Thioglucoside glucohydrolase 1 (TGG1) is one of two known functional myrosinase enzymes in Arabidopsis. The enzyme catalyzes the hydrolysis of glucosinolates into compounds that are toxic to various microbes and herbivores. Transgenic Arabidopsis plants carrying ␤-glucuronidase and green fluorescent protein reporter genes fused to 0.5 or 2.5 kb of the TGG1 promoter region were used to study spatial promoter activity. Promoter activity was found to be highly specific and restricted to guard cells and distinct cells of the phloem. No promoter activity was detected in the root or seed. All guard cells show promoter activity. Positive phloem cells are distributed in a discontinuous pattern and occur more frequent in young tissues. Immunocytochemical localization of myrosinase in transverse and longitudinal sections of embedded material show that the TGG1 promoter activity reflects the position of the myrosinase enzyme. In the flower stalk, the myrosinase-containing phloem cells are located between phloem sieve elements and glucosinolate-rich S cells. Our results suggest a cellular separation of myrosinase enzyme and glucosinolate substrate, and that myrosinase is contained in distinct cells. We discuss the potential advantages of locating defense and communication systems to only a few specific cell types.Arabidopsis ecotype Columbia has been shown to contain 23 different glucosinolates (Hogge et al., 1988). Myrosinase (EC 3.2.3.1), also known as ␤-thioglucoside glucohydrolase, catalyzes the hydrolysis of glucosinolates into Glc and an unstable intermediate that undergoes nonenzymatic rearrangement to form sulfate and isothiocyanates, thiocyanates, nitriles, epithioalkanes, or elementary sulfur dependent on the concentration of H ϩ , metal ions, epithiospecifier protein, and/or other cofactors (Bones and Rossiter, 1996; Foo et al., 2000). The complexity of the myrosinase-glucosinolate system suggests a diverse and multifunctional role in the cruciferous plants.Glucosinolates are a diverse group of sulfurcontaining glycosides that may serve as a sink for nitrogen and sulfur, and the hydrolysis products may have important roles in the defense of the plant against microorganisms and insects (Bones and Rossiter, 1996; Rask et al., 2000). Previous studies have shown that myrosinase is located in idioblasts named myrosin cells, which have been found in several species of Brassicacea, including oilseed rape (Brassica napus), white mustard (Sinapis alba), cauliflower (B. oleracea), and Chinese cabbage (B. campestris; Bones and Iversen, 1985; Thangstad et al., 1990 Thangstad et al., , 1991 Bones et al., 1991; Hö glund et al., 1991 Hö glund et al., , 1992 Geshi et al., 1998). Myrosin cells occur as scattered cells in radicles, stems, leaves, petioles, seeds, and seedlings (Bones and Rossiter, 1996; Rask et al., 2000). The myrosinase enzyme has been localized to myrosin cells by immunocytochemical methods (Bones et al., 1991; Thangstad et al., 1990 Thangstad et al., , 1991 Hö glund et al., 1991; Geshi et al., 1998), a...

Isothiocyanates (ITCs) from biodegradation of glucosinolates comprise a group of electrophiles associated with growth-inhibitory effects in plant- and mammalian cells. The underlying modes of action of this feature are not fully understood. Clarifying this has involved mammalian cancer cells due to ITCs' chemopreventive potential. The binding of ITCs to tubulins has been reported as a mechanism by which ITCs induce cell cycle arrest and apoptosis. In the present study we demonstrate that ITCs disrupt microtubules in Arabidopsis thaliana contributing to the observed inhibited growth phenotype. We also confirmed this in rat bladder cancer cells (AY-27) suggesting that cells from plant and animals share mechanisms by which ITCs affect growth. Exposure of A. thaliana to vapor-phase of allyl ITC (AITC) inhibited growth and induced a concurrent bleaching of leaves in a dose-dependent manner. Transcriptional analysis was used to show an upregulation of heat shock-genes upon AITC-treatment. Transgenic A. thaliana expressing GFP-marked α-tubulin was employed to show a time- and dose-dependent disintegration of microtubules by AITC. Treatment of AY-27 with ITCs resulted in a time- and dose-dependent decrease of cell proliferation and G2/M-arrest. AY-27 transiently transfected to express GFP-tagged α-tubulin were treated with ITCs resulting in a loss of microtubular filaments and the subsequent formation of apoptotic bodies. In conclusion, our data demonstrate an ITC-induced mechanism leading to growth inhibition in A. thaliana and rat bladder cancer cells, and expose clues to the mechanisms underlying the physiological role of glucosinolates in vivo.

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.

The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassicaceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes.

Many plant phytochemicals constitute binary enzyme–glucoside systems and function in plant defence. In brassicas, the enzyme myrosinase is confined to specific myrosin cells that separate the enzyme from its substrate; the glucosinolates. The myrosinase-catalysed release of toxic and bioactive compounds such as isothiocyanates, upon activation or tissue damage, has been termed ‘the mustard oil bomb’ and characterized as a ‘toxic mine’ in plant defence. The removal of myrosin cells and the enzyme that triggers the release of phytochemicals have been investigated by genetically modifying Brassica napus plants to remove myrosinase-storing idioblasts. A construct with the seed myrosin cell-specific Myr1.Bn1 promoter was used to express a ribonuclease, barnase. Transgenic plants ectopically expressing barnase were embryo lethal. Co-expressing barnase under the control of the Myr1.Bn1 promoter with the barnase inhibitor, barstar, under the control of the cauliflower mosaic virus 35S promoter enabled a selective and controlled death of myrosin cells without affecting plant viability. Ablation of myrosin cells was confirmed with light and electron microscopy, with immunohistological analysis and immunogold-electron microscopy analysis showing empty holes where myrosin cells normally are localized. Further evidence for a successful myrosin cell ablation comes from immunoblots showing absence of myrosinase and negligible myrosinase activity, and autolysis experiments showing negligible production of glucosinolate hydrolysis products. The plants where the myrosin defence cells have been ablated and named ‘MINELESS plants’. The epithiospecifier protein profile and glucosinolate levels were changed in MINELESS plants, pointing to localization of myrosinases and a 35 kDa epithiospecifier protein in myrosin cells and a reduced turnover of glucosinolates in MINELESS plants.

The cytological and intracellular localization of myrosinase (EC 3.2.3.1) has been studied by immunochemical techniques using paraffin-embedded sections of radicles and cotyledons from seeds of Brassica napus L. cv. Niklas. For immunolabelling, sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase-and fluorescein-isothiocyanate-conjugated secondary antibodies. Enzyme and fluorescence label was present in typical myrosin cells both in radicles and in cotyledons. With higher magnification, fluorescence label revealed that the intracellular localization of myrosinase was associated with the tonoplast-like membrane surrounding the myrosin grains in the myrosin cells. The results also indicate that a large proportion of the positive myrosin cells are located in the second-outermost cell layer of the peripheral cortex region of the radicles.

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