Nine-year-old blueberry (V. corymbosum “Duke”) plants collected from the small berry garden of the Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences were used as experimental material. Experiments were conducted from March 2019 to February 2022 at the Ministry of Agriculture Key Laboratory of horticultural crop germplasm resources utilization, Institute of Fruit Tree Research, Chinese Academy of Agricultural Sciences (Xingcheng, Liaoning, China) and College of Horticulture Sciences, Shandong Agricultural University (Tai’an, Shandong, China). To ensure the consistency of the material, fruits were collected from the top of the inflorescence (first to mature). Samples were taken from plants under the same growth conditions for organ (root, stem, leaf, flower, and fruit)-specific expression. All samples were rapidly frozen in liquid nitrogen and stored at −80°C.

Blueberry transcriptome data obtained from Illumina and SMRT sequencing were used to identify the gene family members of SnRK2 in blueberry (Tang et al., 2021). The excavated VcSnRK2 protein sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST/) in the National Center for Biotechnology Information (NCBI) database. The results showed that the six VcSnRK2 sequences had high homology with the SnRK2 protein sequences of Solanum tuberosum, Arabidopsis thaliana and Zea mays. The six genes were named according to the Phylogenetic Tree Construction. If VcSnRK2 clusters with Solanum tuberosum, it is named after the same number, including VcSnRK2.1, 2.3, 2.4, and 2.5. For VcSnRK2.2 and 2.6, which do not cluster as closely with Solanum tuberosum, they are named sequentially according to their position on the chromosome in sequence.

The amino acid secondary structure of VcSnRK2 was predicted using the Simple Modular Architecture Research Tool (SMART) software program (http://smart.emb-lheidelberg.de/) through three rounds of PCR. The deduced amino acid sequences of VcSnRK2s were aligned using CLC Sequence Viewer 6.0 software with the default settings.

RNA was extracted from different organs and different fruit stages of blueberry plants, and Arabidopsis leaves using TaKaRa MiniBEST Plant RNA Extration Kit (TaKaRa, Shiga, Japan). qRT-PCR was performed with VcACTIN as a reference gene. Arabidopsis polyubiquitin 10 (AtUBQ10) was used as a reference gene. Single-stranded cDNA was obtained using a reverse transcription kit (TaKaRa, Shiga, Japan). 4 μg RNA was used as a template for cDNA synthesis. The procedure for cDNA synthesis was to incubate the reaction solution at 65°C for five minutes, 42°C for 60 minutes, and 70°C for 5 minutes. qPCR assays were performed on a CFX Connect real-time PCR assay system (Bio-Rad, USA) using the SYBR Green I chimeric fluorescence assay. The amplification reaction procedure was as follows: Stage 1 pre-denaturation, the mixture was reacted at 95°C for 30 sec, one cycle; Stage 2 cyclic reaction, 95°C for 10 sec, 60°C for 30 sec, 40 cycles; Stage 3 melting curve, 95°C for 15 sec, 60°C for 60 sec, 95°C for 15 sec, one cycle. The qPCR results were all analyzed using the comparative CT method. Three biological replicates and three technical replicates were conducted. All primers used in this study are listed in Supplementary Table S1 .

Extraction methods for anthocyanins were based on previous studies (Latti et al., 2010; Pertuzatti et al., 2021). Separation, identification, and quantification of anthocyanins by high-performance liquid chromatography (HPLC) was conducted on an Agilent 1100 Series system (Agilent, Germany), equipped with DAD (G1315B), and an LC/MSD Trap VL (G2445C VL) electrospray ionization mass spectrometry (ESIMSn) system that was coupled to an Agilent ChemStation (version B.01.03) data-processing station (Castillo-Munãoz et al., 2009). Mass spectra data were processed with Agilent LC/MS Trap software (version 5.3). ESI-MSn was used to determine the anthocyanin profiles and the following parameters were employed: positive ionization mode; dry gas, N2, 11 mL/min; drying temperature, 350°C; nebulizer, 65 psi; capillary, –2500 V; capillary exit offset, 70 V; skimmer1, 20 V; skimmer 2, 6 V; compound stability, 100%; scan range, 50–1,200 m/z (Pertuzatti et al., 2016). For the purposes of quantification, DAD chromatograms were extracted at 520 nm for anthocyanins.

Agrobacterium tumefaciens GV3101 transformed the pRI101-AN vector with the open reading frame (ORF) after it had been cloned and inserted. Transformation of Agrobacterium into Arabidopsis by flower dip method. On MS media containing 50 mg L⁻¹ kanamycin, T1 VcSnRK2.3 transgenic plants were selected. Until seed set, kanamycin-resistant T1 seedlings were cultivated in a growth chamber at 22°C and a 16 h day length. Seeds of T1 plants were planted and germinated to obtain T2 seedlings. T2 seedlings were also selected on MS medium containing 50 mg L⁻¹ kanamycin.

Blueberry fruit injection assays were performed with reference to previous studies (An et al., 2019; Tang et al., 2021). The open reading frame of the SnRK2.3 gene was cloned into the pGreenII62-SK vector to obtain the VcSnRK2.3-pGreenII62-SK vector. Blueberry peels were injected with the mixed vector and Agrobacterium solutions.

The Y2H experiment was performed according to Clontech’s instructions. The ORF, N-terminus, and C-terminus of VcSnRK2.3 and the ORF of MdMYB1 were inserted into pGAD and pGBD vectors. The plasmids were co-transformed into yeast strain Y2H Gold (TaKaRa, Beijing, China) by using the lithium acetate method with reference to Veries et al. For transformation, 4-5 μl of plasmid and 50 μg of denatured salmon sperm vector DNA are mixed. Then 500 μl of polyethylene glycol (PEG) lithium acetate solution is added. After incubation 20 μl DMSO is added and incubated again. The transformed yeast strains were grown on medium lacking Leucine and Tryptophan (SD-L/-T), Leucine, Tryptophan, Histidine and Adenine (SD-L/-T/-H/-A) or Leucine, Tryptophan, Histidine, Adenine and X-gal (SD-T/-L/-H/-A+X-gal) at 28°C for 3 days.

The ORF and C-terminus of VcSnRK2.3 and the ORF of MdMYB1 were inserted into YFP^C or YFP^N to generate the VcSnRK2.3-C-YFP^C, VcSnRK2.3-YFP^C and VcMYB1-YFP^N plasmids. The constructs were transformed into Agrobacterium GV3101. The specified plasmid-carrying Agrobacterium solution was incubated with prepared tobacco leaves for 30 minutes. After 2 days of darkness at 24°C, the transformed tobacco leaves were observed under a Confocal microscope (Zeiss LSM 780, Lena, Germany). The excitation wavelength of YFP is 510 nm, and the emission wavelength is 527 nm.

VcDFR promoter sequences were obtained from pre-lab data (Tang et al., 2021). Tobacco transient expression assays were carried out according to previous studies (Shang et al., 2010; An et al., 2017). The VcDFRpro : Luc reporter construct was created by amplifying the VcDFR promoter and cloning it into the pGreenII0800-LUC vector. The ORFs of VcMYB1 and VcSnRK2.3 were cloned into the pGreenII62-SK vector to generate 35Spro : VcMYB1 and 35Spro : VcSnRK2.3, respectively. Transformed leaves were sprayed with 100 mM luciferin and placed in the dark for 5 minutes before being examined for luminescence. The LUC pictures were collected using a charge-coupled device-imaging apparatus (NightOWL LB983 in conjunction with Indigo software).

Effector constructs were generated from Agrobacterium GV3101 strains containing the pRI101-VcSnRK2.3 and pRI101-VcMYB1 genes. The reporter constructs were generated using the promoter sequences of VcDFR cloned upstream of the β-glucuronidase (GUS) reporter gene in the pCAMBIA1301 vector. Agrobacterium GV3101 strains carrying pRI101-VcSnRK2.3 and pRI101-VcMYB1 were co-injected into the abaxial surface of tobacco leaves. Infected leaves were grown in growth chambers for 3-4 days and then analyzed for GUS activity. Proteins were extracted from infected leaves and fluorescence was measured with a fluorometer (VersaFluor Fluorometer, Bio-Rad) (http://www.bio-rad.com) with reference to Jefferson et al., 1987 and Bowling et al., 1994.

Based on the blueberry transcriptome data, we obtained six blueberry SnRK2 gene family members, named VcSnRK2.1 to VcSnRK2.6. We analyzed the physicochemical properties of the blueberry SnRK2 gene family members using the online software ProtParam. Table 1 shows that the amino acid lengths of the six blueberry SnRK2 proteins, ranging from 340 (VcSnRK2.1) AAs to 362 (VcSnRK2.3) AAs, and the molecular weights ranged from 38.56 kDa (VcSnRK2.1) to 41.04 (VcSnRK2.3) kDa. In addition, the pI was in the range of 4.75 (VcSnRK2.6) to 5.62 (VcSnRK2.4). To investigate the structural features of the SnRK2 protein sequences, we conducted a comparative analysis of the amino acid sequences of the blueberry SnRK2 proteins ( Figure 1A ). The results showed that all family members contain ATP binding domains and a protein kinase activation loop and were relatively conserved (Boudsocq et al., 2004; Han et al., 2015).

In addition, to investigate the evolutionary relationship between the blueberry SnRK2 gene and SnRK2 members in other plants, we downloaded the entire SnRK2 gene sequence from the NCBI website for Arabidopsis, Solanum tuberosum, and Zea mays. The Protein BLAST program was used to obtain homologs of Arabidopsis, Solanum tuberosum and Zea mays SnRK2. The evolutionary tree was constructed in MEGA-X software using neighbor- joining, which contained a total of 34 SnRK2 protein sequences. Based on the evolutionary results, VcSnRK2.3 had the highest similarity to StSnRK2.3 and AtSnRK2.6 ( Figure 1B ).

To analyze the expression of each member of the blueberry VcSnRK2 subfamily at different fruiting stages, we examined the expression of VcSnRK2 during the green, pink, and blue fruit stages of blueberry using quantitative real-time PCR analysis (qRT-PCR). According to Figure 2A , the expression of VcSnRK2.3 increased significantly when blueberry fruit ripened from the green fruit stage, indicating a possible positive regulatory relationship between VcSnRK2.3 and blueberry fruit ripening. In contrast, the expression of VcSnRK2.4 decreased significantly with fruit ripening, and the expression of VcSnRK2.1 and VcSnRK2.5 decreased slightly. For VcSnRK2.2 and VcSnRK2.6, we did not observe any correlation with fruit ripening. Thus, we hypothesized that VcSnRK2.3 could positively regulate the ripening of blueberry fruits. And further experiments were designed to verify its function.

Therefore, we investigated the distribution of VcSnRK2.3 in different organs of blueberry, and qRT-PCR analysis was carried out on sample materials. The results showed that VcSnRK2.3 genes were expressed in roots, stems, leaves, flowers and fruits. The expression of VcSnRK2.3 was stem > fruit > root > leaf > flower ( Figure 2B ). Because the relative expression of VcSnRK2.3 was high in blueberry stems and fruits, we speculated that SnRK2.3 may play a relatively important role in stems and fruits.

ABA has been shown to promote anthocyanin accumulation during blueberry fruit ripening. In our study, ABA treatment promoted the synthesis of blueberry anthocyanins. The color of blueberry fruits gradually changed from green to pink as the treatment continued within the 24 h period ( Figure 3A ). In addition, when compared to the control (CK) treatment, the anthocyanin content of ripen blueberry fruits was considerably higher following ABA treatment ( Figure 3B ). To investigate the response of blueberry VcSnRK2.3 to ABA, we measured the expression of VcSnRK2.3 after ABA treatment of blueberry fruits. As shown in Figure 3C , the control (CK) treatment had no significant effect on the expression of VcSnRK2.3 in fruits. The expression of VcSnRK2.3 increased gradually and then decreased slightly with the increase in the ABA treatment time, and the highest value at 12 h was 2.8 times that of the CK treatment ( Figure 3C ). The results indicated that VcSnRK2.3 was strongly induced by ABA, which is in line with other discoveries (Boudsocq et al., 2004; Kulik et al., 2011).

In strawberry, fruit ripening and anthocyanin accumulation were shown to be negatively regulated by FaSnRK2.6 (Han et al., 2015). To study the function of VcSnRK2.3 and validate its effect on anthocyanin biosynthesis, we carried out heterologous expression in Arabidopsis (ecotype Columbia) under CaMV-35S control. Compared with the wild-type (WT), transgenic Arabidopsis had red pigmentation in seeds 8–10 days after flowering ( Figure 4A ). The cotyledons and hypocotyls of Arabidopsis seedlings grown from the above seeds were also pigmented ( Figure 4B ). The total anthocyanin content of WT Arabidopsis was 1.513 mg/kg, while the mean value for total anthocyanin contents of VcSnRK2.3 Arabidopsis (VcSnRK2.3-L1, L2) was 119.91 mg/kg ( Figure 4C ). In addition, RNA was extracted from the above plants and examined using qRT-PCR. The results demonstrated that overexpression of VcSnRK2.3 up-regulated the expression of primary genes involved in the flavonoid biosynthetic pathway, including AtPAP1, At4CL, AtCHS, AtCHI, AtDFR and AtANS ( Figure 4D ). Among them, the transgenic Arabidopsis plants had the highest expression of structural genes AtDFR and AtANS.

To further demonstrate the function of VcSnRK2.3, we carried out Agrobacterium-mediated transient transformation assay in blueberry fruits 40 days after flowering. The control was the pGreenII62-SK empty vector, and the experimental group was the VcSnRK2.3-pGreenII62-SK vector (VcSnRK2.3-L1, L2 and L3). The results showed that overexpression of VcSnRK2.3 facilitated anthocyanin accumulation in blueberry injection sites ( Figure 5A ). And the phenotype can be observed two days after gene overexpression and can last until fruit ripening. The total anthocyanin content, including the levels of delphinidin, cyanidin, petunidin, peonidin, and malvidin, was significantly increased ( Figure 5B ). In addition, compared with the pGreenII62-SK vector, the expression levels of VcMYB1 and anthocyanin genes such as VcF3H, VcDFR, VcANS, and VcUFGT were considerably increased by the overexpression of VcSnRK2.3 ( Figure 5C ). Therefore, based on the above two experiments, we might infer that VcSnRK2.3 can promote not only anthocyanin biosynthesis but also the expression of VcMYB1.

The above results demonstrated that VcSnRK2.3 can promote VcMYB1 expression and anthocyanin accumulation in blueberry fruits ( Figure 5 ). Therefore, Y2H assays were carried out to confirm the interaction between VcSnRK2.3 and VcMYB1. After transforming VcMYB1-pGBD and VcSnRK2.3-pGAD into yeast cells, the results showed that VcMYB1-pGBD and VcSnRK2.3-pGAD as well as VcMYB1-pGBD and VcSnRK2.3-N-pGAD were able to grow on SD/-Leu-Trp -His-Ade medium ( Figure 6A ). This indicated that VcSnRK2.3 interacts with VcMYB1 in yeast cells, and the action site is located at the N-terminus of VcSnRK2.3. In addition, the interaction between VcSnRK2.3 and VcMYB1 was tested by BiFC assay. As shown in Figure 6B , when VcSnRK2.3-YFP^C and VcMYB1-YFP^N were co-expressed, the YFP fluorescent signals could be detected. In contrast, other combinations such as VcSnRK2.3-YFP^C + YFP^N, YFP^C + VcMYB1- YFP^N, VcSnRK2.3-C-YFP^C + VcMYB1-YFP^N, and VcSnRK2.3-C-YFP^C + YFP^N did not detect fluorescent signals ( Figure 6B ). Therefore, it was further demonstrated that VcSnRK2.3 and VcMYB1 interacted with each other.

DFR plays a crucial role in the biosynthesis of anthocyanins. When the expression level of DFR increased, the anthocyanin content in plants increased (An et al., 2020b). A previous study showed that MdMYB1 promotes the synthesis of anthocyanins by directly binding to the promoter sequences of MdDFR and MdUF3GT (An et al., 2018). To verify the action of the VcDFR promoter by VcSnRK2.3 and VcMYB1, we carried out firefly luciferase complementation assay ( Figure 7A ). The results showed that the luciferase signal was highest when the VcDFR promoter was co-injected with VcSnRK2.3 and VcMYB1, and the signal was second highest when the VcDFR promoter was co-injected with VcMYB1. Both were significantly higher than the luciferase signal of the control group ( Figure 7B ).

In a different assay, as shown in Figure 7C , the VcDFR promoter region was fused to the GUS gene as a reporter marker. The GUS activity was highest when the VcDFR promoter was co-injected with VcSnRK2.3 and VcMYB1. And the GUS activity was also significantly higher than control when the VcDFR promoter was injected with VcSnRK2.3 or VcMYB1 alone. These results suggested that the presence of VcSnRK2.3 up-regulated the binding of VcMYB1 to the target promoter and activated the transcription of the anthocyanin biosynthesis genes.