Immunocytochemical localization of vindoline in mesophyll protoplasts of Catharanthus roseus

Phytochemistry,

0031-9422/92 $5 .00+0.00 CC} 1992 Pergamon Press plc

Vol . 31, No . 2, pp . 4 6 5 -470, 1992

Printed in Great Britain.

IMMUNOCYTOCHEMICAL LOCALIZATION OF VINDOLINE IN MESOPHYLL PROTOPLASTS OF CATHARANTHUS ROSEUS L. BRISSON, P . M . CHAREST, * V. DE LUCAt and R . K . IBRAHIM$§ Plant Biology Laboratory, Salk Institute for Biological Studies, San Diego, CA 92138, U .S.A ; *Department de Phytologie, FSAA, Universite Laval, Ste-Foy, Quebec, Canada G1K 7P4 ; tlnstitut de Recherche en Biologie Vegetale, Universite de Montreal, Quebec, Canada H1X 2B2 ; tPlant Biochemistry Laboratory, Department of Biology, Concordia University, 1455 De Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1MB (Received

Key Word Index

Catharanthus roseus;

13 May 1991)

Apocynaceae ; vindoline ; localization ; immunofluorescence ; immunogold

labelling ; cryofixation .

Abstract-Leaves of Catharanthus roseus synthesize the monoterpenoid indole alkaloid, vindoline, while its site of accumulation has not been identified . In order to study the intracellular localization of vindoline, antivindoline antisera were raised in rabbits and used with in situ immunofluorescence and immunogold labelling techniques . While labelling of isolated protoplast preparations showed an intense green fluorescence in the protoplast vacuoles, indicative of the location of vindoline labelled with fluorescein, the high fluorescence intensity did not allow for the recognition of other subcellular compartments . However, the use of cryotechniques coupled with immunogold labelling revealed that the label of vindoline was present in small vesicles and the cytoplasmic area bordering the plasmalemma . A substantial amount of label was also observed in the central vacuole and, to a much lesser extent, in the chloroplasts .

INTRODUCTION

The Madagascan periwinkle, Catharanthus roseus, accumulates the monoterpenoid alkaloid vindoline which is involved in the biosynthesis of the bis-indole alkaloids, vinblastine and vincristine [1] . The latter are well known for their antineoplastic activity [2] . Recent studies of Catharanthus seedlings have indicated that some key enzymes of vindoline biosynthesis are under strict developmental regulation (see [3] for review) . Furthermore, two later steps in vindoline biosynthesis, namely N-methylation and D-acetylation, are catalysed in intact tissues by chloroplastic and cytosolic enzymes, respectively [4] . It is reasonable to assume that the absence of vindoline in cultured cells [5, 6] may be due, in part, to the lack of specialized cells/tissues [7], or of developed chloroplasts that may be required for its biosynthesis and /or storage [8] . The subcellular compartmentation of plant secondary products is believed to play an important role in the regulation of their metabolism [9], although concrete evidence for such role has yet to be shown . To date, only a few reports have focused on the distribution of alkaloids in periwinkle tissues [10 and refs therein] . Radioimmunoassays have indicated that the highest concentration of vindoline occurred in fully mature, non-senescent leaves [11] . However, there have not been any studies of the localization of vindoline at the subcellular level . Despite the availability of antibodies to alkaloids that have been used in several immunoassays (see [12] for review), these antibodies have yet to be

§Author to whom correspondence should be addressed .

utilized for the immunolocalization of their antigens . This prompted us to study the localization of vindoline using immunocytochemical techniques, which are sensitive to and specific for this antigen, in conjunction with light and electron microscopy . In general, immunocytochemical localization of a hapten presents a number of technical problems, especially those related to leaching of the antigen during chemical fixation and dehydration of the material . Nevertheless, immunolocalization of molecules such as plant hormones [13], polymethylated flavonol glucosides [14, 15], polysaccharides [15, 17] and herbicides [18] has been achieved following fixation by either rapid freezing, or the use of an esterifying agent . The high solubility of vindoline presents a major difficulty in its localization when using standard protocols of tissue fixation and processing. However, the recent introduction of cryotechniques [19, 20] has allowed the retention of antigenicity and the preservation of cellular constituents, but not without compromise to ultrastructural details . This report describes the use of low temperature techniques coupled with immunocytochemistry for the localization of vindoline in C. roseus protoplasts with the aim of maintaining cellular integrity, antigenicity of the antibody and retention of the freely diffusible antigen .

RESULTS

Characteristics

of the

antiserum

A vindoline conjugate was synthesized by coupling the glutarate hemiester of the hapten to bovine serum gamma-globulin via EDC, and the resulting conjugate 465



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was purified as described by Westekemper et al. [11] . This conjugate was used to raise antisera in rabbits . The characteristics of the antiserum were found to be similar to those reported previously [11] . The maximum affinity -9 constant of the antibodies was 1 .35 x 10 moll - ', thus indicating the presence of high affinity antibodies in the serum. With an antibody dilution of 1 :150, the lowest detectable amount of vindoline was 2 .42 pmol . Specificity studies of the antiserum were performed using a radioimmunoassay [11] . Cross reactivity was not observed with alkaloids such as catharanthine, tabersonine, ajmalicine, serpentine or vinblastine . However, there was some cross reactivity observed (2 .3-10.5% of vindoline) with a few related alkaloids (Table 1). These results indicate the high specificity of the antiserum to vindoline, and are in agreement with those reported previously [11, 21] .

et al.

indicative of the location of vindoline labelled with fluorescein, was observed in protoplast vacuoles (Fig . 1C). Transient exposure of protoplasts to hypotonic shock was necessary for the permeabilization of cellular membranes, and allowed penetration of the bulky antibodies and the subsequent labelling of antigen in both the cytoplasmic and vacuolar compartments . However, the low resolution of light microscopy, coupled with the high fluorescence intensity, did not permit recognition of other subcellular compartments . Immunogold labelling

In order to optimize the ultrastructural features of vindoline containing cells, cryotechniques were applied to isolated protoplasts, instead of using bulky leaf tissue . Unlike leaf sections, the use of cryofixed protoplasts minimized the damage caused by ice-crystal formation [22] . Moreover, cryofixation of protoplasts was necessary to prevent the leaching of vindoline, while maintaining sufficient ultrastructural features to enable its immunodetection . In comparison with the controls (Table 2), immunogold labelling was observed mainly in the central vacuole and most vesicles within the cytoplasm (Fig . 2) . In addition, some labelling was found associated with, or in vicinity of the chloroplasts (Fig .2, Table 2) . It should be noted, however, that the vesicular

I mmunofluorescence

A preliminary screening of Catharanthus tissues which accumulate vindoline was carried out with cryostat, semithin sections of fresh leaf material after immunolabelling . These studies (results not shown) indicated that immunofluorescence labelling was detected mainly in the mesophyll tissue, Therefore, the immunolocalization of vindoline was investigated further using isolated mesophyll protoplast preparations . Evidence for the accumulation of vindoline in mesophyll cells was obtained by in situ immunolabelling of protoplast preparations (Fig . 1). As compared with the control serum (Fig. 1B), an intense green fluorescence,

Table 2. Morphometric analysis of vindoline labelling*

Cellular compartment Table 1 . Cross reactivity of antivindoline antibody with other related alkaloids*t

Central vacuole Chloroplasts Cytoplasm$ Vesicles Controls§

Relative activity (% of vindoline)

Alkaloid Vindoline Deacetylvindoline Dihydrovindoline Vindorisine

Number of determinations (n)t 3

No . of gold particles per µm2 +mean s.c. 38+26 8±4 107+24 79+38 0.6±0.2

10 5 22

10

100 *Determined from approximately 15 micrographs of immunolabelled material and 10 controls . tDetermined with intact cellular compartments selected from total number of micrographs . $Defined as the area lining the plasmalemma, and excluding the cellular organelles and vesicles . §Average value determined for all cellular compartments .

10.5 7.2 2.3

*Using the radioimmunoassay described in ref . [10]. tCross-reactivity was not observed with ajmalicine, catharanthine, serpentine, tabersonine, or vinblastine .

A

B

C

Fig. 1 . Immunofluorescence labelling of vindoline in C. roseus leaf protoplasts: A, mesophyll protoplast in light microscope (250) ; B, mesophyll protoplast incubated with control serum (200); C, mesophyll protoplast incubated with antivindoline antiserum (250) .

Mesophyll protoplasts of Catharanthus roseus

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Fig. 2. Immunogold labelling of vindoline in membrane vesicles (Ve), chloroplasts (Ch) and the central vacuole (Va) of C. roseus mesophyll protoplast . Note the vindoline labelling on the membranes of some vesicles (arrow heads) . Bar represents 0.2 µm .

membranes exhibited relatively low contrast, which may which survived cryofixation, morphometric analysis of be attributed to the lack of chemical fixation, the latter vindoline labelling (Table 2) is in agreement with the being incompatible with most immunocytochemical distribution of gold particles among the different subwork [22] . Despite the small number of protoplasts cellular compartments . Furthermore, of the protoplasts

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BRISSON

that survived cryofixation, only a few had intact central vacuoles, which explains the reason for the small number analysed (Table 2). It should be also noted that specific labelling of vindoline could not be observed in protoplasts that were fixed by conventional methods, despite the use of EDC for immobilization of the antigen and/or Os04 for membrane preservation . The lack of specific vindoline labelling in non-cryofixed tissue was attributed to the loss of antigen (> 95 % of that found in the intact protoplasts) during tissue processing. This was further confirmed by chromatographic analysis of the methanolic extracts of chemically-fixed protoplasts using standard TLC and HPLC procedures (data not shown) . DISCUSSION

This investigation made use of the high affinity of an antivindoline antibody and the specificity and sensitivity of immunocytochemical techniques to study the localization of vindoline in Catharanthus protoplasts . Although this antibody did not cross-react with the parent alkaloid, tabersonine (Table 1), it did, however, recognize a number of other related alkaloids in the pathway of vindoline biosynthesis [3], albeit to a small extent . However, the combined use of immunofluorescence and immunogold labelling techniques made it possible to recognize a number of subcellular compartments believed to be involved in the biosynthesis and accumulation of vindoline . In situ immunofluorescence labelling of protoplasts could only be achieved after permeabilization of cellular membranes by a transient exposure to hypotonic shock [23] . This, together with the high affinity of the antibody used, allowed for the localization of vindoline in the vacuoles of mesophyll protoplasts (Fig . 1 C). However, the low resolution of light microscopy and the qualitative nature of immunofluorescence technique did not permit the recognition of other fluorescent subcellular compartments. These results are in agreement with those of Mersey and Cutler [7] who demonstrated that protoplast vacuoles of C. roseus were enriched in vindoline and catharanthine, using both HPLC and epifluorescence techniques . Furthermore, several workers have reported the accumulation of various alkaloids, including vindoline, in the cellular vacuoles of intact and cultured tissues [10, 24, 25 and refs cited therein] . Cryotechniques have the dual advantage of excellent retention of antigenicity and high labelling density, in spite of the fact that cryofixed sections usually exhibit low contrast in the electron microscope [19, 20, 26] . Since the specific labelling of vindoline could not be accomplished with chemically fixed tissue, due to the loss of antigen during tissue processing, therefore, we resorted to the use of low-temperature techniques . Unlike glutaraldehyde and Os04 fixation, which involve dehydration at room temperature, the use of cryotechniques [19] prevented leaching of the antigen while maintaining sufficient ultrastructural features to allow vindoline localization . It should be stated, however, that the use of cryotechniques represents a compromise between membrane preservation and the retention of the antigen under investigation [26 and refs cited therein] . Because of their extensive volume, large vacuoles are more liable to break during cryofixation as compared with vesicles, and this may have contributed to the low recovery of intact protoplast vacuoles (Table 2) .

et al.

Morphometric analysis of the distribution of gold particles in cryofixed sections indicates that most of the gold label was associated with small vesicles (79 particles gm -2 ) and the cytoplasm (107 particles µm -2). The latter compartment may represent the vindoline pool prior to its packaging in vesicles, en route to the central vacuole . In addition, a small amount of gold particles was associated with the chloroplasts (Table 2) . Such label distribution may not be unexpected, since the N-methyltransferase and 0-acetyltransferase which catalyse the third and the final steps in vindoline biosynthesis, have been reported to be chloroplastic and cytosolic enzymes, respectively [4]. Therefore, the detection of gold particles in the cytoplasm and, to a lesser extent in the chloroplasts, suggests recognition by the antivindoline antibody of biosynthetic intermediates (e.g. deacetylvindoline, Table 1), which may be the substrates and/or products of the two latter enzymes . Furthermore, the association of relatively high amount of gold label with vesicles implies that the latter compartment may be involved in the packaging or shuttling of vindoline towards the central vacuole . However, not all the vesicles may-necessarily be implicated in these processes, since such an endomembrane system is known to be involved in the secretion of other metabolites, or of enzymes associated with detoxification or degradative processes [27]. Similar vesicular structures have been reported to take up and accumulate exogenous alkaloids in Papaver [28] . In addition, several workers have indicated the importance of vesicles in the transport of alkaloids from the site of biosynthesis to the site of accumulation [9 and refs cited therein] . Except for sanguinarine which has been shown to accumulate on the tonoplast of elicitor-treated Papaver cells [29], most alkaloids, including vindoline, have been reported to accumulate in the cellular vacuole [9, 10, 24, 25, 30] . However, in none of these studies has the exact site of alkaloid biosynthesis or metabolite accumulation been identified with certainity . The results presented here clearly demonstrate the complementarity and sensitivity of both immunofluorescence and immunogold labelling techniques in the localization studies of plant secondary metabolites, especially those present in low abundance . EXPERIMENTAL

Chemicals. Cellulase, driselase and macerozyme RIO were purchased from Caledon Labs . Ltd. (Ontario, Canada), Kyowa Hakko Kogyo Co . (Japan) and Yakult Pharmaceutical Industry Co . Ltd (Japan), respectively . Fluorescein-conjugated antiserum was from Cedar Lane (Ontario, Canada) . Low gelling agarose and goat anti-rabbit gold-conjugated (10 nm) secondary antibody were obtained from Sigma (St . Louis, MO). Preparation of antivindoline antibody. Desacetyl vindoline and glutaric anhydride were reacted in pyridine to produce the glutarate hemiester of vindoline . The latter was coupled to bovine serum gamma-globulin in the presence of (EDC)', as described by Westekemper et al. [11] . The conjugate was purified by dialysis against H2 0, before being used for immunization . Polyclonal antibodies against the vindoline conjugate were produced in New Zealand white rabbits following a previously described protocol [31] . The antisera were characterized by a radioimmunoassay as previously described [11, 21] . Plant material. Catharanthus roseus (L.) G. Don plants were raised from seeds under greenhouse conditions . Leaf protoplasts were prepared from 6-8-week-old plants .

Mesophyll protoplasts of Catharanthus roseus Protoplast preparation . Protoplasts were prepared according to [32] . Briefly, young Catharanthus leaves were deveined, immersed in an osmoticum (0 .7 M sorbital and 3 mM CaCl2 in 3 mM Mes buffer, pH 6.5), then sliced into strips of ca 0 .5 mm . The latter were incubated overnight in the osmotieum containing a mixture of 2% cellulase, 0 .5% driselase and 0 .25% macerozyme (w/v) . Protoplasts were seperated from undigested material by filtration through nylon cloth (300 mesh size) and purified by centrifugation . Immunofluorescence labelling . Indirect immunofluorescence labelling was performed with leaf protoplast suspensions . Protoplasts were fixed in 1 % glutaraldehyde (in the osmoticum) for I h, and the fixative was removed by 3 washes, each of 5 min, in Mes buffer containing 0 .35 M sorbitol and 0.15 mM CaCl2. A transient exposure of protoplasts to hypotonic shock was performed in order to permeabilize both the plasmalemma and tonoplast membranes to the antibodies [23] . Interfering charges in protoplasts were blocked with 1 % gelatin (in the osmoticum, w/v) for 15 min . Of the various dilutions of antisera and incubation times used, the following conditions were found optimal . Samples were incubated for 1 hr with the antivindoline antiserum diluted 1 :30 in PBS . Following antibody binding, the material was rinsed x 3 (in the osmoticum), each for 3 min, then incubated with goat anti-rabbit fluorescein isothiocyanate (FITC-conjugated IgG) that was diluted 1 :60 in PBS . Under these conditions, specific labelling with a low background level was achieved . Finally, after 3 more rinses in PBS and one in H2 O, the immunolabelled material was mounted in 50% glycerol containing 1 mg ml - ' of p-phenylenediamine, an antiquenching reagent [33] . The labelled material was observed in a Zeiss photomicroscope, equipped with epifluorescence and filter sets No. 9 for FITC and No . 17 to block the red fluorescence of chlorophyll . Kodak Tri-X pan 400 was used for photography . Immunogold labelling. Immunogold localization of vindoline was performed with a suspension of protoplasts which were prepared as described before . Protoplasts were prefixed in 1% paraformaldehyde, then embedded in 1 % agarose, both of which were diluted in the osmoticum [34] . Protoplasts were then cryoprotected in 20% glycerol (in the osmoticum) before being cryofixed in liquid propane at -185 " using a Reichert-Jung KF80 system (Vienna, Austria). They were transferred to liquid N 2, cryosubstituted (Reichert-Jung CFC Cryosubstitution System) in dry EtOH at -90" according to ref. [34], and finally embedded in Lowicryl K4M at -20 ' . Ultrathin sections (gold interference colour) were prepared and placed on 200-mesh nickel grids coated with Formvar . The grids were floated on a droplet of `Blotto' (5% nonfat dry milk in PBS, w/v) for 15 min . The sections were incubated for 1 hr with the primary antibody, diluted 1 :30 in PBS, and then rinsed x 3 each for 3 min, in the osmoticum . They were then incubated with goat anti-rabbit gold-conjugated (10 nm) secondary antibody, diluted to 1 :25 . Sections were stained with 2% aq . uranyl acetate, followed by lead citrate before being observed in an electron microscope . Specificity (?flabelling . The specificity of labelling was assessed by using the following controls : (a) an immune serum preabsorbed with its specific antigen, vindoline, (b) a non-immune serum, and (c) the secondary antibody alone . Morphometric analysis. The density of labelling in different cellular compartments was expressed as the number of gold particles per µm 2 of sectioned compartments . The area of the latter was estimated directly on the electron micrographs by the standard stereological technique [35] . Because of the small number of intact protoplasts recovered after cryofixation, morphometric analysis could only be carried out with a limited number of samples, which resulted in a relatively large standard error .

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Acknowledgements-This work was supported in part by a National Research Council of Canada contract (OISG-31964-610) from the Plant Biotechnology Institute, Saskatoon, as well as operating grants from the Natural Sciences and Engineering Research Council of Canada . We thank Dr E . Bleichert for careful reading of the manuscript, and Mr Alain Goulet for technical assistance .

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