Skip to main content
Log in

Identification and expression pattern analysis of the glucosinolate biosynthetic gene BoCYP83B1 from broccoli

  • Original paper
  • Published:
Biologia Plantarum

Abstract

Glucosinolates are a branch of amino acid-derived metabolites, which are specifically found in Brassicales. In Arabidopsis, tryptophan derived indolic glucosinolates are required for plant defense against a wide range of pathogens and herbivores due to their strong antimicrobial activity and potential signaling function. An important enzyme in indolic glucosinolate biosynthesis pathway is CYP83B1, which oxidizes indole-3-acetaldoxime, a precursor of indole-3-acetic acid (IAA). In this study, we reported isolation and expression characterization of a CYP83B1 gene from Brassica oleracea L. var. italica Plenck, which we termed BoCYP83B1. Overexpression of BoCYP83B1 in Arabidopsis resulted in an altered glucosinolate profile and early flowering phenotype. By expressing the reporter gene β-glucuronidase under the control of the BoCYP83B1 promoter in Arabidopsis, we analyzed the spatial expression pattern of BoCYP83B1 under normal growth conditions as well as in response to several hormones and stresses. The BoCYP83B1 was primarily expressed in vascular tissue through the almost whole plant. It was strongly induced by methyl jasmonate, 1-amino-1-cyclopropanecarboxylic acid, salicylic acid (SA), gibberellin, and IAA, suggesting its involvement in complex signaling pathways. Mannitol, NaCl, UV, and Flagelin 22 significantly up-regulated BoCYP83B1 expression, indicating its possible role in stress response. Interestingly, the response of BoCYP83B1 to SA and NaCl showed tissue specificity. Thus, BoCYP83B1 might have different functions in different tissues.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

Abbreviations

ABA:

abscisic acid

ACC:

1-amino-1-cyclopropanecarboxylic acid

CaMV 35S:

cauliflower mosaic virus promoter

CDS:

coding sequence

Flg22:

Flagelin 22

GA:

gibberellic acid

GUS:

β-glucuronidase

IAA:

indole-3-acetic acid

IAOx:

indole-3-acetaldoxime

MeJA:

methyl jasmonate

MS:

Murashige and Skoog

NCBI:

National Center for Biotechnology Information

RACE:

rapid amplification of cDNA ends

RT-PCR:

reverse transcription PCR

SA:

salicylic acid

UTR:

untranslated region

References

  • Agerbirk, N., Olsen, C.E.: Glucosinolate structures in evolution. - Phytochemistry 77: 16–45, 2012.

    Article  PubMed  CAS  Google Scholar 

  • Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J.: Basic local alignment search tool. - J. mol. Biol. 215: 403–410, 1990.

    Article  PubMed  CAS  Google Scholar 

  • Alvarez, S., He, Y., Chen, S.: Comparative investigations of the glucosinolate-myrosinase system in Arabidopsis suspension cells and hypocotyls. - Plant Cell Physiol. 49: 324–333, 2008.

    Article  PubMed  CAS  Google Scholar 

  • Andersson, M.X., Nilsson, A.K., Johansson, O.N., Boztas, G., Adolfsson, L.E., Pinosa, F., Petit, C.G., Aronsson, H., Mackey, D., Tor, M., Hamberg, M., Ellerstrom, M.: Involvement of the electrophilic isothiocyanate sulforaphane in Arabidopsis local defense responses. - Plant Physiol. 167: 251–261, 2015.

    Article  PubMed  CAS  Google Scholar 

  • Ares, A.M., Nozal, M.J., Bernal, J.: Extraction, chemical characterization and biological activity determination of broccoli health promoting compounds. - J. Chromatogr. A 1313: 78–95, 2013.

    Article  PubMed  CAS  Google Scholar 

  • Bak, S., Feyereisen, R.: The involvement of two p450 enzymes, CYP83B1 and CYP83A1, in auxin homeostasis and glucosinolate biosynthesis. - Plant Physiol. 127: 108–118, 2001.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bak, S., Tax, F.E., Feldmann, K.A., Galbraith, D.W., Feyereisen, R.: CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis. - Plant Cell 13: 101–111, 2001.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Barlier, I., Kowalczyk, M., Marchant, A., Ljung, K., Bhalerao, R., Bennett, M., Sandberg, G., Bellini, C.: The SUR2 gene of Arabidopsis thaliana encodes the cytochrome P450 CYP83B1, a modulator of auxin homeostasis. - Proc. nat. Acad. Sci. USA 97: 14819–14824, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Bednarek, P., Osbourn, A.: Plant-microbe interactions: chemical diversity in plant defense. - Science 324: 746–748, 2009.

    Article  PubMed  CAS  Google Scholar 

  • Bhuria, M., Goel, P., Kumar, S., Singh, A.K.: The promoter of AtUSP is co-regulated by phytohormones and abiotic stresses in Arabidopsis thaliana. - Front. Plant Sci. 7: 1957, 2016.

    Article  PubMed  PubMed Central  Google Scholar 

  • Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T.G., Bertoni, M., Bordoli, L., Schwede, T.: SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. - Nucl. Acids Res. 42: W252–W258, 2014.

    Article  PubMed  CAS  Google Scholar 

  • Blom, N., Gammeltoft, S., Brunak, S.: Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. - J. mol. Biol. 294: 1351–1362, 1999.

    Article  PubMed  CAS  Google Scholar 

  • Chen, Y.J., Yu, P., Luo, J.C., Jiang, Y.: Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. - Mammalian Genome 14: 859–865, 2003.

    Article  PubMed  CAS  Google Scholar 

  • Clay, N.K., Adio, A.M., Denoux, C., Jander, G., Ausubel, F.M.: Glucosinolate metabolites required for an Arabidopsis innate immune response. - Science 323: 95–101, 2009.

    Article  PubMed  CAS  Google Scholar 

  • Clough, S.J., Bent, A.F.: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. - Plant J. 16: 735–743, 1998.

    Article  PubMed  CAS  Google Scholar 

  • Delarue, M., Prinsen, E., Onckelen, H.V., Caboche, M., Bellini, C.: Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis. - Plant J. 14: 603–611, 1998.

    Article  PubMed  CAS  Google Scholar 

  • Fahey, J.W., Haristoy, X., Dolan, P.M., Kensler, T.W., Scholtus, I., Stephenson, K.K., Talalay, P., Lozniewski, A.: Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo a pyrene-induced stomach tumors. - Proc. nat. Acad. Sci. USA 99: 7610–7615, 2002.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Fahey, J.W., Zhang, Y., Talalay, P.: Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. - Proc. nat. Acad. Sci. USA 94: 10367–10372, 1997.

    Article  PubMed  CAS  Google Scholar 

  • Frerigmann, H., Pislewska-Bednarek, M., Sanchez-Vallet, A., Molina, A., Glawischnig, E., Gigolashvili, T., Bednarek, P.: Regulation of pathogen-triggered tryptophan metabolism in Arabidopsis thaliana by MYB transcription factors and indole glucosinolate conversion products. - Mol. Plant 9: 682–695, 2016.

    Article  PubMed  CAS  Google Scholar 

  • Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D., Bairoch, A.: ExPASy: the proteomics server for in-depth protein knowledge and analysis. - Nucl. Acids Res. 31: 3784–3788, 2003.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Geourjon, C., Deleage, G.: SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. - C.A.B.I.O.S. 11: 681–684, 1995.

    CAS  Google Scholar 

  • Geu-Flores, F., Nielsen, M.T., Nafisi, M., Moldrup, M.E., Olsen, C.E., Motawia, M.S., Halkier, B.A.: Glucosinolate engineering identifies gamma-glutamyl peptidase. - Nat. chem. Biol. 5: 575–577, 2009.

    Article  PubMed  CAS  Google Scholar 

  • Grubb, C.D., Zipp, B.J., Ludwig-Muller, J., Masuno, M.N., Molinski, T.F., Abel, S.: Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis. - Plant J. 40: 893–908, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Halkier, B.A.: General introduction to glucosinolates. - In: Kopriva, S. (ed.): Advances in Botanical Research. Vol. 80. Pp.1–14. Elsevier, London 2016.

    Article  Google Scholar 

  • Halkier, B.A., Du, L.: The biosynthesis of glucosinolates. - Trends Plant Sci. 2: 425–431, 1997.

    Article  Google Scholar 

  • Halkier, B.A., Gershenzon, J.: Biology and biochemistry of glucosinolates.–Annu. Rev. Plant Biol. 57: 303–333, 2006.

    Article  PubMed  CAS  Google Scholar 

  • Hansen, B.G., Kliebenstein, D.J., Halkier, B.A.: Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis. - Plant J. 50: 902–910, 2007.

    Article  PubMed  CAS  Google Scholar 

  • Hansen, C.H., Du, L., Naur, P., Olsen, C.E., Axelsen, K.B., Hick, A.J., Pickett, J.A., Halkier, B.A.: CYP83B1 is the oxime-metabolizing enzyme in the glucosinolate pathway in Arabidopsis. - J. biol. Chem. 276: 24790–24796, 2001.

    Article  PubMed  CAS  Google Scholar 

  • Hemm, M.R., Ruegger, M.O., Chapple, C.: The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes. - Plant Cell 15: 179–194, 2003.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Hoecker, U., Toledo-Ortiz, G., Bender, J., Quail, P.H.: The photomorphogenesis-related mutant red1 is defective in CYP83B1, a red light-induced gene encoding a cytochrome P450 required for normal auxin homeostasis. - Planta 219: 195–200, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Hull, A.K., Vij, R., Celenza, J.L.: Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis. - Proc. nat. Acad. Sci. USA 97: 2379–2384, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Jefferson, R.A., Kavanagh, T.A., Bevan, M.W.: GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. - EMBO J. 6: 3901–3907, 1987.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S., Madden, T.L.: NCBIBLAST: a better web interface. - Nucl. Acids Res. 36: W5–W9, 2008.

    Article  PubMed  CAS  Google Scholar 

  • Latte, K.P., Appel, K.-E., Lampen, A.: Health benefits and possible risks of broccoli - an overview. - Food Chem. Toxicol. 49: 3287–3309, 2011.

    Article  PubMed  CAS  Google Scholar 

  • Madsen, S.R., Olsen, C.E., Nour-Eldin, H.H., Halkier, B.A.: Elucidating the role of transport processes in leaf glucosinolate distribution. - Plant Physiol. 166: 1450–1462, 2014.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Maharjan, P.M., Dilkes, B.P., Fujioka, S., Pencik, A., Ljung, K., Burow, M., Halkier, B.A., Choe, S.: Arabidopsis gulliver1/superroot2-7 identifies a metabolic basis for auxin and brassinosteroid synergy. - Plant J. 80: 797–808, 2014.

    Article  PubMed  CAS  Google Scholar 

  • Mewis, I., Appel, H.M., Hom, A., Raina, R., Schultz, J.C.: Major signaling pathways modulate Arabidopsis glucosinolate accumulation and response to both phloem-feeding and chewing insects. - Plant Physiol. 138: 1149–1162, 2005.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mewis, I., Khan, M.A.M., Glawischnig, E., Schreiner, M., Ulrichs, C.: Water stress and aphid feeding differentially influence metabolite composition in Arabidopsis thaliana (L.). - PLoS ONE 7: e48661, 2012.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mewis, I., Tokuhisa, J.G., Schultz, J.C., Appel, H.M., Ulrichs, C., Gershenzon, J.: Gene expression and glucosinolate accumulation in Arabidopsis thaliana in response to generalist and specialist herbivores of different feeding guilds and the role of defense signaling pathways. - Phytochemistry 67: 2450–2462, 2006.

    Article  PubMed  CAS  Google Scholar 

  • Mikkelsen, M.D., Hansen, C.H., Wittstock, U., Halkier, B.A.: Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3-acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid. - J. biol. Chem. 275: 33712–33717, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Mikkelsen, M.D., Naur, P., Halkier, B.A.: Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis. - Plant J. 37: 770–777, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Mikkelsen, M.D., Petersen, B.L., Glawischnig, E., Jensen, A.B., Andreasson, E., Halkier, B.A.: Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis by defense signaling pathways. - Plant Physiol. 131: 298–308, 2003.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mizutani, M., Ward, E., Ohta, D.: Cytochrome P450 superfamily in Arabidopsis thaliana: isolation of cDNAs, differential expression, and RFLP mapping of multiple cytochromes P450. - Plant mol. Biol 37: 39–52, 1998.

    Article  PubMed  CAS  Google Scholar 

  • Moldrup, M.E., Salomonsen, B., Geu-Flores, F., Olsen, C.E., Halkier, B.A.: De novo genetic engineering of the camalexin biosynthetic pathway. - J. Biotechnol. 167: 296–301, 2013.

    Article  PubMed  CAS  Google Scholar 

  • Morant, M., Ekstrom, C., Ulvskov, P., Kristensen, C., Rudemo, M., Olsen, C.E., Hansen, J., Jorgensen, K., Jorgensen, B., Moller, B.L., Bak, S.: Metabolomic, transcriptional, hormonal, and signaling cross-talk in superroot2. - Mol. Plant 3: 192–211, 2010.

    Article  PubMed  CAS  Google Scholar 

  • Nafisi, M., Goregaoker, S., Botanga, C.J., Glawischnig, E., Olsen, C.E., Halkier, B.A., Glazebrook, J.: Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis. - Plant Cell 19: 2039–2052, 2007.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Nafisi, M., Sønderby, I.E., Hansen, B.G., Geu-Flores, F., Nour-Eldin, H.H., Nørholm, M.H.H., Jensen, N.B., Li, J., Halkier, B.A.: Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids. - Phytochem. Rev. 5: 331–346, 2006.

    Article  CAS  Google Scholar 

  • Naur, P., Petersen, B.L., Mikkelsen, M.D., Bak, S., Rasmussen, H., Olsen, C.E., Halkier, B.A.: CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis. - Plant Physiol. 133: 63–72, 2003.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Nour-Eldin, H.H., Hansen, B.G., Norholm, M.H.H., Jensen, J.K., Halkier, B.A.: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. - Nucl. Acids Res. 34: e122, 2006.

    Article  PubMed  PubMed Central  Google Scholar 

  • Pang, Q.Y., Chen, S.X., Li, L.X., Yan, X.F.: Characterization of glucosinolate-myrosinase system in developing salt cress Thellungiella halophila. - Physiol. Plant. 136: 1–9, 2009.

    Article  PubMed  CAS  Google Scholar 

  • Pfalz, M., Vogel, H., Kroymann, J.: The gene controlling the Indole Glucosinolate Modifier1 quantitative trait locus alters indole glucosinolate structures and aphid resistance in Arabidopsis. - Plant Cell 21: 985–999, 2009.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Piotrowski, M., Schemenewitz, A., Lopukhina, A., Muller, A., Janowitz, T., Weiler, E.W., Oecking, C.: Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure. - J. biol. Chem. 279: 50717–50725, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Reymond, P., Weber, H., Damond, M., Farmer, E.E.: Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. - Plant Cell 12: 707–720, 2000.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Riach, A.C., Perera, M.V.L., Florance, H.V., Penfield, S.D., Hill, J.K.: Analysis of plant leaf metabolites reveals no common response to insect herbivory by Pieris rapae in three related host-plant species. - J. exp. Bot. 66: 2547–2556, 2015.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rombauts, S., Dehais, P., Van Montagu, M., Rouze, P.: PlantCARE, a plant cis-acting regulatory element database. - Nucl. Acids Res. 27: 295–296, 1999.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rose, P., Faulkner, K., Williamson, G., Mithen, R.: 7-Methyl-sulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes. - Carcinogenesis 21: 1983–1988, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Schultz, J., Milpetz, F., Bork, P., Ponting, C.P.: SMART, a simple modular architecture research tool: identification of signaling domains. - Proc. nat. Acad. Sci. USA 95: 5857–5864, 1998.

    Article  PubMed  CAS  Google Scholar 

  • Smolen, G., Bender, J.: Arabidopsis cytochrome P450 cyp83B1 mutations activate the tryptophan biosynthetic pathway. - Genetics 160: 323–332, 2002.

    PubMed  PubMed Central  CAS  Google Scholar 

  • Stotz, H.U., Sawada, Y., Shimada, Y., Hirai, M.Y., Sasaki, E., Krischke, M., Brown, P.D., Saito, K., Kamiya, Y.: Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum. - Plant J. 67: 81–93, 2011.

    Article  PubMed  CAS  Google Scholar 

  • Textor, S., Gershenzon, J.: Herbivore induction of the glucosinolate-myrosinase defense system: major trends, biochemical bases and ecological significance. - Phytochem. Rev. 8: 149–170, 2009.

    Article  CAS  Google Scholar 

  • Warwick, S.I., Francis, A., Al-Shehbaz, I.A.: Brassicaceae: species checklist and database on CD-Rom. - Plant Syst. E Vol. 259: 249–258, 2006.

    Article  Google Scholar 

  • Weiler, E.W., Jourdan, P.S., Conrad, W.: Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay. - Planta 153: 561–571, 1981.

    Article  PubMed  CAS  Google Scholar 

  • Yang, Y.M., Xu, C.N., Wang, B.M., Jia, J.Z.: Effects of plant growth regulators on secondary wall thickening of cotton fibres. - Plant Growth Regul. 35: 233–237, 2001.

    Article  CAS  Google Scholar 

  • Zang, Y.X., Lim, M.H., Park, B.S., Hong, S.B., Kim, D.H.: Metabolic engineering of indole glucosinolates in Chinese cabbage plants by expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1. - Mol. Cells 25: 231–241, 2008.

    PubMed  CAS  Google Scholar 

  • Zhang, Z.L., Ji, R.H., Li, H.Y., Zhao, T., Liu, J., Lin, C.T., Liu, B.: CONSTANS-LIKE 7 (COL7) is involved in phytochrome B (phyB)-mediated light-quality regulation of auxin homeostasis. - Mol. Plant 7: 1429–1440, 2014.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Z.Y., Ober, J.A., Kliebenstein, D.J.: The gene controlling the quantitative trait locus EPITHIOSPECIFIER MODIFIER1 alters glucosinolate hydrolysis and insect resistance in Arabidopsis. - Plant Cell 18: 1524–1536, 2006.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to J. Li.

Additional information

Acknowledgments: This work was supported by the National Natural Science Foundation of China (NSFC No. 31570298), Science Foundation of Heilongjiang Province (No. C2017031), and Science and Technology Research Project of Education Department of Heilongjiang Province (No. 12531003).

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, R., Kong, W.W., Peng, Y.F. et al. Identification and expression pattern analysis of the glucosinolate biosynthetic gene BoCYP83B1 from broccoli. Biol Plant 62, 521–533 (2018). https://doi.org/10.1007/s10535-018-0797-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10535-018-0797-0

Additional key words

Navigation