The loss-of-function mutation of beta-ketoacyl-CoA synthase (KCS) in tomato fruit, designated as SlCER6 mutant, causes a decrease of alkanes and aldehydes longer than C₃₀ and an increase of intracuticular triterpenoids in the cuticle. An increase in permeability has been observed due to the reduction of the intracuticular VLC aliphatic compounds. These findings  suggest that the transpiration barrier is mainly determined by the proportions of VLC aliphatic constituents and triterpenoids of the intracuticular waxes rather than epicuticular wax’s VLC aliphatic composition ^[1][19].

During tomato fruit development, SlCER6 shows a higher cuticle amount than wild type “MicroTom”. Nevertheless, a reduction in very long chain (VLC) alkanes and an increase in cyclic triterpenoids, together with an increase in water loss, are shown in waxes of SlCER6 ^[2][66]. Positional sterile (ps) mutant of tomato exhibits a similar phenotype to SlCER6, which is defective in the elongation of VLC fatty acids (VLCFA). No difference in cutin composition, cutin accumulation and cuticular wax accumulation between the mutant ps and its wild type is observed. Nevertheless, the wild type exhibits a remarkably higher level of alkanes and aldehydes in cuticular waxes and a remarkably lower level of esters and triterpenoids than ps fruits, with hentriacontane (C₃₁) the most prominent alkane constituent in wild-type tomato waxes. Furthermore, a reduction in growth and weight and an increase in water loss are observed in ps fruits ^[3][67].

Studies with tomato fruit strongly support the statements that (i) cuticle rather than cell wall modifications could have a significant role in the reduction of the softening rate in fleshy fruits ^[4][12], (ii) waxes are the main cuticular structures that regulate the permeability in fleshy fruit peels ^[1][2][19,66], and that (iii) a specific change in cuticle wax composition rather than an increase in thickness or the total amount of cuticle has a more significant effect on the physiological characteristics of fleshy fruits ^[1][2][19,66], especially for the reduction of water rate loss. Furthermore, several studies showed that epicuticular waxes, rather than intracuticular waxes, have a significant effect on fleshy fruit cuticle permeability ^[5][45], although other studies have shown opposite results ^[1][19]. Therefore, more experimental evidence is needed to fully elucidate the role of epicuticular and intracuticular waxes in the permeability properties of the cuticle.

In “Navelate” sweet orange, the total epicuticular wax load significantly increases in the earlier stage of development, breaker (Bk), but remains unchanged in the later stages, namely mature green (MG), colored (C) and full-colored (FC). Terpenoids are mainly present in the Bk stage, and a notorious increase of hentriacontane (C₃₁) happens in this stage. Cuticle transpiration and permeability are lower in the first two stages of development (Bk and MG), suggesting that alkanes and triterpenoids have a relevant function in controlling cuticle permeability during sweet orange fruit development ^[6][22].

It had been suggested that the early rise of epicuticular crystals, alcohols and triterpenoids observed at the beginning of the development of grapevine fruit could be due to a mechanism to protect the underlying cuticle from the exponential increase in volume and rapid surface expansion ^[7][42]. Like sweet orange ^[6][22] and grapevine ^[7][42], during nectarine development, an early active cuticle accumulation process is observed during the first growth period, mainly by triterpenoid deposition and cutin formation. ScholarAuthors suggest that the increase in wax accumulation during the early stages of development allows the fruit to reduce water loss at harvest and during postharvest conditions ^[8][32].

The removal of epicuticular waxes in grape berry induces higher water loss and softening, indicating that epicuticular waxes could play a pivotal role in the postharvest quality of grape berries by reducing the water loss rate ^[5][45].

During postharvest storage, alkanes and aldehydes of Korla pear fruit cuticular waxes negatively correlate with weight loss, whereas fatty acids and alcohols have a positive correlation. The composition and morphological analysis of the Korla pear cuticle suggests that alkanes and aldehydes could contribute to wax crystal formation and consequently fruit water retention in Korla pear during postharvest storage ^[9][68].

Water deficit induces total aliphatic waxes accumulation at the end of grape berry development, accompanied by an obvious increase of VLC esters. This increase in cuticular VLC esters could be due to an adaptation to low water availability in grape berries. Nevertheless, these changes of composition do not cause a significant change in the berry transpiration rate ^[10][44]. On the other hand, it has been observed that water deficit leads to a lower triterpenoids/total aliphatic wax ratio in green and red berries. The apple fruit (M. domestica) cultivar “Florina” shows higher amounts of fatty acids, esters, primary alcohols and aldehydes than the “Prima” cultivar. However, it was not possible to establish a relationship between water permeability and cuticle composition for both cultivars ^[11][29]. It has been reported that the cuticle of physiologically mature guava fruit (P. guajava L.) has a large abundance of cyclic components, epoxy, hydroxy, and carboxyl functional groups, and a relatively smaller amount of average chain length (ACL) of acyclic components than cuticles of other species and organs, which has been related with the low ability to reduce the transpiration of guava fruit cuticle ^[12][48]. In agreement, the cuticle of mature pitahaya fruit (H. polyrhizus) presents a wax/cutin ratio of 0.6 and an ACL of 30.5. ScholarAuthors argue that the ACL is higher than that reported for other petal and fruit cuticles, similar to that reported for leaf cuticles of other plant species. They attribute these changes to the enhancement of the transpiration barrier of the pitahaya cuticle to withstand arid environments ^[13][49]. Cuticle composition analyses of pepper species C. annuum and C. chinense, which have high and low postharvest water loss rates, respectively, showed that postharvest fruit water loss is associated with the cuticle composition and the ratio of the wax constituents rather than the total wax amount. It has been shown that an increase in the total amounts of triterpenoid and sterols in the cuticular waxes of pepper fruits could increase postharvest water loss, while an increase in the amounts of primary alcohols and alkanes could reduce it—specifically, an increase of C₂₉ and C₃₁ alkanes ^[14][46]. In the case of cutin, it has been shown that water loss has a negative association with the C₁₆/C₁₈ ratio. The increase of total C₁₆ monomers and 9(10),16-dihydroxy hexadecenoic acid in the cutin appears to induce postharvest fruit water loss in pepper fruits ^[14][46]. During olive development, a slight increase in the ACL of VLC acyclic compounds was observed, with a slight reduction of the C₁₆/C₁₈ ratio of cutin monomers; nevertheless, no differences were observed in cuticular permeability and water loss during olive fruit development ^[15][47]. The tomato cultivar “Delayed Fruit Deterioration” (DFD) exhibits a remarkable delayed softening at postharvest and remains firm for at least six months at fully ripe stages, but it otherwise undergoes a normal ripening process. No significant differences were found in the patterns of wall polysaccharide modification and the expression of genes related to wall degradation between DFD and the normal softening "Ailsa Craig" (AC) cultivar. However, DFD showed a lower transpiration rate, lower water loss, higher cellular turgor, higher firmness and a thicker and more swelled pericarp at ripening stages than AC ^[4][12]. Although DFD and AC have similar cuticle anatomies, DFD shows a stronger cuticle and a higher total wax amount. Besides, a significant increase in alkadienes in red ripe fruits is shown in DFD, but not in AC. Based on these data, the scholarauthors suggested that the delay of the softening phenotype could be due to the significant increase in alkadienes and the absence of naringenin in the cuticular wax of DFD, which has been associated with both mechanical support and turgor maintenance through water loss reduction ^[4][12]. In agreement, it has been shown that water stress increases fruit firmness and total cuticle, total wax and triterpenoids amounts, whereas it decreases cuticle permeability, transpiration rate and the relative amount of VLC alkanes in AC tomato fruits, which suggests an association between cuticle characteristics, transpiration and fruit firmness. In addition, an increase in total cutin amounts and 9(10),16-dihydroxy hexadecenoic acid was shown in AC ^[16][9]. Water stress does not affect cuticle permeability and thickness in DFD but induces an increase in both characteristics in AC tomato fruits. Actually, after water stress, the cuticle of AC shows similar characteristics to that of DFD, which suggests a change in cuticle metabolism in response to low water availability conditions in tomatoes ^[16][9]. It has been suggested that large amounts of alkanes and low amounts of triterpenoids in cuticles reduce the transpiration of fruit surfaces in fleshy fruits ^[2][3][14][46,66,67]. This Cstudy showed that cuticle permeability was positive and negatively correlated with the total cutin amount and the proportion of VLC alkanes in AC fruits, respectively. Besides, the levels of cyclic triterpenoids were positively correlated with the water loss rate ^[16][9]. The cuticles of mango cultivars “Kent”, “Tommy Atkins”, “Manila”, “Ataulfo”, “Criollo” and “Manila” exhibit different epicuticular wax deposition patterns, architectures and cutin compositions at the mature-green stage and during postharvest, along with different water transpiration rates, firmness and fruit quality appearance. During postharvest, the mango “Tommy” cultivar has a higher wax deposition and cuticle thickness and exhibits a lower percentage of weight loss and less visual deterioration than the “Criollo” cultivar. ScholarAuthors suggest that cuticle characteristics observed in premium cultivars such as “Tommy” are potential factors that could be associated with fruit quality preservation during postharvest storage ^[17][69].

2. Response to Postharvest Storage

The amounts of both epi- and intracuticular waxes of mandarin “Satsuma” fruit (C. unshiu) increase after 20 days of room temperature (25 °C) storage, but they decrease after 40 days. A decrease in terpenoids and fatty acids and an increase in the proportion of alkanes is shown after 40 days of storage. Further, the total cutin amount decreases during postharvest storage, but the proportion of almost all cutin components remains stable ^[18][25]. The navel orange cultivar “Newhall” has a higher total cuticular wax and epicuticular wax amount than mandarin “Satsuma”. Notably, a significantly higher amount of hentriacontane (C₃₁), and C₂₄ and C₂₆ chain length fatty acids and aldehydes is observed. During seven days at 25 °C and 40–50% relative humidity of postharvest conditions, navel orange exhibited a lower weight loss than mandarin ^[19][24], which could be due to the difference in the epicuticular wax content and composition observed. During postharvest, the peach melting cultivar “October Sun” shows a more dramatic firmness loss and weight loss than the non-melting cultivar “Jesca”. At harvest, wax percentages are similar in both cultivars, whereas cutin percentages are significantly higher in “October Sun” than in “Jesca”. Five days after harvest, the total wax and cutin yields remain unchanged in “October Sun”, whereas in the “Jesca” cultivar, both wax and cutin significantly increase. At commercial harvest, the ratio of alkanes to triterpenoids and sterols is 0.31 in “October Sun” and 0.65 in “Jesca”. These data strongly suggest that the larger amounts of  alkanes play a role in maintaining the firmness and reducing weight loss in non-melting “Jesca” cultivars ^[20][31]. The total wax content of Korla pear fruit increases during 30 days of postharvest storage, but it decreases on day 90. Furthermore, alkanes and aldehydes show a negative correlation with the weight loss of Korla pear during postharvest, whereas fatty acids and alcohols have a positive correlation ^[9][68], suggesting that alkanes play an important role in reducing water loss. In apple cultivars “Stark”, “Golden”, “Mutsu”, “Golden Delicious”, “Camachi”, “Huahong”, “Jona Gold”, “Red Star”, “Ralls” and “Mashima Fuji”, a decrease in total cuticular wax amount is observed after 49 days of postharvest storage, with a decrease in alkanes and primary alcohols and an increase in fatty acids proportion. A relationship between weight loss rate and total wax, total alkanes and C₅₄ alkanes are shown in all the 10 cultivars, suggesting that alkane biosynthesis is essential for reducing weight loss during postharvest storage in apples ^[21][59]. Through the cuticle wax analysis of 35 cultivars of pear (Pyrus spp.) mature fruits, it has been shown that the cultivar with the longest postharvest storage period also showed a higher wax concentration ^[22][37]. In berries, lingonberry fruit (V. vitis-idaea), which is characterized as having a longer shelf-life than honeysuckle (Lonicera caerulea), and strawberry tree (Arbutus unedo), a higher content of triterpenoid acids in the cuticle was recorded. It has been suggested that triterpenoid acids might be related to lingonberry surface firmness and durability, probably due to the mechanical properties that they provide and the antimicrobial effect ^[23][64]. The main quality preservation strategies used to maintain the fruit quality at postharvest are based on temperature regulation such as cold storage and the application of ethylene regulators such as 1-methyl cyclopropane (1-MCP). Furthermore, controlled atmospheres are utilized with the same goal ^[24][70]. Nevertheless, during development and postharvest, fleshy fruits are susceptible to phytopathogen attack and the development of physiological disorders, such as cracking, russeting and chilling injury ^[24][70].

3. Response to Cold Storage

Different patterns of changes in response to cold storage have been shown among apple cultivars. The main constituents of the cuticular wax of apple “Maxi Gala” fruit after nine months of cold storage are alkanes and fatty acids, with nonacosane (C₂₉) and cis-13,16-docosadienoic acid (C_22:2) the main compounds of these two fractions, respectively ^[25][72]. The amounts of total cuticular wax and the main alkane constituents nonacosane (C₂₉) and heptacosane (C₂₇) decreased during seven months of postharvest storage at 0 °C in “Red Fuji” apple fruit ^[26][27]. During 140 days of cold storage, the total wax content of apple fruit cultivar “Starkrimson” increases from day 0 to day 80, then decreases at day 140 ^[27][28]. The total epicuticular wax content of sweet orange fruit increases after 30 days of postharvest cold storage (4 °C), then decreases at day 40, whereas at 25 °C, a continuous increase was observed during 40 days of storage. At 4 °C, the total cutin amount decreases continuously, whereas, at 25 °C, an increase is observed at 20 and 40 days of storage. At 4 °C, triterpenoids increase continuously during 20 days and then decrease after 40 days of storage. At the same time, a continuous increase in triterpenoids and a decrease in fatty acid is observed during 40 days of storage at 25 °C. At 4 °C, alkane composition remains stable, whereas, at 25 °C, the alkane fraction increases. Moreover, nonacosane (C₂₉) becomes the main alkane after 40 days of storage at 25 °C ^[28][23]. Changes in cuticle amounts and composition have been observed in response to postharvest at 20 °C and 0 °C for both “Somerset” and “Celeste” sweet cherry fruit cultivars, with a general increase in cuticle amount in response to cold storage ^[29][61]. Ursolic acid content has been positively associated with weight loss and softening of blueberry fruit at postharvest cold storage, whereas a negative association has been reported for oleanolic acid. During 45 days of postharvest storage at 0 °C, blueberry “Duke” was more prone to softening and dehydration than the “Brigitta” variety, which was highly correlated with the higher ursolic acid content in the triterpenoid wax fraction of “Duke” blueberry ^[30][38]. Cold storage at 4 °C for 30 days reduces the total wax content of both “Legacy” (V. corymbosum) and “Brightwell” (V. ashei) blueberry varieties, but differences in wax composition between both cultivars in response to cold storage have been reported. For the “Legacy” cultivar, diketones are the only VLC compound that decreases during the storage at 4 °C, whereas for “Brightwell”, a decrease in the content of all aliphatic VLC compounds was observed ^[31][40].

4. Heat and UV Light Exposure

The quality of fruits is affected by excessive exposure to heat and UV light, mainly due to the oxidation of proteins and enzymes. One of the physiological functions of fruit cuticles is protecting against UV light exposure and extreme temperatures ^[32][33][5,6]. An increase in thickness, cinnamic acid derivatives and chalconaringenin compounds of cuticle appears to play a pivotal role in modulating UV radiation exposure in tomato fruit ^[34][73]. Furthermore, conformational changes leading to the glass transition of the cuticle membrane could serve as an adaptation mechanism in response to a change in environmental temperature ^[34][73]. It has been shown that the heat capacity of cuticle depends on the developmental stages of tomato fruit and that the thermal properties of fruit cuticle could be regulated by phenolic compounds ^[34][73]. Heat treatment increases the wax contents amount of “October Sun” peach fruits, whereas the effect of heat on cutin is less clear ^[35][74]. After a room temperature storage period, peach subjected to heat treatment and cold storage showed a reduction of cutin amount. Furthermore, it has been shown that heat treatment reduces the acyclic/cyclic compounds ratio of peach fruits ^[35][74], which strongly suggests a major role of wax cyclic compounds in response to heat.

5. Ethylene Regulators and Controlled Atmosphere

A controlled atmosphere (CA), dynamic controlled atmosphere (DCA) based on chlorophyll fluorescence (DCA-CF), DCA respiratory quotient (DCA-RQ) and 1-methylcyclopropene (1-MCP) application to apple fruits “Maxi Gala” do not affect the total cuticular wax content during cold storage conditions. Nevertheless, these treatments induce a change in the composition or concentration of specific wax constituents. DCA-CF leads to a higher concentration of nonacosane (C₂₉) and a reduction of mass loss, whereas 1-MCP reduces the concentration of nonacosan-10-ol and specific fatty acid constituents ^[25][72]. Storage of mature apple “Cripps Pink” under CA, DCA-CF and DCA-RQ treatments increase the wax concentration from day 7 to 14 of shelf life at 20 °C. Besides, all treatments led to a general increase of unsaturated fatty acids. Particularly, an increase in cis-11,14-eicosadienoic acid (C_20:2), nonacosane (C₂₉) and tetracosanal (C₂₄) was observed. A controlled atmosphere leads to an increase in ursolic and oleanolic acids, whereas DCA-RQ leads to an increase in 10-nonacosanol (C₂₉) ^[36][71]. In apple fruit cv. “Starkrimson”, the combination of ethephon treatment and storage at 0 °C accelerates total wax and VLC aliphatic deposition, whereas 1-MCP causes the opposite effect. One of the most obvious effects of ethephon and 1-MCP treatments was the increasing and decreasing of octacosanoic acid content, respectively ^[27][28]. In “Red Fuji” apple fruit, the total cuticular wax, nonacosane and heptacosane amounts decrease during seven months of postharvest storage at 0 °C, but 1-MCP treatment slightly suppresses this reduction ^[26][27]. Like “Starkrimson” apple ^[27][28], in “Red Fuji” apple, nonacosan-10-ol, nonacosan-10-one and hexadecanoic acid amounts increased after seven months of cold storage, but when fruits were treated with 1-MCP, their amounts were reduced ^[26][27]. Altogether, these findings support the relevant role of ethylene on the regulation of cuticular wax biosynthesis during the postharvest storage of fleshy fruits.

6. Physiological Disorders

The specific aliphatic composition of cutin influences the mechanical properties of apple fruit cuticles. It has been proposed that microcrack formation is due to low elastic cutin properties due to the low presence of phenolic compounds ^[37][57]. Sweet cherry varieties with a higher amount of nonacosane (C₂₉) are more tolerant to cracking than those with lower levels, suggesting that this wax component could protect sweet cherry from cracking ^[38][60]. There were no differences between alkane contents in both wild-type “Dangshansuli” pear fruit and its russet mutant “Xiusu” at early stages of development. Nevertheless, as the fruit grows, a higher content of alkanes is synthesized in “Dangshansuli” fruits. These differences in wax deposition and composition during pear development could contribute to the russeting formation observed in the “Xiusu” fruit ^[39][35]. The cuticular waxes of jujube fruit (Ziziphus jujuba Mill.) cultivars “Popozao”, “Banzao” and “Hupingzao” are mainly composed of fatty acids, primary alcohols and alkanes. No significant differences were observed in the mass of cuticle or cutin between the jujube fruit cracking-resistant cultivar “Popozao” and the cracking-susceptible cultivar “Hupingzao”. Nevertheless, in the coloring stage of jujube development, “Popozao” shows a higher level of total wax than “Hupingzao”. It has been suggested that the severity of microcracks during fruit development could be related to a lower level of cuticular wax. Furthermore, during the coloring period, “Popozao” cuticular wax contains fewer fatty acids but more alkanes and aldehydes with a chain length greater than 20 carbon atoms than cultivars “Banzao” and “Hupingzao”. Based on the above-mentioned factors, it seems that alkanes and aldehydes with longer chain lengths could contribute to protecting against microcracking during the coloring period of jujube fruit enlargement ^[40][50]. No difference in thickness and total content of epicuticular wax is shown in oleocellosis-damaged lemon fruits, but a significant increase of alkanes (especially C₂₉) and a decrease of the amount of aldehydes (especially C₃₂) are shown, suggesting that oleocellosis can be related to the transformation of VLC aldehydes to VLC alkanes ^[41][26].

7. Pathogen Infection

In Asian pear, a negative association between cuticular wax concentration and the development of Alternaria rot has been shown, with a difference in resistance to Alternaria rot between cultivars ^[42][36].  Despite the lower amount of terpenoids present (1.29%) in cuticular waxes of mature goji berry fruit (L. barbarum) experimental evidence shows an association between their presence and Alternaria alternata infection resistance in goji berry fruit ^[43][51]. It has been proposed that triterpenoids of cuticular waxes have a potential role in maintaining fruit integrity and postharvest quality and extending shelf-life because they provide mechanical toughness and protection against pathogen infections in lingonberry fruit (V. vitis-idaea) ^[23][64]. In agreement, the cuticular wax fraction of pear fruits, mainly composed of triterpenoids, inhibits A. alternata germination and growth in vitro, indicating that these compounds might contribute to antifungal protection against fungal pathogens in pear fruit ^[44][34]. It has been suggested that the increase in wax accumulation in the early stages of nectarine development could play a role in the resistance to fungus infection and water loss at harvest and during postharvest conditions, apparently due to the presence of triterpenoids. Oleanolic and ursolic acids appear to contribute to nectarines’ fruit resistance to brown rot caused by Monilinia laxa in the middle stages of development, but this resistance is not observed at the maturity stage. ScholarAuthors argue that the lack of resistance showed at the mature stage could be due to the presence of microcracks in the fruit epidermis, which affect cuticle integrity, and a higher level of alkanes in the cuticle, which could serve as a carbon source favoring the growth of fungus ^[8][32]. The analysis of epicuticular wax components of mandarin “Satsuma” fruit (C. unshiu) showed that they could promote mycelial growth of Penicillium digitatum, whereas cutin components could inhibit conidial germination at different storage periods ^[18][25]. Furthermore, the tomato mutant DFD exhibits resistance to microbial pathogens even when the cuticular wax has been removed, which suggests a possible role of the cutin structure in the resistance to microbial pathogens in tomato fruit ^[4][12]. The studies mentioned above show that triterpenoids and cutin components, rather than VLC aliphatics, appear to have a more relevant function during the fruit defense to pathogen infection. The biochemical metabolism of fleshy fruit during development and postharvest has been widely studied. Although several studies showed the pivotal role of cuticle biosynthesis in fleshy fruit quality ^[16][45][9,10], studies using genes and proteins to enhance fruit quality through cuticle modification are still scarce. These studies have allowed the identification of genes and proteins that can be modified to extend fleshy fruits’ shelf life ^[46][47][3,75]. Furthermore, this knowledge can contribute to generating technologies to improve fruit quality by increasing postharvest shelf-life, enhancing pathogen resistance, reducing physical disorders and reducing softening and water loss rates.