Cell specific, cross-species expression of myrosinases in Brassica napus, Arabidopsis thaliana and Nicotiana tabacum

Plant Molecular Biology 54: 597–611, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Cell specific, cross-species expression of myrosinases in Brassica napus, Arabidopsis thaliana and Nicotiana tabacum Ole Petter Thangstad1, Bodil Gilde1, Supachitra Chadchawan2,3, Martin Seem1, Harald Husebye1, Douglas Bradley2 and Atle Magnar Bones1,* 1

Department of Biology, The Norwegian University of Science and Technology, Høgskoleringen 5, N-7491 Trondheim, Norway (*author for correspondence; e-mail [email protected]); 2Department of Botany, University of Washington, Seattle, WA 98195, USA; 3Present address: Department of Botany, Chulalongkorn University, Bangkok 10330, Thailand Received 27 February 2002; accepted in revised form 17 March 2004

Key words: glucosinolates, guard cell, GUS (b-glucuronidase), myrosinase (EC 3.2.1.147), myrosinase promoter, signal peptide

Abstract 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 b-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. Abbreviations: DAG, days after germination; DAP, days after pollination; DAS, days after seeding; EM, electron microscopy; ER, endoplasmic reticulum; EST, expressed sequence tags; FITC, fluorescein isothiocyanate; GUS, b-glucuronidase; MUG, 4-methyl umbelliferone glucuronide; NPTII, neomycin phosphotransferase; PCR, polymerase chain reaction

Introduction The myrosinase-glucosinolate system of plants includes myrosinase enzymes (EC 3.2.1.147), interacting proteins like epithiospecifier proteins, myrosinase-binding proteins, myrosinase-associated proteins and nitrilases and a total of more

than 100 glucosinolate substrates (reviewed by Bones and Rossiter, 1996). Glucosinolates (thioglucosides) are compounds consisting of a glucose residue linked by a thioglucoside bond to an amino acid derived R-group of either aliphatic, aromatic or indole types. Myrosinase enzymes hydrolyse glucosinolates and thereby produce

598 glucose, sulphate and isothiocyanates, thiocyanates, nitriles, epithioalkanes or elementary sulphur dependent on pH, metal ions, epithiospecifier proteins and other cofactors (Bones and Rossiter, 1996). Myrosinase (thioglucosidase, EC 3.2.1.147) is the only known S-glycosidase in plants. Glucosinolates or their hydrolysis products have antimicrobial activity (Drobnica et al., 1967; Mithen et al., 1986), affects insect behaviour (Blau et al., 1987; Lambrix et al., 2001) and do also potentially store nitrogen and sulphate (Bones and Rossiter, 1996). Myrosinases are localized to specific cells named myrosin cells or myrosin idioblasts (Bones and Iversen, 1985; Thangstad et al., 1990). Idioblasts are individual cells, differing greatly from its neighbours in regard to size, structure, or contents. The morphology of myrosin idioblasts varies among different organs, tissues and developmental stages. In Sinapis alba L. and Raphanus sativus L., myrosin cells are present in parenchyma tissue of cotyledons (Werker and Vaughan, 1974, 1976; Bones and Iversen, 1985). Such cells are also found in cortex tissue of roots and hypocotyls where they may be several times longer than the normal cortex cells (Bones and Iversen, 1985; Bones et al., 1991). The localization of myrosinase to myrosin cells has been proved using immunocytochemical localization (Thangstad et al., 1990; Bones et al., 1991; Ho¨glund et al., 1991; Geshi and Brandt, 1998; Kelly et al., 1998), and in situ hybridization (Lenman et al., 1993a; Xue et al., 1993, 1995). The subcellular localization in myrosin grains of myrosin cells has been shown using immunogold electron microscopy (EM) techniques (Thangstad et al., 1991; Ho¨glund et al., 1992; Geshi and Brandt, 1998; Kelly et al., 1998). The myrosinases have been shown to be a group of isoenzymes present in all Brassicaceae species examined, and are also found in 14 other plant families (Rodman, 1991). Myrosinases are glycosylated dimeric proteins with subunit molecular weights reported to be from 62 to 75 kDa in plants. Isoenzymes have been purified and characterized from some species and shown to have varying degree of glycosylation, varying degree of ascorbic acid activation and varying hydrolysis rates on different glucosinolates (for review see Bones and Rossiter, 1996). A crystal structure of myrosinase has been made (Burmeister et al., 1997).

The allotetraploid B. napus genome encodes at least 25 different myrosinase (thioglucosidase) genes (Falk et al., 1992; Xue et al., 1992; Thangstad et al., 1993; Beisva˚g, Winge and Bones, unpublished). Based on cDNA sequencing myrosinase genes from B. napus have been divided into three subfamilies, MA, MB and MC, encoding proteins with molecular weights of approximately 75, 65 and 70 kDa, respectively (for review see Rask et al., 2000). The MA and MC types of myrosinase genes have been reported to be exclusively expressed in the developing seed and MB type of genes to be expressed throughout development (Lenman et al., 1993a; Rask et al., 2000). Two functional genomic myrosinase genes from B. napus, Myr1.Bn1 and Myr2.Bn1, have been cloned (Thangstad et al., 1993). Based on homology of several cDNAs (unpublished data), genomic sequencing (Thangstad et al., 1993) and predicted molecular weight of the corresponding proteins, these genes are members of the myrosinase subfamilies MA(Myr1) and MB(Myr1), respectively (Lenman et al., 1993a; Thangstad et al., 1993). The myrosinase genes from A. thaliana have been named thioglucoside glucohydrolase (TGG) (Chadchawan et al., 1993; Machlin et al., 1993). Two functional myrosinase genes, TGG1 and TGG2, and one pseudogene, TGG3 have so far been reported from A. thaliana (Xue et al., 1995). Phylogenetic analyses show that TGG1 and TGG2 are distinct from the myrosinase genes of the MA(Myr1) or MB(Myr1) subfamilies (Chadchawan et al., 1993; Xue et al., 1995). Xue et al., (1995) used in situ hybridization to show that the TGG1 and TGG2 genes are expressed in leaf, stem and floral organs such as sepals and gynoecium. To study the spatial and temporal expression of the myrosinase genes in A. thaliana and B. napus we made promoter constructs with the Myr1.Bn1 and TGG1 promoters fused to the b-glucuronidase (GUS) reporter gene (Jefferson et al., 1987). The Myr1.Bn1 constructs were used to transform B. napus and A. thaliana, and the TGG1 construct to transform A. thaliana and N. tabacum. In A. thaliana and N. tabacum the TGG1 promoter construct direct expression to guard cells and also to phloem cells in A. thaliana. In B. napus the Myr1.Bn1 promoter is specific to myrosin cell idioblasts of developing seed and seedlings. When introduced into A. thaliana the Myr1.Bn1 promoter activity was similar to the

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Materials and methods

the pGEM11 polylinker following the manufacturer’s instructions, with the exception of a lowmelt agarose gel isolation of the deletion products before religation of the vector. These clones were sequenced to produce a full-length sequence from overlapping clones in both directions of the Myr1.Bn1 promoter (Genbank accession AF323020).

Plant material

DNA sequencing

Brassica napus cv. Westar was generously provided by Dr Wilfred Keller (Plant Biotechnology Institute, National Research Council, Saskatoon, Sask., Canada) and A. thaliana L. Columbia ecotype, by Dr Chris Somerville (Carnegie Inst., Stanford, CA, USA). For some experiments, plants were grown aseptically on MS agar plates or vessels (0.5· or 1· MS salts (Sigma), 1.0% sucrose, 0.8% Agar gel (Sigma) under 24 h light (A. thaliana and N. tabacum) or at 16 h day (B. napus) in controlled environment rooms. Otherwise they were grown in soil.

Sequencing reactions were performed as described in Thangstad et al. (1993) and also using dye terminator cycle sequencing on an ABI 373A. Computer sequence analysis was done with the UWGCG package (Devereux et al., 1984). Computer analyses of nucleotide sequence homology were performed at the Genbank and NCBI BLAST network services and non-redundant DNA and protein sequence databases (Altschul et al., 1990).

pattern observed with the TGG1 promoter. Histochemical assays, in situ hybridization, double immunolabelling, myrosinase enzyme assays and quantitative expression analyses confirmed these results.

Bacterial strains, plasmids and molecular methods Standard molecular methods were used (Sambrook et al., 1989). Probes for southern and northern blot analyses and genomic library screening were prepared from gel purified DNA fragments. E. coli DH5a (Gibco-BRL, Paisley, UK), JM109 (Promega, Madison, WI), NM538 (Promega) and Y1090r (Clontech, Palo Alto, CA) were used for plasmid and bacteriophage k manipulations. The plasmids pBluescript II KS (Stratagene, La Jolla, CA), pGEM3, 5 and 11 (Promega) were used for subcloning. Isolation of a Myr1.Bn1 promoter DNA sequence An EMBL-3 genomic library from B. napus cv. Bridger (Clontech Lab.) with insert range from 8 to 21 kb was screened and rescreened by filter hybridization using a 450 bp cDNA fragment according to standard protocols (Sambrook et al., 1989). From one of the lambda clones (Thangstad et al., 1993), a 3.5 kb fragment of the Myr1.Bn.1 promoter sequence was isolated and series of overlapping deletion clones were made using the Erase-a-base system (#E5850, Promega). Exonuclease III deletions were made from the SP6 side of

Promoter-GUS vectors pBIMyr1.Bn1-GUS A 1.2 kb polymerase chain reaction (PCR) fragment was amplified with vector forward T7 and phosphorylated myrosinase reverse primer, 5¢ GGT TAA TTT GTA GAT GTG TGT GC 3¢ using a subclone of the myrosinase gene contained in PGEM5 as template. The resulting PCR fragment was digested with SalI to produce a 404 bp fragment (50 SalI-blunt end 30 , pos. 2520–2924) that was inserted at the SalI and SmaI sites of the pBI101.1 polylinker. The remaining upstream 2.5 kb of the Myr1 promoter was inserted as a HindIII–SalI fragment (pos. 1–2520) into the pBI vector made above, giving the pBIMyr1.Bn1-GUS vector with a full length Myr1 promoter region and 5¢UTR (pos. 1–2924, Figure 1) in frame with the GUS gene ATG. pBIMyr1.Bn1-SPGUS A 1.28 kb SalI–Sca1 fragment (pos. 2520–3806) containing the myrosinase promoter and partial Myr1.Bn1 was inserted at the SalI and SmaI sites of the pBI101.1 polylinker, thus placing the Myr1.Bn1 partial myrosinase promoter, ATG, signal peptide and exons one to four in frame with the GUS-gene ATG. The remaining upstream 2.5 kb of the Myr1.Bn1 promoter was inserted as a HindIII–SalI fragment (pos. 1–2520) into pBI

600 Histochemical localization of GUS activity in transgenic plants

Figure 1. Schematic presentation of the myrosinase promoter constructs Myr1.Bn1-GUS, Myr1.Bn1-SPGUS and TGG1GUS. Shaded boxes I–III represent regions in the Myr1.Bn1 promoter/exons displaying significant homology to the TGG1 gene sequence (accession number X79194). Positions are given relative to the Myr1.Bn1 gene sequence (accession number Z21977). Arrows mark the first codon (ATG) of the respective gene constructs above.

upstream of the SalI–ScaI fragment introduced above, giving the pBIMyr1.Bn1-SPGUS vector (Figure 1). The positions are given relative to the published sequence (Genbank accession AF323020). pBITGG1-GUS Construct contains the 2.5 kb TGG1 myrosinase promoter sequence from A. thaliana (Genbank accession AF079308) (Husebye et al., in press). The myrosinase-GUS gene junctions were verified by sequencing. Plasmids were transformed into Agrobacterium tumefaciens LBA4404 by electroporation. Production and analysis of transgenic plants The pBIMyr1.Bn1-GUS, pBIMyr1.Bn1-SPGUS and TGG1-GUS vectors were transferred into A. thaliana leaf explants via Agrobacterium tumefaciens mediated transformation. Transgenic tobacco plants were generated from leaf disks infected with A. tumefaciens strain LBA4404 carrying the pBITGG1-GUS construct. Cotyledons of B. napus cv. Westar were transformed with A. tumefaciens containing pBIMyr1.Bn1-GUS or pBIMyr1.Bn1-SPGUS vector (Moloney et al., 1989). A total of about 5000 explants were transformed. After regeneration of transgenic shots, transgenic clones were verified using PCR amplification and Southern blot hybridization. After repetitive transfers and hormone induced rooting, plants were transferred to soil and grown in controlled environment rooms. Defined embryo/seedmaturation stages were collected from manually pollinated and tagged flowers.

Different anatomical parts of the transgenic B. napus and A. thaliana and controls were collected and analyzed (Husebye et al., in press). After staining, chlorophyll was extracted with series of ethanol and images taken using a stereomicroscope (Nikon SMZ1500, Tokyo, Japan or Zeiss M2, Carl Zeiss GmbH, Wien, Austria). Whole-mount in situ hybridization Whole-mount in situ hybridization of A. thaliana seedlings was done as described by Ludevid et al. (1992). The TGG1 gene-specific probe (Genbank accession AY058182, position 1462–1794) was tested with dot blots and genomic Southern blots for specificity, before being used as antisense digoxigenin labelled riboprobe. The sense strand was used as a control for background hybridization pattern. Digoxigenin labelling of riboprobes were performed according to manufacturer’s instructions (Roche, Basel, Switzerland). Quantification of GUS activity by fluorescent MUG assay Fluorometric assay of GUS activity was performed essentially as described by Jefferson et al. (1987). Plant tissue was frozen in liquid nitrogen and crushed with alumina in eppendorf tubes using a Teflon pestle and 300 ll 4-methyl umbelliferone glucuronide (MUG) assay buffer. After centrifugation the supernatant was assayed for GUS activity and for total protein. Samples were assayed using 1 mM MUG (4-methyl umbelliferone glucuronide) and the reaction stopped with buffer (0.2 M Na2CO3) after 60 min at 37 C. Fluorescence was measured on a Hoefer TKO 100 fluorometer. Activity was determined from standard and specific GUS activity presented as pmol 4-MU per min and mg protein. Total protein content of samples was measured using Bradford reagent (BioRad, Hercules, CA). Double immunocytochemical localization of myrosinase and GUS Paraffin sections of transgenic and control seeds were made as described by Bones et al. (1991).

601 Sections were sequentially labelled with mouse monoclonal anti-myrosinase (Thangstad et al., 1990) and rabbit anti-GUS (Molecular Probes, Eugene, OR). Secondary antibodies diluted 1:20 (Texas Red anti-mouse and fluorescein isothiocyanate (FITC) anti-rabbit, Dako, Glostrup, Denmark) were added to sections and sections incubated for 20 min. Cover slips were mounted using City Fluor A121 (UKC, Chem. Lab.,

Canterbury, UK) and images taken using the FITC and Texas Red filter-sets. Images from both wavelengths were digitally sampled and merged using Adobe PhotoShopTM . Myrosinase activity assay Myrosinase activity assays were performed as described by Bones and Slupphaug (1989).

Figure 2. Histochemical localization of GUS activity directed by the TGG1 promoter in Arabidopsis thaliana (a)–(g) and Nicotiana tabacum (h)–(i). (a) Imbibed (2 days at 4 C) seed with seed coat removed, (b) seedling 3 DAG, (c) cotyledon from seedling 5 DAG, (d) cotyledon from 14 DAG, (e) stamen, (f) stigma and style, (g) sepal, (S) and, petal (P), (h) leaf of 4 week old N. tabacum plant, (i) microtome cut cross section of fresh leaf of 4 week old N. tabacum. Bars: (a)–(i) ¼ 0.1 mm. DAG ¼ days after germination.

602 Northern blot analysis Northern blots were performed using the TGG1 gene specific probe following standard protocols (Sambrook et al., 1989). Total RNA was extracted from leaves, cauline leaves, bracts, inflorescence stems, and flower buds of A. thaliana and 20 lg of each sample loaded on the gel. A probe for an mRNA encoding ribosomal protein S15 was used as control for loading.

TGG1 promoter directs Guard and phloem cell specific expression in Arabidopsis

In this study we have isolated and used myrosinase gene promoters from Brassica napus (the Myr1.Bn1 promoter) and Arabidopsis thaliana (the TGG1 promoter). The promoter of the Myr1.Bn1 gene was isolated, sequenced and used for construction of the pBIMyr1.Bn1-GUS or pBIMyr1.Bn1-SPGUS vectors. The Myr1.Bn1 and TGG1 genes show significant homology in coding regions starting at the ATG (Pos. 2924; Figure 1, Box I). In the promoter region only two short stretches of homology was found using the Blast engine aligning the two sequences (Tatusova and Madden, 1999), one in the core promoter (pos. 2805–2872; Figure 1, Box II) and another in a region further upstream (pos. 2105–2141; Figure 1, Box III). The TGG1 promoter sequence (GenBank accession no. AF079308) contains a pair of direct repeats ‘ATCAATTGAAAA’, containing a CAN (CANNTG) motif, which may be important for spatially and temporally specific expression (Kawagoe and Murai, 1992). In both promoters many elements of the core sequence (A/T)AAAG are found (13 in TGG1 and 16 in Myr1.Bn1), a recognition sequence of the DOF transcription factors (Yanagisawa and Schmidt, 1999).

In seeds of Arabidopsis GUS expression was found after imbibitions (at 4 C) in the cotyledon (Figure 2a). In seedlings, GUS activity is present mainly in guard cells (Figure 2b and c). In older seedlings, guard cell specific expression was found throughout the developmental stages of A. thaliana. In leaves and cotyledon expression in elongated cells associated with vascular tissue is also seen (Figure 2d). Single guard cells were also found to contain GUS gene expression. In older leaves, the number of guard cells containing GUS was reduced. No GUS activity was detected in roots. In the reproductive organs of A. thaliana, GUS activity was found in sepals and petals (Figure 2g), anthers (Figure 2e), styles (Figure 2f), in the tip of nectary glands and fertilized ovaries in young siliques. In petals, the second floral whorl, no GUS expression was detected. No GUS activity could be detected in pollen and ovules of transgenic A. thaliana. GUS expression in siliques was lower during the seed development, but still present until silique desiccation. Expression of the transgenic GUS reporter are consistent with patterns of TGG activity detected on the native activity gel (Figure 8c) and the northern blot analysis with the TGG1 gene specific probe (Figure 8a and b). All tissues containing guard cells contain GUS activity, including modified guard cells on the epidermis of the nectaries. Some cells of vascular tissues express GUS. After removal of the epidermis, GUS activity was localized to spongy cells around the vascular bundles. These cells could potentially be phloem parenchyma cells, but as a subset containing myrosinase, these cells can be called phloem myrosin cells (phloem idioblast).

Expression analysis of myrosinase promoter directed GUS expression in transgenic plants

Guard cell specific expression of the TGG1 promoter is also observed in N. tabacum

The plants transformed with the promoterGUS vectors showed no alterations in phenotype. Six independent homozygous transgenic lines from A. thaliana, four from B. napus and 10 from N. tabacum were assayed for GUS activity. Although expression levels varied all transgenic lines from each species exhibited the same qualitative expression pattern.

Tobacco, N. tabacum, has no myrosinase, glucosinolate or myrosin cells. However, the specificity of the TGG1 promoter is nevertheless regulated to the same developmental, spatial and cell-specific stringency as in A. thaliana. The A. thaliana TGG1 promoter is able to directed GUS expression specific to guard cells of leaves (Figure 2h and i), stems, sepals, and petals of primary tobacco

Results Promoter sequences

603 transformants. These results indicate a guard cell specific expression from the TGG1 promoter that is independent of species and the myrosin cell properties. The Myr1.Bn1 promoter directs cell specific expression but to different idioblasts in B. napus and in A. thaliana The Myr1.Bn1 promoter directs GUS expression specific to idioblast myrosin cells in seeds of B.

napus (Figure 3). As seen in Figure 3, scattered GUS positive cells are present in the cotyledon and radicle of the embryo. The pattern and distribution observed clearly resembles myrosin cells (Bones et al., 1991). The distribution of myrosin cells in the second outermost cell layer in the radicle is typical (Figure 3c, d and g). Transgenic plants containing the pBIMyr1.Bn1-GUS or pBIMyr1.Bn1-SPGUS vectors show the same typical labelling of GUS in myrosin cells. The embryos containing the pBIMyr1.Bn1-SPGUS vector with

Figure 3. Histochemical localization of GUS activity directed by the Myr1.Bn1 promoter in B. napus embryo, seed and seedling. Blue and black spots represent single cells with GUS activity. (a) Embryo from transgenic B. napus pBIMyr1.Bn1-GUS, 10 DAP, (b) 30 DAP, (c), (d) and (g) mature seed, (h) 3 day old seedling, (e) mature seed from transgenic pBIMyr1.Bn1-SPGUS, (f) control B. napus cv. Westar, mature seed. GUS expression is detectable from approximately 30 DAP in the transgenic clones (b, c, d and g). GUS localization is restricted to idioblast myrosin cells appearing as blue to black spots on the pictures. All pictures are of whole-mount stained material except for (g) which was hand-sliced after staining. Note the typical spatial localization to the second outermost cells in the longitudinal cut radicle (g). No GUS positive guard cells were observed in the seedling (h). DAP ¼ days after pollination.

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Figure 4. Histochemical localization of GUS activity directed by the Myr1.Bn1 promoter in A. thaliana. (a) Cotyledon of transgenic A. thaliana Myr1.Bn1-GUS 7 DAG, (b) flower stalk from 5 week old plant, (c) seedling after 16 days, (d) rosette leaf 3 weeks after seeding. All pictures are of whole-mount stained material except for (b) which was hand-sliced after staining. GUS expression is indistinguishable from TGG1-GUS (Figure 2). DAG ¼ days after germination.

the Myr1.Bn1 signal peptide show significant lower GUS activity (Figure 3e). This is probably due to glycosylation of GUS with resulting decrease in activity, since the myrosinase signal peptide potentially targets the GUS through the endoplasmic reticulum (ER) and Golgi apparatus of the cell. Expression was also observed in a few idioblasts of siliques and flower stalk (data not shown). In A. thaliana the expression from the Myr1.Bn1 promoter appear to be indistinguishable from TGG1 (Figure 4). Clearly visible blue guard cells and one phloem cell can be seen in the cotyledon (Figure 4a). In the flower-stalk cross section (Figure 4b), expression is restricted to guard cells and specific cells in the phloem, likely phloem parenchyma cells. In the rosette leaf both guard cells and phloem cells are expressing GUS (Figure 4c and d). Double immunocytochemical labelling analyses labels both b-glucuronidase and myrosinase in all myrosin cells Using a previously well characterized mouse monoclonal antibody against myrosinase (Thangstad et al., 1990; Bones et al., 1991) and a rabbit anti-GUS antibody, double labelling on paraffin sections of transgenic B. napus seeds was

used to prove the presence of GUS and myrosinase in the same cells. As can be seen in Figure 5a–f, immunolocalization of both GUS (Figure 5a and d) and myrosinase (Figure 5b and e) is restricted to myrosin cells, and the merged composite pictures (Figure 5c and f) show mixed fluorescence in myrosin cells. All myrosin cells (anti-myrosinase labelled) are also labelled with anti-GUS antibody. The control seeds (Figure 5g–i) do not show labelling with the GUS antibody (Figure 5g). Although the resolution in light microscopy is too low to give absolute evidence at subcellular level, most of the labelling in Figure 5d and e appears to be in organelles resembling vacuoles. This shows that the signal peptide of myrosinase directs expression of GUS through the ER and Golgi apparatus to give a subcellular localization in myrosin cells, with an appearance typical of fluorescence labelled myrosin grains (reticular system of protein vacuoles) inside the myrosin cells. Whole mount mRNA in situ hybridization verifies the histochemical localization To confirm the results of the TGG1 promoter specificity, whole mount mRNA in situ hybridization Ludevid et al. (1992) was performed to

605 localize TGG1 mRNA in A. thaliana. As seen from Figure 6, TGG1 mRNA was detected in guard cells of cotyledons from 2 days old seedlings (Figure 6a). The number of positive cells appears to be comparable to the number of GUS positive cells observed in the histochemical analyses of the plants expressing the GUS under control of the TGG1 promoter (Figure 2c and d). Quantitative GUS assay using MUG as substrate Quantification of GUS activity was done to analyze the tissue and developmental stage distribution of GUS activity in the transgenic B. napus plants expressing the pBIMyr1.Bn1-GUS construct. GUS activity could be measured in the

embryo starting just before 20 days after pollination (DAP) (Figure 7). This is in accordance with earlier reported slot blot analyses, myrosinase activity analyses and histochemical GUS analysis. Furthermore immunogold-EM shows that myrosin cells emerge at day 17–19 after pollination (Thangstad, O.P., unpublished). The myrosinase activity then increases during seed maturation and drop slightly at seed maturity. After germination, GUS activity is high in hypocotyl and cotyledon (5 days after seeding (DAS)) and then drops significantly. The observed decrease in GUS activity during development is also observed in root. The observed decrease in GUS activity seems to follow the degradation and disappearance of myrosin cells (Bones and Iversen, 1985; Bones et al., 1991).

Figure 5. Double immunofluorescence co-localization of GUS and myrosinase to idioblast myrosin cells from seeds of transgenic B. napus plants. Antibody immunolabelling of paraffin sections from transgenic B. napus mature seeds with the pBIMyr1.Bn1-GUS (a)–(c) and pBIMyr1.Bn1-SPGUS (d)–(f) constructs, and control plant (g)–(i). Left hand panels, anti-GUS FITC (green fluorescence); middle panels, anti-myrosinase Texas-Red (red fluorescence); right panels; merged composite picture. Genetic constructs are indicated below panels. Both antibodies recognize the same cell-type in the transgenic lines; myrosin cells. The myrosinase signal peptide targets the gene product to myrosin grains of myrosin cells. Notice the vacuole co-localization of Myrosinase and GUS using the pBIMyr1.Bn1-SPGUS construct, (d)–(f) showing the same subcellular localization, directed by the myrosinase gene specific signal-peptide. Bars: (a)–(f) ¼ 50 lm, (g)–(i) ¼ 200 lm.

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Figure 6. In situ hybridization analysis of TGG1 gene expression in rosette leaves of A. thaliana. A TGG1 sense probe was used in (b) and (c) and a TGG1 antisense probe in (a), (d) and (e). Plants were grown aseptically. Arrows indicate guard cells out of focus. The label associated with vascular tissue appeared both with sense and antisense probes as well as in control experiments. Bars ¼ 0.1 mm.

Figure 7. Activity of GUS in transgenic B. napus harbouring the construct pBIMyr1.Bn1-GUS (clone 1131). Expression of GUS high in seeds and tissues of seedlings and much lower in older plants. The distribution and decline in GUS activity with time after germination is following the same pattern as is earlier observed for the myrosinase activity (Bones, 1990). Expression of GUS activity was measured using a MUG assay. Control is non-transgenic B. napus cv. Westar. DAP ¼ days after pollination, DAS ¼ days after seeding. N ¼ 5–10, Lines ¼ SE.

Northern blot analysis of myrosinase expression Expression of TGG1 mRNA was analyzed in A. thaliana plants. Total RNA (20 lg) extracted from leaves, cauline leaves, bracts, inflorescence stems, flower buds of 4 week-old plants grown aseptically, was analyzed by Northern blotting (Figure 8a). TGG1 is highly expressed in young tissues of leaves (Figure 8a, upper part), bracts, and cauline leaves (Figure 8a, lower part). TGG1 gene transcription level is reduced to undetectable levels in older tissues (Figure 8a and b). TGG enzyme activity also declined with ages, but is still detectable in older tissues, although the transcripts are undetectable (data not shown). Northern blot analysis, using a TGG1 gene specific probe, was also used to determine the level of TGG1 transcripts in roots, dry seeds, and imbibed seeds. No TGG1 gene transcripts could be detected in these tissues (Figure 8b). Strong TGG1 expression could be detected in inflorescence (not shown), leaves and bracts, which were used as a positive control of TGG1 expression (Figure 8b). Expression of endogenous myrosinase(s) in A. thaliana L. Native polyacrylamide gel was used to analyze the endogenous expression of TGG (myrosinase) in

tissues of A. thaliana. Ten micrograms of total protein extract from 2-week-old pods, mature dry seeds, 1-day imbibed seeds, cotyledons of 5-dayold plants, leaves of 2-week-old plants, bracts, inflorescence stems, and flower buds from 3-weekold plants were analyzed. TGG activity was visualized with a BaSO4 zymogram method (see Bones and Slupphaug, 1989). TGG activity was found in pods, imbibed seeds, cotyledons, leaves, bracts, inflorescence stems, and flower buds (Figure 8c). No TGG activity was detected in total protein isolated from mature dry seeds.

Discussion Idioblast specific expression Guard cells can be considered to be idioblast of the epidermis. Although other crucifers also express myrosinase in guard cells, the major part of the activity is found in idioblast myrosin cells, in cortex cells of radicles and hypocotyls, and parenchymatic cells of cotyledons and leaves (Bones and Iversen, 1985; Thangstad et al., 1990, 1991; Bones et al., 1991; Ho¨glund et al., 1991, 1992; Lenman et al., 1993a; Xue et al., 1995; Kelly et al., 1998). In leaves

607 also reported the detection of myrosinase and myrosinase transcripts in cells associated with the vascular tissue. Similar to these observations we have observed GUS localization associated with the vascular tissues in leaf, inflorescence, stem, sepal, petal and gynoecium. No expression was observed in the root. Several genes have been reported to have a rather high degree of guard cell specific expression (summarized in Mu¨ller-Ro¨ber et al., 1998). Myrosinases do not easily fit into the current understanding of typical transcripts that participate in the regulation of the stomatal opening/closing. It might be more likely that myrosinase in guard cells is a part of either the defence system activated after destruction of the guard cells and/or is a part of a system generating volatiles from degradation of glucosinolates. TGG1 in A. thaliana is the first gene whose expression is localized in guard cells in every organ. Although current understanding suggest a defence related function for myrosinase, a regulatory role in stomatal regulation should be further explored. The TGG1 and Myr1.Bn1 promoters contain several putative DOF binding motifs. A recent analysis suggests that DOF zinc finger transcription factors controls guard-cell specific gene expression (Plesch et al., 2001). The guard cell and myrosin phloem idioblast specific expression of the TGG1 enzyme in A. thaliana may support a theory that the myrosinase-glucosinolate system is a chemical defence system acting at the site of pathogen invasion. It has been shown that downy mildew and other pathogens can enter plants through the stomatal pores (Mauch-Mani and Slusarenko, 1994). Comparison of the expressed sequence tags (ESTs) from B. campestris guard cells and ESTs from maize leaves indicate a high proportion of defence related genes in guard cells (
20%) compared to the leaves (
10%) (Kwak et al., 1997).

Figure 8. Analysis of myrosinase expression by northern blots and activity by detection of myrosinases after native gel electrophoresis. (a) Northern blots of A. thaliana using a TGG1 specific probe and a probe for an mRNA encoding ribosomal protein S15 as control for loading. Upper part: leaf RNA samples from 2, 3, 4, 5 and 6 weeks old plants, lower part: RNA samples from flower bud, bract, cauline leaf, and inflorescence stem. (b) Northern blot analysis of myrosinase expression in A. thaliana seeds and imbibed seeds using a TGG1 specific probe. RNA samples from 4 week old plants as positive control. (c) Zymogram visualization of myrosinase (TGG) activity after native protein gel electrophoresis separation of proteins from wild type A. thaliana. Plants were grown in greenhouse for 4 weeks and siliques collected 10 days after pollination. Also shown is samples from dry seeds and seeds imbibed for 1 h. Cotyledons were harvested from 5-day-old aseptically grown seedlings.

Embryo and seed myrosin cell specific expression

of older rapeseed plants, the enzyme was no longer localized to guard cells, but in mesophyll cells. This might indicate that TGG1 expression in guard cells of mature A. thaliana plants reflect an early development expression pattern of TGG genes, which in other Brassicaceae only are observed in young seedlings. Bones et al. (1991) and Xue et al. (1995)

The unique expression pattern from the Myr1.Bn1 promoter directing the Myr1.Bn1 expression to idioblast myrosin cells in maturing seeds and mature seeds show that the expression is strictly regulated. Whether this result indicate that the functional genes among the more than 25 myrosinase genes (Beisva˚g, Winge and Bones, unpublished) in B. napus are all developmental, age and

608 cell specific regulated is not yet clear. Present data indicate that at least members of the three subfamilies are differentially regulated (Lenman et al., 1993b; Xue et al., 1993). This may indicate individual regulation of all active myrosinase genes in specific cell types at various developmental stages. Further it might open for differential response to biotic and abiotic stimuli. There are some indications for differential expression. Myrosinase in B. napus has been immunocytochemical localized to phloem cells, guard cells, idioblasts in root cortex and parenchyma cells of cotyledon and leaf (Bones et al., 1991; Ho¨glund et al., 1991). Myrosinase has also been reported to be present in non-idioblast cells of Brassica juncea cotyledons (Kelly et al., 1998) and as cited in Bones and Rossiter (1996) it is evidence for a local induction of myrosinase after fungal infection. Based on the expression patterns observed for the TGG1 and Myr1.Bn1 promoter we expect that other myrosinase promoters may be developmental, organ and idioblast specific. The expression from the Myr1.Bn1 promoter in A. thaliana to guard cells is therefore interesting to note. Why is the promoter active in guard cells in A. thaliana and not in guard cells in B. napus? Myrosinase has also been detected in B. napus guard cells (Ho¨glund et al., 1991). The large number of myrosinase genes in B. napus compared to A. thaliana may have resulted in a more differentiated regulation of the individual gene expression in B. napus. Silencing of protein-coding genes in an allotetraploid species (Arabidopsis suecia) having redundant genes has been reported earlier (Lee and Chen, 2001). Thus one explanation could be that the pMyr1.Bn1-GUS constructs are epigenetically silenced in B. napus. In situ hybridization’s had shown preferential expression of MA myrosinase (Myr1) in subsets of myrosin cells (Lenman et al., 1993a). Using immunocytochemical labelling with antibodies against GUS and myrosinase combined with histochemical localization of GUS we were unable to detect myrosin cells without GUS expression in the transgenic plants. We therefore conclude that all myrosin cells in the B. napus embryo and seed are expressing the MA type myrosinase Myr1. Expression of myrosinase activity in A. thaliana The pattern of TGG activity in various tissues of A. thaliana is consistent with results from Xue

et al. (1995). The TGG1 promoter-GUS gene expression in transgenic A. thaliana, showing the guard cells as a major myrosin cell type in A. thaliana, is, however, in contrast to the in situ hybridization results reported by Xue et al. (1995), who found no TGG1 or TGG2 transcripts in epidermal tissue. Expression of myrosinase activity and GUS activity in B. napus The pattern of GUS activity in the transgenic plants is totally consistent with earlier reports on myrosinase activity in different organs and tissues (Bones, 1990), and mRNA in different tissues of B. napus (Falk et al., 1992). Using RT-PCR it could also be verified that expression of GUS starts at 20 DAP, continues to increase up to maturity, and drops significantly in all other tissues examined (data not shown). Function of the myrosinase-glucosinolate system – a minefield of defence cells The role of myrosinase in plant defence has long been postulated based on the toxic nature of the glucosinolate hydrolysis products, functioning as a binary chemical mine. Glucosinolates has been localized in, or in close proximity of myrosin cells/idioblasts. Feeding with radiolabelled desulpho glucosinolates, has shown a glucosinolate/ desulphoglucosinolate specific transport to cells that are localized in a pattern resembling myrosin cells (Thangstad et al., 2001). Using cell sap and EDX analysis Koroleva et al. (2000) has shown phloem flower stalk ‘S-cells’ to contain glucosinolates, and Husebye et al. (in press) have shown that myrosin cells are neighbours to ‘S-cells’. Recent cell ablation work has also indicated a role for myrosin cells in growth and development (Borgen, 2002). We propose that the A. thaliana myrosin guard cell and myrosin phloem idioblast specific expression of myrosinase represent a simpler version of the more complex system of myrosin cells in Brassica. The longer life cycle of B. napus compared to A. thaliana may explain the need to have a more sophisticated version of the myrosinaseglucosinolate system against pathogens and herbivores. The specialized idioblasts called myrosin cells in B. napus can be viewed as a ‘minefield’ of

609 cells located in ground tissues, where their encounter with pathogens can trigger the release of the volatile hydrolysis products of glucosinolates. Several mechanisms have been postulated (reviewed by Bones and Rossiter, 1996). A likely hypothesis includes a localized drop in cellular redox potential, via ascorbic acid and reduced glutathione, mediated by tissue damage. Oxidation of ascorbate then can free its inhibitory effect on myrosinase activity and trigger the hydrolysis of glucosinolates. The effect would be a local release of toxic hydrolysis products. A support to this view was recently shown by Vercammen et al. (2001), where volatile isothiocyanates were released upon Paraquat spraying of A. thaliana. A likely explanation being Paraquat induced production of reactive oxygen species. A novel thiol methyltransferase from Brassica oleracea, which methylates thiocyanates and isothiocyanates released by degradation of glucosinolates, has recently been found (Attieh et al., 2002), suggesting an endogenous, regulated availability of these compounds. The volatile sulphur components generated by the thiol methyltransferase activity have been suggested to have anti-insect and antipathogen roles (Attieh et al., 2002). Glucosinolate hydrolysis products could also mediate protection against endogenous oxidative damage from other stress factors, like drought and through the rapid germination and fatty acid oxidation in seedlings. Zhu et al. (2000) has shown direct radical scavenging and inhibition of oxidative DNA damage by sinigrin (2-propenylglucosinolate) in Brussels sprouts. A range of binding or associated proteins (myrosinase binding proteins, myrosinase-associated proteins and epithiospecifier proteins) is also involved in the controlled hydrolysis of glucosinolates (Foo et al., 2000; Lambrix et al., 2001; Eriksson et al., 2002). We believe that future studies using knock-out mutants and promoter constructs like these, will elucidate some of these questions, and provide a better understanding of this complex system.

Genbank accession numbers Genbank accession numbers for the sequence of the Myr1.Bn1 promoter is AF323020, and for the TGG1 promoter AF079308.

Acknowledgements This work was supported by grants from the biotechnology program of The Norwegian Research Council. SC was supported by Anunthamahidol Foundation and the research was supported in part by Chulalongkorn University Fund research grant.

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