Hami melon, with its unique flavor, sweet taste, and high nutritional value, is a popular horticultural crop in Xinjiang, Northwest China. The melon fruit is normally harvested in a high temperature and high humidity season, which promotes faster fruit ripening and softening, and in turn reduces the storage life of melon fruit (Bi et al., 2003; Liu et al., 2004). Softening, a complex process is usually associated with the degradation of fruit cell wall components involving an increase in water-soluble pectin (WSP) and a decrease in insoluble and covalently bound pectin (Zerpa-Catanho et al., 2017). Enzymes such as pectinesterase (PE; EC 3.1.11) and polygalacturonase (PG; EC 3.2.1.6.9) and related factors directly participate in fruit cell wall degradation (Deytieux-Belleau et al., 2008). Zhang et al. (2021) demonstrated that cold storage can delay the softening of melon fruit by regulating the transcript abundance of PE and PG genes. Cold storage is a widely used postharvest technology to maintain the quality and prolong the shelf life of fruit and vegetables. Although cold storage slows the decline in melon firmness, the fruit taste, especially sweetness, is negatively affected by long-term cold storage (Wang et al., 2020; Zhang et al., 2021). Furthermore, Hami melon, a cold-sensitive fruit, is susceptible to chilling injury when stored at low temperatures. Typical symptoms of chilling injury in Hami melons include discoloration, surface pitting, brown spots, and decay development (Bi et al., 2003; Fogelman et al., 2011). The development of chilling injury seriously reduces melon quality such as appearance, texture, flavor and nutrition (Zhang et al., 2017; Zhang et al., 2021). Therefore, novel practical methods alleviating chilling injury in melon are urgently needed.

Recent studies showed that edible coatings can preserve fruit quality and prolong the postharvest shelf life of cold-stored fruit by regulating the internal gas environment, reducing water loss, and delaying fruit ripening and senescence (Maringgal et al., 2020). Chitosan, a non-toxic high-molecular-weight cationic polysaccharide, is mainly obtained from the partial deacetylation of natural chitin (Romanazzi et al., 2017). Chitosan, with good biocompatibility, biodegradability, antibacterial activity, and film-forming ability, is widely used as a fruit preservation coating (Maringgal et al., 2020). Chitosan coating improves firmness and soluble solid content (SSC) in fruit (Maringgal et al., 2020; Chen et al., 2021). In guava, chitosan coating delayed fruit ripening and prolonged shelf life by enhancing antioxidant processes (Silva et al., 2018). Similarly, chitosan treatment was shown to retard the degradation of total soluble sugar and sucrose in post-harvested longans (Lin et al., 2020). However, the effects of chitosan treatment on fruit softening, chilling injury, and starch and sucrose metabolism in cold-stored melon are largely unclear.

Starch and sucrose metabolism not only affect fruit quality and sweetness but also are closely related to chilling tolerance during cold storage (Yu et al., 2017; Zhang et al., 2021). Sucrose, apart from being an energy source, also functions as an osmoregulator, cryoprotectant, reactive oxygen scavenger, and signaling molecule, contributing to cell membrane balance and antioxidant systems (Wang et al., 2019). The increase of sucrose level was correlated with the reduction of chilling injury and the enhancement of cold resistance (Zhao et al., 2022). The correlation between fruit quality and key enzymes of sucrose metabolism such as sucrose synthase (SS) and sucrose phosphate synthase (SPS) has been extensively studied (Chen et al., 2019; Duan et al., 2019). Researchers showed that exogenous ATP (Duan et al., 2019), sodium nitroprusside (Chen et al., 2019), and 6-benzylaminopurine (Luo et al., 2017) treatments can effectively maintain the fruit quality by regulating the activity of sucrose metabolism-related enzymes. Likewise, the exogenous application of salicylic acid (Zhao et al., 2021), abscisic acid (Zhao et al., 2022), and glycine betaine (Wang et al., 2019) can mitigate chilling injury by regulating sucrose metabolism in peach fruit.

Starch has multiple cellular functions, including in abiotic stress response (Thalmann and Santelia, 2017). In plants, altering starch structure and content are the two common ways to counter abiotic stress. Cold stress often triggers starch degradation (Dong and Beckles, 2019). β-amylase (BMY), a hydrolytic enzyme, converts starch into maltose and has been related to cold stress response (Zhao et al., 2019). A study showed that cold-tolerant banana cultivars have 3.0 times higher BMY activity than susceptible cultivars (Der Agopian et al., 2011). Cold stress response induced regulation of BMY genes expression promotes starch degradation and in turn increases soluble sugars in cold-stored melon (Zhang et al., 2021). Similarly, overexpression of PbrBMY3 promotes starch degradation and improves cold tolerance in pears (Zhao et al., 2019). Collectively, these studies suggest that regulating starch and sucrose metabolism is an important cold stress countering mechanism and must be investigated in detail. In addition, fruit softening has been linked to starch and sucrose metabolism in banana, mango, and kiwifruit (Nardozza et al., 2013; Zhu et al., 2021). During the process of blueberry fruit softening, the sucrose, glucose, and fructose contents changed accordingly (Wang et al., 2020). The blueberry firmness was positively correlated with sucrose content and SPS activity. However, a possible similar role of starch and sucrose metabolism in melon during postharvest cold storage is yet to be defined. Therefore, it is of great significance to explore the changes in starch and sucrose metabolism accompany the softening of melon, as well as the link between them.

At present, much remains to be determined concerning the regulation mechanism of chitosan treatment in melon fruit, including whether the effect of chitosan to mitigate chilling injury is related to starch and sucrose metabolism. Accordingly, we investigated the effects of chitosan treatment on fruit quality indices and starch and sucrose metabolism with the objective of providing a new perspective on the mechanism of chilling injury in melon fruit, and developing an eco-friendly, safe, and efficient postharvest technology to preserve the quality and prolong the shelf life of harvested melons.

The edible coating is a modified atmosphere method that has been applied to preserve the quality of various fruit, such as tropical fruit, citrus, melon, tomato, and pome fruit (Maringgal et al., 2020). Chitosan, a promising coating material, has been widely used for food preservation and packaging; it is non-toxic, biodegradable, biocompatible, film-forming, and easily available (Maringgal et al., 2020; Kumarihami et al., 2022). Chitosan coating can reduce weight loss, respiration, and softening and increase SSC in post-harvest fruits (Huang et al., 2017; Kumarihami et al., 2020). In our study too, chitosan treatment produced similar results, reducing weight loss, softening, and chilling injury in cold-stored melons. Additionally, the degradation of starch and sucrose was also reduced. We found that chitosan treatment regulated the activities of starch and sucrose metabolism-related enzymes and the expression of corresponding genes.

Fruit softening is a crucial parameter that determines the length of the storage period and commercial value of post-harvest fruit. In our study, chitosan treatment effectively delayed the firmness loss in melons during 30 d of cold storage ( Figure 1A ). Meanwhile, WSP and ISP contents increased and CSP content decreased accompanying the softening of melon ( Figure 2 ). Pectin, a structurally complex polysaccharide, is commonly related to fruit cell wall degradation and softening (Brummell et al., 2004). During fruit softening, insoluble pectin is converted to soluble pectin (Wang et al., 2020). The same was observed in our study. Pectin solubilization is associated with PE and PG enzyme activities (Wang et al., 2020; Win et al., 2021). The main function of PE is to catalyze the demethylation of pectin to generate low methyl-esterified pectin, which makes the cell wall easily decomposed by PG (Khademi et al., 2014). PG further hydrolyzes the galacturonide bond in the main chain of polygalacturonic acid, increasing pectin degradation and in turn fruit softening (Brummell et al., 2004). The correlation between fruit softening and cell wall metabolism-related enzymes has been investigated in many fruits (Lin et al., 2019; Chen et al., 2021). In our study, compared with PE activity, PG activity showed a stronger correlation with softening-related parameters, such as fruit firmness and WSP, ISP, and CSP contents ( Supplementary Table 2 ). This suggests that PG plays a more important role than PE in fruit softening in melon under cold conditions.

Chitosan treatment was shown to delay fruit softening in many fruits (Kumarihami et al., 2022). Chitosan coating forms a protective cover on the fruit surface, which reduces gas exchange and moisture loss, thereby retarding the degradation of cell wall components and retaining fruit firmness (Maringgal et al., 2020; Kumarihami et al., 2022). In our study, chitosan treatment significantly slowed down the degradation of pectin in melon. Consistent with the previous findings (Lin et al., 2019), chitosan-treated fruits had a higher CSP content and lower WSP content than control, which improved fruit quality (Zhao et al., 2018; Lin et al., 2019). ISP, a form of pectin, is involved in fruit ripening. In longan fruit, the ISP content decreased, and the chitosan treated-longan showed higher ISP levels (Lin et al., 2019). However, the opposite change in ISP content was observed in our study ( Figure 2B ). The causes of these differences may be attributed to different roles of ISP in different stages of fruit ripening and senescence in different fruits, and demands further examination. A lower PE and PG activity was observed in chitosan-treated melons compared with control fruits during 0-24 d of cold storage. Although the PE and PG genes showed different expression patterns during storage, their expression was significantly inhibited by chitosan coating ( Figure 3 ). Notably, the expression levels of PE and PG genes are closely related to the firmness of Hami melon (Zhang et al., 2021). Our results indicated that chitosan treatment reduced the enzyme activities and gene expression levels of PE and PG, which reduced the degradation of CSP and maintained the storability of cold-stored melons. This also suggests that pectin degradation contributes to the loss of fruit firmness in melon, which can be countered/regulated by chitosan coating.

Accompany the melon softening, the sucrose and starch content gradually changes. Starch and sucrose metabolism in fruit is the main factor regulating soluble sugar content, which causes the change in SSC, an important sensory parameter of fruit quality. Chitosan treatment could be used to preserve higher SSC in some fruits such as longan (Lin et al., 2020) and orange (Gao et al., 2018). Our result showed that chitosan treatment also increased the SSC compared with the control melon during the entire storage ( Figure 1C ). Potentially, chitosan coating treatment reduced respiration and transpiration rate, consequently retarding the loss of SSC and fruit ripening.

Sucrose content influences the quality, taste, and membrane stability of fruits (Wang et al., 2019; Zhao et al., 2021). Therefore, the regulation of sucrose metabolism through related enzymes is important in maintaining the quality of post-harvest fruits (Stadler et al., 1999). SS and SPS are the two key rate-limiting enzymes that transform fructose and glucose into sucrose (Wang et al., 2020; Zhang et al., 2021). We found that chitosan treatment increased the activities of SS and SPS in cold-stored melons ( Figures 5A, B ), which is consistent with the findings in peach fruit treated with glycine betaine (Wang et al., 2019) and 1-methycyclopropene (Yu et al., 2017). These results suggest that sucrose accumulation in melon was due to the increased activities of SS and SPS. In addition to sucrose accumulation, numerous studies have shown that the improved SS and SPS activities can also lead to the decomposition of fructose and glucose (Sun et al., 2020). Interestingly, in our study, the fructose and glucose contents in chitosan-treated melons were higher than those in control melons, which can be related to the decreased respiration of melons and the degradation of other carbohydrates such as starch.

Starch and sucrose are also key signaling molecules in plant stress response. The relationship between chilling injury and sugar content has been confirmed in several fruits (Wang et al., 2019; Zhao et al., 2021; Zhao et al., 2022). The degradation of starch or accumulation of sucrose has been related to enhanced tolerance to chilling injury under cold stress (Thalmann and Santelia, 2017; Zhao et al., 2021). Der Agopian et al. (2011) found that regulation of starch-to-sucrose metabolism is vital for cold acclimation in banana; increased starch mobilization to sucrose improved cold resistance. Starch is the major storage metabolite in plants, and its degradation is often induced by cold stress (Dong and Beckles, 2019). SS, SPS, AMY, and BMY are the major enzymes involved in the starch and sucrose metabolism in Hami melon (Zhang et al., 2021). AMY, an endo-amylolytic enzyme, cleaves the α-1,4 glycosidic bonds of starch, accelerating starch-to-sucrose metabolism (Der Agopian et al., 2011). SPS also contributes starch to sucrose conversion. BMY, a mediator of starch degradation to downstream sugars, is activated under cold stress (Zhao et al., 2019). We found that the activities of SPS, AMY, and BMY first increased and then decreased in cold-stored melons ( Figures 5A, C, G ), indicating that cold conditions had a significant regulatory effect on starch-to-sucrose metabolism in melon.

Chitosan treatment has been reported to reduce starch degradation in harvested fruits. Cosme Silva et al. (2017) observed a higher starch content in chitosan-treated mango. In our study, chitosan coating inhibited starch degradation and had different effects on the activities of AMY and BMY at different stages of 30 d cold storage in melon ( Figures 5C, G ). Possibly, chitosan coating slowed down transpiration and suppressed the respiration rate in melon. The inhibition of respiration rate coupled with reduced metabolic activity reduces the synthesis and utilization of metabolites, thereby delaying the degradation of starch to sugars (Kumarihami et al., 2022). Similar results were found in kiwifruit (Kumarihami et al., 2021). Our study showed that chitosan coating could alleviate chilling injury by enhancing AMY and BMY activities and accelerating starch degradation in melon during the later stages of cold storage.

Transcriptional regulations play an important role in regulating starch and sucrose metabolism under cold stress. Cold stress increases the expression of several genes related to sucrose and starch metabolism, which improves cold stress resistance (Thalmann and Santelia, 2017; Zhang et al., 2021). In this study, SS (LOC103483781) and SPS (LOC103496894) genes were upregulated by chitosan treatment, resulting in a higher content of sucrose in treated fruit ( Figures 5D, E ), which improves membrane stabilization and cold stress signaling (Zhao et al., 2021; Zhao et al., 2022). Under cold stress, soluble sugars can directly modulate gene expression by mediating sugar signaling pathways (Couee et al., 2006). Thus, chitosan coating-mediated reduction in loss of sucrose content can be the possible mechanism of countering chilling injury in cold-stored melons. Also, BMY (LOC103490827) and AMY (LOC103488899 and LOC103491451) genes were upregulated in chitosan-treated melon during the later stage of cold storage ( Figures 5F, H, I ). Silencing of the BMY gene was shown to increase starch accumulation and decrease soluble sugar levels (Zhao et al., 2019). For example, abscisic acid treatment induced the expression of BMY1 and AMY3 genes in Arabidopsis, increasing starch degradation (Thalmann et al., 2016). Similar results were observed in our study ( Figure 5C ), suggesting that chitosan treatment mitigates chilling injury in melon fruit, at least in part, by upregulating AMY and BMY genes.

In addition to chilling injury, sucrose and starch metabolism are often related to fruit softening (Wang et al., 2020; Zhang et al., 2021). We found a significant positive correlation between melon fruit firmness and sucrose and starch contents ( Table 1 ). Similar results were seen in blueberry fruit treated with ethylene (Wang et al., 2020). In our study, the correlations between starch content and firmness, WSP, ISP and CSP content in chitosan-treated fruit were higher than those in control group. However, opposite results were found in the correlation between sucrose content and fruit softening indicators. Thus, we speculate that the mechanism of chitosan delaying loss in melon firmness maybe firstly regulate the changes of starch metabolism. PE activity was positively correlated with SS activity, while PG and SS activities were negatively correlated. Moreover, in chitosan-treated cold-stored melons, a stronger correlation was observed between PG and the contents of starch, sucrose, glucose, and fructose than PE ( Table 1 ). This could be another point to confirm the aforementioned hypothesis that PG plays a more important role in the softening of cold-stored melon fruit.

The correlation results revealed a certain link between fruit softening and sucrose and starch metabolism in melon during cold storage. These two complex processes involve multiple regulators including metabolites, enzyme activities, and gene regulations, which need to be investigated in future studies.

Our results showed that chitosan coating effectively reduced the conversion of CSP, inhibited the increase of WSP content, and maintained fruit firmness during cold storage of melon at 3°C. Moreover, PE and PG activities were lowered along with the downregulation of related genes. Our results also concluded that chitosan treatment alleviated chilling injury in cold-stored melons by regulating starch and sucrose metabolism. In addition, a strong correlation between melon fruit softening and starch and sucrose metabolism was observed. Altogether, chitosan coating could be a reliable and potential preservation technology for mitigating chilling injury and extending the postharvest shelf life of cold-stored melons. However, further studies are needed to comprehensively elucidate the other possible mechanisms of enhance chilling tolerance after chitosan treatment.

The original contributions presented in the study are included in the article/ Supplementary material . Further inquiries can be directed to the corresponding author.

QZ: Data curation, software, formal analysis, writing - original draft, writing - review & editing. FT: Methodology, project administration. WC: Software. BP: Validation. MN: Validation. CS: Funding acquisition, supervision, visualization. XY: Investigation, conceptualization. All authors contributed to the article and approved the submitted version.