Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol159.Issue2.2000:

Plant Science 159 2000 265 – 272 Functional detection of chemopreventive glucosinolates in Arabidopsis thaliana Heidrun B. Gross 1 , Tim Dalebout 2 , C. Douglas Grubb, Steffen Abel Department of Vegetable Crops, Uni6ersity of California-Da6is, One Shields A6enue, Da6is, CA 95616 , USA Received 18 May 2000; received in revised form 20 July 2000; accepted 20 July 2000 Abstract Natural isothiocyanates, derived from glucosinolates by myrosinase-catalyzed hydrolysis, are potent chemopreventive agents that favorably modify carcinogen metabolism in mammals by inhibiting metabolic activation of carcinogens andor by inducing carcinogen-detoxifying enzymes. Methylsulfinylalkyl isothiocyanates are potent selective inducers of mammalian Phase 2 detoxifi- cation enzymes such as quinone reductase [NADPH:quinone-acceptor oxidoreductase, EC 1.6.99.2]. Members of the Cruciferae family, including the model plant species Arabidopsis thaliana L. Heyhn, synthesize methylsulfinylalkyl glucosinolates. We have adapted a colorimetric bioassay for quinone reductase activity in Hepa 1c1c7 murine hepatoma cells as a versatile tool to rapidly monitor methylsulfinylalkyl glucosinolate content in A. thaliana leaf extracts. Using wild type plants and mutant plants defective in the synthesis of 4-methylsulfinylbutyl glucosinolate glucoraphanin, we have demonstrated that A. thaliana ecotype Columbia is a rich source of Phase 2 enzyme inducers and that methylsulfinylalkyl glucosinolates, predominantly glucoraphanin, account for about 80 of the quinone reductase inducer potency of Columbia leaf extracts. We have optimized leaf extraction conditions and the quinone reductase bioassay to allow for screening of large numbers of plant extracts in a molecular genetic approach to dissecting glucosinolate biosynthesis in A. thaliana. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Arabidopsis thaliana; Bioassay; Chemopreventive glucosinolates; Phase 2 detoxification enzymes; Quinone reductase; Sulforaphane www.elsevier.comlocateplantsci

1. Introduction

Glucosinolates are a diverse class of sulfur- and nitrogen-containing secondary metabolites that have long been the defining chemotaxonomic char- acter of the order of Brassicales and that are mainly found in members of the Brassicaceae Cruciferae and in several other families of di- cotyledonous angiosperms [1]. Glucosinolates con- sist of a common glycone moiety and a variable aglycone side-chain derived from amino acids, most frequently from methionine, phenylalanine, tyrosine, and tryptophane. The glycone is charac- terized by a thioglucose and a sulfonated oxime group, both attached to the a-carbon of the parental amino acid [2]. Upon tissue disruption, glucosinolates are rapidly hydrolyzed by myrosi- nase b-thioglucoside glucohydrolase, EC 3.2.3.1 to unstable intermediates that, as dictated by chemical conditions, spontaneously rearrange to isothiocyanates, thiocyanates, or nitriles [3,4]. Al- though the primary biological function of glucosi- nolates in plants is unknown, glucosinolate breakdown products are proposed to act as allelo- chemicals and to play a role in plant defenses against herbivores, pests, and pathogens [3,4]. Fur- thermore, indolyl glucosinolates can be converted into indoleacetic acid and may thus contribute to active auxin levels in cruciferous plants [5]. As components of food for humans and feed for Corresponding author. Tel.: + 1-530-7525549; fax: + 1-530- 7529659. E-mail address : sabelucdavis.edu S. Abel. 1 Present address: Department of Molecular Biosciences, University of California-Davis, School of Veterinary Medicine, One Shields Avenue, Davis, CA 95616, USA. 2 Present address: Department of Anatomy and Embryology, Uni- versity of Leiden, Medical Center, P.O. Box 9603, 2300 RC Leiden, The Netherlands. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 5 4 - X livestock, the biological activities of glucosinolate hydrolysis products have generated considerable toxicological and pharmocological interest. De- pending on glucosinolate composition and on the prevalence of hydrolysis products, consumption of glucosinolates by mammals has been linked with goitrogenic effects thiocyanates or with a re- duced risk of developing cancer isothiocyanates in experimental animals [6 – 10]. Natural isothiocyanates derived from aromatic and aliphatic glucosinolates are effective chemo- protective agents that block chemical carcinogene- sis and prevent several types of cancer in rodent models [10]. Mechanistic studies have shown that isothiocyanates target mammalian Phase 1 and Phase 2 drug-metabolizing enzymes and their cod- ing genes, resulting in decreased carcinogen-DNA interactions and in increased carcinogen detoxifi- cation [8]. For example, the methionine-derived 4-methylsulfinylbutyl isothiocyanate sul- foraphane inhibits Phase 1 enzyme-mediated acti- vation of procarcinogens [11], induces Phase 2 detoxification enzymes such as quinone reductase QR and glutathione-S transferase in hepatoma cells [12,13], and blocks mammary tumor forma- tion in rats [14,15]. Sulforaphane is the most pow- erful natural inducer of chemoprotective enzymes thus far reported [12] and has become a metabolic target of breeding strategies to enhance the anti- carcinogenic potency of cruciferous vegetables [15,16]. The chemoprotective properties of natural isoth- iocyanates have renewed interest in glucosinolate biosynthesis. While significant progress has been made in understanding the biochemistry and enzy- mology of glucosinolate synthesis, very little is known about the structural and regulatory genes involved [2]. In Arabidopsis thaliana L. Heynh., a member of the Cruciferae family and a premier reference species for plant biology [17], 23 differ- ent glucosinolates have been identified [18]. Inter- estingly, 4-methylsulfinylbutyl glucosinolate glucoraphanin, precursor to sulforaphane, is the major leaf glucosinolate of ecotype Columbia [18,19]. Here, we show that a QR bioassay in murine hepatoma cells reliably reports gluco- raphanin content in A. thaliana. Furthermore, we have optimized the bioassay to allow for high- throughput analysis of leaf extracts. The bioassay for the major chemopreventive glucosinolate in A. thaliana, glucoraphanin, allows for rapid analysis of a large number of samples in an effort to dissect glucosinolate biosynthesis by genetic and molecu- lar genetic approaches.

2. Materials and methods