Báo cáo khoa học: Genome-wide identification of glucosinolate synthesis genes in Brassica rapa potx

Genome-wide identification of glucosinolate synthesis genes in Brassica rapa Yun-Xiang Zang1,2,*, Hyun Uk Kim1,*, Jin A Kim1, Myung-Ho Lim1, Mina Jin1, Sang Choon Lee1, Soo-Jin Kwon1, Soo-In Lee1, Joon Ki Hong1, Tae-Ho Park1, Jeong-Hwan Mun1, Young-Joo Seol1, Seung-Beom Hong3 and Beom-Seok Park1 Genomics Division, Department of Agricultural Bio-resources, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA), Suwon, Korea School of Agricultural and Food Science, Zhejiang Forestry University, Lin’an, Hangzhou, China Department of Biology, San Jacinto College, Houston, TX, USA Keywords bioinformatics; biosynthesis pathway; Brassica rapa; gene identification; glucosinolate Correspondence B S Park, Genomics Division, Department of Agricultural Bio-resources, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA), Suwon 441-707, Korea Fax: +82 31 299 1672 Tel: +82 31 299 1671 E-mail: pbeom@rda.go.kr *These authors contributed equally to this work Database The following have been deposited to the GenBank database Accession numbers are shown in parenthesis: BrBCAT4 (FJ376036– FJ376037), BrMAM (FJ376038–FJ376041), BrBCAT3 (FJ376042–FJ376043), BrCYP79F1 (FJ376044), BrCYP79B2 (FJ376045– FJ376046), BrCYP79B3 (FJ376047), BrCYP79A2-1 (FJ376048), BrCYP83A1 (FJ376049–FJ376050), BrCYP83B1 (FJ376051), BrC-S lyase (FJ376052– FJ376053), BrUGT74B1-1 (FJ376054), BrUGT74C1 (FJ376055–FJ376057), BrST5a (FJ376058–FJ376059), BrST5b (FJ376060– FJ376068), BrST5c-1 (FJ376069), BrFMOGS-OX1 (FJ376070), BrFMOGS-OX5 (FJ376071), BrAOP2 (FJ376073), BrGSL-OH (FJ376074), BrBZO1p (FJ376075), BrDof1.1 (FJ584284– FJ584285), BrIQD1-1 (FJ584286), BrMYB28 (FJ584287–FJ584289), BrMYB29 (FJ584290– FJ584292), BrMYB34 (FJ584293–FJ584295), BrMYB51 (FJ584296–FJ584299), BrMYB122-1 (FJ584300) Glucosinolates play important roles in plant defense against herbivores and microbes, as well as in human nutrition Some glucosinolate-derived isothiocyanate and nitrile compounds have been clinically proven for their anticarcinogenic activity To better understand glucosinolate biosynthesis in Brassica rapa, we conducted a comparative genomics study with Arabidopsis thaliana and identified total 56 putative biosynthetic and regulator genes This established a high colinearity in the glucosinolate biosynthesis pathway between Arabidopsis and B rapa Glucosinolate genes in B rapa share 72–94% nucleotide sequence identity with the Arabidopsis orthologs and exist in different copy numbers The exon ⁄ intron split pattern of B rapa is almost identical to that of Arabidopsis, although inversion, insertion, deletion and intron size variations commonly occur Four genes appear to be nonfunctional as a result of the presence of a frame shift mutation and retrotransposon insertion At least 12 paralogs of desulfoglucosinolate sulfotransferase were found in B rapa, whereas only three were found in Arabidopsis The expression of those paralogs was not tissue-specific but varied greatly depending on B rapa tissue types Expression was also developmentally regulated in some paralogs but not in other paralogs Most of the regulator genes are present as triple copies Accordingly, glucosinolate synthesis and regulation in B rapa appears to be more complex than that of Arabidopsis With the isolation and further characterization of the endogenous genes, health-beneficial vegetables or desirable animal feed crops could be developed by metabolically engineering the glucosinolate pathway (Received 15 February 2009, revised 31 March 2009, accepted 24 April 2009) doi:10.1111/j.1742-4658.2009.07076.x Abbreviations BAC, bacterial artificial chromosome; CDS, coding sequence; EST, expressed sequence tag; LTR, long terminal repeat; MAM, methylthioalkylmalate synthase; NCBI, National Center for Biotechnology Information FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS 3559 Glucosinolate biosynthesis genes in Brassica rapa Glucosinolates, a group of sulfur-rich secondary metabolites, have received much attention because their breakdown products display several potent bioactivities that serve as plant defense, as well as anticarcinogenesis compounds, in mammals [1–3] Upon tissue disruption, the enzyme myrosinase cleaves off the glucose group from a glucosinolate, and the remaining molecule then quickly converts to a bioactive substance (i.e an isothiocyanate, nitrile or thiocyanate) Among the isothiocyanates, sulforaphane, a derivative of glucoraphanin, is known to be the most promising anticancer agent because of its strong and broad spectrum activity against several types of cancer cells [3–10] Indole-3-carbinol, a derivative of glucobrassicin, also comprises a good anticarcinogen Both exhibit their effects by inducing phase II detoxification enzymes, altering estrogen metabolism, blocking the cell cycle or protecting against oxidative damages [11–15] Phenethyl isothiocyanate, a derivative of gluconasturtiin, was reported to be effective for chemoprotection [16–18], although it possesses genotoxic activity [19–21] Crambene (1-cyano-2-hydroxy3-butene), an aliphatic nitrile derived from progoitrin, upregulates the synthesis of glutathione S-transferase in the liver and other organs [22] Glucosinolates are classified into three major groups, namely aliphatic, indolyl and aromatic glucosinolates, based on the amino acids from which they are synthesized [23] Biosynthesis of aliphatic and aromatic glucosinolates generally involves three steps (Fig 1) and begins with the elongation of methionine and phenylalanine, respectively The initial step of aliphatic glucosinolate synthesis is catalyzed by methylthioalkylmalate synthase (MAM) to form the elongated homologs [24,25] The core structures are made via oxidation by cytochrome P450 enzymes, CYP79 and CYP83, followed by C-S cleavage, glucosylation and sulfation Finally, the side chains are modified by oxidation, elimination, akylation or esterification Some of the genes involved in this step, FMOGS-OX15, AOP, GSL-OH and BZO1, have been isolated recently [26–31] Cruciferous vegetables, including broccoli, cabbage, Chinese cabbage, cauliflower, and brussels sprouts, are rich in glucosinolates A high intake of cruciferous vegetables was shown to significantly reduce the risk of certain cancers and cardiovascular diseases [32–34] Chinese cabbage (Brassica rapa ssp pekinensis) is one of the most highly consumed vegetable crops in Asia However, unlike broccoli, many Chinese cabbage cultivars not produce detectable levels of glucoraphanin To date, most of the structural genes responsible for the biosynthesis of the three groups of glucosinolates 3560 Y.-X Zang et al have been identified and characterized in Arabidopsis [23,35] In addition, several regulators that control glucosinolate biosynthesis have been identified recently in Arabidopsis [36–43] However, little is known about the specific genes existing in Brassica crops, except for the MAM and AOP genes in Brassica oleracea [44–46] The glucosinolate profile is highly dependent on genotype, although it is also affected by developmental or environmental changes [47–49] Previously, we reported that the ectopic expression of Arabidopsis glucosinolate synthesis genes altered the glucosinolate profile in Chinese cabbage [50,51] Because most of the Arabidopsis genes encoding glucosinolate biosynthesis pathways have been identified and Chinese cabbage is a close relative of Arabidopsis, comparative genomic studies will allow for the easy identification of relevant genes in Brassicas The identification and characterization of glucosinolate synthesis genes in Chinese cabbage would pave the way for further improvement of agronomic traits via genetic engineering In the present study, we report the genome-wide identification of B rapa glucosinolate synthesis (BrGS) and regulator genes using our B rapa genome sequence in conjunction with the available Arabidopsis sequence We also show that many BrGS genes exist in a small multigene family and that at least 12 desulfoglucosinolate sulfotransferase (BrST) paralogs are present and are differentially expressed Results BrGS gene identification from cDNA and bacterial artificial chromosome (BAC) libraries As part of the B rapa genome sequencing project, we produced 127 143 expressed sequence tags (ESTs) from 28 different cDNA libraries that were released to the National Center for Biotechnology Information (NCBI) database and a new B rapa EST database, BrEMD (http://www.brassica-rapa.org/BrEMD/) with microarray data Furthermore, we determined more than 127 000 BAC end sequences, and approximately 589 seed BACs were sequenced and anchored in Arabidopsis whole chromosomes The 65.8 Mb seed BAC sequence information covered approximately 75.3% of the Arabidopsis genome and 40% of the B rapa euchromatin region [52] On the basis of these databases, homologous genes were identified by a blastn search using the Arabidopsis gene sequence as query All the ESTs that matched each query sequence were aligned to remove the redundant clones, and EST clones containing a start codon were resequenced to generate the full-length cDNA sequence Through FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS Y.-X Zang et al Glucosinolate biosynthesis genes in Brassica rapa Fig Biosynthesis pathways of the three major groups of glucosinolates in B rapa The genes involved in each step are shown Numbers in parenthesis denote gene copy numbers FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS 3561 Glucosinolate biosynthesis genes in Brassica rapa this alignment, a total of 35 different genes was found from ESTs In the same way, blastn searches were performed against the BAC sequence databases, yielding 44 different genes, among which 23 overlapped the EST sequences Thus, a total of 56 individual genes was identified from both EST and BAC clones, of which 44 contained the full-length coding sequence (CDS) (Fig 1, Tables and 2) They contain all the homologs of Arabidopsis except for CYP79F2, FMOGS-OX24, AOP3 and MYB76 In Arabidopsis, AOP2 and AOP3 are tandemly located on chromosome IV [29]; however, AOP2 was only found in B rapa The same observation Y.-X Zang et al was also made in B oleracea [45] This suggests that duplication occurred in Arabidopsis after its divergence from Brassica Four genes, BrUGT74C1-1, BrST5b-6, BrST5b-4 and BrMYB122-1, appear to be nonfunctional as a result of a frame shift or retrotransposon– insertion mutations (Fig 2) To estimate the total number of putative BrGS genes in the whole genome of B rapa, a genomic blot was performed using the CYP79F1 ⁄ F2, CYP79B2 ⁄ B3, CYP83A1 and CYP83B1 genes as probes (see Supporting information, Fig S1) [53] This analysis predicted the presence of a total of eight genes (two, three, two Table Comparison of putative BrGS biosynthetic genes with the Arabidopsis orthologs The nucleotide sequence of the coding region was used for comparison analysis; the BrGS gene sequence is from the partial- or full-length CDS; the single percentage indicates the single B rapa orthologous sequence that was available Most of the genes are full length except those marked with an asterisk Corresponding clones B rapa gene name At3g19710 BrMAM Amino acid side chain elongation Corresponding AGI BrBCAT4 Glucosinolate pathway No of genes found At5g23010 At5g23020 BrBCAT3 At2g22330 At5g05260 At4g13770 1 At4g31500 At2g20610 At1g24100 BrUGT74C1 At2g31790 BrST5a At1g74100 BrST5b 3562 At1g16410 At4g39950 BrUGT74B1 References BrCYP79F1 BrCYP79B2 BrCYP83B1 BrC-S lyase Side chain modification BrCYP79B3 BrCYP79A2 BrCYP83A1 Core structure formation step At3g49680 At1g74090 BrST5c At1g18590 BrFMOGS-OX1 At1g65860 At1g12140 BrFMOGS-OX5 BrAOP2 At4g03060 BrGSL-OH At2g25450 BrBZO1p At1g65880 [24], [25], [26], [27], [28], [29], [31], [57], 1 1 1 [58], [59], [79] BAC EST KBrH046K16 h BR069190 BR005855 BR043724* h BR003821 h BR008244* BR080925* BR007081 BR046183 BR1 20984 BR098069 h BR058540 BR061092 BR091686 BR087395 BR098736 BR100939 BR043439 BR043626 h BR015379 BR082516 BR059286 h h h h BR094491 BR021429 BR116105 BR067797 BR097443 h KBrB010E08F* h KBrH121C04F* KBrH045F08R h KBrB035G16 KBrB022O03 KBrH106H11 h KBrS003K07 KBrH086H05R KBrH009l04 h h KBrH010M17 KBrH015M19 h h KBrH036G21R* KBrB119E11F KBrB056L1 KBrB056L15 KBrH096A10 KBrB041J04 KBrB034H04 KBrB069A23 h KBrS002F02F KBrB032C15 KBrB002P01 KBrH047C14 KBrB083K19 % Identity At and B rapa 83.8–83.9 78.4–87.0 84.7–87.0 85.4 89.0–89.3 89.5 85.6 87.3–87.4 90.1 86.6–87.1 84.5 85.1–88.4 86.6–86.6 76.0–85.9 85.7 83.3 83.3 71.9 85 81.1 FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS Y.-X Zang et al Glucosinolate biosynthesis genes in Brassica rapa A B Fig Structures of the predicted nonfunctional BrGS genes (A) Three of the four carried deletion mutations and (B) the fourth one carried a putative restrotransposon insertion A non-LTR retrotransposon insertion is marked by approximately kb insertion Asterisks indicate the position of a premature stop codon Thick, thin and dotted lines denote the exon, intron and the gap between BrST5b-4 and BrST5b-x, respectively and one copies for each gene, respectively) On the other hand, a total of seven genes was found from our database search for those genes, suggesting that the percentage of BrGS genes identified in the present study is approximately 87.5% Similar to the biosynthetic genes, BrGS regulator genes share 81–94% nucleotide sequence identity with Arabidopsis orthologs and 15 genes exist in a small multigene family (Table 2) Most of the genes are triplicated, indicating that regulator genes are mostly retained after the Brassica genome triplication BrGS gene identity with Arabidopsis and other Brassica orthologs Structure of BrGS genes BrGS biosynthetic genes share 72–90% nucleotide sequence identity with Arabidopsis orthologs and 28 genes exist in a small multigene family (Table 1) This close relatedness is further substantiated by our phylogenetic tree analyses (Fig 3; see also Supporting information, Figs S2–S11) However, most of the BrGS genes share more than 90% identity with other Brassica orthologs (Table 3) This is consistent with the notion that the Brassica species evolved after divergence from the Arabidopsis lineage Notably, BrAOP2 has the lowest sequence identity with the orthologs of Arabidopsis and B oleracea Identities within the BrGS paralogs are usually higher than those with Arabidopsis and other Brassica species All of the BrST5b paralogous genes except BrST5b-4 share more than 80% sequence identity with AtST5b (Table 4) Identities between BrST5b and AtST5b (76–86%) are comparable to those between tandem BrST5b repeats (77–89%) and between nontandem repeats BrST5b-6 and BrST5b-9 (88%) (Fig 4, Table 4) This suggests that duplication occurred after a very recent divergence between Arabidopsis and B rapa One putative benzoate-CoA ligase gene BrBZO1p was identified (see also Supporting information, Fig S11) It has a similarity of 81% compared to both BZO1 and At1g65890 Ordered assembly of the overlapping sequences of BAC and EST clones yielded the overall gene structures shown in Fig The exon and intron structures of the BrGS genes were identical to those of Arabidopsis homologs However, insertion, deletion and intron size variations were commonly noted in BrGS genes One of the two BrC-S lyase genes had a bp deletion at the last exon, which resulted in a 3¢ truncated protein with a 16 amino acid deletion compared to the Arabidopsis homolog The truncation of 3¢ end exon might alter either gene function or the expression pattern in such a way to change feedback regulation, as previously proposed by Gao et al [46] Desulfoglucosinolate sulfotransferase genes did not have any intron in both Arabidopsis and B rapa (Fig 5A) The AOP2 structure of B rapa was compared with that of B oleracea and Arabidopsis All three species contained four exons and three introns, along with considerable changes in intron sizes (Fig 5B) One of the two BrST5a genes contained a bp insertion (Fig 5A), which did not lead to a frame shift mutation Insertion or deletion often gives rise to a frame shift mutation that causes the loss of gene function This type of mutation occurred in two BrGS genes with premature stop codons immediately after the deletion sites (Fig 2A) Among nine BrST5b paralogs, BrST5b-4 FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS 3563 Glucosinolate biosynthesis genes in Brassica rapa Y.-X Zang et al appears to be a pseudogene because it contains an approximately kb insert of a putative non-long terminal repeat (LTR) retrotransposon that encodes a reverse transcriptase (Fig 2B) Transposon insertion mutations in coding sequences or intergenic regions were also previously observed in B oleracea [45] Another gene, BrST5b-x, with only a 150 bp 3¢ end partial sequence, was found to be located in the intergenic region approximately 500 bp downstream of BrST5b-4 (Fig 2B) However, we did not consider this as another copy of BrST5b because of the presence of only a small amount of remainder sequence as a result of a massive deletion event Pseudogenes are assumed to arise frequently during genome evolution and are often regarded as ‘molecular fossils’ in evolutionary genomics [54] Pseudogenes might be the result of natural selection reducing functional redundancy However, the divergent copies of duplicated genes would be further diversified to evolve for neofunctionalization or subfunctionalization [55,56] Fig Nonrooted neighbor-joining phylogenetic tree of B rapa desulfoglucosinolate sulfotransferases and Arabidopsis sulfotransferases Coding sequences of AtST5b were used to identify the orthologs between these two species because some of BrST5b are pseudogenes Bootstrap values with 500 replicates are denoted as percentages Divergent duplication and differential expression of BrST genes In comparison with three orthologs in Arabidopsis, desulfoglucosinolate sulfotransferase exists in a small Table Comparison of putative BrGS regulator genes with the Arabidopsis orthologs The nucleotide sequence of the coding region was used for comparison; the BrGS gene sequences used are either partial or full-length CDS Most of the genes are full length except those marked with an asterisk Corresponding clones Transcription factors B rapa gene name Corresponding AGI No of genes found Nuclear-localized regulators BrDof1.1 At1g07640 BrIQD1-1 BrMYB28 At3g09710 At5g61420 BrMYB29 At5g07690 BrMYB34 At5g60890 BrMYB51 At1g18570 R2R3-Myb transcription factors for aliphatic glucosinolates R2R3-Myb transcription factors for indole glucosinolates References 3564 BrMYB122 At1g74080 [36], [37], [38], [39], [40],[41],[42],[43] BAC EST KBrH010M08 KBrB056G08R* KBrB055G10 KBrB034G03 KBrB051M06 KBrH005L20 KBrBOO1B07 KBrS005P19F* KBrB132A06R* KBrB051M06 KBrB118H07R & KBrH078K01R h KBrH092O19 KBrB065D24 h KBrE052E18F* KBrB056L15 h h h BR046041 h BR078654 BR005887 h h BR012922 BR115967 BR102850* BR104839 BR101256 BR116816* h h % Identity At and B rapa 81.7–81.9 81.4 84.1–85.4 83.2–87.0 82.1–94.3 81.0–89.6 82.4 FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS Y.-X Zang et al Glucosinolate biosynthesis genes in Brassica rapa Table Sequence similarities between BrGS genes and other Brassica orthologs The nucleotide sequence of the coding region was used for comparison analysis; the highest percentages are shown when a gene has several copies B rapa gene Gene of other Brassica species % of homology Sources BrMAM BrCYP79F1 BrCYP79B2 BoGSL-Elong(L) BoCYP79F1 SaCYP79B2 BnCYP79B2 BnUGT74B1 BoUGT74B1 BoGSL-OH BoGSL-ALKa(b) 97.8 97.6 93.9 99 97.4 97.3 84.3 77 Brassica oleracea BAC clone B19N3 [80] Brassica oleracea BAC clone B77C13 (accession EU579455, NCBI) Sinapis alba cytochrome P450 CYP79B1 (accession AF069494) [81] Brassica napus cytochrome P450 CYP79B5 (accession AF453287) [82] Brassica napus thiohydroximate S-glucosyltransferase Brassica oleracea BAC clone B16J1 (accession EU579454) Brassica oleracea BAC clone B67C16 Brassica oleracea BAC clone B21H13 [45] BrUGT74B1 BrGSL-OH BrAOP2 Fig Comparative map of the five BACs containing BrST paralogs and their counterparts in Arabidopsis At Chr1, Arabidopsis chromosome 1; Br R7 (Chr7), B rapa linkage group R7 (chromosome 7); Br R9 (Chr1), B rapa linkage group R9 (chromosome 1); Mb, megabase; cM, centimorgan The loci of AtST5a,b (At1g74100 and At1g74090) and BrST counterparts are indicated by oval-shaped bars The loci that correspond to the five Brassica BACs are all located on Arabidopsis chromosome and are marked by stick bars Colinear and noncolinear genes are indicated by dashed and dotted lines, respectively The location of KBrB034H04 on the B rapa chromosome has not yet been established Table Similarity and divergence among desulfoglucosinolate sulfotransferase genes of Arabidopsis and B rapa Values represent the percentage similarity in the upper triangle area and percentage divergence in the lower triangle area as demarcated by the diagonally aligned black squares; full-length CDS was employed for the analyses using DNASTAR software (DNASTAR Inc., Madison, WI, USA) FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS 3565 Glucosinolate biosynthesis genes in Brassica rapa Y.-X Zang et al are involved in the biosynthesis of indolyl and aliphatic glucosinolates, respectively [57] Nevertheless, they share 80% nucleotide identity and are tandemly located on chromosome (Fig 4) Thus, we examined the expression patterns of BrST genes in different tissues by RT-PCR (Fig 6) BrActin1, an actin gene of B rapa, was used as an internal control to adjust the amount of cDNA template for PCR because it is constitutively expressed in all types of tissues Primers were designed from the gene specific untranslated region (see Supporting information, Table S1) All of the genes except BrST5b-4 were expressed in all six different tissues, although the expression profiles were different Generally, BrST5b was expressed at higher levels than BrST5c but at lower levels than BrST5a Specifically, BrST5b-6 and BrST5b-7 were expressed at the lowest levels because their products were not shown until after 40 cycles of PCR (Fig 6) All of the PCR products were sequenced and were matched to individual gene sequences (data not shown) BrST5a-1 A B Fig Structures of representative BrGS genes (A) Comparison with Arabidopsis orthologs (B) Structural comparison of AOP2 orthologs of Arabidopsis (At), B rapa (Br) and B oleracea (Bo) Representative BrGS gene structures were composed based on the full-length genomic, cDNA, or coding sequences of BAC and EST clones Arabidopsis gene structures were generated according to NCBI sequence information Each pair of genes was aligned in a colinear form Positions of introns are indicated by the triangles, above which intron sizes (bp) are shown as numerals The position and size of the nucleotide insertion and deletion are also marked as In ⁄ Del multigene family with 12 paralogs in which two EST clones are not mapped on B rapa (Table 1, Fig 4) Most of them are clustered in a tandem array, as shown in the chromosomal loci of BAC clones (Fig 4) In addition, they are usually clustered together in the phylogenetic tree (Fig 3) Two Arabidopsis desulfoglucosinolate sulfotransferases, AtST5a (At1g74100) and AtST5b (At1g74090), 3566 Fig RT-PCR analysis of BrST genes in different types of tissues L, mature leaf; R, mature root; FB, floral bud; SL, seedling; S, stamen; C, carpel The PCR products of the BrST genes are approximately kb; BrActin1 is approximately 450 bp, which serves as an internal control FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS Y.-X Zang et al was strongly expressed in all tissues except the stamen By contrast, BrST5a-2 was strongly expressed in the stamen, but weakly in the floral bud and carpel Overall, BrST5b paralogs were expressed at a very low level in the floral bud However, some genes (i.e BrST5b-1 and BrST5b-2) were expressed strongly in the carpel, whereas others (i.e BrST5b-2, BrST5b-8 and BrST5b-9) were expressed strongly in the stamen The results obtained demonstrate that the expression of the paralogs is not tissue-specific but varies greatly depending on tissue type In terms of the overall expression level, mature leaf and root expressed BrST paralogs at higher levels than other tissues, demonstrating functional redundancy for differential expression In seedling tissue, BrST5a paralogs were more strongly expressed than BrST5b paralogs No significant differences in the expression levels of BrST5a paralogs were noted between the seedling and mature leaf and root tissues On the other hand, significant differences between those tissues were found for the expression levels of BrST5b paralogs except BrST5b-1 Thus, expression is developmentally regulated in some BrST5b paralogs but not in BrST5a paralogs Discussion Similarity between B rapa and Arabidopsis in the glucosinolate biosynthesis pathway Our B rapa genome sequence database searches identified the counterparts of most of Arabidopsis glucosinolate synthesis genes, and they are present in various copy numbers (Fig 1) Only a few genes that correspond to Arabidopsis CYP79F2, FMOGS-OX24 and AOP3 were not found in B rapa Thus, a high colinearity in the glucosinolate biosynthesis pathway exists between Arabidopsis and B rapa despite the difference in gene copy numbers As the first step, two different genes, BCAT and MAM, are known to be involved in the chain elongation of Met-derived aliphatic glucosinolate biosynthesis BCAT4 and BCAT3 enzymes catalyze the deamination and transamination, respectively [58,59] B rapa contains two BCAT4 paralogs that have 92% nucleotide sequence identity and are the same size B rapa also carries two BCAT3 paralogs, one of which has a fulllength CDS MAM enzyme catalyzes the condensation of acetyl-coenzyme A with a series of x-methylthio2-oxoalkanoic acids MAM1 ⁄ MAM2, two tandem paralogs found in some of Arabidopsis ecotypes, are responsible for the first two cycles of chain elongation [24] MAM3 enzyme catalyzes all the different cycles of Met chain elongation [25] We identified four MAM Glucosinolate biosynthesis genes in Brassica rapa paralogs in the B rapa genome that share approximately 78–87% identities with the Arabidopsis orthologs, although we were unable to determine which of these is individually equivalent to MAM1, and Two of them are not identical in an approximately 200 bp region of the 3¢ ends This is also the case in the B oleracea MAM (BoGSL-ELONG) gene family [46] and did not affect its enzymatic function equivalent to the Arabidopsis ortholog MAM1 [60] In addition to tissue-dependent differential expression, the members of BrMAM gene family may encode enzymes of different biochemical properties with respect to chain elongation, such as Arabidopsis MAM orthologs Two Arabidopsis genes, IPMS1 and IPMS2, that encode isopropylmalate synthase are similar to the MAM family genes, with 60% similarity in their amino acid sequence [46,61] Nevertheless, they are not involved in Met chain elongation but are involved in leucine biosynthesis Phylogenetic analysis indicates that the BrMAM genes not belong to the IPMS family but, instead, belong to the the MAM gene family We were unable to identify the genes responsible for phenylalanine chain elongation, an initial step of aromatic glucosinolate synthesis, because the corresponding genes have not yet been isolated in Arabidopsis and other Brassica species As the second step, the formation of the glucosinolate core structure is initiated by the conversion of amino acid to the corresponding aldoxime, and this is catalyzed by the CYP79 enzymes [23] Three groups of CYP79 family genes, CYP79F1,2, CYP79B2,3 and CYP79A2, are involved in aliphatic, indolyl and aromatic glucosinolate biosynthesis, respectively Our database searches indicate two copies of the CYP79B2 and C-S lyase genes and three copies of UGT74C1 in B rapa, unlike the single copy genes in Arabidopsis Such duplication may necessate a redundant function for tissue- or development-dependent differential expression Excluding two copies of nonfunctional BrST5 carrying frameshift and transposon insertion mutations, eight copies of BrST5 are actually involved in aliphatic glucosinolate synthesis in B rapa (Table 1, Figs and 4), whereas two copies of the orthologs exist in Arabidopsis The expression level of BrST5 is not only developmentally regulated, but also highly dependent on tissue type (Fig 6) Because sulfonation is a penultimate step of glucosinolate biosynthesis, the expression of BrST5 may play a crucial role in the tissue-specific and developmental accumulation of glucosinolates It remains to be determined whether BrST5 transcript levels are correlated with the accumulated levels of indole and aliphatic glucosinolates The final step of glucosinolate synthesis is side chain modification and, currently, this step is well FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS 3567 Glucosinolate biosynthesis genes in Brassica rapa characterized only for aliphatic glucosinolate in Arabidopsis Glucoraphanin (4-methylsulfinylbutylglucosinolate) is abundant in Columbia but is absent in the Landsberg ecotype of Arabidopsis This difference is attributed to the AOP2 gene, whose expression diverts glucoraphanin into alkenyl-glucosinolate [29] Our genome database search yielded the presence of a single copy of AOP2 in B rapa However, two AOP2 quantitative trait loci, Ali-QTL3.1 and Ali-QTL9.1, were recently reported to be involved in determining the type and concentration of glucosinolates found in B rapa leaves [62] Consistent with this finding, our Southern blot analysis indicated the presence of two copies of AOP2 in B rapa (data not shown) The presence of AOP2 explains why glucoraphanin was not detectable in Chinese cabbage [50,51] In B oleracea, two tandemly repeated copies of AOP2 contain a bp deletion at the third exon, which is responsible for the high accumulation of glucoraphanin (Fig 5B) [45] Brassica napus, a species resulting from interspecific hybridization between B rapa and B oleracea, was reported to be absent in glucoraphanin [63] This is most likely as result of the AOP2 gene introduced from B rapa The content of glucoraphanin in B rapa and B napus could be elevated by inhibiting AOP2 expression via antisense or RNA interference approaches Similarity of the genes controlling glucosinolate biosynthesis between B rapa and Arabidopsis B rapa contains the orthologs of the Arabidopsis glucosinolate regulators, except MYB76 (Table 2) Unlike Arabidopsis, they are normally triplicated, consistent with the Brassica genome triplication event The duplication and divergence of the regulators in a small multigene family along with multiple duplications of their target biosynthesis genes may result in phenotypic variation AOP2 ⁄ AOP3 null accessions of Arabidopsis were shown to accumulate an increased level of the precursor methylsulfinylalkyl glucosinolate but also a considerably lower level of total aliphatic glucosinolates than the accessions with a functional AOP2 allele, which has been explained by the differential feedback regulation of transcript regulators MYB28, 76 and 29 by the side chain modification end products [29,30,64] Similarly, epistatic interactions between AOP2 and transcript regulators MYB28 and MYB29 may exist in B rapa BrGS gene duplication The Brassica genome is believed to have triplicated soon after its divergence from Arabidopsis [65–67] The 3568 Y.-X Zang et al genome sizes of B rapa (550 Mb) and B oleracea (696 Mb) are more than four- and five-fold greater than that of Arabidopsis (125 Mb), respectively [68,69] This could be explained in part by the presence of bigger gene families as a result of genome diploidization, segmentation or gene duplication In B oleracea, genome rearrangement is commonly followed by gene loss, fragmentation and dispersal [70] Many gene duplications arose as a result of the triplication event, and those genes involved in signal transduction or transcriptional control are more extensively retained than others during the evolution process [70] Some tandemly duplicated genes in B rapa and B oleracea are likely to be the result of an unequal crossover during the rearrangement process after Brassica genome triplication [45,46] Approximately 14% of B rapa genome is estimated to consist of transposable elements, the majority of which are retrotransposons [69] It has been proposed that gene duplication also is facilitated by retrotransposon carrying a LTR [71] BrST is a good example of a multigene family with tandem arrays of genes in B rapa The genes adjacent to two tandem repeats, BrST5b-1 and BrST5b-2, were colinear with their Arabidopsis counterparts, and all the other BrST genes jumped to completely new positions (Fig 4) BrST5b-1, 3, and were found to be flanked by LTR Copia-like retrotransposons (data not shown) BrST5b-4 was disrupted by insertion of a putative non-LTR retrotransposon and shares 89% sequence identity with BrST5b-5 in a tandem array They also are tandemly arranged with BrST5b-3 in the same BAC clone, but with lower sequence identities compared to that between them This suggests two consecutive steps of duplication occur at the same locus Sequence comparison of glucosinolate synthesis genes reflects evolution of Brassica lineage Soon after divergence from the Arabidopsis lineage and genome triplication, extensive interspersed gene loss or gain events and large-scale chromosomal rearrangements gave rise to three basic diploid species: B rapa (AA genome), Brassica nigra (BB genome) and B oleracea (CC genome) [66] Our data support this presumptive evolution order; BrGS sequence similarities among the Brassicas (mostly > 90%) are normally higher than those between Brassica and Arabidopsis (mostly 80–90%) Individual tandem repeats or dispersed duplication events are indicative of the self-rearrangements occurring within each species A convincing example is that AOP3 is only present in Arabidopsis and that AOP2 is tandemly duplicated in B oleracea but not in B rapa Even within B oleracea, FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS Y.-X Zang et al AOP2 is a nonfunctional gene in broccoli, but is an active functional gene in collard [44], suggesting a very recent evolutionary event Perspective for metabolic engineering of glucosinolate in B rapa Although a high colinearity in the glucosinolate biosynthetic pathway generally exists between Arabidopsis and B rapa (Fig 1), the glucosinolate profiles of B rapa are quite different from those of Arabidopsis This could be explained in part by increases in BrGS gene number compared to the corresponding gene number of Arabidopsis having an effect on a regulatory circuit controlling the gene expression of multicopy genes Currently, no information is available about the rate-limiting step of metabolic flux and the regulation of glucosinolate biosynthesis at the post-transcriptional and post-translational level Nevertheless, assuming that, overall, the glucosinolate biosynthetic pathway and regulatory networks of B rapa are analogous to those of Arabidopsis, glucosinolate profiles could be quantitatively and qualitatively changed by combining at least three approaches The different enzymes of the CYP79 family are responsible for various types of glucosinolates Thus, altering the endogenous level of CYP79s and introducing exogenous CYP79s may make it possible to generate custom designed glucosinolate profiles The glucosinolate concentration could be changed by altering the side chain modification step because biosynthesis of glucosinolates is shown to be regulated via a feedback mechanism in Arabidopsis, although this remains to be confirmed in B rapa Altered expression of the BrGS regulators, including MYB transcription factors, would provide not only very efficient metabolic engineering tools to manipulate both the content and composition of glucosinolates in B rapa vegetable plants, but also the genetic tools to understand how such plants control the production of these anticarcinogenic, antioxidative and antimicrobial compounds Experimental procedures Construction of the cDNA and BAC libraries B rapa cultivar ‘Chiifu’ was used for library construction based on the agreement of the Multinational Brassica Genome Project Consortium Total RNA was prepared from 28 different kinds of tissues and used to construct individual cDNA libraries Double-stranded cDNA was synthesized from lg of total RNA with the ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA, USA) in accor- Glucosinolate biosynthesis genes in Brassica rapa dance with the manufacturer’s instructions Messenger RNA was reverse-transcribed into cDNA with a hybrid oligo(dT) linker-primer containing a XhoI restriction site In addition, the first-strand cDNA was hemimethylated with 5-methyl dCTP to protect from restriction enzyme digestion The double-stranded cDNA was linked to EcoRI adapter at its 5¢-end and was then digested with XhoI and EcoRI This final product was purified and ligated into several different vectors, such as pBlueScript, pDNR-LIB and pCNS-D2, for the subsequent sequencing project Three large-insert BAC libraries had been constructed for the sequencing project [72] Using high information content fingerprinting, the first BAC-based physical map was recently generated for B rapa ssp Pekinensis [52] Sequencing of EST and BAC clones EST clones were sequenced using the primers for the flanking sites of cloning vectors, such as T3 and T7 primers of pBlueScript vector Most of the clones were subjected to 5¢ end sequencing, which enabled us to quickly identify full-length clones by comparison with Arabidopsis sequences Large size BAC clones were cut into small fragments, which were then inserted into pCUGIblu31 vector as previously described [73–75] All the EST and shotgun clones and both ends of most BAC clones were sequenced using ABI3730 automatic DNA sequencer and BigDye terminator chemistry, version 3.1 (Applied Biosystems, Foster City, CA, USA) Putative full-length cDNA clones were again sequenced for both ends Computational sequence assembly of BAC contigs was conducted using phred ⁄ phrap ⁄ consed software [76] BLAST and data analyses for BrGS gene identification CDS of Arabidopsis glucosinolate biosynthesis and regulatory genes obtained from NCBI database were used to search for B rapa homologs in the sequence databases of BAC and cDNA clones Target sequences with 100% identity with the overlapped region were counted as the same gene Both BAC and cDNA clones often yielded the same homologs In this case, the exon ⁄ intron split pattern was identified by sequence alignment However, for the homologs found only in BAC sequences, gene structures were predicted by sequence comparison with the Arabidopsis CDS Nucleotide insertion, deletion and transposable elements in B rapa homologs were also confirmed by comparison with the corresponding Arabidopsis genes The unrooted neighbor-joining phylogenetic tree was generated using the full-length CDS of BrST5 genes and mega, version 4.1, software (Biodesign Institute, A240, Arizona State University, Tempe, AZ, USA) FEBS Journal 276 (2009) 3559–3574 ª 2009 National Academy of Agricultural Science, RDA, Korea Journal compilation ª 2009 FEBS 3569 Glucosinolate biosynthesis genes in Brassica rapa Y.-X Zang et al Southern blot analysis Genomic DNA was isolated from young leaf tissue of Chinese cabbage cultivar ‘Jangwon’ using the cetyl trimethyl ammonium bromide method [77] Approximately lg of genomic DNA was digested with BamHI, DraI, EcoRI, HindIII, EcoRV, XbaI and ScaI, electrophoresized in 0.8% agarose gel at 30 V overnight, and transferred to a nylon membrane (Hybond N-Filter; Amersham Pharmacia, Piscataway, NJ, USA) The probe DNA either comprised restriction enzyme fragments of EST clones or PCR products of genomic DNA or BAC clones All of the probes were labeled with radioactive P32 using the Ladderman kit (Takara Bio Inc., Shiga, Japan) Hybridization and detection were carried out as described previously [78] RT-PCR Total RNA was prepared using RNeasy mini kit (Qiagen GmbH, Hilden, Germany) and treated with RNase-free DNase to remove any genomic DNA contaminants All RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and were adjusted to the same concentration with diethylpyrocarbonate-treated water The first strand cDNA was synthesized using lg of total RNA and SprintÔ PowerScriptÔ PrePrimed Single Shots (Clontech Laboratories, Inc., a Takara Bio company, Mountain View, CA, USA) with oligo(dT)18 primers in accordance with the manufacturer’s instructions PCR was then performed using gene-specific primers and ExTaq DNA polymerase (Takara, Shiga, Japan) The reaction was initiated by predenaturating at 94 °C for min, followed by 35 cycles of denaturation (94 °C for 30 s), annealing (53 °C for 30 s) and extension (72 °C for 1.5 min), 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Halkier BA (2008) Subclade of flavin-monooxygenases involved Glucosinolate biosynthesis genes in Brassica rapa 29 30 31 32 33 34 35 36 37 38 39 in aliphatic glucosinolate biosynthesis Plant Physiol