Comparative Transcriptome Analysis of Purple and Green Non-Heading Chinese Cabbage and Function Analyses of BcTT8 Gene

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis) is an important vegetative crop in the south of China. As an antioxidant, anthocyanin is the major quality trait for vegetables with purple leaves or petioles. However, the molecular biosynthetic mechanism of anthocyanin in non-heading Chinese cabbage has not been explained exclusively. In this study, two non-heading Chinese cabbage with contrasting colors in the leaves were used as the materials for RNA-seq. A total of 906 DEGs were detected, and we found that the anthocyanin and flavonoid biosynthetic pathways are significantly enriched in the purple NHCC. The transcriptome result was verified by RT-qPCR. Though bioinformatics analysis, BcTT8 was selected as the candidate gene for the regulation of anthocyanin synthesis, and the characterization of BcTT8 was elucidated by the functional analyses. The results proved that BcTT8 is a nucleus protein and phylogenetically close to the TT8 protein from Brassica. After silencing BcTT8, the total anthocyanin content of pTY-BcTT8 plants decreased by 42.5%, and the relative expression levels of anthocyanin pathway genes BcDFR, BcLODX and BcUF3GT-1 were significantly downregulated, while the transcription level of BcFLS was significantly upregulated. Compared with the wild type, the transgenic Arabidopsis showed obvious violet in the cotyledons part, and the anthocyanin biosynthetic genes such as AtDFR and AtLODX were significantly upregulated. In conclusion, BcTT8 is critical in the anthocyanin synthesis process of non-heading Chinese cabbage. Our findings illustrated the molecular mechanism of anthocyanin biosynthesis in non-heading Chinese cabbage.


Introduction
Anthocyanins are a type of water-soluble pigment that belongs to the flavonoid family, and they play a role in plant color, development, and reactions to their biotic and abiotic environments [1]. Except for these biological functions in the plant, they are also involved in human health, since they can be employed to prevent cardiovascular and neurological illnesses [2]. However, the biosynthesis of anthocyanins is also influenced by the abiotic stress, such as temperature, high light intensity, sucrose, UV irradiation, and drought [3][4][5][6][7].
In many plant species, the anthocyanin production pathway is conserved and well elucidated [8][9][10]. There are four steps for anthocyanin biosynthesis. The first key step is that chalcone synthase (CHS) catalyzes the production of naringenin chalcone from one molecule of 4-coumaroyl CoA and three molecules of malonyl CoA. Then, the production of naringenin, dihydrokaempferol, dihydroquercetin, or dihydromyricetin is catalyzed by the chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3 -hydroxylase (F3 H)

Plant Materials
The experimental materials were planted and located on the 15th September of 2020 in Jiangsu Agricultural Expo Park (119 • 01 N, 31 • 09 E). After growing for two months, we collected the leaves of purple NHCC 'HP072 and green NHCC 'HG072 to perform the experiment. Three biological replicates were quickly put in the liquid nitrogen and then sequenced by the company Biomarker (Beijing, China).

Determination of Total Anthocyanin Content
The total anthocyanin content was detected by the pH differential method [28]. First of all, 100 mg of fresh leaves were dipped in 3.4 mL methanol (40% acetic acid) and sonicated for 30 min; then, they were centrifuged for 10 min at 3000 rpm. All supernatants were filtered with a 0.45 µm filter. The filtrate was diluted 20 times, taken in two 1 mL samples, and reacted with 4 mL KCl (pH 1) and 4 mL NaAc (pH 4.5), respectively. After incubation for 30 min at room temperature, we measured the absorbance at 510 nm and 700 nm in an Enzyme Linked Immunosorbent Assay (ELISA) and calculated the total anthocyanin content.

Transcriptome Analysis of Green and Purple NHCC
An mRNA isolation kit was utilized to isolate the total mRNA from the leaves (Aidlab, Beijing, China). The quality of mRNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). The NEBNext UltraTM RNA Library Prep Kit for Illumina was used (NEB, Ipswich, MA, USA) to generate the sequencing libraries. The Illumina HiSeq2500 platform (San Diego, CA, USA) at Biomarker (Beijing, China) was applied to sequence the libraries. The FPKM (fragments per kilobase of transcript per million fragments mapped) method was applied to calculate the gene expression levels. The NHCC001 genome was used as the reference genome [29].

Expression of Anthocyanin-Related Pathway Genes in Green and Purple NHCC
To verify the results of transcriptome analysis, RT-qPCR was carried out for anthocyanin pathway genes. The reverse transcription of mRNA was used the Evo M-MLV RT Kit II (Accurate Biotechnology, Hunan, China) as directed by the protocols. RT-qPCR was carried out on the ABI StepOne (Applied Biosystems, Waltham, MA, USA) with Hieff ® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) in triplicate. Data were normalized with BcACTIN gene of NHCC, and the 2 −∆∆CT method was employed for analysis [31]. The gene-specific primer sequences are listed in Supplementary Table S1.

Subcellular Localization Assays and Analysis of Phylogenetic Tree
We amplified the coding sequence of BcTT8 using the gene-specific primers (Supplementary Table S1) and then cloned it into the PRI101 vector with a CaMV35S promoter. The construct was transformed into A. tumefaciens strain GV3101, and we resuspended the overnight cultures of A. tumefaciens strains with infiltration buffer (10 mM MgCl 2 , 10 mM MES, and 0.1 mM acetosyringone) to OD 600 at 0.8 and incubated them at room temperature for 4 h. The suspension was infiltrated into Nicotiana. benthamiana leaves. The injected plants were grown under the appropriate growth condition for about 60 h; next, the leaf samples were observed using the Laser Scanning Confocal Microscope (Zeiss LSM780); 35S:GFP alone served as the control. A neighbor-joining phylogenetic tree was constructed with MEGA X (1000 bootstrap replicates).

Silencing of BcTT8 through VIGS System
To silence of BcTT8, we designed a self-hybridizing palindromic oligonucleotide of 80 nt (Supplementary Table S1) following the protocol [32]. The primers p-TYMV-F and p-TYMV-R were used to identify the pTY-BcTT8 plasmid with the expected size (1566 nt). The total of 50µg purified pTY-BcTT8 plasmid was diluted with 50µL ddH 2 O; then, we mixed the plasmid with 0.1 M spermidine, 10 µL gold power and 0.1 M CaCl 2 in the 2 mL tubes on ice for 20 min. The mixture was centrifuged at 12,000 rpm for 15 s, and it was washed 4 times using the ethanol (100%). For infecting, we utilized the particle bombardment, and the empty VIGS vector (pTY-S) plasmid was inoculated as a control.

Overexpression of BcTT8 in Arabidopsis
The coding sequence of BcTT8 was cloned into vector PRI101-GFP; BcTT8-GFP plasmid was transformed into Agrobacterium tumefaciens strain GV3101 and cultured in LB liquid medium with antibiotics (50 mg·L −1 kanamycin and 50 mg·L −1 rifampicin). We conducted this experiment by the floral dip method [33]. Overnight cultures of A. tumefaciens strains were resuspended and diluted using the 5% sucrose solution buffer (pH 5.8) containing 0.01-0.05% (vol/vol) Silwet L-77 to OD 600 ≈0.8. Then, we dipped the Arabidopsis inflorescences for 60 s until the resuspended Agrobacterium cells carrying the BcTT8 gene were transferred. To obtain the transformants, the treated plants were selected with the solid medium with 50 mg·L −1 kanamycin and 160 mg·L −1 timentin.

Statistical Analysis
We analyzed the data through Microsoft Excel 2021 and the statistical significance of the differences between the two cultivars was determined with by an unpaired t-test with SPSS 22.0. Significant differences (p < 0.05) were indicated with different letters.

Samples Expression Pattern and Differentially Expressed Genes Clustering
In our study, we measured the total anthocyanin content in the two non-heading Chinese cabbage varieties; the total anthocyanin content of purple NHCC is 3.5 folds higher than the green one, which is 7.57 mg·100 g −1 and 2.26 mg·100 g −1 , respectively ( Figure S1). Based on the anthocyanin difference between the two cultivars, we performed comparative transcriptome analysis. The sequencing results contained a total of 906 DEGs, of which 520 DEGs showed upregulation and 386 DEGs showed downregulation ( Figure 1 and Supplementary Table S2) Among these, we annotated 11 classes of transcription factor family protein, and the bHLH family was comprised four genes, of which only BcTT8 was upregulated (Tables 1 and S3). The volcano map tells us the expression trends of these DEGs between green and purple samples; the red dots and green dots present the upregulation and downregulation of DEGs, respectively, while the black dots mean genes without a significant difference in expression between the two samples.

Differentially Expressed Genes GO Enrichment
In our result, the DEGs genes were enriched in the GO terms and further classified into three categories: the cell component category, biological process as well as molecular function process (Figure 2A and Supplementary Table S4). A total of 377 upregulated and 271 downregulated unigenes were annotated to GO terms in the biological process, of which most of the DEGs were mainly linked to the metabolic process, cellular process and single-organism process (Supplementary Table S5 Table S6). In the molecular function process, a total of 581 DEGs were enriched into this classification, and most of them were mainly related to the catalytic activity and binding terms (Supplementary Table S7).

Differentially Expressed Genes GO Enrichment
In our result, the DEGs genes were enriched in the GO terms and further classified into three categories: the cell component category, biological process as well as molecular function process (Figure 2A and Supplementary Table S4). A total of 377 upregulated and 271 downregulated unigenes were annotated to GO terms in the biological process, of which most of the DEGs were mainly linked to the metabolic process, cellular process and single-organism process (Supplementary Table S5 Table S6). In the molecular function process, a total of 581 DEGs were enriched into this classification, and most of them were mainly related to the catalytic activity and binding terms (Supplementary Table S7).   For the GO functional enrichment, the top 20 GO functional process was annotated ( Figure 2B). The 'anthocyanin-containing compound biosynthetic' process (GO:0009718) was not in the top20 GO biological terms, while it was also significantly enriched (2.71 × 10 −6 , p < 0.05) (Supplementary Table S8). A total of 15 DEGs involved in the 'anthocyanincontaining compound biosynthetic' process and the upregulated DEGs were comprised of the anthocyanin accumulation genes BcCHI-1, BcCHI-2, BcDFR, BcLODX, BcUF3GT-1, BcUF3GT-2, BcUF75C1, BcTT19-1, BcTT19-2, Bc5MAT and transcription factors BcTT8, BcMYBL2-1, and BcMYBL2-2 ( Table 2).

Verification of Transcriptome Result by RT-qPCR
In order to verify the results, several genes related to anthocyanin biosynthesis were selected and measured by RT-qPCR (Figure 4). The result indicated that the transcript expression levels of BcDFR (BraC09g018850), BcLODX (BraC03g052160) and BcUF3GT-1 (BraC06g022480) in HP072 were remarkably more upregulated than those in HG072. Similarly, the relative expression levels of transcription factors BcTT8 (BraC09g027820) and BcMYBL2-1 (BraC07g035800) were also significantly higher in HP072 than in HG072. However, the expression levels of the early anthocyanin biosynthesis genes (EBGs) BcCHS2 (BraC10g026540), BcF3H (BraC02g029180) and BcF3 H (BraC08g015770) showed no difference between these two samples ( Table 4). The relative expression levels of these genes were consistent with the transcriptome analysis result. BcMYBL2-1 (BraC07g035800) were also significantly higher in HP072 than in HG072. However, the expression levels of the early anthocyanin biosynthesis genes (EBGs) BcCHS2 (BraC10g026540), BcF3H (BraC02g029180) and BcF3′H (BraC08g015770) showed no difference between these two samples ( Table 4). The relative expression levels of these genes were consistent with the transcriptome analysis result.

Characterization and Phylogenetic Analysis of BcTT8
The BcTT8 homologous clone result showed that it encodes a 1566 bp nucleotide sequence and the ORF encodes a full function protein with 521 amino acids. Structure analysis results demonstrated that BcTT8 belongs to the bHLH family, which contains the conserved bHLH-MYC-N and the bHLH superfamily domains ( Figure S2). Multiple sequences analysis for BcTT8 and other homologous proteins ( Figure S3). A phylogenetic

Characterization and Phylogenetic Analysis of BcTT8
The BcTT8 homologous clone result showed that it encodes a 1566 bp nucleotide sequence and the ORF encodes a full function protein with 521 amino acids. Structure analysis results demonstrated that BcTT8 belongs to the bHLH family, which contains the conserved bHLH-MYC-N and the bHLH superfamily domains ( Figure S2). Multiple sequences analysis for BcTT8 and other homologous proteins ( Figure S3). A phylogenetic tree was performed to analyze the homologous relationship between BcTT8 and similar bHLH proteins in other species. The result showed that BcTT8 had the closest phylogeny with BoTT8 (Brassica oleracea var. botrytis) ( Figure 5).

Subcellular Localization of BcTT8
We constructed a 35S:BcTT8-GFP fusion vector to analyze the subcellular localization of BcTT8 protein. The suspension was infiltrated into N. benthamiana leaves. In the cell nucleus, we observed the BcTT8-GFP fusion protein while the empty vector GFP protein was observed in both the nucleus and the cytoplasm, which indicated the BcTT8 functions in the cell nucleus ( Figure 6).

Expression Analysis of Structural Genes after Silencing of BcTT8
In this study, we obtained from these plants emerged color fading, which was one of the viral symptoms. However, the color variations among control plants, infected pTY-S plasmid plants and the infected pTY-BcTT8 plants were obviously different. Both the viral

Subcellular Localization of BcTT8
We constructed a 35S:BcTT8-GFP fusion vector to analyze the subcellular localization of BcTT8 protein. The suspension was infiltrated into N. benthamiana leaves. In the cell nucleus, we observed the BcTT8-GFP fusion protein while the empty vector GFP protein was observed in both the nucleus and the cytoplasm, which indicated the BcTT8 functions in the cell nucleus ( Figure 6).

Subcellular Localization of BcTT8
We constructed a 35S:BcTT8-GFP fusion vector to analyze the subcellular localization of BcTT8 protein. The suspension was infiltrated into N. benthamiana leaves. In the cell nucleus, we observed the BcTT8-GFP fusion protein while the empty vector GFP protein was observed in both the nucleus and the cytoplasm, which indicated the BcTT8 functions in the cell nucleus ( Figure 6).

Expression Analysis of Structural Genes after Silencing of BcTT8
In this study, we obtained from these plants emerged color fading, which was one of the viral symptoms. However, the color variations among control plants, infected pTY-S plasmid plants and the infected pTY-BcTT8 plants were obviously different. Both the viral plants appeared to have color fading, but the one inoculated with pTY-BcTT8 presented

Expression Analysis of Structural Genes after Silencing of BcTT8
In this study, we obtained from these plants emerged color fading, which was one of the viral symptoms. However, the color variations among control plants, infected pTY-S plasmid plants and the infected pTY-BcTT8 plants were obviously different. Both the viral plants appeared to have color fading, but the one inoculated with pTY-BcTT8 presented barely violet (Figure 7A), and the silencing efficiency of BcTT8 expression was about 50% compared with control ( Figure 7B). We performed the RT-qPCR assay for analyzing the transcription expression levels of anthocyanin synthesis-related genes. The expression levels of BcCHS, BcCHI and BcF3H were significantly increased in pTY-BcTT8 plants, while BcF3 H showed no difference between pTY-S and pTY-BcTT8 plants. FLS (flavonol synthase) is regarded as the key gene for the biosynthesis of flavonols, and in the present study, the BcFLS showed significantly high expression in pTY-BcTT8 plants. The expression levels of BcDFR, BcLODX and BcUFG3T-1 were significantly declined in pTY-BcTT8 plants compared with pTY-S plants ( Figure 7C).  Figure 7C). We determined the total amount of anthocyanin content; the content of pTY-BcTT8 silencing plants was about 57.5% for the content of pTY-S plants, which was 1.48 mg·100 g −1 and 2.57 mg·100 g −1 , respectively ( Figure 7D). We proposed that the silencing of BcTT8 caused the redirection of metabolism flux to flavonol synthase that reduced the anthocyanin accumulation.

Heterologous Expression Analysis of BcTT8 in Arabidopsis
In order to elucidate the function of BcTT8, we constructed a 35S:BcTT8 vector using an Agrobacterium-mediated floral dip method. The coding sequence of BcTT8 was 1566 bp, and three transgenic plants were selected from the MS solid medium ( Figure S4). Compared with wild-type plants, BcTT8-overexpressed plants had increased transcription levels of anthocyanin biosynthesis pathway genes. AtCHS, AtCHI, AtF3H, AtF3′H, AtDFR, AtLODX, and AtUF3GT were all significantly upregulated ( Figure 8A). The cotyledons of transgenic plants presented obviously violet, but the WT still appeared green ( Figure 8B), which demonstrated that BcTT8 promoted anthocyanin synthesis in Arabidopsis. We determined the total amount of anthocyanin content; the content of pTY-BcTT8 silencing plants was about 57.5% for the content of pTY-S plants, which was 1.48 mg·100 g −1 and 2.57 mg·100 g −1 , respectively ( Figure 7D). We proposed that the silencing of BcTT8 caused the redirection of metabolism flux to flavonol synthase that reduced the anthocyanin accumulation.

Heterologous Expression Analysis of BcTT8 in Arabidopsis
In order to elucidate the function of BcTT8, we constructed a 35S:BcTT8 vector using an Agrobacterium-mediated floral dip method. The coding sequence of BcTT8 was 1566 bp, and three transgenic plants were selected from the MS solid medium ( Figure S4). Compared with wild-type plants, BcTT8-overexpressed plants had increased transcription levels of anthocyanin biosynthesis pathway genes. AtCHS, AtCHI, AtF3H, AtF3 H, AtDFR, AtLODX, and AtUF3GT were all significantly upregulated ( Figure 8A). The cotyledons of transgenic plants presented obviously violet, but the WT still appeared green ( Figure 8B), which demonstrated that BcTT8 promoted anthocyanin synthesis in Arabidopsis.

Discussion
Transcriptome analysis is a powerful tool for selecting the differentially expressed genes (DEGs) with our samples, which are useful to find the candidate genes. Contrasting transcriptome analysis had been performed in two Pak-Choi, and they found that in the purple variety, 'flavonoid biosynthesis' was the only KEGG significantly enriched pathway, which comprises structural genes BrDFR, BrLODX, BrUF3GT-1, BrUF3GT-2, and BrUF75C1 [26]. As for the release of the NHCC001 genome [29], we identified several enriched anthocyanin-related pathways and further explain the mechanism of anthocyanin regulation. Our results have many differences with the previous studies, except for the 'flavonoid' pathway, the 'anthocyanin biosynthesis', 'starch and sucrose metabolism', and 'biosynthesis of secondary metabolites' pathways, which were also significantly enriched ( Figure 3). Phenylpropane and flavonoid pathway genes participate in synthesizing the precursors of anthocyanin, which is also a subgroup of flavonoid [15], so that genes that participate in the 'flavonoid' and 'phenylalanine metabolism' pathways were significantly enriched. What is more, catalyzing anthocyanin synthesis requires ample enzymes, and encoding these products costs a large amount of energy by starch hydrolysis [34]; thus, it makes sense that the 'starch and sucrose metabolism' pathway genes were significantly enriched in the purple NHCC HG072. The KEGG pathway enrichment results confirmed that several structural genes, BcDFR, BcLODX, BcUF3GT-1, BcUF3GT-2, and BcUF75C1, which are related to the flavonoid and anthocyanin pathways, showed significantly expression in purple leaves (Table 3). Our analyses are in accordance with the transcriptome profiling in Pak-Choi and red Chinese cabbage (Brassica Rapa), of which the BrDFR, BrLDOX, BrUF3GT, and BrUGT75C1-1 are highly expressed [26,35], and relevant studies have revealed that these genes are critical in the process of anthocyanin biosynthesis [36].
Utilizing comparative RNA sequencing, researchers found that MYB and bHLH TFs are involved in the anthocyanin biosynthetic pathway [37,38]. MYB and bHLH TFs could finely tune the expression of anthocyanin pathway genes, so it is crucial to analyze transcription factor expression levels that could provide thorough insights into the regulatory mechanism of anthocyanin synthesis. In our study, we identified that BcTT8 was more significantly expressed in purple leaves (Figure 4), indicating that BcTT8 functions as an anthocyanin biosynthetic regulator. Earlier studies reported that NnTT8 recovered anthocyanin accumulation in Arabidopsis tt8 mutant [18], and other bHLH family proteins were also proved to regulate anthocyanin biosynthesis in other higher plants [39][40][41]. In our study, both the pTY plants and pTY-BcTT8 plants presented color fading ( Figure 7A), which was a symptom of virus injection [42], but the silencing of BcTT8 led to more severe symptoms. BcTT8-silenced non-heading Chinese cabbage showed a notable downregula-

Discussion
Transcriptome analysis is a powerful tool for selecting the differentially expressed genes (DEGs) with our samples, which are useful to find the candidate genes. Contrasting transcriptome analysis had been performed in two Pak-Choi, and they found that in the purple variety, 'flavonoid biosynthesis' was the only KEGG significantly enriched pathway, which comprises structural genes BrDFR, BrLODX, BrUF3GT-1, BrUF3GT-2, and BrUF75C1 [26]. As for the release of the NHCC001 genome [29], we identified several enriched anthocyanin-related pathways and further explain the mechanism of anthocyanin regulation. Our results have many differences with the previous studies, except for the 'flavonoid' pathway, the 'anthocyanin biosynthesis', 'starch and sucrose metabolism', and 'biosynthesis of secondary metabolites' pathways, which were also significantly enriched ( Figure 3). Phenylpropane and flavonoid pathway genes participate in synthesizing the precursors of anthocyanin, which is also a subgroup of flavonoid [15], so that genes that participate in the 'flavonoid' and 'phenylalanine metabolism' pathways were significantly enriched. What is more, catalyzing anthocyanin synthesis requires ample enzymes, and encoding these products costs a large amount of energy by starch hydrolysis [34]; thus, it makes sense that the 'starch and sucrose metabolism' pathway genes were significantly enriched in the purple NHCC HG072. The KEGG pathway enrichment results confirmed that several structural genes, BcDFR, BcLODX, BcUF3GT-1, BcUF3GT-2, and BcUF75C1, which are related to the flavonoid and anthocyanin pathways, showed significantly expression in purple leaves (Table 3). Our analyses are in accordance with the transcriptome profiling in Pak-Choi and red Chinese cabbage (Brassica Rapa), of which the BrDFR, BrLDOX, BrUF3GT, and BrUGT75C1-1 are highly expressed [26,35], and relevant studies have revealed that these genes are critical in the process of anthocyanin biosynthesis [36].
Utilizing comparative RNA sequencing, researchers found that MYB and bHLH TFs are involved in the anthocyanin biosynthetic pathway [37,38]. MYB and bHLH TFs could finely tune the expression of anthocyanin pathway genes, so it is crucial to analyze transcription factor expression levels that could provide thorough insights into the regulatory mechanism of anthocyanin synthesis. In our study, we identified that BcTT8 was more significantly expressed in purple leaves (Figure 4), indicating that BcTT8 functions as an anthocyanin biosynthetic regulator. Earlier studies reported that NnTT8 recovered anthocyanin accumulation in Arabidopsis tt8 mutant [18], and other bHLH family proteins were also proved to regulate anthocyanin biosynthesis in other higher plants [39][40][41]. In our study, both the pTY plants and pTY-BcTT8 plants presented color fading ( Figure 7A), which was a symptom of virus injection [42], but the silencing of BcTT8 led to more severe symptoms. BcTT8-silenced non-heading Chinese cabbage showed a notable downregulation of anthocyanin biosynthetic genes BcDFR, BcLODX, and BcUF3GT, while the transcription level of BcFLS increased considerably ( Figure 7C). Previous studies had proved that in other plant species, bHLH transcription factors could activate the expression of DFR, ANS, and UFGT, which improve the anthocyanin content [16,17]; thus, we proposed that in nonheading Chinese cabbage, transcription factor BcTT8 also facilitates the similar function, which could explain the downregulation of the LBGs and the decrease in anthocyanin production. We should mention that the production of flavonols and anthocyanins share the same biosynthesis pathway and compete for the same precursors. Flavonol synthase (FLS) may direct the dihydroflavonol precursors to the flavonol route [8]. In our study, BcFLS exhibited significant upregulation in the BcTT8-silencing plants as the anthocyanin content decreased dramatically. The metabolic flux redirection was also observed in other higher plants. Mutations in ScbHLH17 prevented the biosynthesis of anthocyanins in white Seneclo cruentus cultivars, and the RNAi silencing lines of anthocyanidin reductase (ANR) induced a redirection of the proanthocyanidin as well as the flavonol biosynthesis pathway, causing a reduction in anthocyanin synthesis in strawberry [43,44].
In Caryophyllales plants, the suppression of DFR and ANS resulted in the lack of anthocyanin, but the ectopic overexpression of these two genes induced anthocyanin accumulation [45]. In the BcTT8-overexpressed lines, the relative expression levels of anthocyanin structural genes showed significant upregulation, especially the LBGs AtDFR, AtLODX and AtUF3GT, causing the transgenic plants to present obviously violet ( Figure 8A,B).
Brassicaceous vegetables have been receiving scientific attention for many years because numerous studies reported that eating these vegetables would reduce the risk of some chronical diseases and kinds of cancer [46,47]. The main reason for that is that brassicaceous vegetables contain various phytonutrients such as the polyphenol, glucosinolates, carotenoid or terpenoid groups. Currently, purple brassicaceous vegetables, including nonheading Chinese cabbage, Chinese cabbage, Zicaitai, and kale have become increasingly popular not only for their attractive colors but also for the benefits they bring to the public. An increasing number of studies have pointed out that diets in anthocyanins help lower the risk of cancer, cardiovascular diseases, diabetes, oxidative stress, inflammation, and related diseases [48][49][50]. Non-heading Chinese cabbage is a nutrition-rich vegetable that is widely consumed worldwide, but the molecular mechanism of anthocyanin synthesis is under explored. In this study, we identified that BcCHI-1, BcCHI-2, BcDFR, BcLODX, BcUF3GT-1, BcUF3GT-2, BcUF75C1, and one bHLH transcription factor BcTT8 were significantly upregulated in purple NHCC, and functional analyses demonstrated that BcTT8 could positively promote anthocyanin accumulation. Our findings illustrated the anthocyanin molecular regulation of non-heading Chinese cabbage, which could provide the theoretical basis for breeding high anthocyanin content non-heading Chinese cabbage cultivars.

Conclusions
In the present study, we have a further understanding of the anthocyanin biosynthetic pathway in non-heading Chinese cabbage through the comparative transcriptome analysis. A number of DEGs related to anthocyanin and flavonoid biosynthesis pathways were identified, indicating their important roles in the anthocyanin biosynthesis in NHCC. In addition, we explained the function of BcTT8 gene and demonstrated that BcTT8 is of great importance in anthocyanin synthesis.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13060988/s1. Figure S1: Photographs of green NHCC 'HG072 and purple NHCC 'HP072 (A) and total anthocyanin content of these two cultivars (B); Figure S2: Conversed domains analysis of BcTT8; Figure S3: Amino acids sequences blast of bHLH proteins; Figure S4: Agarose gel electrophoresis picture of BcTT8 transgenic plants verification by RT-PCR. Table S1: Primer pairs used in this study; Table S2: All the DEGs from the transcriptome result; Table S3: Differentially expressed transcription factors list from the transcriptome result;  Table S4: Classification of GO terms; Table S5: GO classification of biological process; Table S6: GO classification of cell component; Table S7: GO classification of molecular function; Table S8: Result of GO enrichment annotation: Table S9: Result of KEGG pathway annotation.
Author Contributions: L.T. completed the experiments and wrote the manuscript; D.X. participated in plotting; Y.Y. helped data collection and analysis; H.W. was involved in the transcriptome data analyses; J.W. and T.L. helped the revision of this manuscript; X.H. provided materials used in this study; Y.L. designed the study and provided financial support. All authors have read and agreed to the published version of the manuscript.