SmMYB113 Is a Key Transcription Factor Responsible for Compositional Variation of Anthocyanin and Color Diversity Among Eggplant Peels

To understand the color formation mechanism in eggplant (Solanum melongena L.) peel, a metabolomic analysis was performed in six cultivars with different peel colors. A total of 167 flavonoids, including 16 anthocyanins, were identified based on a UPLC-MS/MS approach. Further analysis revealed that the delphinidins/flavonoids ratio was consistent with the purple coloration of eggplant peels, and SmF3′5′H expression level was consistent with the delphinidin 3-O-glucoside and delphinidin 3-O-rutinoside contents, the main anthocyanins in the purple-peels eggplant cultivars identified in this study. SmMYB113 overexpression promoted anthocyanins accumulation in eggplant peels and pulps. Metabolomic analysis revealed that delphinidins were still the main anthocyanins class in the peels and pulps of SmMYB113-OE4, but most anthocyanins were glycosylated at the 5-position of the B-ring. Our results provide new insights into the anthocyanin composition of eggplant peels and demonstrate the importance of SmMYB113 in stimulating anthocyanin biosynthesis in eggplant fruits.


INTRODUCTION
Anthocyanins are colored water-soluble pigments that give flowers, fruits, and tubers blue, red, or purple coloration. Studies have reported that anthocyanins can have anti-diabetic, anti-cancer, antiinflammatory, anti-microbial, and anti-obesity effects and prevent cardiovascular diseases (Khoo et al., 2017). Therefore, anthocyanin-rich flowers, fruits, and tubers, such as red rose, blue chicory, purple mint, red cabbage, purple eggplant, and purple potato are popular with consumers.
The technology behind transgenic crops in breeding programs constantly develops and elevates many excellent genotypes. MYB transcription factors (TFs) have been identified to be one of the most important transcriptional regulators in anthocyanins biosynthesis, acting as activators or repressors (Ma and Constabel, 2019;Yan et al., 2021). Anthocyanins content increases when activators MYBs are overexpressed through transient transactivation experiments or transgenic calluses in tobacco leaves and strawberry. Previous studies have reported that SmMYB113, orthologous to AtMYB75, promote anthocyanins biosynthesis in tobacco leaves in transient expression assays (Li et al., 2017) and eggplant calluses through experimental transformation (Shi S. et al., 2021). However, whether the anthocyanins profile induced by SmMYB113 is consistent with the original profile in wild-type plants has been rarely reported.
In this study, we report the metabolic profiles of flavonoids that may contribute to different pigmentation in eggplants. Six eggplant cultivars with different peel colors representing all possible eggplant colors were examined (Duan et al., 2021). In addition, the anthocyanin composition of eggplant peels and pulps activated by transgenic overexpression of SmMYB113 were also investigated using metabolome technology. This study aimed to improve our understanding of the mechanisms of eggplant coloration and to explore whether overexpression of SmMYB113 alters their anthocyanin profiles.

Sampling
Six eggplant cultivars (Numbers 44,64,76,108,109,and 133) (Duan et al., 2021) including five S. melongena and one Solanum aethiopicum (mainly cultivated and popular in Africa), and SmMYB113-OE transgenic eggplants were planted in the solar greenhouse at Shandong Agricultural University. Peels of the six eggplant cultivars and peels and pulps of SmMYB113-OE transgenic eggplants were harvested from nine representative fruits of each type in triplicate. The samples were immediately frozen in liquid nitrogen after harvest, and the frozen samples were subjected to anthocyanin content measurement, gene expression analysis, and flavonoids metabolomic analysis.

Anthocyanins Content Analysis
The anthocyanin contents were extracted using methods detailed in Neff and Chory (1998).

RNA Extraction and Quantitative Real-Time PCR Analysis
The total RNA was extracted from each sample using TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Otsu, Shiga, Japan). Next, 1 µg RNA was reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). Quantitative real-time PCR (QRT-PCR) analysis was performed using SYBR Premix Ex Taq II Kit (TaKaRa) and LightCycler 96 system (Roche, Basel, Switzerland). The Actin gene (GU984779.1) was used as a standard. The relative expression was calculated using the 2 − Ct method (Livak and Schmittgen, 2001).

Plasmid Construction and Plant Transformation
The SmMYB113 coding sequence was inserted into the pRI 101 vector containing the 35S-CaMV promoter. The fusion vector was transferred into Agrobacterium strain LBA4404 and introduced into cultivar No. 108. The cut-cotyledons were precultured on MS medium in the dark for 2 days. Then the cotyledons were infected with Agrobacterium (OD600 about 0.6) for 15-20 min. The infected-cotyledons were placed on the differentiation MS medium without Kan and co-cultured in the dark for 2-3 days. The differentiation MS medium consisted of 200 mg L −1 carbenicillin (Cb), 3.0 mg L −1 6-benzylaminopurine (6-BA), 0.1 mg L −1 thidiazuron (TDZ), 7 g L −1 agar, 30 g L −1 sucrose, and 100 mg L −1 kanamycin. After the shoots were differentiated, the explants were placed on MS medium with 200 mg L −1 carbenicillin (Cb) for roots differentiation.

The Anthocyanin Contents in the Peels of Six Different Eggplant Cultivars
Eggplant is a widespread vegetable bearing different colored peels (Koley et al., 2019). Anthocyanins are the main phenolic compounds in eggplant peels. To explore the relationship between anthocyanin composition and color in eggplant, six eggplant cultivars representing all eggplant cultivar colors were examined (Duan et al., 2021). As shown in Supplementary Figure 1A, the six peel colors covered lavender in No. 44,white in No. 108,orange in No. 109,and green in No. 133. Peels samples were collected during fruit setting (fruiting), rapid growth (growth), and commodity maturity (maturity), and their relative anthocyanin contents were measured (Supplementary Figure 1B). Anthocyanin content increased along with fruit development. The anthocyanin contents were highest in cultivar No. 76, followed by No. 64,and No. 44. The anthocyanin contents of cultivars No. 133,No. 109,and No. 108 were the lowest, and did not differ significantly. These results indicated that higher anthocyanin contents corresponded to a deeper purple peel color, consistent with results in the distinct purple leaves of the novel tea cultivar 'Ziyan' (Lai et al., 2016).

The Delphinidin/Flavonoid Ratio Could Better Explain the Purple Peel Color of Eggplants
Anthocyanin biosynthesis is a branch of flavonoid biosynthesis, and colored flavonoids (flavones, flavanols, and isoflavonoids) and their glycosides contribute to the diversity of colors in leaves, fruits, and flowers (Ono et al., 2010;Zhang et al., 2014;Shi J. et al., 2021). In eggplant, anthocyanins accumulated with fruit development and peaked at fruit maturity (Supplementary Figure 1). Therefore, flavonoidtargeted metabolism analysis was conducted on methanolic extracts of mature eggplant peels using UPLC-MS/MS. A total of 167 flavonoids were identified and divided into eight categories, including proanthocyanidins, anthocyanins, catechin derivatives, flavanone, flavone, flavone C-glycosides, flavonol, and flavonolignan (Supplementary Table 1 and Figure 1A). Among the 167 flavonoids, 16 anthocyanins, including 10 in No. 44,10 in No. 64,14 in No. 76,9 in No. 108,12 in No. 109,and 11 in No. 133, were identified. These results suggested that the eggplant cultivars without purple peels can also synthesize anthocyanins. Further analysis showed that the relative total flavonoid content ranked from highest to lowest as No. 109 > No. 76 > No. 64 > No. 44,No. 133,No. 108. The relative anthocyanin content was highest in No. 76, followed by No. 64,and No. 44,No. 133,No. 109,No. 108 ( Figure 1B). Notably, the relative flavonoid content of No. 109 was greater than the other eggplant cultivars, but it contained almost no anthocyanin. Approximately 50% of the flavonoids in No. 64 and 70% of the flavonoids in No. 76 were anthocyanins ( Figure 1C), resulting in purple peels. According to the "anthocyanins biosynthesis pathway (00942)" in the Kyoto Encyclopedia of Genes and Genomes (KEGG), the identified anthocyanins consisted of "cyanidins" (including cyanidins and peonidin derivatives), "delphinidins" (including delphinidins, malvidins, and petunidin derivatives), and "pelargonidins." The delphinidin/flavonoid ratios of No. 76,No. 64,and No. 44 (purple peels), were higher than those of No. 133,No. 109,and No. 108 (no purple peels), particularly No. 76 and No. 64 ( Figure 1D). Differently, the cyanidin/flavonoid ratios of No. 133,No. 109,and No. 108 were higher than No. 64 but lower than No. 44. The cyanidin/flavonoid ratio of No. 76 was higher than No. 109 but lower than No. 133 and No. 108. Together, a correlation was found between the delphinidin/flavonoid ratio and purple peel color among the six eggplant cultivars, meaning that the delphinidin/flavonoid ratio could better explain the purple coloration of eggplant peels.
Considering that No. 109 had the highest relative content of total flavonoids, but the lowest anthocyanin/flavonoid ratio, it was speculated that orange peel color resulted from flavones, flavanols, and isoflavonoids (Ono et al., 2010;Shi J. et al., 2021). As shown in Supplementary Figure 2A, the relative contents of flavones, flavanols, and isoflavonoids were highest in No. 109, followed by No. 76 and No. 64,No. 44 and No. 133,and No. 108. In addition, the ratio of flavones, flavanols, and isoflavonoids in the total flavonoids [(flavones and flavanols and isoflavonoids) / flavonoids)] was highest in No. 109 (Supplementary Figure 2B).

SmF3 5 H Is Responsible for Purple Peel of Eggplant at the Transcriptional Level
To find the direct mechanism underlying the different purple peel colors of No. 76,No. 64,and No. 44, the 16 anthocyanin structures were further analyzed according to the anthocyanin biosynthesis pathway in the website of KEGG. Eight anthocyanins were found in the six eggplant cultivars, while "pelargonin" was found only in No. 76 and Values are means ± SD (n = 3), same as following. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan's multiple range test.
"cyanidin 3,5-O-diglucoside" was found both No. 76 and No. 44 (Figure 2A). Therefore, it was speculated that the eight anthocyanin structures cause purple peel colors. In the peels of No. 76 and No. 64, delphinidin 3-O-glucoside (Mirtillin) and "Tulipanin" contents were highest ( Figure 2B and Supplementary Table 1). In the peels of No. 44, "Cyanidin 3-O-glucoside (Kuromanin)" content was highest, followed by "Mirtillin, " "Cyanidin, " and "Pelargonidin 3-O-beta-Dglucoside." Based on the anthocyanin biosynthesis pathway in the KEGG website, we concluded that the "delphinidin" biosynthetic branch was significantly more active than the "cyanidin" and "pelargonidin" biosynthetic branches in No. 76 and No. 64, while the cyanidin biosynthetic branch activities were the strongest in No. 44. Flavonoid 3 -hydroxylases (F3 Hs) and flavonoid 3 ,5 -hydroxylases (F3 5 Hs) competitively control the biosynthesis of cyanidin and delphinidin (Bogs et al., 2005;Castellarin et al., 2005). Castellarin et al. (2007) found that F3 H expression did not show a clear pattern associated with anthocyanin accumulation, but variation in the F3 5 H/F3 H expression ratio was consistent with anthocyanin biosynthesis in grape varieties. Therefore, the expression levels of SmF3 H and SmF3 5 H in the peels of eggplant fruits were analyzed using qRT-PCR. The expression ratio between SmF3 5 H and SmF3 H was also calculated. The SmF3 5 H/SmF3 H expression ratio was associated with the anthocyanin profiles variation among the six eggplant cultivars (Figure 2C). Notably, no significant difference in the relative expression level of SmF3 H was found among the six eggplant cultivars. However, SmF3 5 H expression level during the growth stage was highest in No. 76, followed by No. 64,No. 44,No. 133 and No. 109,and No. 108, consistent with the purple peel color pattern. Altogether, we speculated that the variation in purple peels of eggplant were mainly determined by the "Mirtillin" and "Tulipanin" contents, and that SmF3 5 H expression was a critical factor.

Anthocyanins Accumulate in Eggplants After SmMYB113 Overexpression
SmMYB113 is an important regulator promoting anthocyanin biosynthesis in eggplant (Li et al., 2017;Shi S. et al., 2021). The eggplant SmMYB113 gene was grouped with homologous R2R3MYBs from other plant species, including AtPAP1 from Arabidopsis (Borevitz et al., 2000), AmRosea1 and AmDelila from Antirrhinum majus (Sharma et al., 2020), AN2 from petunia (Quattrocchio et al., 1999), DcMYB113 from carrots (Xu et al., 2019), and OsMYB3 from black rice cultivar (Zheng et al., 2021). Deletion of R2R3MYBs homologous results in tissue color loss (without anthocyanin enrichment or decrease), while overexpression results in more vivid coloration (anthocyanin enrichment or increase). Here, qRT-PCR analysis showed that SmMYB113expression levels in cultivars No. 108,No. 109,and No. 133 were significantly lower than those in No. 44,No. 64,and No. 76, corresponding to their anthocyanin contents (Supplementary Figure 3A). The SmMYB113full-length coding sequence was constructed into the overexpression pRI vector with the CaMV 35S promoter, obtaining 35S:SmMYB113 transgenic eggplant lines ( Figure 3A and Supplementary Figure 3C). PCR and qRT-PCR analysis were used to confirm SmMYB113 integration into the transgenic eggplant lines (Figures 3B,C). Compared with the WT, all the aboveground plant parts were purple. Anthocyanin contents measurement confirmed that the SmMYB113-OE plants produced more anthocyanins (Figure 3D). These results suggested that the SmMYB113function on regulating anthocyanin biosynthesis in eggplant is similar to that of R2R3MYB in other plant species.

The Flavonoid Profiles in the Peels and Pulps of SmMYB113-OE Eggplants
To reveal the flavonoid profiles regulated by SmMYB113 in eggplant fruits, UPLC-MS/MS analysis was performed on the peels and pulps of WT and SmMYB113-OE4 plants. A total of 228 flavonoids were identified from peels and pulps, which could be classified into eight classes, including 23 flavonoids, 27 anthocyanins, 2 proanthocyanidins, 11 polyphenols, 91 flavones, 40 flavonols, 14 isoflavones, and 20 flavanones (Supplementary Table 2). Based on |log 2 (foldchange) | ≥ 1 and VIP ≥ 1, 147 (in the peels) and 128 (in the pulps) flavonoids differed significantly between SmMYB113-OE4 and WT plants. Most flavonoids contents were primarily increased via SmMYB113 overexpression, except for 10 in peels and 11 in pulps which were downregulated ( Figure 4A). More than 70% of anthocyanins and flavonols changed significantly via SmMYB113 overexpression in both peels and pulps (Figure 4C), including 95 flavonoids changed in both peels and pulps, 52 in peels only, and 33 in pulps only ( Figure 4B). Compared with WT, the delphinidin/flavonoid ratio in peels and pulps increased significantly in SmMYB113-OE4 ( Figure 4D). Delphinidin 3,5-diglucoside (Delphin chloride) and "Tulipanin" were the most significantly increased by SmMYB113, and their contents were highest in both peels and pulps (Supplementary Table 2). In addition, SmF3 H and SmF3 5 H relative expression levels in peels and pulps of SmMYB113-OE4 and WT were analyzed using qRT-PCR (Supplementary Figure 4). The SmF3 5 H/SmF3 H expression ratio was significantly increased by overexpressing SmMYB113, associated with the increased delphinidin/flavonoid ratio. According to the KEGG pathway, anthocyanins and flavonols are two neighboring branches of flavonoid biosynthesis, catalyzing dihydroflavonols via DFR and FLS, respectively ( Figure 4E). Strikingly, most anthocyanins and flavonols biosynthesis depends on SmMYB113. Similarly, following the knockout of OsMYB3 in black rice, 12 anthocyanins were completely undetected (Zheng et al., 2021). These results implied that R2R3MYBs play a critical role in anthocyanin biosynthesis. In addition, the anthocyanins in SmMYB113-OE4 were primarily glycosylated at the 5-position of the B-ring, leading to the formation of highly stable and soluble anthocyanidin 3,5diglucoside, such as delphinidin 3,5-diglucoside, malvidin 3,5-diglucoside, peonidin 3,5-diglucoside, and petunidin 3,5diglucoside. Similarly, Sharma et al. (2020) reported that cyanidin 3-xylosyl(sinapoylglucosyl)galactoside was the main anthocyanin present in AmRosea1-and AmDelila-overexpressed taproots, while cyanidin 3-xylosyl(feruloylglucosyl)galactoside was the main anthocyanin present in the black carrot cultivar 'Deep Purple'. The study of R2R3MYBs promoting anthocyanin biosynthesis has mainly focused on regulating structural gene expression (Karppinen et al., 2021), however, little is known about the regulation of modified genes.
The genes encoding glycosylation-modifying enzymes, including Sm3GT and Sm5GT, were analyzed according to the KEGG pathway annotation. Firstly, Sm3GT and Sm5GT relative expression levels in the peels and pulps of SmMYB113-OE4 and WT lines were analyzed by RT-qPCR (Supplementary Figures 5A,B). The results showed that Sm3GT and Sm5GT expression levels in the peels and pulps of the SmMYB113-OE4 line were significantly increased compared to WT. Subsequently, yeast one-hybrid assays (YIH) assay were performed between SmMYB113 and Sm3GT and Sm5GT promoters. However, no transcriptional regulation was found (Supplementary Figure 5C).

Comprehensive Comparison of the Two Metabolomic Data in Eggplant
Although the two metabolomic analyses were not performed simultaneously, the peels of No. 108 were included in both, and the metabolomics data could be connected by calculating the relative content of each sample to reference No. 108. The relative anthocyanin contents of purple peel cultivars No. 44,No. 64,and No. 76 were approximately 2-, 63-, and 109-fold greater than the anthocyanin content of No. 108. However, the relative anthocyanin contents in the peels and pulps of SmMYB113-OE4 were approximately 249-and 183-fold greater than the anthocyanin content of No. 108 ( Figure 5A). This implied that molecular genetic breeding technology could make supernatural phenomena, one major advantage of molecular genetic breeding. However, the contents of the other flavonoids (flavonoids without   Table 1). Since cyanidin is an upstream product in anthocyanin biosynthesis, and the "Tulipanin" content was the highest in the peels of No. 64, we speculated that "Tulipanin" directly causes the reddish-purple peel coloration in No. 64. Further analysis showed that the delphinidins were the main anthocyanins in the peels of No. 64,No. 76,, and the pulps of SmMYB113-OE4 (Figures 5C,D), indicating that SmMYB113 was indeed a critical transcript factor regulating anthocyanin biosynthesis in eggplant. However, the delphinidins structures differed between No. 64/No. 76 and SmMYB113-OE4 (Figures 2B, 4E). Compared with No. 64 and No. 76, SmMYB113 overexpression induced anthocyanins glycosylation at the 5-position of the B-ring. Similar results were reported by Sharma et al. (2020), who reported that overexpression of AmRosea1 and AmDelila (R2R3-MYB and bHLH TFs from A. majus) in taproots resulted in cyanidin 3-xylosyl(sinapoylglucosyl)galactoside accumulation and not cyanidin 3-xylosyl(feruloylglucosyl)galactoside accumulation. Although R2R3-MYB played a critical role in anthocyanin accumulation in plant tissues, other anthocyanin regulators also establish anthocyanin biosynthesis equilibrium. R2R3-MYB overexpression in plants cannot change the main anthocyanin class. However, it can disrupt their equilibrium by activating or enhancing the expression of downstream modifier genes, resulting in anthocyanin structure change.

CONCLUSION
Anthocyanins are important chemical components leading to the purple coloration of eggplant peels. This study used targeted metabolic profiling of flavonoids to investigate the flavonoid and anthocyanin structures of six eggplant cultivars with different peel colors. This method detected 167 flavonoid metabolites, including 16 anthocyanins, with various modifications. The 16 anthocyanins could be classified in cyanidins, delphinidins, and pelargonins. According to our data, the purple color of eggplant peels was positively correlated with the delphinidin/flavonoid ratio, and "Mirtillin" and "Tulipanin" were the major anthocyanins in the purple eggplant peels. In addition, SmF3 5 H expression level in eggplant peels was sufficient to explain the purple color, so the SmF3 5 H/SmF3 H expression ratio was unnecessary. Simultaneously, targeted metabolic profiling of flavonoids was performed on the fruit peels and pulps of SmMYB113 overexpressing eggplant lines. SmMYB113 overexpression significantly increased the anthocyanins and flavonols contents in peels and pulps. Compared with WT, the delphinidin/flavonoid ratio was significantly increased, while no difference or small decreases in the cyanidin/flavonoid and pelargonin/flavonoid ratios were found. The delphinidins significantly accumulated as a result of SmMYB113 overexpression in this study differed slightly from the data reported in other studies. Altogether, our data provide a glimpse into the flavonoid metabolites in eggplant peels with different colors and SmMYB113-overexpressed eggplant peels.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
GY: investigation and data curation. LL: investigation. MW: project administration. JL: project administration, data curation, software, and writing -original draft, review and editing. FY: project administration, and writing -review and editing.
All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We are very grateful for the reviewers for their comments and suggestions, which is helpful for improving the quality of our manuscript.