Four years old rabbiteye blueberry “Brightwell” were grown in experimental base of Baima district, Nanjing city, China. Blueberries of same size and growing condition were selected to do the treatment. Each treatment was arranged in a randomized complete block design with 6 replications and each block replication contains 6 shrubs.

The (+)-Abscisic Acid (Purity 95%, Coolaber company, China) was dissolved in double distilled H₂O containing 5% (v/v) ethanol and 0.1% Tween 80. When most of blueberries fruits were in a growing stage of “green mature” and the fruit at the top of the branch has just begun to turn red, 0, 500, and 1,000 mg/L ABA solutions were sprayed on fruit clusters. Tiny sprayer were used to spray the ABA solutions on the peels untill the peels are wetted. The fruit is guaranteed to be sprayed 2–3 times on all sides. Although the mock treatment was performed on blueberry shrubs, leaves, and branches were carefully avoided.

In different development stage of fruits, 30 fruits were immediately frozen in liquid nitrogen and stored at −80°C for later experiments.

The developmental stage of cv “Brightwell” from ripening initiation to maturity was divided into six stages (Figure 1A) based on fruit color (Stage 1, the whole fruit was green; Stage 2, the top turned red; Stage 3, the side turned red; Stage 4, the whole fruit was red; Stage 5, the whole fruit turned purple; and Stage 6, the whole fruit was purple) and size (Supplementary Table 1). Application of 1,000 mg/L ABA resulted in a significant acceleration of fruit coloration (Figure 1B). From S3 to S4, the a^∗ value suggests that the top, side and bottom of fruit from the 1,000 mg/L ABA group turned red earlier than those from the 0 to 500 mg/L ABA groups. At S5, the top and bottom of the fruits turned green earlier. From S5 to S6, the b^∗ value suggests that the side and bottom of the fruit from the 1,000 mg/L ABA group turned blue earlier than those from the 0 to 500 mg/L ABA groups. From the a^∗ and b^∗ values, there were no notable differences among the three groups at S6. Anthocyanin accumulation was consistent with the change in fruit coloration (Figure 1C). At S1–S3, almost no anthocyanins were detected. At S4, although the average anthocyanin content of the 1,000 mg/L treatment was higher than that of the control group, there was no statistically significant difference. At S5 and S6, the total anthocyanin content of the 1,000 mg/L ABA treatment group was obviously higher than that of the 0 and 500 mg/L treatment groups. From S5 to S6, the anthocyanin content of the 0 mg/L treatment group nearly doubled but was still lower than that in the 1,000 mg/L group. Application of 1,000 mg/L ABA also changed the fruit quality (Supplementary Figure 1). Compared to 0 and 500 mg/L ABA treatment, fruits hardness of 1,000 mg/L ABA treatment declined more sharply from S4 and become softer in S6 (Supplementary Figure 1A). Consist with fruit hardness, fruits of 1,000 mg/L ABA treatment contains more soluble solid in S6 (Supplementary Figure 1B). Application of ABA did not significantly change the fruit size, which was within the range of Supplementary Table 1. Therefore, blueberry fruits treated with 0 and 1,000 mg/L ABA were selected for transcriptome analysis.

The fruits in Stages 1–6 with 0 and 1,000 mg/L ABA treatment were further subjected to transcriptome analysis. The raw bases of each sample varied from 7.04 G to 10.91 G with a 0.03% error ratio and 46% GC content. In total, 2218553864 clean reads and 2176923110 raw reads were obtained (Supplementary Table 2). The raw reads of 12 samples with three biological repeats were uploaded to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (all samples and corresponding explanations can be found in PRJNA664011). For each sample, 87.43–89.19% of the reads were mapped to the highbush blueberry genome. Approximately 87% of reads were mapped to exonic regions, 7% of reads were mapped to intergenic regions, and 6% of reads were mapped to intronic regions. The clean reads were assembled into 128,559 unigenes. A total of 88,532 unigenes were annotated by the Swiss-Prot database, accounting for 68.86% of the total (Supplementary Table 3).

Principal component analysis (PCA) was used to analyze the overall variation in transcripts among 32 samples. The 3D plot shows that the three repetitions are well clustered together, and the differences between the sample points are obvious (Figure 2A). The FPKM value of genes in the ABA-treated group was compared with that in the control group. A Log₂(fold change) value greater than 1.3 or less than −1.3 indicates upregulation and downregulation of gene expression, respectively. At S2, the number of genes with significant changes was approximately twice as high as that of the control. The number of differentially expressed genes decreased sharply at S3 and then gradually increased at S4–S6 (Figure 2B). Volcano maps were used to represent the overall changes in gene expression between two groups (Figure 2C). The results showed that the number of genes with drastic changes in expression decreased continuously from S1 to S6.

The total amount of anthocyanins in the ABA treatment group was higher than that in the control group from S4, so the transcripts of fruits at S4 were functionally categorized and analyzed using the GO and KEGG databases (Figure 3). The results showed that the anthocyanin synthesis pathway had changed significantly.

The transcripts of all key enzymes involved in the anthocyanin pathway were analyzed (Figure 4). Green indicates that the expression of transcripts in the ABA-treated group was lower than that in the control group, while red indicates the opposite. In the whole process, the transcripts of CHS and CHI, two important enzymes upstream, all showed consistent downregulation at S1 and S2 and increased at S5 and S6. Another key enzyme, LDOX/ANS, showed a similar expression pattern and was highly expressed at S3 and S4. The transcripts of the three enzymes F3H, F3′H and F3′5′H, which are found on different branches, showed different expression patterns across the six stages. Similarly, the expression of UFGT transcripts was inconsistent. Although the transcripts of DFR differed to some extent across the six stages, they were generally highly expressed after S3. In general, the transcript expression patterns of CHS, CHI, DFR, and LDOX were consistent with the changes in the anthocyanin content in the fruits.

The expression pattern of several key structural genes was the same, which indicates that the expression of transcription factors may affect the expression of downstream genes. Published transcription factors from different species were used to construct a phylogenetic tree with homologous transcription factors in blueberry. According to the classification of MYB transcription factors (Jiang and Rao, 2020) and their functions, MYB transcription factors involved in anthocyanin synthesis in blueberry were identified (Figure 5). Five blueberry MYB transcripts belong to the branch SG VIII-e2κ, and the expression of MYB transcription factors on this branch has been proven to promote anthocyanin synthesis. A blueberry transcript belonging to the SG V δ branch that promotes anthocyanin synthesis was also identified. For the negatively regulated SG VIII-E e1β branch, two transcripts were identified. For bHLH and WD40 transcription factors involved in anthocyanin regulation, four homologous genes were identified (Supplementary Figures 2, 3).

Among the identified transcription factors, six MYB transcription factors, one bHLH transcription factor and four WD40 transcription factors showed significant differences in specific stages (Figure 6). The expression pattern of these transcription factors was verified by qRT-PCR (Supplementary Figure 4). The expression of two MYB transcription factors on the SG VIII-e2κ branch was positively correlated with anthocyanin content. At the same time, both transcripts are homologous genes of VcMYBA that have been proved to regulate anthocyanin synthesis. Two homologous negatively regulated MYB transcription factors showed upregulated and downregulated differential expression at S1. The transcript belonging to the SG V δ branch showed a high increase from S4 to S6, which was earlier than the change in the anthocyanin content. The bHLH transcript homologous to AtGL3 was downregulated at S1–S2 but highly expressed at S4–S5. There was no correlation between the expression patterns of the four WD40 transcripts. The results showed that transcripts belonging to the SG VIII-e2κ branch and homologous to VcMYBA were most likely related to the increase in anthocyanins. Through promoter element analysis, 2-4 ABREs (ABA-responsive elements) was found to appear in the promoter region of four copies of VcMYBA (Supplementary Table 5). This shows that VcMYBA responds in the ABA pathway and may therefore be associated with the changes of PYL (Encodes regulatory components of ABA receptor), SnRK2 (protein kinases involved in ABA signaling), and ABF (bZIP transcription factor with specificity for ABRE) gene families (Supplementary Figure 6). After ABA treatment, seven genes of ABF family were up-regulated in S1–S6, but the up-regulation degree decreased gradually. In S4, the expression of four ABF genes in the treatment group was 1.27–1.5 times higher than that in the control group. PYL and SnRK2 gene families did not show an obvious overall change trend, but one gene in PYL family changed 1.79 times in S3 and one gene in SnRK2 family changed 1.59 times in S4. From the expression of these genes, VcMYBA has a positive correlation with their expression.

Through the genome, a total of five copies of VcMYBA were identified. One copy had transposon insertion, two copies had frameshift and could not be translated into protein, one copy of TCA base sequence was mutated into the termination codon TGA and only one copy could be fully translated into protein (Supplementary Figure 5). Because of their high sequence similarity, transient silencing was designed in exon and intron regions, respectively. The results showed that in the control group, the anthocyanin synthesis in the injection wound area increased, and the anthocyanin synthesis in the fruit treated with TRV: VcMYBA-exon decreased, while the anthocyanin accumulation in the wound treated with TRV: VcMYBA-intron was not obvious, and the anthocyanin synthesis in the fruit did not decrease significantly (Figure 7A). The expression of VcMYBA in TRV:VcMYBA-exon group was also significantly lower than that in control group and TRV:VcMYBA-intron group (Figure 7B). The contents of six main anthocyanins were detected by UPLC-MS/MS (Figure 7C). The contents of petunidin, cyanidin, delphinidin, and peonidin in TRV: VcMYBA-exon group were significantly lower than those in the other two groups (Figure 7D). There was no significant change in anthocyanin content in TRV:VcMYBA-intron compared with the control group.

Many studies have reported that exogenous ABA could increase the anthocyanin content in non-climacteric fruits (Jia et al., 2011; Sandhu et al., 2011). The anthocyanin content of plants will also increase under many abiotic stresses, such as drought (Nakabayashi et al., 2014), low temperature (Li et al., 2017), and nitrogen deficiency (Zhang et al., 2017). The response of plants to these stresses is also related to the ABA signaling pathway. Previous studies in blueberry only focused on the 12 days after ABA application at the late green fruit stage (Oh et al., 2018). The results showed that the content of anthocyanins increased and the color of blueberry fruits changed faster after 1,000 mg/L ABA treatment, which was consistent with the results of other non-climacteric fruits. There was no significant difference in anthocyanin content, fruit quality and size between 500 mg/L ABA treatment and the control group. The effective ABA concentration of increasing anthocyanin accumulation in blueberry is about three times higher than that of grape (Vitis vinifera L.) It may be that fruit pruina blocks part of ABA.

For the mechanism by which ABA increases the anthocyanin content, only transcriptome analysis at 24 and 48 h after ABA treatment was carried out (Pilati et al., 2017). It takes approximately 35 days for late green fruits to ripen, and the key genes involved in this process need to be studied in detail. Therefore, we divided blueberry fruits into six stages from late green to mature and analyzed the changes in gene expression throughout the whole process in detail. The transcriptome analysis results showed that the expression of structural genes involved in anthocyanin synthesis was inconsistent. In ABA-treated fruits, the transcripts of CHS, CHI, DFR, and LDOX/ANS, which were proven to be the key structural genes in anthocyanin biosynthesis, were generally expressed at low levels at S1–S2 and at high levels at S5–S6. Previous studies only involved the changes of several structural genes in anthocyanin pathway in mature fruits (Jia et al., 2011; Li et al., 2011). We analyzed the structural gene changes of the whole anthocyanin pathway through transcriptome system to clarify the direct reason for the increase of anthocyanin content during fruit color transformation.

Combined with previous studies on transcription factors upstream of these structural genes (Gonzalez et al., 2008), we speculate that the MBW complex is involved in the anthocyanin regulation of the ABA pathway. Homologous genes of MBW transcription factors published in important plants were identified by database annotation and phylogenetic tree construction in blueberry. Two VcMYBA (AtMYB75/90 homologous gene) copies, one AtMYB56 homologous copy and one AtGL3 homologous copy were highly correlated with changes in anthocyanin contents. Overexpression of VcMYBA (Plunkett et al., 2018) and AtMYB75/90 (Gonzalez et al., 2008) can enhance anthocyanin synthesis. In addition, DFR and CHS were also proven to be downstream of AtMYB75/90, and DFR was downstream of VcMYBA. Our results are consistent with those of previous studies. The high expression of VcMYBA at S5–S6 was consistent with the change in anthocyanins, and the same structural genes, DFR and CHS, were highly expressed. Atmyb56 needs sucrose induction, and its anthocyanin increasing effect is relatively low (Jeong et al., 2018). The homologous gene of AtMYB56 in blueberry began to be highly expressed at S3, suggesting that this gene may not play a major role. AtGL3 can enhance downstream anthocyanin expression by interacting with AtMYB75/90 (Zhang et al., 2003). Therefore, VcMYBA may play a key role in ABA pathway.

Grape, which is also a non-climacteric fruit similar to blueberry, can increase anthocyanin accumulation under drought (Castellarin et al., 2007), low temperature (Cohen et al., 2012) and ABA treatment (Zhai et al., 2017). DFR, LDOX, UFGT, and VvMYBA1 are upregulated under water deficit (Castellarin et al., 2007), and the promoters of both VvMYBA1-2 and VvMYBA2 can be activated by ABA, but only VvMYBA2 can be activated by ethylene (Zhai et al., 2017). White grapes arose through the mutation of VvMYBA1-2 and VvMYBA2 (Walker et al., 2007). These findings indicate that VvMYBA plays a role in the ABA pathway of non-climacteric fruits and that DFR, LDOX, and UFGT are downstream of VvMYBA. For VcMYBA, heterologous expression of VcMYBA in Nicotiana benthaminana could induce anthocyanin accumulation (Plunkett et al., 2018). Through promoter element analysis, there are four promoter elements ABRE responding to ABA in the promoter region of VcMYBA (Supplementary Table 5). Transient silencing of the exon region of vcmyba in blueberry fruit significantly reduced the synthesis of anthocyanin in blueberry fruit, while silencing the intron region did not significantly reduce the synthesis of anthocyanin in blueberry fruit. Therefore, it can be considered that the copy of VcMYBA that can be translated into protein can effectively regulate anthocyanin synthesis, and whether other copies play a role as lncRNA remains to be further studied.

This study is based on many previous studies, and the results are consistent with the previous conclusions. These results preliminarily explain the mechanism by which ABA increases the anthocyanin content in non-climacteric fruits and broadens the MYB pathway in response to ABA. As ABA is a stress response hormone, these results also provide a reference for explaining the stress-induced increase in the anthocyanin content in plants. This result can provide a basis and new target for anthocyanin breeding in blueberry.