Characterization of Highbush Blueberry (Vaccinium corymbosum L.) Anthocyanin Biosynthesis Related MYBs and Functional Analysis of VcMYB Gene

As one of the most important transcription factors regulating plant anthocyanin biosynthesis, MYB has attracted great attentions. In this study, we identified fifteen candidate anthocyanin biosynthesis related MYB (ABRM) proteins, including twelve R2R3-MYBs and three 1R-MYBs, from highbush blueberry. The subcellular localization prediction results showed that, with the exception of VcRVE8 (localized in chloroplast and nucleus), all of the blueberry ABRMs were nucleus-localized. The gene structure analysis revealed that the exon numbers of the blueberry ABRM genes varied greatly, ranging between one and eight. There are many light-responsive, phytohormone-responsive, abiotic stress-responsive and plant growth and development related cis-acting elements in the promoters of the blueberry ABRM genes. It is noteworthy that almost all of their promoters contain light-, ABA- and MeJA-responsive elements, which is consistent with the well-established results that anthocyanin accumulation and the expression of MYBs are influenced significantly by many factors, such as light, ABA and JA. The gene expression analysis revealed that VcMYB, VcMYB6, VcMYB23, VcMYBL2 and VcPH4 are expressed abundantly in blueberry fruits, and VcMYB is expressed the highest in the red, purple and blue fruits among all blueberry ABRMs. VcMYB shared high similarity with functionally proven ABRMs from many other plant species. The gene cloning results showed that VcMYB had three variable transcripts, but only the transient overexpression of VcMYB-1 promoted anthocyanin accumulation in the green fruits. Our study can provide a basis for future research on the anthocyanin biosynthesis related MYBs in blueberry.


Introduction
Anthocyanins, which are natural polyphenols that are widely found in many foods, possess many biological and health-beneficial abilities, such as anti-inflammatory, visualimproving and so on [1][2][3][4]. All of these abilities are achieved through their high antioxidant activity [5,6] and some other biological effects, such as their anti-proliferative effect [7], hepatoprotective effect [8] and anti-depressant behavior [9].
The MYB transcription factor plays an important role in regulating the spatio-temporal expression of anthocyanin biosynthesis related structural genes and anthocyanin accumulation, and is one of the most important and widely investigated transcription factors involved in anthocyanin biosynthesis regulation [17,22]. The first plant MYB gene, C1, was discovered from maize [23], whose encoded protein was required for anthocyanin biosynthesis in the aleurone layer [24]. Since then, MYBs have been isolated from many plant species. Most MYB proteins contain conserved MYB domain/domains at their Nterminus. According to the number of their MYB domains, MYBs can be further classified into four types, i.e., 1R-MYB/MYB-related, R2R3-MYB, 3R-MYB and 4R-MYB. It is noteworthy that most of the reported anthocyanin biosynthesis related MYBs (ABRMs) were R2R3-MYBs, followed by 1R-MYB/MYB-related MYBs [25]. In blueberry, most of the reported anthocyanin metabolism regulatory transcription factors were MYBs [26]. For example, the up-regulated expression of VcMYB21 and VcR2R3MYB was confirmed to be associated with the UV-B-induced anthocyanin accumulation in blueberry pericarp [27]; VcMYB21 played a negative regulatory role in anthocyanin accumulation [28]; the transient overexpression of blueberry MYBA in an Antirrhinum majus MYB mutant restored the anthocyanin accumulation, and its co-expression with a heterologous bHLH could induce anthocyanin accumulation in tobacco leaves [29]; the pink fruit mutation phenotype of 'Pink Lemonade' was caused by the mutation of MYB1 [20]; the expression of VcUFGT in highbush blueberry was positively regulated by VcMYBA1 and negatively regulated by VcMYBC2 [30].
Although some studies have been conducted on blueberry ABRMs [31,32], their applications are restricted as almost all these reported ABRMs were obtained based on de novo assembled transcriptome data. The publication of the draft genome of blueberry [33,34] greatly facilitated the genome-wide identification of the functional and regulatory genes involved in anthocyanin biosynthesis [35]. Based on the genome data, Wang et al. [31] identified a total of 229 MYB members that could be further divided into 23 subfamilies, but they did not focus on the anthocyanin biosynthesis related members; Zhao et al. [17] identified 11 MYBs from blueberry fruits through homologous protein searching, using Arabidopsis, apple, grape and strawberry MYBs belonging to the MBW complex as queries. Recently, many ABRMs have been identified and functionally approved in various plant species, such as Arabidopsis [36], Helianthus tuberosus [37], monkeyflower [38], Eutrema salsugineum [39], Freesia hybrida [40], grape hyacinth [41], apple [42] and so on. In this study, for the exploration and characterization of blueberry ABRMs, we identified the blueberry ABRMs by homologous searches against the blueberry protein data provided by the highbush blueberry genome project using the reported ABRMs from some other plant species as queries, characterized their sequences, and investigated their corresponding genes' expression patterns in blueberry fruits at five different ripening stages, based on our previously obtained transcriptome data and quantitative real time PCR (qRT-PCR) analysis. Moreover, the function of VcMYB (VaccDscaff1486-snap-gene-0.3), whose encoded protein showed high similarity with ten functionally proved ABRMs from other plant species (including Arabidopsis AtMYB114, AtMYB90, AtMYB75 and AtMYB113, H. tuberosus HtMYB2, monkeyflower PELAN, E. salsugineum EsMYB90, F. hybrida FhPAP1, grape hyacinth MaAN2 and apple MdMYB10), was further studied by transient overexpression in young blueberry fruits. The results obtained in this study will provide a foundation for the functional analysis and applications of blueberry anthocyanin biosynthesis related MYB genes, and will lay the foundations for research on the high-anthocyanin aimed blueberry breeding in the future.

Plant Materials
The plant materials used in this study were the fruits of four-year-old southern highbush blueberry 'FL03' at five different stages (green fruit (GF), pink fruit (PiF), red fruit (RF), purple fruit (PF) and blue fruit (BF)) [22]. After harvesting, the fruits were quickly taken back to the laboratory, washed with distilled water three times, immediately frozen in liquid nitrogen after draining the fruit surface with sterilized filter paper, and stored in the refrigerator at −80 • C for further use.

Identification of ABRM Proteins in Blueberry
The gDNA, cDNA and protein sequence files of the blueberries were downloaded from https://www.vaccinium.org/analysis/49 (accessed on 3 March 2021). To identify ABRM proteins in the blueberries, homologous protein sequence alignment was performed against the blueberry protein data, using recently reported ABRM protein sequences as queries with e-value ≤ 1 × 10 −5 and similarity >50% as criteria. The screened sequences with the highest bit score were selected as candidate MYBs and were named according to their homologous proteins sharing the highest similarity with them. One exception is that one candidate MYB (VaccDscaff1486-snap-gene-0.3) was named as VcMYB to distinguish it from blueberry VcMYBA (MH105054) [32]. A phylogenetic tree was constructed by Maximum Likelihood method using MEGAX (Possion mode, complete deletion, and 1000 bootstrap values) to show the relationships among the blueberry ABRMs and the reported ABRMs from some other plants.

Bioinformatic Analysis of Blueberry ABRM Genes and Their Encoded Proteins
The physicochemical properties, conserved motifs (motif number set as 10) and domains, as well as the subcellular localization of the blueberry MYB proteins, were analyzed according to the method described by Zhang et al. [22]. Gene structure analysis was performed using GSDS (http://gsds.gao-lab.org/, accessed on 3 March 2021). The ClustalW program in MEGA 6.06 software was used for multiple protein sequence alignment, and the alignment results were incorporated into MEGA 6.06 for the construction of the phylogenetic tree using the neighbor-joining method under the criteria of the Poisson model, complete deletion and bootstrap value = 1000. The 2000 bp sequences to upstream the start codon of the MYBs were extracted from the blueberry genome database and considered as promoter sequences, and were then subjected to cis-acting element analysis using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 3 March 2021).

Gene Expression Analysis
In our previous study, we sequenced the transcriptome of 'FL03' blueberry fruits at five different ripening stages (GF, PiF, RF, PF and BF). To study the expression patterns of these identified blueberry ABRMs, their FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values were first extracted from the transcriptome data and transformed into Log 2 (FPKM + 1) for heatmap drawing using TBtools software [43].
To validate the expression of the blueberry ABRMs, four highly expressed ABRMs, including VcMYB6, VcMYB23, VcMYBL2 and VcMYB, were selected and subjected to quantitative real time PCR analysis. Primers were designed according to their CDS sequences using Primer 3.0 (Table 1). A Trizol RNA Extraction Kit (TaKaRa, Dalian, China) was used to isolate the total RNA from the blueberry fruits at five different ripening stages. Then, high quality RNA was used for cDNA synthesis using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit (TransGen, Beijing China). The qRT-PCR reactions were performed on a Bio-Rad CFX96 TM real-time quantitative fluorescent PCR instrument using a TB Green Premix Ex Taq II (Tli RNaseH Plus; TaKaRa, Dalian, China) kit according to the manual. The relative expression levels of the selected blueberry ABRM genes in the fruits at different ripening stages were calculated using the 2 −∆∆Ct method with GAPDH (Genbank ID: AY123769) as the internal reference gene [22]. Three biological and three technical replications were made for the qRT-PCR analysis of the selected genes.

Gene Cloning, Vector Construction and Transient Overexpression Analysis
A RevertAid First-strand cDNA synthesis Kit (Thermo Scientific, Shanghai, China) was used to synthesize the cDNA for gene cloning. The primers for VcMYB gene amplification were designed using Primer 3 ( Table 1). The 25 µL PCR system contained 1 µL cDNA, 1 µL each of the forward and reverse primer, 12.5 µL 2 × Green mix and 9.5 µL ddH 2 O. The PCR conditions were set as follows: predenaturation at 95 • C for 3 min; 35 cycles of denaturation at 95 • C for 30 s, annealing at 59 • C for 30 s, and extension at 72 • C for 2.5 min; and final extension at 72 • C for 8 min. The electrophoresis detection results showed that there were three variable transcripts for VcMYB. The PCR products of the three transcripts were separately gel extracted, ligated into an 18-T vector and transformed into Escherichia coli DH5a. Positive clones were selected and sent to Beijing Liuhe Huada Gene Technology Co., Ltd. (Beijing, China) for sequencing verification. According to the sequencing results, the primers for the VcMYB gene vector construction were further designed and used for the amplification of the three variable transcripts ( Table 1). The cloned fragments were ligated into the pBI123 vector using the Ready-to-use Seamless Cloning Kit (Sangon Biotech, Shanghai, China), and transformed into Agrobacterium tumefaciens GV3101. A. tumefaciens GV3101 carrying empty pBI123 (as control), pBI123-VcMYB-1, pBI123-VcMYB-2 and pBI123-VcMYB-3 vectors were cultured in the dark at 180 rpm and 28 • C until OD 600 = 0.6~0.8 they were centrifuged at 5000 rpm for 4 min, resuspended using an MES solution (2-(N-Morpholino) and ethanesulfonic acid hydrate) solution (containing 10 mM MES, 10 mM MgCl 2 ·6H 2 O and 200 µM acetosyringone) to a final concentration of OD 600 = 0.6. Then, they were injected into the green fruits (at about 15 days post flowering (dpf), to further confirm the function of VcMYB-1, green fruits at 20 dpf were also used) of the 'Legacy' blueberry, cultured in the dark for 1 day and removed to normal light for fruit color change observation. At three days post treatment, the colour parameters (L*, a* and b* values) of the fruit pericarps were measured using a CR8 Portable Colorimeter (Shenzhen Threenh Technology Co., Ltd., Shenzhen, China), and the anthocyanin was extracted using the acidified ethanol [44] from the blueberry fruit pericarps. The anthocyanin content in the blueberry pericarps was measured and calculated according to the method of Zhuang et al. [45].

Physiobiochemical Properties and Sequence Characteristics of Blueberrry ABRMs
The identified blueberry ABRM proteins consisted of between 73 and 365 aa, with molecular weights ranging between 8407.08 and 40,095.85 Da, and isoelectric points (pI) ranging between 4.31 and 9.72. Their instability coefficients ranged between 36.10 and 79.67. All of these proteins were predicted to be hydrophilic proteins. The subcellular localization prediction results showed that all of these blueberry ARBMs were nucleus-localized, with the exception of VcRVE8 (localized to both chloroplast and nucleus) ( Table 3). By analyzing the amino acid sequences, we found that VcMYB12, VcMYB85, VcMYB340, VcPL, VcMYB6, VcMYB23, VcMYBC1, VcMYB24L, VcMYB123, VcMYB, VcPH4 and VcMYBL2 have conserved R2 and R3 domains, indicating that they belong to the R2R3-MYB (2R-MYB). The remaining three ARBMs belong to 1R-MYB, of which VcMYBATV and VcCPC contain a R3 domain, and VcRVE8 contains a R1/2 domain ( Figure 1).

Conserved Motifs in Blueberry ABRM Proteins and Gene Structures of Their Corresponding Genes
In total, we identified five conserved motifs from the blueberry ABRM proteins ( Figure 2A). Among these motifs, Motif1 was found in all of the ABRMs. The R2R3 type blueberry ABRMs all contained Motif1~3, VcMYB23 contained two Motif3, and VcMYB contained an extra Motif5. VcPCP and VcMYBATV contained only one Motif1, VcRVE8 contained only one Motif1 and one Motif4.  The gene structure analysis results showed that the exon numbers of the blueberry ARBM genes ranged between one and eight ( Figure 2B). VcRVE8 had the largest number of exons with eight, VcMYB24L had four exons, VcPH4 and VcMYB123 contained two exons, and VcMYB6 had only one exon. All the other blueberry ABRMs contained three exons.

Cis-Acting Elements in Promoters of Blueberry ABRM Genes
We further analyzed the cis-acting elements in the promoters of the blueberry ABRM genes. The results showed that there were many light-responsive, phytohormone-responsive, stress-responsive, and growth and development related elements in their promoters ( Figure 3). In total, we identified ten types of light-responsive elements in their promoters. All of the blueberry ABRMs, with the exception of VcMYB, contained a light-responsive Box4 element in their promoter regions, and the promoters of 12 blueberry ABRM genes (except VcMYB24L, VcMYB6 and VcMYBATV) contained the light responsive G-box elements (Figure 3).

Conserved Motifs in Blueberry ABRM Proteins and Gene Structures of Their Correspondi Genes
In total, we identified five conserved motifs from the blueberry ABRM proteins (F ure 2A). Among these motifs, Motif1 was found in all of the ABRMs. The R2R3 type blu berry ABRMs all contained Motif1~3, VcMYB23 contained two Motif3, and VcMYB co tained an extra Motif5. VcPCP and VcMYBATV contained only one Motif1, VcRVE8 co tained only one Motif1 and one Motif4.
The gene structure analysis results showed that the exon numbers of the blueber ARBM genes ranged between one and eight ( Figure 2B). VcRVE8 had the largest numb of exons with eight, VcMYB24L had four exons, VcPH4 and VcMYB123 contained two ons, and VcMYB6 had only one exon. All the other blueberry ABRMs contained three ons. In total, six kinds of stress-related elements, including low temperature-responsive element LTR, anaerobic-induction related element ARE, MYB drought-inducibility related element MBS, defense and stress related element TC-rich elements, anoxic specific inducibility related element GC-motif and wounding related element WUN-motif, were identified in the blueberry ABRM promoters (Figure 3). Moreover, there were eleven, nine, six, five, four and three ABRMs contained ARE, LTR, MBS, GC-motif, TC-rich repeats and WUN-motif in their promoters, respectively.
tained an extra Motif5. VcPCP and VcMYBATV contained only one Motif1, VcRVE8 contained only one Motif1 and one Motif4.
The gene structure analysis results showed that the exon numbers of the blueberry ARBM genes ranged between one and eight ( Figure 2B). VcRVE8 had the largest number of exons with eight, VcMYB24L had four exons, VcPH4 and VcMYB123 contained two exons, and VcMYB6 had only one exon. All the other blueberry ABRMs contained three exons.  Additionally, we also identified many growth and development related cis-acting elements in the promoters of the blueberry ABRM genes (Figure 3). Eight ABRMs contained the regulatory A-box element in their promoters; seven ABRMs' promoters contained the zein metabolism regulation related O2-site element; seven ABRMs' promoters contained the MYBHV1-binding site element CCAAT-box; four ABRMs' promoters contained the meristem expression related element CAT-box; the promoters of VcMYB24L and VcMYB340 contained the endosperm expression related GCN4_motif element; the promoter of VcMYBL2 contained the circadian control related element. Moreover, the VcMYB24L promoter specifically contained the flavonoid biosynthetic related MBSl element.

Expression Analysis of Blueberry ABRM Genes
According to our transcriptome data, the expression patterns of the blueberry ABRM genes in fruits at different ripening stages were studied. It was found that VcMYB6, VcMYB23, VcMYB, VcMYBL2 and VcPH4 are expressed highly in blueberry fruits, but other ABRMs, such as VcMYB12, VcMYB123, VcPL, VcMYB24L and VcMYB340, showed either low expression (FPKM < 4) or are not expressed in the fruits ( Figure 5). The expression level of VcMYB was low in GF (FPKM < 2), gradually increased in PiF (FPKM > 10), and then maintained at an abundant level in RF, PF and BF (FPKM > 40). The expression levels of VcMYB in RF and PF were both higher than that in BF. Moreover, its expression in the late three stages was found to be the highest among all the blueberry ABRMs and very significantly higher than that in GF and PiF, indicating that it might play an important role in regulating anthocyanin biosynthesis, particularly at the late fruit ripening stages. The expression level of VcMYB6 in fruits at all stages was high (FPKM > 10), and its expression level changed slightly during blueberry fruit ripening. The expression of VcMYB23 was the highest in RF and PF. VcMYBL2 showed a 'fall-rise' expression pattern, and its expression level in PiF was the lowest, but there was no significant expression difference at the late three ripening stages. VcPH4 also showed a 'fall-rise' expression profile during fruit ripening, but no significant difference was found among the fruits at different ripening stages.
Curr. Issues Mol. Biol. 2023, 3, FOR PEER REVIEW 10 Figure 3. The identified cis-acting elements in the promoters of blueberry anthocyanin biosynthesis related MYB genes.

Expression Analysis of Blueberry ABRM Genes
According to our transcriptome data, the expression patterns of the blueberry ABRM genes in fruits at different ripening stages were studied. It was found that VcMYB6, VcMYB23, VcMYB, VcMYBL2 and VcPH4 are expressed highly in blueberry fruits, but other ABRMs, such as VcMYB12, VcMYB123, VcPL, VcMYB24L and VcMYB340, showed either low expression (FPKM < 4) or are not expressed in the fruits ( Figure 5). The expression level of VcMYB was low in GF (FPKM < 2), gradually increased in PiF (FPKM > 10), and then maintained at an abundant level in RF, PF and BF (FPKM > 40). The expression levels of VcMYB in RF and PF were both higher than that in BF. Moreover, its expression in the late three stages was found to be the highest among all the blueberry ABRMs and very significantly higher than that in GF and PiF, indicating that it might play an important role in regulating anthocyanin biosynthesis, particularly at the late fruit ripening stages. The expression level of VcMYB6 in fruits at all stages was high (FPKM > 10), and its expression level changed slightly during blueberry fruit ripening. The expression of VcMYB23 was the highest in RF and PF. VcMYBL2 showed a 'fall-rise' expression pattern, and its expression level in PiF was the lowest, but there was no significant expression difference at the late three ripening stages. VcPH4 also showed a 'fall-rise' expression For the validation of the expression changes of the blueberry ABRM genes in fruits during ripening, quantitative real time PCR (qRT-PCR) analysis of four selected genes, including VcMYB6, VcMYB23, VcMYBL2 and VcMYB, was performed. The results showed that the expression change patterns of these blueberry ABRMs during fruit ripening were mostly consistent with our transcriptome data (Figure 6), indicating that our transcriptome data is believable. Among them, the expression of VcMYB was found to be the lowest in GF, and its expression in PiF, RF, PF and BF was approximately 1.44-fold, 1.79-fold, 2.1-fold and 1.75-fold of GF, respectively. that the expression change patterns of these blueberry ABRMs during fruit ripening we mostly consistent with our transcriptome data (Figure 6), indicating that our transcri tome data is believable. Among them, the expression of VcMYB was found to be the lowe in GF, and its expression in PiF, RF, PF and BF was approximately 1.44-fold, 1.79-fold, 2. fold and 1.75-fold of GF, respectively.

Transient Overexpression Analysis of Three VcMYB Variable Transcripts
Given the high similarity with more than ten reported ABRM genes and its high expression in fruits, particularly at the late ripening stages, VcMYB was proposed to play a key role in regulating blueberry anthocyanin biosynthesis. To confirm its function, the gene was successfully cloned by reverse transcription PCR (RT-PCR). The electrophoresis detection results showed that this gene had three variable transcripts, which were all shorter than the reference cDNA sequence (VaccDscaff1486-snap-gene-0.3) ( Figure 7A). The sequencing results showed that the lengths of these three transcripts were 786 bp, 704 bp and 568 bp, respectively, and they were termed as VcMYB-1, VcMYB-2 and VcMYB-3, in descending order of sequence length.
The three transcripts were separately inserted into the pBI123 vector, transformed into A. tumefaciens GV3101, and then transiently overexpressed in the young blueberry fruits. The results showed that only the transient overexpression of VcMYB-1 triggered anthocyanin accumulation in the young blueberry fruits ( Figure 7B). For the green blueberry fruits, at approximately 15 days post-flowering (dpf), at three days post-treatment, the areas injected with A. tumefaciens GV3101 carrying VcMYB-1 became purple-red, and the L*, a* and b* values of the fruit pericarps overexpressing VcMYB-1 were all significantly lower than the control check group (CK) ( Figure 7C). For the green fruits, at about 20 dpf, at three days post-treatment, the injected areas became much redder than the fruits at 15 dpf. By measuring the anthocyanin content, we found that the anthocyanin content in the VcMYB-1 overexpressing fruit pericarps was approximately 1.56-fold of the CK ( Figure 7D-F). However, no obvious pigmentation was found in the areas injected with A. tumefaciens GV3101 carrying VcMYB-2 and VcMYB-3, indicating that their transient overexpression could not trigger anthocyanin accumulation in the blueberry fruit pericarps.

Transient Overexpression Analysis of Three VcMYB Variable Transcripts
Given the high similarity with more than ten reported ABRM genes and its high expression in fruits, particularly at the late ripening stages, VcMYB was proposed to play a key role in regulating blueberry anthocyanin biosynthesis. To confirm its function, the gene was successfully cloned by reverse transcription PCR (RT-PCR). The electrophoresis detection results showed that this gene had three variable transcripts, which were all shorter than the reference cDNA sequence (VaccDscaff1486-snap-gene-0.3) ( Figure 7A). The sequencing results showed that the lengths of these three transcripts were 786 bp, 704 bp and 568 bp, respectively, and they were termed as VcMYB-1, VcMYB-2 and VcMYB-3, in descending order of sequence length.
The three transcripts were separately inserted into the pBI123 vector, transformed into A. tumefaciens GV3101, and then transiently overexpressed in the young blueberry fruits. The results showed that only the transient overexpression of VcMYB-1 triggered anthocyanin accumulation in the young blueberry fruits ( Figure 7B). For the green blueberry fruits, at approximately 15 days post-flowering (dpf), at three days post-treatment, the areas injected with A. tumefaciens GV3101 carrying VcMYB-1 became purple-red, and the L*, a* and b* values of the fruit pericarps overexpressing VcMYB-1 were all significantly lower than the control check group (CK) ( Figure 7C). For the green fruits, at about 20 dpf, at three days post-treatment, the injected areas became much redder than the fruits at 15 dpf. By measuring the anthocyanin content, we found that the anthocyanin content in the VcMYB-1 overexpressing fruit pericarps was approximately 1.56-fold of the CK (Figure 7D-F). However, no obvious pigmentation was found in the areas injected with A. tumefaciens GV3101 carrying VcMYB-2 and VcMYB-3, indicating that their transient overexpression could not trigger anthocyanin accumulation in the blueberry fruit pericarps.

Discussion
According to their R repeat numbers and sequences, MYBs could be classified into four major types, i.e., 1R-MYB/MYB-related, R2R3-MYB, 3R-MYB and 4R-MYB [25]. In plants, R2R3-MYBs and 1R-MYBs are the major types of MYBs. It was frequently discovered that most of the ABRMs belong to the R2R3 type, followed by the 1R type [62]. In this study, we identified fifteen ABRMs from the blueberry genome data through the homologous protein sequence alignment method. Among these ABRMs, twelve were R2R3-MYBs and three were 1R-MYBs. The subcellular localization prediction results showed that almost all of these ABRMs were nucleus-localized, which was consistent with the localization characteristics of the transcription factors. The gene structure analysis revealed that the exon number of blueberry ABRMs varied greatly, ranging between one and eight. The gene expression analysis showed that only VcMYB6, VcMYB23, VcMYB, VcMYBL2 and VcPH4 were expressed highly in fruits at different ripening stages, suggesting that they may play more important roles in regulating anthocyanin biosynthesis in blueberry.
MYBs function in regulating anthocyanin biosynthesis by associating with bHLH, WD40 and anthocyanin biosynthesis structural genes. Wang et al. [49] reported that exogenous cytokinin treatment could induce anthocyanin accumulation in red-fleshed apple callus by promoting the expression of MdDFR, MdUFGT, MdMYB10 and MdbHLH3 genes, and by suppressing the expression of MYBL2. Moreover, they found that MdMYBL2 could interact with MdbHLH3. In the callus overexpressing MdMYBL2, anthocyanin accumulation decreased, and the expression levels of MdDFR, MdUFGT, MdMYB10 and MdbHLH3 were all significantly inhibited. Actinidia arguta AaMYBC1 could interact with AabHLH42. The virus-induced gene silencing of AaMYBC1 resulted in decreased anthocyanin accumulation and the reduced expression of anthocyanin biosynthesis structural genes in 'HB' kiwifruit [57]. Eggplant SmMYB86, a negative regulator of anthocyanin biosynthesis, can inhibit the expression of SmCHS, SmF3H and SmANS by binding to their promoters [63]. The down-regulation of SmMYB86 resulted in anthocyanin content reduction and SmCHS, SmF3H and SmANS gene expression up-regulation, and its overexpression resulted in a significant decrease in the anthocyanin content in eggplant.
In this study, we predicted the protein-protein interaction network of blueberry ARBMs. According to their interacting proteins, it was predicted that the functions of these MYBs varied. VcCPC, VcMYB6 and VcMYBATV were found to be highly homologous to AtCPC, AtMYBR1 and AtTT2, respectively. AtCPC is a MYB that negatively regulates anthocyanin accumulation [64], suggesting that VcCPC might play a negative role in regulating the anthocyanin biosynthesis in blueberry. AtMYBR1 (also called AtMYB44) is involved in the abiotic stress responses of A. thaliana and exhibited a negative regulatory role in anthocyanin biosynthesis. Sweet potato IbMYB44 could interact with IbMYB340 and IbNAC56a/b, thereby inhibiting the formation of the MYB340-BHLH2-NAC56 complex and negatively regulating the accumulation of anthocyanins [52]. Apple MdMYB6 can bind to the promoters of MdANS and MdGSTF12, and the overexpression of MdMYB6 in red apple callus would reduce anthocyanin accumulation and inhibit the expression of MdANS and MdGSTF12 [51]. RCAR3, the interacting protein of AtMYBR1, is an ABA receptor regulator and plays a pivotal role in activating ABA signaling [65], suggesting that VcMYB6 might play a role in the ABA-regulated anthocyanin biosynthesis in blueberry. AtTT2 (AtMYB123) is mainly responsible for the biosynthesis regulation of tannins, such as proanthocyanidins [66]. VcMYB23 and VcRVE8 are homologous to AtMYB15 and AtRVE8, respectively. In Arabidopsis, the AtMYB15 interacts with AtICE1, and AtRVE interacts with AtLNK2 and AtLNK2. ICE1 plays an important role in plants' responses to low temperatures [67], while LNK1 and LNK2 play roles in the integrated regulation of light signal responses and circadian regulation [68,69], and they can also act as corepressors of phenylpropanoid metabolism by interacting with MYB [70], suggesting that VcMYB23 and VcREV8 might be involved in the low temperature and light triggered anthocyanin biosynthesis in blueberry, respectively.
It is worth noting that VcMYB shared the highest similarity with one reported blueberry MYB (VcMYBA) [32] and was identified as a homologous protein of many positive anthocyanin regulatory MYB proteins, such as Arabidopsis AtMYB114, AtMYB90 (PAP2), AtMYB75 (PAP1) and AtMYB113, Helianthus tuberosus HtMYB2, monkeyflower PELAN, Eutrema salsugineum EsMYB90, Freesia hybrida FhPAP1, grape hyacinth MaAN2 and apple MdMYB10. In Arabidopsis, the AtMYB75/90/113/114 members of the SG6 subfamily of the MYB family have been proven to be involved in the regulation of anthocyanin biosynthesis; the SG7 subfamily members AtMYB11/12/111 are involved in the flavonol biosynthesis regulation; the AtMYB5/123 are involved in the regulation of tannin biosyn-thesis, and AtMYB3/4/7/32 encode the transcriptional repressors [36]. The expression levels of HtMYB2 in the root, stem, leaf and tuber epidermis of the red-skinned tubers variety 'QY1' are higher than those of the white-skinned tubers variety 'QY3'. The heterologous overexpression of HtMYB2 in tobacco activates the anthocyanin biosynthesis pathway and accumulated pigments in leaves [37]. PyMYB114 and PyMYB10 can activate anthocyanin biosynthesis by interacting with PybHLH3; PyWRKY26 and PybHLH3 can synergistically target the promoter of PyMYB114 and participate in the regulation of anthocyanin biosynthesis and transportation [71]. The co-expression of the PalbHLH1 and PalMYB90 genes in Populus alba could improve its disease resistance by promoting flavonoid synthesis [72]. The overexpression of MdMYB90-like upregulated the expression of the anthocyanin biosynthesis related structural genes and regulatory genes (including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3 and MdMYB1) and induced the accumulation of pigments in transgenic callus and pericarps [73]. The overexpression of onion MYB1 can restore anthocyanin accumulation and the cyanic petal phenotype of the A. majus R2R3-MYB mutant, and heterologous co-expression of MYB1 and bHLH can promote ectopic red pigmentation in garlic plants [74]. VcMYB is highly homologous to AtMYB114, which is a SG6 subfamily member of the Arabidopsis MYB family that positively regulates anthocyanin biosynthesis. Moreover, based on our transcriptome data, we found that the expression levels of VcMYB in RF, PF and BF were the highest among all members. Our qRT-PCR results also revealed that VcMYB was expressed significantly higher in the fruits at the red, purple and blue stages, accounting for 1.79-fold, 2.1-fold and 1.75-fold of that in GF, respectively. These results suggested that VcMYB may be a pivotal positive regulator of anthocyanin biosynthesis in blueberry. To verify the function of VcMYB in anthocyanin biosynthesis in blueberry, it was further cloned and functionally analyzed. Similar to sweet potato IbMYB1 [75], the VcMYB also has three transcripts. The transient transformation results showed that only the longest VcMYB transcript, VcMYB-1, could promote anthocyanin accumulation in young blueberry fruit pericarps.
These results have demonstrated that the expression of anthocyanin regulatory MYBs was greatly influenced by light, ABA and MeJA [76][77][78][79]. Consistently, in this study, we identified many light-, ABA-and MeJA-elements in the promoters of blueberry ABRMs. Light quality and quantity greatly and widely affect the biosynthesis of anthocyanins or flavonoids in various plants [79,80]. UV-B treatment could upregulate the expression of the HY5 gene that encodes the UV receptor at the green fruit stage, and HY5 promoted anthocyanin accumulation by upregulating the expression of VcMYBA1, while inhibiting the expression of VcMYBC2 [30]. However, the expression of VcMYBC2 was induced by UV-B treatment in the mature fruits, which could inhibit the excessive accumulation of anthocyanins. JA is an important signal for the biosynthesis of plants' secondary metabolites, and its induction role in anthocyanin accumulation has also been found in many plants [77]. Exogenous JA treatment can activate the expression of anthocyanin biosynthesis related structural genes and regulatory transcription factor genes, and enhance anthocyanin accumulation in Arabidopsis under far-red light [81]. In apple, JA can induce anthocyanin and proanthocyanin accumulations by regulating the JAZ-TRB1-MyB9 complex [78]. In addition, MeJA treatment could induce the expression of MdMYB9 and MdMYB11, whose overexpression in apple callus could improve anthocyanin and proanthocyanin accumulation, and this promotion effect could be further enhanced by MeJA [82]. ABA is considered to be one of the most important positive regulators of the ripening of non-climacteric fruits and anthocyanin biosynthesis [32,83,84]. Karppinen et al. [85] found that exogenous ABA treatment could promote the accumulation of anthocyanins in V. myrtillus. Han et al. [32] found that the expression levels of CHS, CHI, DRF, LDOX/ANS and some other anthocyanin biosynthesis related genes were significantly up-regulated in blueberries when treated with ABA at the late ripening stages, and were highly positively correlated with the anthocyanin content. Consistently, in our present study, we found that 16 of the 19 identified blueberry ABRM genes contained the ABA-responsive ABRE element in their promoters, indicating that ABA have a significant function in influencing the expression of blueberry ABRMs and in regulating anthocyanin biosynthesis.

Conclusions
In this study, we identified fifteen candidate ABRM proteins from blueberry. Among them, twelve were R2R3-MYBs and three were 1R-MYBs, which could be well supported by their conserved motif types and numbers. With the exception of VcRVE8, which was localized in the chloroplast and nucleus, all of the blueberry ABRMs were predicted to be nucleus-localized. The exon numbers of the blueberry ABRM genes varied significantly. The promoters of ABRMs contain a large number of light-, ABA-and MeJA-responsive elements, indicating that the influences of these factors on anthocyanin accumulation were achieved, at least partially, by regulating the expression of the ABRM genes. VcMYB was highly expressed in blueberry fruits at the late ripening stages. This gene had three transcripts; however, only the transient overexpression of its longest transcripts (VcMYB-1) could promote anthocyanin accumulation in young blueberry fruits. Our study will provide a basis for the applications of the blueberry anthocyanin biosynthesis related MYB genes, and will lay the foundation for the high-anthocyanin aimed blueberry selection and breeding in the future.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cimb45010027/s1. Figure S1: Phylogenetic analysis result of anthocyanin biosynthesis related MYB proteins from blueberry and some other plant species.