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Article

bZIP Transcription Factor PavbZIP6 Regulates Anthocyanin Accumulation by Increasing Abscisic Acid in Sweet Cherry

by
Shilin Gai
,
Bingyang Du
,
Yuqin Xiao
,
Xiang Zhang
,
Maihemuti Turupu
,
Qisheng Yao
,
Xinyu Wang
,
Yongzhen Yan
and
Tianhong Li
*
Frontiers Science Center for Molecular Design Breeding, College of Horticulture, China Agricultural University, Beijing 100193, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(18), 10207; https://doi.org/10.3390/ijms251810207
Submission received: 13 August 2024 / Revised: 18 September 2024 / Accepted: 19 September 2024 / Published: 23 September 2024
(This article belongs to the Section Molecular Genetics and Genomics)
Browse Figures
Review Reports Versions Notes

Abstract

:
Basic leucine zipper (bZIP) transcription factors (TFs) play a crucial role in anthocyanin accumulation in plants. In addition to bZIP TFs, abscisic acid (ABA) increases anthocyanin biosynthesis. Therefore, this study aimed to investigate whether bZIP TFs are involved in ABA-induced anthocyanin accumulation in sweet cherry and elucidate the underlying molecular mechanisms. Specifically, the BLAST method was used to identify bZIP genes in sweet cherry. Additionally, we examined the expression of ABA- and anthocyanin-related genes in sweet cherry following the overexpression or knockdown of a bZIP candidate gene. In total, we identified 54 bZIP-encoding genes in the sweet cherry genome. Basic leucine zipper 6 (bZIP6) showed significantly increased expression, along with increased anthocyanin accumulation in sweet cherry. Additionally, yeast one-hybrid and dual-luciferase assays indicated that PavbZIP6 enhanced the expression of anthocyanin biosynthetic genes (PavDFR, PavANS, and PavUFGT), thereby increasing anthocyanin accumulation. Moreover, PavbZIP6 interacted directly with the PavBBX6 promoter, thereby regulating PavNCED1 to promote abscisic acid (ABA) synthesis and enhance anthocyanin accumulation in sweet cherry fruit. Conclusively, this study reveals a novel mechanism by which PavbZIP6 mediates anthocyanin biosynthesis in response to ABA and contributes to our understanding of the mechanism of bZIP genes in the regulation of anthocyanin biosynthesis in sweet cherry.

1. Introduction

Sweet cherry (Vitis vinifera L.) is an economically valuable fruit with increased consumer demand. Notably, skin colour, sweetness, firmness, and acidity are key quality attributes of sweet cherries. Particularly, skin colour, ranging from deep red to light yellow, is crucial, as it influences the fruit’s economic value and consumer purchasing decisions [1]. Therefore, cherry colour plays a critical role in determining the commercial viability of grape products [2].
Anthocyanins play a crucial role in determining the colour of sweet cherries. Importantly, anthocyanins are essential for plant function and have beneficial effects on human health, including antioxidative, antimutagenic, and anticancer effects [3,4,5]. Therefore, enhancing anthocyanin accumulation in plants is a major research focus. Anthocyanins are important secondary metabolites belonging to the flavonoid class and are synthesised through the glycosylation of anthocyanidins. Additionally, anthocyanins are widely distributed in plant roots, stems, flowers, and fruits. Notably, the biosynthetic pathway of anthocyanins involves over 20 enzymatic steps that are catalysed by a series of enzymes, which are regulated by structural and regulatory genes, including chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonoid 3-O-glucosyltransferase (UFGT) [6]. Sweet cherries are characterized by their anthocyanin profile, which prominently features two major compounds: cyanidin-3-glucoside and cyanidin-3-rutinoside. Additionally, they contain smaller amounts of peonidin-3-rutinoside and pelargonidin-3-rutinoside as minor anthocyanins [7,8]. In many plants, these structural genes are regulated by the MBW regulatory complexes composed of R2R3-MYB transcription factors, bHLH proteins, and WD40 repeat proteins [9]. Research indicates that MYB TFs are critical in regulating anthocyanin biosynthesis [10].
In addition to the MYB TF family, research evidence indicates that the basic region-leucine zipper (bZIP) TF family plays a significant role in anthocyanin biosynthesis. The conserved bZIP domain consists of approximately 40–80 amino acid residues and comprises two parts: a highly conserved DNA-binding basic region composed of 20 amino acids and a relatively diverse leucine zipper region [11]. The basic region, which is rich in basic amino acids, is located at the C-terminus and binds to DNA sequences, specifically through a fixed N-x7-R/K structure. The leucine zipper region at the N-terminus consists of several heptad repeats or hydrophobic amino acid residues such as leucine, isoleucine, and valine. The primary function of this domain is the formation of dimers via leucine zipper structures [12,13]. In addition to the bZIP domain, bZIP TFs contain other conserved domains that serve as transcription activation factors. For example, two conserved motifs, R/KxxS/T and S/TxxD/E, have been identified as phosphorylation sites for Ca2+-independent protein kinases and casein kinase II, respectively [12]. Domains rich in proline, glutamine, and acidic residues have also been implicated in the transcriptional activation of bZIP genes [14].
HYPOCOTYL5 (HY5) is a member of the bZIP gene family and is activated in a light-dependent manner to promote pigment accumulation. HY5 directly binds to the G-box or ACE-box motifs of MYB factors, including those producing anthocyanins, such as PIGMENT1 (PAP1), flavonol glycosides (MYB12 and MYB111), and MYB-like structures (MYBD), thereby enhancing their gene expression [15,16,17,18,19]. Anthocyanin accumulation is promoted by MdbZIP44, which enhances the interaction between MdMYB1 and downstream gene promoters [20]. Notably, the overexpression of PgbZIP16 and PgbZIP34 from pomegranate promoted anthocyanin accumulation in the leaves of Arabidopsis [21]. Additionally, VvbZIP36 plays a crucial role in regulating anthocyanin synthesis in grapes by ensuring a balance in the production of stilbenes, lignans, flavonols, and anthocyanins [22]. Moreover, HlbZIP1A and HlbZIP2 expression increased flavonol glycosides, phenolic acids, and anthocyanin contents through the activation of the CHS_H1 promoter in Humulus lupulus [23]. Although the bZIP protein family has been shown to play key roles in anthocyanin synthesis in some plant species [22,24,25], its role in the regulation of anthocyanin synthesis in sweet cherry remains unclear.
ABA plays a distinct role in promoting seed and bud dormancy, as well as in regulating stress responses in various plant species [26,27,28]. ABA is crucial for anthocyanin accumulation, which enhances colour richness during fruit ripening [29]. ABA treatment increased the transcription of FaMYB10 and promoted anthocyanin accumulation by upregulating genes involved in anthocyanin biosynthesis in strawberries [30]. Additionally, MdMYB1 regulated ABA-induced anthocyanin biosynthesis in apples [24]. Moreover, PacMYBA plays a key role in ABA-mediated anthocyanin accumulation in sweet cherries [31]. However, the regulatory pathways of ABA signalling in anthocyanin biosynthesis in sweet cherry require further elucidation.
Therefore, this study aimed to investigate whether bZIP TFs are involved in ABA-induced anthocyanin accumulation in sweet cherry and elucidate the underlying molecular mechanisms. Specifically, we identified members of the bZIP TF family in sweet cherries and analysed their conserved domains, evolutionary relationships, and cis-acting elements using bioinformatic approaches.

2. Results

2.1. Identification and Characterization of the bZIP TF Family in Sweet Cherry

In total, 54 members of the bZIP family were identified in the genome of the sweet cherry cultivar ‘Tieton’. For subsequent analyses, the sequences were renamed according to their chromosomal positions (Table 1). Additionally, we analysed the physicochemical properties of the bZIP TFs using ExPASy online tools and found that the molecular weights of the proteins range from 7872.53 to 111,776.79 Da, with theoretical isoelectric points of 3.73–11.1. Moreover, the protein lengths varied from 74 to 1000 amino acids, with the shortest being PavbZIP41 (74 aa) and the longest being PavbZIP10 (1000 aa). Overall, these findings provide a theoretical basis for further research on the purification, activity, and function of PavbZIP. Subcellular localisation predictions for each member indicated that all members of the bZIP gene family were expressed in the nucleus. Based on these results, we investigated the role of PavbZIP proteins in transcriptional regulation and their potential involvement in anthocyanin synthesis in sweet cherry.

2.2. Phylogenetic Analysis of PavbZIP Proteins in Sweet Cherry

To explore homologous evolutionary relationships and classifications within the bZIP family, we constructed a phylogenetic tree using bZIP members from sweet cherry and Arabidopsis. Notably, bZIP gene family members in sweet cherry were divided into 13 groups, labelled A, B, C, D, E, G, H, I, K, M, and S. Additionally, these groups vary greatly in size, with the K group consisting of only one member and the F and J groups having no members at all. Importantly, the largest group was Group A, which included 13 members (Figure 1).

2.3. PavbZIP6 Is Closely Associated with Anthocyanin Synthesis in Sweet Cherry

To investigate the role of the bZIP TFs in anthocyanin synthesis, we examined bZIP mRNA expression and anthocyanin content in sweet cherry at three developmental stages: the big green (BG), yellow white (YW), and full red (FR) stages (Figure 2A). It’s important to note that the anthocyanin content in sweet cherries rises significantly as the fruit matures, with the content at the FR stage being 26.2 times higher than that at the BG stage (Figure 2B). Additionally, an expression analysis of the 54 identified bZIP genes across the three stages showed significant upregulation of only bZIP6, with its expression at the FR stage being 4.5 times higher than that at the BG stage (Figure 2C). Collectively, these findings suggest that bZIP6 plays a crucial role in regulating anthocyanin synthesis in sweet cherry.

2.4. Functional Characterization of PavbZIP6

To further investigate the function of PavbZIP6, we transiently expressed the fusion protein of PavbZIP6 and green fluorescent protein (GFP) in tobacco epidermal cells and observed the GFP’s fluorescence signals 48 h later. Notably, the fluorescence signal of the PavbZIP6 fusion protein in sweet cherry was localised to the nucleus, indicating its role as a nuclear-localised transcription factor (Figure 3A). Therefore, we validated PavbZIP6 transcriptional self-activation in a yeast system. Fusion with the LexA DNA-binding domain activated the transcription of the LacZ reporter gene in the presence of PavbZIP6, confirming that PavbZIP6 is a transcriptional activator (Figure 3B).

2.5. PavbZIP6 Promotes Anthocyanin Accumulation in Sweet Cherry Fruits

To further investigate the function of PavbZIP6, an Agrobacterium-mediated transient injection system was employed for the transient overexpression and silencing of PavbZIP6 in sweet cherry ‘Rainier’ fruits (Figure 4A). Real-time PCR revealed a significant increase in PavbZIP6 expression in fruits overexpressing the gene compared with that in the control group, whereas PavbZIP6 silencing showed the opposite effect (Figure 4B). Phenotypic observations indicated that PavbZIP6 overexpression caused a darker fruit colouration relative to that of the controls, whereas silencing resulted in a lighter colouration. Consistent results were obtained from the anthocyanin content measurements (Figure 4C).
Considering that the transient overexpression of PavbZIP6 enhanced anthocyanin accumulation in fruits, an RT-qPCR analysis was conducted to assess changes in the expression patterns of anthocyanin biosynthetic genes in transgenic sweet cherry fruits. PavbZIP6 overexpression significantly upregulated anthocyanin biosynthesis-related genes, including PavCHS, PavF3H, PavF3′H, PavDFR, PavANS, and PavUFGT (Figure 4D), whereas PavbZIP6 knockdown significantly downregulated the genes in sweet cherry, consistent with the observed phenotypes (Figure 4E). Collectively, these findings indicate that PavbZIP6 overexpression promotes anthocyanin accumulation in sweet cherry fruits by modulating the expression of anthocyanin biosynthetic genes.

2.6. PavbZIP6 Regulates the Expression of Structural Genes in Anthocyanin Biosynthesis

To further investigate how PavbZIP6 regulates the accumulation of anthocyanins in sweet cherry, we examined significantly altered genes in PavbZIP6-overexpressing fruit, including PavCHS, PavF3H, PavF3′H, PavDFR, PavANS, and PavUFGT. Specifically, we cloned their full-length promoters into the pLacZi2μ vector as reporters, while the PavbZIP6 sequence was cloned into the pB42AD vector as effectors. A yeast one-hybrid analysis revealed that co-transforming empty effectors with PavDFR, PavANS, and PavUFGT reporters did not result in a blue colour in the selection plates. In contrast, the co-transformation of effectors containing PavbZIP6 with PavDFR, PavANS, and PavUFGT reporters consistently yielded blue colonies (Figure 5A). Overall, these results demonstrate that PavbZIP6 can directly bind to the promoters of the anthocyanin biosynthesis genes PavDFR, PavANS, and PavUFGT.
To further analyse the regulatory effects of PavbZIP6 on the promoters of PavDFR, PavANS, and PavUFGT, we examined LUC activity in tobacco using a dual-luciferase reporter assay. Live fluorescent imaging showed weak or negligible fluorescence signals in tobacco cells co-injected with empty effectors and PavDFR, PavANS, and PavUFGT promoters. In contrast, strong fluorescence signals were observed in cells co-injected with effectors containing PavbZIP6 with these promoters. Notably, results consistent with the fluorescence imaging were obtained in the LUC enzyme activity assays (Figure 5B,C). Collectively, these findings indicate that PavbZIP6 enhances the expression of the anthocyanin biosynthesis genes PavDFR, PavANS, and PavUFGT.

2.7. PavbZIP6 Regulates Anthocyanin Synthesis by Controlling ABA Levels through the Modulation of PavBBX9 Expression

ABA plays a significant role in anthocyanin biosynthesis in sweet cherry fruits. For example, PavBBX6 and PavBBX9 promote ABA accumulation in sweet cherry fruits, thereby enhancing anthocyanin accumulation [32]. To investigate whether PavbZIP6 regulates anthocyanin biosynthesis via ABA, we examined the mRNA expression of PavBBX6, PavBBX9, and HY5. The RT-qPCR revealed that PavbZIP6 overexpression significantly upregulated the mRNA expression of PavbZIP6, PavBBX6, PavBBX9, and HY5. In contrast, PavbZIP6 silencing significantly downregulated PavBBX6, PavBBX9, and HY5 mRNA levels (Figure 6A). Overall, these findings suggest that PavbZIP6 promotes anthocyanin accumulation in sweet cherry fruits through the expression of these genes.
Furthermore, we explored the regulatory effects of PavbZIP6 on PavBBX6, PavBBX9, and HY5. Yeast one-hybrid assays showed that the co-transformation of the effector construct containing PavbZIP6 with the reporter constructs PavBBX9 and HY5 did not result in blue colouration on the selection plates. However, the co-transformation of the effector construct containing PavbZIP6 with the reporter construct PavBBX6 alone resulted in blue colouration (Figure 6B). Moreover, the dual-luciferase reporter assay and LUC enzyme activity analysis confirmed that PavbZIP6 positively regulated PavBBX6 (Figure 6C,D), indicating that PavbZIP6 may modulate anthocyanin biosynthesis by regulating PavBBX6 expression. Additionally, we measured the ABA content in fruits following PavbZIP6 overexpression or knockdown. PavbZIP6 overexpression significantly increased ABA content, whereas PavbZIP6 knockdown markedly decreased ABA content in fruits (Figure 6E). Moreover, the RT-PCR showed that PavbZIP6 overexpression significantly upregulated ABA biosynthesis-related genes (PavNCED1 and PavNCED3) in sweet cherry fruits compared with those in the control group. In contrast, PavbZIP6 knockdown significantly downregulated PavNCED1 expression. Collectively, these findings suggest that PavbZIP6 regulates ABA levels and influences anthocyanin biosynthesis by modulating the expression of PavBBX6 and PavNCED1 genes.

3. Discussion

In this study, we identified 54 bZIP genes in the sweet cherry genome database (Table 1). Notably, the number of bZIP genes varies across plant species, with Arabidopsis having 78 [33], humans having 53 members [34], rice having 92 [35], and maize having 125 [36]. Differences in the number of bZIP genes may be attributed to variations in genome size and complexity between these species. Based on multiple clustering analyses of Arabidopsis bZIP proteins, PavbZIP proteins were classified into 13 subgroups (Figure 1). Group S represented the largest cluster of PavbZIP proteins in sweet cherry, similar to findings for Arabidopsis [33]. In Arabidopsis, the S subgroup is the largest bZIP cluster (17 members), typically comprising intronless genes encoding small TF proteins of approximately 20 kDa. All the members of the S subgroup preferentially form heterodimers, collectively known as the C/S1-bZIP network [33]. Whether bZIP proteins in the S subgroup of sweet cherries also function via heterodimerisation remains to be explored.
Currently, bZIP TFs play crucial roles in plant growth, development, and responses to abiotic stresses, such as seed maturation, flower development, and stress responses [37,38,39]. Importantly, the bZIP gene family has been extensively studied in plants, including AtbZIP11/18 in Arabidopsis [40,41], GsbZIP67 in soybean [42], CabZIP25 in pepper [39], TabZIP15 in wheat [43], and MdbZIP44 in apples [24]. Despite the completion of whole-genome sequencing of sweet cherry, little is known about the bZIP gene family, particularly in relation to anthocyanin synthesis. Comprehensive studies have been undertaken on the functions of the bZIP genes HY5 and HYH in Arabidopsis, tomatoes, and apples. For example, HY5 and HYH act as downstream photoreceptors in the light signalling pathway in Arabidopsis by directly activating the expression of early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) and positively regulating the transcriptional activation of AtPAP1 [38,44,45]. In apples, the bZIP TF gene MdHY5 directly promoted the expression of MdMYB10 and MdMYB1 and positively regulated anthocyanin accumulation by enhancing interactions with downstream target genes [24,42,46]. SlHY5 knockdown in tomatoes downregulated anthocyanin accumulation [47]. In tomatoes, the bZIP transcription factor SlAREB1 regulates anthocyanin biosynthesis in seedlings under low temperatures through an ABA-dependent pathway [48]. The MpbZIP9 transcription factor regulates the synthesis of anthocyanins in fruit skin and acts as a positive regulator by promoting anthocyanin biosynthesis through the activation of MpF3′H expression [49]. The ABA-induced NAC transcription factor MdNAC1 interacts with bZIP-type transcription factors to promote anthocyanin synthesis in red-fleshed apples [50]. In the present study, we examined the expression of the 54 identified bZIP TFs at different developmental stages of sweet cherries. Although anthocyanin accumulation increased in the skin of the ‘Tieton’ cultivar along with PavbZIP6 upregulation, HY5 expression was not enhanced (Figure 2). A further analysis of PavbZIP6 revealed its nuclear localisation and transcriptional self-activation (Figure 3), suggesting that PavbZIP6 may function as a transcriptional regulator of other genes in the nucleus. The transient overexpression of PavbZIP6 in ‘Rainier’ cherry caused a darker fruit colour compared to that of the controls, whereas PavbZIP6 silencing resulted in fruits with a lighter colour (Figure 4). Collectively, these results indicate that PavbZIP6 overexpression enhances anthocyanin accumulation in fruits. Yeast one-hybrid and dual-luciferase assays further demonstrated that PavbZIP6 enhanced the expression of the anthocyanin biosynthesis genes PavDFR, PavANS, and PavUFGT, thereby increasing anthocyanin accumulation.
Recent studies have demonstrated that bZIP TFs play crucial and diverse roles in the ABA-mediated regulation of plant growth, development, and abiotic stress responses [48]. ABA-induced anthocyanin accumulation has also been reported in many species, such as apple [20], Arabidopsis [51], wolfberry [52], and maize [53]. In Arabidopsis, bZIP16 directly regulates ABA- and light-responsive genes to promote seed germination and hypocotyl elongation [54]. In apples, MdbZIP44 enhances anthocyanin biosynthesis in an ABA-dependent manner [24]. MdNAC1 forms a complex with MdbZIP23 in the ABA signalling pathway, promoting the expression of MdMYB10 and MdUFGT and the accumulation of anthocyanins [50]. As a key regulatory factor in signal transduction, ABA is closely associated with anthocyanin biosynthesis in various species [28,55,56,57,58]. However, the molecular mechanisms by which the bZIP TFs influence ABA-induced anthocyanin synthesis in sweet cherry remain unclear. In the present study, PavbZIP6 overexpression in cherry fruit significantly upregulated PavBBX6, PavBBX9, and HY5, whereas PavbZIP6 knockdown significantly downregulated these genes (Figure 6A). Yeast one-hybrid and dual-luciferase reporter assays confirmed that PavbZIP6 positively regulates PavBBX6 (Figure 6B–D), indicating that PavbZIP6 regulates anthocyanin biosynthesis by modulating PavBBX6 expression. A previous study showed that PavBBX6 promoted ABA accumulation in sweet cherry fruits by regulating the ABA biosynthesis gene PavNCED1, thereby enhancing anthocyanin accumulation [29]. Notably, our measurements of ABA content and PavNCED1, PavNCED2, and PavNCED3 expression in fruits following PavbZIP6 overexpression or silencing confirmed these findings (Figure 6E,F).
This study identified all bZIP genes in sweet cherry fruits and identified PavbZIP6 as a TF closely associated with anthocyanin synthesis. Additionally, we demonstrated that the protein encoded by PavbZip6 binds directly to the promoters of the anthocyanin biosynthesis genes PavDFR, PavANS, and PavUFGT, thereby promoting their expression and regulating anthocyanin biosynthesis (Figure 5). Moreover, PavbZIP6 can bind directly to the promoter of PavBBX6 to promote ABA synthesis (Figure 6). Conclusively, this study confirmed that PavbZIP6 is a positive regulatory factor that mediates anthocyanin synthesis in sweet cherry, laying the groundwork for further explorations of the molecular mechanisms underlying anthocyanin biosynthesis in sweet cherry.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The ‘Tieton’ sweet cherry (Prunus avium L.) was cultivated at the Beijing Forestry Fruit Tree Research Institute. Fruit samples were collected at three developmental stages: BG (approximately 15 days after flowering), YW (approximately 22 days after flowering), and FR (approximately 28 days after flowering). The collected fruits were visually similar in appearance, with uniform size and without bruising. Thirty fruits were selected for anthocyanin analysis, while the remaining fruits were frozen in liquid nitrogen and subsequently stored at −80 °C for future analysis. Each developmental stage was replicated three times, with each replicate consisting of 10 cherry fruits.

4.2. Mining of PavbZIP Genes in the Prunus avium Genome and Phylogenetic Tree Analysis

To identify the bZIP transcription factor family members in cherry (Prunus avium), we used the BLAST method to compare the amino acid sequences of all bZIP genes from Arabidopsis thaliana with the cherry genome (http://cherry.kazusa.or.jp/, accessed on 16 October 2023). Candidate genes were identified and screened using the CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 3 October 2023) and the Pfam database (http://pfam.xfam.org/search accessed on 12 November 2023). Molecular weight and isoelectric point (pI) calculations were performed using ExPASy (https://web.expasy.org/protparam/, accessed on 25 November 2023).
Additionally, we constructed a phylogenetic tree using the neighbour-joining method in MEGA 6.0 software to compare PavbZIP proteins with bZIP proteins from Arabidopsis, with bootstrap values set to 1000 replications.

4.3. Determination of Anthocyanin Content

After grinding sweet cherry fruit in liquid nitrogen, 1.0 g of the sample was soaked in 5 mL of methanol:hydrochloric acid (999:1, v/v) solution. Anthocyanins were extracted from the sample overnight at −20 °C in darkness, followed by centrifugation to obtain the supernatant. Finally, anthocyanin content was determined using the pH differential method. The absorbance of each sample was measured at 510 and 710 nm using a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan) in buffers at pH of 1.0 and pH of 4.5. The anthocyanin content was calculated as follows: A = {[(A510–A710) pH1.0] − [(A510–A710) pH4.5]}.

4.4. RT-qPCR

Total RNA was extracted using a plant total RNA extraction kit (Huayueyang Biotechnology, Beijing, China). cDNA synthesis was performed with a reverse transcription kit (TaKaRa Biotechnology, Beijing, China). All primer sequences used in the experiments are listed in Table S1. qRT-PCR was conducted using SYBR Premix Ex Taq (Kangwei Century Biotechnology, Beijing, China). Each experiment was performed in triplicate. Relative gene expression was calculated using the 2−ΔΔCt method, with PavActin as the reference gene.

4.5. Subcellular Localization

The full-length open reading frame of PavbZIP6 was amplified from sweet cherry fruit and cloned into the plant expression vector pCAMBIA1302, which contains a green fluorescent protein (GFP) tag under the control of the 35S promoter of cauliflower mosaic virus CaMV35S promoter. Green fluorescence was observed using a laser scanning confocal microscope (FV31-HSD, OLYMPUS, Tokyo, Japan).

4.6. Self-Activation Activity Analysis

Briefly, EGY48 yeast competent cells were mixed with 3 μg of carrier DNA, 300 ng of LexA vector plasmid, and 300 ng of H18 LacZ-2μ reporter plasmid. After adding 300 mL of PEG lithium acetate solution (PEG/LiAc), the mixture was vortexed and incubated in a 45 °C water bath for 30 min, with intermittent vortexing every 10 min. Thereafter, the cells were plated on SD-Trp-Ura medium and cultured at 30 °C for 3 days. Colonies were selected based on size and streaked on SD-Trp-Ura medium containing X-Gal at 30 °C for 1–2 days to observe colour development.

4.7. Field Transient Infiltration of Sweet Cherry Fruit

Briefly, the promoter of PavbZIP6 was amplified and cloned into the pCAMBIA1301 and pFGC5941 vectors to generate the overexpression vector, PavbZIP6-pCAMBIA1301, and the RNAi interference vector, PavbZIP6-pFGC5941. Recombinant vectors were transformed into Agrobacterium tumefaciens, which were then used to infiltrate sweet cherry ‘Rainier’ fruits at the large green fruit stage (approximately 20 days after flowering). Specifically, the respective bacterial solutions and corresponding empty vector solutions were slowly injected into the fruit near the pedicel to ensure thorough infiltration of the fruit using a microinjector. After injection, the fruits were bagged and labelled, and the bags were removed every other day. Samples were collected at the ripening stage of sweet cherry ‘Rainier’ fruits for analysis.

4.8. Yeast One-Hybrid Assay

Briefly, the coding region of PavbZIP6 was cloned into the pB42AD vector. Additionally, the promoter regions of the genes involved in the experiment were cloned into the pLacZ-2μ vector. Thereafter, yeast cells were transformed with these constructs, and larger colonies were selected for identification. Finally, colonies on SD-Trp-Ura medium containing X-Gal were incubated at 30 °C for 1–2 days to observe colour development.

4.9. Transient Expression Assay in Tobacco Leaves

The promoter regions of the relevant genes were amplified and cloned into the pGreen II 0800-LUC vector. The full-length open reading frame (ORF) of PavbZIP6 was inserted into the effector vector pGreen II 0029 62-SK to create a recombinant vector. This recombinant vector was then transformed into Agrobacterium tumefaciens strain GV3101 (Psoup) and used to infiltrate tobacco leaves. After 2 days of infiltration, luciferin was uniformly sprayed onto the leaves for luminescence detection (Tanon-5200Multi, Tanon, Shanghai, China). Tobacco leaf samples were collected, and their fluorescence activity was assessed using a fluorescence detection kit (Promega, Beijing, China).

4.10. Measurement of ABA Content

Briefly, the ABA contents of the sweet cherry varieties ‘Tieton’ and ‘Rainier’ were measured using enzyme-linked immunosorbent assay (ELISA). All samples were ground in liquid nitrogen, and 0.5 g of each sample was extracted in 1× PBS buffer. The extraction followed the procedures outlined in the instructions of Abscisic Acid Assay Kit (Shanghai Jingkang, Shanghai, China), and all samples were analysed in triplicate.

4.11. Statistical Analysis

All statistical analyses were performed using SPSS 20.0 software. Statistical significance was determined using one-way ANOVA followed by Duncan’s multiple range test or Student’s t-test.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms251810207/s1.

Author Contributions

Conceptualization, S.G. and T.L.; methodology, Y.X.; software, X.Z.; validation, M.T., Q.Y. and X.W.; formal analysis, B.D.; investigation, Y.Y.; All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the National Key Research and Development Program (2022YFD1200501), the National Natural Science Foundation of China (Grant No. 32272686), and the Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects (Grant no. CEFF–PXM2019_014207_000032).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Acknowledgments

We are grateful to Kaichun Zhang (Beijing Institute of Forestry and Pomology, Beijing Academy of Agricultural and Forestry Sciences, Beijing, China) for their material support.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Crisosto, C.H.; Crisosto, G.M.; Metheney, P. Consumer acceptance of ‘Brooks’ and ‘Bing’ cherries is mainly dependent on fruit SSC and visual skin color. Postharvest Biol. Technol. 2003, 28, 159–167. [Google Scholar] [CrossRef]
  2. Liu, Y.; Liu, X.Y.; Zhong, F.; Tian, R.R.; Zhang, K.C.; Zhang, X.M.; Li, T.H. Comparative Study of Phenolic Compounds and Antioxidant Activity in Different Species of Cherries. J. Food Sci. 2011, 76, C633–C638. [Google Scholar] [CrossRef] [PubMed]
  3. He, J.; Giusti, M. Anthocyanins: Natural colorants with health-promoting properties. Annu. Rev. Food Sci. Technol. 2010, 1, 163. [Google Scholar] [CrossRef] [PubMed]
  4. Khoo, H.E.; Azlan, A.; Tang, S.T.; Lim, S.M. Anthocyanidins and anthocyanins: Colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food Nutr. Res. 2017, 61, 1361779. [Google Scholar] [CrossRef] [PubMed]
  5. Pojer, E.; Mattivi, F.; Johnson, D.; Stockley, C.S. The Case for Anthocyanin Consumption to Promote Human Health: A Review. Compr. Rev. Food. Sci. Food Saf. 2013, 12, 483–508. [Google Scholar] [CrossRef] [PubMed]
  6. Dooner, H.K.; Robbins, T.P.; Jorgensen, R.A. Genetic and developmental control of anthocyanin biosynthesis. Annu. Rev. Genet. 1991, 25, 173–199. [Google Scholar] [CrossRef]
  7. Ballistreri, G.; Continella, A.; Gentile, A.; Amenta, M.; Fabroni, S.; Rapisarda, P. Fruit quality and bioactive compounds relevant to human health of sweet cherry (Prunus avium L.) cultivars grown in Italy. Food Chem. 2013, 140, 630–638. [Google Scholar] [CrossRef]
  8. Gao, L.; Mazza, G. Characterization, quantitation, and distribution of anthocyanins and colorless phenolics in sweet cherries. J. Agric. Food Chem. 1995, 43, 343–346. [Google Scholar] [CrossRef]
  9. Tanaka, Y.; Sasaki, N.; Ohmiya, A. Biosynthesis of plant pigments: Anthocyanins, betalains and carotenoids. Plant J. 2008, 54, 733–749. [Google Scholar] [CrossRef]
  10. Dubos, C.; Gourrierec, L.J.; Baudry, A.; Huep, G.; Lanet, E.; Debeaujon, I.; Routaboul, J.M.; Alboresi, A.; Weisshaar, B.; Lepiniec, L. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J. 2008, 55, 940–953. [Google Scholar] [CrossRef]
  11. Talanian, R.V.; Mcknight, C.J.; Kim, P.S. Sequence-specific DNA-binding by a short peptide dimer. Science 1990, 249, 769–771. [Google Scholar] [CrossRef] [PubMed]
  12. Landschulz, W.H.; Johnson, P.F.; McKnight, S.L. The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science 1998, 240, 1759–1764. [Google Scholar] [CrossRef] [PubMed]
  13. Ellenberger, T.E.; Brandl, C.J.; Struhl, K.; Harrison, S.C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted a helices: Crystal structure of the protein-DNA complex. Cell 1992, 71, 1223–1237. [Google Scholar] [CrossRef] [PubMed]
  14. Furihata, T.; Maruyama, K.; Fujita, Y.; Umezawa, T.; Yoshida, R.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc. Natl. Acad. Sci. USA 2006, 103, 1988–1993. [Google Scholar] [CrossRef] [PubMed]
  15. Liao, Y.; Zou, H.F.; Wei, W.; Hao, Y.J.; Tian, A.G.; Huang, J.; Liu, Y.F.; Zhang, J.S.; Chen, S.Y. Soybean GmbZIP44, GmbZIP62 and GmbZIP78 genes function as negative regulator of ABA signaling and confer salt and freezing tolerance in transgenic Arabidopsis. Planta 2008, 228, 225–240. [Google Scholar] [CrossRef]
  16. Shin, J.; Park, E.; Choi, G. PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis. Plant J. Cell Mol. Biol. 2007, 49, 981–994. [Google Scholar] [CrossRef]
  17. Stracke, R.; Favory, J.J.; Gruber, H.; Bartelniewoehner, L.; Bartels, S.; Binkert, M.; Funk, M.; Weisshaar, B.; Ulm, R. The Arabidopsis bZIP transcription factor HY5 regulates expression of the PFG1/MYB12 gene in response to light and ultraviolet-B radiation. Plant Cell Environ. 2010, 33, 88–103. [Google Scholar]
  18. Shin, D.H.; Choi, M.; Kim, K.; Bang, G.; Cho, M.; Choi, S.B.; Choi, G.; Park, Y.I. HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis. FEBS Lett. 2013, 587, 1543–1547. [Google Scholar] [CrossRef]
  19. Nguyen, N.H.; Jeong, C.Y.; Kang, G.H.; Yoo, S.D.; Hong, S.W.; Lee, H. MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis. Plant J. 2015, 84, 1192–1205. [Google Scholar] [CrossRef]
  20. An, J.P.; Zhang, X.W.; Liu, Y.J.; Wang, X.F.; You, C.X.; Hao, Y.J. ABI5 regulates ABA-induced anthocyanin biosynthesis by modulating the MYB1-bHLH3 complex in apple. J. Exp. Bot. 2021, 72, 1460–1472. [Google Scholar] [CrossRef]
  21. Wang, S.; Zhang, X.H.; Li, B.B.; Zhao, X.Q.; Shen, Y.; Yuan, Z.H. Genome-wide identification and characterization of bZIP gene family and cloning of candidate genes for anthocyanin biosynthesis in pomegranate. BMC Plant Biol. 2022, 22, 170. [Google Scholar] [CrossRef] [PubMed]
  22. Tu, M.X.; Fang, J.H.; Zhao, R.K.; Liu, X.Y.; Yin, W.C.; Wang, Y.; Wang, X.H.; Wang, X.P.; Fang, Y.L. CRISPR/Cas9-mediated mutagenesis of VvbZIP36 promotes anthocyanin accumulation in grapevine. Hortic. Res. 2022, 9, uhac022. [Google Scholar] [CrossRef] [PubMed]
  23. Matousek, J.; Kocábek, T.; Patzak, J.; Stehlík, J.; Füssy, Z.; Krofta, K.; Heyerick, A.; Roldán-Ruiz, I.; Maloukh, L.; De Keukeleire, D. Cloning and molecular analysis of HlbZip1 and HlbZip2 transcription factors putatively involved in the regulation of the lupulin metabolome in hop (Humulus lupulus L.). J. Agric. Food Chem. 2010, 58, 902–912. [Google Scholar] [CrossRef] [PubMed]
  24. An, J.P.; Yao, J.F.; Xu, R.R.; You, C.X.; Wang, X.F.; Hao, Y.J. Apple bZIP transcription factor MdbZIP44 regulates abscisic acid-promoted anthocyanin accumulation. Plant Cell Environ. 2018, 41, 2678–2692. [Google Scholar] [CrossRef] [PubMed]
  25. Zhou, P.; Li, J.W.; Jiang, H.Y.; Jin, Q.J.; Wang, Y.J.; Xu, Y.C. Analysis of bZIP gene family in lotus (Nelumbo) and functional study of NnbZIP36 in regulating anthocyanin synthesis. BMC Plant Biol. 2023, 23, 429. [Google Scholar] [CrossRef]
  26. Nambara, E.; Okamoto, M.; Tatematsu, K.; Yano, R.; Seo, M.; Kamiya, Y. Abscisic acid and the control of seed dormany and germination. Seed Sci. Res. 2010, 20, 55–67. [Google Scholar] [CrossRef]
  27. Sano, N.; Marion-Poll, A. ABA Metabolism and Homeostasis in Seed Dormancy and Germination. Int. J. Mol. Sci. 2021, 22, 5069. [Google Scholar] [CrossRef]
  28. Pan, W.; Liang, J.; Sui, J.; Li, J.; Liu, C.; Xin, Y.; Zhang, Y.; Wang, S.; Zhao, Y.; Zhang, J.; et al. ABA and bud dormancy in perennials: Current knowledge and future perspective. Genes 2021, 12, 1635. [Google Scholar] [CrossRef]
  29. Leão, P.C.D.; Lima, M.A.C.; Costa, J.P.D.; da Trindade, D.C.G. Abscisic acid and ethephon for improving red color and quality of crimson seedless grapes grown in a tropical region. Am. J. Enol. Vitic. 2015, 66, 37–45. [Google Scholar] [CrossRef]
  30. Kadomura-Ishikawa, Y.; Miyawaki, K.; Takahashi, A.; Masuda, T.; Noji, S. Light and abscisic acid independently regulated FaMYB10 in Fragaria × ananassa fruit. Planta 2015, 241, 953–965. [Google Scholar] [CrossRef]
  31. Shen, X.J.; Zhao, K.; Liu, L.L.; Zhang, K.C.; Yuan, H.Z.; Liao, X.; Wang, Q.; Guo, X.W.; Li, F.; Li, T.H. A role for PacMYBA in ABA-regulated anthocyanin biosynthesis in red-colored sweet cherry cv. Hong Deng (Prunus avium L.). Plant Cell Physiol. 2014, 55, 862–880. [Google Scholar] [CrossRef] [PubMed]
  32. Wang, Y.Y.; Xiao, Y.Q.; Sun, Y.T.; Zhang, X.; Du, B.Y.; Turupu, M.; Yao, Q.S.; Gai, S.L.; Tong, S.; Huang, J.; et al. Two B-box proteins, PavBBX6/9, positively regulate light-induced anthocyanin accumulation in sweet cherry. Plant Physiol. 2023, 192, 2030–2048. [Google Scholar] [CrossRef] [PubMed]
  33. Dröge-Laser, W.; Snoek, B.L.; Snel, B.; Weiste, C. The Arabidopsis bZIP transcription factor family—An update. Curr. Opin. Plant Biol. 2018, 45, 36–49. [Google Scholar] [CrossRef] [PubMed]
  34. Rodríguez-Martínez, J.A.; Reinke, A.W.; Bhimsaria, D.; Keating, A.E.; Ansari, A.Z. Combinatorial bZIP dimers display complex DNA-binding specificity landscapes. eLife 2017, 6, e19272. [Google Scholar] [CrossRef] [PubMed]
  35. Corrêa, L.G.G.; Riaño-Pachón, D.M.; Schrago, C.G.; dos Santos, R.V.; Mueller-Roeber, B.; Vincentz, M. The role of bZIP transcription factors in green plant evolution: Adaptive features emerging from four founder Genes. PLoS ONE 2008, 3, e2944. [Google Scholar] [CrossRef] [PubMed]
  36. Zhou, Y.; Xu, D.X.; Jia, L.D.; Huang, X.H.; Ma, G.Q.; Wang, S.X.; Zhu, M.C.; Zhang, A.X.; Guan, M.W.; Lu, K.; et al. Genome-wide identification and structural analysis of bZIP transcription factor genes in brassica napus. Genes 2017, 8, 288. [Google Scholar] [CrossRef]
  37. Toh, S.; McCourt, P.; Tsuchiya, Y. HY5 is involved in strigolactone-dependent seed germination in Arabidopsis. Plant Signal Behav. 2012, 7, 556–558. [Google Scholar] [CrossRef]
  38. Strathmann, A.; Kuhlmann, M.; Heinekamp, T.; Dröge-Laser, W. BZI-1 specifically heterodimerises with the tobacco bZIP transcription factors BZI-2, BZI-3/TBZF and BZI-4, and is functionally involved in flower development. Plant J. 2001, 28, 397–408. [Google Scholar] [CrossRef]
  39. Gai, W.X.; Ma, X.; Qiao, Y.M.; Shi, B.H.; ul Haq, S.; Li, Q.H.; Wei, A.M.; Liu, K.K.; Gong, Z.H. Characterization of the bZIP transcription factor family in pepper (Capsicum annuum L.): CabZIP25 positively modulates the salt tolerance. Front. Plant Sci. 2020, 11, 139. [Google Scholar] [CrossRef]
  40. Gibalová, A.; Steinbachová, L.; Hafidh, S.; Bláhová, V.; Gadiou, Z.; Michailidis, C.; Muller, K.; Pleskot, R.; Dupl’áková, N.; Honys, D. Characterization of pollen-expressed bZIP protein interactions and the role of ATbZIP18 in the male gametophyte. Plant Reprod. 2017, 30, 1–17. [Google Scholar] [CrossRef]
  41. Weiste, C.; Pedrotti, L.; Selvanayagam, J.; Muralidhara, P.; Fröschel, C.; Novák, O.; Ljung, K.; Hanson, J.; Dröge-Laser, W. The Arabidopsis bZIP11 transcription factor links low-energy signalling to auxin-mediated control of primary root growth. PLoS Genet. 2017, 13, e1006607. [Google Scholar] [CrossRef] [PubMed]
  42. Wu, S.Y.; Zhu, P.H.; Jia, B.W.; Yang, J.K.; Shen, Y.; Cai, X.X.; Sun, X.L.; Zhu, Y.M.; Sun, M.Z. A Glycine soja group S2 bZIP transcription factor GsbZIP67 conferred bicarbonate alkaline tolerance in Medicago sativa. BMC Plant Biol. 2019, 19, 25. [Google Scholar] [CrossRef] [PubMed]
  43. Bi, C.; Yu, Y.; Dong, C.; Yang, Y.; Zhai, Y.; Du, F.; Xia, C.; Ni, Z.; Kong, X.; Zhang, L. The bZIP transcription factor TabZIP15 improves salt stress tolerance in wheat. Plant Biotechnol. J. 2021, 19, 209–211. [Google Scholar] [CrossRef] [PubMed]
  44. Lee, J.; He, K.; Stolc, V.; Lee, H.; Figueroa, P.; Gao, Y.; Tongprasit, W.; Zhao, H.Y.; Lee, I.; Deng, X. Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development. Plant Cell 2007, 19, 731–749. [Google Scholar] [CrossRef]
  45. Zhang, H.Y.; He, H.; Wang, X.C.; Wang, X.F.; Yang, X.Z.; Li, L.; Deng, X.W. Genome-wide mapping of the HY5-mediated genenetworks in Arabidopsis that involve both transcriptional and post-transcriptional regulation. Plant J. 2011, 65, 346–358. [Google Scholar] [CrossRef]
  46. Yoshida, T.; Fujita, Y.; Sayama, H.; Kidokoro, S.; Maruyama, K.; Mizoi, J.; Shinozaki, K.; Yamaguchi-Shinozaki, K. AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. Plant J. 2010, 61, 672–685. [Google Scholar] [CrossRef]
  47. Liu, C.C.; Chi, C.; Jin, L.J.; Zhu, J.; Yu, J.Q.; Zhou, Y.H. The bZip transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato. Plant Cell Environ. 2018, 41, 1762–1775. [Google Scholar] [CrossRef]
  48. Xu, Z.; Wang, J.; Ma, Y.; Wang, F.; Wang, J.; Zhang, Y.; Hu, X. The bZIP transcription factor SlAREB1 regulates anthocyanin biosynthesis in response to low temperature in tomato. Plant J. 2023, 115, 205–219. [Google Scholar] [CrossRef]
  49. Chen, M.; Cao, X.; Huang, Y.; Zou, W.; Liang, X.; Yang, Y.; Wang, Y.; Wei, J.; Li, H. The bZIP transcription factor MpbZIP9 regulates anthocyanin biosynthesis in Malus ‘Pinkspire’ fruit. Plant Sci. 2024, 342, 112038. [Google Scholar] [CrossRef]
  50. Liu, W.; Mei, Z.; Yu, L.; Gu, T.; Li, Z.; Zou, Q.; Zhang, S.; Fang, H.; Wang, Y.; Zhang, Z.; et al. The ABA-induced NAC transcription factor MdNAC1 interacts with a bZIP-type transcription factor to promote anthocyanin synthesis in red-fleshed apples. Hortic. Res. 2023, 10, uhad049. [Google Scholar] [CrossRef]
  51. Shi, W.; Wang, X.; Liu, H.; Cai, Z.; Lu, C.; Chen, Y. A novel ABA-insensitive mutant in Arabidopsis reveals molecular network of ABA-induced anthocyanin accumulation and abiotic stress tolerance. J Plant Physiol. 2022, 278, 153810. [Google Scholar] [CrossRef] [PubMed]
  52. Li, G.; Zhao, J.; Qin, B.; Yin, Y.; An, W.; Mu, Z.; Cao, Y. ABA mediates development-dependent anthocyanin biosynthesis and fruit coloration in Lycium plants. BMC Plant Biol. 2019, 19, 317. [Google Scholar] [CrossRef] [PubMed]
  53. Kim, J.S.; Lee, B.H.; Kim, S.H.; Oh, K.H.; Cho, K.Y. Responses to environmental and chemical signals for anthocyanin biosynthesis in non-chlorophyllous corn (Zea mays L.) leaf. J. Plant Biol. 2006, 49, 16–25. [Google Scholar] [CrossRef]
  54. Hsieh, W.P.; Hsieh, H.L.; Wu, S.H. Arabidopsis bZIP16 transcription factor integrates light and hormone signaling pathways to regulate early seedling development. Plant Cell 2012, 24, 3997–4011. [Google Scholar] [CrossRef] [PubMed]
  55. Loreti, E.; Povero, G.; Novi, G.; Solfanelli, C.; Alpi, A.; Perata, P. Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis. New Phytol. 2012, 193, 289. [Google Scholar] [CrossRef]
  56. Guo, S.H.; Yang, B.H.; Wang, X.W.; Li, J.N.; Li, S.; Yang, X.; Ren, R.H.; Fang, Y.L.; Xu, T.F.; Zhang, Z.W.; et al. ABA signaling plays a key role in regulated deficit irrigation-driven anthocyanins accumulation in ‘Cabernet Sauvignon’ grape berries. Environ. Exp. Bot. 2021, 181, 104290. [Google Scholar] [CrossRef]
  57. Singh, S.P.; Saini, M.K.; Singh, J.; Pongener, A.; Sidhu, G.S. Preharvest application of abscisic acid promotes anthocyanins accumulation in pericarp of litchi fruit without adversely affecting postharvest quality. Postharvest Biol. Technol. 2014, 96, 14–22. [Google Scholar] [CrossRef]
  58. Han, T.Y.; Wu, W.L.; Li, W.L. Transcriptome analysis revealed the mechanism by which exogenous ABA increases anthocyanins in blueberry fruit during veraison. Front. Plant Sci. 2021, 12, 758215. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic analysis of PavbZIP6 protein in sweet cherry (Prunus avium). The protein indicated by the marked red dot is PavbZIP6.
Figure 1. Phylogenetic analysis of PavbZIP6 protein in sweet cherry (Prunus avium). The protein indicated by the marked red dot is PavbZIP6.
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Figure 2. Analysis of anthocyanin content and bZIP gene family expression in sweet cherry fruits at different developmental stages. (A) Phenotypes of sweet cherry ‘Tieton’ fruit at different developmental stages. Scale bar: 1 cm. (B) Anthocyanin content of sweet cherry during three stages of fruit development: big green (BG); yellow white (YW); and full red (FR). Data are expressed as means ± standard deviation (SD) of three measurements from at least 10 sweet cherry fruits, and significant differences were assessed (* p < 0.05, ** p < 0.01). (C) Expression analysis of the PavbZIP gene family in sweet cherry ‘Tieton’ fruits at different developmental periods.
Figure 2. Analysis of anthocyanin content and bZIP gene family expression in sweet cherry fruits at different developmental stages. (A) Phenotypes of sweet cherry ‘Tieton’ fruit at different developmental stages. Scale bar: 1 cm. (B) Anthocyanin content of sweet cherry during three stages of fruit development: big green (BG); yellow white (YW); and full red (FR). Data are expressed as means ± standard deviation (SD) of three measurements from at least 10 sweet cherry fruits, and significant differences were assessed (* p < 0.05, ** p < 0.01). (C) Expression analysis of the PavbZIP gene family in sweet cherry ‘Tieton’ fruits at different developmental periods.
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Figure 3. Subcellular localisation and self-activation activity analysis of PavbZIP6. (A) Subcellular localisation analysis of PavbZIP6 protein; Scale bar = 50 μm. Green represents GFP and the fusion protein of PavbZIP6 with GFP. Yellow indicates the color resulting from the combination of nuclear-localized mCherry and GFP fluorescence. (B) Analysis of the self-activating activity of the PavbZIP6 protein.
Figure 3. Subcellular localisation and self-activation activity analysis of PavbZIP6. (A) Subcellular localisation analysis of PavbZIP6 protein; Scale bar = 50 μm. Green represents GFP and the fusion protein of PavbZIP6 with GFP. Yellow indicates the color resulting from the combination of nuclear-localized mCherry and GFP fluorescence. (B) Analysis of the self-activating activity of the PavbZIP6 protein.
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Figure 4. Generation of PavbZIP6 overexpression and knockdown fruits. (A) Phenotypic analysis of sweet cherry following transient overexpression and silencing of PavbZIP6; scale bar: 1 cm. (B) Expression analysis of PavbZIP6 in transgenic fruits. (C) Determination of anthocyanin content in sweet cherry fruit following PavbZIP6 overexpression and silencing. (D) Expression analysis of genes related to anthocyanin synthesis in fruits overexpressing PavbZIP6. (E) Expression analysis of genes related to anthocyanin synthesis in fruits following PavbZIP6 silencing. Data are expressed as means ± standard deviation (SD) of three measurements from at least 10 sweet cherry fruits, and significant differences were assessed (* p < 0.05, ** p < 0.01).
Figure 4. Generation of PavbZIP6 overexpression and knockdown fruits. (A) Phenotypic analysis of sweet cherry following transient overexpression and silencing of PavbZIP6; scale bar: 1 cm. (B) Expression analysis of PavbZIP6 in transgenic fruits. (C) Determination of anthocyanin content in sweet cherry fruit following PavbZIP6 overexpression and silencing. (D) Expression analysis of genes related to anthocyanin synthesis in fruits overexpressing PavbZIP6. (E) Expression analysis of genes related to anthocyanin synthesis in fruits following PavbZIP6 silencing. Data are expressed as means ± standard deviation (SD) of three measurements from at least 10 sweet cherry fruits, and significant differences were assessed (* p < 0.05, ** p < 0.01).
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Figure 5. PavbZIP6 regulates the expression of PavDFR, PavANS and PavUFGT. (A) Yeast one-hybrid assay to verify that PavbZIP6 binds the promoter of the anthocyanin synthesis gene. (B) Transient LUC imaging assays showing that PavbZIP6 activate the transcription of the report gene. Representative images of LUC activity in N. benthamiana leaves 48 h after infiltration. (C) Dual-luciferase assay showing promoter activity expressed as the ratio of luciferase (LUC) to 35S::Renilla (REN). Data are expressed as means ± standard deviation (SD; n = 3), and significant differences were assessed (** p < 0.01).
Figure 5. PavbZIP6 regulates the expression of PavDFR, PavANS and PavUFGT. (A) Yeast one-hybrid assay to verify that PavbZIP6 binds the promoter of the anthocyanin synthesis gene. (B) Transient LUC imaging assays showing that PavbZIP6 activate the transcription of the report gene. Representative images of LUC activity in N. benthamiana leaves 48 h after infiltration. (C) Dual-luciferase assay showing promoter activity expressed as the ratio of luciferase (LUC) to 35S::Renilla (REN). Data are expressed as means ± standard deviation (SD; n = 3), and significant differences were assessed (** p < 0.01).
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Figure 6. PavbZIP6 regulates the expression of PavBBX6 and the accumulation of abscisic acid (ABA). (A) mRNA expression of PavBBX6/9 and PavHY5 in transgenic fruits. (B) Yeast one-hybrid assay to verify the binding of PavbZIP6 to the promoter of PavBBX6/9 and PavHY5. (C) Transient LUC imaging assays showing that PavbZIP6 activate the transcription of the report gene. Representative images of LUC activity in N. benthamiana leaves 48 h after infiltration. (D) Dual-luciferase assay showing promoter activity expressed as the ratio of luciferase (LUC) to 35S::Renilla (REN). (E) ABA contents in transgenic sweet cherry fruit and control. (F) mRNA expression of PavNCED1, PavNCED2, and PavNCED3 in transgenic fruits. Data are expressed as means ± standard deviation (SD; n = 3), and significant differences were assessed (* p < 0.05, ** p < 0.01, n.s.: no signifcant difference).
Figure 6. PavbZIP6 regulates the expression of PavBBX6 and the accumulation of abscisic acid (ABA). (A) mRNA expression of PavBBX6/9 and PavHY5 in transgenic fruits. (B) Yeast one-hybrid assay to verify the binding of PavbZIP6 to the promoter of PavBBX6/9 and PavHY5. (C) Transient LUC imaging assays showing that PavbZIP6 activate the transcription of the report gene. Representative images of LUC activity in N. benthamiana leaves 48 h after infiltration. (D) Dual-luciferase assay showing promoter activity expressed as the ratio of luciferase (LUC) to 35S::Renilla (REN). (E) ABA contents in transgenic sweet cherry fruit and control. (F) mRNA expression of PavNCED1, PavNCED2, and PavNCED3 in transgenic fruits. Data are expressed as means ± standard deviation (SD; n = 3), and significant differences were assessed (* p < 0.05, ** p < 0.01, n.s.: no signifcant difference).
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Table 1. The identified PavbZIP genes and their corresponding information.
Table 1. The identified PavbZIP genes and their corresponding information.
Gene NameGene IDGroupCDSAAMW (Da)pISubcellular Localization
PavbZIP1Pav_sc0000861.1_g130.1.mkI132043947,164.036.51Nucleus
PavbZIP2Pav_sc0000478.1_g270.1.mkH68422725,359.724.57Nucleus
PavbZIP3Pav_sc0000588.1_g350.1.brS61520423,314.856.51Nucleus
PavbZIP4Pav_sc0000567.1_g900.1.mkD139546451,644.696.42Nucleus
PavbZIP5Pav_sc0000085.1_g210.1.mkA43514416,328.3610.65Nucleus
PavbZIP6Pav_sc0002208.1_g520.1.mkS50416719,048.157.1Nucleus
PavbZIP7Pav_sc0000912.1_g090.1.mkS60320023,108.1410.16Nucleus
PavbZIP8Pav_sc0000852.1_g810.1.mkA126041944,868.410.33Nucleus
PavbZIP9Pav_sc0000800.1_g1460.1.mkC104434737,768.465.42Nucleus
PavbZIP10Pav_sc0001476.1_g130.1.mkH30031000111,776.797.77Nucleus
PavbZIP11Pav_sc0000030.1_g770.1.mkA95731835,694.136.97Nucleus
PavbZIP12Pav_sc0001196.1_g670.1.mkM114638142,932.378.63Nucleus
PavbZIP13Pav_sc0001106.1_g630.1.mkA82827530,123.375.13Nucleus
PavbZIP14Pav_sc0001109.1_g050.1.mkD119139643,881.266.81Nucleus
PavbZIP15Pav_sc0001836.1_g030.1.mkA96932235,962.588.17Nucleus
PavbZIP16Pav_sc0005746.1_g050.1.mkI108336039,846.276.36Nucleus
PavbZIP17Pav_sc0000051.1_g030.1.brS60920223,407.846.68Nucleus
PavbZIP18Pav_sc0000051.1_g020.1.brS62720824,093.686.87Nucleus
PavbZIP19Pav_sc0000600.1_g560.1.mkK101733837,409.074.42Nucleus
PavbZIP20Pav_sc0003492.1_g420.1.mkE100533437,716.816.68Nucleus
PavbZIP21Pav_sc0002986.1_g270.1.mkE93631134,822.556.44Nucleus
PavbZIP22Pav_sc0000754.1_g090.1.mkG199266373,814.148.61Nucleus
PavbZIP23Pav_sc0000358.1_g970.1.brS53117620,146.547.81Nucleus
PavbZIP24Pav_sc0000638.1_g900.1.mkB87629132,697.94.55Nucleus
PavbZIP25Pav_sc0000638.1_g910.1.mkM117339042,986.999.72Nucleus
PavbZIP26Pav_sc0000206.1_g660.1.mkI132043947,164.036.51Nucleus
PavbZIP27Pav_sc0001801.1_g090.1.mkA115238342,651.345.12Nucleus
PavbZIP28Pav_sc0001801.1_g110.1.mkD139246350,544.46.76Nucleus
PavbZIP29Pav_sc0009346.1_g010.1.mkA78326029,071.566.95Nucleus
PavbZIP30Pav_sc0002914.1_g170.1.mkD109236341,013.386.89Nucleus
PavbZIP31Pav_sc0005841.1_g030.1.mkS63921224,188.5111.1Nucleus
PavbZIP32Pav_sc0001341.1_g060.1.mkB51016918,978.0810.31Nucleus
PavbZIP33Pav_sc0000396.1_g1160.1.mkC134444748,357.086.51Nucleus
PavbZIP34Pav_sc0000110.1_g150.1.mkD135945249,961.858.44Nucleus
PavbZIP35Pav_sc0008321.1_g030.1.mkD163554461,074.377.19Nucleus
PavbZIP36Pav_sc0000363.1_g920.1.mkA200166673,099.788.78Nucleus
PavbZIP37Pav_sc0001175.1_g270.1.mkG94531432,954.16.06Nucleus
PavbZIP38Pav_sc0000099.1_g260.1.mkH2769110,271.534.25Nucleus
PavbZIP39Pav_sc0000044.1_g090.1.mkA66322024,133.769.22Nucleus
PavbZIP40Pav_sc0000863.1_g670.1.mkA179159665,091.386.8Nucleus
PavbZIP41Pav_sc0001518.1_g090.1.mkS225747872.533.73Nucleus
PavbZIP42Pav_sc0002234.1_g130.1.mkA132644147,583.3410.23Nucleus
PavbZIP43Pav_sc0000407.1_g140.1.mkE57619122,444.7810.16Nucleus
PavbZIP44Pav_sc0000983.1_g310.1.mkC213671177,608.539.48Nucleus
PavbZIP45Pav_sc0000130.1_g590.1.mkS42914216,109.175.32Nucleus
PavbZIP46Pav_sc0000008.1_g430.1.mkG151250354,115.738.97Nucleus
PavbZIP47Pav_sc0000322.1_g160.1.mkI104134637,843.87.51Nucleus
PavbZIP48Pav_sc0000627.1_g120.1.mkM90630133,743.999.68Nucleus
PavbZIP49Pav_sc0003905.1_g010.1.mkB221773979,096.16.43Nucleus
PavbZIP50Pav_sc0005505.1_g010.1.mkA43514416,328.3610.65Nucleus
PavbZIP51Pav_sc0010299.1_g010.1.mkS63921224,188.5111.1Nucleus
PavbZIP52Pav_sc0012575.1_g010.1.mkI128142646,510.516.46Nucleus
PavbZIP53Pav_co4033649.1_g010.1.mkA91830534,317.718.45Nucleus
PavbZIP54Pav_co4037981.1_g010.1.brS60920223,331.756.68Nucleus
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MDPI and ACS Style

Gai, S.; Du, B.; Xiao, Y.; Zhang, X.; Turupu, M.; Yao, Q.; Wang, X.; Yan, Y.; Li, T. bZIP Transcription Factor PavbZIP6 Regulates Anthocyanin Accumulation by Increasing Abscisic Acid in Sweet Cherry. Int. J. Mol. Sci. 2024, 25, 10207. https://doi.org/10.3390/ijms251810207

AMA Style

Gai S, Du B, Xiao Y, Zhang X, Turupu M, Yao Q, Wang X, Yan Y, Li T. bZIP Transcription Factor PavbZIP6 Regulates Anthocyanin Accumulation by Increasing Abscisic Acid in Sweet Cherry. International Journal of Molecular Sciences. 2024; 25(18):10207. https://doi.org/10.3390/ijms251810207

Chicago/Turabian Style

Gai, Shilin, Bingyang Du, Yuqin Xiao, Xiang Zhang, Maihemuti Turupu, Qisheng Yao, Xinyu Wang, Yongzhen Yan, and Tianhong Li. 2024. "bZIP Transcription Factor PavbZIP6 Regulates Anthocyanin Accumulation by Increasing Abscisic Acid in Sweet Cherry" International Journal of Molecular Sciences 25, no. 18: 10207. https://doi.org/10.3390/ijms251810207

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