Anthocyanin biosynthetic genes in Brassica rapa
© Guo et al.; licensee BioMed Central Ltd. 2014
Received: 20 December 2013
Accepted: 27 May 2014
Published: 4 June 2014
Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level.
In total, we identified 73 genes in B. rapa as orthologs of 41 anthocyanin biosynthetic genes in A. thaliana. In B. rapa, the anthocyanin biosynthetic genes (ABGs) have expanded and most genes exist in more than one copy. The anthocyanin biosynthetic structural genes have expanded through whole genome and tandem duplication in B. rapa. More structural genes located upstream of the anthocyanin biosynthetic pathway have been retained than downstream. More negative regulatory genes are retained in the anthocyanin biosynthesis regulatory system of B. rapa.
These results will promote an understanding of the genetic mechanism of anthocyanin biosynthesis, as well as help the improvement of the nutritional quality of B. rapa through the breeding of high anthocyanin content varieties.
KeywordsComparative genomics Anthocyanin biosynthetic genes Whole genome duplication Brassica rapa Cruciferae
Flavonoids comprise a major group of secondary metabolites, which exhibit a wide range of biological functions in plants [1, 2]. Anthocyanin pigments and flavonol co-pigments are the two major flavonoid compounds, which serve as attractants of pollinators and seed dispersers. They also play an important role in protecting plants against abiotic and biotic stresses . Anthocyanins, like other flavonoid compounds, are known as potent antioxidants . The beneficial health roles of anthocyanins have received considerable attention as they are potentially protective factors against cancer and heart disease . Therefore, a comprehensive understanding of anthocyanin biosynthesis is important for developing foods that are rich in anthocyanins to meet the increasing demand for health-promoting components in our daily diet.
The biosynthetic pathways of anthocyanins have been well characterized  and the corresponding genes have been isolated from various plants. In the model plant A. thaliana, the biosynthesis, regulation and transport of anthocyanins, specifically most of the structural genes and regulatory proteins involved in anthocyanin synthesis, have been identified and functionally characterized in the last two decades [7–9]. These researches have played important roles in the comprehensive understanding of anthocyanin biosynthesis and revealed the accumulation and metabolic profiles of anthocyanins in A. thaliana.
Brassica rapa comprises a variety of vegetables, among which Chinese cabbage (Brassica rapa L. ssp. pekinensis) and pakchoi (Brassica rapa L. ssp. chinensis) are the two most consumed vegetables in China and throughout East Asia. B. rapa vegetables provide dietary fiber, vitamin C, and anti-cancer glucosinolates , and are also a potentially important source of dietary flavonols . Several varieties of B. rapa are red or purple, such as some kinds of pakchoi, turnip (Brassica rapa ssp. rapa) and Zicaitai (Brassica rapa L. ssp. chinensis var. purpurea). The purple pigments in B. rapa have been identified as anthocyanins . Nevertheless, the genetic mechanism of this purple phenotype is unclear. Because of the previous absence of genome information, little is known about the genes involved in the anthocyanin biosynthetic pathways in B. rapa[13, 14]. A complete understanding of the structural and regulatory genes involved is very important for elaborating the mechanism of anthocyanin biosynthesis in B. rapa, as well as for the breeding of new B. rapa varieties rich in anthocyanins. With the reference genome and gene annotation information for B. rapa ‘Chiifu’ , we now have the chance to systematically study the anthocyanin biosynthetic genes (ABGs) in B. rapa.
Both B. rapa and A. thaliana belong to the cruciferae family. B. rapa (A genome) has undergone whole genome triplication since its divergence from A. thaliana, followed by extensive gene loss [15, 16]. In B. rapa, the level of gene loss among the three subgenomes is unequal, with one subgenome, the Least Fractionated (LF) subgenome, having retained roughly 70% of its genes during fractionation after triplication, while the other two subgenomes, termed the Medium and Most Fractionated (MF1 and MF2, respectively) subgenomes, have retained much fewer genes [15, 16].
Whole genome duplication (WGD) is a major way of gene copy number expansion in plants. As reported previously, gene families expanded by WGD can maintain the proper balance in biological networks or cascades . After WGD and subsequent gene fractionation, the number of genes that respond to abiotic and biotic stresses or with membrane protein functions tends to be increased . In B. rapa, the genes expanded through whole genome triplication (WGT) tend to come from functional categories such as transcriptional regulation, ribosomes, response to abiotic or biotic stimuli, response to hormonal stimuli, cell organization, and transporter functions . However, no detailed information about the status of ABGs after the WGT in B. rapa has been available till now. To obtain comprehensive information on the anthocyanin biosynthetic pathway in B. rapa and look into the effect of WGT on these genes, comparative genomic analysis between B. rapa and A. thaliana was performed here based on the reference genome of B. rapa. We present several interesting observations about the evolutionary history of ABGs in B. rapa, the similarities between the anthocyanin biosynthetic pathways in A. thaliana and B. rapa, the synteny of ABGs between A. thaliana and B. rapa, and the expansion and retention of ABGs in B. rapa. The results of our systematic analysis of the complete set of ABGs in B. rapa will promote the understanding of the genetic mechanism of anthocyanin biosynthesis and the anthocyanin profiles/accumulation in B. rapa crops.
Results and discussion
ABGs in B. rapaidentified by comparative genomic analysis
Anthocyanin biosynthetic genes (ABGs) identified in B. rapa
Biosynthetic genes in phenylpropanoid pathway
BrC4H2 (Bra021636) a
Early biosynthetic genes
BrCHS 3 (Bra023441)
Late biosynthetic genes
Regulatory genes (Transcription factor)
Independent regulatory genes
Regulation by forming MBW complex
Single-Repeat R3 MYB
LATERAL ORGAN BOUNDARY DOMAIN (LBD)
Among the 73 BrABGs, 67 were syntenic orthologs of 38 AtABGs; only 8.2% of BrABGs had no syntenic relationship. Multiple copies of BrABGs in B. rapa syntenic to genes in A. thaliana were generated from the WGT, whereas 28 of the 38 AtABGs had less than three syntenic orthologs as a result of gene fractionation following triplication as described above. Furthermore, the amino acid sequence identities for pairs of syntenic genes (80.35%) and non-syntenic genes (80.43%) did not show a statistical difference. The BrABGs of different functions in the anthocyanin biosynthesis pathway had different sequence identities to their counterparts in A. thaliana (Additional file 1: Table S1). The homologous pairs of structural genes encoding anthocyanin biosynthesis enzymes shared significantly higher amino acid identity (82.10%) than those encoding transcriptional factors (76.79%), which regulate the expression of structural genes (P < 0.05), indicating that the structural genes were more highly conserved than regulatory genes. It is reasonable that excessive mutation of structural genes would likely block the biosynthesis of anthocyanins, reducing the fitness of the plant.
ABGs are over-retained in B. rapa
Comparison of the number of genes involved in the anthocyanin biosynthetic pathways in B. rapa and A. thaliana
A. thaliana a
Number and ratio of single copy to multiple copy paralogs of AtABGs
No. of paralogs with different copies a
Ratio of single to multiple copies b
The anthocyanin biosynthetic structural genes have expanded through whole genome and tandem duplication in B. rapa
Gene copy numbers can be expanded in four major ways: WGD, tandem duplication (TD), segmental duplication, and gene transposition duplication . We found that the structural BrABGs were expanded through both WGD and TD. We identified 46 BrABG structural genes as homologs of 24 AtABG structural genes. The almost-doubled number of homologs indicated that anthocyanin biosynthetic structural genes were expanded in B. rapa. Of the 46 structural genes, 41 were located at 33 loci that had syntenic relationships to 21 AtABG structural gene loci. Among the 41 syntenic orthologs, 14 BrABGs came from six tandem arrays, which accounted for about 30% of all structural genes in B. rapa. These data showed that both TD and WGT contributed to the expansion of anthocyanin biosynthetic structural genes in B. rapa. The 46 genes were distributed in different subgenomes of B. rapa, with 19, 12 and 14 genes located on LF, MF1 and MF2, respectively, and one gene not anchored on any chromosome in the current version of the B. rapa genome.
Anthocyanins are derived from branches of the flavonoid pathway, which starts with phenylalanine via the general phenylpropanoid pathway. The phenylpropanoid pathway contains three major genes: PAL, C4H and 4CL. Two types of correlated structural genes can be distinguished in the flavonoid biosynthetic pathway: Early Biosynthetic Genes (EBGs) and Late Biosynthetic Genes (LBGs) . The EBGs, which include CHS, CHI, F3H, F3’H, and FLS, lead to the production of flavonols and other flavonoid compounds, while the LBGs, which include DFR, ANS/LDOX, and UFGT, lead to the production of anthocyanins . The phenylpropanoid pathway genes and EBGs are upstream genes while the LBGs are downstream genes in the anthocyanin biosynthetic pathway. The downstream genes are specifically for anthocyanin biosynthesis .
The upstream structural genes have been expanded not only by WGD, but also TD. There are 21 homologs of the nine A. thaliana phenylpropanoid pathway genes in B. rapa (Table 1). AtC4H has five syntenic orthologs in B. rapa. BrC4H2 and BrC4H3, BrC4H4 and BrC4H5 are from two tandem arrays located in MF1 and MF2, respectively, while BrC4H1 is in LF (Table 1). This shows that BrC4Hs have expanded by both WGD and TD. At4CL2 and At4CL5 have not expanded by whole genome duplication, but their homologs in B. rapa form two tandem arrays, with one array containing four genes and the other containing two genes (Table 1).
DFR, LDOX/ANS and UFGTs are the major LBGs (downstream structural genes) of the anthocyanin biosynthetic pathway. DFR (dihydroflavonol-4-reductase) catalyzes the first committed reaction to generate anthocyanins. In A. thaliana, the gene AtDFR is known to encode a functional DFR . DFR was not expanded in B. rapa; only one gene, BrDFR, was identified as an ortholog of AtDFR. Leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS) catalyzes the formation of anthocyanidin, the first colored compound in the anthocyanin biosynthetic pathway . Two syntenic orthologs in B. rapa, BrANS1 and BrANS2, were identified by comparative genomic analysis with A. thaliana.
The gene copy number ratios between B. rapa and A. thaliana were 2.3 (21:9), 2 (18:9) and 1.4 (7:5) for the phenylpropanoid pathway genes, EBGs and LBGs, respectively. In addition to WGT, some phenylpropanoid pathway genes and EBGs (which are the upstream structural genes) were also expanded by TD (Table 1). These results show that more upstream structural genes were retained than downstream structural genes of the anthocyanin biosynthetic pathway. The redundancy of the upstream genes may guarantee products for successful downstream anthocyanin synthesis.
More negative regulatory genes are retained in the anthocyanin biosynthesis regulatory system of B. rapa
Anthocyanin biosynthesis is regulated mostly by the coordinated transcriptional control of the structural genes. The transcriptional control of ABG structural genes has been intensively studied . In A. thaliana, anthocyanin biosynthetic regulatory genes can be divided into two groups: positive and negative regulatory genes. For positive regulation of anthocyanin biosynthesis in A. thaliana, the spatial and temporal expression of structural genes is mainly determined by R2R3-MYB, basic helix-loop-helix (bHLH) and WD40-type transcriptional factors and their interaction . Four R2R3-MYB (PAP1, PAP2, MYB113 and MYB114) transcription factors and three bHLH (TT8, GL3 and EGL3) proteins combine with the WD40 repeat protein (TTG1) to form ternary transcriptional complexes that activate several anthocyanin biosynthetic structural genes, especially in the later steps (LBGs) of the flavonoid pathway [27–30]. Other R2R3-MYB proteins (MYB11, MYB12, and MYB111) regulate the structural genes of the anthocyanin biosynthetic pathway independently, especially the early steps (EBGs) of the flavonoid pathway [31, 32]. Two single-repeat R3-MYB transcription factors, MYBL2 and CPC (CAPRICE), and three members of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, LBD37, LBD38, and LBD39, are negative regulators of anthocyanin biosynthesis in A. thaliana[33–36]. The regulatory genes in anthocyanin biosynthetic pathway of B. rapa were identified as well as the duplication and retention of these genes were analyzed. Detailed information on these biosynthetic regulatory genes in B. rapa is presented below.
MYB11, MYB12, and MYB111 activate EBGs of the anthocyanin biosynthetic pathway in A. thaliana. In B. rapa, there were two syntenic orthologs of AtMYB12 and three syntenic orthologs of AtMYB111 (Table 1), but no homologs of AtMYB11 were found. These results show that the homolog of AtMYB11 was lost, while the homologs of AtMYB12 and AtMYB111 were over-retained after the WGT of B. rapa.
Another important group of transcriptional factors is the basic helix-loop-helix (bHLH) gene family, which regulates anthocyanin biosynthesis through formation of MBW ternary complexes. In A. thaliana, the bHLH transcriptional factors TT8 (TRANSPARENT TESTA 8), GL3 (GLABROUS 3) and EGL3 (ENHANCER OF GLABRA 3) interact with TTG1 (WD40 protein) and PAP1, PAP2, MYB113, or MYB114 to form multiple MBW complexes that then activate LBGs. In B. rapa, there are four BrbHLH genes, BrTT8, BrGL3, BrEGL3.1 and BrEGL3.2, corresponding to three bHLH genes in A. thaliana. The BrbHLH genes in B. rapa seem to have undergone relatively extensive fractionation after WGT.
There are several transcription factors including two R3-type single MYB proteins, MYBL2 and CPC, and three N/NO3− induced members of the LBD gene family that act as negative regulators of anthocyanin biosynthesis in A. thaliana. AtMYBL2 is a transcriptional repressor that negatively regulates anthocyanin biosynthesis by interacting with TT8 and MBW complexes [33, 34]. In B. rapa, there are two syntenic orthologs, BrMYBL2.1 and BrMYBL2.2, found in LF and MF1, respectively. AtCPC, another single repeat R3-MYB transcription factor, works as a negative regulator of anthocyanin biosynthesis . The AtCPC gene has two syntenic orthologs in B. rapa, BrCPC1 and BrCPC2, which both share more than 90% amino acid sequence identity with their At ortholog. LBD37, LBD38 and LBD39 negatively regulate the late anthocyanin-specific steps by repressing PAP1 and PAP2 under N/NO3− induction . There are seven syntenic orthologs of AtLBD37, AtLBD38 and AtLBD39 in B. rapa (Table 1).
The regulatory genes have expanded mainly through WGT in B. rapa. In total, 25 BrABG regulatory genes were identified as homologs of 16 AtABG regulatory genes. Fourteen BrABG positive regulatory genes were identified as homologs of 11 AtABGs, while 11 BrABG negative regulatory genes were identified as homologs of 5 AtABGs. The copy numbers of the positive regulatory genes did not show a significant change between B. rapa and A. thaliana, but the number of negative regulatory genes was almost doubled in B. rapa. Comparing the copy numbers of the regulatory genes, we found that more negative than positive regulatory genes were retained in the B. rapa genome.
Anthocyanins play important roles in responding to abiotic and biotic stresses for plants. B. rapa comprises a variety of vegetables with rich morphological diversity. Several varieties accumulate variant kinds and contents of anthocyanins in different tissues, while more varieties of B. rapa present green with no anthocyanins accumulated (data not shown in this paper). So the copy numbers of negative and positive regulatory genes will help us to understand the metabolic characteristics of anthocyanin in B. rapa.
Anthocyanin biosynthetic genes (ABGs) were identified based on whole-genome comparative analysis between A. thaliana and B. rapa. Anthocyanin biosynthetic pathways including 73 genes were established in B. rapa. Multiple copies of the BrABGs were generated by WGD and retained synteny with their orthologs in A. thaliana, but most genes appeared to comprise less than three copies because of gene loss following WGT. More upstream structural genes of the anthocyanin biosynthetic pathway have been retained than downstream. Based on the presence of these homologous structural genes, the anthocyanin biosynthetic pathway in B. rapa was then established. More negative regulatory genes have been retained than positive by comparing the copy numbers. The composition of BrABGs could help us to explain the metabolic profiles of anthocyanin accumulation and elaborate the genetic mechanism of anthocyanin biosynthesis in B. rapa.
There has been little research on anthocyanin biosynthetic genes in B. rapa. Here, we identified anthocyanin biosynthetic genes systematically at the whole genome level. The determination of a complete set of anthocyanin biosynthetic genes in B. rapa provides a valuable resource for the study of anthocyanin-related traits and genetic improvement of the anthocyanin nutritional quality of B. rapa.
Database for ABGs identification in B. rapa
The complete sets of gene sequences in A. thaliana involved in the anthocyanin biosynthetic pathway were downloaded from the TAIR database (http://www.arabidopsis.org/). The B. rapa genome sequence (version 1.5) and gene sequences from BRAD (http://brassicadb.org/brad/)  were used to identify the ABGs in B. rapa.
Identification of homologous genes between B. rapa and A. thaliana
We used the anthocyanin biosynthetic gene and protein sequences of A. thaliana to align with the genome and protein sequences of B. rapa using BLASTN and BLASTP with a cut off E-value ≤ 1E−10 and coverage ≥ 0.75. We identified syntenic orthologs between A. thaliana and B. rapa from BRAD (http://brassicadb.org/brad/), which were determined by both sequence similarity (cutoff: E ≤ 10−20 ) and the collinearity of flanking genes .
Phylogenetic analysis of gene sequences
A phylogenetic tree was constructed by the neighbor-joining method using MEGA4 . The stability of tree nodes was tested by bootstrap analysis with 1000 iterations.
Availability of supporting data
All the anthocyanin biosynthetic genes of A. thaliana referred in this paper were retrieved from the TAIR database (http://www.arabidopsis.org/).
The B. rapa genome sequence (version 1.5), as well as CDS and protein sequences of BrABGs were retrieved from the Brassica database (BRAD) (http://brassicadb.org/brad/).
The protein sequences of VvFLS (BAE75808) and ZmP1 (P27898) used as outgroups to construct the phylogenetic trees in this paper were downloaded from GeneBank (NCBI) (http://www.ncbi.nlm.nih.gov/).
Whole genome duplication
Whole genome triplication
Anthocyanin biosynthetic genes
Early biosynthetic genes
Late biosynthetic genes
Anthocyanin 3-O-glucoside: 2 -O-xylosyltransferase
Production of anthocyanin pigment 1/2
Transparent testa 8/19
Enhancer of glabrous 3
Transparent testa glabrous 1
Lateral organ boundary domain.
The work was funded by the National High Technology R&D Program of China (2012AA100101), the National Program on Key Basic Research Projects of China (The 973 Program: 2012CB113900, 2013CB127000 and 2013CB127006) and International Joint Research Grant of Ministry of Science and Technology, P. R. China (2011DFR31180), as well as National Natural Science Foundation of China (NSFC grant: 31301771 and 31201628). Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, P. R. China and the Sino-Dutch Joint Lab of Horticultural Genomics Technology in Beijing.
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