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Genome-wide identification of glucosinolate synthesis genes in Brassica rapa: Glucosinolate biosynthesis genes in Brassica rapa

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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
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  Genome-wide identification of glucosinolate synthesisgenes in  Brassica rapa  Yun-Xiang Zang 1 , 2, *, Hyun Uk Kim 1, *, Jin A Kim 1 , Myung-Ho Lim 1 , Mina Jin 1 , Sang Choon Lee 1 ,Soo-Jin Kwon 1 , Soo-In Lee 1 , Joon Ki Hong 1 , Tae-Ho Park 1 , Jeong-Hwan Mun 1 , Young-Joo Seol 1 ,Seung-Beom Hong 3 and Beom-Seok Park 1 1 Genomics Division, Department of Agricultural Bio-resources, National Academy of Agricultural Science (NAAS), Rural DevelopmentAdministration (RDA), Suwon, Korea2 School of Agricultural and Food Science, Zhejiang Forestry University, Lin’an, Hangzhou, China3 Department of Biology, San Jacinto College, Houston, TX, USA Keywords bioinformatics; biosynthesis pathway; Brassica rapa  ; gene identification;glucosinolate Correspondence B. S. Park, Genomics Division, Departmentof Agricultural Bio-resources, NationalAcademy of Agricultural Science (NAAS),Rural Development Administration (RDA),Suwon 441-707, KoreaFax: +82 31 299 1672Tel: +82 31 299 1671E-mail: pbeom@rda.go.kr*These authors contributed equally to this work Database The following have been deposited to theGenBank database. Accession numbers areshown 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),  BrFMO  GS-OX1 (FJ376070),  BrFMO  GS-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)(Received 15 February 2009, revised 31March 2009, accepted 24 April 2009)doi:10.1111/j.1742-4658.2009.07076.x Glucosinolates play important roles in plant defense against herbivores andmicrobes, as well as in human nutrition. Some glucosinolate-derived isothi-ocyanate and nitrile compounds have been clinically proven for their anti-carcinogenic activity. To better understand glucosinolate biosynthesis in Brassica rapa , we conducted a comparative genomics study with  Arabidopsisthaliana  and identified total 56 putative biosynthetic and regulator genes.This established a high colinearity in the glucosinolate biosynthesis path-way between  Arabidopsis  and  B. rapa . Glucosinolate genes in  B. rapa  share72–94% nucleotide sequence identity with the  Arabidopsis  orthologs andexist in different copy numbers. The exon   ⁄   intron split pattern of   B. rapa  isalmost identical to that of   Arabidopsis , although inversion, insertion, dele-tion and intron size variations commonly occur. Four genes appear to benonfunctional as a result of the presence of a frame shift mutation andretrotransposon insertion. At least 12 paralogs of desulfoglucosinolatesulfotransferase were found in  B. rapa , whereas only three were found in Arabidopsis . The expression of those paralogs was not tissue-specific butvaried greatly depending on  B. rapa  tissue types. Expression was alsodevelopmentally regulated in some paralogs but not in other paralogs.Most of the regulator genes are present as triple copies. Accordingly, gluc-osinolate synthesis and regulation in  B. rapa  appears to be more complexthan that of   Arabidopsis . With the isolation and further characterization of the endogenous genes, health-beneficial vegetables or desirable animal feedcrops could be developed by metabolically engineering the glucosinolatepathway. 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  Glucosinolates, a group of sulfur-rich secondarymetabolites, have received much attention becausetheir breakdown products display several potent bio-activities that serve as plant defense, as well as anti-carcinogenesis compounds, in mammals [1–3]. Upontissue disruption, the enzyme myrosinase cleaves off the glucose group from a glucosinolate, and theremaining molecule then quickly converts to a bioac-tive substance (i.e. an isothiocyanate, nitrile or thiocya-nate). Among the isothiocyanates, sulforaphane, aderivative of glucoraphanin, is known to be the mostpromising anticancer agent because of its strong andbroad spectrum activity against several types of cancercells [3–10]. Indole-3-carbinol, a derivative of gluco-brassicin, also comprises a good anticarcinogen. Bothexhibit their effects by inducing phase II detoxificationenzymes, altering estrogen metabolism, blocking thecell cycle or protecting against oxidative damages[11–15]. Phenethyl isothiocyanate, a derivative of gluconasturtiin, was reported to be effective forchemoprotection [16–18], although it possesses geno-toxic activity [19–21]. Crambene (1-cyano-2-hydroxy-3-butene), an aliphatic nitrile derived from progoitrin,upregulates the synthesis of glutathione  S  -transferasein 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 synthe-sized [23]. Biosynthesis of aliphatic and aromatic gluc-osinolates generally involves three steps (Fig. 1) andbegins with the elongation of methionine and phenyl-alanine, respectively. The initial step of aliphaticglucosinolate synthesis is catalyzed by methylthioalkyl-malate synthase (MAM) to form the elongatedhomologs [24,25]. The core structures are made viaoxidation by cytochrome P450 enzymes, CYP79 andCYP83, followed by C-S cleavage, glucosylation andsulfation. Finally, the side chains are modified byoxidation, elimination, akylation or esterification.Some of the genes involved in this step,  FMO GS-OX1  5 , AOP ,  GSL-OH   and  BZO1 , have been isolated recently[26–31].Cruciferous vegetables, including broccoli, cabbage,Chinese cabbage, cauliflower, and brussels sprouts, arerich in glucosinolates. A high intake of cruciferousvegetables was shown to significantly reduce the riskof certain cancers and cardiovascular diseases [32–34].Chinese cabbage ( Brassica rapa  ssp.  pekinensis ) is oneof the most highly consumed vegetable crops in Asia.However, unlike broccoli, many Chinese cabbage culti-vars do not produce detectable levels of glucoraphanin.To date, most of the structural genes responsible forthe biosynthesis of the three groups of glucosinolateshave been identified and characterized in  Arabidopsis [23,35]. In addition, several regulators that controlglucosinolate biosynthesis have been identified recentlyin  Arabidopsis  [36–43]. However, little is known aboutthe specific genes existing in  Brassica  crops, except forthe  MAM   and  AOP  genes in  Brassica oleracea  [44–46].The glucosinolate profile is highly dependent ongenotype, although it is also affected by developmentalor environmental changes [47–49]. Previously, wereported that the ectopic expression of   Arabidopsis glucosinolate synthesis genes altered the glucosinolateprofile in Chinese cabbage [50,51]. Because most of the Arabidopsis  genes encoding glucosinolate biosynthesispathways have been identified and Chinese cabbage isa close relative of   Arabidopsis , comparative genomicstudies will allow for the easy identification of relevantgenes in  Brassicas . The identification and characteriza-tion of glucosinolate synthesis genes in Chinese cab-bage would pave the way for further improvement of agronomic traits via genetic engineering. In the presentstudy, we report the genome-wide identification of  B. rapa  glucosinolate synthesis ( BrGS  ) and regulatorgenes using our  B. rapa  genome sequence in conjunc-tion with the available  Arabidopsis  sequence. We alsoshow that many  BrGS   genes exist in a small multigenefamily and that at least 12 desulfoglucosinolatesulfotransferase ( BrST  ) paralogs are present and aredifferentially expressed. Results BrGS   gene identification from cDNA and bacterialartificial chromosome (BAC) libraries As part of the  B. rapa  genome sequencing project, weproduced 127 143 expressed sequence tags (ESTs) from28 different cDNA libraries that were released to theNational Center for Biotechnology Information(NCBI) database and a new  B. rapa  EST database,BrEMD (http://www.brassica-rapa.org/BrEMD/) withmicroarray data. Furthermore, we determined morethan 127 000 BAC end sequences, and approximately589 seed BACs were sequenced and anchored in Arabidopsis  whole chromosomes. The 65.8 Mb seedBAC sequence information covered approximately75.3% of the  Arabidopsis  genome and 40% of the B. rapa  euchromatin region [52]. On the basis of thesedatabases, homologous genes were identified by a blastn  search using the  Arabidopsis  gene sequence asquery. All the ESTs that matched each query sequencewere aligned to remove the redundant clones, andEST clones containing a start codon were resequencedto generate the full-length cDNA sequence. Through Glucosinolate biosynthesis genes in  Brassica rapa   Y.-X. Zang  et al. 3560  FEBS Journal  276  (2009) 3559–3574  ª  2009 National Academy of Agricultural Science, RDA, Korea. Journal compilation  ª  2009 FEBS  Fig. 1.  Biosynthesis pathways of the three major groups of glucosinolates in  B. rapa  . The genes involved in each step are shown. Numbersin parenthesis denote gene copy numbers.Y.-X. Zang  et al.  Glucosinolate biosynthesis genes in  Brassica rapa  FEBS Journal  276  (2009) 3559–3574  ª  2009 National Academy of Agricultural Science, RDA, Korea. Journal compilation  ª  2009 FEBS  3561  this alignment, a total of 35 different genes was foundfrom ESTs. In the same way,  blastn  searches were per-formed against the BAC sequence databases, yielding44 different genes, among which 23 overlapped the ESTsequences. Thus, a total of 56 individual genes wasidentified from both EST and BAC clones, of which44 contained the full-length coding sequence (CDS)(Fig. 1, Tables 1 and 2). They contain all the homologsof   Arabidopsis  except for  CYP79F2 ,  FMO GS-OX2  4 , 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 observationwas also made in  B. oleracea  [45]. This suggests thatduplication occurred in  Arabidopsis  after its divergencefrom  Brassica . Four genes,  BrUGT74C1-1 ,  BrST5b-6 , BrST5b-4  and  BrMYB122-1 , appear to be nonfunc-tional as a result of a frame shift or retrotransposon– insertion mutations (Fig. 2).To estimate the total number of putative  BrGS   genesin the whole genome of   B. rapa , a genomic blot wasperformed using the  CYP79F1   ⁄   F2 ,  CYP79B2   ⁄   B3 , CYP83A1  and  CYP83B1  genes as probes (see Support-ing information, Fig. S1) [53]. This analysis predictedthe presence of a total of eight genes (two, three, two Table 1.  Comparison of putative  BrGS   biosynthetic genes with the  Arabidopsis   orthologs. The nucleotide sequence of the coding regionwas 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.Glucosinolate pathway B. rapa   genenameCorrespondingAGINo. ofgenesfoundCorresponding clones% Identity  At  and  B. rapa  BAC ESTAmino acid side chainelongation BrBCAT4   At3g19710 2 KBrH046K16 BR069190 83.8–83.9 h  BR00585578.4–87.0 BrMAM   At5g23010 4 BR043724*KBrB010E08F*  h At5g23020  h  BR003821KBrH121C04F*  h BrBCAT3   At3g49680 2 KBrH045F08R BR008244* 84.7–87.0 h  BR080925*Core structure formationstep BrCYP79F1  At1g16410 1 KBrB035G16 BR007081 85.4 BrCYP79B2   At4g39950 2 KBrB022O03 BR046183 89.0–89.3KBrH106H11 BR1 20984 BrCYP79B3   At2g22330 1  h  BR098069 89.5 BrCYP79A2   At5g05260 1 KBrS003K07  h  85.6 BrCYP83A1  At4g13770 2 KBrH086H05R BR058540 87.3–87.4KBrH009l04 BR061092 BrCYP83B1  At4g31500 1  h  BR091686 90.1 BrC-S lyase   At2g20610 2  h  BR087395 86.6–87.1KBrH010M17 BR098736 BrUGT74B1  At1g24100 1 KBrH015M19 BR100939 84.5 h  BR043439 BrUGT74C1  At2g31790 3  h  BR043626 85.1–88.4KBrH036G21R*  h BrST5a   At1g74100 2 KBrB119E11F BR015379 86.6–86.6KBrB056L1 BR082516KBrB056L15 BR059286KBrH096A10  h BrST5b   At1g74090 9 KBrB041J04  h  76.0–85.9KBrB034H04  h KBrB069A23  h BrST5c   At1g18590 1  h  BR094491 85.7Side chain modification  BrFMO  GS-OX1  At1g65860 1 KBrS002F02F BR021429 83.3 BrFMO  GS-OX5   At1g12140 1 KBrB032C15 BR116105 83.3 BrAOP2   At4g03060 1 KBrB002P01 BR067797 71.9 BrGSL-OH   At2g25450 1 KBrH047C14 BR097443 85 BrBZO1p   At1g65880 1 KBrB083K19  h  81.1References [24], [25], [26], [27], [28], [29], [31], [57], [58], [59], [79] Glucosinolate biosynthesis genes in  Brassica rapa   Y.-X. Zang  et al. 3562  FEBS Journal  276  (2009) 3559–3574  ª  2009 National Academy of Agricultural Science, RDA, Korea. Journal compilation  ª  2009 FEBS  and one copies for each gene, respectively). On theother hand, a total of seven genes was found from ourdatabase search for those genes, suggesting that thepercentage of   BrGS   genes identified in the presentstudy is approximately 87.5%. BrGS   gene identity with  Arabidopsis   and other Brassica   orthologs BrGS   biosynthetic genes share 72–90% nucleotidesequence identity with  Arabidopsis  orthologs and 28genes exist in a small multigene family (Table 1). Thisclose relatedness is further substantiated by our phy-logenetic tree analyses (Fig. 3; see also Supportinginformation, Figs S2–S11). However, most of the BrGS   genes share more than 90% identity with other Brassica  orthologs (Table 3). This is consistent withthe notion that the  Brassica  species evolved afterdivergence from the  Arabidopsis  lineage. Notably, BrAOP2  has the lowest sequence identity with theorthologs of   Arabidopsis  and  B. oleracea . Identitieswithin the  BrGS   paralogs are usually higher thanthose with  Arabidopsis  and other  Brassica  species. Allof 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 nontandemrepeats  BrST5b-6  and  BrST5b-9  (88%) (Fig. 4,Table 4). This suggests that duplication occurred aftera very recent divergence between  Arabidopsis  and B. rapa . One putative benzoate-CoA ligase gene BrBZO1p  was identified (see also Supporting informa-tion, Fig. S11). It has a similarity of 81% comparedto both  BZO1  and At1g65890.Similar to the biosynthetic genes,  BrGS   regulatorgenes share 81–94% nucleotide sequence identity with Arabidopsis  orthologs and 15 genes exist in a smallmultigene family (Table 2). Most of the genes are trip-licated, indicating that regulator genes are mostlyretained after the  Brassica  genome triplication. Structure of  BrGS   genes Ordered assembly of the overlapping sequences of BAC and EST clones yielded the overall gene struc-tures shown in Fig. 5. The exon and intron structuresof the  BrGS   genes were identical to those of   Arabidop-sis  homologs. However, insertion, deletion and intronsize variations were commonly noted in  BrGS   genes.One of the two  BrC-S   lyase genes had a 2 bp deletionat the last exon, which resulted in a 3 ¢  truncated pro-tein with a 16 amino acid deletion compared to the Arabidopsis  homolog. The truncation of 3 ¢  end exonmight alter either gene function or the expression pat-tern in such a way to change feedback regulation, aspreviously proposed by Gao  et al.  [46]. Desulfogluco-sinolate sulfotransferase genes did not have any intronin both  Arabidopsis  and  B. rapa  (Fig. 5A). The  AOP2 structure of   B. rapa  was compared with that of   B. oler-acea  and  Arabidopsis . All three species contained fourexons and three introns, along with considerablechanges in intron sizes (Fig. 5B). One of the two BrST5a  genes contained a 3 bp insertion (Fig. 5A),which did not lead to a frame shift mutation.Insertion or deletion often gives rise to a frame shiftmutation that causes the loss of gene function. Thistype of mutation occurred in two  BrGS   genes with pre-mature stop codons immediately after the deletion sites(Fig. 2A). Among nine  BrST5b  paralogs,  BrST5b-4 AB Fig. 2.  Structures of the predicted nonfunctional  BrGS   genes. (A) Three of the four carried deletion mutations and (B) the fourth one carried aputative restrotransposon insertion. A non-LTR retrotransposon insertion is marked by approximately 6 kb insertion. Asterisks indicate the posi-tion of a premature stop codon. Thick, thin and dotted lines denote the exon, intron and the gap between  BrST5b-4   and  BrST5b-x  , respectively.Y.-X. Zang  et al.  Glucosinolate biosynthesis genes in  Brassica rapa  FEBS Journal  276  (2009) 3559–3574  ª  2009 National Academy of Agricultural Science, RDA, Korea. Journal compilation  ª  2009 FEBS  3563
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