Genome‐wide identification of glucosinolate synthesis genes in Brassica rapa

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.

[1]  U. Flügge,et al.  HAG 2 / MYB 76 and HAG 3 / MYB 29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis thaliana , 2008 .

[2]  C. N. Stewart,et al.  Constitutive and herbivore-inducible glucosinolate concentrations in oilseed rape (Brassica napus) leaves are not affected by Bt Cry1Ac insertion but change under elevated atmospheric CO2 and O3 , 2007, Planta.

[3]  B. Berger,et al.  The R2R3-MYB transcription factor HAG1/MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana. , 2007, The Plant journal : for cell and molecular biology.

[4]  R. Wing,et al.  Construction of Various Copy Number Plasmid Vectors and Their Utility for Genome Sequencing , 2004 .

[5]  Daniel J. Kliebenstein,et al.  Linking Metabolic QTLs with Network and cis-eQTLs Controlling Biosynthetic Pathways , 2007, PLoS genetics.

[6]  D. Williams,et al.  Mechanisms of indole-3-carbinol (I3C) anticarcinogenesis: inhibition of aflatoxin B1-DNA adduction and mutagenesis by I3C acid condensation products. , 1995, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[7]  J. Tokuhisa,et al.  Two Arabidopsis Genes (IPMS1 and IPMS2) Encode Isopropylmalate Synthase, the Branchpoint Step in the Biosynthesis of Leucine1[W][OA] , 2006, Plant Physiology.

[8]  A. Force,et al.  Preservation of duplicate genes by complementary, degenerative mutations. , 1999, Genetics.

[9]  Hans-Peter Mock,et al.  The transcription factor HIG1/MYB51 regulates indolic glucosinolate biosynthesis in Arabidopsis thaliana. , 2007, The Plant journal : for cell and molecular biology.

[10]  J. Finley,et al.  Cruciferous Vegetables: Cancer Protective Mechanisms of Glucosinolate Hydrolysis Products and Selenium , 2004, Integrative cancer therapies.

[11]  K. Gunderson,et al.  Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana. , 2007, The Plant journal : for cell and molecular biology.

[12]  The Arabidopsis Genome Initiative Analysis of the genome sequence of the flowering plant Arabidopsis thaliana , 2000, Nature.

[13]  R. Dashwood,et al.  Chemoprotection by sulforaphane: keep one eye beyond Keap1. , 2006, Cancer letters.

[14]  K. Guru,et al.  Consumption of Raw Cruciferous Vegetables is Inversely Associated with Bladder Cancer Risk , 2008, Cancer Epidemiology Biomarkers & Prevention.

[15]  J. Juvik,et al.  Genetic and Environmental Variation of Glucosinolate Content in Chinese Cabbage , 2006 .

[16]  M. Koornneef,et al.  Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves. , 2008, The New phytologist.

[17]  M. Koch,et al.  Chromosome triplication found across the tribe Brassiceae. , 2005, Genome Research.

[18]  Jung Sun Kim,et al.  The first generation of a BAC-based physical map of Brassica rapa , 2008, BMC Genomics.

[19]  F. Schroeder,et al.  Identification of indole glucosinolate breakdown products with antifeedant effects on Myzus persicae (green peach aphid). , 2008, The Plant journal : for cell and molecular biology.

[20]  Y. Lim,et al.  The Korea Brassica Genome Project: a Glimpse of the Brassica Genome Based on Comparative Genome Analysis With Arabidopsis , 2005, Comparative and functional genomics.

[21]  Barbara Ann Halkier,et al.  Biology and biochemistry of glucosinolates. , 2006, Annual review of plant biology.

[22]  W. McCombie,et al.  Comparative analysis of a Brassica BAC clone containing several major aliphatic glucosinolate genes with its corresponding Arabidopsis sequence. , 2004, Genome.

[23]  W. F. Thompson,et al.  Rapid isolation of high molecular weight plant DNA. , 1980, Nucleic acids research.

[24]  C. P. Hong,et al.  Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kbp gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5 , 2005, Molecular Genetics and Genomics.

[25]  D. Potter,et al.  Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana , 2006, Plant Cell Reports.

[26]  M. Eun,et al.  The semidwarf gene, sd-1, of rice (Oryza sativa L.). II. Molecular mapping and marker-assisted selection , 1994, Theoretical and Applied Genetics.

[27]  Yasuko Takahashi,et al.  Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events , 2022 .

[28]  B. Berger,et al.  The R 2 R 3-MYB transcription factor HAG 1 / MYB 28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana , 2007 .

[29]  U. Flügge,et al.  HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis thaliana. , 2008, The New phytologist.

[30]  E. Ho,et al.  Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apcmin mice , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[31]  F. Kassie,et al.  Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC). , 2000, Chemico-biological interactions.

[32]  Lingyun Wu,et al.  Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Ecker,et al.  Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3. , 2002, Genes & development.

[34]  Yiwei Li,et al.  Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells , 2001, Oncogene.

[35]  B. Haas,et al.  Comparative Genomics of Brassica oleracea and Arabidopsis thaliana Reveal Gene Loss, Fragmentation, and Dispersal after Polyploidy[W][OA] , 2006, The Plant Cell Online.

[36]  P. Karplus,et al.  A Novel Mechanism of Chemoprotection by Sulforaphane , 2004, Cancer Research.

[37]  Seung-Beom Hong,et al.  Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1. , 2008, BMB reports.

[38]  Stefan Binder,et al.  BRANCHED-CHAIN AMINOTRANSFERASE4 Is Part of the Chain Elongation Pathway in the Biosynthesis of Methionine-Derived Glucosinolates in Arabidopsis[W] , 2006, The Plant Cell Online.

[39]  Michele D. Sobolewski,et al.  Sulforaphane induces cell type–specific apoptosis in human breast cancer cell lines , 2007, Molecular Cancer Therapeutics.

[40]  Søren Bak,et al.  The presence of CYP79 homologues in glucosinolate-producing plants shows evolutionary conservation of the enzymes in the conversion of amino acid to aldoxime in the biosynthesis of cyanogenic glucosides and glucosinolates , 1998, Plant Molecular Biology.

[41]  M. Hirose,et al.  Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats , 1998, International journal of cancer.

[42]  Jung Sun Kim,et al.  Isolation of circadian-associated genes in Brassica rapa by comparative genomics with Arabidopsis thaliana. , 2007, Molecules and cells.

[43]  C. Quirós,et al.  In planta side-chain glucosinolate modification in Arabidopsis by introduction of dioxygenase Brassica homolog BoGSL-ALK , 2003, Theoretical and Applied Genetics.

[44]  Jerry D. Cohen,et al.  The effect of temperature, photoperiod, and light quality on gluconasturtiin concentration in watercress (Nasturtium officinale R. Br.). , 2006, Journal of agricultural and food chemistry.

[45]  P. Green,et al.  Consed: a graphical tool for sequence finishing. , 1998, Genome research.

[46]  Jeannie T. Lee Molecular biology: Complicity of gene and pseudogene , 2003, Nature.

[47]  J. Tokuhisa,et al.  MAM3 Catalyzes the Formation of All Aliphatic Glucosinolate Chain Lengths in Arabidopsis1[W][OA] , 2007, Plant Physiology.

[48]  Rachel E. Kerwin,et al.  A Novel 2-Oxoacid-Dependent Dioxygenase Involved in the Formation of the Goiterogenic 2-Hydroxybut-3-enyl Glucosinolate and Generalist Insect Resistance in Arabidopsis[C][W][OA] , 2008, Plant Physiology.

[49]  D. Kliebenstein,et al.  A Systems Biology Approach Identifies a R2R3 MYB Gene Subfamily with Distinct and Overlapping Functions in Regulation of Aliphatic Glucosinolates , 2007, PloS one.

[50]  Genying Li,et al.  Genetic analysis, expression and molecular characterization of BoGSL-ELONG, a major gene involved in the aliphatic glucosinolate pathway of Brassica species. , 2002, Genetics.

[51]  Miroslav Strnad,et al.  DOF transcription factor AtDof1.1 (OBP2) is part of a regulatory network controlling glucosinolate biosynthesis in Arabidopsis. , 2006, The Plant journal : for cell and molecular biology.

[52]  M. Osborne,et al.  Multifunctional Aspects of the Action of Indole‐3‐Carbinol as an Antitumor Agent , 1999, Annals of the New York Academy of Sciences.

[53]  E. Rosen,et al.  Suppression of breast cancer invasion and migration by indole-3-carbinol: associated with up-regulation of BRCA1 and E-cadherin/catenin complexes , 2000, Journal of Molecular Medicine.

[54]  C. P. Hong,et al.  Progress in Understanding and Sequencing the Genome of Brassica rapa , 2008, International journal of plant genomics.

[55]  D. Kim,et al.  Metabolic engineering of indole glucosinolates in Chinese cabbage plants by expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1. , 2008, Molecules and cells.

[56]  P. Taylor,et al.  Determination of sinigrin and glucoraphanin in Brassica species using a simple extraction method combined with ion-pair HPLC analysis , 2002 .

[57]  M. Hirai,et al.  Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis , 2007, Proceedings of the National Academy of Sciences.

[58]  Barbara A Halkier,et al.  Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis. , 2007, The Plant journal : for cell and molecular biology.

[59]  Pengfei Li,et al.  Mechanism of Sulforaphane-Induced Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells , 2004, Nutrition and cancer.

[60]  C. Rao,et al.  Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate. , 2000, Carcinogenesis.

[61]  J. Gershenzon,et al.  Genetic control of natural variation in Arabidopsis glucosinolate accumulation. , 2001, Plant physiology.

[62]  S. Abel,et al.  Glucosinolate metabolism and its control. , 2006, Trends in plant science.

[63]  E. Stockinger,et al.  A Retrotransposon-Mediated Gene Duplication Underlies Morphological Variation of Tomato Fruit , 2008, Science.

[64]  J. Celenza,et al.  The Arabidopsis ATR1 Myb Transcription Factor Controls Indolic Glucosinolate Homeostasis1 , 2005, Plant Physiology.

[65]  N. Eckardt Identification of Rust Fungi Avirulence Elicitors , 2006, The Plant Cell Online.

[66]  Guillaume Blanc,et al.  Functional Divergence of Duplicated Genes Formed by Polyploidy during Arabidopsis Evolution , 2004, The Plant Cell Online.

[67]  Bjarne Gram Hansen,et al.  Subclade of Flavin-Monooxygenases Involved in Aliphatic Glucosinolate Biosynthesis1[W] , 2008, Plant Physiology.

[68]  L. Caltavuturo,et al.  Genetic and metabolic effects of gluconasturtiin, a glucosinolate derived from cruciferae. , 2004, Mutation research.

[69]  A. Müller,et al.  Desulfoglucosinolate Sulfotransferases from Arabidopsis thaliana Catalyze the Final Step in the Biosynthesis of the Glucosinolate Core Structure* , 2004, Journal of Biological Chemistry.

[70]  P. Naur,et al.  CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime. , 2003, Archives of biochemistry and biophysics.

[71]  E. Jeffery,et al.  The cruciferous nitrile, crambene, induces rat hepatic and pancreatic glutathione S-transferases. , 1998, Toxicological sciences : an official journal of the Society of Toxicology.

[72]  M. Reichelt,et al.  Gene Duplication in the Diversification of Secondary Metabolism: Tandem 2-Oxoglutarate–Dependent Dioxygenases Control Glucosinolate Biosynthesis in Arabidopsis , 2001, Plant Cell.

[73]  M. Reichelt,et al.  Arabidopsis Branched-Chain Aminotransferase 3 Functions in Both Amino Acid and Glucosinolate Biosynthesis1[W][OA] , 2007, Plant Physiology.

[74]  Jung Sun Kim,et al.  A Sequence-Tagged Linkage Map of Brassica rapa , 2006, Genetics.

[75]  E. Jeffery,et al.  The synergistic upregulation of phase II detoxification enzymes by glucosinolate breakdown products in cruciferous vegetables. , 2001, Toxicology and applied pharmacology.

[76]  Weiya Ma,et al.  Essential role of p53 in phenethyl isothiocyanate-induced apoptosis. , 1998, Cancer research.

[77]  J. Tokuhisa,et al.  A gene controlling variation in Arabidopsis glucosinolate composition is part of the methionine chain elongation pathway. , 2001, Plant physiology.

[78]  Wen-Hsiung Li,et al.  Rates of Nucleotide Substitution in Angiosperm Mitochondrial DNA Sequences and Dates of Divergence Between Brassica and Other Angiosperm Lineages , 1999, Journal of Molecular Evolution.

[79]  S. Abel,et al.  Arabidopsis IQD1, a novel calmodulin-binding nuclear protein, stimulates glucosinolate accumulation and plant defense. , 2005, The Plant journal : for cell and molecular biology.

[80]  S. Yao,et al.  c‐jun/AP‐1 activation does not affect the antiproliferative activity of phenethyl isothiocyanate, a cruciferous vegetable‐derived cancer chemopreventive agent , 2006, Molecular carcinogenesis.

[81]  Genying Li,et al.  Comparative analysis of a transposon-rich Brassica oleracea BAC clone with its corresponding sequence in A. thaliana , 2005, Theoretical and Applied Genetics.

[82]  B. Halkier,et al.  Altering glucosinolate profiles modulates disease resistance in plants. , 2006, The Plant journal : for cell and molecular biology.

[83]  T. Kensler,et al.  Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. , 1994, Proceedings of the National Academy of Sciences of the United States of America.