Red Pigment Content and Expression of Genes Related to Anthocyanin Biosynthesis in Radishes ( Raphanus sativus L . ) with Different Colored Flesh

Radish with red skin and red flesh (Raphanus sativus) is a unique vegetable containing large amounts of a red pigment, which is widely used in foods, wine, and cosmetics. To investigate the gene or genes that play a key role in anthocyanin biosynthesis in radish with red skin and red flesh, the red pigment content and expression of genes involved in anthocyanin biosynthesis of 16 varieties with different colored flesh were studied. The expression level of the genes RsPAL, RsCHS, RsCHI, RsDFR, RsF3H, RsF3'H, and RsANS in radish with red skin and red flesh are all significantly higher than that of radish with white skin and white flesh, radish with red skin and white flesh, radish with green skin and pinky flesh, and radish with red skin and pinky flesh. Correlation analysis indicated that the gene expression level of RsDFR, RsF3H, RsCHS, RsANS, RsF3'H, RsCHI, and RsPAL showed remarkable positive and significant correlation to red pigment content of radish. Stepwise regression analysis showed that the gene expression level of RsDFR had the highest and significant direct effect (0.8932) on red pigment content of radish. The results indicated that (1) the red pigment content of radish is closely related to the increased expression of a number of structural genes in anthocyanin biosynthesis, (2) the RsDFR gene plays a key role in anthocyanin biosynthesis in red radish with red flesh, and (3) RsDFR might be one of the best targets in genetic engineering for anthocyanin production from radish and other plants.


Introduction
Chinese radishes possess the richest genetic resources in the world, which can be further differentiated by fleshy root size, shape, and color, as well as leaves (Wang et al., 2015).Traditionally, Chinese radishes have been classified into five types: radish with white skin and white flesh (Figure 1A), radish with red skin and white flesh (Figure 1B), radish with green skin and pinky flesh (Figure 1C), radish with red skin and pinky flesh (Figure 1D), and radish with red skin and red flesh (Figure 1E) (Chen et al., 2015).Of these types, the radish with red skin and red flesh is a unique ecotype which contains large amounts of a red pigment which is widely used in foods, wine, and cosmetics.A previous study on red radish with red skin and red flesh detected that the red pigment content of radish with red skin and red flesh was 6.4-28.8‰ with an average of 16.13 ‰, indicating that the radish with red skin and red flesh is a valuable source of material for the red pigment industry (Chen et al., 2015).The red pigment content and production of radish with red skin and red flesh are eight and four times higher than that of radish with green skin and pinky flesh, respectively (Tong et al., 2013).In recent years, the physiological and biochemical characteristics (Fang et al., 2012), inheritance of traits (Lv et al., 2015) and the extraction and separation of red pigment (Tong et al., 2013) from radish with red skin and red flesh have been well studied.However, despite these studies, little is known about the molecular mechanism of anthocyanin accumulation in radish with red skin and red flesh.biosynthesis, metabolic pathways, and regulatory mechanisms of anthocyanins have received increased attention.The accumulation of anthocyanin in plants is closely related to the expression level of genes associated with anthocyanin biosynthesis (Zhang et al., 2015).There are two types of genes that control anthocyanin biosynthesis in plants: one is the structural gene that encodes the anthocyanin biosynthetic pathway enzymes, and the other is a regulatory gene that regulates spatiotemporal expression of the structural gene (Rouholamin et al., 2015;Zhao et al., 2015).The chalcone synthase gene (CHS) plays an essential role in anthocyanin biosynthesis and regulates the relative amount of specific flavonoids (Zhang et al., 2015).The change of expression in the CHS gene can significantly affect anthocyanin accumulation (Yin et al., 2015).The interference decreased CHS expression and anthocyanin content by 88.8% and 87% in Prunus persica, respectively (Zhang et al., 2015).Overexpression of FhCHS1 in Arabidopsis thaliana changed the pigmentation phenotype of the seed coats, cotyledons, and hypocotyls (Sun et al., 2015).Chalcone isomerase (CHI) catalyzes the stereospecific isomerization of chalcones into their corresponding (2S)-flavanones (Guo et al., 2015).IbCHI of sweet potato is responsible for the activation of anthocyanin biosynthesis in the early stage of root development (Guo et al., 2015).Flavanone 3-hydroxylase (F3H) catalyzes flavanone into flavonol and one to two F3H genes were found to be cloned in most plants, and F3H gene has two introns with 350-380 amino acids in protein (Laura, 2013;Kumar et al., 2015).Flavonoid 3′-hydroxylase (F3'H) and flavonoid 3′5′-hydroxylase (F3'5'H), both of which belong to the cytochrome P450 superfamily, catalyze hydroxylation at the 3′ and 3′, 5′ positions of the B-ring of the flavonoids (Forkmann et al., 2014;Nakamura et al., 2015).Dihydroflavonol 4-reductase (DFR), only expressed in the colored organ responsible for anthocyanidin production, catalyzes flavanonols into leucoanthocyanidin (Xu et al., 2014).The total anthocyanin content has a positive correlation between the level of DFR gene expression and the accumulation of anthocyaninsin in Punica granatum (Rouholamin et al., 2015).Anthocyanidin synthase (ANS) catalyzes the conversion of colorless leucoanthocyanins into colored anthocyanins (Shi et al., 2015a).The transcript level of MsANS of Magnolia sprengeri was 26-fold higher in red petals than in white petals (Shi et al., 2015b).Meanwhile, the anthocyanin accumulation is associated with the expression of several structural genes in the anthocyanin biosynthetic pathway (Zhao et al., 2015b).In Morus alba, MaCHS, MaDFR, and MaANS genes control the anthocyanin biosynthesis in mulberry and upregulation of them greatly increases the anthocyanin content (Li et al., 2014).CHS and DFR play important roles in the process of the color changes of rose petals from yellow to red (Luo et al., 2013).R2R3-MYB, bHLH, and WD40 proteins are the principal regulatory factors involved in anthocyanin biosynthesis (Shoeva et al., 2015).The combination and interaction amongst the regulatory factors determine the expression of structural genes.The different co-expression of MYB10 and bHLH33 genes and the different expressions of WD40 are involved in the differential regulation mechanisms of anthocyanin biosynthesis and the coloration pattern between occidental and oriental pears (Yang et al., 2015).LcMYB1 controls anthocyanin biosynthesis in litchi, and LcUFGT might be the structural gene that is targeted and regulated by LcMYB1 (Lai et al., 2014).Concerning the anthocyanin biosynthesis in radish, Park et al. (2011) reported that RsDFR and RsANS were found to accumulate in the flesh or skin, and radish skin contained higher CHS, CHI, and F3H transcript levels than radish flesh.Lim et al. (2015) isolated the RsMYB1 gene from red radish (red skin) plants and suggested that RsMYB1 is an actively positive regulator for anthocyanin biosynthesis in radish plants.To summarize, although the mechanism of anthocyanin accumulation in many plants have been reported, little is known about the genes associated with the anthocyanin accumulation in radish with red skin and red flesh because of its specific distribution and the red pigment content is remarkably reduced when the radish be cultivated in other locations.
With the trend of synthetic colorants progressively being replaced by natural colorants, the need for natural pigments has dramatically increased.As a unique agricultural resource, there is a great prospect for the application of radish with red skin and red flesh.Hence, it is necessary to study the molecular mechanisms of anthocyanin accumulation in radish with red skin and red flesh.In this study, the red pigment content, expression of related genes in anthocyanin biosynthesis of 16 radish (Raphanus sativus L.) varieties with different colored flesh, and the relationships between red pigment content and gene expression level were studied.The goal of this study was to estimate the key gene or genes associated with anthocyanin biosynthesis for future study of the mechanisms of anthocyanin accumulation in radish with red skin red flesh.

Plant Materials
In total, 16 radish varieties were sampled, including four radishes white skin and white flesh, three radishes with red skin and white flesh, one radish with green skin and pinky flesh, five radishes with red skin and pinky flesh, and three radishes with red skin and red flesh (Table 1 and Figure 1).The seeds were abtained from Crop Genetics and Breeding Research Center, Yangtze Normal University, China.jas.ccsenet.

Radish
A field tri University single row plants wer mixed tog and juice w the supern high-perfo content wa using the s Total root   Total genomic RNA was extracted from the flesh of the root before bolting using a Column Plant Total RNA Extraction Kit SK8661 (Sangon Biotech Shanghai Co. Ltd., Shanghai, China).Agarose electrophoresis and spectrophotometry were used to determine the quality and content of total RNA, respectively.The cDNA were acquired by using an AMV First Strand cDNA Synthesis Kit SK2445 (Sangon Biotech Shanghai Co. Ltd., Shanghai, China).The first strand synthesis of cDNA was performed in a 0.2 mL PCR tube containing 5 µL of total RNA, 1 µL of random primer p(dN) 6 (0.2 µg/µL), and 5 µL of RNase-free ddH 2 O.The mixed liquor was measured with thermostatic bath at 70 °C for 5 min, followed by freezing for 10 sec.After centrifugation, 4 µL of 5 × reaction buffer, 2.0 µL of dNTP mix (10 mmol/L), 1.0 µL of RNase inhibitor (20 U/µL), and 2.0 µL AMV reverse transcriptase (10 U/µL) were added to the PCR tube.After a thermostatic bath at 37 °C for 5 min followed by 42 °C for 60 min, the reaction was stopped in the thermostatic bath at 70 °C for 10 min.The mixtures were stored at -20 °C for post-study.

Fluorescence Quantitative PCR Analysis
The

Statistical Analyses
Variance analysis was carried out to evaluate differences between radish varieties for red pigment content and gene expression with SPSS software (Verma, 2013) followed by multiple comparisons using Duncan's new multiple range method.When significant treatment differences occurred, the correlation coefficients among red pigment content and gene expression and stepwise regression analysis were also calculated in this program.

Differences in Genes Expression with Different Colored Flesh
The results of the F-test of gene expression in 16 radishes are listed in  Note.* and ** represent the significant levels at 0.05 and 0.01, respectively.
In order to better determine the relationship between the related gene expression and the red pigment content of radishes, we carried out a correlation analysis with the red pigment content considered as the dependent variable and the gene expression level of RsPAL, RsCHS, RsCHI, RsDFR, RsF3H, RsF3'H, RsANS and RsMYB genes considered as the independent variable.Correlation analysis (Table 5, coefficient of determination R 2 = 0.9335) indicated that the correlation coefficients of the gene expression level to the red pigment content, in order of large to small, were RsDFR, RsF3H, RsCHS, RsANS, RsF3'H, RsCHI, RsPAL, and RsMYB.Among the correlation coefficients, the gene expression level of RsDFR, RsF3H, RsCHS, RsANS, RsF3'H, RsCHI, and RsPAL showed positive remarkable significant correlation to red pigment content, and the gene expression level of RsPAL showed positive significant correlation to red pigment content.The gene expression level of RsMYB showed negative and insignificant correlation to red pigment content.Moreover, 20 correlation coefficients among the gene expression level were determined to be significant.

Path Correlation Analysis among Red Pigment Content and Gene Expression
Correlation analysis revealed that there are seven genes that show a significant correlation to red pigment content, indicating that the red pigment content trait is controlled by multigenes and that the relationship among the gene expression levels are close.Therefore, stepwise regression analysis was carried to further determine the importance of these gene expressions on red pigment content.Stepwise regression analysis indicated that the contribution frequency of RsDFR gene expression level (X 4 ) to red pigment content is 87.23%.The regression equation is as follows: Y = 2.10 + 3.77 X 4 (t = 7.4331, p = 0.0001).The gene expression level of RsDFR had the highest and significant direct effect (0.8932) on red pigment content of radish.

Discussion
Radish with red skin and red flesh, an intense source of red pigment, has been the subject of much scientific interest owing to its large amount of a red pigment that is widely used in foods, wine, and cosmetics.Radish with red skin and red flesh is indigenous to the city of Chongqing, and the red pigment content is remarkably reduced when cultivated in other locations.The data on the molecular mechanism of anthocyanin accumulation in radish with red skin and red flesh can be applied to better protecting and using the resource, and also for genetic engineering breeding which may lead to the creation of new radish materials.Although the mechanism of anthocyanin accumulation in some plants has been studied extensively, little is known about the molecular mechanism of anthocyanin accumulation in radish with red skin and red flesh.Investigations have revealed that the accumulation of anthocyanin in plants is controlled by two types of genes and is closely related to the expression level of genes associated with anthocyanin biosynthesis (Zhao et al., 2015a).Our research showed that the average gene expression level of RsPAL, RsCHS, RsCHI, RsDFR, RsF3H, RsF3'H, and RsANS in red radish with red flesh are significantly higher than in four other types of radishes, and the expression level of these genes all showed positive significant correlation to red pigment content.The results indicated that the red pigment content is closely related to the expression level of genes associated with anthocyanin biosynthesis.The results were consistent with the previously reported data by Louarn et al. (2007) that anthocyanin accumulation coincides with a coordinated increase in the expression of a number of structural genes (RsPAL,RsCHS,RsCHI,RsDFR,RsF3H,RsF3'H,and RsANS) in the anthocyanin biosynthetic pathway.However, the gene expression level of the regulatory gene (RsMYB) in radish with green skin and pinky flesh is significantly higher than in the other four types of radishes, and there is no insignificance among the other four types, while the gene expression level of RsMYB showed negative and insignificant correlation to red pigment content.By contrast, Lim et al. (2015) suggested that RsMYB is an actively positive regulator for anthocyanins biosynthesis in radish plants (radish with red skin and pinky flesh), Zhao et al. (2015b) reported that both RhMYBs4-1 and RhMYBs6-1 were highly expressed in red petals and might be important regulators of anthocyanin biosynthesis and coloration in rose petals.It may be due to the fact that the regulation mechanism of the RsMYB genes is different.
Previous studies suggest that the key genes controlling anthocyanin accumulation in plants are different.Zhang et al. (2015), Yin et al. (2015), and Sun et al. (2015) suggested that the CHS gene plays an essential role in anthocyanin biosynthesis.Guo et al. (2015) suggest that IbCHI is a key enzyme in the in the anthocyanin biosynthesis of sweet potato.Rouholamin et al. (2015) found that total anthocyanin content measurement showed a positive correlation between the level of DFR gene expression and accumulation of anthocyanins in different genotypes in Punica granatum.Shi et al. (2015b) reported that the transcript level of MsANS of Magnolia sprengeri was 26-fold higher in red petals than in white petals.Li et al. (2014) suggested that MaCHS, MaDFR, and MaANS genes may control the anthocyanin biosynthesis in mulberry and up-regulation of them may greatly increase the anthocyanin content in Morus alba.Zhao et al. (2015a) suggested that the onset of anthocyanin synthesis during berry development coincides with a coordinated increase in the expression of a number of genes (such as F3′HPAL, CHI1, and DFR) in the anthocyanin biosynthetic pathway.Luo et al. (2013) proved that the genes CHS and DFR play important roles in the process of anthocyanin synthesis.Our data also showed that gene expression level of RsDFR, RsF3H, RsCHS, RsANS, RsF3'H, RsCHI, and RsPAL showed positive significant correlation to red pigment content.Meanwhile, most of the correlation coefficients among the gene expression level of the seven genes appeared to be significant.Therefore, stepwise regression analysis was carried out to further determine the importance of the expression of these genes on red pigment content.It is interesting that the contribution frequency of the RsDFR gene expression level to red pigment content is 87.23% and the gene expression level of RsDFR had the highest and significant direct effect (0.8932) on red pigment content of radish.The average gene expression level of RsDFR in radish with red skin and red flesh is 57.99-fold higher than in radish with white skin and white flesh.We hypothesized that the RsDFR gene plays a key role in anthocyanin biosynthesis in radish with red skin and red flesh.RsDFR might be one of the best targets for anthocyanin production by single gene manipulation that is applicable in radish and other plants.
Figure 1.Diff white flesh, B ed skin and pin

Table 2 .
primers for radish RsPAL,RsCHS, RsCHI, RsDFR, RsF3H, RsF3'H, RsANS, andRsMYB were designed from the conserved sequences of known orthologous sequences from GenBank (https://www.ncbi.nlm.nih.gov/genbank/).The primers were synthesized by Sangon Biotech (Shanghai, China), and are listed in Table2.The fluorescence quantitative PCR was performed in a reaction mixture (20 µL) containing 10 µL of 2 × ABI SybrGreen PCR Master Mix (Sangon Biotech Shanghai Co. Ltd., Shanghai, China), 1 µL of each primer (10 µmol/mL), 2 µL (50 ng) of cDNA, and 7 µL of ddH 2 O.The ABI StepOne Plus™ System (Applied Biosystems, USA) was programmed to run for 2 min at 95 °C at the initial step, followed by 40 cycles of melting for 10 sec at 95 °C, then annealing and extending for 40 sec at 60 °C.The fluorescence quantitative PCR experiments were performed with three replications for each sample.The primers designed from the homologous sequences

Table 3 .
It is shown that the variance of gene expression level is significant at the 1% level in the RsPAL,RsCHS, RsDFR, RsF3H, RsF3'H, RsANS and RsMYB genes and significant at the 5% level in the RsCHI gene.The average gene expression level of RsPAL, RsCHS, RsCHI, RsDFR, RsF3H, RsF3'H, RsANS and RsMYB genes are 1.12, 1.40, 0.75, 1.57, 1.10, 0.66, 1.91, and 2.14, respectively.It was indicated that the expression level of RsCHS, RsCHI, RsDFR, RsF3H, RsF3'H, and RsANS genes in radish with red skin and red flesh were all highest, while the expression level of RsPAL, RsCHS, RsDFR, RsF3H, RsF3'H, and RsANS genes in radish with white skin and white flesh are all lowest.

Table 5 .
Correlation analysis among red pigment content and gene expression in the 16 radishes