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Anthocyanin Accumulation, Antioxidant Ability and Stability, and a Transcriptional Analysis of Anthocyanin Biosynthesis in Purple Heading Chinese Cabbage (Brassica rapa L. ssp. pekinensis) Qiong He,† Zhanfeng Zhang,‡ and Lugang Zhang*,† †

College of Horticulture, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, People’s Republic of China ‡ College of Plant Protection, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, People’s Republic of China S Supporting Information *

ABSTRACT: Heading Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a significant dietary vegetable for its edible heading leaves in Asia countries. The new purple anthocyanin-rich pure line (11S91) was successfully bred, and the anthocyanins were mainly distributed in 2−3 cell layers beneath the leaf epidermis, whereas siliques and stems accumulated only a cell layer of anthocyanins. The anthocyanins of 11S91 were more stable at pHs below 3.0 and temperatures below 45 °C. The total antioxidant ability was highly positive correlated with the anthocyanin content in 11S91. Thirty-two anthocyanins were separated and identified, and 70% of them were glycosylated and acylated cyanidins. The four major anthocyanins present were cyanidin-3sophoroside(p-coumaroyl)-5-glucoside(malonyl), cyanidin-3-sophoroside(ferulyl)-5-glucoside(malonyl), cyanidin-3sophoroside(sinapyl-p-coumaroyl)-5-glucoside(malonyl), and cyanidin-3-sophoroside-(sinapyl-ferulyl)-5-glucoside(malonyl). According to the expression of biosynthetic genes and the component profile of anthocyanins in 11S91 and its parents, regulatory genes BrMYB2 and BrTT8 probably activate the anthocyanin biosynthesis but other factors may govern the primary anthocyanins and the distribution. KEYWORDS: anatomical feature, anthocyanin, antioxidant ability, Chinese cabbage, gene expression, LC-MS/MS, purple heading

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INTRODUCTION

examined the anthocyanin accumulation of Chinese cabbages with purple or red edible head. Colorful, nutritious, and low-cost vegetables are needed to meet demand and improve human health. Heading Chinese cabbage (Brassica rapa L. ssp. pekinensis), a crop native to China, is an important fresh and cheap market vegetable in Asian countries. In this report, a new purple heading Chinese cabbage pure line was examined for anthocyanin biosynthetic gene expression, composition, stability, distribution, and antioxidant ability. Our results will enable the creation of more economically valuable vegetable sources, supply new insights into the molecular mechanisms of anthocyanin biosynthesis, and provide a new anthocyanin-rich functional food in Brassica crops.

Anthocyanins, the main water-soluble pigments that accumulate in leaves, petals, sepals, and fruits of plants, endow color, attract insects for pollination, and protect against biotic and abiotic stresses.1 Moreover, anthocyanins are well-known for their applications in human health to protect against cardiovascular disease, diabetes, cancer, and vision loss due to their free radical scavenging abilities.2 Anthocyanins in high concentrations have no toxic, teratogenic, or mutagenic characteristics and are used in the food industry.3 To date, many anthocyanin biosynthesis genes have been isolated.4 The activation of structural genes by transcription factors is the major mechanism of anthocyanin biosynthesis in plants, and an MYB-bHLH-WD40 (MBW) complex usually formed by MYB, bHLH, and a WD40 factor is involved in this pathway.4,5 Furthermore, the activation of biosynthesis can be regulated by only an R2R3-MYB factor6,7 and induced by sugar, light, etc.8,9 The main anthocyanidins in Brassica plants are cyanidins.10 Anthocyanin biosynthesis in Brassica crops is thought to be regulated by the MYB and bHLH transcription factors.11−14 In particular, in the red Brassica rapa (with red outer leaves), BrMYB2-2 and BrTT8 activate BrANSs and BrDFRs to control anthocyanin synthesis.15,16 Results from mapping of the purple genes vary, such as the linkage groups A09 and A03.17,18 Currently, much attention has been paid to breeding new purple/red Chinese cabbages,19 preliminarily mapping the purple genes, developing molecular markers, and identifying anthocyanins in Brassica genus. However, few reports have © 2015 American Chemical Society

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MATERIALS AND METHODS

Plant Materials. An inbred line [94S17 (94S, Figure 1B)] of Chinese cabbage with white heading leaves was utilized as the female parent. The male parent, 95T2 (95T, Figure 1C), was an inbred line of flowering Chinese cabbage with deep purple leaves and stems. A new pure line [11S91 (11S, Figure 1A,D)] with stable inheritance of purple heading leaves was bred from the F1 of 94S and 95T, with subsequent continuous self-crossing for 10 generations from selected single plant. All materials were grown outdoors in autumn in Yangling, Shaanxi Province, China. When the samples matured (about 4 months after

Received: Revised: Accepted: Published: 132

September 24, 2015 November 30, 2015 December 3, 2015 December 28, 2015 DOI: 10.1021/acs.jafc.5b04674 J. Agric. Food Chem. 2016, 64, 132−145

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Journal of Agricultural and Food Chemistry

described.22 The results were expressed as the average of three biological replicates. Effects of Temperature and pH on the Stability of Anthocyanins. Temperature and pH are two important factors that affect the stability of anthocyanins in processing, storage, and cooking. The effects of temperature and pH on the stability of anthocyanins were analyzed as in a previous report23 with slight modifications. Prepared extracts of 95T and the P01 of 11S were transferred into phosphate buffered solutions (at pH 1.0, 3.0, 5.0, 7.0, and 9.0) with consistent dilution. Each pH was tested with 6 temperature treatments in the dark (at 4, 25, 45, 65, 85, and 100 °C). After 2 h, λmax absorbance of the anthocyanins was measured in triplicate using the UV−visible spectrophotometer. Measurement of Total Antioxidant Ability. The oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), and DPPH assays are often used to evaluate the total antioxidant abilities of vegetables and fruits in the food industry.24 Thus, to better assess the total antioxidant ability of Chinese cabbages, these three methods were performed as previously described24 with some modifications. All of the operations were performed in the dark. The results were expressed as the average of three biological replicates. The total antioxidant ability was expressed as the Trolox equivalent antioxidant capacity (TEAC, μM TE g−1 fresh mass). ORAC Assay. An automated microplate reader (TECAN, Mannedorf, Switzerland) and black 96-well plates without covers (Nunc, Roskilde, Denmark) were used in the ORAC procedure. All the reactions were conducted in phosphate buffer (75 mM, pH 7.4) at 37 °C in a total volume of 300 μL. Peroxyl radicals were generated using 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH, SigmaAldrich, USA), which was freshly prepared for each run with a reaction concentration of 25 mM. Fluorescein sodium (FL, Sigma-Aldrich, USA) was used as the substrate with a reaction concentration of 63 nM. A Trolox standard (10 μM), 100-fold diluted prepared extracts, FL, and AAPH solutions were prepared in phosphate buffer. The reaction mixture contained the sample (Trolox or prepared extracts), FL, phosphate buffer, and AAPH in a volume ratio of 1:1:1:7, and phosphate buffer was used as a blank sample. To collect data, the plate was read at excitation and emission wavelengths of 485 and 538 nm, respectively, at 37 °C in 2 min intervals for 70 cycles. The Trolox standard curve was linear between 0 and 10 μM. FRAP Assay. 10-fold-diluted prepared extracts (200 μL) were allowed to react with 2.8 mL of FRAP solution for 1 h in the dark at 37 °C. The formation of a ferrous tripyridyltriazine complex was measured at 596 nm using the UV−visible spectrophotometer. The standard curve was linear between 0 and 300 μM Trolox. DPPH Assay. The working solution was acquired by mixing 0.1 mL of 2-fold-diluted sample extract and 1.9 mL of 2,2-diphenyl-1picrylhydrazyl (DPPH, Sigma-Aldrich, USA) solution (0.1 mM), and the mixture was incubated at room temperature in the dark for 1 h. Then, the absorbance was measured at 517 nm using the spectrophotometer. The standard curve was linear between 0 and 800 μM Trolox. Liquid Chromatography Tandem Mass Spectrometry (LCMS/MS) Analysis of Anthocyanins. The extraction and purification of anthocyanins were performed according to a previous report.25 Mass spectra were acquired using an electrospray ionization mass spectroscopy method as described in a previous report25 with slight modifications. Briefly, the purified sample collected from the C-18 Sep-Pak cartridge (Waters, Milford, MA, USA) was diluted with a 0.1% formic acid aqueous solution. The purified anthocyanins were analyzed using an LTQ XL linear ion trap mass spectrometer (Thermo, Wilmington, MA, USA). Liquid chromatography separations were performed with an XTerra MS C18 column (125 Å pore size, 5 μm, 150 mm × 2.1 mm; Waters, Milford, MA, USA). Anthocyanin elution was performed with the following gradient: 5% mobile phase A for 7 min, 5−6% linear A for 1 min, 6−20% linear A for 24 min, 20−30% linear A for 6 min, 30−40% linear A for 4 min, 40−95% linear A for 1.5 min, and a system hold at 95% A for 5 min. Mobile phase A was acetonitrile with 0.1% formic acid, and mobile phase B was an aqueous solution containing 5% acetonitrile and 0.1%

Figure 1. Appearance of Chinese cabbages. (A) Longitudinal cutting view about the head of 11S. (B) Longitudinal cutting view about the head of 94S. (C) 95T. (D) Sequence numbering of the heading leaves of 11S. Interior leaves less than 8 cm in length were labeled as P01. The scale bar is 10 cm. sowing), they were picked, frozen in liquid nitrogen, and immediately stored in a −80 °C freezer (Sanyo, Osaka, Japan). In particular, the innermost heading leaves less than 8 cm in length were labeled as P01, and the other leaf samples of 11S were classified with serial numbers from the interior P01 to the external P21 leaves of the edible head (Figure 1D) because the head of 11S showed different degrees of purple coloring. Samples of 94S and 95T were mixed whole edible tissues. Each sample was analyzed in triplicate, and three biological replicates were conducted. Anatomical Observation of Plant Tissues. Samples of leaves, flowering stalks, and siliques were prepared for anatomical observation by free-hand sectioning as previously described.20 The leaf samples were picked at the heading mature stage. Then, the plant was stored to overwinter and bloom in the coming spring (about 9 months after sowing). The flowering stalks and siliques were picked from the same plant in the same farm. Sample observation was performed with a fluorescent microscope (Olympus, Tokyo, Japan) at an applicable magnification in a bright field. Determination of Pigments. Chinese cabbages include three main pigments, namely, anthocyanins, chlorophylls, and carotenoids. Total anthocyanins were determined using a UV−visible spectroscopy method as previously described with some modifications.21 Briefly, every frozen sample (1 g) was crushed into powder in a mortar with liquid nitrogen, transferred into a 10 mL centrifuge tube with a cover, combined with up to 10 mL of 1% hydrochloric acid in methanol, and incubated overnight in the dark at 4 °C. The mixtures were subsequently centrifuged at 12,000 rpm for 15 min at 4 °C. Anthocyanin absorbance, which differs according to the components and plant origin, ranges from 510 to 530 nm. Thus, the samples were collected and diluted for direct measurement at the λmax (530 nm, Figure S1) and at 700 nm using a UV−visible spectrophotometer (Thermo, Wilmington, MA, USA) in a 0.2 M potassium chloride buffer (pH 1.0) and a 0.45 M sodium acetate buffer (pH 4.5), respectively. These dilutions were equilibrated for 3 h at 4 °C in the dark. Total anthocyanins were calculated using the equation in a previous report.21 Total contents of chlorophylls and carotenoids were measured using a UV−visible spectroscopy method as previously 133

DOI: 10.1021/acs.jafc.5b04674 J. Agric. Food Chem. 2016, 64, 132−145

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Figure 2. Free-hand cross sections of different tissues from Chinese cabbages. Samples A, B, C, samples D, E, F, and samples G, H, I are from 11S, 95T, and 94S, respectively. Samples A, D, G, samples B, E, H, and samples C, F, I were cut from the leaves, flowering stalks, and siliques, respectively. The scale bar is 200 μm. Arrowheads indicate anthocyanins. formic acid. The flow rate was 0.2 mL/min. The mass spectrometer was used in the positive electrospray ionization mode. Nitrogen was used as the sheath gas (18.0 arbitrary units) and auxiliary gas (3.0 arbitrary units). The spray voltage was set to 4.5 kV, and the ion transfer capillary temperature was 275 °C. The anthocyanins were scanned and fragmented using data-dependent MS/MS. The data were acquired and processed using the Xcalibur 2.1 software (Thermo, USA). Samples were pooled from three biological replicates, and relative quantification was achieved using an external standard, cyanidin-3-O-glucoside (50 μg mL−1, Must, Chengdu, China). Real-Time Quantitative PCR (qRT-PCR) Analysis. Total RNA was extracted using the Bizol reagent according to the manufacturer’s instructions (Bioer, Hangzhou, China). To remove contaminating genomic DNA, all the RNA extracts were treated with DNaseI according to the manufacturer’s instructions (TaKaRa, Japan). Subsequently, the first-strand cDNA was synthesized from the total RNA (500 ng) using the PrimeScript RT reagent kit according to the manufacturer’s instructions (TaKaRa, Japan). The synthesized cDNAs were diluted to 50 ng μL−1 in doubledistilled water (ddH2O) using a microspectrophotometer, and the concentrations were normalized according to the amplification of BrACTIN. The cDNAs were pooled from three biological replicates. The primers (listed in Table S1) for the amplification of anthocyanin biosynthetic structural genes and regulatory genes were diluted to 10 μM.

Each reaction mixture (20 μL) contained 7 μL of ddH2O, 2 μL of cDNA templates, 0.5 μL of forward primers, 0.5 μL of reverse primers, and 10 μL of SYBR Premier Ex TaqII mix (TaKaRa, Japan). PCR amplification was performed using three-step cycling conditions at 95 °C for 1 min, followed by 40 cycles of 95 °C for 20 s, 58 to 60 °C for 30 s, and 72 °C for 30 s. A melt curve analysis of the qRT-PCR samples revealed that only a single product was obtained for every gene primer reaction, confirming specific amplification. Additionally, each gene from Chinese cabbage typically shared high homology with an Arabidopsis gene (Table S1). To design genespecific amplification primers, Arabidopsis anthocyanin biosynthetic gene sequences were retrieved from the National Center for Biotechnology Information Database (http://www.ncbi.nlm.nih.gov/ ). Homologous genes from Chinese cabbages were screened from the Brassica Database (BRAD, http://brassicadb.org/brad/) using the Syntenic Genes or Annotations functions. All the primers were designed using Primer Premier 5.0 (Premier, Canada). All the qRT-PCR reactions were normalized using the CT value that corresponds to BrACTIN. The relative expression levels of the target genes were calculated according to the formula 2−ΔΔCT26 using the IQ5 optical system software (Bio-Rad, California, USA) according to the manufacturer’s recommendations. Statistical Analysis. Data were subjected to a one-way analysis of variance (ANOVA) using SPSS version 13.0 (SPSS, Chicago, IL, USA), and a bivariate analysis of Pearson correlation coefficients was 134


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Figure 3. Total anthocyanin, carotenoid, and chlorophyll contents and the total antioxidant activity of Chinese cabbages as determined by UV− visible spectroscopy assays. P01 to P21 are the heading leaves of 11S. The values are means ± SD. The different letters above each column are significantly different at p < 0.05 by Duncan’s test. calculated in a two-tailed test. A Duncan’s multiple-range test was determined for all the data at the 0.05 confidence level. Hierarchical Clustering Analysis. Gene expression that showed statistically significant changes related to anthocyanin biosynthesis and different anthocyanins in various leaves were grouped through a twoway hierarchical clustering methodology using the PermutMatrix software.27 The Euclidean distance and McQuitty’s method were used for gene aggregation; however, the Euclidean distance and Ward’s algorithm were used for anthocyanin aggregation.

under the epidermis result in a bright magenta color, which makes the purple heading Chinese cabbage a desirable vegetable for daily diet and landscaping. Analysis of Anthocyanins, Carotenoids, Chlorophylls Content, and Total Antioxidant Ability. Anthocyanins, chlorophylls, and carotenoids are three main pigments in Chinese cabbages but are not equally distributed. Carotenoids and anthocyanins are treated as dietary antioxidants.29 Both 95T and P01 had significantly higher levels of anthocyanins (Figure 3A), while the carotenoid and chlorophyll contents in 94S and 95T were both significantly higher than in 11S91 (Figures 3B,C). The line 95T had the highest content of anthocyanins (634.001 mg kg−1 FW), and 94S had a lower content of anthocyanins (30.615 mg kg−1 FW) (Figures 3A). The total anthocyanin contents of leaves from 11S differed greatly. The highest content was 394.279 mg kg−1 FW in the innermost leaves, which was less than that of 95T. Total anthocyanin content dropped dramatically from 394.279 mg kg−1 FW (in P01) to 17.812 mg kg−1 FW (in P13) in the internal heading leaves and then gradually and generally increased to 94.813 mg kg−1 FW (in P21) in the external heading leaves of 11S, which correlated with the head’s color change from deep purple to light purple (Figure 1D). In addition, lower concentrations of chlorophylls and carotenoids were observed in the heading leaves of 11S. In contrast, both of them were abundant in 94S, 95T, and the functional external leaf P21 of 11S (Figure 3B,C). Compared with the anthocyanin contents of the parents (Figure 3) and other plants, such as red cabbage (90 to 1820 mg kg−1 FW),14 blueberry (250 to 4950 mg kg−1 FW),21 black raspberry (4000 mg kg−1 FW), grape (250 to 2600 mg kg−1 FW),30 and jaboticaba (44 to 163 mg kg−1 FW),31 the purple heading Chinese cabbage 11S had relatively high levels of anthocyanins in the head.

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RESULTS AND DISCUSSION Distribution Features of Anthocyanins in Different Tissues. As important flavonoids, anthocyanins confer colorful appearances to plants. Microscopic observation of sections from the leaves of 11S revealed that the purple pigment mainly resided in three cell layers under the upper epidermis and two cell layers under the lower epidermis (Figures 2A and S2A). In contrast, the green chlorophyll layers were detected in 94S at the same locations (Figure 2G). Interestingly, in 95T, the number of purple layers decreased in both the upper and lower epidermises (Figures 2D and S2D). Previous reports have shown that anthocyanins accumulated in the top cell layer or contiguous two layers in leaves.12,14,28 Another report found that anthocyanins accumulated only in the upper epidermis in purple nonheading Chinese cabbage.18 Transections of flowering stalks and siliques showed that purple pigment areas were only a layer under the epidermis. Higher-level anthocyanin accumulation underneath the epidermal layers provides gorgeous color in the purple Chinese cabbage. In contrast, Chinese cabbages with green chlorophyll layers exhibited the common green color in different tissues (Figures 2G−I). In the same plant, anthocyanin accumulation is organ and development specific. However, in different species from the same genus, differences in anthocyanin distribution may be due to genetic discrepancies. Anthocyanins distributed in these cell layers 135


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Figure 4. Absorbance and extracts at different pHs and temperatures. Anthocyanins from 11S were collected from P01. The values are means ± SD. The letters above the white columns were analyzed for 95T, while letters contained in the gray columns were analyzed for 11S only. The different letters are significantly different at p < 0.05 by Duncan’s test.

FRAP, and DPPH, respectively (Table S2). In contrast, both carotenoid and chlorophyll contents were poorly correlated with the antioxidant activity (Table S2). In addition, the correlations among the three assays were extremely high (Table S3). The line 95T, which exhibited the highest anthocyanin content, had the highest TEAC (28.402 μM TE g−1 FW in the ORAC assay, 24.527 μM TE g−1 FW in the FRAP assay, and 13.898 μM TE g−1 FW in the DPPH assay; Figure 3D−F). The inner leaves of 11S, P01, had antioxidant values of 20.239 μM TE g−1 FW in the ORAC assay, 16.728 μM TE g−1 FW in the

The antioxidant activities of all samples were measured using ORAC, FRAP, and DPPH assays. As expected, the extracts from leaves with higher anthocyanin contents exhibited higher antioxidant abilities in the three different assays, and the antioxidant activity correlated well with the anthocyanin content (Figure 3). Further correlation analysis showed that the total antioxidant capacities obtained using the three different methods were extremely highly correlated with anthocyanin content, and the correlations coefficients were 0.978, 0.981, and 0.965 with the TEAC obtained using ORAC, 136


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Journal of Agricultural and Food Chemistry FRAP assay, and 6.667 μM TE g−1 FW in the DPPH assay (Figure 3D−F). In contrast, P09, P13, and P17 of 11S and 94S had much lower antioxidant abilities, ranging from 8.894 to 11.259 μM TE g−1 FW (ORAC), 4.964 to 7.214 μM TE g−1 FW (FRAP), and 1.743 to 2.896 μM TE g−1 FW (DPPH). However, compared with the total antioxidant capacity of the parents and other plants (Figure 3D−F), such as guava fruit (18.03 to 25.5 μM TE/g FW in an ORAC assay, 15.5 to 33.3 μM/g FW in a FRAP assay),24 the purple heading Chinese cabbage 11S had relatively high levels of antioxidant abilities. The free radical theory posits that free radical reaction is widely extant in living organisms and it is a dangerous killer to biological molecules, cells, and tissues.32 Anthocyanins are strong scavengers of reactive oxygen,33 which explains their high correlation with antioxidant capacity. As such, the high correlation between antioxidant capacity and anthocyanin content in the new purple heading Chinese cabbage implies that it may be a good and cheap resource with natural anthocyanins to human health. Effects of Temperature and pH on the Stability of Anthocyanins. Temperature and pH are two important factors that affect the stability and color of anthocyanins. Anthocyanin extracts of 11S and 95T had a maximum absorption peak at 530 nm in the visible spectrum and high absorbance readings at 330 nm (Figure S1). A pH jump from 1.0 to 9.0 directly induced bathochromic shifts from 530 to 610 nm of maximum absorbance in the visible range, with a concomitant fade in red coloring and enhanced blue/green coloring (Figures S3−6 and 4F−I). These results were similar to those in previous reports that the color of anthocyanins changes with pH from crimson (pH less than 3) to dark blue (pH greater than 7).34 In acidic solutions, the absorbance and color of 11S and 95T extracts were nearly identical at 4, 25, and 45 °C (Figure 4A−C), particularly at pH = 1.0. When the temperature exceeded 85 °C at pH ⩾ 7.0, the maximum absorption peak of anthocyanins from 11S and 95T disappeared, and the green/blue color faded to light yellow or colorless (Figures S7,8 and 4J,K). More than 90% of anthocyanins remained stable up to pH 3.3, whereas a pH above 4.5 rapidly decreased the stability.35 In acidic conditions, anthocyanin degradation is greater between pH 1.5 and 3.0 than at pHs below 1.5.23 Instability and color change are caused by reversible structural transformations. The main structure in acidic conditions (at pH 1−3), the red flavylium cation, transforms into a pseudobase (at pH 4−5, colorless) or a quinonoidal base (at pHs up to 9, blue).34 When heated, the blue quinonoidal base is in equilibrium with a chalcone form, and it is time-consuming and difficult to convert back to the original form.36 Therefore, at pHs below 3.0, these reports provide evidence that the anthocyanins of the purple heading Chinese cabbage 11S and its male red-colored parent were due to anthocyanins in the red flavylium cation form. However, the anthocyanin extracts at a pH of 5.0 were colorless (Figure 4F−K), which might have been caused by the transformation of anthocyanins into colorless pseudobase forms.34 When the pH exceeded 7.0 and the temperature was below 65 °C, a blue color appeared because of the formation of blue quinonoidal base.34 As the temperature was raised above 85 °C, the blue quinonoidal base was degraded to a faintly yellow chalcone. To sum up, extremely acidic (pH ⩽ 3.0) and moderate temperature (⩽45 °C) conditions protect anthocyanins of the purple heading Chinese cabbage from irreversible

degradation, which is meaningful for their utilization in food processing and storage. LC-MS/MS and Hierarchical Clustering Analysis of Anthocyanins. There are six common anthocyanins in plants, each with different colors due to the number of hydroxyl groups attached to the skeleton.34 Anthocyanins exist primarily as glycosidic conjugates of anthocyanins in vegetables, and glucose is the most prevalent glycosylated sugar in anthocyanins.37 Additionally, other monosaccharides (i.e., rhamnose, galactose, xylose, and arabinose) and disaccharides (i.e., sophorose, rutinose) are present in the anthocyanins. Furthermore, the glycosides are frequently acylated by aromatic and aliphatic acids such as p-coumaric, caffeic, ferulic, gallic, acetic, malonic, and malic acids.37,38 The identification of anthocyanins in Chinese cabbages initially relied on the anthocyanic profile from previous references.39−41 In this study, a total of 32 anthocyanins were found in the samples, and their relevant names are displayed (Table 1, Figures 5 and S9). Four types of anthocyanins were found in the purple Chinese cabbage: cyanidins, delphinidins, peonidins, and petunidins with parent ions at m/z 287, 303, 301, and 317, respectively. Glycosylated and acylated anthocyanins were the primary forms accumulated in 11S (Table 1 and Figure 5). Over 70% of the anthocyanins were cyanidins. The hierarchical clustering analysis in Figure 5 showed that 95T and the innermost leaves (P01) of 11S shared high similarity in anthocyanin content, as indicated by the purple color their grids shared, while 94S was more similar to the external leaves of 11S. These anthocyanins were further classified into four main categories. The first category was the cyanidin family in 95T and the inner leaves of 11S. This category included four predominant cyanidins at relatively high levels (ranging from 19.37 mg kg−1 FW to 71.69 mg kg−1 FW): cyanidin-3-sophoroside(p-coumaroyl)-5-glucoside(malonyl) (m/z = 1005; 46.657 mg kg−1 FW in P01, 25.967 mg kg−1 FW in P05, 13.258 mg kg−1 FW in P09, and 12.378 mg kg−1 FW in P013; 29.516 mg kg−1 FW in 95T), cyanidin-3-sophoroside(ferulyl)-5-glucoside(malonyl) (m/z = 1035; 71.691 mg kg−1 FW in P01, 28.956 mg kg−1 FW in P05, 17.909 mg kg−1 FW in P09, and 12.467 mg kg−1 FW in P013; 33.165 mg kg−1 FW in 95T), cyanidin-3-sophoroside(sinapyl-p-coumaroyl)-5glucoside(malonyl) (m/z = 1211; 19.369 mg kg−1 FW in P01, 11.507 mg kg−1 FW in P05, 3.815 mg kg−1 FW in P09, and 1.667 mg kg−1 FW in P013; 64.099 mg kg−1 FW in 95T), and cyanidin-3-sophoroside-(sinapyl-ferulyl)-5-glucoside(malonyl) (m/z = 1241; 27.348 mg kg−1 FW in P01, 16.160 mg kg−1 FW in P05, 6.356 mg kg−1 FW in P09, and 2.248 mg kg−1 FW in P013; 62.831 mg kg−1 FW in 95T). It is noteworthy that levels of the two former compounds were higher in P01 than in 95T and vice versa for the two latter compounds. In addition to the cyanidins, the second category was composed of three delphinidins and two cyanidins that displayed higher levels in purple Chinese cabbages, especially in 95T: delphinidin-3-sophoroside-5-glucoside (m/z = 789, 40.308 mg kg−1 FW in 95T), delphinidin-3-glucoside(sinapyl)-5-glucoside-7-glucoside (m/z = 995, 34.258 mg kg−1 FW in 95T), delphinidin-3-glucoside-5-glucoside (m/z = 627, 23.280 mg kg−1 FW in 95T), cyanidin-3-glucoside(malonyl)-5sophoroside (m/z = 859, 11.438 mg kg−1 FW in P01 and 18.398 mg kg−1 FW in 95T), and cyanidin-3-glucoside-5glucoside(malonyl) (m/z = 697, 9.148 mg kg−1 FW in P01 and 17.194 mg kg−1 FW in 95T). The differences in the principal 137


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(Table 1). Compared to the parents, 94S included a low content of anthocyanins in its white head (except for cyanidin3-sophoroside(caffeoyl)-5-sophoroside with an m/z = 1097 and a relatively high content of 7.993 mg kg−1 FW, and cyanidin-3glucoside-5-glucoside(malonyl) with an m/z = 697 at the content of 9.532 mg kg−1 FW; other compounds were less than 2.0 mg kg−1 FW), whereas the female parent 95T exhibited more types of anthocyanins and a deep purple appearance. The skeleton of the principal anthocyanins in 11S and 95T was cyanidin-3-sophoroside-5-glucoside. In recent reports, approximately 20 anthocyanins have been identified in purple bok-choy (Brassica rapa L. ssp. chinensis), and its main anthocyanins are cyanidin-3-sophoroside(malonyl)-5-arabinose(p-hydroxybenzoyl) and cyanidin-3rutinoside(caffeoyl-sinapoyl)-5-glucoside.13,18 In red cabbage (Brassica oleracea L. var. capitata), over 30 anthocyanins have been identified,3,42 and cyanidin-3-diglucoside-5-glucose comprises the core skeleton of these major anthocyanins for subsequent acylation. Approximately 10 anthocyanins have been found in violet cauliflower (Brassica oleracea L. var. botrytis), however, cyanidin-3-sophoroside(p-coumaryl)-5-glucoside is the main component.43 In purple tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee), cyanidin 3sophoroside(feruloyl-sinapoyl)-5-glucoside(malonyl) and 3sophoroside(feruloyl)-5-glucoside(malonyl) are the most abundant anthocyanins.44 Moreover, delphinidin and malvidin have been found in red cabbage.45 In this study, new delphinidins were found in 11S when it was compared to the red nonheading Chinese cabbage (Brassica rapa L. ssp. chinensis, with a purple color only in its rosette leaves).18 In Brassica vegetables, the predominant anthocyanins are cyanidins that are primarily glycosylated with glucose or disaccharides and acylated with sinapyl and malonyl groups. However, the extent and type of modification and the primary composition of anthocyanins vary among varieties, and this may be due to genetic differences that govern which acyl or glycosyl transferase participates in the modification. Anthocyanin Biosynthetic Gene Expression and Correlation with Total Anthocyanin Content. An MBW complex participates in the transcriptional regulation of anthocyanin biosynthesis.46 The simultaneous activation of four MYB proteins (PAP1, PAP2, MYB113, and MYB114) and three bHLH proteins (TT8, GL3, and EGL3) mediates the anthocyanin pathway in plants.46,47 Several reports have verified that overexpression of anyone of these MYBs leads to increased anthocyanin content in Arabidopsis.46,47 Accoding to the data in BRAD, these four MYBs have three highly homologous MYB genes in Brassica rapa genome, namely BrMYB1, BrPAP1, and BrMYB2 (Table S1). To study the molecular mechanism of anthocyanin synthesis in 11S, the transcription levels of genes in the pathway were compared with the parents. Except for BrPAL and BrCHI, all the early biosynthesis genes (EBGs; BrCHS, BrF3H, and BrF3′H) exhibited high expression levels in the high anthocyanin content leaves of 95T and the P01 and P05 leaves of 11S (Figure 6A). The expression of all the late biosynthesis genes (LBGs; BrDFR, BrANS, BrLDOX, BrUF3GT1, BrUF3GT2, Br5GT, Br5MAT, BrGST1, and BrGST2) was particularly highly correlated with anthocyanin content (Figures 3A and 6A). The expression of almost all the LBGs declined dramatically from P01 to P13 in 11S, and the expression levels of some LBGs increased moderately from P13 to P21, such as BrDFR, BrANS, BrUF3GT2, Br5GT, Br5MAT, and BrGST1 (Figure 6A). To further clarify the relationship

Table 1. Composition of Anthocyanins in Chinese Cabbages Obtained by LC-MS/MS Analysisa peak

rt (min)

[M]+ (m/z)

1 2

15.39 15.46

465 787

303 301, 463, 625

3

15.46

789

303, 465, 627

4 5 6

15.46 16.12 16.57

773 859 627

287, 449, 611 287, 611, 535 303, 465

7

16.57

1097

8 9 10 11 12 13 14 15

16.98 17.02 17.02 17.08 17.08 17.28 17.38 17.48

449 935 965 611 965 995 727 1065

16 17 18

17.61 17.77 17.77

641 979 1373

19 20

17.86 18.31

949 1021

21

18.34

1403

22 23

18.57 19.09

919 1005

24 25 26 27 28

19.09 19.41 19.41 19.45 19.9

1035 1155 1125 1317 1241

29

19.93

1211

30 31 32

20.22 20.22 21.49

697 1181 565

MS/MS (m/z)

287, 324, 449, 486, 611, 935 287 287, 449 287, 449 287, 449 303, 465, 627, 803 303, 465, 627 317, 565 287, 449, 535, 817

identification Del-3-glu Peo-3-sop-5-glu or Peo-3glu(Ca)-5-glu Del-3-sop-5-glu or Del-3glu(Ca)-5-glu Cya-3-sop-5-glu Cya-3-sop-5-glu(Ma) Del-3-glu-5-glu or Del-3glu(Ca)-5-glu Cya-3-sop(Ca)-5-sop

Cya-3-glu Cya-3-sop(Ca)-5-glu Cya-3-sop(Ox-pH)-5-glu Cya-3-glu-5-glu Del-3-glu(Fe)-5-glu-7-glu Del-3-glu(Si)-5-glu-7-glu Pet-3-glu(Ma)-5-glu Cya-3-glu(Ma)-5-glu(Si)7-glu 317, 479 Pet-3-glu-5-glu 287, 449, 817 Cya-3-sop(Si)-5-glu 287, 535, 1125 Cya-3-glu(Ma)-5-rut(pH)7-sop(Ma) 287, 449, 787 Cya-3-sop(Fe)-5-glu 287, 535, 773, 977 Cya-3-glu(Ma)-5-(pH)glu7-glu(Ac) 449, 535, 1155, 993, Cya-3-sop(Si-Fe)-51241 sop(Ma) 287, 449, 757 Cya-3-sop(pCo)-5-glu 287, 535, 757, 961 Cya-3-sop(pCo)-5glu(Ma) 287, 535, 757, 991 Cya-3-sop(Fe)-5-glu(Ma) 287, 449, 993 Cya-3-sop(Si-Fe)-5-glu 287, 449, 963 Cya-3-sop(Si-pCo)-5-glu 449, 993, 1155 Cya-3-sop(Si-Fe)-5-sop 449, 535, 993 Cya-3-sop(Si-Fe)-5glu(Ma) 449, 535, 963, 1167 Cya-3-sop(Si-pCo)-5glu(Ma) 287, 449, 551 Cya-3-glu-5-glu(Ma) 535, 933, 1137 Cya-3-glu(Ma)-rut(Ca-Fe) 317 Pet-3-glu(Ma)

a The rt data were collected from P01. Ac = acetyl; Ca = caffeoyl; Cya = cyanidin; Del = delphinidin; Fe = ferulyl; glu = glucoside; Ma = malonyl; Ox = oxalic acid acyl; pCo = p-coumaroyl; Peo = peonidin; Pet = petunidin; pH = p-hydroxybenzoyl; rut = rutinoside; Si = sinapyl; sop = sophoroside.

components between 11S and 95T are probably due to the modifications of the cyanidin sinapyl transferase or factors that govern delphinidin accumulation (Figure 8). The third class, from the whole head of 11S and its parents, included a few anthocyanins ranging from approximately 0.1 to 7.5 mg kg−1 FW in both 95T and the anthocyanin-rich P01 heading leaves, with m/z values of 449, 465, 565, 935, 1317, 1097, 641, 919, and 1021. The levels of the remaining components that comprised the fourth class were much higher in 95T and P21, such as peonidin-3-sophoroside-5-glucoside (m/z = 787) and cyanidin-3-glucoside-5-glucoside (m/z = 611). Interestingly, a few glycosylated and acylated peonidins, petunidins, and delphinidins were detected in the new purple phenotype 138


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Figure 5. Hierarchical clustering analysis of anthocyanins from Chinese cabbages. The clustering analysis was performed on anthocyanin contents (mg kg−1) using the PermutMatrix graphical software and analyzed with Euclidean distance and Ward’s algorithm. In accordance with the color scale, magenta color brightness is directly proportional to the level of anthocyanin content.

between the expression patterns of anthocyanin biosynthetic genes and anthocyanin accumulation, correlation coefficients were calculated. Extremely significant correlations were observed with EBGs’ expression: BrCHS (r = 0.920) and BrF3′H (r = 0.941). Apart from BrLDOX, all the LBGs displayed extremely high correlations between expression and anthocyanin concentration, with correlation coefficients ranging from 0.880 to 0.992. To compare the possible regulation differences during anthocyanin biosynthesis in 11S, the expressions of three types of regulatory genes were investigated: the MYB, bHLH, and WD40 gene families. R2B2-MYB plays an important role in the direct association and activation of structural genes or the bHLH factor in anthocyanin biosynthesis.4 As shown, the regulatory genes BrMYB2, BrMYB12, and BrTT8 exhibited high levels of expression both in 95T and in the P01 and P05 of 11S, which possessed large quantities of anthocyanins (Figure 7A). BrMYB111, BrEGL3-1 and BrEGL3-2 were upregulated in the

white heading leaves of 94S but not in 95T or 11S. In particular, BrTTG1 expression fluctuated in 94S, whereas BrMYB0 and BrMYB1 had no expression in 95T and 11S, respectively. From the anthocyanin accumulation’s correlation with the expression data, it is evident that the regulatory genes BrMYB2 (r = 0.990), BrMYB12 (r = 0.977), and BrTT8 (r = 0.987) showed an extremely high positive correlation with purple pigment content (Figure 7B), which was a trend similarly demonstrated by the EBGs BrCHS and BrF3′H and all the LBGs (except BrLDOX). In Arabidopsis, a complex regulatory network of negative and positive feedback mechanisms controls the expression of anthocyanin genes.4 TT8 positively regulates through the MBW complex, and an initial MBW complex activates TT8, which can then form an MBW complex that autoactivates TT8 and strongly induces the biosynthesis genes.4,48,49 In red cabbages, red cauliflower, purple bok-choy, and several other species of the Brassica genus, TT8 and MYB2 are thought to activate the expression of 139


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Figure 6. Expression analysis of anthocyanin biosynthetic structural genes (A) and their correlation with anthocyanin concentration (B) in Chinese cabbages. The BrACTIN gene was the reference gene. The values are means ± SD. P01 to P21 are the heading leaves of 11S in panel A, and the vertical labels in the x-axis are the concentrations of anthocyanins in panel B. The relative coefficient r with “*” and “**” indicates significant and highly significant correlations at the level of 0.05 and 0.01, respectively.

structural genes and constitutively upregulate anthocyanin accumulation.11−15 These results imply that MYB and bHLH factors are critical factors in the activation of anthocyanins biosynthesis in plants. As visually grouping genes with similar expression patterns can reveal associations of interest, genes with differential expression were subjected to a two-way hierarchical clustering analysis. The clustering of columns reflects the distance between the different samples, whereas the row indicates the various genes (Figure S10). It is clear that gene expression varied greatly between P01 and P05 in 11S. The line 95T shared similarity with P01. Excluding the innermost leaves of 11S, the other leaves can be divided into two classes. P05 and P09 (near P01) showed higher gene expression levels. However, P21, the outermost functional leaves of the head, displayed higher levels of gene upregulation, higher contents of

anthocyanins, and higher antioxidant ability than the P13 and P17. Taken together, these genes (except BrMYB0 and BrMYB1, which were barely upregulated) can be separated into two classes. The first class was composed of genes expressed more or less in 11S and 95T. Specifically, this class included nearly all the highly upregulated genes in 95T and the head of 11S, i.e., the regulatory genes BrMYB2 and BrTT8 and the LBGs BrDFR, BrLDOX, BrUF3GT2, BrUF5GT, Br5MAT, and BrGST1 (Figure S10). The second class was composed of those genes in 95T and the P01 of 11S that are almost expressed at high levels, i.e., the regulatory genes BrMYB12, BrMYB111, BrPAP1, BrTT2, BrTTG1, BrEGL3-1, BrEGL3-2, and BrGL3; the EBGs BrPAL, BrCHS, BrCHI, BrF3H, and BrF3′H; and the LBGs BrANS, BrUF3GT1, and BrGST2 (Figure S10). These results elucidate that there was a significant change in anthocyanin140


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Figure 7. Expression analysis of anthocyanin regulatory genes (A) and their correlation with anthocyanin concentration (B) in Chinese cabbages. The BrACTIN gene was the reference gene. The values are means ± SD. P01 to P21 are the leaves of 11S in panel A, and the vertical labels in the xaxis are the anthocyanin concentrations in panel B. The relative coefficient r with “*” and “**” indicates significant and highly significant correlations at the level of 0.05 and 0.01, respectively.

anthocyanins may depend on other regulators and the relative expression levels of glycosylases (i.e., sophoroside transferase) and acylases (i.e., sinapyl transferase and ferulyl transferase). Despite the differences in anthocyanin compositions between 11S and 95T, they are surprisingly similar regarding the expression patterns of anthocyanin synthesis genes. In this report, samples with higher anthocyanin contents had higher levels of expression of EBGs BrCHS, BrF3H, and BrF3′H and the LBGs BrDFR, BrANS, BrUF3GT1, BrUF3GT2, Br5GT, Br5MAT, BrGST1, and BrGST2. Furthermore, 11S and 95T shared extremely significant correlations between expression of the biosynthetic genes and the anthocyanin concentration, which was similar to the regulatory genes BrMYB2 and BrTT8. Though there were three MYB genes (which were highly homologous with the PAP1, PAP2, MYB113, and MYB114 in Arabodipsis), the key MYB gene in controlling the purple characterization in both purple parent 95T and the heading Chinese cabbage 11S was the BrMYB2. However, BrMYB1 and BrPAP1 upregulated in 95T but the expression tendencies of them were not identical to 11S (Figure 7). Hence, it is speculated that the activation of anthocyanin biosynthesis

related gene expression between the P01 and P05/P07 of 11S and these genes exhibited the same trend with probably similar function in 95T. There are sophisticated regulatory mechanisms to ensure anthocyanin accumulation in response to developmental and environmental signals. Additionally, anthocyanins require appropriate modifications.4,5 Many genes have been confirmed to encode enzymes for the modification of anthocyanins, such as UF3GT1 (UGT78D2), UF3GT2 (UGT79B1), At3ATs, UF5GT (UGT75C1), and At5MAT.50 Because acylated anthocyanins have more chemical stability, they are thought to be better candidates for food coloration than nonacylated anthocyanins.37 It has been shown that modification of the anthocyanin skeleton involves a complicated pathway that results in the diversity and stability of anthocyanins. In this report, all the high-content anthocyanins were sophisticated glycosylated and acylated cyanidins. BrUF3GT1, BrUF3GT2, Br5MAT, and Br5GT displayed high correlations and expression levels. Thus, these genes are likely involved in the modification of cyanidins to form various anthocyanins in 11S and 95T. The different degrees of modification of these 141


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Figure 8. Potential biosynthetic pathway of anthocyanins in the purple heading Chinese cabbage.

structural genes (such as BrCHS, BrF3′H, BrDFR, BrANS, BrUFGTs, and BrGSTs) induced by the expression upregulation of BrMYB2 (a gene with high homology to AtPAP2 in Arabidopsis) and BrTT8 (a gene with high homology to AtTT8 in Arabidopsis) is the main mechanism for governing anthocyanin biosynthesis in the purple heading Chinese cabbage (Figure 8) and its purple male parent. Nonetheless, it was noteworthy that the offspring 11S displayed only partial purple color in the head and it was not similar to its purple male parent, the red cabbage (with whole red head) and red

cauliflower, which were all with whole systemic purple color. Maybe it was caused by these regulators BrMYB1, BrPAP1, or other unknown factors of the differences in genetic background. In addition, the main compositions and degree of glycosyl and acyl modifications may be governed by other factors. One possible explanation is that some other regulators control the anthocyanins’ modification genes which can lead to the difference of individual anthocyanins’ accumulation. Another possibility is that these modification genes have the difference in their promoter areas, which may determine the gene 142


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Journal of Agricultural and Food Chemistry Funding

expression level. These results preliminarily clarify the molecular and metabolic mechanisms governing the anthocyanin synthesis in the purple Chinese cabbage, which supplies valuable insights for the innovation of functional germplasm resources. However, the new purple heading Chinese cabbage has been first acquired and the purple proportion was small. Hence, it requires the breeders to breed a heading Chinese cabbage with higher proportion of purple pigments, and this paper may help us to develop molecular markers for markerassisted breeding or use genetic engineering modification of the key genes in anthocyanins’ accumulation in the purple heading Chinese cabbage. In conclusion, anthocyanins mainly accumulate in 1−3 cell layers under the epidermis of purple organs of the purple heading Chinese cabbage, both in edible tissues and in the reproductive organ, which makes this vegetable remarkable and beautifies the environment. This cheap and essential vegetable with high antioxidant abilities has anthocyanins with good stabilities under acidic conditions and temperatures below 45 °C, which provides useful evidence for storage and cooking of this new resource. Glycosylated and acylated cyanidins were characterized as the main anthocyanins in the purple heading Chinese cabbage. Cyanidin-3-sophoroside(p-coumaroyl)-5glucoside(malonyl), cyanidin-3-sophoroside(ferulyl)-5glucoside(malonyl), cyanidin-3-sophoroside(sinapyl-p-coumaroyl)-5-glucoside(malonyl), and cyanidin-3-sophoroside-(sinapyl-ferulyl)-5-glucoside(malonyl) were identified as the primary anthocyanins in edible leaves of 11S. Besides these four cyanidins in the purple parent 95T, delphinidin-3glucoside-5-glucoside, delphinidin-3-glucoside(sinapyl)-5-glucoside-7-glucoside and delphinidin-3-sophoroside-5-glucoside were also identified as the major anthocyanins. In the molecular mechanism of anthocyanin accumulation, regulatory genes BrMYB2 and BrTT8 probably activate the expression of anthocyanin structural biosynthesis genes (such as BrCHS, BrF3′H, BrDFR, BrANS, BrUFGTs, and BrGSTs), but other factors may affect the formation of major anthocyanins and the distribution of anthocyanins. The diversity of cyanidins, new delphinidins, and the exploration of mechanism of anthocyanin accumulation and metabolism of this new purple heading Chinese cabbage pure line may provide a foundation and experimental evidence for developing Chinese cabbage with high level of anthocyanins to Brassica vegetable breeders. Thus, it is necessary for breeders to make great efforts to find when these anthocyanin biosynthetic genes are upregulated, to verify key genes’ function, to develop related molecular markers for marker-assisted breeding, and to breed heading Chinese cabbage with higher proportion of anthocyanins in subsequent study.

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This work was supported by the National Science Foundation of China (Grant No. 31171965) and the 863 plan of China (Grant No. 2012AA100105). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Jing Zhang for assistance in IQ5 software operation, Dr. Mingke Zhang for help in primer design, and Master degree student Zeping Yang for sample collection. We appreciate suggestions for writing the manuscript from Prof. Pengmin Li and Dr. Junxiang Zhang. We are grateful to Prof. Haijun Gong for revising the manuscript.

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ABBREVIATIONS USED 5MAT, anthocyanidin 5-O-glucoside-6″-O-malonyltransferase; ANOVA, analysis of variance; ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; EBGs, early biosynthesis genes; EGL3, enhancer of glabra3; F3H, flavanone 3-hydroxylase; F3′H, flavanone 3′-hydroxylase; FRAP, ferric reducing antioxidant power; GL3, glabra3; GST, glutathione S-transferase; LBGs, late biosynthesis genes; LC-MS/MS, liquid chromatography tandem mass spectrometry; LDOX, leucoanthocyanidin dioxygenase; MBW, MYB-bHLH-WD40; MW, molecular weight; ORAC, oxygen radical absorbance capacity; PAL, phenylalanine ammonia-lyase; PAP, production of anthocyanin pigment protein; qRT-PCR, real-time quantitative PCR; TEAC, Trolox equivalent antioxidant capacity; TT2, transparent testa2; TT8, transparent testa8; TTG1, transparent testa glabra1; UF3GT1, anthocyanidin-3-O-glucosyltransferase; UF3GT2, anthocyanin-3-O-glucoside-2″-O-xylosyl-transferase; UF5GT, anthocyanidin-5-O-glucosyltransferase

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04674. Full-wave scans, visible spectra, LC-MS/MS chromatogram, hierarchical clustering analysis, primers for the qRT-PCR, and correlation analysis (PDF)

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