Article pubs.acs.org/JAFC
Accumulation and Molecular Regulation of Anthocyanin in Purple Tumorous Stem Mustard (Brassica juncea var. tumida Tsen et Lee) Qiaoli Xie,† Zongli Hu,† Yanjie Zhang,† Shibing Tian,‡ Zhijin Wang,‡ Zhiping Zhao,† Yang Yang,‡ and Guoping Chen*,† †
Bioengineering College, Campus A, Chongqing University, 174 Shapingba Main Street, Chongqing 400030, People’s Republic of China ‡ The Institute of Vegetable Research, Chongqing Academy of Agricultural Sciences, Chongqing 401329, People’s Republic of China S Supporting Information *
ABSTRACT: Tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee) is an economically and nutritionally important dietary vegetable in Asian countries. Purple tumorous stem mustard contains inflated tumorous stem and abundant anthocyanin accumulation in leaves. Here, 20 anthocyanins were separated and identified from the purple tumorous stem mustard by highperformance liquid chromatography−electrospray ionization−tandem mass spectrometry (HPLC−ESI−MS/MS). In order to investigate the regulatory anthocyanin production in purple tumorous stem mustard, the expression of anthocyanin biosynthetic and regulatory genes in leaves from purple and green cultivars were examined. Regulatory gene BjTT8 and all biosynthetic genes were dramatically upregulated in the purple variety. Moreover, the transcript level of BjTT8 and all structural genes, except BjPAL, were all significantly higher in light-treated sprouts than in the dark. These results indicate that transcriptional activation of BjTT8 is associated with upregulation of most anthocyanin biosynthetic genes, to produce anthocyanins in purple tumorous stem mustard. KEYWORDS: purple tumorous stem mustard, anthocyanin biosynthesis, transcriptional regulation, gene expression, HPLC−ESI−MS/MS
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INTRODUCTION
increasing demand for health-promoting components in our diet. Anthocyanin biosynthesis has been studied extensively.4,12 All of the anthocyanin biosynthetic genes have been isolated from plant species, such as PAL, C4H, CHS, CHI, F3H, F3′H, DFR, ANS, and UFGT.13 The increased expression of some structural genes is directly linked with enhanced levels of anthocyanin accumulation, and the major mechanism in controlling anthocyanin biosynthesis appears to be transcriptional regulation.14 Regulatory genes, including R2R3-MYB transcription factors, basic helix−loop−helix (bHLH) transcription factors, and WD40 proteins, have been cloned from plants, such as Arabidopsis, Petunia, maize, gentian, and other species. They are found to always interact with each other and form a ternary complex to activate the expression of anthocyanin biosynthetic genes.14−16 Despite WD-40 repeat proteins playing an ubiquitous role in the regulation of anthocyanin biosynthesis, R2R3-MYB and bHLH transcription factors represent the two major families of anthocyanin regulatory proteins. For example, Rosea1 and Rosea2 of Antirrhinum and Venosa MYB genes interacting with the Mutabilis and Delila bHLH genes activate genes in the anthocyanin biosynthetic pathway.17,18 The activation of BoMYB2 and BoTT8 upregulates a subset of anthocyanin
Anthocyanins are a large group of water-soluble natural pigments that are widely distributed in higher plants. They are responsible for the red, purple, and blue colors found in many flowers, fruits, seeds, and vegetables.1,2 They are glycosylated forms of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavylium salts. The sugar residues of many anthocyanins are further modified by one or more acylations with aliphatic or aromatic acids.3 As a group of flavonoid compounds, anthocyanins fulfill important biological functions in protecting plants against various biotic and abiotic stresses and in furnishing flowers and fruits with distinct colors to attract insects and animals for pollination and seed dispersal.4 Anthocyanins are also valuable color agents for the food industry. In particular, anthocyanins as natural food additives have many significant advantages, such as good water solubility, high security, etc. Thus, anthocyanin is an ideal natural pigment, recognized internationally as an alternative to synthetic food coloring.5,6 The health beneficial roles of anthocyanins have been most frequently associated with their high antioxidant activity, such as protection against cardiovascular disease, certain cancers, and some other chronic diseases.1,7−9 Furthermore, anthocyanins provide a good foundation for colorful breeding, new ornamental species, health vegetables, and development of a colorful germplasm resource.10,11 Therefore, a complete understanding of anthocyanin biosynthesis, regulation, composition, and content is important to develop anthocyanin-rich foods to meet the © 2014 American Chemical Society
Received: Revised: Accepted: Published: 7813
April 15, 2014 June 27, 2014 June 30, 2014 July 23, 2014 dx.doi.org/10.1021/jf501790a | J. Agric. Food Chem. 2014, 62, 7813−7821
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Figure 1. Phenotype of purple cultivar “Zi Ying” and green cultivar “Lv Ying” and total anthocyanin content. (A) Phenotype of purple tumorous stem mustard, at 4 months of growth. (B) Phenotype of green tumorous stem mustard, at 4 months of growth. (C and D) Free-hand section of leaf samples of panels A and B examined under a light microscope, respectively. (E and F) Phenotype of cotyledons (14 days) and young leaves (45 days) of purple cultivar “Zi Ying” and green cultivar “Lv Ying”, respectively. (G) Total anthocyanin content of cotyledons (14 days), young leaves (45 days), and mature leaves (120 days) of purple cultivar “Zi Ying” and green cultivar “Lv Ying”. (H) Total anthocyanin content of seedlings (3, 6, 9, and 12 DAS) of purple cultivar. The data represent the mean from three replicates with three biological repeats. (∗∗) p < 0.01 between the wild type and others by the t test. Error bars indicate standard error (SE).
strongly affected the mechanisms of flavonoid and anthocyanin biosynthesis.35 Along with economic development, demand by people for high-quality vegetables is increasing. In addition to common leaf color vegetables, especially those vegetables with novel leaf or flower color have broad prospects for development and use. Tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee), a native cruciferous vegetable crop in Asia countries, is a very important fresh and processed vegetable in winter and spring. In the domestication history of mustard, lots of morphological and physiological changes have been preserved on the basis of human selection. The purple tumorous stem mustard (B. juncea var. tumida Tsen et Lee) is a mutation of the B. juncea (Cruciferae) crop containing an inflated tumorous stem and abundant anthocyanin accumulation in leaves. The swollen succulent stems are used as raw material for pickled products, which are very popular for their special flavor and nutritional value. 36 In addition, because of abundant anthocyanins in its leaves, purple tumorous stem mustard can be also used in many important fields, such as the health protection, pharmaceutical, and cosmetic industries. Although radishes and other root crops containing anthocyanins are used as plants for natural pigment extraction, it will take a long time to collect the fully mature organs with anthocyanins, which is limited by the plant developmental stages. However, leaves of purple tumorous stem mustard remain bright fuchsia in the whole life of plants. Therefore, purple tumorous stem mustard can be used as a better resource for natural pigment. In summary, purple tumorous stem mustard attracts more and more attention because of its wide applications in food, pharmaceutical, and cosmetic industries. In recent years, most studies about purple tumorous stem mustard are focused on the cultivations. The molecular regulatory mechanism of antho-
biosynthetic genes, leading to the anthocyanin accumulation in purple cauliflower and red cabbage.19,20 In Arabidopsis, anthocyanin accumulation in vegetative tissues has been shown to be mediated by four MYB proteins, production of anthocyanin pigment 1 (PAP1), PAP2, MYB113, and MYB114, as well as bHLH protein, transparent testa 8 (TT8).21−23 Furthermore, MYB transcription factors are central players in the coordinated activation of sets of genes specific for the anthocyanin and tannin pathways.24 R2R3-MYB and bHLH transcription factors regulate anthocyanin biosynthesis in gentian flowers.25 In Petunia, R2R3-MYB transcription factors are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning.26 Rc encodes a bHLH protein conditioning red pericarp in rice.27 Although the MYB transcription factors require a bHLH in regulating anthocyanin biosynthesis in Arabidopsis,28 MYB proteins can act directly in activating transcription in maize.29 The anthocyanin biosynthetic pathway is also regulated by environmental factors, such as light and temperature, as well as internal factors, such as plant hormones, other secondary metabolites, and nutrients.30 Light acts as an essential stimulus and also modulates the intensity of the pigment by affecting the regulatory and structural genes involved in anthocyanin biosynthesis.31 Research in eggplant showed that anthocyanin synthesis was induced in the hypocotyl tissues when the seedlings were grown under purple light with an ultraviolet (UV) light supplement.32 The apple gene MdMYB1 was shown to regulate anthocyanin biosynthesis in response to sunlight.33 In Arabidopsis, a genome-wide analysis suggests that PAP1 and PAP2 play a major role in the induction of the anthocyanin biosynthesis transcriptional cluster by high light stress.34 Recently, Cevallos-Casals and Cisneros-Zevallos observed that the duration and amount of light applied during sprouting 7814
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extracted and separated by HPLC following the improved procedure described by Wu and Prior.38 Samples (100 mg) were extracted in 1 mL of methanol/H2O/acetic acid (85:15:0.5) for 10 min at room temperature and centrifuged at 12000g for 10 min. The supernatants were collected and filtered through a 0.2 μm polytetrafluoroethylene (PTFE) syringe filter to remove cell debris. The samples were then analyzed by Waters 2795 HPLC (Waters, Milford, MA), equipped with a variable wavelength detector. The results were analyzed by Waters 2795 HPLC ChemStation software. The chromatographic separation was performed on a Zorbax Stablebond Analytical SB-C18 column (4.6 × 150 mm, 5 μm, Waters). The injection volume was 10 μL. Elution was performed using mobile phase A (aqueous 2% formic acid solution) and mobile phase B (methanol). The detection was at 520 nm, and the column oven temperature was set at 40 °C. The flow rate was 1 mL/min. The gradient program is described as follows: 0−2 min, 10−20% B; 2−40 min, 20−55% B; 40−45 min, 55−60% B; 45− 60 min, 90% B; and 60−65 min, 10% B. Quantification of the different anthocyanins was based on peak areas and calculated as equivalents of the external standards. All contents were expressed as milligrams per gram of dry weight. Low-resolution electrospray mass spectrometry was performed with a solariX ion trap mass spectrometer (Bruker Daltoniks, Billerica, MA). The experimental conditions were as follows: ESI interface, nebulizer, 50 psi; dry gas, 15.0 psi, dry temperature, 320 °C; MS/MS, scan from m/z 200 to 2000; ion trap, scan from m/z 200 to 2000; source accumulation, 50 ms; ion accumulation time, 300 ms; flight time to acquire cell, 1 ms; smart parameter setting (SPS), compound stability, 50%; and trap drive level, 60%. Total Anthocyanin Analysis. The total anthocyanin content in samples was measured with a pH-differential spectrum method.39 The mature leaves or seedlings (100 mg) were frozen in liquid nitrogen and immediately crushed into powder. Samples were treated separately with 2 mL of pH 1 buffer solution (50 mM KCl and 150 mM HCl) and 2 mL of pH 4.5 buffer solution (400 mM sodium acetate and 240 mM HCl). The mixtures were centrifuged at 12000g for 20 min at 4 °C. Then, absorbance of the solutions was measured at 520 nm using diluted supernatants. The anthocyanin content was calculated according to the equation: amount [mg g−1 of fresh weight (FW)] = (A1 − A2) × 484.8/24 825 × dilution factor, where A1 represents the absorbance of supernatants gathered from pH 1.0 buffer solution at 520 nm, A2 represents the absorbance of supernatants gathered from pH 4.5 buffer solution at 520 nm, 484.8 represents the molecular mass of cyanidin-3-glucoside chloride, and 24 825 is its molar absorptivity at 520 nm. Each sample was analyzed in triplicate, and the results were expressed as the average of the three measurements. Statistical Analysis. Data were analyzed by Origin 8.6 (OriginLab) software, using the t test to assess significant differences among the means.
cyanin biosynthesis and the anthocyanin composition and content in purple tumorous stem mustard remain unclear. In this study, the anthocyanin content and composition of the mature leaf of purple tumorous stem mustard were detected. To investigate the mechanism of the anthocyanin biosynthesis in the purple tumorous stem mustard, the expression of structural and regulatory genes were detected in two genotype cultivars, purple cultivar “Zi Ying” and green cultivar “Lv Ying”. The effect of light on anthocyanin accumulation of “Zi Ying” was also investigated.
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MATERIALS AND METHODS
Plant Materials and Growth Conditions. To analyze the influence of genotype on anthocyanin productions, two cultivars of tumorous stem mustard, “Zi Ying” and “Lv Ying”, were selected in this study based on different phenotype appearances. The seeds were sowed on September 1, 2012, transplanted into plastic pots after 20 days, colonized on October 10, 2012, and managed routinely. For light and dark treatment, the seeds were surface-sterilized using 70% (v/v) ethanol for 1 min, rinsed 3 times with sterile water, then washed in 1% sodium hypochlorite solution for 12−15 min, and rinsed 8 times with sterile water. The sterilized seeds were divided into two sets germinated on 1/2MS medium in a growth chamber under light (16 h of light/8 h of dark) or dark (24 h of dark) conditions at 25 °C, 60% humidity, and 440 μmol m−2 s−1 light intensity. For biological replicates, we used three plastic boxes for each single treatment, placed 80 seeds per box, and harvested sprouts in each plastic box at 3, 6, 9, and 12 days after sowing (DAS). Microscopic Analysis of Anthocyanin Distribution in Tumorous Stem Mustard. Young fresh leaves of the “Zi Ying” cultivar underwent microscopic observation and analysis [Figure 1C with an optical microscope (Leica-DMI4000B)]. Observations were repeated three times for each sample. RNA Extraction and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis. When the color of “Zi Ying” leaf turned to brilliant purple, the mature leaves of both ‘‘Zi Ying” and “Lv Ying” cultivars were collected for the treatments and sprouts in each box at 3, 6, 9, and 12 days after sowing were harvested for RNA isolation. Total RNA was extracted from samples for three biological repeat using RNAiso Plus (TaKaRa). Then, 1−2 μg of total RNA was used to synthesis the complementary DNA (cDNA) through reverse transcriptase (M-MuLV RT) (Promega) with Oligo-dT18 primer. The synthesized cDNAs were diluted 1 times with RNase/DNase-free water. qRT-PCR analysis was carried out using the CFX96 real-time system (C1000 Thermal Cycler, Bio-Rad). All reactions were performed using the SYBR Premix Go Taq II kit (Promega, China) in a 10 μL total sample volume (5.0 μL of 2× SYBR Premix Go Taq II, 0.5 μL of primers, 1.0 μL of cDNA, and 3.5 μL of ddH2O). To remove the effect of genomic DNA and the template from the environment, no template control (NTC) and no reverse transcription control (NRT) were performed. Three replications for each sample were used, and standard curves were run simultaneously. Melting curve analysis of qRT-PCR samples revealed that there was only one product for each gene primer reaction. The PCR products were sequenced to confirm the specific amplification. A house-keeping gene BjEF-1-α37 was used as an internal standard in tissues. Expression of genes from the green cultivar were set to 1. PCR primers are shown in Table 1S of the Supporting Information. Chemicals. The anthocyanin cyanidin-3-glucoside chloride for external standards was purchased from Phytolab (Germany). Highperformance liquid chromatography (HPLC)-grade methanol (MeOH) and formic acid were bought from Sigma. HPLC-grade water was prepared from distilled water using a Milli-Q system (Millipore Laboratory, Bedford, MA). All of the other solvents were provided from Aldrich (St. Louis, MO). Anthocyanin Extraction and High-Performance Liquid Chromatography−Electrospray Ionization−Tandem Mass Spectrometry (HPLC−ESI−MS/MS) Analysis. Anthocyanins were
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RESULTS AND DISCUSSION
Influence of Genotype on Anthocyanin Concentrations. Two cultivars, “Zi Ying” and “Lv Ying”, were investigated to study the influence of genotype on anthocyanin biosynthesis. During the seedling stage, cotyledons of the “Zi Ying” cultivar began to show a punctate purple and the purple area gradually increased with plant growing. When four true leaves were present, 80−90% area of the leaves showed purple. Later all leaves turned purple gradually. Under the same growth conditions, the leaves of “Lv Ying” cultivar remain green in the whole growth process, no purple was seen (panels A and B of Figure 1). The anthocyanin content in “Zi Ying” and “Lv Ying” was measured with a pH-differential spectrum method. At the seedling stage, the anthocyanin content of cotyledon of “Zi Ying” was up to 0.61 mg g−1 of FW. Higher amounts of anthocyanins were detected in young leaves of “Zi Ying” (1.52 mg g−1 of FW). The highest level of anthocyanin accumulation (1.929 mg g−1 of FW) was found in mature leaves of “Zi Ying” 7815
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Table 1. Anthocyanin Levels (mg g−1 of Dry Weight) in the Two Cultivars (Purple and Green) (n = 3) cultivar peak numbera 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 total
anthocyanin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin cyanidin
b
3-feruloylsophoroside-5-glucoside 3-caffeoylsinapoylsophoroside-5-glucosideb 3-hydroxyferuloylcaffeoylsophoroside-5-malonylglucoside 3-feruloylsophoroside-5-malonylglucoside 3-caffeoylsophoroside-5-malonylglucoside 3-feruloylsophoroside-5-malonylglucoside 3-p-coumaroylsophoroside-5-glucosideb 3-caffeoylferuloylsophoroside-5-gIucosideb 3-caffeoylsinapoylsophoroside-5-malonylglucoside 3-diferuloylsophoroside-5-glucosideb 3-p-coumaroylsophoroside-5-malonylglucoside 3-feruloylcaffeoylsophoroside-5-malonylglucoside 3-p-coumaroylferuloylsophoroside-5-glucosideb 3-feruloylsinapoylsophoroside-5-malonyldiglucoside 3-p-coumaroylsinapoylsophoroside-5-malonyldiglucoside 3-feruloylsinapoylsophoroside-5-malonylglucosideb 3-p-coumaroylferuloylsophoroside-5-malonyldiglucosid 3-diferuloylsophoroside-5-malonylglucoside 3-p-coumaroyltriglucoside-5-malonylglucoside 3-di-p-coumaroylsophoroside-5-malonylglucoside
purple
green
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
ndc nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd
0.042 0.01 0.070 0.286 0.063 0.090 0.031 0.006 0.032 0.236 0.308 0.270 0.037 0.076 0.127 0.858 0.214 0.694 0.398 0.068 3.916
0.000 0.001 0.001 0.002 0.002 0.001 0.001 0.001 0.002 0.002 0.003 0.001 0.002 0.001 0.001 0.001 0.001 0.001 0.002 0.001 0.025
a
Peak number corresponds to elution order by HPLC analysis in Figure S1 of the Supporting Information. bKnown Brassica anthocyanins (most were identified with the reference anthocyanin in the database). cnd = not detected.
side-5-malonyldiglucoside (m/z 1373) were found as the major anthocyanins in red mustard greens. Different numbers and types of anthocyanins identified in purple tumorous stem mustard are different from those in red mustard greens, which might be owing to the different varieties and methods. Analysis of Sprouts Grown under Light and Dark Conditions. In our study, we find that the color of the upper epidermis is deeper than the lower epidermis, which may be attributed to the different light intensities in the upper and lower surfaces of the “Zi Ying” leaves (Figure 1C). Previous studies on apple and pear showed that anthocyanin biosynthesis was light-induced and restricted in fruit skin.33,44 Therefore, the experiment of light and dark treatments of “Zi Ying” seedlings was performed. Cotyledons grown under light conditions were purple, whereas cotyledons grown under dark conditions were yellow. The anthocyanin contents in seedlings of “Zi Ying” grown under light conditions were all significantly higher than the anthocyanin contents in seedlings of “Zi Ying” grown under dark conditions from 3 to 12 DAS (Figure 1H). These results are consistent with those findings in radish and buckwheat sprouts,31,45 which indicates that the light is essential for anthocyanin synthesis in the “Zi Ying” cultivar. Expression of Anthocyanin Biosynthetic Genes. To determine the relationship between anthocyanin accumulation and the expression of anthocyanin biosynthetic genes, the transcripts of anthocyanin structural genes were examined in the mature leaves between the purple cultivar “Zi Ying” and the green cultivar “Lv Ying”. The expressions of phenylpropanoid pathway genes PAL and C4H and anthocyanin biosynthetic genes CHS, CHI, F3H, F3′H, DFR, ANS, and UFGT are shown in Figure 2. All structural genes in “Zi Ying” were dramatically upregulated in comparison to those in “Lv Ying”. Early structural gene CHS was upregulated about 149-fold, while transcript levels of late structural genes F3H, F3′H, DFR, and ANS in the purple
(panels E−G of Figure 1), a level comparable to that found in blueberries.40 On the contrary, low amounts of anthocyanins were detected at various stages in the “Lv Ying” grown under the same conditions, which is consistent with their phenotype. Microscopic examination of sections of young leaf tissues revealed that the purple pigments accumulated in both upper and lower epidermal layers in “Zi Ying” samples, while no pigments were present in the “Lv Ying” samples (panels C and D of Figure 1). Qualitative and Quantitative Analyses of Anthocyanins in Purple Tumorous Stem Mustard. To examine the composition and relative content of anthocyanins in “Zi Ying”, the anthocyanins were extracted and analyzed by HPLC−ESI− MS/MS. The elution profile of purple cultivar is shown in Figure 1S of the Supporting Information. There were 20 major peaks detected in the extracts of “Zi Ying”. The individual peaks were putatively identified through comparison of the retention time and spectrum character to the published data38,41−43 and listed in Table 1S of the Supporting Information. The content of each individual anthocyanin in “Zi Ying” was calculated and shown in Table 1. All of these anthocyanins were cyanidin 3diglucoside-5-glucoside and derivatives with different acylated groups bound to diglucoside (Table 1 and see Figure 1S of the Supporting Information). The anthocyanins showing the highest levels were cyanidin 3-feruloylsinapoylsophoroside-5malonylglucoside (m/z 1241) and cyanidin 3-diferuloylsophoroside-5-malonylglucoside (m/z 1211) of 0.858 and 0.694 mg g−1 of dry weight, respectively. It was reported that 67 anthocyanins were identified from red mustard greens (B. juncea Coss variety) using an ultrahigh-performance liquid chromatography−photodiode array−electrospray ionization− high-resolution tandem mass spectrometry operated in the MSn mode (UHPLC−PDA−ESI−HRMS/MSn) profiling method,43 and cyanidin 3-feruloylsinapoylsophoroside-5-malonyldiglucoside (m/z 1403) and cyanidin 3-p-coumaroylsinapoylsophoro7816
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Figure 2. Expression analysis of anthocyanin synthesis genes under normal growth conditions. Expression analysis of (A) BjPAL, (B) BjC4H, (C) BjCHS, (D) BjCHI, (E) BjF3H, (F) BjF3′H, (G) BjDFR, (H) BjANS, and (I) BjUFGT in mature leaves of purple cultivar “Zi Ying” and green cultivar “Lv Ying”, respectively. The data represent the mean from three replicates with three biological repeats. (∗∗) p < 0.01 between the wild type and others by the t test. Error bars indicate SE. Relative expression was normalized using the housekeeping gene BjEF1 (GO479260). The expression level of genes in the green cultivar were set to 1.
anthocyanin accumulation to those high anthocyanin accumulating plants. Furthermore, previous studies have reported that RsCHS, RsCHI, and RsDFR mRNAs were not expressed in the dark, but their expression was induced in seedlings exposed to light up to day 6.50 FtPAL, Ft4CL, FtF3H, FtDFR, and FtANS show higher transcript levels in both seeds and sprouts from 2 to 10 DAS, and most genes in the flavonoid biosynthetic pathway are upregulated at 2 DAS under light/dark or dark culture.45 In our light-treated seedlings, except the upstream pathway gene BjPAL, the transcript levels of structural genes were all higher in light-treated sprouts than those in the dark (Figure 4). The transcript levels of all of these genes were highest at nearly 3 and 6 DAS, except BjC4H, which had no change in light conditions from 3 to 12 DAS. It is noteworthy that, in lighttreated sprouts, the transcript levels of BjF3′H and BjANS were about 1200- and 120-fold higher than those in the dark treated at 6 DAS, respectively. At 3 DAS, the transcript levels of BjF3H of light-treated sprouts were about 60-fold higher than those in the dark. In addition, except BjPAL, all other genes are hardly detected in the dark-treated seedlings. Moreover, anthocyanin contents in light-treated seedlings of “Zi Ying” were all significantly higher than those in dark-treated seedlings from 3 to 12 DAS (Figure 1H). These results are completely
cultivar were approximately 1020-, 660-, 572-, and 3450-fold higher than those in the green cultivar, respectively. In addition, the expression levels of PAL, C4H, CHI, and UFGT in “Zi Ying” were about 3.3-, 5.5-, 22.6-, and 6.2-fold higher than those in the green cultivar, respectively. Despite the absence of visual anthocyanin pigmentation in green cultivars, low expression levels of all of these structural genes were observed (Figure 2), which was consistent with the previous reports in other plant species.46,47 These results illustrate that the high expression levels of biosynthetic genes are strongly associated with the high intensity of anthocyanin pigments in purple tumorous stem mustard. A number of plants that show constitutive accumulation of high levels of anthocyanins have been studied. In purple-fleshed apple, the transcript levels of anthocyanin biosynthetic genes were significantly higher throughout fruit development than those found in white-fleshed apple.48 Similarly, examination of seven sweet potato cultivars revealed that the high anthocyanin accumulation in purple sweet potato also showed high-level expression of the structural genes.49 In pap1-D Arabidopsis, the increased anthocyanin production throughout the plant was associated with upregulation of the entire biosynthetic pathway genes.19,23 It appears that purple tumorous stem mustard shares very similar upregulation of structural genes in mediating 7817
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Figure 3. Expression analysis of anthocyanin synthesis genes of seedlings under light and dark growth conditions. Expression analysis of (A) BjPAL, (B) BjC4H, (C) BjCHS, (D) BjCHI, (E) BjF3H, (F) BjF3′H, (G) BjDFR, (H) BjANS, and (I) BjUFGT in seedlings of 3−12 DAS of purple cultivar “Zi Ying” grown in light and dark conditions. The data represent the mean from three replicates with three biological repeats. (∗∗) p < 0.01 between the wild type and others by the t test. Error bars indicate SE. Relative expression was normalized using the housekeeping gene BjEF1 (GO479260).
“Zi Ying”. These results suggest that upregulation of BjMYB1, BjMYB2, BjMYB3, BjMYB4, and BjTT8 expression is likely to activate the structural genes (F3H, F3′H, DFR, and ANS) of anthocyanin biosynthesis, and BjTT8 is likely a critical gene to control the anthocyanin biosynthetic pathway in “Zi Ying”. This is likely the general mechanism underlying anthocyanin production responsible for the purple leaves of purple tumorous stem mustard. This regulation mechanism is similar to that in red cabbage.19 Moreover, in the light- and dark-treated seedlings, the transcript levels of BjMYB1, BjMYB2, BjMYB3, and BjMYB4 were 16.5-, 14.2-, 1.73, and 15.5-fold higher in the light than those in the dark treated at 9 DAS, respectively (Figure 5). BjTTG1 was also induced in the seedling stage, but there was no significant difference between the light and dark conditions. These results indicated that MYB transcription factors may participate in the regulation of anthocyanin accumulation in seedlings of purple cultivar and BjTTG1 might not account for the transcriptional activation of anthocyanin structural genes in purple tumorous stem mustard in the light. In addition, the transcript levels of BjTT8 in the light-treated seedlings were approximately 85-, 80-, and 240-fold higher than those in the
consistent with their phenotype and further illustrate that the expression level of structural genes is closely related to the anthocyanin accumulation in purple tumorous stem mustard. Expression of Anthocyanin Biosynthetic Regulatory Genes. On the extensive studies of Arabidopsis, grape, apple, Petunia, pears, and strawberry, more and more evidence show that plants are often fond of the way of regulating anthocyanin biosynthesis by transcriptional control of anthocyanin biosynthetic genes.4,12 MYB transcription factors, bHLH proteins, and WD40 proteins are known to participate in regulating anthocyanin biosynthesis. In this work, the transcripts of BjMYB1, BjMYB2, BjMYB3, BjMYB4, BjTT8, and BjTTG1 (homologues of AtPAP1, AtPAP2, AtMYB113, AtMYB114, AtTT8, and AtTTG1, respectively) were analyzed. As shown in Figure 4, these genes had a consistent expression trend, and their expression was elevated in the “Zi Ying” variety. Among them, the expression levels of MYB transcription factors BjMYB1, BjMYB2, BjMYB3, and BjMYB4 were increased by approximately 2.9, 2.3, 1.8, and 5.9 times, respectively. BjTTG1 exhibited only a small increase (1.4-fold) in the purple cultivar. It is noteworthy that BjTT8 was increased by up to 80-fold in 7818
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Figure 4. Expression analysis of anthocyanin regulatory genes under normal growth conditions. Expression analysis of (A) BjTT8, (B) BjMYB1, (C) BjMYB2, (D) BjMYB3, (E) BjMYB4, and (F) BjTTG1 in mature leaves of purple cultivar “Zi Ying” and green cultivar “Lv Ying”, respectively. The data represent the mean from three replicates with three biological repeats. (∗) p < 0.05 and (∗∗) p < 0.01 between the wild type and others by the t test. Error bars indicate SE. Relative expression was normalized using the housekeeping gene BjEF1 (GO479260). The expression level of genes in the green cultivar were set to 1.
Figure 5. Expression analysis of anthocyanin regulatory genes of seedlings under light and dark growth conditions. Expression analysis of (A) BjTT8, (B) BjMYB1, (C) BjMYB2, (D) BjMYB3, (E) BjMYB4, and (F) BjTTG1 in seedlings of 3−12 DAS of purple cultivar “Zi Ying” grown in light and dark conditions, respectively. The data represent the mean from three replicates with three biological repeats. (∗) p < 0.05 and (∗∗) p < 0.01 between the wild type and others by the t test. Error bars indicate SE. Relative expression was normalized using the housekeeping gene BjEF1 (GO479260).
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dark-treated seedlings at 6, 9, and 12 DAS, respectively. The upregulation of BjTT8 in purple cultivar is as extreme as that in purple cauliflower and red cabbage,19,20 suggesting that BjTT8 may be a very important regulatory factor, regulating accumulation of anthocyanins in purple tumorous stem mustard. On the basis of these results, we can speculate that mustard, cabbage, and cauliflower may have the same way of regulating the anthocyanin accumulation. In conclusion, our research investigated the anthocyanin composition and transcriptional analysis of flavonoid structural and regulatory genes in purple tumorous stem mustard. The results suggest that cyanidin is characterized as the major anthocyanin in the purple tumorous stem mustard, and the upregulation of BjMYB genes and BjTT8 likely triggers anthocyanin accumulation. Because of its extreme upregulation, BjTT8 is likely a key gene to control the anthocyanin accumulation in the “Zi Ying” cultivar. To clarify the role of BjMYB genes and BjTT8 in anthocyanin production in purple tumorous stem mustard, future studies through genetic analysis and candidate gene approach should be carried out.
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ASSOCIATED CONTENT
S Supporting Information *
Major structure of anthocyanins and HPLC profiles of anthocyanins in purple tumorous stem mustard (Figure 1S), sequence alignment and phylogenetic analysis of Brassica TT8s (Figure 2S), primers used for qRT-PCR (Table 1S), and data on anthocyanin identification in purple tumorous stem mustard (Table 2S). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Telephone/Fax: 0086-23-65112674. E-mail: chenguoping@ cqu.edu.cn. Funding
This work was supported by the National Natural Science Foundation of China (30871709, 31100089, and 31171968) and the Fundamental Research Funds for the Central Universities (CDJXS10232209). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED PAL, phenylalanine ammonia lyase; C4H, cinnamate 4hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; HPLC, high-performance liquid chromatography; ESI−MS/MS, electrospray ionization−tandem mass spectrometry; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction
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