In this study, the fruit of rabbiteye blueberry cultivar ‘Brightwell’ was used as the experimental material. Four-year-old blueberry trees in the Lishui District Baima Base of Institute of Botany, Jiangsu Province and Chinese Academy of Sciences were selected for sampling. The experiment was conducted in a completely randomized block design, with 6 replicates for each treatment and 6 plants for each block. At the late stage of green fruit development, the fruit was treated with 0 (control), 500 mg/L or 1000 mg/L ABA (95% purity, Coordinator Company, China) once, and the fruit was sprayed until the surface was wet. According to previous research results of this research group, blueberry fruits are divided into six stages from the late green fruit stage to the mature stage (Han et al., 2021). Specifically, in the first stage S1, the fruit turned green; in the second stage S2, the top of the fruit began to turn red, while the sides and bottom remained green. In the third stage S3, the side of the fruit begins to turn red; in the fourth stage S4, the fruit turns red as a whole. In the fifth stage S5, the entire fruit turns purplish red; in the sixth stage S6, the fruit turns completely dark purple ( Figure 1A ). At each fruit development stage, two groups of S1-S6 blueberry fruits treated with 0 or 1000 mg/L ABA were selected for six biological replications and stored at -75°C for further metabolomics experiments and analysis.

Reducing power assays were adapted from the method described by Shen et al. (2017), and ascorbic acid (0.1 mg/mL) was used as a positive control. Briefly, 0.15 g samples were homogenized with 1.35 mL of phosphate buffer (0.2 M, pH 6.6) and centrifuged at 5000 × g for 10 min. The supernatant (1 mL) was mixed with 1 mL of potassium ferricyanide solution (1%, w/v). The mixtures were then heated at 50°C for 20 min. Then, 1 mL of trichloroacetic acid (10%, w/v) solution was added to stop the reaction immediately. After centrifugation, 1 mL of supernatant was mixed with 1 mL of distilled water and 0.2 mL of ferric chloride solution (0.1%, w/v). After 10 min, the 96 plates were shaken sufficiently and measured spectrophotometrically at 700 nm.

DPPH radical scavenging activity was measured according to Cerezo et al. (2010), and ascorbic acid (0.5 mg/mL) was used as a positive control. DDPH (0.1 mM) was dissolved in ethanol. The sample slurry was added to 3 DPPH reaction systems. Absorbance was measured at 515 nm after 30 min to reach a steady state.

The scavenging activity of the DPPH radical was calculated as follows:

where Ai is 1 mL ethanol + 1 mL DDPH solution, Aj is 1 mL sample slurry + 1 mL DDPH, and Ao is 1 mL sample slurry + 1 mL ethanol.

Approximately 5 g of blueberry fruits from each treatment (a total of 72 fruit samples) was thoroughly ground with liquid nitrogen. Then, 100 mg of homogenate was collected and resuspended in prechilled 80% methanol and 0.1% formic acid (FA) by vortexing. The samples were incubated on ice for 5 min and then centrifuged at 15000 rpm and 4°C for 5 min. Equal amounts of supernatant were diluted to a final concentration containing 60% methanol with LC−MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube with a 0.22 μm filter and then centrifuged at 15000 × g at 4°C for 10 minutes. Finally, the filtrate was injected into the LC−MS/MS system for analysis.

LC−MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected onto a Hypersil Gold column (100 mm × 2.1 mm, 1.9 μm) using a 16-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in water) and eluent B (methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; and 2% B, 16 min. The Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, a capillary temperature of 320°C, a sheath gas flow rate of 35 arb and an aux gas flow rate of 10 arb.

The raw data files generated by UHPLC−MS/MS were processed using Compound Discoverer 3.0 (CD 3.0, Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite. The main parameters were set as follows: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity, 100000. Then, the peak intensities were normalized to the total spectral intensity. The normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. Then, peaks were matched with the mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) databases to obtain accurate qualitative and relative quantitative results. In addition, significantly different metabolites were selected, and their relative abundances were used as indicators for further analysis.

Significant differences were calculated by one-way ANOVA tests or T tests in SPSS 19 (IBM, USA). Different letters and asterisks (*) in the figure indicate statistical significance (P< 0.05). Unsupervised principal component analyses (PCA) and partial least squares discriminant analysis (PLS-DA) clustering methods were performed by MetaboAnalyst (http://www.metaboanalyst.ca/). Variable importance in the projection (VIP) is the weighted sum of squares of the PLS-DA analysis, which represents the contribution rate of metabolite differences in different groups. The fold change (FC) is the ratio of the mean of all replicate quantitative values for each metabolite in the comparison group. Combined with the T test, differential metabolites were screened by the following criteria: VIP >1.0, FC >1.2 or FC<0.833 and P value<0.05.

Based on the previous experiment, the ripening of blueberry fruit can be divided into six stages (S1-S6) ( Figure 1A ). Then, after 20 and 25 days of spraying ABA treatment, the number of mature fruits (S6) was counted, and the proportion of mature fruits was calculated ( Figure 1B ). The results showed that 20 days after treatment, the fruit maturity rate (percentage of mature fruit) of the 1000 mg/L ABA treatment group was approximately 36%, which was significantly higher than that of the control group and 500 mg/L ABA treatment group (24%). On the 25th day after treatment, the fruit maturity rate of the 1000 mg/L ABA treatment group was approximately 53%, and that of the control group and 500 mg/L ABA treatment group was approximately 32% and 34%, respectively. The results showed that ABA treatment could promote blueberry fruit ripening.

In addition, we also found that under three treatments (control group, 500 mg/L and 1000 mg/L ABA treatment group), the hardness of blueberry fruits at six developmental stages gradually declined ( Figure 1C ). From stages S1 to S3, there was no significant difference in fruit hardness between the treatment group and the control group (the highest fruit hardness was 0.68 kg·cm^-2 in the control group at the S1 stage). Notably, from the S4 stage, the fruit hardness of the treatment group and the control group began to decrease significantly, and the fruit hardness of the 1000 mg/L treatment group was significantly reduced by 14.29% and 9.52% compared to the control and 500 mg/L treatment groups at the S4 stage, respectively. In addition, fruit hardness at the S5 and S6 stages in the 1000 mg/L treatment group was significantly reduced by 13.88% and 8.65%, respectively, compared to the control group. According to the change in fruit hardness, it was speculated that the flesh tissue changed. Therefore, mature fruit slices at the S6 stage of the 0 and 1000 mg/L ABA treatment groups with significant differences in hardness were selected for microscopic observation by the semithin section method ( Figure 1D ). Through comparison, it was found that the flesh tissue of the 1000 mg/L ABA treatment group was obviously fragmented, while the flesh tissue of the control group was complete and full, with less fragmentation. The fragmentation of pulp tissue in the 1000 mg/L ABA treatment group may indicate higher fruit maturity, which is consistent with the hardness results.

The 0 and 1000 mg/L ABA treatments were selected for nonbiased global metabolomics (nontarget metabolomics) based on UHPLC−MS/MS. Quality control (QC) analysis showed that the quality of metabolomics was stable and adequate ( Figure 2 ). A total of 1145 metabolites and 575 metabolites were detected and annotated in positive mode ( Table S1 ) and negative mode ( Table S2 ), respectively. Principal component analysis (PCA) ( Figure 3 ) and partial least squares discrimination analysis (PLS-DA) ( Figure 4 ) were performed to systematically profile metabolic variances between the two groups. Score plots of samples are well separated in S1, S2 and S3, suggesting a clear distinction in metabolite accumulation. In contrast, the scores plotted in S4, S5 and S6 intersect to different degrees. A volcano map can directly show the overall distribution of differential metabolites ( Figure 5 ). Compared with the S1-S3 stages, the number of different metabolites in blueberry fruits during the S4-S6 stages was significantly reduced. The total amount of upregulated and downregulated differential metabolites of blueberry fruit S1-S6 in the 0 and 1000 mg/L ABA treatment groups at each stage showed that the total amount of differential metabolites increased continuously at the S1-S3 stages and peaked at the S3 stage ( Figure 6A ). The upregulation and downregulation of differential metabolites detected in the two modes were irregular. Differential metabolites were annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) ( Figure 6B ) and Lipid metabolites and pathways strategy (LIPID MAPS) ( Figure 6C ). In the LIPID MAPS annotation, there were 33 metabolites in positive mode and 10 metabolites in negative mode involved in flavonoid metabolism. During the S3 period, when the most differential metabolites were found, the flavonoid biosynthesis pathway and flavone and flavonol biosynthesis pathway were significantly enriched in blueberry fruits under positive mode ( Figure 6D ). In addition, the top 10 upregulated ( Table S3 ) and downregulated ( Table S4 ) metabolites were separated. Two compounds were noteworthy: gallic acid was strikingly upregulated from S1-S3, and chlorogenic acid was significantly and continuously downregulated from S1 to S6.

The ABA relative content in the control group continued to increase from S1 to S4 but began to decrease from S4 to S5 and then increased from S5 to S6 ( Figure 7A ). The mean ABA relative content in the ABA group was higher than that in the control group at the S1-S3 stage but lower than that in the control group at the 4-6 stage ( Figure 7A ). From stages S1-S3, the L-proline relative content in the 1000 mg/L group was significantly higher than that in the control group, and the trehalose-6-phosphate relative content in the 1000 mg/L group was significantly lower than that in the control group ( Figure 7B ). The change in metabolite levels of the two stress resistance markers was consistent with that of endogenous ABA. During the S4-S6 period, the two stress-resistance metabolites of the treatment group and the control group tended to be the same, showing that the effect of exogenous ABA was reduced.

For the flavonoid pathway, the 1000 mg/L group differed from the control group at the important metabolite level, and the differences were found and annotated by KEGG ( Figure 8 ). Cyanindin is a precursor of red anthocyanins. Its content is consistent with the coloration change, and its content increased more than that of the control group from S3. Similarly, the level of blue anthocyanin petunidin-3-glucoside increased more in the treatment group than in the control group when the blueberry fruits turned purple. Quercetin, catechin and epicatechin are downstream metabolites of flavonoid 3’-hydroxylase (F3’H), showing a persistently lower content from S1 to S6. Myricetin and epigallocatechin are downstream metabolites of flavonoid flavonoid-3’,5’-hydroxylase (F3’5’H) and were present at lower levels than in the control group in S5 and S3, respectively.

Due to the significant changes in metabolites in the flavonoid biosynthesis pathway, we further analyzed the changes in other flavonoid metabolites in blueberry fruits treated with ABA. The representative metabolites among catechins, flavonols, tannins and phenolic acids in mature fruits are shown ( Figure 9 ). Overall, most of their contents were reduced to some extent under the 1000 mg/L ABA treatment. The two metabolites were noteworthy: gallic acid was higher at the S1-S3 stages and returned to normal levels in the ripe fruit, but chlorogenic acid remained at low levels in the ripe fruit. Because of the changes in bioactive metabolites, we further studied the antioxidant capability of fruits. The reducing ability of the 1000 mg/L ABA treatment was higher than that of the 0 mg/L treatment ( Figure S1A ). In addition, the DPPH radical scavenging activity assay ( Figure S1B ) also showed the same result.

According to previous research by our research group, the total anthocyanins of fruits can be detected from the S4 stage, and the total anthocyanin content increases with the development of fruits (Han et al., 2022). Based on this, the anthocyanin components in blueberry fruits of the 0 (CK) and 1000 mg/L ABA treatment groups with significant differences in anthocyanin content were analyzed by LC−MS. In each of the S3-S6 stages, five pigments, petunidin, cyanidin, delphinidin, pelargonidin and peonidin, were detected in blueberry fruits, and the content of cyanidin and pelargonidin in the fruits at the S3 stage of the 1000 mg/L ABA treatment group was significantly increased by 10.10% and 15.43%, respectively, compared to the control group. Meanwhile, the content of each pigment in the fruits of the 1000 mg/L ABA treatment group under the S4-S6 stage was significantly higher than that of the control fruits in the same period, except for cyanidin and pelargonidin. Notably, the content of cyanidin and delphinidin was higher at the beginning of blueberry fruit development (S3), with a decreasing trend at the later stage, while their content showed a certain increase after fruit ripening. In addition, except for cyanidin and pelargonidin, the contents of which decreased sharply from S3-S5, the other pigments all showed a steady increasing trend during fruit development and maturity and reached the highest content when the fruit was fully mature at S6 ( Table 1 ).