Plant Physiol. (1982) 70, 1162-11680032-0889/82/70/1 162/07/$00.50/0

Fluorescence Immunohistochemical Localization of MalateDehydrogenase Isoenzymes in Watermelon Cotyledons'A DEVELOPMENTAL STUDY OF GLYOXYSOMES AND MITOCHONDRIA

Received for publication January 19, 1982 and in revised form June 5, 1982

CHRISTOF SAUTTER AND BERTOLD HOCKDepartment of Botany, Faculty ofAgriculture and Horticulture, Technical University of Munich, D-8050Freising 12, West Germany

ABSTRACT

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic Imalate dehydrogenase were used for the fluorescence immunohistochemi-cal localization of these isoenzymes in dark-grown watermelon (Citrlusvulgaris Schrad.) cotyledons. It was demonstrated that, with cell organeliesisolated by sucrose density gradient centrifugation, antibodies to glyoxy-somal malate dehydrogenase were specific markers for glyoxysomes, andsimilarly, antibodies to mitochondrial malate dehydrogenase were markersfor mitochondria. The time course of the glyoxysomal malate dehydroge-nase appearance and decline was not synchronous for the individual tissuesand differed completely from that of the mitochondria. The cytosolic malatedehydrogenase I was confined to restricted regions of the lower epidermis.The activity which was definitively localized outside the cell organeilesdecreased during the first days of germination.

The MDH2 isoenzymes in cotyledons of dark-grown fatty seed-lings are involved in different metabolic functions, ie. the glyox-ylate pathway, the tricarboxylic acid cycle, and the malate/aspar-tate shuttle (1 1). The individual forms exhibit different molecularproperties (19, 20), and they can be easily separated by PAGE,isoelectric focusing, or other techniques (16). The association ofthe isoenzymes with different cellular compartments has beenestablished by electrophoretic analyses of cell organelles whichwere purified by sucrose density gradient centrifugation (8).The individual isoenzymes which are numbered consecutively

from the anode to the cathode are distributed in the followingway: MDH V is located in the glyoxysomes; MDH III in themitochondria; whereas MDH I, II, and IV are cytosolic isoen-zymes.Up to now, an in situ localization of the MDH isoenzymes in

plant tissues has not been reported. Clearly, histochemical stainswhich assay enzyme activities would not achieve this purposesince they do not discriminate between the different isoenzymes.The availability of antibodies directed against individual isoen-zymes which do not cross-react with the other isoenzymes (17, 18)

' Supported by the Deutsche Forschungsgemeinschaft (Grant Ho 383/19).

2 Abbreviations: gMDH, glyoxysomal malate dehydrogenase; mMDH,mitochondrial malate dehydrogenase; cMDH, cytoplasmic malate dehy-drogenase; PAGE, polyacrylamide gel electrophoresis; FP, 0.25% (w/v)formaldehyde (freshly prepared from paraformaldehyde in 0.5 M K-phos-phate (pH 7.0); FITC, fluoresceine isothiocyanate.

overcame this difficulty.This report presents information on the intracellular distribution

ofgMDH, mMDH, and cMDH I in the cotyledons ofwatermelonsduring seed germination by the aid of immunofluorescence mi-croscopy. For this purpose, indirect immunolabeling was usedwith monospecific antibodies against the isoenzymes and FITC-coupled goat-anti-rabbit immunoglobulin G's. The data confirmthe hypothesis of the strict and precise compartmentation of theMDH isoenzymes and provide new information on their tissue-specific distribution.

MATERIALS AND METHODS

Plant Material. Watermelon seed (Citrullus vulgaris Schrad.,var. Stone Mountain, harvest 1978) were obtained from Vaughan'sSeed Company (Ovid, MI). They were germinated at 300C in thedark under sterile conditions on 0.8% agar as described before (7).When indicated, 2-d-old seedlings were exposed to continuouswhite light (36 tuE/m2. s) at 25°C.

Preparation of Monospecific Anti-MDH Antisera. Anti-gMDHand anti-mMDH antisera were produced according to the methodsof Walk and Hock (17, 18). For the production of anti-cMDH Iantiserum, the isoenzyme was purified according to Kaiser (10),involving DEAE-cellulose (Serva) chromatography; ammoniumsulfate fractionation; followed by chromatography on the Phar-macia gels Sephadex G-25, Sephacryl S-200, CM-Sephadex C-50,5'-AMP-Sepharose 4B, QAE-Sephadex A-50, Blue Sepharose CL-6B, and isoelectric focusing. The immunization schedule was thesame as with gMDH and mMDH as antigens. All antisera werefractionated by ammonium sulfate precipitation (12) in order torecover the immunoglobulin G (IgG) fractions.

Separation of Cell Organelles. Glyoxysomes and mitochondriawere purified by sucrose density gradient centrifugation of a crudeparticulate fraction (crude 10,000g pellet after a 10-min centrifu-gation, corresponding to 30 cotyledons from 3-d-old dark-grownseedlings) as described before (8).

Immunofluorescence Localization of MDH Isoenzymes. Thetissue processing followed in general the procedures of Baumgart-ner et al. (1) and Tokuyasu and Singer (15). One mm thick cross-sections were handcut from cotyledons and fixed with FP underslight evacuation at 20°C for 1 h. The samples were carriedthrough a series of increasing sucrose concentrations (0.25, 0.5, 1.0M) in FP, each step for 30 min. The sections were mounted oncopper rods and frozen in melting nitrogen.

Glyoxysomal and mitochondrial fractions were fixed in thefractionation medium with final concentrations of 0.25% (w/v)formaldehyde and 25 mm K-phosphate (pH 7.0) for 15 min at4°C. Before centrifugation (5 min at 20,000g), the volume of thefractions was doubled by the addition of FP. The pellets were

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IMMUNOHISTOCHEMICAL MDH LOCALIZATION

embedded in 2% (w/v) agar, and frozen in melting nitrogen.The frozen samples (tissue slices or organelle pellets, respec-

tively) were cut with a Leitz 'Grundschlittenmikrotom' into sec-tions of 40 pam thickness using a knife angle of 20 and a temper-ature of -30°C. The sections were collected at the surface of alarge droplet containing 1% (w/v) gelatin, 0.3% (w/v) agarose,and 10 mm K-phosphate (pH 7.0). The antibody binding wascarried out at 20°C for 10 min following precisely the procedureof Tokuyasu and Singer (15) using 5 pl IgG from antiserum orcontrol serum, respectively, per droplet of buffered gelatin andagarose solution. To remove unbound antibodies, the sectionswere washed three times with 10 mM glycine in 0.8% NaCl (w/v)and 10 mm K-phosphate (pH 7.0), and then carried through threedroplets of the same buffer without glycine. The subsequentincubation with fluorescent-labeled secondary antibodies (goatanti-rabbit FITC-labeled IgG obtained from Behringwerke AG,Marburg) was similar. Afterwards, the sections were mounted inFP with the labeled side pointing upwards, and covered with acover slip.The sections were examined in a Zeiss Photomicroscope II

equipped with epifluorescence (filter combination 450-490, FT510, LP 520). Micrographs were taken on Ilford PanF, Ilford HP5,and Agfachrome 50 L.

Histochemical Localization of MDH Activity. Two-d cotyle-dons were fixed and cryosectioned as described above. For thehistochemical localization of MDH activity, the slices were incu-bated for 45 min at 37°C in a freshly prepared medium, describedby Hanker et al. (6) except for 10 mM L-malate substituting forlactate. In the controls, malate was omitted. The sections weremounted in 0.5 M K-phosphate (pH 7.0).

Electron Microscopy. The preparation of organelle fractions forelectron microscopy was carried out according to Sautter et al.(13). The sections were counterstained with uranylacetate andlead citrate and examined with a Zeiss EM 10.

RESULTSThe use of monospecific antibodies as cytochemical markers for

the intracellular localization of glyoxysomes and mitochondriawas demonstrated with anti-gMDH and anti-mMDH immuno-globulin fractions, respectively. To exclude any artificial bindingof the antibodies, the assays were first carried out with isolatedglyoxysomes and mitochondria. For this purpose, the organellesfrom 3-d cotyledons were separated by sucrose density gradientcentrifugation and identified by measuring the isocitrate lyase andfumerase activities as markers for glyoxysomes and mitochondria,respectively. The gradients were loaded to the limits of theircapacity by an equivalent of 30 pairs of cotyledons per gradientand yielded glyoxysomes at a density of 1.24 g/cmn and mito-chondria at a density of 1.19 g/cm3. The two organelle fractionswere prefixed, embedded in agar and frozen. After sectioning, theprimary immunoreaction was carried out with anti-MDH anti-bodies as indicated, followed by treatment with secondary FITC-labeled antibodies.

Figure 1 shows the immunofluorescence of the glyoxysomal (A,C) and the mitochondrial fractions (B, D) challenged with anti-gMDH (A, B) and anti-mMDH antibodies (C, D) respectively.The fluorescence patterns correspond to the electron microscopicanalysis (E, F). A high level of specific staining was observedwhen glyoxysomes were treated with anti-gMDH antibodies andmitochondria with anti-mMDH antibodies. In addition to thestrong fluorescence of the particles, there was in both cases adistinct background fluorescence which was due to the leakage ofthe isoenzymes out of the organelles into the medium duringpreparation. When the antisera were substituted by control serumobtained prior to the immunization, a much weaker backgroundwithout any particulate staining was observed. In spite of theabsence of isocitrate lyase activity in the mitochondrial fraction,

there was a slight cross-contamination of the mitochondrial frac-tion by glyoxysomes (Fig. 1B) which was typical for the highgradient loads. The reverse case shown in Figure IC is notrepresentative; moreover, the weak fluorescence was due to anunspecific background staining, resulting from the loss of FITCmarker from the secondary antibody. These data prove the suita-bility of the antibody labels for the immunohistochemical detec-tion of cell organelles.

Figure 2 shows the distribution of glyoxysomes in frozen sec-tions obtained from dark-grown cotyledons of different age. Onthe left, representative sections of the palisade parenchyma areshown, while on the right, corresponding sections of the storageparenchyma are seen. In l-d cotyledons (Fig. 2, A and B), occa-sionally a few glyoxysomes could be detected, usually close to thevascular bundles. The main appearance of the organelles did notoccur before day 2 (Fig. 2, C and D). The first places where theglyoxysomes could be seen were in the surroundings of the vas-cular bundles and in the lower epidermis and in a few neighboringlayers of the future spongy parenchyma which at this time servesas a storage parenchyma. The diameters of the globular organelleswere between 1 and 2 ,um. On day 3, a distinctive progression oforganelle production was observed. In the spongy parenchyma(Fig. 2F), the glyoxysomes reached their maximal number andtheir most intensive fluorescence, whereas in the palisade layers,the maximum was achieved 1 d later (Fig. 2G). At this time, adramatic decline in glyoxysomal number and fluorescence hadalready taken place in the spongy parenchyma (Fig. 2H). For acomparison of the cell types, fluorescence micrographs of thesections are shown in Figure 2, G and H, with the correspondingbright field micrographs in Figure 2, 1 and K. With control serum,shown here for 4-d cotyledons, no fluorescence could be detected(Fig. 4E). From these data, a characteristic developmental patternfor glyoxysomes is. inferred with an increase from almost zerolevels to a maximum in organelle numbers and fluorescenceintensities followed by a fast decline. The analysis of a largenumber of slices has shown that the different cotyledonary tissuesexhibit shifted time courses, beginning with the lower epidermis,followed by the spongy parenchyma, especially in the neighbor-hood of vascular bundles, and later by the palisade parenchyma.The differences in organelle appearance and number at different

developmental stages were not due to an artificial covering ofavailable antibody binding sites, e.g. by changing concentrationsof cell constituents such as fat, etc. This possibility was ruled outby the immunocytochemical labeling of mitochondria with anti-mMDH antibodies which exclusively bind mMDH. Here, anentirely different labeling pattern was seen. Figure 3 shows thetime course of fluorescence labeling in the storage parenchyma of1- to 4-d cotyledons. By direct microscopic examination of fluo-rescent organelles which permits a quick evaluation of severalfocusing planes, the different appearance of mitochondria withtheir smaller and often curled forms became evident. Most impor-tantly, there was already a significant and specific labeling in l-dcotyledons (Fig. 3A) which increased to a high level at day 2 (Fig.3B). Three- and 4-d cotyledons exhibited a further increase which,however, was much smaller than the increase in the glyoxysomalnumber during the same period (Figs. 3, C and D). The othercotyledonary tissues (not shown) yielded comparable patterns ofmitochondrial development. These experiments have shown thatthe immunohistochemical localization of organelles is feasibleeven in early stages of fatty cotyledons. The organelle numberscorrespond roughly to the enzyme activities ofgMDH and mMDHextracted from cotyledons of different developmental stages (17,18).The availability ofmonospecific antibodies against one cytosoic

form ofMDH, cMDH I, which is representative for the cotyledonsand the embryo axis, raises the intriguing problem of the tissue-specific localization of this isoenzyme. This isoenzyme was con-

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SAUTTER AND HOCK

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FIG. 1. Immunofluorescent labeling of glyoxysomes (A, C) and mitochondria (B, D) by rabbit anti-gMDH antibodies (A, B) and anti-mMDHantibodies (C, D), respectively, followed by FITC-labeled goat anti-rabbit antibodies, after sucrose density gradient centrifugation of a crude organellefraction (lO,OOOg pellet from watermelon cotyledons). Electronmicrographs from glyoxysomal (E) and mitochondrial (F) fractions are shown for controlpurposes.

fined to the lower epidermis (Figs. 4, A, C, and D): it was entirelylacking in the upper epidermis (Fig. 4B). The most intense fluo-rescence was observed in l-d cotyledons (Fig. 4A), and it decreasedas germination progressed (Fig. 4, C and D). During the laterstages, the cytosolic localization of the isoenzyme became evident.The various organelles were observed as dark spots. The fluores-

cence label was never uniformly distributed within the tissue butwas confined to small groups of cells within the lower epidermis.When the tissue of 2-d cotyledons was histochemically stained forgeneral MDH activity, the most intensive concentration of the dyewas again observed within small groups of the lower epidermiswhich contained the differentiating guard mother cells, followed

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IMMUNOHISTOCHEMICAL MDH LOCALIZATION

'" A'FIG. 2. Fluorescent immunohistochemical localization of glyoxysomes in frozen sections of dark-grown watermelon cotyledons by anti-gMDH

antibodies followed by FITC-labeled secondary antibodies, 1 d (A, B), 2 d (C, D), 3 d (E, F), and 4 d (G, H) after germination. Brightfield micrographsof sections G and H are shown in plates I and K. Left column, palisade parenchyma; right column, spongy parenchyma. Bar, 10 ,tm.

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Plant Physiol. Vol. 70, 1982

FIG. 3. Fluorescent immunohistochemical localization of mitochondria in frozen sections from spongy parenchyma of dark-grown watermeloncotyledons by anti-mMDH antibodies followed by FITC-labeled secondary antibodies, 1 d (A), 2 d (B), 3 d (C), and 4 d (D) after germination. Bar,IoOLm.

by the neighboring layers of the storage parenchyma and thesurroundings of the vascular bundles (Fig. 4F). It is tempting toenvisage a functional connection between the future stomatalapparatus, which are not completely differentiated in these earlystages, and the lower epidermal groups stained for cMDH activity.

DISCUSSIONImmunocytochemistry is the method of choice for the intracel-

lular localization of isoenzymes when the enzyme reaction doesnot permit discrimination. The use of gMDH and mMDH asmarkers for glyoxysomes and mitochondria, respectively, wasdemonstrated by immunofluorescent labeling of organelles pre-viously purified by sucrose density gradient centrifugation andidentified by isocitrate lyase or fumarase activity as well as electronmicroscopy. By this technique, the cross-contamination of the twoorganelle fractions was checked; it confirmed the high purity ofthe glyoxysomal fraction in contrast to the mitochondrial fraction,which contained some glyoxysomes in the case of high organelleloads. Considering the sensitivity of the serological tests, it is likelythat antibodies to glyoxysomal membranes will detect determi-nants of glyoxysomal origin -in mitochondrial fractions which were

previously judged as pure on the basis of marker enzymes (9).This type of experiment, therefore, does not provide clues to thequestion of common determinants in the glyoxysomal and theouter mitochondrial membrane. For this purpose, ultrastructuralstudies combined with immunochemical investigations are re-quired.The immunofluorescent labeling ofgMDH and mMDH in cell

organelle fractions and tissue sections yielded a considerableconcentration of the dye in the organelles. These must haveremained relatively intact, since particles cut prior to fixation onlycontribute to the background staining (C. Sautter, unpublished).The penetration of primary and secondary antibodies through theorganelle envelopes without a prior leakage ofthe isoenzymes intothe surrounding areas is not a contradiction. The treatment withlow concentrations of formaldehyde fixed the original isoenzymelocation without causing a destruction of the antibody bindingsites. The subsequent passage through increasing sucrose concen-trations provided the necessary cryoprotection during deep freez-ing and sectioning and also changed the permeability of theorganelle membranes during thawing of the cut sections. This hasbeen noticed before and was confirmed by electron microscopy

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FIG. 4. Fluorescent immunohistochemical localization ofcMDH I in frozen sections from the lower (A, C, D) and the upper epidermis (B) of dark-grown watermelon cotyledons by anti-cMDH I antibodies followed by FITC-labeled secondary antibodies, 1 d (A, B), 3 d (C), and 4 d (D) aftergermination. E, 4-d spongy parenchyma treated with control serum instead of antiserum. F, 2-d cotyledon, histochemically stained for MDH activity.Bar, 10 ,tm (A-E); F, 100 ttm.

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SAUTTER AND HOCK

(C. Sautter, unpublished). The globular shape of the glyoxysomesdeviated from their original conditions in vivo where the organellesappear as irregularly lobed and invaginated particles squeezedinto the spaces, especially those between oleosomes (14). It wasthe infiltration by sucrose which allowed easy access to the matrixof the glyoxysomes and caused this conspicuous change. Sphericalforms were also seen with other histochemical stains, e.g. malatesynthase (2).

Antibody labeling of the organelles within the tissues waslimited to cut cells. Again, the cryocutting of prefixed tissuesections guaranteed the preservation of the original antibodybinding sites. The choice of thick cryosections with a diameter of40 ,um and the use of epifluorescence equipment resulted from theneed to view large areas. The additional advantages of this pro-cedure lie in a uniform staining over the entire surface and in theshort time required. The results were available in less than 4 hafter chopping the cotyledons.

It was verified by different controls that the developmentalcourse of fluorescence labeling of gMDH in the cotyledonarytissues reflected the true pattern of glyoxysomal development. Thelack of fluorescent labeling in early stages of seed germination wasnot due to the inability of the anti-gMDH antibodies to reachtheir binding sites, since the infiltration and labeling ofthe sectionswith anti-mMDH did not present any problem. The labelingpatterns were in accordance with the results of serological isoen-zyme determination in crude extracts (17, 18). Moreover, in hy-pocotyls which were known to lack gMDH, no glyoxysomes couldbe detected by immunohistochemical techniques (not shown). Onthe other hand, fluorescent labeling of cMDH I was restricted toareas outside the organelles, which was to be expected in the caseof a cytosolic isoenzyme.

Evidence for the different temporal and spatial pattern ofglyoxysomal development in contrast to mitochondrial is basedon the analysis of a large number of slices, of which only a fewhave been presented in this paper. This difference reflects thefunctional separation of fat degradation and respiration. Thesequential appearance of glyoxysomes in the different cotyledon-ary tissues is closely correlated to the fat degradation which isknown to be nonsimultaneous in the different parts of the coty-ledons (5). The increase in mitochondria during germination asdetected by fluorescent labeling ofmMDH appeared to be almostsimultaneous in the different tissues. This is in contrast to thefindings of Flinn and Smith (3) who analyzed in pea cotyledonsthe distribution pattern of Cyt oxidase and succinate dehydroge-nase by enzyme specific staining. This difference might be due toa contrast in fat and starch storing seedlings.The immunochemical demonstration of cMDH I provides the

first tissue-specific localization of a cytosolic MDH. The choice ofthis isoenzyme was governed by its occurrence in the embryo axisand the cotyledons, whereas the cMDH II and IV are restricted tothe cotyledons (8). The association ofcMDH I with distinct groupsof epidermal cells, probably meristemoids giving rise to the sto-mata, raises the question of the metabolic role of this isoenzyme.

Biochemical studies in our laboratory (4) have shown that incontrast to cMDH I, the organelle-bound MDH isoenzymes aresynthesized as higher mol wt precursors which are processedduring the importation into their organelles. This event is probablydue to a posttranslational transport mechanism. It would be mostimportant to study the intracellular route of the isoenzymes fromthe site of synthesis to their final destination. The immunochem-ical localization at the electron microscopic level provides thebasis for these efforts, and significant contributions are to beexpected using this technique.

Acknowledgment--The competent technical assistance of Mrs. Danuta Weberis gratefully acknowledged. We thank Dr. R. Youngman (Technical University ofMunich) for his comments on the manuscript.

LITERATURE CITED

1. BAUMGARTNER B, KT TOKUYASU, MJ CHRUSPEELS 1980 Immunocytochemicallocalization of reserve protein in the endoplasmic reticulum ofdeveloping bean(Phaseolus vulgaris) cotyledons. Planta 150: 419-425

2. BuRKE JJ, RN TRELEASE 1975 Cytochemical demonstration of malate synthaseand glyoxylate oxidase in microbodies of cucumber cotyledons. Plant Physiol56: 710-717

3. FLINN AM, DL SMITH 1967 The localization of enzymes in the cotyledons ofPisum arvense L. during germination. Planta 75: 10-22

4. GiETL C, B HOCK 1982 Organelle-bound malate dehydrogenase isoenzymes aresynthesized as higher molecular weight precursors. Plant Physiol 70: 484-487

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6. HANKER JS, CJ KuSYK, FE BLOOM, AGE PEARSE 1973 The demonstration ofdehydrogenases and monoamine oxidase by the formation of osmium at thesites of Hatchett's brown. Histochemie 33: 205-230

7. HOCK B 1969 Die Hemmung der Isocitratlyase bei Wassermelonenkeimlingendurch Weisslicht. Planta 85: 340-350

8. HOCK B 1973 Kompartimentierung und Eigenschaften der Malatdehydrogenase-Isoenzyme aus Wassermelonenkeimblattern. Planta 112: 137-148

9. HOCK B 1974 Antikorper gegen Glyoxysomenmembranen. Planta 115: 271-28010. KAISER S 1978 Reinigung der cytoplasmatischen Malatdehydrogenase I aus

Wassermelonenkeimlingen. Master thesis. Ruhr University, Bochum, WestGermany

11. METTLER IJ, H BEEVERS 1980 Oxidation ofNADH in glyoxysomes by a malate-aspartate shuttle. Plant Physiol 66: 555-560

12. NOVOTNY A 1969 Basic Exercises in Immunochemistry. Springer-Verlag, NewYork, pp 3-5

13. SAUTrER C, HC BARTSCHERER, B HOCK 1981 Separation of plant cell organellesby zonal centrifugation in reorienting density gradients. Anal Biochem 113:179-184

14. SCHOPFER P, 0 BAJRACHARYA, R BERGFELD, H FALK 1976 Phytochrome-me-diated transformation of glyoxysomes into peroxisomes in the cotyledons ofmustard (Sinapis alba L.) seedlings. Planta 133: 73-80

15. TOKUYASU KT, SJ SINGER 1976 Improved procedures for immunoferritin label-ing of ultrathin frozen sections. J Cell Biol 71: 894-896

16. WALK RA, B HOCK 1976 Separation of malate dehydrogenase isoenzymes byaffinity chromatography on 5'-AMP-sepharose. Eur J Biochem 71: 25-32

17. WALK RA, B HOCK 1976 Mitochondrial malate dehydrogenase of watermeloncotyledons: time course and mode of enzyme activity changes during germi-nation. Planta 129: 27-32

18. WALK RA, B HOCK 1977 Glyoxysomal malate dehydrogenase of watermeloncotyledons: de novo synthesis on cytoplasmic ribosomes. Planta 124: 277-285

19. WALK RA, B HOCK 1977 Glyoxysomal and mitochondrial malate dehydrogenaseof watermelon (Citrullus vulgaris) cotyledons. II. Kinetic properties of thepurified isoenzymes. Planta 136: 221-228

20. WALK RA, S MICHAELI, B HOCK 1977 Glyoxysomal and mitochondrial malatedehydrogenase of watermelon (Citrullus vulgaris) cotyledons. I. Molecularproperties of the purified isoenzymes. Planta 136: 211-220

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