Characterization of the fruit post-harvest ripening under low temperature
The fully developed and physiological matured mangoes were harvested at color change stage. The fruit attains its full color development after 4 days of storage at 30°C, reaching a stage where it is ready for consumption, with typical taste and firmness (Fig. 1). However, low temperature inhibited the skin color development (Fig. 1A), and delayed the fruit softening (Fig. 1B). Even at 10 d, the skin color only partially turned to yellow whereas the TSS and flesh firmness were comparable to that of under 30°C storage at 4 d (Fig. 1B and C). In order to gain a further understanding of how delayed ripening at low temperatures affects the quality of mango fruit, we conducted a comprehensive analysis of the transcriptome and metabolome. This analysis focused on dynamic changes observed in mango fruits stored at 13°C for 10 days, with a positive control involving a treatment of 30°C for 4 days representing the conventional post-harvest ripening, and a negative control at 0 days.
Metabolome and differential metabolites analysis
The metabolome of 3 groups of mango samples were profiled via untargeted UPLC-MS/MS. A total of 640 metabolites were identified, including 20 vitamins, 26 saccharides and alcohols, 5 sesquiterpenoids, 6 monoterpenoids, 18 triterpene, 3 terpenes, 25 flavonols, 9 flavonoids, 67 organic acids, 53 free fatty acids, and 128 phenolic acids, etc.
To evaluate the differences in the metabolite changes caused by the two storage temperatures, HCA was used to analyze intra-treatment homogeneity and inter-treatment variability. Different storage temperatures and extension of storage time increased the differences in metabolites between groups (Fig. 2A). PCA was performed on the samples by using UPLC-MS/MS data (Fig. 2B). The PCA showed that the variation of the same sample was small, and 3 groups of samples effectively separated, which indicated that different storage temperatures and duration times significantly affect metabolites profiles. The total differential metabolites detected at 13°C storage (249) is more than at 30°C storage (206) (Fig. 2C) which mainly contributed by the down regulated metabolites at low temperature (Fig. 2D).
Pathways associated with the DEGs under low temperature storage
To inspect the changes of gene expression in mango fruits during post-harvest at different temperatures, KEGG pathway enrichment analysis was performed based on the results of differential expressed genes (Table 1). The highly expressed genes in positive control (30°C 4 d) are dominantly involved in respiratory metabolism and nutrient metabolism including “carbon metabolism”, “glycolysis/gluconeogenesis”, “pyruvate metabolism”, “metabolic pathways”, “thiamine metabolism”, “glycosaminoglycan degradation”, “biosynthesis of secondary metabolites”, “selenocompound metabolism”, etc., in comparison to 0-day control.
However, low temperature not only weakened the respiratory, but also suppressed the biosynthesis processes focused on secondary metabolites. These compounds, notably responsible for imparting flavor and nutritional substances like flavonoids and amino acids (Table 1), exhibited a parallel pattern with the findings of enriched differential metabolites (Fig. 3). The citrate cycle, also known as the TCA cycle, maintained elevated activity even after 10 days of storage at 13°C. This increased metabolic activity led to increased carbohydrate consumption, providing a partial explanation for the observed decrease in TSS during low-temperature storage (Fig. 1B).
Table 1
Significantly enriched KEGG pathways of DEGs.
Pathway Description | 0d vs 30°C 4 d | 0dvs 13°C 10 d | 30°C 4 d vs 13°C 10 d |
Thiamine metabolism | √ | - | - |
Glycosaminoglycan degradation | √ | - | - |
Biosynthesis of secondary metabolites | √ | - | √ |
Selenocompound metabolism | √ | - | - |
Glycolysis / Gluconeogenesis | √ | - | - |
Biosynthesis of cofactors | √ | √ | - |
Metabolic pathways | √ | √ | √ |
Carbon metabolism | √ | √ | - |
Pyruvate metabolism | √ | √ | - |
Propanoate metabolism | - | √ | - |
Citrate cycle (TCA cycle) | - | √ | - |
Tyrosine metabolism | - | - | √ |
Ubiquinone and other terpenoid − quinone biosynthesis | - | - | √ |
Flavonoid biosynthesis | - | - | √ |
Valine, leucine and isoleucine degradation | - | - | √ |
Glycine, serine and threonine metabolism | - | - | √ |
Note: ‘√’ indicates the difference is significant (P < 0.05). ‘-’ shows that no significant difference was detected. |
Low temperature storage enhanced the accumulation of the organic acids
Accompanied the sweetness increasing, the fruit acidity is decreasing during post-harvest ripening. The acidity of mango fruit is predominantly influenced by the levels of citric acid and malic acid [23]. In this study, we found that the content of citric acid decreased during ripening under 13°C and 30°C storage, and a more obvious reduction was observed in 13°C storage. On the contrary, low temperature storage led to an increased the accumulation of malic acid. Moreover, low temperature storage also slowed down the ascorbic acid degradation (Fig. 4).
Genes involved in low temperature induced starch and sucrose conversion
During the ripening of mango fruit, starch undergoes active hydrolysis, serving not only as an energy source but also contributing to the increased TSS content in the flesh (Fig. 1B). Key enzymes involved in this process include acid invertase (Al), neutral invertase (NI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) as substantiated by previous studies [20, 21]. Through transcriptome analysis, we identified one AI gene, eight NI genes, and eight SS genes, comparing the expression patterns of different gene members within each family. One AI (mango006181) and one NI (mango012278) exhibited upregulation at 10d under 13°C, whereas a SS (mango003315) and an SPS gene (mango011412) showed an earlier stage, which rose up at 4d (Fig. 5). Above results suggest that low temperature storage may accelerate respiration, and only specific gene members were involved in this process.
Low temperature storage stimulates the accumulation of flavonoids
The KEGG pathway enrichment analysis (Table 1) shows the “flavonoid biosynthesis” pathway is more active at low temperature. There are 16 flavonoids were detected, including 7-O-galloyltricetiflavan, quercetin-7-O-glucoside, gallocatechin-3-O-gallate, epitheaflavicacid-3-O-gallate, di-O-galloyl-methyl gallate, (+)-catechin-hydrate, dihydroquercetin, epicatechin-6-C-β-D-glucopyranoside, pinobanksin, epicatechin-glucoside, epicatechin-3'-O-B-D-glucopyranoside, syringe, and naringenin, etc. Among which, only kaempferol-3-O-glucuronide showed down regulation trend (Fig. 6A). Upon examining the expression of key genes involved in the biosynthesis of these flavonoids, it was observed that several members of the (F3H, F3’H, CHI) gene families were highly expressed at low temperature maturation. This increased expression may be responsible for the synthesis of flavonoids induced by low temperatures (Fig. 6B).
Regulation of ethylene receptor gene expression by low temperature storage
Ethylene, a pivotal plant hormone, is perceived by ethylene receptors (ETRs), which in turn regulate various physiological processes. Notably, an increased expression of ETR has been observed during the ripening of mango fruit [22]. In the present study, we conducted a comprehensive screening of ETR genes in mango fruits exposed to different storage temperatures and durations, identifying 6 differentially expressed genes. The analysis revealed that the expressions of all six ETR genes increased at 4 days under 13°C storage conditions, with only one gene (mango016667) maintaining high expression levels until 10 days. Interestingly, under 30°C storage, a distinct member (mango007369) showed upregulation (Fig. 7). Though the TSS and flesh firmness was comparable when stored at 4°C 10 d as 30°C 4 d, the member that functioned in ethylene signaling pathway may different.
Low temperature storage inhibited VOCs producing
Aromas are one of the most attractive features of mango, among which, terpenes occupy the most important position in terms of VOCs [24]. These VOCs belong to low molecular weight (< 400 Da) compounds and can be quickly dispersed into the surroundings [25]. However, low temperature storage not only affect the process of post-harvest ripening but also dramatically change the VOCs biosynthesis [26].
Analysis of VOCs in mango flesh using GC-MS was conducted during ripening at both 30°C and 13°C (Fig. 8). The main volatile substances in the fruit including terpinolene, 3-carene, γ-terpinene, (-)-β-citronellene and ethyl butyrate. However, low temperature storage greatly inhibited the biosynthesis of these VOAs, among which, ethyl butyrate even disappeared (Fig. 8E). When looking into the transcriptomic results, the expressions of the genes involved in the biosynthesis of terpenoids were not in accordance with the content of VOCs (Fig. 9). This suggests that the biosynthesis of these aromas may not be influenced by transcriptional regulation.