Center for Molecular Biology, Department of Botany, AVH, University of Trondheim, ... Technology Center (D.B. and T.M.-O.), the University of Washington.
Plant Physiol. (1993) 103: 671-672
Plant Gene Register
Ambidopsis cDNA Sequence Encoding Myrosinase' Suprachitra Chadchawan, JohnBishop, Ole P. Thangstad, Atle M. Bones, Thomas Mitchell-Olds, and Douglas Bradley* Department of Botany, AJ-30, University of Washington, Seattle, Washington 98195 (S.C., J.B., D.B.); UNIGENCenter for Molecular Biology, Department of Botany, AVH, University of Trondheim, N-7005 Trondheim, Norway (O.P.T., A.M.B.); and Division of Biological Sciences, University of Montana, Missoula, Montana 5981 2 (T.M.-O.)
in Arabidopsis myrosinase is encoded by a small gene family of two to three genes (Xue et al., 1992; S. Chadchawan and D. Bradley, unpublished results) (Table I). This makes Arabidopsis an attractive Crucifer species in which to study the molecular biology of myrosinase gene expression and to study the effects of myrosinase gene manipulation on glucosinolate metabolism and pathogen resistance.
Myrosinase (0-thioglucosidase, thioglucoside glucohydrolase, EC 3.2.3.1) catalyzes the hydrolysis of glucosinolates, a class of sulfur-containing glycosides present in a11 Crucifers. Although intact glucosinolates are relatively nontoxic, their breakdown products (isothiocyanates, nitriles, or thiocyanates) have important biological influences on mammals, insects, and microbial pathogens (for review, see Fenwick et al., 1983). Myrosinase has been purified from a number of Crucifer species and found to be a glycoprotein of 135,000 mo1 wt consisting of two subunits of 65,000 mo1 wt (Bjorkman, 1976; Bones and Slupphaug, 1989). In intact plant tissues, myrosinase is sequestered in specialized cells called myrosin cells. When plant tissues are damaged as the result of pathogen or herbivore attack, myrosinase and glucosinolates come into contact, causing hydrolysis of glucosinolates to the biologically active compounds. Myrosinase cDNA sequences have been reported for Sinapis alba and Brassica napus (Falk et al., 1992; Xue et al., 1992). Southern blot analysis of genomic DNA isolated from S. alba (Xue et al., 1992), B. napus (Falk et al., 1992), and Brassica rapa (S. Machlin and D. Bradley, unpublished results) has shown that in these species myrosinase is encoded by large multigene families consisting of 6 to 14 genes. However,
Received May 3, 1993; accepted May 18, 1993. Copyright Clearance Center: 0032-0889/93/103/0671/02. The GenBank accession number for the sequence reported in this article is L11454.
LITERATURE CITED
Bjorkman R (1976) Properties and function of plant myrosinases. Zn JG Vaughan, AJ MacLeod, BMG Jones, eds, The Biology and Chemistry of the Cruciferae. Academic Press, New York, pp 191-205 Bones AM, Slupphaug G (1989) Purification, characterization, and partia1 amino acid sequencing of P-thioglucosidase from Brassica napus L. J Plant Physioll34 722-729 Falk A, Xue J, Lenman M, Rask L (1992) Sequence of a cDNA clone encoding the enzyme myrosinase and expression of myrosinase in different tissues of Brassica napus. Plant Sci 8 3 181-186 Fenwick GR, Heaney RK, Mullin WJ (1983) Glucosinolates and their breakdown products in food and food plants. CRC Crit Rev Food Sci Nutr 18: 123-201 Xue J, Lenman M, Falk A, Rask L (1992) The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family. Plant Mo1 Biol 18: 387-398
'This work was supported by funding from the Washington Technology Center (D.B. and T.M.-O.), the University of Washington Graduate Research Fund (D.B. and J.B.), and the National Science Foundation, BSR-9022025 (T.M.-O. and D.B.). S.C. was supported by the Ananthamahidol Foundation. * Corresponding author; fax 1-206-543-3262.
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Table 1. Characteristics of the cDNA sequence encoding an Arabidopsis myrosinase Organism: Arabidopsis thaliana L. Columbia. Location on Chromosome: Unknown. Cene Product; Pathway: Thioglucoside glucohydrolase (EC 3.2.3.1); hydrolysis of glucosinolates to isothiocyanates, nitriles, or thiocyanates, D-CIC,and sulfate. Designation: tal. Cloning and Sequencing Techniques: A. thaliana cDNA library, catalog no. FL1002b, was purchased from Clontech Laboratory, Inc. This cDNA library was made in bacteriophage vector Xgtll using mRNA prepared from A. thaliana L. Columbia. Two pools of mixed oligonucleotides were synthesized corresponding to two amino acid regions within the 25 amino acid residues determined from the N terminus of the purified Arabidopsis myrosinase protein. A labeled polymerase chain reaction product was used to screen the cDNA library. Restriction fragments were subcloned into pBluescriptll (Stratagene), and both strands were completely sequenced using the dideoxy chain termination method. Sequence Identification: DNA and amino acid sequence comparison with S. alba MB3 (Xue et al., 1992) and B. napus (Falk et al., 1992). Similarity at the amino acid leve1 is 67% for both species. Regulation and Expression: A 1.9-kb transcript as detected on northern blots using Arabidopsis total RNA from young leaves, stems, and floral tissues. No message was observed in total RNA from dry seeds. Gene Copy Number: Southern blot analysis of genomic DNA indicated that tggl is part of a small gene family consisting of two to three genes. Features of Gene Sequence: An open reading frame of 1623 bp. Three putative poly(A) addition sites in the 3’ noncoding region. (G C) Content: 42% in protein coding region. Features of Protein Sequence: The 1623-bp open reading frame encodes a polypeptide of 522 amino acids (including a putative signal peptide of 19 amino acids) with a predicted M, of 61,130. There are nine potential N-linked glycosylation sites. TGGl is more similar to the MB family of myrosinase proteins as described in S. alba (Xue et al., 1992). Anti bodies: Not available. Subcellular Localization: In other Crucifers, myrosinase is reported to localize within myrosin grains that are themselves within the vacoules of myrosin cells.
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Plant Physiol. Vol. 103, 1993
