PgMYB1 Positively Regulates Anthocyanin Accumulation by Activating PgGSTF6 in Pomegranate

The peel color of pomegranates is an important exterior quality that determines market value. Anthocyanins are biosynthesized in the cytosol and then transported to the vacuole for storage. However, the molecular mechanism that determines the color variation between red and white pomegranates remains unclear. In this study, we identified an R2R3-MYB protein (PgMYB1) that interacts with the PgGSTF6 promoter and regulates its transcriptional expression, thus promoting the accumulation of anthocyanins in pomegranate. The expression of PgMYB1 and PgGSTF6 was positively correlated with the anthocyanin content in red and white pomegranates. Further investigation showed that the knockdown of PgMYB1 in red pomegranate ‘Taishanhong’ (TSH), by the virus-induced gene-silencing system, inhibited anthocyanin accumulation. Together, our results indicate that PgMYB1 controls the transport of anthocyanin via PgGSTF6 and thus promotes anthocyanin accumulation in red pomegranates. Our results have a certain reference value for further clarifying the regulation of anthocyanin synthesis and transport in pomegranate fruits.


Introduction
Pomegranate (Punica granatum L.) originated in central Asian countries [1,2]. In China, many high-genetic-diversity pomegranate varieties have been cultivated for about 2000 years [3]. The exterior fruit color of pomegranates, with red and white variability, reflects differences in anthocyanin concentrations [4]. Anthocyanins play an essential role in seed dispersal, resistance to pathogens, and protection against ultraviolet radiation [5][6][7]. They can also be beneficial for humans in protecting against diseases [8][9][10]. The presence of anthocyanins in pomegranate has important effects on fruit quality; however, the molecular genetics of anthocyanin biosynthesis and transport in pomegranate is still sparse. Many studies have focused on the components and content of pomegranate anthocyanins in fruit peels and juices [11,12].
Currently, the mechanisms that determine anthocyanin synthesis and transport in other plants are well known, but little is known specifically regarding pomegranate. In our study, the R2R3-MYB transcription factor PgMYB1 was found to play a key role in anthocyanin accumulation in pomegranate. We hypothesized that PgMYB1 directly activates the expression of PgGSTF6 and is positively correlated with the expression level of PgANS, thus promoting anthocyanin accumulation. We aimed to investigate the key role that PgMYB1 plays in anthocyanin synthesis and transport in pomegranate.

Phylogenetic Analysis and Subcellular Localization of PgMYB1
To identify the putative MYB1 gene in the pomegranate genome, BlastP was used to search the pomegranate genome database based on apple MYB1 (NP_001288045.1) protein. To explore the homology of the PgMYB1 gene among different plant species, a phylogenetic tree was constructed. We found that PgMYB1 was clustered within the R2R3-MYB clade, which includes MdMYB1 (apple), CmMYB6 (chrysanthemum), and other proteins that have been identified as being involved in anthocyanin synthesis (Figure 1a). The comparative analysis of the amino acid sequence of PgMYB1 and its close homologs showed that the PgMYB1 protein is an R2R3-MYB transcription factor ( Figure S1). We constructed a translational fusion protein, 35S:PgMYB1-GFP, to determine the subcellular localization of PgMYB1. The 35S:PgMYB1-GFP fusion protein showed a fluorescent signal in the nucleus (Figure 1b,c), suggesting that PgMYB1 was translated in the nucleus.

Identification and Analysis of GSTF Genes in Pomegranate
To identify the putative GSTF family genes in the pomegranate genome, BlastP was used to search against the pomegranate genome database based on 15 apple GSTF proteins. A total of 18 GSTF genes were identified in the pomegranate genome. The comparative analysis of the amino acid sequences of 18 GSTF proteins and their close homologs showed that the pomegranate GSTF proteins are highly conserved ( Figure S2). To explore the homology of GSTF genes among different plant species, a phylogenetic analysis was conducted to compare GSTF proteins from pomegranate, Arabidopsis, apple, dragon's blood tree, and maize. PgGSTF6 was clustered within the GSTF clade, which includes MdGSTF6 and other proteins identified as being involved in anthocyanin transport (Figure 2), suggesting that PgGSTF6 may be involved in pomegranate anthocyanin transport.

Expression Patterns of PgMYB1, PgANS, and PgGSTFs in Different Developmental Stages of TSH and SBT
Previous research has shown that MdMYB1 plays a key role in anthocyanin transport via MdGSTF6 [34]. To further confirm that PgMYB1 is involved in anthocyanin accumulation via PgGSTFs in pomegranate, samples of two pomegranate cultivars, TSH, with red fruit peels, and 'Sanbaitian' (SBT), with white fruit peels at maturity, were screened as experimental materials (Figure 3a). We observed higher expression levels of PgMYB1 (GenBank accession number: OWM81764.1) in S1-S4 of TSH than those in SBT (Figure 3b). The expression level trends of PgGSTF9 (GenBank accession number: NC_045127.1) and other PgGSTF genes were not obvious in different developmental stages of TSH and SBT (Figures 3b and S3). We also observed lower expression levels of PgANS (GenBank accession number: AHZ97874.1) and PgGSTF6 (GenBank accession number: NC_045131.1) in S1-S6 of SBT than in TSH, indicating that PgMYB1, PgANS, and PgGSTF6 play a key role in anthocyanin synthesis and transport in pomegranate (Figure 3b). This finding prompted us to explore the mechanism leading to high anthocyanin levels in red pomegranate.  parative analysis of the amino acid sequences of 18 GSTF proteins and their close hom-ologs showed that the pomegranate GSTF proteins are highly conserved ( Figure S2). To explore the homology of GSTF genes among different plant species, a phylogenetic analysis was conducted to compare GSTF proteins from pomegranate, Arabidopsis, apple, dragon's blood tree, and maize. PgGSTF6 was clustered within the GSTF clade, which includes MdGSTF6 and other proteins identified as being involved in anthocyanin transport (Figure 2), suggesting that PgGSTF6 may be involved in pomegranate anthocyanin transport.

Figure 2.
Phylogenetic tree of GSTF proteins in pomegranate, Arabidopsis, apple, dragon's blood tree, and maize. The 18 pomegranate, 15 apple, 14 Arabidopsis, 5 dragon's blood tree, and 7 maize GSTF protein sequences were aligned using ClustalW. The long red rectangular box represents PgGSTF6. The phylogenetic tree was constructed based on the neighbor-joining algorithm.

Expression Patterns of PgMYB1, PgANS, and PgGSTFs in Different Developmental Stages of TSH and SBT
Previous research has shown that MdMYB1 plays a key role in anthocyanin transport via MdGSTF6 [34]. To further confirm that PgMYB1 is involved in anthocyanin accumulation via PgGSTFs in pomegranate, samples of two pomegranate cultivars, TSH, with red fruit peels, and 'Sanbaitian' (SBT), with white fruit peels at maturity, were Phylogenetic tree of GSTF proteins in pomegranate, Arabidopsis, apple, dragon's blood tree, and maize. The 18 pomegranate, 15 apple, 14 Arabidopsis, 5 dragon's blood tree, and 7 maize GSTF protein sequences were aligned using ClustalW. The long red rectangular box represents PgGSTF6. The phylogenetic tree was constructed based on the neighbor-joining algorithm.

PgMYB1 Positively Regulates Anthocyanin Accumulation in Pomegranate
Previous studies have clarified that MdMYB1 positively regulates anthocyanin accumulation in apple [34]. To demonstrate whether PgMYB1 is essential for pomegranate fruit coloration, we used a virus-induced gene-silencing system to conduct a transient expression assay. The injection sites on the pomegranate peel turned lighter red after transformation with pTRV2-PgMYB1 and pTRV1. However, there was no obvious phenotypic change observed after the transformation with the pTRV2 vector ( Figure 4a). Additionally, the anthocyanin content in pTRV2-PgMYB1 pomegranate peels was lower than that in pTRV2 pomegranate peels (Figure 4b).
Knockdown of PgMYB1 strongly downregulated the expression of PgMYB1. Furthermore, we performed qRT-PCR to further study the expression patterns of other genes in the anthocyanin biosynthesis pathway in pTRV2-PgMYB1 pomegranate peels. We observed that PgGSTF6 and PgANS were less expressed in pTRV2-PgMYB1 pomegranate peels ( Figure 4c). Knockdown of PgMYB1 affected the expression level of PgGSTF6 and PgANS. Together, our results show that the lower expression of PgMYB1 may affect anthocyanin accumulation via downregulated expression of PgGSTF6 and PgANS, indicating that PgMYB1 plays a key role in anthocyanin accumulation in pomegranate.

PgMYB1 Directly Regulates PgGSTF6 Transcription
To further understand the mechanism by which PgMYB1 regulates anthocyanin accumulation in pomegranate, yeast one-hybrid (Y1H) assays were performed. These assays showed that PgMYB1 binds to the PgGSTF6 promoter and activates its expression (Figure 5a). To further investigate whether PgMYB1 activates the transcriptional expression of PgGSTF6, transient expression assays were conducted. As expected, the PgMYB1 plus proPgGSTF6:GUS treatments showed significantly higher GUS activity (Figure 5b,c). Furthermore, we performed qRT-PCR to further study the expression level of GUS in tobacco leaves. We found higher GUS expression levels in PgMYB1 plus proPgGSTF6:GUS tobacco leaves than in the proPgGSTF6:GUS leaves (Figure 5d). These results show that PgMYB1 could bind to the PgGSTF6 promoter and activate its expression in pomegranate. screened as experimental materials (Figure 3a). We observed higher expression levels of PgMYB1 (GenBank accession number: OWM81764.1) in S1-S4 of TSH than those in SBT ( Figure 3b). The expression level trends of PgGSTF9 (GenBank accession number: NC_045127.1) and other PgGSTF genes were not obvious in different developmental stages of TSH and SBT (Figure 3b, Figure S3). We also observed lower expression levels of PgANS (GenBank accession number: AHZ97874.1) and PgGSTF6 (GenBank accession number: NC_045131.1) in S1-S6 of SBT than in TSH, indicating that PgMYB1, PgANS, and PgGSTF6 play a key role in anthocyanin synthesis and transport in pomegranate (Figure 3b). This finding prompted us to explore the mechanism leading to high anthocyanin levels in red pomegranate.

PgMYB1 Positively Regulates Anthocyanin Accumulation in Pomegranate
Previous studies have clarified that MdMYB1 positively regulates anthocyanin accumulation in apple [34]. To demonstrate whether PgMYB1 is essential for pomegranate fruit coloration, we used a virus-induced gene-silencing system to conduct a transient expression assay. The injection sites on the pomegranate peel turned lighter red after transformation with pTRV2-PgMYB1 and pTRV1. However, there was no obvious phe- in the anthocyanin biosynthesis pathway in pTRV2-PgMYB1 pomegranate peels. We observed that PgGSTF6 and PgANS were less expressed in pTRV2-PgMYB1 pomegranate peels (Figure 4c). Knockdown of PgMYB1 affected the expression level of PgGSTF6 and PgANS. Together, our results show that the lower expression of PgMYB1 may affect anthocyanin accumulation via downregulated expression of PgGSTF6 and PgANS, indicating that PgMYB1 plays a key role in anthocyanin accumulation in pomegranate. . qRT-PCR was conducted in three biological replicates and three technical replicates, and one representative biological repeat is shown. ** p-values < 0.01 were considered statistically significant.

PgMYB1 Directly Regulates PgGSTF6 Transcription
To further understand the mechanism by which PgMYB1 regulates anthocyanin accumulation in pomegranate, yeast one-hybrid (Y1H) assays were performed. These assays showed that PgMYB1 binds to the PgGSTF6 promoter and activates its expression (Figure 5a). To further investigate whether PgMYB1 activates the transcriptional expression of PgGSTF6, transient expression assays were conducted. As expected, the PgMYB1 plus proPgGSTF6:GUS treatments showed significantly higher GUS activity (Figure 5b,c). Furthermore, we performed qRT-PCR to further study the expression level of GUS in To further reveal how the transcriptional regulation mechanism of PgMYB1 regulates the PgGSTF6 gene expression, the promoter sequence of PgGSTF6 was analyzed. We observed that there was a potential MBS binding site (CTGTTG) in the PgGSTF6 gene promoter region (−638-633) (Figures 5e and S4). To further determine if PgMYB1 could directly bind to the binding site, yeast one-hybrid (Y1H) assays were performed. As expected, PgMYB1 could interact with MBS (CTGTTG), while PgMYB1 could not bind when the MBS (CTGTTG) site was mutated to mMBS (CGGTGG) (Figure 5f). Together, these data indicate that PgMYB1 directly binds to the CTGTTG motif of the PgGSTF6 promoter region and activates its expression.
promoter region (−638-633) (Figure 5e, Figure S4). To further determine if PgMYB1 could directly bind to the binding site, yeast one-hybrid (Y1H) assays were performed. As expected, PgMYB1 could interact with MBS (CTGTTG), while PgMYB1 could not bind when the MBS (CTGTTG) site was mutated to mMBS (CGGTGG) (Figure 5f). Together, these data indicate that PgMYB1 directly binds to the CTGTTG motif of the PgGSTF6 promoter region and activates its expression.

Discussion
Fruit color is one of the most important agronomic traits of fruit quality and is an important attribute in terms of consumer preference. Anthocyanins in fruit not only confer different bright colors but are also beneficial for human health. Therefore, it is of great significance to study the regulation of anthocyanin biosynthetic pathways. Glutathione S-transferase (GST) has been shown to play a key role in anthocyanin transport [20][21][22][23][24].
Previous studies have shown that the abundance of GSTs in several horticultural crops, including peach, lychee, grape, and strawberry, is related to the pigmentation of the fruits [20,21,33,36]. MdGSTF6 is highly expressed in red apple fruits, and its expression abundance is positively correlated with anthocyanin content [34]. To date, the transcriptional regulation mechanism of GSTs in pomegranate has not been well investigated. In this study, we identified that PgGSTF6, a GST gene, was clustered within the GSTF clade, including MdGSTF6, which is positively correlated with anthocyanin content and is highly expressed in red apple fruits (Figure 2). We found a lower expression level of PgGSTF6 in S1-S6 of SBT than that in TSH, indicating that PgGSTF6 plays a key role in anthocyanin transport in pomegranate (Figure 3b). This finding provides new insight into anthocyanin transport and prompted us to explore the mechanism leading to high anthocyanin levels in red pomegranates.
MdMYB1 is an anthocyanin accumulation gene that responds to light signals in apple [37]. We found that PgMYB1 was clustered within the R2R3-MYB clade, including MdMYB1 and CmMYB6 (Figure 1a), which are involved in anthocyanin accumulation. Subcellular localization analysis of PgMYB1 showed that the 35S:PgMYB1-GFP fluoresced in the nucleus (Figure 1b,c), suggesting that PgMYB1 may be involved in pomegranate anthocyanin accumulation as a transcription factor. However, how PgMYB1 regulates the process of anthocyanin synthesis and transport in pomegranate needs further exploration.
In apple, MdMYBA and MdMYB1 regulate anthocyanin biosynthesis in fruit peel. Additionally, MdMYBA binds to the MdANS promoter, while MdMYB1 activates the transcription of MdDFR and MdUFGT [34,35]. We found that the anthocyanin content in pTRV2-PgMYB1 pomegranate peels was lower than that in pTRV2 pomegranate peels. Previous studies have shown that the GSTs play key roles in anthocyanin accumulation in Arabidopsis, petunia, and maize [38][39][40]. In our study, the lower expression levels of PgANS in peels of white fruits suggest that the lack of PgANS expression may be the main factor responsible for the absence of anthocyanins in white pomegranate. Furthermore, knockdown of PgMYB1 strongly downregulated the expression of PgGSTF6 and PgANS but did not affect the transcription of the other genes in the anthocyanin biosynthesis pathway (Figure 4c). We found that PgMYB1 could activate the expression of PgGSTF6 in pomegranate ( Figure 5). Together, these results show that knockdown of PgMYB1 affects anthocyanin accumulation via downregulated expression of PgGSTF6 and PgANS.
Anthocyanin accumulation in fruit is a key quality trait in pomegranate. However, there have been few studies on anthocyanin accumulation in pomegranate. Therefore, how anthocyanin accumulation is regulated is important for clarifying the mechanism underlying pomegranate fruit coloration. In this study, our results reveal that the lower expression of PgMYB1 may affect anthocyanin accumulation via downregulated expression of PgGSTF6 and PgANS in pomegranate. This is because PgMYB1 directly activates the expression of the glutathione S-transferase (GST) gene PgGSTF6 and affects the expression level of the encoding anthocyanidin synthase (ANS) gene, PgANS, thus promoting anthocyanin accumulation ( Figure 6). Our results reveal the mechanism leading to anthocyanidin accumulation in red peel pomegranate and provide new insights that are useful in the cultivation of red peel pomegranate with high contents of anthocyanins, which are beneficial to human health.

Plant Materials and Growth Conditions
The two pomegranate cultivars, TSH, with red fruit peels, and SBT, with white fruit peels at maturity, were grown in Tai'an, Shandong Province, China. Fruit peels were collected on the following six sampling dates, 10 July, 25 July, 9 August, 24 August, 8 September, and 23 September, and were designated as S1, S2, S3, S4, S5, and S6, respectively. We collected nine fruits from each cultivar at each stage. Peels from these fruits were pared with a knife and then immediately frozen in liquid nitrogen and stored at −80 °C until use.

RNA Extraction and qRT-PCR Analysis
Total RNA was extracted from 'TSH' and 'SBT' pomegranate peels using a Total RNA Isolation System (Tiangen, Beijing, China). The RT-PCR reactions were performed using qPCR SuperMix (TransGen Biotech, Beijing, China) with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, New York, NY, USA). Expression data were normalized to those of the pomegranate actin gene. Transcription levels were calculated using the cycle threshold (Ct) 2 -ΔΔCt method, according to Livak et al. (2001) [41]. All primers used in this study are listed in Table S1.

Sequence Alignments and Phylogenetic Analysis
The sequence alignment was performed as described by Wang et al. (2019) [13]. Two phylogenetic trees were constructed using the full-length amino acid sequences of 18 pomegranate, 15 apple, 14 Arabidopsis, 5 dragon's blood tree, and 7 maize GSTF protein sequences and 8 R2R3-MYB transcription factor protein sequences using MEGA 7.0 and the neighbor-joining (NJ) method as described by Li et al. (2022) [15].

Plant Materials and Growth Conditions
The two pomegranate cultivars, TSH, with red fruit peels, and SBT, with white fruit peels at maturity, were grown in Tai'an, Shandong Province, China. Fruit peels were collected on the following six sampling dates, 10 July, 25 July, 9 August, 24 August, 8 September, and 23 September, and were designated as S1, S2, S3, S4, S5, and S6, respectively. We collected nine fruits from each cultivar at each stage. Peels from these fruits were pared with a knife and then immediately frozen in liquid nitrogen and stored at −80 • C until use.

RNA Extraction and qRT-PCR Analysis
Total RNA was extracted from 'TSH' and 'SBT' pomegranate peels using a Total RNA Isolation System (Tiangen, Beijing, China). The RT-PCR reactions were performed using qPCR SuperMix (TransGen Biotech, Beijing, China) with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, New York, NY, USA). Expression data were normalized to those of the pomegranate actin gene. Transcription levels were calculated using the cycle threshold (Ct) 2 −∆∆Ct method, according to Livak et al. (2001) [41]. All primers used in this study are listed in Table S1.

Sequence Alignments and Phylogenetic Analysis
The sequence alignment was performed as described by Wang et al. (2019) [13]. Two phylogenetic trees were constructed using the full-length amino acid sequences of 18 pomegranate, 15 apple, 14 Arabidopsis, 5 dragon's blood tree, and 7 maize GSTF protein sequences and 8 R2R3-MYB transcription factor protein sequences using MEGA 7.0 and the neighbor-joining (NJ) method as described by Li et al. (2022) [15].

Yeast One-Hybrid Assays
According to the manufacturer's instructions, Y187 (Clontech, Palo Alto, CA, USA) was used for Y1H assays. The full-length PgMYB1 CDS was cloned into the pGADT7 vector to generate the construct AD-PgMYB1. The 2000 bp promoter sequences of PgGSTF6 were inserted into the pHIS2 vector (Clontech, Palo Alto, CA, USA) to generate the construct pHIS2-proPgGSTF6. AD-PgMYB1 and pHIS2-proPgGSTF6 were co-transformed into yeast Y187. The negative control was the pHIS2 empty vector. All primers used in this study are listed in Table S1.

Transient Expression Assays
Transient expression assays were conducted using tobacco leaves. The 2000 bp promoter sequences of PgGSTF6 were cloned into pCAMBIA1300 (Takara, Dalian, China) to generate the construct proPgGSTF6:GUS. The CDS of PgMYB1 was inserted into pCAM-BIA1300 to generate the construct 35S:PgMYB1. The different combinations were cotransformed into tobacco leaves via an agrobacterium-mediated method. The injected tobacco grew for 2-3 days under normal conditions. The GUS activity was detected via GUS staining and GUS expression quantity. All primers used in this study are listed in Table S1.

Virus-Induced Gene Silencing of PgMYB1 in Pomegranate
A 459 bp fragment of PgMYB1 was amplified and cloned into the pTRV2 vector to generate the construct pTRV2-PgMYB1, and then the pTRV2, pTRV2-PgMYB1, and pTRV1 were each transformed into the GV3101 A. tumefaciens strain for the next VIGS experiments. The pTRV2 empty vector was used as the control. The pTRV2-PgMYB1 and pTRV2 vectors were injected into pomegranate peel along with pTRV1. The fruits of the 'TSH' cultivar were selected to inject the VIGS constructs. A total of 80 fruits were selected and divided into two groups, with one to three injections for each single fruit. The expression analysis was conducted on the infected area. The VIGS analysis was performed as described by Jiang et al. (2019) [34]. All primers used for VIGS experiments are listed in Table S1.

Measurement of Total Anthocyanins
The total anthocyanins of the fruit peels of TSH fruits after PgMYB1 silencing and pTRV2 empty vector were extracted using the HCl-methanol method. Then, 0.5 g of each sample was fully ground in liquid nitrogen and incubated with 5 mL cold 1% (v/v) methanol-hydrochloric acid in the dark for 24 h. The absorbances of samples at 650, 620, and 530 nm were measured using a UV-1600 spectrophotometer (Shimadzu, Kyoto, Japan). We used the formula OD=(A530-A620)-0.1(OD650-OD620) to calculate the anthocyanin contents of fruit peels of TSH fruits after PgMYB1 silencing and pTRV2 empty vector control treatment, according to Li et al. (2020) [42].

Subcellular Localization Analyses
PgMYB1 was cloned into the pRI101 vector (Takara, Dalian, China) to construct the 35S:PgMYB1-GFP recombinant vector. The vector was then transformed into Agrobacterium tumefaciens LBA4404 competent cells. Transgenic onion epidermal cells of 35S:PgMYB1-GFP were obtained as described by Wang et al. (2017) [43], while 35S:GFP was used as a negative control. All primers used in this study are listed in Table S1.