LOCALIZATION OF LECTINS IN LEGUME COTYLEDONSMethyl a-D-glucopyranoside, a-D-galactose, and...

J. Cell Sci. 19, 157-167 (i975) 157Printed in Great Britain

LOCALIZATION OF LECTINS IN LEGUME

COTYLEDONS

A. E. CLARKE AND R. B. KNOXSchool of Botany, University of Melbourne

AND M. A. JERMYNCSIRO Division of Protein Chemistry, Parkville 3052 Victoria, Australia

SUMMARYHigh-resolution techniques for the localization of lectins are described. Concanavalin A

(Con A) and phytohaemagglutinin (PHA) are localized using a fluorescent method with(FITC)-labelled immunoglobulins which bind to the lectins in sections of jack and red kidneybean cotyledons. Specificity is defined by the use of specific sugar inhibitors. Both Con A andPHA are found in cytoplasmic sites.

Lectins with /?-glycoside specificity are detected with red-coloured artificial carbohydrateantigens. The /?-galactosyl and /9-glucosyl antigens bind specifically to clustera of sphericalbodies in the intercellular spaces, to cell wall sites, and to the periphery of the cytoplasmassociated with the cell membrane.

INTRODUCTION

Plant lectins are soluble proteins or glycoproteins which can be extracted fromplants and which interact in a variety of ways with mammalian cells by virtue of theirproperty of specific binding to carbohydrates. The configuration of the carbohydratemoiety required for binding to a lectin has been well defined for a number of lectins(see Lis & Sharon, 1973); and most lectins have multiple carbohydrate-binding sites.A consequence of these properties is that lectins may precipitate glycoproteins andpolysaccharides containing accessible sugar sequences which fulfil their specificityrequirements. Also, lectins may interact with the carbohydrates of mammalian cellplasma membranes, and with isolated plant protoplasts (Glimelius, Wallin & Eriksson,1974); this may result in agglutination of the cells, or in certain cases, to changes inthe growth pattern of the cells. All these effects can be inhibited by the simple sugarsfor which the particular lectin is specific.

The best known lectin, Concanavalin A (Con A), comprises up to 3 % of the totalprotein of the jack bean (Canavalia ensiformis): its sugar-binding requirements areknown in detail, thus it will bind a-D-mannopyranose, a-D-glucopyranose and steri-cally related structures (So & Goldstein, 1967). The lectin from red kidney bean isknown as phytohaemagglutinin (PHA) because of its very potent agglutinating effectson red blood cells. It is also a powerful mitogen and both these effects can be inhibitedby high concentrations of iV-acetyl-D-galactosamine.

Recently, Jermyn (1974) and Jermyn & Yeow (1975) have described a further classof lectins of widespread occurrence in plants, the 'all-/?' lectins. These substances are

158 A. E. Clarke, R. B. Knox and M. A. Jermyn

glycoproteins which interact with the /?-glycosyl determinants of artificial carbo-hydrate antigens (Yariv, Rapport & Graf, 1962). These antigens are prepared bycoupling diazotized 4-amino phenyl glycosides with phloroglucinol. The resultingproducts are highly aggregated and provide a red-coloured, high mol. wt. moleculewith multiple carbohydrate-determinant groups of a clearly defined configuration.

Despite the prominence of plant lectins such as Con A in contemporary cell biology,little is known of the role of these lectins in plants. A number of suggestions have beenput forward, and these have been summarized by Lis & Sharon (1973). Lectins mayact as plant antibodies produced in response to soil bacteria, or as agents of plantprotection from microbial attack through the inhibition of fungal cell-wall-hydrolysingenzymes. Alternatively, they may be involved in sugar transport and storage, or evenin the binding of glycoprotein enzymes into batteries of multienzyme systems. Themitogens may act as controlling agents of cell differentiation and division, particularlyat germination. In approaching this problem, we have suggested that certain lectinsmay well be involved in the determination of self-recognition between plant cells(Knox, 1975). In order to fulfil such a role, lectins would presumably have to belocated at the interface between cells. We have accordingly developed high-resolutionmethods to locate lectins in plant cells precisely; this may provide an insight into theirpossible functions.

MATERIALS AND METHODS

Source of seeds

Jack beans, Canavalia ensiformis, were obtained from the Genetic Resources section ofCSIRO Division of Plant Industry (CPI No. 59505). Red kidney beans, Phaseolus vulgaris(CPI No. W245), were obtained from Dr R. Biggs, CSIRO Division of Plant Industry; pigeonpea, Cajanus cajan, from Watters & Sons, Melbourne, and lima beans, Phaseolus lunatus cv.Early Thorogreen from CSIRO Division of Plant Industry (CPI No. N934).

Seeds were soaked between 2 layers of moist cotton wool for 24-48 h, at room temperature,before sectioning.

Reagents

Methyl a-D-glucopyranoside, a-D-galactose, and iV-acetyl-a-D-galactosamine were fromCalbiochem, San Diego, Calif., U.S.A. Salicin was from Sigma Chemical Co., St Louis,Mo., U.S.A. Dimethyl sulphoxide was from Unilab, Sydney, Australia. The fluorescein iso-thiocyanate (FITC)-labelled globulin preparation used was from Behringwerke, Marburg-Lahn,Germany (goat anti-rabbit IgG, OTKF 04/05).

Localization of Con A and PHA

Both these lectins will precipitate serum glycoproteins in Ouchterlony tests. This has pre-vented us from using high-titre antiserum raised in rabbits against the lectins for immuno-fluorescence localization, since the binding of normal serum components to the lectins obscuresany specific antibody binding. However, we have developed a fluorescence technique for thelocalization of these lectins which relies on binding of the lectins to serum glycoproteins andinhibition of this binding by simple sugars to define staining specificity. We have used a FITC-labelled globulin preparation which was shown to interact with both Con A and PHAin Ouchterlony gel diffusion tests. These interactions were abolished by the presence ofmethyl a-D-glucoside and JV-acetyl-D-galactosamine respectively, the sugars which fulfil the

Lectin localization in cotyledons 159

lectin-binding requirements, but were not affected by the presence of the sugars galactose ora-methyl glucose respectively, which do not fulfil the lectin-binding requirements.

Freehand razorblade sections of jack or red kidney bean were placed in the FITC-labelledglobulin preparation diluted 1:2 or 1:10 with phosphate-buffered saline (0-15 M NaCl, 005 Mphosphate buffer pH 72), for 20 min at room temperature, rinsed in saline for 10 min, andmounted in saline for fluorescence microscopy. As controls of specificity, sections were pre-treated with specific sugars. Jack bean sections were treated for 10 min with 10%, w/v,methyl a-D-glucoside or galactose, red kidney bean sections with 1 %, w/v, A^-acetyl galactos-amine or 10 %, w/v, glucose. Other sections were pretreated in saline in which the sugars wereprepared. Sections were then exposed to the labelled IgG containing the same sugar.

Fluorescence-microscope observations were carried out using reflected light in the blueregion, with a Carl Zeiss reflected-light incident fluorescent illuminator.

Azo-dye technique for localization of (3-lectins

Freehand razorblade sections of the various bean seeds were placed in solutions of artificialcarbohydrate antigens, prepared as described by Jermyn & Yeow (1975) either fresh or afterfixation for 4 or 12 h in 2-5 % glutaraldehyde in phosphate-buffered saline at 4 °C. The fixedmaterial was extensively washed in saline at 4 CC before staining. For these experiments, the/7-glucosyl, /?-galactosyl, a-galactosyl and a-mannosyl derivatives were used. Stock solutions(10 mg/ml) in dimethyl sulphoxide (DMSO) were prepared and diluted 1:10 with phosphate-buffered saline just before use. The final solution contained antigen (1 mg/ml) in 10 % DMSO.Sections were stained for 30 min at room temperature, washed in saline for 10 min, andviewed by bright-field microscopy. As controls of staining specificity, 2 methods were adopted.First, parallel sections were treated with artificial antigens of a-glycosyl as well as /?-glycosylspecificity. Secondly, the effect of a simple /?-glycoside, salicin, on the reaction was examined.Sections of jack bean were placed in a salicin solution (25 % in saline) for 15 min before reactionwith the /9-glucosyl antigen in the presence of salicin (2-5%).

OBSERVATIONS

Con A has proved to be cytoplasmic in its cellular localization in 24-h-soaked jackbean cotyledons (Fig. IA). It occurs throughout the cotyledons, including the epi-dermal cells which are frequently heavily stained. In the cotyledon parenchyma, thefluorescence is associated with the protein bodies and also the outer surface of thestarch grains (Fig. IB). Occasionally, some staining has been found in the cell wallsand intercellular spaces, but usually these sites are negative. Binding occurs to thecell walls at the sites of pit fields on the inner faces of the parenchyma cell walls, butsuch binding also occurs in the control sections incubated with a-methyl glucose.Pretreatment with saline alone or with galactose had no effect on staining, but it wasalmost completely abolished by methyl a-D-glucoside (Fig. 1 c).

PHA localization in red kidney beans has proved to be similar to Con A, in thatspecific fluorescence is entirely associated with cytoplasmic sites, confirming theobservations of Mialonier et al. (1973) using immunoperoxidase methods. JV-acetylgalactosamine pretreatment abolished staining, while pretreatment with a-methylglucoside had no effect.


Localization of fl-lectins

Jackbean. Both y?-galactosyl and /?-glucosyl artificial antigens gave similar results,and intense deposits of orange-red reaction product were obtained in the intercellularspaces of the cotyledon parenchyma and at the periphery of the cytoplasm associatedwith the cell membrane (Fig. 2). No differences in staining patterns could be detectedbetween the fresh and the glutaraldehyde-fixed sections, after either 4 or 12 hfixation. At higher magnification, the intercellular spaces were seen to be filled withclusters of spherical bodies, varying in size up to a maximum of 2-3 /(tn (Fig. 2c).The reaction was also apparent in the cell walls, especially in the central middlelamella zone. Treatment of the cotyledon sections with 2-5% salicin in 0-15 M NaClfor 15 min prior to staining with the Yariv antigen, abolished all staining at the inter-cellular spaces, but did not inhibit the binding at the cell walls (Fig. 3). In a controlexperiment, pretreatment of the sections in the saline did not affect staining either atthe intercellular spaces or at the cell walls.

Red kidney bean. Antigen binding in the intercellular spaces was generally foundonly in the outer parenchyma. Most binding was associated with the greatly thickenedand fluted cell walls of the parenchyma cell (Fig. 7). Binding of antigen to sitesassociated with the circular pit field on the inner wall face were also present in controls.

Lima bean. Some binding of antigen was evident in the intercellular spaces, butthe most prominent was associated with the fluted margins of the cell walls adjacentto the cell membrane (Fig. 8).

Pigeon pea. Similar patterns were obtained, though the intercellular spaces weresmaller and the reaction product less easy to detect (Fig. 9). Reaction product was alsoprominent, apparently in the inner layer of the parenchyma cell walls adjacent to thecytoplasm.

Controls of staining specificity

For each tissue, control sections were prepared using both a-galactosyl anda-mannosyl artificial antigens. The a-galactosyl antigen gave no staining in the cyto-plasm or intercellular spaces (Fig. 4) of any of the beans tested except red kidneybean, which showed faint binding to cytoplasmic sites. This is due to binding toPHA which will precipitate a-galactosyl antigen in gel diffusion tests. This confirmsthe cytoplasmic localization defined by the fluorescent method. Some binding to thepit fields was observed, especially in jack bean. This is interpreted as non-specificstaining, since toluidine blue rapidly stains these sites a red-purple colour, indicativeof the presence of phenolic complexes (Feder & O'Brien, 1968). The a-mannosylantigen bound strongly to the cytoplasmic sites of jack bean sections (Fig. 5), but notdetectably to any of the other bean sections. Con A binds preferentially to a-D-mannose, so such a result confirms the localization of Con A obtained with thefluorescent method. No reaction product was associated with the cell walls or inter-cellular spaces, so that results were almost the converse of those with the /?-glycosylantigens.

The patterns obtained in these various tests is characteristic of total proteins within


the tissue, as seen by staining patterns following incubation in ponceau 2R (Fig. 6)using the method of Flint & Moss (1970). The cytoplasm is intensely stained, withconspicuous staining of material in the intercellular spaces. Similar fresh sections,exposed to the periodic acid-Schiff method for carbohydrate localization, showedstaining in the cell walls and starch grains.

DISCUSSION

Cellular sites of Con A and PHA

Con A localization in plant cells has not previously been reported. The fluorescentmethod for Con A and PHA localization provides a very sensitive tool that is inde-pendent of non-specific binding problems. Even with very high-titre antisera, wehave found it difficult to interpret normal serum control slides in immunofluorescenceprocedures because of the high level of binding shown by normal serum to both theselectins. The method described in this paper is likely to have wide applications inlocating lectin activity during differentiation, and in tissues other than seeds.

Using immunoperoxidase methods, Mialonier et al. (1973) detected PHA in thecytoplasm of Phaseolus cotyledon parenchyma, with a suggestion also of some activityassociated with the cell walls. Our experiments confirm this predominantly cytoplasmiclocalization. The fluorescent method has the additional advantage of greater sensitivityand specificity, since peroxidases occur widely in plants and would make the inter-pretation of controls more difficult. Peroxidase, itself a glycoprotein, binds readily toCon A, and this reaction has been used to locate Con A binding to mammalian cellsurfaces using enzyme cytochemical techniques to reveal the peroxidase (Bernhard &Avrameas, 1971). However, both in the thick sections used in this study, and on cellsurfaces, reflected-light fluorescence microscopy has advantages, since the excitinglight does not pass through the tissue. The fluorescence procedure is applicable to awide range of plant tissues, where the specific fluorescence is clearly differentiatedfrom background autofluorescence. Both Con A and PHA appear, on the presentevidence and that of Mialonier et al. (1973), to be located at cytoplasmic sites. Thispresumably means that their functions are likely to involve cytoplasmic components.It is perhaps significant that Con A is present at greatest activity in developing jackbean cotyledons at the peak of synthesis of storage protein, possibly suggesting somerole in the laying down of storage materials. However, it is also present and active ingerminating seeds, so a role in the utilization of storage products is also possible.

Extracellular location of fi-lectins

A novel feature of this work has been the discovery that lectins with /?-glycosidespecificity are located in the intercellular spaces, cell walls, and peripheral cytoplasmof cotyledon parenchyma cells. It is highly unlikely that this localization has beeninfluenced by diffusion artifacts, particularly since toluidine-blue staining has shownthat phenolic materials are absent from these particular sites. The possibility that theobserved extracellular location of the /Mectin is due to non-specific binding or pre-cipitation of lectin which might have been solubilized in the staining solution is

I I CEL 19


excluded, since fixation under conditions which would immobilize the lectin does notalter the staining pattern.

Opik (1966) investigated the fine structure of germinating Phaseolus cotyledons, andobserved electron-lucent vesicles frequently filling the intercellular spaces. The sizesof these vesicles correspond well to the clusters of bodies staining for /Mectin activityin jack bean and red kidney bean. The nature of these spherical structures needs to befully investigated cytochemically during seed development, and the jack bean wouldbe ideal material. A question of particular interest is whether these extracellularmaterials originate from the cells surrounding the intercellular channels, or whetherthey may be products of special secretory cells located elsewhere in the cotyledon.This is of some importance, since Yomo & Taylor (1973) have shown that proteasescan move through the intercellular spaces of Phaseolus cotyledons during germinationto bring about hydrolysis of the storage proteins. They assume that later the productsof hydrolysis - the amino acids and sugars - will themselves be transported throughthese intercellular channels to the developing seedling apex. It is of some importanceto establish whether /Mectins occupy similar sites during germination, and work isin progress to determine the fate of lectins during cotyledon development in both jackbean and red kidney bean.

Role of lectins in plant cells

The present results indicate that lectins with quite different substrate specificity,and presumably function, occupy different cellular sites in legume cotyledons. Thebiologically active lectins, Con A and PHA, apparently occur exclusively in cyto-plasmic sites. In contrast, the /?-Iectins, defined on their ability to precipitate the Yarivartificial /?-glycosyl antigens, are associated with the cell membrane and concentratedin the cell walls and intercellular spaces. We suggested earlier that some lectins mayfunction as self-recognition factors in plant cells, determining the relationshipsbetween vegetative cells in tissues and organs. To function in this way, they pre-sumably need to be located in extracellular sites in the cell walls, or at the cell mem-brane. This has been found to be the case for the pollen-wall proteins which functionin recognition reactions on the female stigma (see reviews by Knox, Heslop-Harrison &Helsop-Harrison, 1975; Heslop-Harrison, Knox, Heslop-Harrison & Mattsson, 1975).The /?-lectins certainly appear to be strategically sited to fulfil some role in cellcommunication in cotyledon cells. Jermyn & Yeow (1975) have demonstrated theirwidespread occurrence throughout the plant kingdom, and in various tissues andorgans of individual plants. This suggests such a communication role need not beconfined to cotyledons.

Several other lectins are not exclusively located in seeds (see Lis & Sharon, 1973).Mialonier et al. (1973) record that an immunologically related form of PHA occursin the young shoots of Phaseolus vulgaris. Pokeweed mitogen occurs in leaves, stemsand roots of Phytolacca americana, sometimes in even higher concentrations than inthe seed (Fames, Barker, Brownhill & Fanger, 1964). Hamblin & Kent (1973) havepreliminary evidence for the presence of PHA on young root primordia acting asbinding sites for symbiotic bacteria. SBA has recently been shown to have a similar


function in soy beans (Bohlool & Schmidt, 1974). Pseudomonas and other leaf-infectingmicro-organisms may enter the host leaf via the intercellular spaces, and suscepti-bility or resistance - a recognition event - is determined from these sites (Nelson &Dickey, 1970). The/?-lectins so far examined comprise about 80% carbohydrate and20% protein, so it is not inconceivable that they could carry determinants controllingboth self-recognition and disease resistance. As Burnet (1971) pointed out in relationto the human immune system, it is likely that defence mechanisms have evolved assecondary elaborations to a basic system evolved for self-recognition. The presentobservations of the extracellular localization of certain lectins are consistent withsuch a role.

This work is supported by a grant to R.B.K. from the Australian Research Grants Com-mittee, and was carried out during the tenure by A.E.C. of a University of Melbourne ResearchFellowship. We thank Professors J. Heslop-Harrison and B. A. Stone for helpful comments onthe manuscript, and Miss Eve Muchnicky, B.Sc, for her helpful assistance.

REFERENCESBERNHARD, W. & AVRAMEAS, S. (1971). Ultrastructural visualization of cellular carbohydrate

components by means of concanavalin A. Expl Cell Res. 64, 232-236.'BOHLOOL, B. B. & SCHMIDT, E. L. (1974). Lectins: a possible basis for specificity in the

Rhizobium-legume root nodule symbiosis. Science, N.Y. 185, 269—271.BURNET, F. M. (1971). 'Self-recognition' in colonial marine forms and flowering plants in

relation to the evolution of immunity. Nature, Lond. 232, 230-235.FARNES, P., BARKER, B. E., BROWNHILL, L. E. & FANGER, H. (1964). Mitogenic activity in

Phytolacca americana (pokeweed). Lancet ii, Nov. 21, 1100-1101.FEDER, N. & O'BRIEN, T. P. (1968). Plant microtechnique: some principles and new methods.

Am. J. Bot. 55, 123-134.FLINT, F. O. & Moss, R. (1970). Selective staining of protein and starch in wheat flour and

products. Stain Technol. 45, 75-79.GLIMELIUS, K., WALLIN, A. & ERIKSSON, T. (1974). Agglutinating effects of concanavalin A

on isolated protoplasts of Daucus carota. Physiol. PL 31, 225-230.HAMBLIN, J. & KENT, S. P. (1973). Possible role of phytohaemagglutinin in Phaseolus vulgaris L.

Nature, New Biol. 245, 28-30.HESLOP-HARRISON, J., KNOX, R. B., HESLOP-HARRISON, Y. & MATTSSON, O. (1975). Pollen-

wall proteins: emission and role in incompatibility responses. In The Biology of the MaleGamete (ed. J. G. Duckett & P. A. Racey). Biol. J. Linn. Soc. 6, Suppl. 1 (in Press).

JERMYN, M. A. (1974). A class of lectins widespread in the seeds of flowering plants. Proc.Aust. biochem. Soc. 7, 32.

JERMYN, M. A. & YEOW, Y. M. (1975). A class of lectins present in the tissues of floweringplants. Aust. J. Plant Physiol. 2 (in Press).

KNOX, R. B. (1975). Cell recognition and pattern formation in plants. In A Textbook of Develop-mental Biology (ed. P. F. Wareing & C. Grahame), ch. 10. Oxford: Blackwell (in Press).

KNOX, R. B., HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1975). Pollen-wall proteins:localization and characterization of gametophytic and sporophytic fractions. In The Biologyof the Male Gamete (ed. J. G. Duckett & P. A. Racey). Biol. J. Linn. Soc. 6, Suppl. 1 (inPress).

Lis, H. & SHARON, N. (1973). The biochemistry of plant lectins (phytohaemagglutinins).A. Rev. Biochem. 42, 541-574.

MIALONIER, G., PRIVAT, J.-P., MONSIENY, M., KAHLEM, G. & DURAND, R. (1973)- Isolement,propri^tes physicochimique et localisation in vivo d'une phytoh&nagglutine (lectine) dePhaseolus vulgaris L. (var. rouge). Physiol. vig. 11, 519-537.

NELSON, P. E. & DICKEY, R. S. (1970). Histopathology of plants infected with vascular bacterialpathogens. A. Rev. Phytopath. 8, 259-280.


OPIK, H. (1966). Changes in cell fine structure in the cotyledons of PhaseolusvulgarisL,. duringgermination. J. exp. Bot. 17, 427-439.

So, L. L. & GOLDSTEIN, I. J. (1967). Protein and carbohydrate interaction. Application of thequantitative hapten inhibition test to polysaccharide-concanavalin A interaction. J. Ivimun.99, 158-163.

YARIV, J., RAPPORT, M. M. & GRAF, L. (1962). The interaction of glycosides and saccharideswith antibody to the corresponding phenylazo glycosides. Biochem. J. 85, 383-388.

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{Received 20 January 1975)

Figs. 1, 2. Hand sections of 48-h-imbibed cotyledons of jack bean, Canavalia evsi-formis.

Fig. 1. Localization of concanavalin A using FITC-labelled globulin, A, sectionthrough pair of cotyledons showing reaction of epidermal and parenchyma cells.Fluorescence (white in print) is intense in cytoplasmic sites, x 190. B, detail showingintense cytoplasmic fluorescence, especially around starch grains (s). x 300. c, controlsection treated with 10% a-methyl glucoside prior to staining with FITC-labelledglobulin showing absence of cytoplasmic fluorescence, x 190.

Fig. 2. Localization of /?-lectin in sections treated with /?-glucosyl Yariv antigen(1 mg/ml) in 10% DMSO. A, dense reaction product in intercellular spaces, andassociated with plasma membrane on inner face of walls. Dark rings around starchgrains due to refractivity. x 190. B, detail showing intercellular spaces filled withreaction product, x 480. c, intercellular space reaction product is associated withspherical vesicles, x 750.


Figs. 3-6. Hand sections of 48-h-imbibed cotyledons of jack bean, Canavaliaensiformis.

Fig. 3. Section treated with salicin (25 % in 0-15 M NaCl) for 15 min before stainingwith /?-glucosyl Yariv antigen (1 mg/ml) in 10% DMSO. Staining is absent in theintercellular spaces, except within cell wall sites (arrows), x 190.

Fig. 4. Section treated with a-galactosyl Yariv antigen (1 mg/ml) in 10% DMSO.No reaction product is present, x 190.

Fig. 5. Section treated with oc-mannosyl Yariv antigen (1 mg/ml) in 10% DMSO.Reaction product is present in cytoplasmic sites but is absent from the intercellularspaces, x 300.

Fig. 6. Section stained with Ponceau 2R for localization of total protein, showingdense staining reaction in intercellular spaces (arrows), and cytoplasmic sites, x 190.Figs. 7-9. Hand sections of 48-h-imbibed cotyledons of other beans, treated with/?-glucosyl Yariv antigen (1 mg/ml) in 10% DMSO.

Fig. 7. Section of red kidney bean, Phaseolus vtdgaris, showing reaction product inintercellular spaces, and associated with plasma membrane, x 190.

Fig. 8. Section of lima bean, Phaseolus lunatus; localization as in Fig. 7. x 190.Fig. 9. Section of pigeon pea, Cajanus cajan, showing similar localization pattern.

Note that dark rings around starch grains are due to refractivity. x 190.

LOCALIZATION OF LECTINS IN LEGUME COTYLEDONSMethyl a-D-glucopyranoside, a-D-galactose, and...

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