The cuticle is a barrier that was first developed in plants during their colonization to dry land. Due to its lipidic composition, the cuticle not only prevents dehydration but it is also a protective barrier against biotic and abiotic stress, and plays a role in preventing damage from mechanical stress (Domínguez et al., 2011). The cuticle is synthesized by the epidermal cell layer and is composed of a cutin matrix, in contact with the cell wall, and a complex group of different cuticular waxes that embed and cover the cutin matrix, constituting intracuticular and epicuticular waxes, respectively, (Ensikat et al., 2006; Koch and Ensikat, 2008; Buschhaus and Jetter, 2011). Cuticular waxes consist of a mixture of very-long chain fatty acids (VLCFAs) and its derivatives, which are synthesized by two pathways: the alcohol-forming pathway, which generates primary alcohols and esters, and the alkane-forming pathway, which produces aldehydes, alkanes, secondary alcohols, and ketones (Yeats and Rose, 2013). The biosynthesis of cuticular wax by one metabolic pathway or another may be regulated by different compounds, such as abscisic acid (Wang et al., 2015a; Romero and Lafuente, 2020). The predominant components of the waxes in the cuticle of many fruits are VLC-alkanes, which account for 50–80% of total wax content, and nonacosane (C29) or hentriacontane (C31), which are the majority. In addition, fatty acid alcohols, aldehydes, ketones, and triterpenoids have also been detected (Bauer et al., 2004; Parsons et al., 2012; Wang et al., 2015a; Wu et al., 2018).

In fleshy fruits, cuticular waxes play a crucial role in minimizing water loss/uptake through the surface, providing mechanical support, preventing fruit softening, and increasing pathogen resistance (Martin and Rose, 2013; Tafolla-Arellano et al., 2017; Trivedi et al., 2019). In blueberry fruits, wax removal decreased postharvest fruit quality during cold storage, accelerating water loss and decay (Chu et al., 2018). The cuticle in fruits is usually thicker than in leaves, and the structure and composition of cuticular wax has been shown to be closely related to the postharvest quality of fruit (Lara Ayala et al., 2014, 2019). The composition of cuticular wax varies widely among fruit species and cultivars (Trivedi et al., 2019), and many of the cuticular properties are affected by wax composition. The presence of long-chain alkanes and aldehydes has been found to increase water impermeability of fruit cuticles. With regard to this, a positive correlation between cuticle composition and water loss has been reported, finding that the alkane content was a significant determinant of water permeability in pepper (Parsons et al., 2012).

With respect to the biosynthetic pathway of the cuticular wax, most of the studies have been conducted in Arabidopsis thaliana, and many of the genes involved in alkane biosynthesis have been identified from eceriferum mutants (McNevin et al., 1993). Among them, CER2, CER26, CER1, and CER3 have been found to be determinant genes for the proper development of the cuticle, and shown to be involved in alkane biosynthesis. CER2 mutants were shown to have a severe wax deficiency and accumulated 26C wax components, being deficient for components longer than 28C (Haslam et al., 2017). CER26 mutants were shown to have a similar behavior to CER2, being specifically affected in the VLC-fatty acid elongation process (Pascal et al., 2013). Mutants with a CER1 loss-of-function have cuticles with a reduced number of alkanes and its derivatives (Bourdenx et al., 2011). CER3/WAX2 is also involved in the production of wax, with the wax2 mutants having a decreased wax content of about 80% compared to wild type plants (Chen et al., 2003). WAX2 catalyzes the reduction of VLC-acyl-CoAs to intermediate VLC-aldehydes, which are then converted to alkanes through decarbonylation by CER1 (Bernard et al., 2012). The most important genes belonging to the alkane-forming pathway that lead to the formation of VLC-alkanes are CER1 and CER3. They interact physically, and it has been proposed that they are the core components of a VLC-alkane synthesis complex (Bernard et al., 2012). CER3 catalyzes the reduction of VLC-acyl-CoAs, forming aldehydes that are decarbonylated by CER1 to VLC-alkanes (Bernard et al., 2012). Homologues to these genes have been characterized in other species; in cucumber, the importance of CER1 and CER3 in wax biosynthesis and its implication in stress and water permeability has been investigated. The CsCER1 transcript was induced by abiotic stresses, and transgenic RNAi lines with a knock down of the transcript were altered in cuticular wax biosynthesis (Wang et al., 2015a). CsWAX2 (CER3) also played a significant role in the plant’s response to biotic and abiotic stresses (Wang et al., 2015b). Along with the genes encoding for enzymes responsible for the biosynthesis of wax components, several transcription factors have been identified as regulators of the cuticular wax biosynthesis (Hen-Avivi et al., 2014), such as members of the SHINE clade of AP2-domain transcription factors, which are responsible for cuticle formation and deposition in several fruits, such as tomato, sweet cherry, apple, and mango (Alkio et al., 2012; Shi et al., 2013; Lashbrooke et al., 2015; Tafolla-Arellano et al., 2017).

During the postharvest cold storage of Cucurbita pepo, the fruits experience chilling injury (CI), a disease triggered by low temperatures, which affects fruit quality and is responsible for economic losses, with the intensity of the damages dependent on the variety (Carvajal et al., 2011). The search for resistance and improvement of zucchini fruit quality during postharvest cold storage has resulted in the selection of varieties that are more resistant to CI, such as ‘Natura’, and this resistant variety has been compared to a ‘Sinatra’, a very sensitive variety. The biochemical and genetic differences that make these two varieties resistant and sensitive, respectively, have been thoroughly investigated (Palma et al., 2014a,b; Carvajal et al., 2017), and different treatments have been applied to search for improvements in the quality of the fruit during cold storage. Fruit from the cold sensitive ‘Sinatra’, when preconditioned at 15°C for 48 h before storage at 4°C (PCT) were able to resist cold storage and improved in quality during the postharvest period (Carvajal et al., 2015). In a transcriptomic study, several genes that were differentially expressed during cold storage were selected (Carvajal et al., 2018b), and among them genes related to cuticular wax deposition were found. Since one of the differences between ‘Natura’ and ‘Sinatra’ was the lower weight loss in the resistant ‘Natura’, one of the possibilities for cold resistance was a difference in the cuticle between these varieties. Based on this hypothesis, in a first approach, we detected that long-term cold storage diminished the expression of genes belonging to the fatty acid elongation complex and the ECERIFERUM proteins 1 and 3 (CER1 and CER3; Carvajal et al., 2018a). To shed light on the involvement of the cuticle in the maintenance of postharvest quality of zucchini fruit, the aim of the present work was to analyze the cuticle properties and the differences in cuticular waxes between the varieties ‘Natura’ and ‘Sinatra’, as well as in PCT ‘Sinatra’ fruit. Changes in permeability, structure, composition, and expression of cuticle-related genes will be discussed in terms of cold resistance.

The implication of the cuticular waxes in the resistance to cold stress during postharvest storage of zucchini was investigated in the present study. A summary of the low temperature effects on the main quality parameters, weight loss, chilling-injury (CI) index, and firmness, throughout storage at 4°C is provided in Supplementary Table S3. These data show that Natura fruit, as well as fruit of Sinatra under a preconditioned treatment, were more tolerant to cold storage that Sinatra fruit, that were more sensitive and with more chilling injuries. Fruits from the more resistant variety ‘Natura’ showed a well-developed cuticle, whereas fruits from the very sensitive ‘Sinatra’ showed a slight stain that was not uniform, indicating a less developed cuticle in fruits that were more prone to developing CI. Cuticular waxes have been suggested to play an important role in fruit quality, postharvest storability, and pathogen susceptibility during postharvest of horticultural crops (Lara Ayala et al., 2014; Chu et al., 2018), being related to fruit postharvest water loss and resistance to environmental and biotic stresses. In zucchini fruit during cold storage after postharvest, the variety ‘Natura’ lost a smaller amount of water than the variety ‘Sinatra’ (Supplementary Table S3), which was also consistent with a better developed cuticle in ‘Natura’. The chlorophyll leaching assay also showed that the cold-tolerant variety had a more reduced cuticular permeability that the cold-sensitive variety, as was also observed in leaves of Arabidopsis that overexpressed genes involved in the biosynthesis of VLCFAs (Yang et al., 2021). Transgenic cucumber overexpressing a gene involved in alkane synthesis also had a lower cuticular transpiration and chlorophyll leaching than the wild type (Wang et al., 2015b). Contradictory results have been reported as to the relevance of cuticle thickness in weight loss during storage, but most of the studies published reveal a positive relation between thickness and a decrease in weight loss, for example in pepper (Parsons et al., 2012). In blueberry, the removal of cuticular wax accelerated postharvest water loss and decay, and hence fruit quality (Chu et al., 2018). In zucchini, we conclude that cuticle thickness is related to chilling tolerance and helps to reduce water loss.

From a previous transcriptomic approach contrasting Natura and Sinatra fruits (Carvajal et al., 2018b), important differences among genes related to cuticle have been detected. Among the up-regulated DEGs in ‘Natura’, genes for wax and cutin synthesis were included. Homologous of these genes have been reported to be implicated cuticle development and also in water maintenance, such as GDSL lipase occluded stomatal pore 1 (At2g04570) from Arabidopsis (Tang et al., 2020), or the cytochrome P450, with a high identity with Arabidopsis CYP86A2, whose att1 mutants showed a reduction of all cutin monomers and a consequent increase in cuticle permeability (Xiao et al., 2004). On the other hand, the only DEG up-regulated in ‘Sinatra’ with respect to ‘Natura’ at harvest corresponded with a MBOAT, a homolog to At5g55340, which catalyzes the final step in the synthesis of long-chain linear esters. The differences found at the gene expression level are related with the higher development of the cuticle in ‘Natura’, as observed at the microscopic level as well as its lower permeability; it also highlights the importance of the pre-harvest transcriptomic status on the subsequent postharvest behavior during cold storage.

In fruit of the varieties ‘Natura’ and ‘Sinatra’, differences in cuticular wax load were also detected, both at harvest, with a higher amount of waxes per surface area in ‘Natura’ than in ‘Sinatra’, and a sharp increase after 14 days of cold storage in ‘Natura’. In fruits of the preconditioned ‘Sinatra’, a higher amount was also observed after 14 days at 4°C. These results are supported by the SEM microscopy images, where higher wax crystal deposition was detected in ‘Natura’ and in PCT ‘Sinatra’ fruit surfaces. The preconditioned fruit showed images similar to those found in ethylene-treated orange fruit, where the formation of a new wax cover has been described (Cajuste et al., 2010). In that case, ethylene improved the postharvest behavior related to non-chilling peel pitting and increased cuticular wax yields. These results, together with the higher resistance to cold in ‘Natura’ and the great improvement in the postharvest performance of preconditioned ‘Sinatra’ fruit, point to an involvement of cuticular wax on the defense to cold stress conditions and the maintenance of the fruit water status (Supplementary Table S3).

With respect to the components of the cuticular wax, changes in composition and content during postharvest storage have been characterized in fruits such as apple (Chai et al., 2020), blueberry (Chu et al., 2018), and pear (Wang et al., 2021). These studies revealed that triterpenes and alkanes were, in general, the most prominent wax components of fruit cuticles in many species. The predominance of a wax compound has been associated to the taxonomic family of the species (Lara Ayala et al., 2014). We did not detect triterpenes in zucchini cuticular waxes. In fruits of Cucumis sativus, the main components were alkanes, aldehydes, and fatty acids (Wang et al., 2015a), similar to the components found in Cucurbita pepo. The most abundant alkanes, in our case, were heptacosane (C27), nonacosane (C29), and hentriacontane (C31); with the last two being the most abundant alkanes in fruit cuticles of many species (Wang et al., 2015a; Wu et al., 2018; Romero and Lafuente, 2020). The amount of alkanes increased in the cold-resistant fruit of ‘Natura’ during the storage at 4°C after 14 days, but remained unchanged in sensitive ‘Sinatra’ fruits, also increasing in PCT ‘Sinatra’ fruit, which indicates a mechanism to overcome chilling damages in which cuticular wax synthesis would be involved. The involvement of the alkanes in the defense to stresses has been reported for other species. In a study conducted with 50 pepper accessions, Parsons et al. (2012) found a positive correlation between water loss and alkane content/composition. When analyzing 10 apple cultivars during cold storage, the lowest alkane content was found in the ‘Red Star’ cultivar., which showed the highest weight loss rate during the storage period (Chai et al., 2020). In apple fruit peel, low temperatures induced the alkane-forming pathway and resulted in the accumulation of VLC-alkanes (C22, C27, C29, and C31; Hao et al., 2017), the same accumulation of C27, C29, and C31 alkanes detected in ‘Natura’ fruit and also in preconditioned fruit of ‘Sinatra’, which points to the implication of these compounds in chilling tolerance of zucchini fruit.

We have detected that the content of esters was higher in ‘Natura’ than in ‘Sinatra’ at harvest, whereas the fatty acids content showed the contrary behavior. This could be due to a higher rate of ester hydrolysis in the cold-sensitive variety. In apple fruit, an increase in the content of fatty acids has been described due to the hydrolysis of esters (Veraverbeke et al., 2001). Samuels et al. (2008) have described that most cuticular waxes are generated from the elongation of C16 and C18 free fatty acids, including two well characterized metabolic pathways for the formation of alkanes and for the production of primary alcohols and wax esters. In our analysis, we detected only two esters in zucchini fruit: 1-monopalmitin and 1-monostearin. These two esters were also the only glyceride compounds identified in all 35 pear cultivars studied when the chemical composition of the cuticular wax in mature fruits was analyzed (Wu et al., 2018). The content of 1-monopalmitin and 1-monostearin diminished during the cold storage in both zucchini varieties, observing a greater decrease in ‘Natura’ than ‘Sinatra’ fruit. This behavior is indicative of an induction of the alkane biosynthesis pathway and the inhibition of the ester’s biosynthesis pathway in the cold-tolerant fruit kept at 4°C. This finding is supported by different studies such as that of Wang et al. (2015a), where the knocked-down expression of CsCER1 in transgenic cucumber plants caused a decrease in alkanes content as well as an increase in esters, whereas overexpression of CsCER1 showed the opposite response. In a similar way, transgenic cucumber lines with an abnormal expression of CsWAX2, an Arabidopsis CER3 homolog involved in alkane synthesis, showed an increased amount of esters in cuticular waxes, which decreased with the overexpression of this gene (Wang et al., 2015b). The alternation between the formation of alkanes and wax esters also seems to be regulated by abscisic acid (ABA). In cucumber, the expression of CsCER1 was shown to be induced by this phytohormone (Wang et al., 2015a), whereas the ABA deficiency orange mutant Pinalate showed an induction in the gene CsWSD1-like, responsible for wax ester formation (Romero and Lafuente, 2020). These results suggest that ABA could act as a negative regulator of wax ester formation, and a positive regulator of alkane formation. In a previous work, we described that ‘Natura’ fruit drastically increased the ABA content during the first days of exposure to low temperature, whereas ‘Sinatra’ fruit did not show significant differences in ABA content during storage (Carvajal et al., 2017). This ABA response could be associated with the changes observed in the content of alkanes and esters in the two zucchini varieties, and should be investigated in the future.

It is obvious from this research that cuticular waxes, specifically alkanes, play an important role in defense against low temperature stress in fruits of Cucurbita pepo. Fruits of ‘Natura’, a cold resistant variety, and preconditioned ‘Sinatra’ had a higher content of alkanes after cold storage, and this correlated with a lower weight loss and CI damage during postharvest, as well as greater firmness. The biosynthetic pathway of cuticular waxes and regulatory genes have been researched and described in model species and in some fruits, but little is known in zucchini fruit. In this study, we analyzed the expression of 4 genes involved in proper cuticle development, and specifically involved in VLC-alkane biosynthesis. CpCER2-like only showed differences in the cold-tolerant variety ‘Natura’ at the end of the storage period. On the contrary, CpCER26-like gene expression was significantly higher in ‘Natura’ at harvest and after 1 day of cold exposure. Both genes, CER2 and CER26, are important components of the VLCFA elongation process (Haslam et al., 2017), with the Arabidopsis cer2 mutant showing an impaired production of wax components longer than 28 carbons, and cer26 mutant affected in the production of wax components longer than 30 carbons (Pascal et al., 2013). The higher expression of both genes in Natura fruit during storage, explains the accumulation of nonacosane (C29) and hentriacontane (C31).

The expression of the zucchini homolog genes, CpCER1-like and CpCER3-like, has been studied in this work. CpCER1-like mRNA was highly accumulated when ‘Natura’ fruit were harvested, whereas in ‘Sinatra’, very low levels were measured. Our results are supported by those found in Arabidopsis cer1 insertional mutants, which showed a reduction in heptacosane (C27), nonacosane (C29), hentriacontane (C31), and tritriacontane (C33), with its overexpression resulting in an accumulation of these components (Bourdenx et al., 2011). The expression of CpCER1-like in zucchini decreased with storage at 4°C, similar to the results found by (Bourdenx et al., 2011), which reported a decreased expression of the CER1 gene in Arabidopsis plants subjected to dark and cold treatments. This behavior is in agreement with the trend described in previous works (Wang et al., 2015b) for its homolog CsWAX2, which was induced after 24 h of cold stress in cucumber plants. These authors also described a significant reduction in the concentration of the alkanes, pentacosane (C25), heptacosane (C27), nonacosane (C29), and hentriacontane (C31), in fruit from CsWAX2 RNAi lines, as well as an increase of them in the CsWAX2 overexpression lines. In zucchini fruit, the large difference found in CpCER1-like and CpCER3-like gene expression between varieties at harvest time and after 1 day of storage could explain, in part, the accumulation VLC-alkanes during cold stress.

Regarding the possible role of transcriptional regulators in wax biosynthesis, it has been reported that several members of the APETALA2/ETHYLENE-RESPONSIVE ELEMENTBINDING PROTEINS (AP2/EREBP) family regulate cuticle-related genes. In this work, we analyzed the expression of two cuticle-related AP2/EREBP transcription factors, CpSHINE2 and CpWIN1-like. Cold storage induced CpSHINE2 mRNA accumulation in both varieties in the short-term, with the increase being greater in the transcription detected in ‘Natura’. On the contrary, this induction occurred in the long-term in PCT ‘Sinatra’ fruit. However, CpWIN1-like only showed a sharp increase in the cold-tolerant ‘Natura’ fruit after being exposed to low temperature, whereas no changes were detected in ‘Sinatra’. Recently, Zhang et al. (2019) reported that the ectopic expression of the apple gene MdSHINE2 in Arabidopsis resulted in a higher accumulation of wax crystals, and an increase in the alkanes, alcohols, aldehydes, and fatty acids wax components. These results correlated with an induction of CER1, CER3, and WIN1 gene expression, as well as an increase in drought tolerance. WAX INDUCER1/SHINE1 has been associated with the induction of different wax biosynthesis genes such as CER1 and CER2 (Broun et al., 2004). Arabidopsis plants overexpressing WIN1/SHN1 also showed enhanced drought tolerance (Aharoni et al., 2004). These results provide evidence that these genes play an important role on the regulation of the cuticular wax biosynthesis in zucchini fruit subjected to stress due to low temperatures.

Several MADS-box genes have been associated with the ripening process in tomato fruit, such as the FRUITFULL homologues, which may play a role in cuticle formation (Bemer et al., 2012; Shima et al., 2013; Fujisawa et al., 2014). In zucchini fruit, the homolog to tomato TDR4/FUL1, CpFUL1-like, was significantly expressed in the cold-tolerant ‘Natura’ at harvest and after a short-term storage at 4°C. This response could be related with the lower percentage of weight loss observed in these fruits according to Bemer et al. (2012), who described an increase in the water loss rate in harvested tomato FUL1/2 RNAi fruits.

This study is the first comprehensive analysis of the cuticular wax structure, load, and composition in Cucurbita pepo fruit during postharvest, and its involvement in the defense against chilling injury, proving the importance of the biosynthesis of VLC-alkanes and its transcriptional regulation during the adaptation of the zucchini fruit to low temperatures. Overall, the results obtained can be the basis of future functional studies, and could serve in the identification of markers for the selection of cold-resistant varieties in zucchini breeding programs.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

DG and MJ designed the project and were responsible for funding acquisition. FC and FP conducted the research and analyzed the results. RJ-M and AC-C collaborated in part of the investigation. FC, FP, and DG wrote the original draft with contributions by MJ. All authors have read and accepted the final manuscript.

This work was supported by the Ministerio de Ciencia, Innovacion y Universidades (Project AGL2017-82885-C2-2-R and Project PID2020-118080RB-C22). AC-C is being funded by FPI Grants (MEC).

The authors declare that the investigation was conducted in the absence of any financial interests that could be interpreted as a potential conflict of interest.

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