Labelling was achieved using monoclonal antibodies (MAbs) produced against myrosinase. Myrosinase was extracted and purified from seeds of rape {Brassica ...
Journal of Experimental Botany, Vol. 42, No. 245, pp. 1541-1549, December 1991
Fate of Myrosin Cells: Characterization of Monoclonal Antibodies Against Myrosinase A . M . BONES', O. P. THANGSTAD 1 - 4 , O. A. HAUGEN 2 and T. ESPEVIK3 1 UNIGEN—Centre lor Molecular Biology, Department of Botany, University of Trondheim, Medisinsk Teknisk Senter, N-7005 Trondheim, Norway 2
Department of Pathology, University of Trondheim, Regionsykehuset, N-70O0 Trondheim, Norway
3
Institute for Cancer Research, University of Trondheim, Medisinsk Teknisk Senter, N- 7005 Trondheim, Norway
Received 21 March 1991; Accepted 27 June 1991
ABSTRACT Immunofluorescence labelling of myrosinase in paraffin sections was used to study the fate, appearance, and distribution of myrosin cells in Brassica napus after seeding. Labelling was achieved using monoclonal antibodies (MAbs) produced against myrosinase. Myrosinase was extracted and purified from seeds of rape {Brassica napus L.) to homogeneity. Mice were immunized with highly purified myrosinase and after fusion and cloning of the hybridoma cells, specificity was tested with highly purified myrosinase and the antibodies were characterized. One monoclonal antibody inhibited myrosinase activity. Precipitation of myrosinase activity was achieved with antibodies coupled to monosized magnetic polymer particles (immunomagnetic precipitation). Labelled cells were found in all organs and also in vascular tissue. A large proportion of the labelled cells were found in the marginal zone, both in radicles and hypocotyl, supporting the theory that myrosinase and its substrates, the glucosinolates, take part in the defence system of the plants. A description of the morphological development of myrosin cells during seedling growth is presented. Key words: |3-thioglucosidase, Brassica, monoclonal antibodies, myrosinase, myrosin cell, thioglucoside glucohydrolase.
INTRODUCTION Myrosinase (/3-thioglucosidase, thioglucoside glucohydrolase, E.C. 3.2.3.1) catalyses the hydrolysis of glucosinolates, a group of sulphur containing glycosides present in all Brassicaceae species examined. The glucosinolates are not deleterious themselves, but the cleavage products isothiocyanates, nitriles or thiocyanates (depending upon substrate and pH of the hydrolysis) can have undesirable effects in animal feedstuff's owing to their pungency and goitrogenic activity (Wilkinson, Rhodes, and Fenwick, 1984). Although a model for the compartmentation of glucosinolates and myrosinases was reported by Luthy and Matile (1984), little definitive evidence was available concerning how the glucosinolate-myrosinase system was held latent until the tissue was disrupted. Myrosinase has long been thought to be localized in myrosin cells (Bones and Iversen, 1985), and this has recently been shown to
be the case (Thangstad, Iversen, Slupphaug, and Bones, 1990; Thangstad, Evjen, and Bones, 1991). This study deals with the morphological changes and the appearance of immunofluorescence-labelled myrosin cells during seedling growth. The purification and characterization of myrosinase from seeds of Brassica napus and Sinapis alba are reported in two recent reports (Bones and Slupphaug, 1989; Bones and Thangstad, 1991). Brassica napus has been reported to contain at least four different isoenzymes of myrosinase (Lonnerdal and Janson, 1973). The glycoprotein myrosinase C from rape consists of three forms Ca, Cb, and Cc with varying carbohydrate content. The molecular mass of the enzyme is approximately 154 kD with two subunits and isoelectric points of 4-94, 4-96, and 500 for Ca, Cb, and Cc, respectively (Bones and Slupphaug, 1989). We describe here the generation, identification, and
* To whom correspondence should be addressed. Abbreviations: ELISA = Enzyme-linked immunosorbent assay; FITC = Fluorescein isothiocyanate; MAb = monoclonal antibody; PAGE-polyacrylamide gel electrophoresis; SDS = sodium dodecyl sulphate. © Oxford University Press 1991
1542
Bones et al.—Fate of Myrosin Cells
characterization of monoclonal antibodies against myrosinases present in Brasssica napus L. In addition we use the monoclonal antibody UNI288 as a cytochemical marker for a study of distribution and morphological changes of myrosin cells during seedling growth. MATERIALS AND METHODS Enzyme preparation Seeds of Brassica napus L. cv. Niklas (spring rape, Svalof, Sweden) were crushed in a coffee mill and the myrosinase extracted with distilled water. Myrosinase was purified as described by Bones and Slupphaug (1989). Briefly this method includes the following steps; affinity chromatogrpahy on a Con A-Sepharose (Pharmacia) column, ion-exchange chromatography on a DEAE-cellulsoe (DE-52, Whatman) column followed by fast protein liquid chromatography (FPLC) on a Mono Q HR 5/5 column. Rechromatography of the three active fractions obtained, was performed on the same column after dilution of the samples, but with a less steep gradient. Isoelectric focusing and PAGE were carried out with the PhastSystem (Pharmacia) on PhastGel IEF 4-6-5 and gradient 8-25, respectively. All gels were silver stained. Monoclonal antibody production Balb/c mice (6-8 weeks old) were immunized at day 1, 14, 90, and 92 with 9-44 ftg of the highly purified myrosinase obtained after individual rechromatography of each of the three collected peaks from thefirstFPLC separation. The first immunization was given with 50% Freund's complete adjuvant and the following immunizations with Freund's incomplete adjuvant (Difco), respectively. Three days after the last immunization, spleens were taken out and the spleen cells were fused with NSO myeloma cells (Clark et al., s.a. unpublished results cited in Galfre and Milstein, 1981) as described by Eshhar (1985). Following fusion the cell suspension was plated into microtitre plates (Costar) and grown in Dulbecco's modified Eagle's medium (DMEM, Gibco), supplemented with 10% fetal calf serum (Hyclone) and 100 ^M hypoxanthine, 16fiM thymidine, 0-4 fiM aminopterin (HAT, Sigma). After 14 d more than 600 hybridomas were obtained from the fusion of NSO myeloma cells with spleen cells from the two mice used for fusion. Hybridoma supernatants were removed and screened for antibodies against myrosinase in an ELISA assay as described below. The primary screening gave 113 positive hybridomas. The hybridomas which gave the highest reading in the ELISA assay were cloned by limiting dilution. Enzyme-linked immunosorbent assay of antibody binding After each of the following incubation steps, plates were washed three times with PBS (phosphate-buffered saline) supplemented with 0-05% Tween (PBS-T). All steps were performed at 37 °C and for 30 min when otherwise not stated. ELISAgrade, flat bottom plates (96 wells/plate, Nunc) were incubated overnight at 4°C with 100 mm3 per well of the myrosinase preparation diluted to 4-6 fig cm" 3 with PBS, washed, and incubated with 0-5% bovine serum albumin (RIA-grade, Sigma) in PBS. Each well was incubated with 100 mm3 of each serum dilution, supernatant or ascites, followed by incubation with 50 mm3 of Biotin-F(abr)2 fragment of rabbit x mouse Ig(G + A + M + H + L) (Zymed) diluted 1:2000 with PBS. Plates were thereafter washed and incubated with 50 mm3 streptavidinbiotin diluted in PBS (SABC-solution, Zymed) at room temper-
ature for 30 min, washed and 100 mm3 substrate solution consisting of 2-8 cm3 01 M citric acid, 2-2 cm3 0-2 M Na2HPO4, 5-0 cm3 distilled H2O, 100 mg ABTS (2,2-azinodi(3-ethylbenzthiazoline sulphonic acid)) and 5-0 mm3 H2O2 solution (30% H2O2), added. The reaction was stopped by adding 100 mm3 0-2 M citric acid to each well and results recorded using an automatic MicroELISA reader (Dynatech) at 410 or 490 nm. Alternatively OPD (1,2-phenylenediamine, dihydrochloride) was used as substrate and results recorded at 490 nm. The antibody class was determined with a monoAb-ID EIA kit A from Zymed, as described in their general procedure, using myrosinase as capture antigen or goat anti-mouse Ig(G + A + M) as capture antibody. In both cases visualization was performed with the alkaline phosphatase system. The production of ascites fluid for obtaining concentrated immunoglobulins was performed by injection of hybridoma cells (106) intraperitoneally into pristane-treated 43-d-old male syngeneic mice. After 14 d ascites fluid was removed by tapping. Inhibition of myrosinase activity Inhibition of myrosinase activity was determined by adding 25 or 50 mm3 hybridoma supernatant to a myrosinase test solution consisting of 29 mm3 0-05 M citrate buffer (pH 5-5), 15 mm3 sinigrin (15 mg cm" 3 ), 10 mm3 myrosinase solution and 140 mm3 GOD-Perid-reagent. Monoclonal antibodies from cell culture supernatants for the above analysis, were used after thorough dialysis against PBS to remove glucose which will interfere in the myrosinase assay based upon the glucose reagent. Test of specificity and cross reactivity Highly purified myrosinase derived from two repetitive runs on FPLC was used to determine the specificity of the generated monoclonal antibodies by ELISA and immunoblotting. Controls were run with fractions from the separation without myrosinase activity. The cross reactivity against bovine serum albumin (RIA-grade) and gelatine were determined by ELISA. Anti-myrosinase ascites from clone UNI288 were added to monosized magnetic polymer particles, Dynabeads M450 (Dynal, Norway), conjugated with 50fig sheep anti-mouse IgG per mg beads in PBS with 0 1 % BSA and incubated for 2 h. After washing in PBS/BSA the particles (009-60mg) were added to solutions of partially purified myrosinase. The solutions were incubated for 2 h at room temperature. The monosized, magnetic polymer particles were collected at the bottom of the tubes with a magnetic particle concentrator (Amersham). Supernatants were collected and myrosinase activity in the supernatant and precipitate containing the monobeads measured by the God-Perid myrosinase assay (Bones and Slupphaug, 1989). Controls were treated as the samples, with the exception that another mouse IgGl antibody was substituted for the anti-myrosinase antibody. Western blotting of myrosinase Proteins were concentrated by the method of Saul and Don (1984) prior to electrophoresis or isoelectric focusing. After electrophoresis of crude extracts, partially and highly purified myrosinase in gradient polyacrylamide gels (8-25%) or isoelectric focusing in polyacrylamide gels (pH 4-6-5), proteins were blotted to nitrocellulose membranes (0-45 /im, Bio-Rad) by diffusion blotting as described by Beisiegel (1986). Myrosinase was detected by specific monoclonal antibodies followed by rabbit anti-mouse Ig(G + A + M) (Dako P161) conjugated with peroxidase and visualized by diaminobenzidine.
Bones et al.—Fate of My rosin Cells Detection of myrosinase in polyacrylamide gels Myrosinase activity after PAGE was also detected by the barium sulphate assay as described by Bones and Slupphaug (1989). Immunocytochemistry Seeds of Brassica napus L. cv. Niklas were surface-sterilized in a 15% (v/v) sodium hypochlorite solution for 45 min, washed four times in sterilized water, and germinated in an environmental chamber under aseptic conditions on 1% (w/v) agar (Bacto-agar, Difco) containing 2% (w/v) sucrose and 500 mg CaCl2.2H2O. Seeds were imbibed for 4h and germinated from 1 to 14 d. Ethanol-fixation, paraffin embedding, sectioning, and immunocytochemical detection and immunoassay were performed as described by Thangstad et al. (1990). Micrographs were taken with a Nikon Microphot-FXA. RESULTS Isolation of hybridoma cell lines and antibody screening Hybridoma cultures were screened for the production of antibodies which bound to purified myrosinase as detected in an ELISA. The primary screening gave 113 positive hybridomas. In the ELISA test, non-immune mouse IgG gave low background readings. Thirty-four hybridomas with high score in the ELISA, were chosen for further expansion. Following cloning by limiting dilution, more than twenty cell lines were, obtained that were stable antibody producers. Four hybridomas (Unil78, Uni288, Uni388, Uni488) were chosen for expansion and passages into mice for ascites production. Immunoglobulin classes and subclasses were determined in an ELISA by using a capture antibody. In a separate assay highly purified myrosinase was used as capture antigen giving identical results. Using mouse subclass specific rabbit antisera from Zymed, MAbs were classified as IgM (85-3%), IgGl (5-9%) and IgG2b (8-8%). The four chosen hybridomas produced antibodies of IgGl (2), IgG2b (1) and IgM (1) class and subclass (Table 1). The MAbs were examined for cross reactivity with crude extracts and with susbtances used for blocking non-specific binding in ELISA and/or immunoblotting. None of the MAbs described here showed a significant reaction with bovine serum albumin or gelatine. However, they bound
1543
to crude extracts from seeds. This reactivity is due to myrosinase in the crude extract as shown in the immunoblotting, where only bands corresponding to myrosinase Ca, Cb, and Cc could be detected after isoelectric focusing and transfer to nitrocellulose membranes (Fig. 1). Immunodetection of myrosinase on Western blots The three myrosinase forms Ca, Cb, and Cc could not be resolved by SDS-PAGE, but they form distinct bands in isoelectric focusing (Fig. 1). The MAbs were tested for their ability to recognize intact or denatured and reduced myrosinase after PAGE or SDS-PAGE and Western blotting. A comparison of silver-stained polyacrylamide gels and nitrocellulose membranes developed by immunological detection of myrosinases is shown in Fig. 1. A protein band with molecular weight identical to that of purified myrosinase was detected from the crude extracts by the MAbs after PAGE (data not shown). The same band was also found when purified myrosinase was used in the immunoblotting. None of the 11 MAbs tested recognized myrosinase after reduction and denaturation. Immunoblotting after isoelectric focusing revealed bands corresponding to the three myrosinase forms Ca, Cb, and Cc (Fig. 1) which shows that the M Ab Uni288 detects the three forms of the major myrosinase in rape. Inhibition of myrosinase activity MAb Unil78 was found to inhibit the myrosinase activity by 50% when sinigrin was used as substrate
TABLE 1. Characterization of selected monoclonal antibodies against myrosinase from B. napus Antibody class, subclass, and reactivity against the three forms Ca, Cb, and Cc of myrosinase obtained after the fast protein liquid chromatography, expressed as mutual binding capacity ( + + + high, + + medium and + low binding capacity).
Clone
Unil78 Uni288 Uni388 Uni488
Class/ subclass
lgG2b IgGl IgGl IgM
Reactivity with myrosinase form Cc
Ca
Cb
r
::: H:
1 2
3
4 5
6
7
8 9
FIG. I. Isoelectric focusing of crude extract from seeds of B. napus (2), the three myrosinase forms obtained after FPLC separation (3-5) as compared to Western blots of crude extracts and purified myrosinase Cb and Cc with the MAb Uni288 (6-9). Pharmacia low pi calibration kit (I), crude extract from seeds (2), myrosinase Cabc (3), myrosinase Cb (4), myrosinase Cc (5), Western blots of crude extract (6), myrosinase Cabc (7), myrosinase Cb (8) and myrosinase Cc (9). Isoelectric points of markers are indicated. Proteins were transferred to nitrocellulose membranes by diffusion blotting.
1544
Bones et al.—Fate of Myrosin Cells
PLATE 1. Distribution and morphology of myrosin cells in radicles, roots and cotyledons of Brassica napus seedlings. (A-B) Cross-section of radicle imbibed for 4 h. (A) Immunofluorescence labelling of myrosinase. Most of the labelled cells in the cross-section are at the periphery of the radicle. (B) Phase contrast microscopy of the same region. (C-F) Immunoperoxidase-labeUed sections from root (C-D) and cotyledon (E-F). A-Bx 100, C x 240, D x 2000, E x 40, F x 240.
Bones et al.—Fate of My rosin Cells
1545
PLATE 2. Morphological changes of myrosin cells in roots 4 h (A-B) after imbibition to 14 d after seeding (F). (A, C, and E-G) Immunofluorescence labelling of myrosinase. (B-D) DIC micrographs showing the same region as shown on (A) and (C). Note that the spots detected by the antibody apparently are within the vacuoles (cf. A-D). A-D x 770, E x 390, F x 300, G x 345.
1546
Bones et al.—Fate of My rosin Cells
(Fig. 2). This result indicates that the antibody Unil78 binds to a site associated with the function of the enzyme. No significant effects on the enzyme activity was found when other MAbs were tested for inhibition of myrosinase activity. Precipitation of myrosinase with magnetic monodispersive polymer particles As shown in Fig. 3, approximately 95% of the myrosinase activity could be precipitated from a solution of partly purified myrosinase when the monosized magnetic polymer particles coated with the MAb Uni288 were used. The myrosinase activity in crude extracts from seeds
50
Supernatant (ul) FIG. 2. Inhibition of myrosinase activity by MAb from clone Unil78. The values are given as per cent of the activity in the control samples, which contained the same amounts of supernatant as the test samples, but from a non-inhibiting clone.
0,00
0,10
0,20
0,75
1,503,00 6 , 0 0
Monodispersive magnetic particles (mg) FIG. 3. Precipitation of myrosinase from solutions by magnetic monosized polymer particles coated with MAb Uni288. Activity in the supernatant. was measured by the GOD-Perid method. As shown precipitation of 95% of the myrosinase activity was obtained.
could also be precipitated (data not shown). The myrosinase bound to the washed, magnetic monosized polymer particles was active, and no loss of myrosinase activity was observed when measured in a myrosinase assay. No precipitation of myrosinase activity was obtained when monosized magnetic polymer particles coated with a nonreactive mouse IgGl were used as controls. Localization and distribution of my rosin cells As can be seen on fluorescence micrographs (Plate 1A) a specific localization of myrosinase to a limited number of cells is typical. These myrosin cells are different from the surrounding cells, especially when osberved in phase contrast or DIC (differential interference contrast) microscopy (Plate 2B and D) and show a distinct restricted fluorescence. A large proportion of the myrosin cells are located to the outer part of both cotyledon, hypocotyl, and root (Plate 1A-F). During seedling growth the fluorescence labelling decreases with age of the organs. As the cells differentiate and increase in size the label is found in the periphery of the cells (Plate2F). Positive labelled cells are also found in vascular tissue (Plate 3A-D), and the root hair zone (Plate 2E). Immunoperoxidase gives a less distinct labelling than immunofluorescence (Plate 1C-F). This is probably due to diffusion of the labelling produced. Myrosin cell development Myrosin cells appear in all parts of the radicle after 4 h of imbibition (Plate 1A-D). When the hypocotyl and root can be separated and the root hair zone appears, myrosin cells can be found in all parts except for the root tip. The development of myrosin cells in roots during early seedling growth are described in Plates 1A-D and 2A-E. As can be seen from these micrographs the internal structures undergo considerable changes as the plants germinate. After 4 h of imbibition uniformly labelled myrosin grains can normally be observed (Plate 1A). Three days after germination the appearance of the myrosin cells varies from cells containing spots labelled with antibodies (Plate 2C) to a more even but weaker labelling of the vacuoles. During days 4 to 8 after seeding, an emptying of the myrosinase from the myrosin cells was normally observed (Plate 2E-F). However, even after 10 d a few cells with the spotted labelling pattern were present. Fourteen days after seeding the myrosin cells in roots have expanded and the labelling can mainly be found in the periphery of the cells (Plate 2F). The small amount of myrosinase still present, was observed at the periphery of the cells. The number of myrosin cells in roots clearly declines during the first days of seedling growth. Example of a myrosin cell in hypocotyl is shown on Plate 2G. As for myrosin cells in roots some of the myrosin cells in hypocotyls appear elongated in the
Bones et al.—Fate of Myrosin Cells 1547 direction of the axis when compared to the other cortex cells (Plate 2G). In cotyledons a similar pattern of development of myrosin cells as in roots and hypocotyls can be observed (micrographs not shown). After 4 h of imbibition myrosin cells show a labelling corresponding to the typical myrosin grains found in myrosin cells. Two to more than twenty myrosin grains can be observed in each section of a cell. Labelling with FITC conjugated antibodies gives at some stages a denser labelling in the outermost part of the myrosin grains. In cotyledons there are homogeneous myrosin grains distributed in the cells in the beginning. Two days after seeding some of the myrosin grains appear less homogeneous and with larger vacoules. The spotted pattern of labelling was observed at a later developmental stage in cotyledons as compared to roots and hypocotyls. Eight days after seeding most myrosin cells in cotyledons appears with the spotted pattern. Twelve days after seeding the labelling appears as more evenly distributed small dots. In general, the decline in myrosinase detectable by the antibody was more rapid in roots and hypocotyls compared to cotyledons. It should also be pointed out that myrosinase also has been localized in cells associated with the vascular tissue (Plate 3A-D). Myrosinase containing cells associated with the vascular tissue were observed both in cotyledons, radicles, hypocotyls, and roots. These cells are most likely phloem companion cells. DISCUSSION The aim of this study was to develop a panel of MAbs which could serve as probes for immunological studies of myrosinase and to study the fate of myrosin cells during early seedling growth. We describe here the characterization of four monoclonal antibodies against myrosinase and their use as markers for a study of the myrosin cell distribution and development during seedling growth. Although myrosinase is generally found in species from the Brassicaceae (Bones and Iversen, 1985; Bones, Evjen, and Iversen, 1989; Bones, 1990), there are differences between myrosinases within one plant and between different species. This is clearly shown, for example, after isoelectric focusing in polyacrylamide gels (Henderson and McEwen, 1972). Given this background, it seemed possible that the monoclonal antibody technique could be used to examine further the characteristic features of myrosinases. Four out of eleven MAb tested, reacted positively with the myrosinase after electrophoresis or isoelectric focusing in polyacrylamide gels, and after Western blotting on a nitrocellulose membrane. The reaction with three close bands in the immunoblotting after isoelectric focusing (Fig. 1), was expected. As shown earlier these three forms of myrosinase have different carbohydrate contents, but
most likely identical amino acid sequences (Bones and Slupphaug, 1989). The MAb Unil78 causes an inhibition of the myrosinase activity and reacts similarly with the myrosinase forms Ca, Cb, and Cc. This indicates that binding of Unil78 interferes with a site associated with the function of myrosinase. In contrast, no significant effects were found to occur when other MAbs were tested for inhibition of myrosinase activity. Immunoprecipitation of antigens by coupling of MAbs to magnetic monosized polymer particles was used to show that the antibodies bound to myrosinase. A similar strategy was very recently reported by Worlock, Sidgwick, Horsburgh, and Bell (1991). Worlock et al. (1991) used paramagnetic beads for precipitation and detection of major histocompatibility complex class I and class II antigens. No reduction of myrosinase activity was observed when the enzyme was bound to the MAb Uni288 on these particles. This shows that the MAb Uni288 used binds to a myrosinase epitope not associated with the active site of the enzyme. One advantage of using magnetic monosized polymer particles is that no centrifugation is necessary, therefore saving time and equipment. Isoelectric focusing combined with densitometry is the most frequently used method for determining the distribution of myrosinases in extracts. This method provides adequate resolution of some of the enzyme forms but can not be regarded as more than semi-qualitative and -quantitative, because of possible differences in substrate affinity of the different forms (isoenzymes) and possible lack of resolution. At least four myrosinases have been reported in seeds of rape (Henderson and McEwen, 1972; Lonnerdal and Janson, 1973). The MAbs Uni288 and Uni388 reacted with three myrosinase forms (Ca, Cb, and Cc) after isoelectric focusing and after Western blotting on a nitrocellulose membrane. Results in this paper shows that the morphology of myrosin cells changes dramatically during seedling growth. By employing the monoclonal antibody Uni288 a specific labelling of protein bodies in myrosin cells was obtained. Earlier studies of myrosin cells and their development have relied on general staining techniques (Bones and Iversen, 1985). Due to a dilution of the content of the myrosin grains and unspecific stains it has only been possible to follow the development of the myrosin cells during early seedling growth (Bones and Iversen, 1985). Immunocytochemical techniques using antibodies with high affinity are much better suited for this type of investigation. As shown in the present paper the fate of myrosin cells seems to follow a similar pattern in all organs, although at a different time after sowing in cotyledons, hypocotyl, and root. This development seems to include fission followed by formation of small myrosinase-containing grains. The spotted pattern obtained at certain developmental stages seems to reflect both that
1548
Bones et al.—Fate of Myrosin Cells
PLATE 3. Visualization of myrosinase containing cells in vascular tissue. (A-B) Positive labelled cells in seeds imbibed for 4 h. (C) Differential interference contrast micrograph of a myrosinase containing cell in hypocotyl 4 d after seeding. (D) Immunofluorescence labelling of the same section. A x 410, B x 230, C-D x 775.
Bones et al.—Fate of Myrosin Cells 1549 the myrosinase is not uniformly distributed in the vacuoles formed as a result of the extensive fusion processes and by the formation of small vesicles containing myrosinase. At later stages the myrosinase can be observed at the periphery of the vacuole (cf. Plate 2F). Details in the development of myrosin cells at the subcellular level are reported by Bones and Iversen (1985) and Werker and Vaughan (1974, 1976). In general, the labelling of myrosinase in the myrosin cells decreased during the investigated period of seedling growth. This supports our earlier reported results which showed that specific myrosinase activity declined during seedling growth (Bones, 1990). Myrosin cells are not restricted to the cortex and parenchyma tissue. As shown in Plate 3A-D they can also be found associated to vascular tissue. Similar results were reported by Werker and Vaughan (1976). They observed phloem companion cells with a density similar to myrosin cells in the cortex. The localization of myrosinase to vascular tissue and the outermost cell layers of the organs support earlier reports which suggest that the myrosinase-glucosinolate system has a role in the defence system against micro-organisms. However, the large amount of substrates, myrosinases, and cleavage products reported may indicate that this system has more than one function in the plant. In addition to the defence function, nutrition storage and a potential role as a supplier of hormone precursors are some of the most obvious possibilities. A library of MAbs against different forms of the enzyme should make it possible to make a more detailed study of the enzymes for, e.g. determination of conserved regions, catalytic sites, and occurrence of different forms at different developmental stages. Furthermore, the antibodies may also be used to study the details during the formation of the myrosin cells in maturing seeds.
intact plants, cell and tissue cultures and regenerant plants of Brassica napus L. Journal of Experimental Botany, 41, 737^44. EVJEN, K., and IVERSEN, T. H., 1989. Characterization and
distribution of dilated cisternae of the endoplasmic reticulum in intact plants, protoplasts and microcalli of Brassicaceae. Israel Journal of Botany, 38, 177-92. and IVERSEN, T.-H., 1985. Myrosin cells and myrosinase. Ibid. 34, 351-76. and SLUPPHAUG, G., 1989. Purification, characterization and partial amino acid sequencing of 0-thioglucosidase from Brassica napus L. Journal of Plant Physiology, 134, 722-9. and THANGSTAD, O. P., 1991. Preparative purification of myrosinase from Sinapis alba L.-characterization of polyclonal antibodies against myrosinase. Proceedings of the 8th International Rapeseed Congress, Saskatoon, Canada, (in press). ESHHAR, Z., 1985. Monoclonal antibody strategy and techniques. In Hybridoma technology in the biosciences and medicine. Ed. T. Springer. Plenum Press, New York. Pp. 3—41. GALFRE, G., and MILSTEIN, C , 1981. Preparation of monoclonal
antibodies: strategies and procedures. Methods in Enzymology, 73, 3-46. HENDERSON, H. M., and MCEWEN, T. J., 1972. Effect of ascorbic
acid on thioglucosidases from different Crucifers. Phytochemistry, 11, 3127-33. LONNERDAL, B., and JANSON, J.-C, 1973. Studies on myrosinase.
II. Purification and characterization of a myrosinase from rapeseed {Brassica napus L). Biochimica et biophysica acta, 315, 421-9. LUTHY, B., and MATILE, PH., 1984. The mustard oil bomb:
Rectified analysis of the subcellular organization of the myrosinase system. Biochemie und Physiologic der Pflanzen, 179, 5-12. SAUL, A., and DON, M., 1984. A rapid method of concentrating proteins in small volumes with high recovery using Sephadex G-25. Analytical Biochemistry, 138, 451-3. THANGSTAD, O. P., EVJEN, K.., and BONES, A. M., 1991. Immuno-
gold-EM localization of myrosinase in Brassicaceae. Protoplasma, 161, 85-93. IVERSEN, T. H., SLUPPHAUG, G., and BONES, A., 1990.
Immunocytochemical localization of myrosinase in Brassica napus L. Planta, 180, 245-8. WERKER,
ACKNOWLEDGEMENTS Finanical support from the Norwegian Research Council for Science and the Humanities (NAVF) and the Norwegian Agricultural Research Council (NLVF) is gratefully acknowledged. LITERATURE CITED BEISIEGEL, U., 1986. Protein blotting. Electrophoresis, 7, 1-18. BONES, A., 1990. Distribution of /J-thioglucosidase activity in
E., and VAUGHAN, J. G.,
1974. Anatomical
and
ultrastructural changes in aleurone and myrosin cells of Sinapis alba during germination. Ibid. 116, 243-55. 1976. Ontogeny and distribution of myrosin cells in the shoot of Sinapis alba L. A light and electron microscope study. Israel Journal of Botany, 25, 140-51. WILKINSON, A. P., RHODES, M. J. C. and FENWICK, R. G., 1984.
Myrosinase activity in Cruciferous vegetables. Journal of the Science of Food and Agriculture, 35, 543-52. WORLOCK, A. J., SIDGWICK, A., HORSBURGH, T., and BELL,
P. R. F., 1991. The use of paramagnetic beads for the detection of major histocompatibility complex class I and class II antigens. Bio techniques, 10, 310-15.
