarboxylic acids, and a wide variety of organic solvent-soluble lipids including n-alkanes or primary alcohols to name a few (Yeats and Rose, 2013; Fernández et al., 2016). The cuticle structure has been divided in multiple domains composed by (i) an internal cuticular layer linked to the cell wall, which is rich in cutin polymer with embedded polysaccharides, such as pectins and cellulose, (ii) an external cuticle (cuticle proper) with lower amount of polysaccharides but enriched in embedded intracuticular waxes and finally (iii) the epiculticular waxes which accumulate in the form of crystals or films responsible for the glossy phenotype of the apple fruit skin (Yeats and Rose, 2013; Fernández et al., 2016). Notably, a second non-saponifiable polymer called cutan has been observed in some species including apple (in low extent, Johnson et al., 2007). Cutan is rich in ether and carbon-carbon bounds but its structure remains poorly understood until now (Yeats and Rose, 2013; Fernández et al., 2016). Russeting is mainly due to the accumulation of suberin in the inner part of the primary cell wall. This complex biopolymer has been widely studied in other plant models, such as Arabidopsis thaliana, Solanum tuberosum L., and Quercus suber L. (Graça and Pereira, 2000a,b; Franke et al., 2005; Molina et al., 2006). Although recent advances strongly improved the knowledge of the complex metabolic cascade resulting in the deposition of suberin in the primary cell wall, factors driving its biosynthesis remain still uncharacterized in apple and, more generally, in plants (Graça, 2015; Legay et al., 2015; Vishwanath et al., 2015). Since the 60's, russeting has been extensively studied at the agronomical and histological levels, and key factors favoring the development of russeted skin in apple have been identified (Sironval and Clijsters, 1962; Walter, 1966; Tukey, 1969; Faust and Shear, 1972; Skene, 1982; Knoche and Grimm, 2008; Lashbrooke et al., 2015). Among these, the development and the integrity of the cuticle seems to be one of the most critical points in the regulation of russeting (Lashbrooke et al., 2015). Indeed, the development a thinner cuticle is correlated with the occurrence of skin russeting. This altered cuticle appears to be linked with the appearance of cuticle microcracking on the apple fruit surface and is thought to be one of the major causes of russeting in apple (Knoche et al., 2011; Curry, 2012). The cuticle is particularly sensitive to microcracking during early stages of fruit development, due to the extreme tangential tensions on the surface generated by the expansive growth (Khanal et al., 2012). Moreover, rheological studies showed that the skin of the non-russeted apple is less elastic compared to the russeted periderm, suggesting that the cuticle might be more sensitive to microcracking compared to the suberized tissue (Khanal et al., 2012). Scanning electron microscopy (SEM) analyses performed on the “Golden Delicious” variety showed that gibberellins (GA₄₊₇) application during the early stage of fruit development decreases the epidermal cell surface and fruit surface tensions, leading to a lower cuticle microcracking rate and russeting (Curry, 2012). In addition to the developmental factors, environmental factors strongly impact russeting in apple. As an example, the long term moisture exposure has been also associated with cuticle microcracking and russeting, suggesting that the causes leading to russeting are multifactorial (Tukey, 1969).

In our previous bulk transcriptomic analysis comparing russeted and waxy apples, a large number of genes involved in the cutin, wax and suberin biosynthesis pathway have been identified and new putative candidate genes implicated in these metabolic processes have been highlighted too (Legay et al., 2015). Interestingly, a strong decreased expression of the cutin and wax biosynthetic genes, with a concomitant increased expression of suberin biosynthetic and cell modification transcripts, was observed in russeted skin. Cutin and suberin share a similar backbone composed of α,ω-dicarboxylic acids and ω-hydroxyacids esterified with glycerol (Bernards, 2002). However, strong differences occur between these biopolymers, which might explain their differential rheological properties observed in apple (Khanal et al., 2012). As an example, cutin displays higher relative content in ω-hydroxyacids, which are composed by a majority of C16 and C18 aliphatic chains. Conversely, suberin is constituted by distinct polyphenolic and polyaliphatic domains, which contains significant amount of hydroxycinnamic acids, mainly ferulic acid, which are esterified within the suberin structure (Graça and Pereira, 2000b; Bernards, 2002). The suberin polyaliphatic domain also displays characteristic very long chains ω-hydroxyacids and α,ω-dicarboxylic acids, with a higher relative content in these latter compounds (Graça and Pereira, 2000a,b; Franke et al., 2005; Molina et al., 2006). Suberin composition displays variable distribution of these suberin precursors among plant species (Graça and Pereira, 2000a; Franke et al., 2005; Ranathunge and Schreiber, 2011; Vishwanath et al., 2015). In Q. suber, C22, and C24 ω-hydroxyacids and α,ω-dicarboxylic acids are predominant, while a majority of C16-C18 chains is observed in Pseudotsuga menziesii (Graça and Pereira, 2000a). In the potato tuber periderm, in addition to the long chain alkanoic acids (C24-C28), a majority of 18:1 derived ω-hydroxyacids and α,ω-dicarboxylic acids is found (Graça and Pereira, 2000b). Moreover, a variable composition of suberin is also observed between tissues of the same species. As an example, a differential suberin composition has been observed between Arabidopsis root and seed coat. Root suberin displays a large majority of C16, C18:1, and C22 α,ω-dicarboxylic acids, whereas seed coat suberin shows longer chains (Franke et al., 2005; Molina et al., 2006). In plants, cutin and suberin are complexed with non-covalently linked waxes which differ in composition between these two types of polymers (Millar et al., 1999; von Wettstein-Knowles, 2001; Schreiber et al., 2005; Rowland et al., 2007; Kosma et al., 2012). Waxes found in the cuticle are composed by very long chain fatty acids, primary and secondary alcohols, aldehydes and ketones, alkanes, wax esters and alicyclic components, such as triterpenes, which are deposited on the cutin surface (epicuticular waxes), or embedded within the polymer matrix (intraculticular waxes) (Aarts et al., 1995; Rowland et al., 2007; Lü et al., 2009; Zeisler and Schreiber, 2015; Jetter and Riederer, 2016). Recent studies showed that the transpiration barrier property of the cuticle is highly dependent to the fatty acyl content and to their intracuticular localization (Zeisler and Schreiber, 2015; Jetter and Riederer, 2016). Conversely, the suberin-associated waxes comprise very long chain alkanes, primary alcohols, fatty acids, some sterol derivatives and alkyl-hydroxycinnamates. Alkyl-hydroxycinnamates, which are composed by hydroxycinnamic acid esterified with primary alcohols, can reach 60% of the total waxes weight in A. thaliana (Kosma et al., 2012). Moreover, in A. thaliana, a recent study showed that the large majority of the primary alcohols is non-covalently linked to suberin is in the form of alkyl-hydroxycinnamates (Delude et al., 2016).

The apple waxy and russeted (suberized) skins present some important specificities in their wax composition compared to A. thaliana. Indeed, in the waxy apples, massive contents of the pentacyclic triterpenes, ursolic and oleanolic acids are found and can reach 30–60% of the total content depending on the variety (André et al., 2012, 2013). Conversely, russeted skins contain a lower amount of these compounds, but accumulate betulinic acid and lupeol (André et al., 2012, 2013). Moreover, betulinic acid-3-trans-caffeate also specifically accumulates in this suberized tissue (André et al., 2013). This suggests that, in apple, there might be a close interplay between the suberin, cutin and triterpene biosynthesis pathways, although no evidence has been established so far.

The purpose of this study is to characterize for the first time, in apple, the differential distribution of the cutin, suberin, waxes, and triterpenes, which occur between waxy (normal) and russeted (suberized) tissues. For this, a deep investigation of the lipid surface composition was performed from normal waxy skin and russeted patches sampled from the semi-russeted fruits of the “Cox Orange Pippin” variety. “Cox Orange Pippin” is a famous and well-studied apple variety, which has been used as parent in a large number of available commercial varieties (Sironval and Clijsters, 1962; Knee, 1972; Skene, 1981, 1982; Irving and Drost, 1987; Evans et al., 2011; Chagné et al., 2012; Legay et al., 2015). The comparison of the russeted and waxy areas of the same variety provides a first picture of the differential accumulation of cutin, suberin, waxes and triterpenes, discarding genotypic factors that might occur when such a comparison is performed on different russeted/waxy varieties. The composition of these polymers and free lipid compounds was also compared with previous works performed in suberin model plants, such as A. thaliana, S. tuberosum and Q. suber (Graça and Pereira, 2000a,b; Franke et al., 2005; Molina et al., 2006). Finally, a targeted gene expression profiling of biosynthetic genes previously identified in apple has been performed using RT-qPCR. These results will be merged in order to decipher the key players involved in the differentiation of these two contrasting tissue types.

Russeting is a major concern for the apple fruit production. Since the 60's, the complex relationship occurring between cuticle deposition and russeting has been extensively reported by multiple research teams. The current model postulates that russeting occurrence is tightly linked with defects and/or lower deposition of the cuticle on the fruit surface (Sironval and Clijsters, 1962; Walter, 1966; Faust and Shear, 1972; Skene, 1982; Knoche and Grimm, 2008; Lashbrooke et al., 2015). Our previous bulk transcriptomic analysis showed that genes involved in cutin and wax biosynthesis displayed a decreased expression in russeted apple skin, with a concomitant increased expression of the suberin biosynthetic genes (Legay et al., 2015). Compared to the fully russeted varieties, which represent an extreme case of russeting, the use of the semi-russeted “Cox Orange Pippin” provides a model which is closer to the genetic and phenotypic germplasm used in the apple production. Indeed, russeting in commercial varieties usually appears as patches, like those observed in “Cox Orange Pippin,” which gradually appear during the fruit development, whereas old heirloom fully russeted apples exhibit this strong phenotype at very early stages of fruit growth (Sironval and Clijsters, 1962; Walter, 1966; Faust and Shear, 1972).

In the present study, the total wax concentration observed in the waxy tissue (537 μg/cm², 20.5% of the cuticle sample) is in accordance with previous reports, which described total wax concentrations ranging from 366 to 1,038 μg/cm² depending on the variety (Belding et al., 1998). This suggests that the non-russeted skin surface of Cox Orange Pippin might be considered as moderately rich in wax. Alternatively, russeted apple tissues showed a massive decrease in this total wax concentration, which reach only 87.7 μg/cm² (3% of the cuticle sample). This supports the hypothesis that a thinner and/or damaged cuticle is a crucial factor leading to russeting in apple (Skene, 1982; Knoche and Grimm, 2008; Lashbrooke et al., 2015). This decrease was observed in nearly all components of waxes and included the very long chain primary alcohols, aldehydes, fatty acids and alkanes, with carbon chains longer than 24 carbons (Table 2, Figure 3) and suggests that a decreased very long chain fatty acid (VLCFA) biosynthesis occurs in these russeted areas. This was also supported by the decreased expression of genes coding for KCS2, KCS6, and KCS10, which are implicated in the elongation of C16 and C18 acyl-CoA into very long chain acyl-CoA, the latter being used as precursors for the different classes of metabolites comprised in these cuticle waxes (Millar et al., 1999; Lee et al., 2009; Lee and Suh, 2013). Finally, decreased concentrations in C16 and C18 fatty acid precursors were also observed suggesting that the core fatty acid biosynthesis which occurs in the plastids is also altered in the russeted tissues.

The following steps of the cuticular wax biosynthesis are still under investigation. However, it is suggested that the wax biosynthesis pathway follows two distinct branches (Millar et al., 1999; von Wettstein-Knowles, 2001). The reductive pathway produces even number carbon chain numbers as found in aldehydes and primary alcohols. Aldehydes are considered as intermediate compounds between the acyl-CoAs and primary alcohols (von Wettstein-Knowles, 2001). On the other side, the decarbonylation of fatty acyl-CoAs might be involved in the biosynthesis of classes sharing odd carbon chain number, as observed for the alkanes. These latter compounds are further hydroxylated into secondary alcohols and ketones. In the present work, it is noteworthy that the major primary alcohols and aldehydes were composed of 26- and 28-carbon chains whereas alkanes showed C27- and C29-carbon chains. Thirty-carbon chains were only observed for the free fatty acids, which strongly accumulated in the waxy skin of “Cox Orange Pippin” (Table 2, Figure 3). As VLCFAs are synthesized from very long chain acyl-CoA, the C29 n-alkanes biosynthesis might result from the decarbonylation of a C30 acyl-CoA common precursor. CER1, CER3 and CER8 are also involved in the alkane biosynthesis (Aarts et al., 1995; Rowland et al., 2007; Lü et al., 2009). Apple orthologous genes of CER1 and CER3, which were found in our previous RNA-Seq data, were more expressed in the waxy tissues of “Cox Orange Pippin,” suggesting that they might indeed play a role in the biosynthesis of apple skin waxes. We noticed some trace of n-alkanes in the depolymerized fraction, which might (i) remained after the wax removal or (ii) have been confined in the polymer matrix, probably in the cutin, and released during the depolymerization process.

As opposed to the different wax compounds described above, the “Cox orange Pippin” russeted fruit skin specifically accumulated some alkyl-hydroxycinnamates (Table 2, Figure 3). These metabolites, which are composed of hydroxycinnamic acids conjugated with primary alcohols, have been previously associated with the suberization process in A. thaliana and S. tuberosum to name a few (Schreiber et al., 2005; Serra et al., 2010; Kosma et al., 2012, 2015; Molina and Kosma, 2015; Delude et al., 2016). In our work, a majority of coumarate conjugates have been found, followed by ferulates and caffeates. Primary alcohols esterified with these hydroxycinnamic acids are mostly represented by 22- and 24-carbon chains length (Table 2, Figure 3). This was not in accordance with the primary alcohols distribution observed in the free lipids extract, which displayed predominantly 26- and 28- carbon chains. However, primary alcohols found in the depolymerized extract showed a completely different pattern with an enhanced accumulation of C22 and C24 chains in the russeted skin (Table 3, Figure 3). In A. thaliana roots, the large majority of the primary alcohols found in the suberized tissues are in the form of alkyl-hydroxycinnamates (Delude et al., 2016). In apple, it might be conceivable that a similar process occurred and explains the concordant distribution of primary alcohols observed in the alkyl-hydroxycinnamates and those identified in the depolymerized extract. This suggests that the differentially expressed KCS2 and KCS4 genes, observed in this work and in our previous transcriptomic data (Legay et al., 2015), might be responsible for the specific synthesis of these C22 and C24 acyl-CoA. These are partly converted into primary alcohols by fatty acyl CoA reductase enzymes, such as FAR5, which expression was enhanced in the present work (Domergue et al., 2010). In A. thaliana, the kcs2 loss-of-function mutant displayed a significant decreased content in C22 and C24 VLCFAs (Lee et al., 2009). The suberin biosynthetic process occurring in the russeted skin might induce KCS2 genes which produce essentially C22 and C24 chains for further synthesis of these alkyl-hydroxycinamates. Inversely, the KCS2, KCS6/CER6/CUT1 and KCS10 genes, which are up-regulated in the waxy skin, might produce longer carbon chain acyl-CoA, which are further used for the cutin wax production, including the C26 and C28 primary alcohols observed in the present work. As an example, the cut1-loss of function mutant led to an over-accumulation of C24 chain-length wax components, suggesting that KCS6 is responsible for the elongation of the C24 acyl-CoA into longer precursors (Millar et al., 1999).

Pentacyclic triterpenes are major components of the fruit skin waxes in apple (André et al., 2012). In waxy varieties, the total triterpene content can reach up to 70% of the total waxes content depending on the genetic background of the variety and the environmental factors (André et al., 2012). Three main triterpene series, namely the ursane, oleane, and lupane series, have been identified in the apple skin, so far. The distribution of the different series of triterpene seems to be tightly linked with the degree of skin russeting. Indeed, recent studies showed that the ursane and oleane series strongly accumulate in the waxy tissues, whereas the lupane series seems to be specifically synthesized in the russet skin (Brendolise et al., 2011; André et al., 2012, 2016). Ursolic and oleanolic acids concentrations strongly decreased in the russeted skin, suggesting that in addition to the fatty acyl derivatives, the alicyclic component of apple waxes is also altered in this tissue (Table 2). However, a recent study showed that the transpiration barrier efficiency is mainly driven by the fatty acyl derivatives content in the intracuticular waxes (Jetter and Riederer, 2016). This suggests that in russeted apple tissues, decreased concentrations in ursolic and oleanolic acids might not have a strong impact on the transpiration barrier efficiency, but further are needed to validate this hypothesis. This observation was also supported by the decreased expression of the MdOSC1 and MdOSC3 genes in the russeted skin. MdOSC1 produces mainly α-amyrin, which is further converted into ursolic acid by the CYP716A145 oxidase. MdOSC3, which is highly homologous to MdOSC1, probably because of the ancestral genome duplication of the Pyrae ancestor, has a similar function (Brendolise et al., 2011; André et al., 2016). Conversely, MdOSC5, which has been recently described as a crucial enzyme involved in the synthesis of lupeol (the betulinic acid precursor), was up-regulated in russeted tissues (André et al., 2016). Previous works showed that betulinic acid predominantly accumulates in the russeted tissues (André et al., 2012). However, betulinic acid was only detected in the depolymerized fraction. To our knowledge, triterpenes are not covalently linked to the cutin and suberin polymers (André et al., 2012, 2016). Thus, we can speculate that it might be embedded in this polymeric matrix (probably suberin). Finally, it is noteworthy that other minor unidentified pentacyclic triterpenes, which display differential accumulations, have been detected in both fractions. However, standards are currently lacking to identify them properly and it is not yet possible to identify them. Finally, the CYP716A145 gene displays a slight increase (43%) in the waxy skin. This multifunctional enzyme is responsible for the conversion of α-amyrin, β-amyrin and lupeol into ursolic, oleanolic and betulinic acids, respectively (André et al., 2016). Altogether, these observations strengthened the present knowledge about the tight relationship between the suberization process and the pentacyclic triterpenes synthesis. As ursolic and oleanolic acids are major component of the non-russeted skin waxes, their decreased accumulation might also have an impact on the integrity of the cuticle. Thus, it would be interesting, in further works, to evaluate their role in the development of cuticle microcracking.

The boron trifluoride-methanol (BF₃/MeOH) transesterification method is commonly used to depolymerize either cutin or suberin (Franke et al., 2005; Gou et al., 2009; Panikashvili et al., 2009). It is worth noting that, in the present experimental design, these two polymers are likely to be present in the russeted skin of the “Cox Orange Pippin” variety, whereas a large majority of cutin monomers are expected in the waxy tissue. The total amount of released monomers did not significantly changed between waxy and russeted tissues with 80.2 μg/cm² (3.06% of the starting material) and 73.3 μg/cm² (2.27% of the starting material), respectively. This suggests that other polymers and compounds, such as polysaccharides, cutan or lignin, which are recalcitrant to the transesterification process might remain in the skin tissue. As example, the polyaromatic domain of suberin, which is considered as a lignin-like polymer, might probably remain in this fraction (Bernards, 2002). Moreover, cutan, which is also a lipidic polymer found in the apple cuticle, might also remain in the residual fraction as its reticulation seems to be caused by ether and C-C bounds, the latter being recalcitrant to the BF3/MeOH treatment (Johnson et al., 2007; Yeats and Rose, 2013). Finally, cell wall polysaccharides, which are embedded in the cuticular layer or linked to the polyaromatic domain of suberin might also remain in this fraction.

The analysis of the depolymerized extract showed strong differences between the metabolic profiles obtained from the russeted and waxy skin of “Cox Orange Pippin.” As example, the distribution of fatty acids was particularly interesting and differed from the results obtained in the free lipid extract. Suberized tissues displayed increased concentrations of VLCFAs ranging from C20 to C24 chains, whereas a decreased accumulation of C16, C18, C18:1, and C18:2 was observed (Table 3, Figure 3). In plants, the composition of suberin-associated fatty acids seems to vary among species and tissues. In A. thaliana roots, the analysis of the suberin monomers revealed a majority of C20 and C22 fatty acids whereas the S. tuberosum suberin exibits longer carbon chains with a majority of C24–C28 fatty acids (Graça and Pereira, 2000b; Franke et al., 2005). A. thaliana seed coat suberin is composed of a majority of C16, C20, and C24 chains (Molina et al., 2006). Finally, the bark suberin monomeric contents of P. menziesii and Q. suber present higher proportions of the C20–C24 fatty acids as observed in our work (Graça and Pereira, 2000a). Caution should be taken with the present results because these fatty acids are probably released from both cutin and suberin. However, as observed for the alkyl-hydroxycinnamate-associated primary alcohols, our results suggest a suberin specific biosynthesis of C20–C24 (predominantly C22) very long chain acyl-CoA precursors in the russeted tissues (Lee et al., 2009; Legay et al., 2015). Interestingly, small amount of C20, C22, and C24 precursors were also observed in the waxy tissues and might be due to the presence of suberized lenticels on the whole surface of the fruit. Lenticels are small pores spread over the fruit surface, which have been first linked to high transpiration rate. However, more recent studies showed that this transpiration rate was not correlated with the size and the number of lenticels due to either suberin or wax deposition on their surface (Martin and Rose, 2014).

In A. thaliana, the analysis of the cutin composition indicated a quasi-exclusive occurrence of C16 and C18 monomers, suggesting that the shorter carbon chain fatty acids, found in a higher amount in the waxy apple skin, might be part of the cutin polymer and that their synthesis is impaired in the russeted tissue (Bonaventure et al., 2004). The composition of the ω-hydroxyacids is more complex but seemed to follow a similar trend compared to fatty acids. Indeed, waxy skin contained increased amount of C16, C18, C18:2 whereas a specific accumulation of C18:1, C20, C22, and C24 ω-hydroxyacids was observed in the suberized skin (Table 3). In A. thaliana roots, ω-hydroxyacids are mainly composed of C16, C18, C20, and C22 aliphatic chains with a strong predominance of the 18-hydroxyoctadec-9-enoic acid (C18:1 ω-hydroxyacid, Franke et al., 2005), whereas C18:1, C18:2, C22, and C24 ω-hydroxyacids are mostly represented in the A. thaliana seed coat suberin (Molina et al., 2006). In the potato periderm, a massive amount of C18:1 ω-hydroxyacid has been also found and, to a lower extent, some C16, C22, C24, C26, and C28 ω-hydroxyacids (Graça and Pereira, 2000b). Finally, the bark of Q. suber displays a majority of C22, C24 ω-hydroxyacids (Graça and Pereira, 2000a). In our study, the specific accumulation of these very long chain ω-hydroxyacids in the apple suberized skin was supported by the increased expression of the VLCFA hydroxylase (CYP86B1) gene (Compagnon et al., 2009). Moreover, an increased expression of GPAT5 was observed in the russeted tissues. In A. thaliana, GPAT5, which synthetizes the suberin ω-hydroxyacyl- and α,ω-diacyl-glycerol building blocks, has a strong affinity for the very long chain (>C18) ω-hydroxyacids and α,ω-dicarboxylic acids (Beisson et al., 2007). Altogether, these data support the idea that C20, C22, and C24 ω-hydroxyacids might constitute specific precursors of suberin in apple. Additionnally, the C18:1 ω-hydroxyacid, which strongly accumulates in the A. thaliana roots (Franke et al., 2005) and the russeted potato periderm (Graça and Pereira, 2000b), is the only short chain ω-hydroxyacid displaying an increased concentration in the russeted apple skin. We might speculate that this compound might be produced by the CYP86A1 fatty acid hydroxylase (Höfer et al., 2008), which showed a significant increased expression in our RT-qPCR results. Some mid-chain hydroxylated ω-hydroxyacids showed a higher concentration in the waxy skin, suggesting that they might be part of the cutin polymer. These might be synthesized by the preliminary action of some cutin specific epoxidase, such as the A. thaliana CYP77A4 (Sauveplane et al., 2009), which was also more expressed in the waxy skin in a previous work (Legay et al., 2015). Epoxidized fatty acids might be further hydroxylated by some epoxide hydrolase to yield mid-chain hydroxylated fatty acids, but no candidate has been found yet (Yeats et al., 2012a).

The core structural backbone of both cutin and suberin is composed of α,ω-dicarboxylic acids esterified to glycerols. These are crucial components of the cutin and suberin building blocks despite reduced amounts were found in cutin (Moire et al., 1999; Beisson et al., 2007; Yang et al., 2010, 2016). Surprisingly, we found increased amount of free glycerol in the waxy tissues. This might be explained an enhanced activity of the CD1 (cutin synthase) polymerization enzyme, which release a glycerol moiety from each MAG condensed (Yeats et al., 2012b). Previous studies showed that suberin shows a high content in α,ω-dicarboxylic acids, as compared to cutin (Franke et al., 2005). A similar tendency was observed in apple, which showed an increased accumulation in the majority of the identified α,ω-dicarboxylic acids. Interestingly, the highest contents were observed for the shorter carbon chains α,ω-dicarboxylic acids, such as hexadecanedioic, octadecanedioic and 9,10-dihydroxyoctadecanedioic acids, whereas eiocosanedioic, docosanedioic and tetracosanedioic acids were less abundant. Again, the apple suberization process seemed to specifically produce C20, C22, and C24 α,ω-dicarboxylic acids, whereas shorter carbon chains α,ω-dicarboxylic acids were predominant in the waxy apple skin (Table 3, Figure 3). The same trend is observed in S. tuberosum, A. thaliana roots and P. menziesii (Graça and Pereira, 2000a,b; Franke et al., 2005). Only suberin monomers obtained from the Quercus suber bark and A. thaliana seed coats displayed predominant or large amount of very long chain α,ω-dicarboxylic acids (Graça and Pereira, 2000a; Molina et al., 2006). The synthesis of these α,ω-dicarboxylic acids remains unclear, some authors argue that the consecutive action of alcohol dehydrogenase and aldehyde dehydrogenase on ω-hydroxyacids precursors might support the synthesis of these crucial compounds (Kurdyukov et al., 2006; Franke and Schreiber, 2007). The second putative DCA synthesis path might involve cytochrome P450-based enzymes being able to perform both steps of this carboxylation process. As example, a CYP94A5 isolated from Nicotiana benthamiana and expressed in Saccharomyces cerevisiae is able to convert the 18-hydroxy-9,10-epoxystearic acid into its corresponding DCA (Le Bouquin et al., 2001).

Finally, coumarate and ferulate methyl esters were detected in both russeted and waxy tissues, suggesting that they were covalently linked within the cutin and/or suberin polymeric matrix, or conjugated into other compounds, such as alkyl-hydroxycinnamates or triterpene conjugates (Kosma et al., 2012; André et al., 2013; Table 3, Figure 3). Higher concentration of ferulic acid in the russeted skin is obvious as it is one of the major suberin structure signature (Bernards, 2002; Franke et al., 2005; Molina et al., 2006; Graça, 2015; Vishwanath et al., 2015). Hydroxycinnamic acids are thought to be part of the suberin polyphenolic domain of suberin which is anchored in the cell wall (Bernards et al., 1995; Bernards, 2002). Moreover, ASFT is responsible for the linkage of ferulic acid with ω-hydroxyacids resulting in feruloyl-acyl esters, which are considered as crucial suberin building blocks (Molina et al., 2009). An apple orthologous gene of ASFT, which has been found in our previous transcriptomic profiling of the russet trait, displayed a strong increased expression in the russeted tissues of “Cox Orange Pippin” (Legay et al., 2015). Covalently linked coumaric acid, possibly to cutin, has been already described in apple, but not ferulic acid (Riley and Kolattukudy, 1975); this might be explained by the fact that this study did not comprise russeted varieties. Conversely, a fairly detectable amount of coumaric acid was found in the russeted skin and even more in the waxy skin, suggesting that coumaric acid might be more related to the cuticle development but its exact involvement in this matrix remains unclear (Riley and Kolattukudy, 1975).

As a whole, the present data depict for the first time the complex structure of the waxy and russeted apple skins. Our previous transcriptome profiling study showed an impaired expression of genes involved in the cuticle deposition in the russeted varieties with a concomitant increased expression of genes involved in the suberin biosynthetic pathway. Recent studies postulated that an efficient cuticle is crucial to prevent any occurrence of suberization on the fruit surface (Knoche and Grimm, 2008; Khanal et al., 2012; Lashbrooke et al., 2015). Our data clearly support this hypothesis. Indeed, in the russeted skin patches of the “Cox Orange Pippin,” a massive decrease of common wax components was observed. These included primary alcohols, alkanes, fatty acids and aldehydes, along with the pentacyclic triterpenes ursolic and oleanolic acids. This decrease in the wax content is also accompanied by a reduced synthesis of cutin, which is composed by a majority of 16- and 18-carbon chain ω-hydroxyacids and α,ω-dicarboxylic acids. The concomitant decreased concentrations of cutin and wax components, together with the expression pattern of their biosynthetic genes, suggest that a common regulatory network might regulate these pathways. It would be interesting to investigate whether some cuticle deposition master regulators, such as MdSHN3 and/or a AtMYB94 apple orthologous genes (Lashbrooke et al., 2015; Lee and Suh, 2015), are able to alter the triterpenic acid contents in the apple fruit skin.

Suberin, which accumulates in russeted patches, was similar to the previously reported composition investigated in other species (Graça and Pereira, 2000a,b; Franke et al., 2005; Molina et al., 2006). In addition to the characteristic ferulic acid, C20, C22, and C24 very long chain backbone precursors, which are further used for the synthesis of primary alcohols, fatty acids, ω-hydroxyacids, α,ω-dicarboxylic and possibly the suberin specific non-covalently linked alkyl-hydroxycinnamates, constituted the main characteristic of the apple suberin. The tight interplay between the decreased cuticle synthesis and suberin deposition is evident and the central remaining question now relates to the causes leading part of the fruit epidermis to a decreased cuticle deposition. Further studies have therefore to be performed to answer this crucial question.

SL, GG, CA, and JH designed the experimental setup. SL, EC, and GG performed the sampling. SL and GG performed the transriptomic work (RNA extraction, QC control, RT-qPCR). SL, EC, CA, and CG performed the analytical chemistry work, the GC-MS data extraction, metabolite identification. SL and GG performed the statistical analysis. SL, GG, and EC wrote the article. SL, EC, CA, CG, JH, and GG involved in the manuscript refinement.