In this study, M. nana peel was irradiated with a dose of 0.02 KJ/m² for 0–18 days under controlled temperature and humidity. We observed that, when banana was stored under the UV-C dose for a longer time i.e., 18 days, a few black spots appeared and the peel was shiny, while the CK peel had more black spots and its color was light yellow (Figure 1). There was no decay from 0 to 8 days, while from day 12–18, the decay rate of the UV-C group showed upward trend, while that of CK group was highly significantly low (p < 0.01) (Figure 2A). The weight loss rate of the CK was higher (significant at p < 0.01) than UV-C group at all time points (Figure 2B). These two observations suggest that UV-C treatment up to 8 days controls the banana peel decay rate, while the weight loss rate remains lower for UV-C treated bananas. Another indicator of fruit peel quality is the activity of cellulase activity, where it is known that its activity affects the hardness and the quality of fruits and vegetables (Kannahi and Elangeswari, 2015). We observed an increased cellulase activity with an increase in time. The CK cellulase activity was higher after 4 days, while that of UV-C treated bananas did not differ significantly till 8 days. Interestingly, from the eighth day till the end of the experiment (18th day), the cellulase activity of CK and UV-C at the same sampling time was significantly different; the activity was significantly higher in CK as compared to UV-C group (Figure 2C). These observations suggest that after 8 days of UV-C treatment, the cellulase activity is reduced as compared to CK and it possibly maintains the hardness and the quality of the banana peels.

It is known that the total phenol content of the banana increases with maturity. In our experiment, we observed that the total phenol contents of the UV-C treated peels were higher than CK. The total phenol contents showed an upward accumulation content in the UV-C treated banana as well as CK. Overall, we observed that the phenolics contents didn’t differ significantly between UV-C and CK except on eighth day. These observations suggest that total phenols content increases with maturity and that the UV-C treatment has minor effect on the total phenols content and such an effect can be seen from day 8 (Figure 2D). On the contrary, a sharp increase in the total flavonoids content was observed with the increase in storage time (Figure 2E). UV-C treatment significantly affected (increased) the total flavonoids content in M. nana peels except for day 12 of storage.

Phenylalanine ammonia lyase (PAL) plays important roles in resistance to stresses (low temperature, drought, UV irradiation) in plants. The activity of PAL increased with the prolongation of the storage time. Considering the effect of UV-C treatment, we noticed that PAL activity of the treatment group was significantly higher than control group at all time points particularly at day 8 (Figure 2F). It could be stated that UV-C treatment causes an increase in PAL activity and therefore protects banana from abiotic stresses. On the other hand, we also studied the effect of UV-C on the activity of superoxide dismutase (SOD), and peroxidase (POD), which are indispensable enzymes for anti-oxidation and anti-aging systems. The SOD activity of the two groups of banana fruits decreased briefly in the early stage of storage, and then increased significantly. During the storage period, the SOD activity of the treatment group was always higher than that of the control group, and the difference was extremely significant (p < 0.01). The difference was the largest on the eighth day. The SOD activity of the treatment group was 4.75 times that of the control group. (Figure 2G). The results show that UV-C treatment can effectively induce the increase of SOD activity of banana fruits, which is beneficial to the elimination of oxygen free radicals generated by the ripening and senescence during storage. The activity of the key enzyme in the antioxidant system of plants i.e., POD was increased with UV-C treatment however, the differences were not significant until day 8; significant differences were observed from day 12 and later (Figure 2H). These observations suggest that the UV-C treatment enhances the activity of POD and hence activates the antioxidant system in banana peels.

In this experiment, three indicators i.e., 2, 20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) value, DPPH, and FRAP were used to evaluate the antioxidant capacity of banana peels before and after UV-C treatment. The ABTS clearance rate of the two groups of bananas continued to increase with the days of storage. The ABTS clearance rate of the UV-C treated peels was significantly higher than that of the CK at 4, 8, and 18 days (Figure 2I). The DPPH clearance rate of the two groups of bananas continued to increase during the storage period, though on the day 0, the differences between the treated and CK group were non-significant. These observations suggest that DPPH as like ABTS clearance rate is affected by storage time as well as the UV-C treatment (Figure 2J). The FRAP values only differed significantly between UV-C and CK groups after 8 and 18 days, however, the FRAP value of the two groups of bananas increased continuously during the storage period, and that the FRAP value of the treatment group was always higher than that of the control group (Figure 2K). These observations indicate a positive impact of UV-C treatment on the antioxidant capacity of the tested bananas.

Based on the effect of UV-C treatment on banana peel appearance, quality, and other physiological parameters, it was concluded that storage under the experimental conditions for 8 days were suitable for maintenance of banana quality and hardness. Therefore, we further processed the banana peels treated with UV-C for 8 days (Figures 1, 2).

The transcriptome sequencing of six banana peel samples resulted in 48.14 Gb clean data with Q30 base % >90%. The clean reads ranged from 50,539,726 to 58,994,130 reads per sample (average 53,803,030 reads) and the average GC content per sample was 51.61% (Supplementary Table S1). Quantification of gene expression results for the UV-C treated and CK samples is shown in Figure 3A; the Fragments Per Kilobase of Transcript per Million fragments mapped (FPKM) of UV-C treated banana peels was slightly higher than CK (Figure 3A). The gene expression quantification results were highly reliable evident from higher Pearson Correlation Coefficient (PCC) between the expression of treatments and replicates (Figure 3B).

The screening conditions for selection of the differentially expressed genes (DEGs) was log2 Fold Change ≥1 and false discovery rate (FDR) < 0.05. The screening resulted in the identification of 425 DEGs; 94 and 331 genes were downregulated and upregulated, respectively, in UV-C treated banana as compared to CK. Top-5 highly downregulated genes in UV-C treated banana were Protein of unknown function (DUF1191; Ma11_g18320), MYB44-like transcription factor (TF) Ma07_g11110, ethylene-responsive transcription factor ERF057 (Ma04_g24880), auxin efflux carrier component 3a-like (Ma07_g18160), and LOB domain-containing protein 18 (Ma04_g39080) (Supplementary Table S2). The changes in the expression of these genes indicate that UV-C treatment affects ethylene responses, auxin transport, responses to abiotic stresses (Jaradat et al., 2013), and possibly regulates cell wall loosening (Lee and Kim, 2013). Top-5 genes that showed higher log two fold change values (higher expression in UV-C treated banana) were glutathione S-transferase (Ma05_g07440), isoleucyl-tRNA synthetase (Ma09_g10880), Wound-induced protein (Ma11_g17160), alcohol-forming fatty acyl-CoA reductase (Ma09_g30440), and histone H3 (Ma05_g00960) (Supplementary Table S2). These increased expressions indicate that UV-C treated banana has a stronger wounding response (Yen et al., 2001), increased detoxification of toxic substances by their conjugation with glutathione (Yen et al., 2001), higher protein synthesis (Saga et al., 2020), higher fatty alcohol synthesis (Metz et al., 2000), and cell cycle and development (Otero et al., 2014).

KEGG pathway enrichment analysis indicated that the DEGs were significantly enriched in taurine and hypotaurine metabolism, cutin, suberine and wax biosynthesis, aminoacyl-tRNA biosynthesis, phenylpropanoid biosynthesis, flavonoid biosynthesis, and stilbenoid, diarylheptanoid and gingerol biosynthesis pathways. We also found that the DEGs were enriched in Flavone and flavonol biosynthesis, starch and sucrose metabolism, plant-hormone signal transduction, MAPK signaling pathway-plants, plant-pathogen interaction, and carotenoid biosynthesis. Detailed changes in each pathway are presented below (Supplementary Figure S1).

a. Differential regulation of signaling and defense related pathways

Our results showed that the DEGs were enriched in two signaling related pathways i.e., plant-hormone signaling (17 DEGs) and MAPK signaling pathway (13 DEGs). Four DEGs i.e., auxin response factor (ARF), two jasmonate ZIM domain-containing protein (JAZ), and a protein brassinosteroid insensitive 1 (BRI1) had lower expression in UV-C treated bananas. The expression of three JAZs; two downregulated and one upregulated in UV-C treated bananas, indicate that Jasmonic acid (JA) signaling is affected by UV-C irradiation. Other genes in JA signaling i.e., coronatine-insensitive protein 1 (COI1), JA-amino synthetase (JAR1), and two MYC2 genes were also upregulated in UV-C treated banana peels. The UV-C irradiation on banana resulted in the increased expression of gibberellin (gibberellin receptor GID1 and DELLA protein), abscisic acid (ABA, PYR/PYL family protein and protein phosphatase 2C, PP2C), ethylene (ethylene insensitive protein 3, EIN3 and ethylene-responsive transcription factor 1, ERF1), and salicylic acid (SA, pathogenesis-related protein 1, PR1) signaling related genes. Therefore, it could be suggested that the UV-C irradiation activates the phytohormone signaling mechanisms in banana peels. These changes affect fruit ripening and senescence possibly due to changes in the expression of EIN3 and ERF1 (Lado et al., 2015). Additionally, the UV-C treatment impart changes in anthocyanin accumulation in fruits in response to differential expression of PYR/PYL family protein (Gao et al., 2018; Yang et al., 2019). The delay in softening is possibly due to changes in the expression of gibberellin signaling related genes (Wongs-Aree et al., 2014). Finally, UV-C treatment may also help in maintaining fruit quality by activating SA related genes (Ennab et al., 2020) (Supplementary Table S3).

The MYC2 TFs, EIN3, ERF1, PP2C, PYR/PYL, and PR1 genes were also significantly enriched in the MAPK signaling. Among other genes that showed increased expression in response to UV-C irradiation, we found two calmodulin (CaM4, Ma00_g02460 and Ma00_g06660), a WRKY22 (Ma04_g08390), respiratory burst oxidase (RbohD, Ma05_g29360), LRR receptor-like serine/threonine-protein kinase ERECTA (Ma10_g00450), and transmembrane protein 222 (Ma04_g08810). Increased expression of WRKY22 suggests that reactive oxygen species (ROS) are induced in response to UV-C treatment in banana peel as compared to CK (Adachi et al., 2015). While the increased expressions of CaM4s and RbohD maintains the homeostasis of ROS in UV-C treated peels (Taj et al., 2010; Pathak et al., 2013) (Supplementary Table S3). Thus, UV-C treatment protects bananas from decay.

Nineteen DEGs were enriched in plant-pathogen interaction pathway; only two DEGs (3-ketoacyl-CoA synthases, Ma04_g08880 and Ma03_g01910) showed reduced expression after UV-C treatment as compared to control. However, two other genes annotated as 3-ketoacyl-CoA synthase (Ma02_g23450 and Ma03_g02070) were upregulated. Genes that were enriched in the MAPK signaling pathway were also enriched in the plant-pathogen interaction pathway i.e., RbohD, WRKY22, CaM4, and PR1. Most importantly, we noticed the UV-C treatment increased the expression of pattern recognition receptors (PRRs i.e., EIX44198 and CERK1 (chitin elicitor receptor kinase 1)), genes related to hypersensitive response (PBS1, serine/threonine-protein kinase PBS1, and HSP90, molecular chaperone HtpG), and pathogenesis-related genes transcriptional activator PTI5 (Supplementary Table S3). These expression changes suggest that UV-C treatment induced a hypersensitive response in banana peels as compared to CK and thus protects banana during storage.

Other enzymes that are parts of antioxidant and defense responses i.e., PAL (Ma01_g04420 and Ma05_g03720) and POD (Ma10_g05180, Ma07_g23680, and Ma11_g04630) also showed increased expressions after UV-C treatment. These observations also confirm the activity of PAL and POD as presented in Figure 2. Thus, UV-C treatment activates a defense response in banana peels.

b. Effect of UV-C treatment on secondary metabolites related pathways

It has been reported that secondary metabolite biosynthesis increases when tomato is treated with UV-C irradiation (Liu et al., 2018). To this regard, our findings that DEGs were enriched in many secondary metabolite related pathways is quite relevant. Seven DEGs annotated as cysteamine dioxygenases (enriched in taurine and hypertaurine biosynthesis metabolism) showed increased expression in UV-C treated banana peels as compared to CK. These genes are known for their role in enhanced plant survival under abiotic stresses (Licausi et al., 2011) by increasing the biosynthesis of taurine (Kotzamanis et al., 2020). Hence, it is possible that UV-C treatment affected their expression in order to respond to changes in O₂ levels in peels, since cysteamine dioxygenases have also been shown to be sensitive to O₂ levels (White et al., 2018). Their function is directly linked with ERFs (White et al., 2020), thus it could be suggested that together, ERFs and cysteamine dioxygenases are activated under the influence of UV-C in banana peel and protect it during storage. Among other pathways, we found that 13 DEGs were enriched in the phenylpropanoid biosynthesis pathway. Only one gene i.e., shikimate O-hydroxycinnamoyltransferase (Ma03_g26620) was downregulated in response to UV-C treatment, while all other genes showed higher expression in the UV-C group; three PODs, three trans-cinnamate 4-monooxygenases, two cinnamoy-CoA reductases, and two PALs (Figure 4). The higher expression of all these genes leads towards increased biosynthesis of lignins (p-hydroxyphenyl lignin, guaiacyl lignin, coniferin, and syringyl lignin). The activity of callose (Figure 2C) is also in line with these observations. The three trans-cinnamate 4-monooxygenases were also enriched in flavonoid biosynthesis pathway and stilbenoid, diarylheptanoid and gingerol biosynthesis (Figure 5; Supplementary Table S3). Furthermore, the UV-C treatment resulted in increased expression of two chalcone synthases, a shikimate O-hydroxycinnamoyltransferase (also enriched in stilbenoid, diarylheptanoid and gingerol biosynthesis), and a flavonoid 3′,5′-hydroxylase. All these genes are located in the upstream of anthocyanin biosynthesis, dihydrotricetin and dihydromyricetin biosynthesis. These expression changes suggest that UV-C treatment increases the production of these compounds which are substrates for production of unstable leucopelargonidin, leucocyanidin, and leucodelphinidin. However, we did not detect the differential expression of any of the genes in anthocyanin biosynthesis pathway (Hua et al., 2013). Thus, specifically, overproduction of dihydrotricetin and dihydromyricetin in banana peels under UV-C treatment might be for different protective activities. However, we detected the increased expression of zeaxanthin epoxidase (Ma07_g03980) and (+)-abscisic acid 8′-hydroxylase (Ma07_g07210). Zeaxanthin epoxidase converts zeaxanthin into antheraxanthin and then to violaxanthin and protects plants against damage by excess light (Hieber et al., 2000).

c. Effect of UV-C treatment on other important pathways

The KEGG enrichment analysis also revealed that the DEGs were significantly enriched in cutin, suberine and wax biosynthesis and aminoacyl-tRNA biosynthesis. Seven DEGs were enriched in cutin, suberine and wax biosynthesis. A gene annotated as fatty acid omega-hydroxylase (Ma03_g26150) was upregulated in UV-C treated peels. It converts 9,10-epoxy-18-hydroxystearate into 9,10,18-Trihydroxystrearate (an unsaturated fatty acid, UFA). Previously, it was shown that the proportion of UFAs in peels is closely related with membrane fluidity and tolerance against abiotic stresses. A higher proportion of UFAs is found in plants that show tolerance to cold (Liang et al., 2020). Thus, it could be proposed that UV-C irradiation increases the UFA content in banana peel. Further, we found four DEGs (three alcohol-forming fatty acyl-CoA reductases and an aldehyde decarbonylase) that were upregulated in response to UV-C irradiation. Both genes control essential steps in the biosynthesis of wax (Cheesbrough and Kolattukudy, 1984; Metz et al., 2000). Higher wax production in fruit offers a better shelf-life; cuticle is known as a modulator of postharvest quality of fruits (Lara et al., 2014; Lara et al., 2019).

Apart from wax and UFA biosynthesis, we found that 10 DEGs were enriched in aminoacyl-tRNA biosynthesis pathway (Supplementary Table S3). Interestingly, all the genes were annotated as isoleucyl-tRNA synthetases. These genes are known to play essential roles in plants e.g., (together with dirigent proteins) maintenance of plant cell wall integrity (Paniagua et al., 2017). Thus, it is possible that UV-C treatment increases the expression of isoleucyl-tRNA synthetases, which in turn helps banana to maintain cell wall to avoid being flaccid.

The qRT-PCR analysis of 17 M. nana genes showed that the expression profiles were consistent with the transcriptome sequencing results in M. nana peels treated with UV-C as compared to CK (Figure 6). These results validated the RNA sequencing data and confirmed its reliability.

The metabolome analysis resulted in the identification of 38 differentially accumulated metabolites (DAMs) (Table 1). The DAMs belonged to a range of metabolite classes i.e., alkaloids, amino acids and derivatives, anthocyanins, diterpenoids, flavanols, flavonoids, flavonols, free fatty acids, glycerol ester, phenolic acids, tannin, and xanthone. Eight of the 38 metabolites were accumulated in lower quantities in UV-C treated bananas as compared to CK. These DAMs included two amino acids i.e., L-tryptophan and L-valyl-L-Phenylalanine, three free fatty acids, a glycerol ester, a phenolic acid (disooctyl phthalate), and a tannin (cinnamtannin A2). Interestingly, all alkaloids, flavonoids, and terpenoids showed increased accumulation in banana peels treated with UV-C irradiation (Table 1). These observations are in accordance with the results of total phenols and flavonoids contents (Figures 2D,E). Top five DAMs that were accumulated in response to UV-C treated are 4-p-Cumaroyl-rhamnosyl-(1→6)-D-glucose, 5-Aminovaleric acid, 6,8-dihydroxy-1,2,3-trimethoxyxanthone, quercetin-3-O-(2'''-p-coumaroyl) sophoroside-7-O-glucoside, and Hydroxyanigorufone (Table 1).

We found that the DAMs were significantly enriched in eighteen different pathways (Supplementary Figure S2). Significant enrichment of DAMs was observed in anthocyanin biosynthesis (cyanidin-3-O-(6″-O-p-Coumaroyl) glucoside and delphinidin-3-O-(6″-O-p-coumaroyl) glucoside) and flavone and flavonol biosynthesis (quercetin-3-O-rutinoside (Rutin) and quercetin-3-O-(6″-feruloyl) glucoside-7-O-rutinoside) pathways. The changes in the accumulation pattern of the anthocyanin biosynthesis pathway related metabolites are consistent with the increased expression of the genes that were present upstream in this pathway (Figure 5). Thus, the changes in the expression of phenylpropanoid pathway genes and accumulation of these metabolites might be a reason for the color differences between the UV-C and CK bananas. Similarly, the higher accumulation of the two metabolites in flavone and flavonol biosynthesis pathway is consistent with the observed higher flavonoid contents in UV-C treated bananas (Figure 2E). The enrichment of DAMs in other pathways is consistent with that of DEGs’ enrichment in KEGG pathways (Supplementary Figures S1-2; Figure 7A).

In this experiment, the fruit decay rate and weight loss rate were used to evaluate the storage effect, which were calculated according to formulas (1) and (2) respectively. The decay rate is calculated as rot, which is based on the visible rot spots of the stored banana fruit (Gol and Ramana Rao, 2011). D e c a y   r a t e = N u m b e r   o f   r o t t e n   f r u i t s T o t a l   n u m b e r   o f   ⁡ e x p e r i m e n t a l   f r u i t s × 100 (1) W e i g h t   l o s s   r a t e = Q u a l i t y   b e f o r e   s t o r a g e − q u a l i t y   a f t e r   s t o r a g e Q u a l i t y   b e f o r e   s t o r a g e × 100 (2)

Callose synthase and PAL activity was measured as reported earlier by Cao, et al. (Cao et al., 2007). Similarly, the activities of SOD and POD enzymes were assayed according to modified method of Oracz, et al. (Oracz et al., 2009) and Sun, et al. (Sun et al., 2011).

Methanol extract was prepared according to the modified method described by (Silva and Sirasa, 2018). 2ml 80% methanol was added to 0.50 g of banana fruit peel powder and placed in a water bath at 60°C at 50 r/min for 25 min, then centrifuge at 5,000 r/min for 10 min, collect the supernatant, and repeat the above steps twice. The supernatant of both extractions was combined, diluted to 10 ml with 80% methanol and stored at -20°C. The extract was then used to determine total phenols, total flavonoids, DPPH free radical scavenging ability, ABTS free radical scavenging ability, and ferric reducing antioxidant power (FRAP) value. Care was taken while storing and processing the extract by covering the tubers with aluminum foil to prevent the extract from being damaged by light. Total phenols content and DPPH free radical scavenging ability was measured according the method described by Khaliq, et al. (Khaliq et al., 2016). Total flavonoid content, ABTS free radical scavenging ability, and FRAP value were measured according to the methods described by Al Amri and Hossain (Amri and Hossain, 2018), Thaipong, et al. (Thaipong et al., 2006), and Wu, et al. (Wu et al., 2006), respectively.

Triplicate banana samples treated with UV-C for 8 days and the respective controls were used for RNA-sequencing. Total RNA extraction, determination of purity of extracted RNAs, and RNA quantification was done as reported earlier (Chen et al., 2019).

First strand cDNA was prepared from the purified RNA of each of three replicates of the control and the experimental groups. The cDNA synthesis, and library preparation was completed as reported earlier (Chen et al., 2019). The Agilent 2,100 Bioanalyzer was used to detect the size and concentration of the library fragments. Single-stranded circular DNA molecules were used to generate DNA nanospheres (DNB), which contained more than 200 copies. The DNBs were added into the mesh holes on the chip using high-density DNA nanochip technology. Sequencing was performed by the combined probe anchored polymerization technology (cPAS) to obtain a sequencing read length of 150 bp.

The raw reads were filtered, sequencing error rate distribution check was performed, followed by performing a GC content check. HISAT2 was then used to compare the clean reads with the reference genome (Busche et al., 2020). Gene expression was quantified as FPKM, which was then used for subsequent analyses. The overall distribution of the FPKM was visualized and used to calculate PCC between treatments and replicates. We employed DESeq2 to obtain DEGs between UV-C and CK groups. FeatureCounts was used for count of gene reads, and then Benjamini-Hochberg method was used to perform multiple hypothesis test correction on the hypothesis test probability (p value) to obtain the FDR. The screening conditions for differential genes were |log2 Fold Change| ≥ 1, and FDR <0.05. Visualization, cluster analysis, and heatmap representation of the gene expression was done as reported earlier (Chen et al., 2019).

Functional annotation and enrichment of DEGs in KEGG (Kyoto Encyclopedia of Genes and Genome; https://www.genome.jp/kegg) pathways was done as reported earlier (Deng et al., 2021).