Abstract

   In this study, we analyzed the effects of treatments with titanium
   dioxide nanoparticles (NPs-TiO[2]) and ethylene on anthocyanin
   biosynthesis and reactive oxygen species (ROS) metabolism during light
   exposure in ripe ‘red delicious’ apples. Both treatments led to
   improved anthocyanins biosynthesis in detached mature apples, while the
   NPs-TiO[2] had less impact on the fruit firmness, TSS, TA, and TSS/TA
   ratio. Furthermore, the effects of both treatments on the expression of
   anthocyanin-related enzymes and transcription factors in the apple peel
   were evaluated at the gene level. The differentially expressed genes
   induced by the two treatments were highly enriched in the
   photosynthesis and flavonoid biosynthesis pathways. The expression of
   structural genes involved in anthocyanin biosynthesis and ethylene
   biosynthesis was more significantly upregulated in the ethylene
   treatment group than in the NPs-TiO[2] treatment group, and the
   opposite pattern was observed for the expression of genes encoding
   transcription factors involved in plant photomorphogenesis pathways. In
   addition, the ROS levels and antioxidant capacity were higher and the
   membrane lipid peroxidation level was lower in fruit in the NPs-TiO[2]
   treatment group than in the ethylene treatment group. The results of
   this study reveal differences in the coloration mechanisms induced by
   NPs-TiO[2] and ethylene in apples, providing new insights into
   improving the color and quality of fruits.

   Keywords: apple coloration, nanoparticle, NPs-TiO[2], reactive oxygen
   species, ethylene

1. Introduction

   The apple is an economically important fruit crop that is widely
   consumed. According to the FAO, the worldwide apple production quantity
   is about 82 million tons, of which China produces more than 42 million
   tons [[36]1,[37]2]. Consumers usually associate the red color of apple
   fruit with ripeness and a sweet taste, thereby establishing red apple
   cultivars as particularly favored among consumers [[38]3]. The
   biosynthesis of anthocyanins in the apple peel is affected by a variety
   of environmental factors and plant hormones [[39]4], with solar
   illumination being one of the most important environmental factors.
   Reflective mulch is usually used to promote apple fruit coloration and
   improve the utilization effect of light [[40]5,[41]6]. In addition,
   ultraviolet (UV) radiation and low temperatures are particularly
   important for regulating the production of anthocyanins in apples
   [[42]7,[43]8].

   The typical approaches employed to artificially improve the deposition
   of pigments in the apple epidermis involve the use of cultivation
   techniques that facilitate efficient light utilization to promote
   anthocyanin biosynthesis, as well as the application of chemically
   synthesized biostimulants or the like to promote ripening and
   accelerate anthocyanin accumulation [[44]9]. A variety of cultivation
   strategies have been used to promote the deposition of pigments in
   apple fruits, such as approaches that increase the transmissibility of
   light [[45]5,[46]10], micro-sprinkler irrigation systems [[47]11],
   fruit bagging [[48]12], delayed harvesting [[49]13], and spray
   applications of chemicals such as exogenous ethylene [[50]14,[51]15].
   However, spray applications of chemically synthesized materials promote
   defoliation, which improves the penetration of sunlight and accelerates
   the ripening of fruits, which reduces their storage tolerance
   [[52]16,[53]17]. In addition, consumers might be discouraged to
   purchase fruits that have been treated with ethylene-releasing
   compounds because of their potential health hazards [[54]18].

   Nanotechnology is expected to provide important alternatives to
   conventional cultivation methods [[55]19]. Engineered nanomaterials
   have been widely used in agriculture for improved crop quality and
   efficiency and to enhance the ability of crops to deal with
   environmental stress [[56]20,[57]21]. Titanium dioxide nanoparticles
   (NPs-TiO[2]) are some of the most widely used engineering nanomaterials
   [[58]22]. NPs-TiO[2] are chemically stable, non-toxic, and highly
   reactive, and have been shown to effectively absorb UV radiation
   [[59]23]. When NPs-TiO[2] are exposed to UV light with a wavelength
   equal to or less than 387.5 nm, reactive oxygen species (ROS) can be
   generated via the photocatalysis of H[2]O [[60]24,[61]25]. These
   photocatalytically generated ROS can attack and degrade organic
   material (e.g., plant organs, microorganisms, viruses, and organic
   compounds) adsorbed on the surfaces of NPs-TiO[2] in both aqueous and
   gaseous phases [[62]26,[63]27,[64]28,[65]29]. A previous study has
   shown that the attachment of NPs-TiO[2] to the surface of the exocarp
   can accelerate the coloring of the exocarp by absorbing UV light, and
   the NPs-TiO[2] particles attached to the fruit’s epidermal surface can
   be washed off using a common tap process [[66]30]. In addition,
   Surround™ WP (95% kaolin) was applied to reduce sunburn on fruit by
   reflecting more light [[67]31]. The mechanisms by which NPs-TiO[2]
   treatment regulate anthocyanin metabolism in apple fruit during light
   exposure remain unclear. Here, differences in the coloring mechanisms
   of apples and the regulation of anthocyanin biosynthesis under
   treatments with NPs-TiO[2] and the conventional plant growth regulator
   ethylene were investigated. The results subsequently revealed
   differences in the coloring mechanisms of apples induced by NPs-TiO[2]
   and ethylene, which have implications for the development of new
   agricultural applications of photocatalytic nanoparticles.

2. Results

2.1. Changes in Peel Color and Fruit Quality during Light Exposure

   [68]Figure 1 shows the changes in apple fruit phenotype and skin color
   during light exposure. The rind coloring process was significantly
   accelerated in both treatments compared with the control ([69]Figure
   1A). The color parameters ([70]Figure 1B–E) measured included the
   brightness (L*), redness (a*), yellowness (b*), and hue angle (h*).
   During light exposure, the a* value was highest in the ethylene
   treatment, followed by the NPs-TiO[2] treatment and the control
   ([71]Figure 1C). The h* value of the control decreased more slowly than
   that of the NPs-TiO[2] and ethylene treatments, and there was a
   significant difference (p < 0.05) on the second day of treatment
   ([72]Figure 1E). The results indicated that the utilization of
   NPs-TiO[2] and ethylene treatments led to noticeable changes in the
   color and quality of the fruit epidermis during light exposure.

Figure 1.

   [73]Figure 1
   [74]Open in a new tab

   Effects of NPs-TiO[2] and ethylene treatments on color and fruit
   physiological quality of “Red Delicious” apple during light exposure.
   (A) Fruit coloring phenotype after treatment, (B) L* value (black to
   white), (C) a* value (green to red), (D) b* value (yellow to blue), (E)
   h* value (hue angle), (F) Firmness, (G) Total soluble solids (TSS), (H)
   Total titratable acid (TA), and (I) Solid–acid ratios (TSS/TA ratio).
   Error bars, mean ± standard error (n = 6). Different letters indicate
   significant differences among treatments in the same period using
   Duncan’s test (p < 0.05), n.s. represents no significant difference (p
   ≥ 0.05).

   Fruit quality at the end of the 8-day treatment period was measured.
   The firmness of fruit was significantly lower in the ethylene treatment
   (p < 0.05) than in the NPs-TiO[2] treatment and control ([75]Figure
   1F). The total soluble solids (TSS) ([76]Figure 1G) and solid–acid
   ratio ([77]Figure 1I) were significantly (p < 0.05) higher in the
   ethylene treatment than in the NPs-TiO[2] treatment and the control;
   however, there were no significant differences (p ≥ 0.05) between the
   NPs-TiO[2] treatment and the control. In addition, the fruit
   respiration rate ([78]Figure S2A) and ethylene production ([79]Figure
   S2B) were significantly higher (p < 0.05) in the ethylene treatment
   than in the other two treatments at 8 days. These findings indicate
   that the fruits in the ethylene treatment were more mature after they
   were harvested at the end of the in vitro light culture.

2.2. Effects of NPs-TiO[2] and Ethylene Treatments on the Secondary
Metabolites in Exocarp Tissue

   The color of the skin is closely related to pigment deposition, and the
   most common pigments in the skin of apple fruits are anthocyanins.
   Flavonoids and phenolic compounds can also affect the coloration of
   apple fruits [[80]32]. The content of anthocyanins, proanthocyanins,
   flavonoids, and total phenols was determined during light exposure
   ([81]Figure 2). The anthocyanin and proanthocyanin content of the
   exocarp treated with the NPs-TiO[2] and ethylene treatments
   significantly differed (p < 0.05) from that in the control on the
   second day of treatment ([82]Figure 2A,B). After 8 days of light
   exposure, the anthocyanin content of fruits sprayed with ethephon
   reached 293.44 ± 18.01 μg g^−1, which was 1.11 times and 1.46 times
   higher than that in the NPs-TiO[2] treatment and the control,
   respectively ([83]Figure 2A). Variation in the content of flavonoids
   and anthocyanins among treatments was similar, and the content of
   flavonoids and anthocyanins was significantly higher in the ethylene
   treatment (p < 0.05) than in the NPs-TiO[2] treatment and the control
   on the sixth day ([84]Figure 2C). The content of total phenolics in the
   fruit epidermis continued to increase over time, and the content
   measured in the NPs-TiO[2] treatment group was significantly higher (p
   < 0.05) than that of the remaining two treatments on the sixth day. On
   the eighth day of treatment, the total phenolics content of fruit in
   NPs-TiO[2] group finally reached 1661.17 ± 77.87 μg g^−1, which was
   1.10 times and 1.24 times of ethylene and control, respectively
   ([85]Figure 2D).

Figure 2.

   [86]Figure 2
   [87]Open in a new tab

   Effects of two treatments on (A) anthocyanin content, (B)
   proanthocyanidin content, (C) total flavonoids content and (D) total
   phenolics content during light cultivation of ‘Red Delicious’ apples.
   Error bars, mean ± standard error (n = 3). Different letters indicate
   significant differences among treatments in the same period using
   Duncan’s test (p < 0.05), n.s. represents no significant difference (p
   ≥ 0.05).

2.3. Transcriptome Analysis of the Differently Expressed Genes (DEGs)

   To identify DEGs between treatment groups, transcriptomic analyses were
   conducted using samples collected during 8 days of treatment.
   Correlation analysis of the data in each group, principal component
   analysis scores, and a Venn diagram of DEGs between treatments revealed
   that the transcriptome data were reliable ([88]Figure 3A,B). Venn
   diagram showed that 1124 DEGs were overlapped between clusters of
   control vs. NPs-TiO[2] and control vs. ethylene, which was much higher
   than the number of DEGs compared between the other two groups
   ([89]Figure 3C). A volcano plot was constructed to clarify the
   distribution of DEGs among the control, NPs-TiO[2] treatment, and
   ethylene treatment (log[2](Fold Change) > 1, q-value < 0.05). The
   results revealed 3416 DEGs between the control and NPs-TiO[2]
   treatment, including 2298 up-regulated DEGs and 1118 down-regulated
   DEGs ([90]Figure 3D). A total of 1719 DEGs were detected between the
   control and ethylene treatment, including 1264 up-regulated DEGs and
   455 down-regulated DEGs ([91]Figure 3E). A total of 934 genes were
   detected between the NPs-TiO[2] and ethylene treatments, including 379
   up-regulated DEGs and 555 down-regulated DEGs ([92]Figure 3F).

Figure 3.

   [93]Figure 3
   [94]Open in a new tab

   Differentially expressed genes (DEG) in apple after NPs-TiO[2] and
   ethylene treatment. (A) Correlation heatmap of different treatment
   groups, (B) PCA score diagram of DEGs, (C) Venn diagram showing the
   shared and distinct DEGs among the three compared groups, (D–F) Volcano
   plots displaying the up-regulated and down-regulated genes between
   different treatment groups. ck represents Control, TiO[2] represents
   NPs-TiO[2], and Eth represents Ethylene.

2.4. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
Pathway Enrichment Analyses of the DEGs

   GO analysis of 2143 (Control vs. NPs-TiO[2]), 1493 (Control vs.
   Ethylene), and 1153 (NPs-TiO[2] vs. Ethylene) DEGs revealed that the 30
   most significantly enriched GO terms were in three categories:
   biological process, cell component, and molecular function. DEGs in the
   Control and the two treatments encoded enzymes with high catalytic
   activity, and they were mainly enriched in ‘chloroplast thylakoid
   membrane’, ‘thylakoid and chloroplast’, and ‘chloroplast’ ([95]Figure
   4A,B). DEGs in the NPs-TiO[2] vs. Ethylene comparison group were mainly
   enriched in GO terms in the biological process and molecular function
   categories; these DEGs were also enriched in several GO terms in the
   cellular component category. Within the molecular function category,
   the most highly enriched terms were ‘UDP-glucosyltransferase activity’
   and ‘enoyl-[acyl-carrier-protein] reductase (NADH) activity’
   ([96]Figure 4C).

Figure 4.

   [97]Figure 4
   [98]Open in a new tab

   GO classification and KEGG pathway enrichment analyses of
   differentially expressed genes (DEGs) under the different treatments.
   Top 30 GO classification analysis of the genes identified under the
   biological process, molecular function, and cellular component
   categories between (A) NPs-TiO[2] vs. Control, (B) Ethylene vs. Control
   and (C) NPs-TiO[2] vs. Ethylene. Top 20 KEGG enrichment analysis of the
   DEGs between (D) NPs-TiO[2] vs. Control, (E) Ethylene vs. Control and
   (F) NPs-TiO[2] vs. Ethylene. Number of DEGs is represented by the size
   of the circle. ck represents Control, TiO[2] represents NPs-TiO[2], and
   Eth represents Ethylene.

   Next, KEGG pathway enrichment analysis was performed on selected DEGs.
   DEGs in the Control vs. NPs-TiO[2] and Control vs. Ethylene comparison
   groups were significantly enriched in the ‘photosynthesis’ and
   ‘flavonoid biosynthesis’ pathways ([99]Figure 4D–F). We performed
   real-time quantitative polymerase chain reaction (qRT-PCR) and
   transcription fragment per kilobase/million fragment profiling to
   validate the expression patterns of 12 candidate DEGs enriched in the
   above KEGG signaling pathways ([100]Figure S3). The expression patterns
   of nine of the 12 DEGs inferred by the qRT-PCR analyses were consistent
   with those inferred by the transcriptome analysis.

2.5. Differential Expression of Genes and Enzyme Activity Associated with
Anthocyanin Biosynthesis in Exocarp

   [101]Figure 5 shows the interaction expression of the major genes
   involved in anthocyanin metabolism in apple peel under different
   treatments, based on their corresponding positions in the apple
   anthocyanin biosynthesis pathway for 30 DEGs. The levels of gene
   expression in fruit treated with both treatments were significantly
   up-regulated compared to control.

Figure 5.

   [102]Figure 5
   [103]Open in a new tab

   The regulation of flavonoids and anthocyanins biosynthesis pathways in
   apple under different treatments during light exposure. Heatmaps of
   DEGs in transcripts from flavonoids and anthocyanins biosynthesis
   pathway. PAL, phenylalanine ammonia-lyase; CHS, chalcone synthase; CHI,
   chalcone isomerase; F3H, flavanone-3-O-hydroxylase; DFR,
   dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, UDP
   glucose: flavonoid-3-O-glucosyltransferase; FLS, flavonoid
   3′-monooxygenase. Red five-pointed stars represent significant DEGs in
   signaling pathways.

   To verify the effects of the NPs-TiO[2] and ethylene treatments on the
   expression levels of genes involved in anthocyanin production in apple
   during light exposure, we examined the expression of eight genes that
   showed significant changes in expression over the treatment period
   ([104]Figure 6). The relative expression levels of seven structural
   genes involved in the anthocyanin synthesis pathway, MdPAL, MdCHS,
   MdCHI, MdF3H, MdDFR, MdUFGT, MdLDOX, and the key transcription factor
   MdMYB1, are shown in [105]Figure 6A–H. The expression of MdPAL, MdF3H,
   MdLDOX, and MdMYB1 first increased and then decreased in the NPs-TiO[2]
   and ethylene treatments. The expression of MdCHS, MdCHI, MdDFR, and
   MdUFGT continuously increased in the NPs-TiO[2] and ethylene treatment
   groups.

Figure 6.

   [106]Figure 6
   [107]Open in a new tab

   Expression of genes (A–H) and enzyme activities related to anthocyanin
   biosynthesis (I–K) in the ‘Red Delicious’ apple exocarp tissues during
   light exposure with NPs-TiO[2] and ethylene treatments. Error bars,
   mean ± standard error (n = 3). Different letters indicate significant
   differences among treatments in the same period using Duncan’s test (p
   < 0.05), n.s. represents no significant difference (p ≥ 0.05). PAL,
   phenylalanine ammonia-lyase; CHS, chalcone synthase; CHI, chalcone
   isomerase; F3H, flavanone-3-O-hydroxylase; DFR, dihydroflavonol
   4-reductase; UFGT, UDP glucose: flavonoid-3-O-glucosyltransferase;
   LDOX, leucoanthocyanidin dioxygenase; MYB, R2R3-MYB transcription
   factor.

   To clarify the effects of the NPs-TiO[2] and ethylene treatments on the
   activities of enzymes involved in anthocyanin synthesis, the activities
   of three enzymes involved in anthocyanin synthesis were measured. As
   shown in [108]Figure 6I, significant differences (p < 0.05) in
   phenylalanine ammonia-lyase (PAL) activity were observed in peels
   treated with NPs-TiO[2] and ethylene in the early stage of the
   treatment period (0–24 h). The activity of PAL in the NPs-TiO[2]
   treatment increased rapidly at 2 d, and PAL activity was 2.16 times
   higher at 2 d than at 24 h. The PAL activity was also significantly
   higher (p < 0.05) in the NPs-TiO[2] treatment than in the ethylene
   treatment (1.25 times) and control (1.43 times). Changes in the
   activity of dihydroflavonol 4-reductase (DFR) in NPs-TiO[2] and
   ethylene treatments first increased and then decreased, and the
   activity of DFR was significantly (p < 0.05) higher in the two
   treatments than in the control at 12 h during the light exposure
   ([109]Figure 6J). The activity of UDP-glucose:
   flavonoid-3-O-glucosyltransferase (UFGT) was higher in the ethylene
   treatment than in the NPs-TiO[2] treatment and the control at 24 h;
   UFGT activity peaked at day 6 and decreased at day 8 ([110]Figure 6K).

2.6. Differential Expression of Genes Related to Ethylene and UV Light
Signals Pathway

   Both ethylene and UV light can promote apple pigment accumulation. We
   also analyzed the expression of genes involved in the ethylene pathway
   during light exposure ([111]Figure 7A–D). The results showed that the
   expression of MdACS1, MdACO1, MdERF3 and MdEIL1 were markedly
   up-regulated in the ethylene-treated fruit compared to the other two
   groups. Meanwhile, there were no significant (p ≥ 0.05) changes in the
   expression patterns of ethylene biosynthesis and signaling related
   genes under NPs-TiO[2] treatment. We further analyzed the expression
   levels of genes involved in UV-B signaling ([112]Figure 7E–G). The
   expression levels of MdCOP1, MdHY5, and MdUVR8 in the NPs-TiO[2]
   treatment were highest in the early and middle stages of the treatment
   period (24 h–4 days), and were markedly higher (p < 0.05) than those in
   the other two treatments.

Figure 7.

   [113]Figure 7
   [114]Open in a new tab

   Expression of genes related to ethylene (A–D) and UV light signals
   pathway (E–G) in the ‘Red Delicious’ apple exocarp tissues during light
   exposure with the NPs-TiO[2] and ethylene treatments. Error bars, mean
   ± standard error (n = 3). Different letters indicate significant
   differences among treatments in the same period using Duncan’s test (p
   < 0.05), n.s. represents no significant difference (p ≥ 0.05). ACS1,
   1-aminocyclopropane-1-carboxylate synthase 1; ACO1:
   1-aminocyclopropane-1-carboxylate oxidase 1; EIL1: ethylene
   insensitive3-like 1; ERF3: ethylene response factors 3; COP1,
   constitutively photomorphogenic 1; HY5, elongated hypocotyl 5; UVR8, UV
   resistance locus 8.

2.7. Changes in ROS Levels and Membrane Lipid Peroxidation during Light
Exposure

   To clarify the effects of the NPs-TiO[2] and ethylene treatments on
   oxidative damage to the cell membrane and the degree of membrane lipid
   peroxidation in the fruit epidermis during light exposure, we measured
   the cell membrane permeability ([115]Figure 8A) and malondialdehyde
   (MDA) content ([116]Figure 8B) of the exocarp. At 12 h, the cell
   membrane permeability of the exocarp treated with the two treatments
   increased notably (p < 0.05), and the cell membrane permeability was
   3.07% higher in the ethylene treatment than in the NPs-TiO[2]
   treatment. Treatment with NPs-TiO[2] resulted in a rapid increase in
   the cell membrane permeability of the peel on day 4, and the cell
   membrane permeability of the exocarp was significantly higher (p <
   0.05) in the NPs-TiO[2] treatment than in the ethylene treatment and
   control ([117]Figure 8A). The content of MDA increased during the light
   exposure, and the increase was most pronounced in the ethylene
   treatment, followed by the NPs-TiO[2] treatment and the control
   ([118]Figure 8B); at the end of the treatment period, the content of
   MDA was markedly higher (p < 0.05) in both treatments than in the
   control.

Figure 8.

   [119]Figure 8
   [120]Open in a new tab

   Effects of two treatments on (A) Cell membrane permeability, (B) MDA
   content, (C) DPPH, (D) O[2]^− production rate and (E) H[2]O[2] content,
   (F) LOX activity, (G) SOD activity and (H) CAT activity during light
   cultivation of the ‘Red Delicious’ apples. Error bars, mean ± standard
   error (n = 3). Different letters indicate significant differences among
   treatments in the same period using Duncan’s test (p < 0.05), n.s.
   represents no significant difference (p ≥ 0.05).

   The total antioxidant capacity of the exocarp tissue during light
   exposure was determined via a 2,2-diphenyl-1-picryl-hydrazyl-hydrate
   (DPPH) assay. The content of DPPH in the peel decreased slightly at 12
   h and then increased over the treatment period ([121]Figure 8C). The
   relative content of DPPH was 2.17 times higher in the NPs-TiO[2]
   treatment on the eighth day than at the start of the treatment period;
   the content of DPPH was significantly higher (p < 0.05) in the
   NPs-TiO[2] treatment than in the ethylene treatment (1.87 times) and
   control (1.30 times). Changes in the O[2]^− production rate and the
   H[2]O[2] content were similar over the treatment period ([122]Figure
   8D,E). At 24 h, the H[2]O[2] content was significantly higher in the
   NPs-TiO[2] treatment (p < 0.05) than in the ethylene treatment and
   control, and the H[2]O[2] content increased continuously after 24 h.

   The activity of lipoxygenase (LOX) increased in all fruit after 24 h of
   treatment; LOX activity was significantly higher (p < 0.05) in the
   ethylene treatment than in the NPs-TiO[2] treatment and control, and
   LOX activity peaked in the ethylene treatment on the sixth day at 26.04
   ± 3.42 U g^−1. The activity of LOX on the eighth day was 1.27 and 1.95
   times (p < 0.05) higher in the NPs-TiO[2] treatment than in the
   ethylene treatment and control, respectively ([123]Figure 8F). Changes
   in the activity of catalase (CAT) and superoxide dismutase (SOD) in
   fruit in the NPs-TiO[2] treatment were similar over the treatment
   period, and CAT and SOD activity was higher in the NPs-TiO[2] treatment
   than in the ethylene treatment and the control ([124]Figure 8G,H). In
   the NPs-TiO[2] treatment group, the activity of SOD peaked (59.80 ±
   9.97 U g^−1) on the fourth day, and the activity of CAT peaked (1603.91
   ± 272.66 U g^−1) on the sixth day.

3. Discussion

   The appearance quality of fruit has a major effect on its economic
   value, and dysplasia and instability of the color of fruit during
   cultivation and production have negative economic effects. NPs-TiO[2]
   are semiconductor materials with a band gap of approximately 3.2 eV,
   corresponding to a wavelength of approximately 390 nm, and the crystal
   surface of the anatase phase is the most photocatalytically active
   [[125]33]. Therefore, UV absorption by the apple peel is enhanced after
   application with NPs-TiO[2] nano-colloids resulting in higher UV
   effective absorption. This consequently leads to the accumulation of
   more pigments. In this study, we found that the effect of the
   NPs-TiO[2]treatment on fruit quality was weaker than that of the
   ethylene treatment. NPs-TiO[2] treatment had less impact on fruit
   quality during the accelerated pigmentation process as reflected by the
   insignificant (p ≥ 0.05) reduction in fruit firmness, and the
   insignificant increase in TSS and the solid–acid ratio. In addition,
   the ethylene release rate and fruit respiration rate also indicated
   that the effects of NPs-TiO[2] treatment alleviated the post-ripening
   process during the post-harvest treatment period and was weaker
   compared with the effects of the ethylene spray treatment. These
   results indicate that NPs-TiO[2] promoted peel color and had a weaker
   effect on fruit quality than the ethylene treatment. Light plays an
   important role in fruit development. Light can affect physiological
   changes in fruits, especially the synthesis of phenolic substances in
   the later ripening stage [[126]34]. Primary phenolics in apples are
   secondary metabolites with antioxidant properties, and the total phenol
   content and total flavonoid content in some apple varieties are
   significantly positively correlated with levels of antioxidant activity
   [[127]35]. Anthocyanins and proanthocyanidins are produced through the
   flavonoid pathway and are important secondary metabolites that control
   apple appearance quality, and flavonoids are components of the
   phenylpropane system [[128]36,[129]37]. Interactions between many
   important enzymes in the flavonoid pathway are assumed to be the cause
   of anthocyanin accumulation [[130]38]. In our study, changes in the
   content of anthocyanins and flavonoids of fruit were more gradual in
   the NPs-TiO[2] treatment than in the ethylene treatment, and decreases
   in the content of proanthocyanidins in the ethylene and NPs-TiO[2]
   treatments were similar during the two treatments. This suggests that
   the activity of the enzymes and transcription factors involved in the
   flavonoid synthesis pathway were higher under ethylene treatment than
   in the NPs-TiO[2] treatment.

   Light induces anthocyanin biosynthesis by promoting the expression of
   key enzyme genes involved in the anthocyanin biosynthesis pathway in
   apples, and the induction of UV light is extremely important
   [[131]4,[132]7,[133]8,[134]39]. In this study, KEGG analysis revealed
   that the DEGs in the two treatments were primarily enriched in
   ‘Flavonoid synthesis’ pathways, which also corresponded to changes in
   the content of anthocyanins and flavonoids over the treatment period.
   This is consistent with the results of previous studies of apple fruits
   that were treated with UV light, which promoted the accumulation of
   anthocyanins in the flavonoid metabolic pathway [[135]40,[136]41].

   Anthocyanins are derived from the phenylpropanol biosynthesis pathway,
   and several enzymes are involved in anthocyanin biosynthesis; the
   functions of anthocyanins have been elucidated [[137]42]. These
   structural genes are regulated by several transcription factors at the
   transcriptional and translational levels under different environmental
   conditions [[138]4]. The proteins encoded by MYB family genes are
   components of the MYB-bHLH-WD40/WDR protein complex, and MdMYB1 plays
   an essential role in this process [[139]43,[140]44,[141]45]. We
   examined seven structural genes involved in anthocyanin production and,
   with the exception of MdPAL, the expression of these genes was higher
   in the ethylene treatment than in the NP-TiO[2] treatment. In addition,
   the transcript levels of the MYB, bHLH, and WD40 group genes were also
   typically quite different under the two treatments compared to the
   control. The activity of the three anthocyanin-related enzymes PAL,
   DFR, and UFGT was determined, and changes in their activity were
   consistent during the treatment period. Ethylene biosynthesis begins
   with the synthesis of ACC by 1-aminocyclopropane-1-carboxylate synthase
   (ACS) from S-adenosyl methionine, which is then oxidized to ethylene by
   ACC oxidase (ACO). The ACS and ACO genes have been identified as
   important factors in the ethylene production in fruit and in the
   ripening of fruit [[142]46,[143]47]. Ethylene is an important plant
   hormone, and numerous studies have shown that it can promote
   anthocyanin accumulation in apple [[144]44,[145]48,[146]49,[147]50]. In
   apples, MdEIL1 promotes anthocyanin biosynthesis by directly binding to
   the MdMYB1 promoter, and MdERF3 interacts with MdMYB1 to positively
   regulate ethylene production in the fruits [[148]44]. The expression of
   genes related to ethylene biosynthesis was remarkably higher (p < 0.05)
   in the ethylene treatment (0.1% ethephon, w/v) than in the NPs-TiO[2]
   treatment and the control. Correspondingly, the ethylene productions
   were higher in the ethylene treatment, and RNA-Seq data also indicated
   that the expression levels of structural genes during ethylene
   biosynthesis were more active in ethylene-treated fruit
   ([149]Supplementary Table S3). All the abovementioned findings showed
   that ethylene treatment promoted anthocyanin accumulation mainly
   dependent on the ethylene pathway. In addition, we found that there
   were no significant changes in the expression patterns of ethylene
   production and signaling-related genes under NPs-TiO[2] treatment. UV
   light can promote apple anthocyanin accumulation [[150]7,[151]8].
   Meanwhile, photocatalytic nanomaterials promote fruit coloration by
   improving the absorption of UV light [[152]30]. Therefore, we further
   analyzed the expression levels of relevant gene expression in the light
   signaling pathway. In apple, MdCOP1 and MdHY5 encode key transcription
   factors involved in anthocyanin biosynthesis and plant photosynthesis
   [[153]51,[154]52]. In addition, MdUVR8 is a UV-B photoreceptor that can
   interact with MdCOP1 and plays an essential role in photomorphogenesis
   [[155]53]. Interestingly, we found the expression levels of these three
   genes were higher in fruit treated with NPs-TiO[2] than in fruit
   treated with ethylene. These findings further indicate that anthocyanin
   biosynthesis in fruit is induced by UV light signals under NPs-TiO[2]
   treatment, which differ from those induced by ethylene.

   ROS are signaling molecules that are crucial for controlling plant
   development, senescence, and growth. Recent investigations have shown
   that ROS may play a role in controlling the production of anthocyanins
   in apples [[156]54,[157]55]. According to previous studies, UV
   radiation stimulates the production of ROS by regulating the activity
   of NADPH oxidase in the cytoplasmic membrane of the exocarp, thereby
   enhancing anthocyanin biosynthesis [[158]56,[159]57]. Elevated levels
   of ROS can induce lipid peroxidation of the cell membrane, which is
   mainly catalyzed by LOX, and MDA is one of the end products of lipid
   peroxidation of the cell membrane. Intracellular ROS levels and cell
   membrane permeability were higher in the NPs-TiO[2] treatment than in
   the ethylene treatment and the control during the treatment period.
   However, the MDA content was slightly higher in the ethylene treatment
   than in the NPs-TiO[2] treatment during the treatment period; a sharp
   increase in LOX enzyme activity was observed at 4 days and 6 days. We
   evaluated levels of antioxidant activity in fruit during the treatment
   period and found that the levels of antioxidant enzymes and the DPPH
   content were higher in the NPs-TiO[2] treatment than in the ethylene
   treatment and the control during the treatment period. This also
   explains why the MDA content was lower in the NPs-TiO[2] treatment
   group than in the ethylene treatment group. In sum, treatment with
   NPs-TiO[2] and ethylene induced the accumulation of anthocyanins in
   fruit during light exposure. The antioxidant capacity and ROS levels of
   fruits were higher and levels of membrane lipid peroxidation were lower
   in the NPs-TiO[2] treatment than in the ethylene treatment. Additional
   research is needed to determine whether the ROS generated via the
   photocatalytic activity of NPs-TiO[2] or the absorption and use of UV
   light by NPs-TiO[2] are the principal drivers of anthocyanin
   biosynthesis in fruit.

4. Materials and Methods

4.1. Preparation and Characterization of NPs-TiO[2]

   NPs-TiO[2] were thermally synthesized following the methods described
   in previous studies [[160]30,[161]58]. The prepared NPs-TiO[2] were
   characterized by scanning electron microscopy (SEM), transmission
   electron microscopy (TEM), dynamic light scattering (DLS) and X-ray
   crystal diffraction (XRD). The obtained NPs-TiO[2] had an irregular
   spherical shape and a crystal conformation with a particle size of
   10.34 ± 3.34 nm ([162]Figure S1). After obtaining dry NPs-TiO[2]
   powder, NPs-TiO[2] colloidal solution was prepared via an ultrasonic
   process [[163]30].

4.2. Fruit Material and Experimental Treatments

   Apple fruit (Malus domestica Borkh. ‘Red Delicious’) was collected from
   an orchard in Tai‘an, Shandong Province, China. The fruits were bagged
   at the young fruit stage (40 days after the flowering), and the orchard
   was managed using standard practices during growth and development. The
   fruits used in this study were harvested at 150 days after flowering
   (DAF). Apples of the same size and with no obvious signs of
   physiological disease were collected and stored in soft-bottom foam
   boxes. They were then transported to the laboratory of Shandong
   Agricultural University the same day that they were collected for
   subsequent experimental treatments. The apples were divided into three
   groups, with 30 apples in each group, and transferred into bags in
   low-light environments. Following previous studies [[164]44,[165]59],
   0.1% (w/v) ethephon solution and 0.1% (w/v) NPs-TiO[2] colloids were
   sprayed on apples; in the control treatment, the same amount of
   deionized water was applied. After the surface water dried naturally,
   the cells were placed into three independent light incubators (25 °C,
   70 μmol m^−2 s^−1 intensity of illumination. and 330 ± 20 nm
   wavelength) for light cultivation for 8 days. The peel tissue was
   sampled at 0 h, 12 h, 24 h, 2 days, 4 days, 6 days and 8 days. Three
   fruit skin samples were taken from each treatment group at each time
   period and discarded immediately after the skin samples were taken. The
   samples were rapidly frozen with liquid nitrogen and stored at −80 °C
   in a refrigerator for subsequent experiments.

4.3. Determination of Fruit Physiological Quality

   The exocarp color of the apple was determined using a chroma meter at
   the indicated times (0 h,12 h, 24 h, 2 days, 4 days, 6 days and 8
   days). Fruit treated with light for 8 days were measured for firmness,
   total soluble solids (TSS), total titratable acid (TA) and solid–acids
   ratio physiological qualities as described in the previous study
   [[166]59]. The respiration rate and ethylene production of the
   experiment fruit at the indicated times (0 h, 4 days, 8 days) were
   measured using a CO[2] detector (Testo535-CO[2], Testo Inc., Shanghai,
   China) and a gas chromatograph (GC-2014, Shimadzu, Toyko, Japan),
   respectively, as described in previous studies [[167]60]. A total of
   six biological replicates were performed for each treatment in the
   above experiments.

4.4. Determination of Secondary Metabolites

   The anthocyanin content in exocarp was determined using the
   hydrochloric acid–methanol method with reference to the previous study
   [[168]61]. The supernatant’s absorbance was calculated at 530, 620, and
   650 nm. Anthocyanin content was expressed as 0.1 change in optical
   density (μg g^−1 FW).

   The content of proanthocyanidins was determined according to the method
   of previous research with slight modification [[169]62]. At a
   wavelength of 640 nm, the absorbance readings of reaction system were
   recorded. The standard curve was made of catechin (μg g^−1 FW).

   The flavonoids and the total phenolics content were determined with
   reference to previous studies [[170]63]. For the flavonoids content,
   the absorbance value of reaction solution at 430 nm wavelength was
   recorded. The rutin-based standard curve was used (μg g^−1 FW). For the
   total phenolics content, the absorbance value of reaction solution at
   725 nm wavelength was recorded. The gallic-acid-based standard curve
   was used (μg g^−1 FW).

4.5. RNA-Seq Analysis

   RNA was extracted from exocarp tissue exposed to 8 days of light, and
   purified RNA was collected in triplicate for each treatment group. OE
   Biotech Co., Ltd. carried out the sequencing and analysis of the
   transcriptome (Shanghai, China). Trimmomatic was used to process the
   raw data [[171]64]. To achieve the clean reads, the low-quality reads
   and reads containing ploy-N were eliminated. A total of 60.47 G of
   clean data was obtained. Effective data for each sample ranged from
   6.57G to 6.96 G, with Q30 bases ranging from 91.2% to 91.98%, and GC
   fractions ranging from 44.45% to 47.62% ([172]Supplementary Table S1).
   Following that, hisat2 was used to map the clean reads to the reference
   genome [[173]65]. By using the approaches from previous research
   [[174]66,[175]67,[176]68,[177]69,[178]70], quantitative analysis of
   gene expression, differentially expressed genes analyses (DEGs), Gene
   Ontology analyses (GO) and Kyoto Encyclopedia of Genes and Genomes
   (KEGG) enrichment analyses were carried out. The threshold for
   significantly altered expression was chosen at p < 0.05 and foldchange
   > 2 or foldchange < 0.5. The RNA-seq raw data were submitted to NCBI
   with the following ID number: PRJNA947803.

4.6. Real-Time qPCR Expression Analysis

   RNA plant plus Reagent kit (Tiangen, Beijing, China) was used to
   extract RNA from apple exocarp tissue according to the corresponding
   experimental methods. After obtaining the RNA of the pure sample, the
   PrimeScript cDNA synthesis kit (Tiangen, Beijing, China) was used for
   subsequent reverse transcription experiments to obtain the cDNA
   template of the sample. The cDNA template was subsequently diluted to a
   concentration of approximately 2 ng μL^−1. The qRT-PCR was performed
   using the 2^−ΔΔCt calculation method, and the results were normalized
   using the MdActin gene of apple as the reference gene. The qRT-PCR
   procedures were set according to the experimental methods of previous
   studies [[179]52]. The primer sequences for all genes are provided in
   [180]Supplementary Table S2.

4.7. Determination of Malonaldehyde (MDA) Content, Cell Membrane Permeability
and Reactive Oxygen Species (ROS) Levels

   The relative cell membrane permeability of the exocarp tissue was
   detected using an electrical conductivity meter (DDS-307A, INESA
   Scientific Inc., Shanghai, China) at the indicated times (0 h, 12 h, 24
   h, 2 days, 4 days, 6 days and 8 days). The relative cell membrane
   permeability is expressed as % [[181]60].

   The content of MDA in exocarp was determined by trichloroacetic acid
   (TBA) method with slight modification according to the previous study
   [[182]71]. The sample’s absorbance value at wavelengths of 600 nm, 532
   nm, and 450 nm was calculated (μmol g^−1 FW).

   The production rate of superoxide anion and the content of hydrogen
   peroxide (H[2]O[2]) in exocarp were determined by Superoxide Anion kit
   and Hydrogen Peroxide detection kit (Suzhou Keming Biotechnology Co.,
   Ltd., Suzhou, China), respectively. The absorption value of the
   reaction system at a wavelength of 530 nm was recorded by a microplate
   reader to determine the superoxide anion production rate. One hour
   passed without any static reaction occurring in the centrifuge tubes of
   the control group. The result is denoted as nmol g^−1 min^−1 FW. For
   H[2]O[2] content, the absorbance value of reaction solution at 415 nm
   wavelength was recorded (μmol g^−1 FW).

4.8. Determination of Total Antioxidant Capacity

   The scavenging activity of 2,2-diphenyl-1-picryl hydrazide (DPPH) free
   radical in exocarp tissues was measured by using the previous method
   [[183]72]. The same mass fraction of ethanol solution was used to
   generate the standard solution curve of Trolox. At a wavelength of 515
   nm, the reaction solution’s absorbance was recorded. The results of
   DPPH relative content are expressed as μmol Trolox g^−1 FW.

4.9. Determination of Enzyme Activity

   For LOX, one gram of frozen materials was crushed in 10 mL of 0.05 mM
   of phosphatic buffer solution (PBS, PH 7.8, containing 50 mM
   β-mercaptoethanol), and the homogenate was centrifuged for 10 min (4
   °C, 12,000 rpm). The substrate for the enzymatic reaction was prepared
   by adding 0.5 mL linoleic acid, 0.25 mL Tween 20, and 1 mL NaOH (1 M)
   to 20 mL borate buffer, followed by adjusting the pH to 9.0 and
   diluting to 500 mL. The reaction system was prepared by mixing 0.2 mL
   of the supernatants with 0.8 mL of the enzyme substrates [[184]73]. The
   absorbance value of reaction system at 234 nm wavelength was detected
   using a microplate reader (U g^−1 FW)

   The extraction and determination methods of superoxide dismutase (SOD)
   and catalase (CAT) were referred to previous studies [[185]63]. The
   ability of the samples to inhibit the photo-chemical reduction of
   Nitro-Blue Tetrazolium (NBT) was analyzed to assay the enzyme activity
   of SOD. The absorbance value of reaction solution at 560 nm wavelength
   was detected by microplate reader (U g^−1 FW). For CAT, the absorbance
   value of reaction solution at 240 nm wavelength was detected by
   microplate reader. A unit of enzyme activity was defined as 1 nmol
   H[2]O[2] degradation per minute per gram of tissue catalyzed (U g^−1
   FW).

   The enzyme activities of l-Phenylalanine Ammonia-Lyase (PAL),
   Dihydroflavonol 4-Reductase (DFR) and UDP-glycose flavonoid
   glycosyltransferase (UFGT) enzyme activity were determined by enzyme
   activity detection kit (Hefei Laier Biotechnology, Inc., China). For
   PAL, the absorbance of reaction solution at a wavelength of 290 nm was
   measured using a microplate reader. Each gram of tissue in each
   milliliter reaction system changes the absorbance value at 290 nm by
   0.1 per minute, which is one unit of enzyme activity (U g^−1 FW). For
   DFR, the absorbance value of reaction solution at a wavelength of 500
   nm was measured using a microplate reader. A unit of enzyme activity
   was defined as 1 mmol catechin per gram of tissue per minute of tissue
   catalyzed (U g^−1 FW). For UFGT, the absorbance value of reaction
   solution at 340 nm wavelength was detected by microplate reader (U g^−1
   FW). A unit of enzyme activity was defined as 1 nmol NADH per minute
   per gram of tissue catalyzed.

4.10. Statistical Analysis

   The experiment was conducted in a completely random manner. The data
   were evaluated by One-way analysis of variance (ANOVA) using Excel 2019
   and Origin 2021 software. At least three biological replicates were
   used in each experiment. All data were reported as mean ± standard
   error (SE). The ANOVA was performed by p < 0.05. The difference was
   significant by least partial square test (LSD).

5. Conclusions

   We analyzed the effects of treatment with NPs-TiO[2] and ethylene on
   the anthocyanin biosynthesis and ROS metabolism in ripe ‘Red Delicious’
   apples under light exposure. The application of both treatments
   significantly promoted the deposition of pigments in the peel during
   light exposure, and NPs-TiO[2] had a minor effect on the quality of the
   fruit during the treatment period. Furthermore, ethylene treatment
   promoted fruit coloring mainly by up-regulating the expression of
   ethylene-related genes, whereas NPs-TiO[2] promoted fruit coloring
   mainly by up-regulating the expression of UV-radiation-related genes.
   The ROS levels and antioxidant capacity of the treated fruit were
   higher in the NPs-TiO[2] treatment group than in the ethylene treatment
   group during the treatment period, and the degree of membrane lipid
   peroxidation was lower in fruit in the NPs-TiO[2] treatment than in the
   ethylene treatment. These results imply the potential utility of
   NPs-TiO[2] in enhancing apple coloration. Moreover, the transcriptomic
   data offer novel insights into the molecular mechanisms underlying the
   promotion of apple coloration via NPs-TiO[2] treatment.

Supplementary Materials

   The following supporting information can be downloaded at:
   [186]https://www.mdpi.com/article/10.3390/foods12163137/s1, Figure S1:
   NPs-TiO[2] characterization. (A) and (B) SEM. (C) TEM. (D) Particle
   size distribution. (E) X-RD; Figure S2: Effects of NPs-TiO[2] and
   ethylene treatments on (A) respiratory rate and (B) ethylene production
   during light exposure in apples. Error bars, mean ± standard error (n =
   3). Different letters indicate significant differences among treatments
   in the same period using Duncan’s test (p < 0.05), n.s. represents no
   significant difference (p ≥ 0.05); Figure S3: qRT-PCR validation of
   DEGs in the peels of different treatments detected by RNA-Seq. Error
   bars, mean ± standard error (n = 3). Different letters indicate
   significant differences among treatments in the same period using
   Duncan’s test (p < 0.05); Figure S4: Heatmap representation of the
   expression patterns of genes associated with the MYB-bHLH-WD40 protein
   complex. Rows and columns in the heat map represent genes and samples
   collected. The color bar represents the FPKM average values. MYB, MYB
   family members; bHLH, basic helix–loop–helix; WD40, WD40-repeat
   proteins; Table S1: Primers for qRT-PCR; Table S2: Quality evaluation
   of RNA-Seq data; Table S3: DEGs related to ethylene biosynthesis
   pathway in apple.
   [187]Click here for additional data file.^ (10.8MB, zip)

Author Contributions

   Y.W.: Conceptualization, Methodology, Investigation, Data curation,
   Writing—original draft. G.C., D.W., J.Z., C.Y. and X.W.: Validation,
   Writing—review and editing, Supervision. H.L.: Supervision,
   Writing—review and editing. All authors have read and agreed to the
   published version of the manuscript.

Data Availability Statement

   The data used to support the findings of this study can be made
   available by the corresponding author upon request.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This study was supported by the National Natural Science Foundation of
   China (32172538), the major basic research projects of Shandong
   Province (ZR2020ZD43), the China Agriculture Research System of MOF and
   MARA (CARS-27), the Shandong Province Key R&D Program (2021CXGC010802),
   and Taishan scholar project (LJNY202026).

Footnotes

   Disclaimer/Publisher’s Note: The statements, opinions and data
   contained in all publications are solely those of the individual
   author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI
   and/or the editor(s) disclaim responsibility for any injury to people
   or property resulting from any ideas, methods, instructions or products
   referred to in the content.

References