Plant Physiol. (1976) 58, 309-314

Isolation of Intact Chloroplasts and Other Cell Organelles fromSpinach Leaf Protoplasts1

Received for publication April 2, 1976 and in revised form May 21, 1976

MiKio NISHIMURA, DOUGLAS GRAHAM,2 AND TAKASHI AKAZAWAResearch Institute for Biochemical Regulation, School ofAgriculture, Nagoya University, Chikusa, Nagoya464, Japan

ABSTRACT

Freshly prepared spinach leaf protoplasts were gently ruptured bymechanical shearing followed by sucrose density gradient centrifugationto separate constituent cell organelles. The isolation of intact Class Ichloroplasts (d = 1.21) in high yield, well separated from peroxisomesand mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydro-genase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the frac-tions. A clear separation of chloroplastic ribosomes from the solublecytoplasmic ribosomes was also demonstrated by the band patterns ofconstituent RNA species in the polyacrylamide gel electrophoresis. Lo-calization of several enzyme activities specific to leaf peroxisomes, e.g.catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate re-ductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC2.6.1.4), seine glyoxylate aminotransferase, and alanine glyoxylateaminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25),was demonstrated. Overall results show the feasibility of the method forthe isolation of pure organelle components in leaf tissues.

A number of recent investigations have been increasinglyshowing the potential usefulness of plant protoplasts for physio-logical as well as biochemical research (25). In previous papers(20, 21) we have reported the value of spinach leaf protoplastsfor studying the mechanism of photosynthesis and photorespira-tion, the biggest advantage being the manipulation of homoge-neous "naked leaf cell" preparations for such studies. We havealso emphasized that the maintenance of interorganellar rela-tionships in protoplasts is useful for the analysis of the cellularcompartmentation mechanism in photorespiration which is be-lieved to proceed in multiorganellar fashion (28). As a next step,the separation of individual cell organelles having sufficientlyhigh activities of enzyme reactions operating in photorespirationand the subsequent reassembling are crucial to understandingthe overall processes. However, both structural complexity andfragility of organelles in leaf cells pose an obstacle for this task,and breakage of structural organization of organelles duringisolation procedures is frequently encountered. Despite inten-

I This is Paper No. XXXV in the series "Structure and Function ofChloroplast Proteins." The research is supported in part by the grantsfrom the Ministry of Education of Japan, the Toray Science Foundation(Tokyo), and the Naito Science Foundation (Tokyo).2Permanent address: Plant Physiology Unit, Commonwealth Scien-

tific and Industrial Research Organization, Division of Food Researchand School of Biological Sciences, Macquarie University, North Ryde,N. S. W. 2113, Australia. Recipient of Award from the Japan Societyfor the Promotion of Science under the Visiting Professorship Pro-gramme (1974).

sive trials, the isolation of pure chloroplast fractions retainingboth structural and functional integrity has reached only a lim-ited success (31). A clear separation of pure leaf peroxisomesfree from other components has barely been accomplished (29).Since cell wall materials are enzymically degraded during thepreparation of protoplasts, it is obvious that gentle breakage ofthe latter surrounded only by the plasmalemma is ideal forisolating the constituent organelles. In the work reported in thiscommunication we have undertaken the isolation of intact chlo-roplasts and other organelles from spinach leaf protoplasts in anattempt to investigate the nature of the interorganellar relation-ship of photorespiration.

MATERIALS AND METHODS

Protoplasts. Protoplasts were prepared from freshly harvestedspinach leaves (Spinacia oleracea L var. Kyoho) essentially fol-lowing the method reported previously (20). The only modifica-tion employed was that the concentration of mannitol was low-ered to 0.7 M. The final preparations were shown to havephotosynthetic activities of 35 to 70 ,umol CO2 fixation/mgChl hr. The protoplasts (approximately 4 mg Chl) suspended in2 ml of 0.05 M Tricine-NaOH buffer (pH 7.5) containing 0.5 Msucrose and 0.1 % BSA were then ruptured through a syringe(0.5 x 4 cm, Termo). A small piece of Miracloth (Calbiochem)was placed in the bottom of the needle (0.7 x 32 mm), and aftereach stroke samples released from the needle were examined bylight microscope. Usually protoplasts were completely broken bythree strokes (cf. Fig. 1).

Sucrose Density Gradient Centrifugation. The ruptured pro-toplast preparations (0.5 ml) were directly layered on top of 15ml of the linear sucrose gradient (35-60%, w/w) dissolved in0.02 M Tricine-NaOH buffer (pH 7.5) and run at 24,000 rpm for3 hr at 4 C using a Beckman-Spinco SW 25-3 rotor. At the endof the run, 0.5-ml fractions were collected and aliquots wereused for measuring the enzymic activities as well as the contentof Chl, protein, RNA, and DNA (see below).Enzyme Assays. (a) RuDP3 carboxylase (EC 4.1.1.39) activ-

ity was measured as described by Nishimura et al. (22). (b)NADP triose-P (EC 1.2.1.9) and NAD triose-P dehydrogenases(EC 1.2.1.12) activities were determined by measuring the de-crease in absorbance at 340 nm due to the oxidation of NADPHor NADH by 3-PGA following the method of Heber et al. (10).(c) Carbonic anhydrase (EC 4.2.1.1) activity was measured aspreviously described by Atkins et al. (4) using veronal-pH indi-cator buffer at 0C and pH 8.2. (d) Catalase (EC 1.11.1.6)activity was assayed by measuring the disappearance of H202spectrophotometrically at 240 nm following the method of Luck(18). (e) Glycolate oxidase (EC 1.1.3.1) assay was carried out

3Abbreviations: RuDP: ribulose 1,5-bisphosphate; DCPIP: 2,6-di-chlorophenolindophenol; 3-PGA: 3-P-glyceric acid.

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anaerobically by measuring the reduction of DCPIP at 600 nmfollowing the method of Tolbert et al. (30). (f) NADH-glyoxyl-ate reductase (EC1..1.1.26) activity was assayed by measuringthe decrease in absorbance at 340 nm due to the oxidation ofNADH by glyoxylate following the method of Zelitch and Gotto(33). (g) Cytc oxidase (EC 1.9.3.1) activity was determined bymeasuring the decrease of reduced Cyt c at 550 nm according tothe method of Smith (26). (h) NAD-isocitrate dehydrogenase(EC 1.1.1.41) activity was assayed by measuring the increase ofabsorbance at 340 nm of NADH by isocitrate following themethod of Cox (6). (i) NAD-malate dehydrogenase (EC1.1.1.37) activity was measured as described by Asahi andNishimura (3).(j) Glutamate glyoxylate aminotransferase (EC2.6.1.4), serine glyoxylate aminotransferase, alanine glyoxylateaminotransferase (EC 2.6.1.12), and aspartate a-ketoglutarateaminotransferase (EC 2.6.1.1) activities were determined fol-lowing the procedures described by Rehfeld and Tolbert (23).

Except in the case of carbonic anhydrase, enzyme assays werecarried out at 25 C for appropriate reaction periods, and activi-ties are expressed as,mol substrates utilized or productsformed/minm tube (0.5 ml) (cf. Figs. 3 and 4).

Analytical Methods. Chl content was determined according tothe method of Arnon (2). Protein content was determined by thespectrophotometric method of Lowry et al. (17) using BSA as astandard.The content of RNA and DNA in the fractionated samples

was determined according to the method of Fleck and Munro(7).Each fraction (0.5 ml) was made to 5 ml with distilled H20and 2.5 ml of cold 2.1 N HClI4 were added. After 15 min theprecipitate collected by centrifugation was washed twice with 0.1ml of 0.7 N HC104. The precipitate was digested in an incubatorat 37C using 2 ml of 0.3 N KOH for 1 hr. At the end ofincubation, the samples were neutralized with10 N HClO4 andacidified with 1 volume of 1 N HClO4. The precipitate (DNAfraction) separated by centrifugation was washed twice with1 mlof cold 0.5 N HCl04. The combined supernatant fluid andwashings (RNA fraction) were subjected to absorbance meas-urement at 260 nm and 275 nm. The RNA content was esti-mated by:

RNA (jig/ml) = 11.87 A26onm -A275nm

The DNA fraction (precipitate) which was dissolved in 0.1 ml of1 N KOH was assayed for DNA content by the diphenylaminemethod.

Polyacrylamide Gel Electrophoresis.Each of the chloroplast(No. 14 to 18 in Figure Sa) and the supernatant fractions (No.27 to 31) were mixed with 1 volume of 90% phenol and stirredvigorously for 15 min at room temperature. Afterward thesamples were thoroughly dialyzed against phenol-saturated wa-ter to remove sucrose. Then the samples were centrifuged andthe water phase fraction was carefully collected and 2 volumes ofethanol were added. The precipitate formed was centrifuged andwashed twice with 4 ml of 70% ethanol. The precipitate wasdissolved in 100,ul of electrophoretic buffer and applied to thepolyacrylamide gel electrophoresis following the method ofLoening and Ingle (16). The whole leaf RNA fraction wasextracted from spinach leaves by 50% phenol and treated in thesame way. After electrophoresis (5 mamp/tube, 2 hr), the gelwas stained with methylene blue. A Joyce Loeble Chromo-Scanwas used to obtain the densitometric tracings of the gels stainedwith dye.

RESULTS

Isolation of Intact Chloroplasts. The primary purpose of thepresent investigation was to isolate preparations of intact chloro-plast after breakage of protoplasts. Several procedures forbreakage were examined such as osmotic shock and mechanical

breakage by Teflon homogenizer or passage through a syringe.The gentle mechanical shearing using a syringe was found to bemost satisfactory. Figure 1 shows the release of a homogeneouspopulation of chloroplasts examined by light microscope afterthe syringe treatment. A single passage is compared with a triplepassage preparation. By phase contrast microscopy the chloro-plasts appeared brightly refractive, with a halo around the outermargin of the particles (27). A clear separation of a single bandof chloroplasts (d = 1.21), practically free from stripped chloro-plasts, was achieved by sucrose density gradient centrifugation(Fig. 2), a nearly complete recovery of chloroplast fractions wasachieved by this procedure (93% recovery from starting proto-plasts on Chi basis).

Cellular Localization of Enzyme Activities in Chloroplasts andOther Organelles. A satisfactory resolution of chloroplasts andother constituent organelles can be supported from experimentalresults examining the enzyme localization in separated fractions.Figure 3 shows a sedimentation profile of the broken protoplastspreparations after sucrose density gradient centrifugation. Theprominent peaks of chloroplastic marker enzyme exactly coin-cide with the peak of Chl (d = 1.21) (Fig. 3, a and b). Someactivities of RuDP carboxylase and NADP triose-P dehydrogen-ase are detectable in the soluble supernatant fraction, but theycomprise only a small proportion of the total. Sizable activities ofcarbonic anhydrase were found to be clearly associated withchloroplasts, but much activity remained in the top, solublefractions. Presumably enzyme activities in each fraction repre-sent the chloroplastic and cytoplasmic carbonic anhydrase (8),but that in the soluble fraction is so high that contamination byenzyme leached out, or from the surface, of chloroplasts must beconsidered.Marker enzyme activities of peroxisomes (28, 30, 32), i.e.

catalase, glycolate oxidase, and glyoxylate reductase, are clearlypresent in peroxisome fractions (d = 1.25), although much ofthese activities (approximately 70%) are also present in thesupernatant fractions (Fig. 3c). This is probably due to thefragility of the single membranous organelles and leaching of theenzyme during the separation procedure. Tolbert reported (29)that leaf peroxisomes comprise 1 to 1.5% of the total protein,and the present results show that the content of peroxisomesrecovered is approximately 3% on a protein basis (Fig. 3a).Employing the osmotic treatment of protoplasts followed bysucrose density gradient centrifugation, both glycolate oxidaseand glyoxylate reductase activities were almost exclusively lo-cated in the top, soluble fractions of the centrifuged samples,although chloroplasts and mitochondria were fairly well pre-served.

While not much data are available regarding the separation ofmitochondria from other organelles in leaf tissues, reportedresults from several laboratories are somewhat conflicting (11,23, 24, 32). We have consistently observed that when the proto-plast preparations suspended in sucrose solution without BSAwere subjected to mechanical breakage followed by gradientcentrifugation, the mitochondrial fractions (d = 1 .22) over-lapped with chloroplasts (d = 1.21), probably because of theabsorption of solute (sucrose) through the outer membranes ofmitochondria (T. Asahi, personal communication). Activity ofCyt c oxidase was found in the leading edge fractions of chloro-plasts (data not shown). These results are judged to be basicallysimilar to those of Rocha and Ting (24) and Huang and Beevers(1 1); in such studies the density of mitochondria (d = 1.21) wasshown to be larger than that of stripped chloroplasts (d = 1 .17).On the other hand, we have found that inclusion of BSA in thesuspending media exhibits a preservative effect on mitochondria,thereby the density of mitochondria becoming distinctly smallerthan that of the chloroplasts. Figure 3d shows the specific locali-zation of both Cyt c oxidase and NAD-isocitrate dehydrogenasein the fractions of d = 1.18. Figure 3e shows that activities of

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ORGANELLE SEPARATION FROM PROTOPLASTS

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FIG. 1. Photomicrographs of gently ruptured protoplasts. a and b show the spinach protoplasts ruptured by gentle shearing through syringe onceand three times, respectively. In a arrows indicate unbroken protoplasts. Experimental procedures are described in the text.

NAD-malate dehydrogenase are detected in peroxisomes, mito-chondria, and chloroplasts. In agreement with results of previousinvestigators (28, 32), the specific activity is highest in peroxi-somes, but it must be pointed out that the enzyme activity isclearly detected in the chloroplastic fractions.

Activities of four aminotransferases which are reported to beoperative in the glycolate pathway were assayed in the separatedfractions and results are shown in Figure 4. In accordance withthe results of Tolbert and his associates (23, 28, 32), a tightassociation of three aminotransferases, i.e. glutamate glyoxylate(Fig. 4a), serine glyoxylate (Fig. 4b), and alanine glyoxylateaminotransferases (Fig. 4c), with peroxisomes was clearly dem-onstrated. However, for each of these enzymes some activity was

detected in the soluble supernatant fractions and small activitiesin other particulate fractions as well. Activity of aspartate a-ketoglutarate aminotransferase was detected in all three organ-elles: chloroplasts, peroxisomes, and mitochondria (Fig. 4d),presumably representing each individual isozyme (23, 32).

Isolation of Ribosomal RNA. An additional proof for theisolation of intact chloroplasts can be seen from the separation ofchloroplastic and cytoplasmic ribosomal RNA. The profile ofRNA analysis in the separated fractions is given in Figure 5a. Inorder to characterize the RNA components of the chloroplastand soluble cytoplasmic fractions, RNA sepcies were resolved bypolyacrylamide gel electrophoresis according to the method ofLoening and Ingle (16). The stained electrophoresed gel samples

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Plant Physiol. Vol. 58, 1976 311

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NISHIMURA, GRAHAM, AND AKAZAWA

trifugation of gently homogenized leaf cells using isotonic sorbi-tol solution (12, 31). It is inevitable that all such preparationsare mixed populations of various organelles. It is unavoidablethat these methods cause some structural damage of chloroplastsand other organelles during the step of breaking the leaf tissues.The use of leaf protoplasts as a starting material is different inprinciple from the previous commonly used methods, and thepresent investigation clearly shows that the gentle breakage ofnaked leaf cells (protoplasts) followed by sucrose density centrif-ugation is advantageous for separating three major organelles,namely chloroplasts, mitochondria, and peroxisomes, in pureform. The specific localization of some enzyme activities as wellas the ribosomal RNA in the distinctly separated chloroplast

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FIG. 2. Photograph of the separation of chloroplasts by sucrose den-sity gradient centrifugation. Mechanically broken protoplasts (Fig. lb)(approximately 2 mg Chl) were applic to the sucrose density gradientcentrifugation as explained in the text

and their scan tracing are given in Figure 6 (b and c), in compari-son with the picture of the total RNA fraction obtained from thewhole spinach leaf (Fig. 6a). The pictures clearly show that thechloroplastic RNA (23s, 16s) is well separated from the cyto-plasmic RNA (25S, 18s), indicating that the chloroplastic andcytoplasmic fractions contain chloroplastic rRNA and cytoplas-mic rRNA, respectively. Percentage distribution of each RNAspecies in the individual fraction was calculated from the figureand the values are summarized in Table I. A small amount ofcross-contamination between the two fractions can be seen, butwe cannot exclude a possibility that cytoplasmic rRNA is associ-ated with the chloroplastic outer membranes (1). The same typeof association is clearly demonstrated in yeast mitochondria(13). Also, the presence of small amounts of unknown RNAspecies having smaller molecular size can be seen in Figure 6, band c, and is probably due to the degradation of each respectiverRNA species during the preparation of samples (16).

Figure Sb shows the results of DNA analysis. There are twomajor DNA-containing fractions, I and II, but none of them isfully characterized. Although we found that fraction I is positiveto the Feulgen staining, the sedimented pellets in centrifugetubes are also positive to this stain. It will be noted that since theabsorption spectra of peak II fractions analyzed by the diphenyl-amine method showed the presence of two prominent peaks at650 nm and 600 nm, there is a possibility that non-DNA sub-stances are present in these fractions.

DISCUSSION

The hitherto reported structurally intact and functionally ac-tive chloroplasts were preparations obtained by low speed cen-

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FIG. 3. Localization of enzyme activities in the separated fractionsafter sucrose density gradient centrifugation of mechanically rupturedprotoplast preparations. Mechanically ruptured protoplasts were sub-jected to sucrose density gradient centrifugation as shown in Fig. 2, andthe 0.5-ml fractions separated were used for the following assays: a:protein and Chl; b: RuDP carboxylase, NADP triose-P dehydrogenase,and carbonic anhydrase; c: catalase, glycolate oxidase, and glyoxylatereductase; d: Cyt c oxidase and NAD-isocitrate dehydrogenase; and e:NAD-malate dehydrogenase and NAD triose-P dehydrogenase. Experi-mental details for the enzyme activity measurements are described in thetext. Except in the case of carbonic anhydrase all other enzyme activitiesare expressed as ,umol substrates utilized or products formed/minm tube(0.5 ml).

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ORGANELLE SEPARATION FROM PROTOPLASTS

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FIG. 4. Localization of various aminotransferase activities in the sep-arated fractions after sucrose density gradient centrifugation of mechani-cally ruptured protoplast preparations. The preparation used for me-chanical breakage and sucrose density gradient centrifugation was thesame as that shown in Fig. 3. Aliquots of separated fractions weresubjected to the following enzyme assays: a: glutamate glyoxylate ami-notransferase; b: serine glyoxylate aminotransferase; c: alanine glyoxyl-ate aminotransferase; and d: aspartate a-ketoglutarate aminotrans-ferase. Experimental details for the enzyme activity measurements aredescribed in the text, and all enzyme activities are expressed as ,umolproducts formed/min- tube (0.5 ml).

deterioration of their biochemical activities. The light-dependentCO2 fixation activity is usually low. The final chloroplast prepa-rations (d = 1.21) obtained in the present study exhibitedfixation of about 5 Mumol C02/mg Chl - hr. Therefore, in spite ofthe fact that chloroplasts appear to retain their structural integ-rity microscopically, the type of preparations isolated can beclassified as Class I according to Spencer and Unt (27). Anadditional factor causing the decline of photosynthetic activity islikely to be the time lapse during preparation of protoplasts (4hr), probably resulting in the consumption of photosyntheticintermediates during this step. It will be recalled that previously

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FIG. 5. Localization of RNA and DNA in the separated fractionsafter sucrose density gradient centrifugation of mechanically rupturedprotoplast preparations. The preparation used for mechanical breakageand sucrose density gradient centrifugation was the same as that of Fig.2. Aliquots of separated fractions were analyzed for (a) RNA and (b)DNA.

fractions (d = 1.21) is evidence that the isolated fractions are

pure. However, the present method has its own limitations. As isgenerally recognized, gradient separation of chloroplasts on a

dense sucrose solution for a prolonged period (3 hr) causes

© Fraction 27-31 25s8s

FIG. 6. Polyacrylamide gel electrophoresis of RNA species. (a)Whole leaf RNA, (b) chloroplastic RNA obtained from pooled fraction(No. 14 to 18 of Fig. Sa), and (c) soluble cytoplasmic RNA obtainedfrom pooled fractions No. 27 to 31 of Fig. 5a were subjected to poly-acrylamide gel electrophoresis according to the method of Loening andIngle (16). After separation of the constituent RNA species, the gelswere stained with methylene blue and the density tracing was made.Experimental details are described in the text.

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314 NISHIMURA, GRAHAI

Table I. Percentage Distribution of RNA SpeciesValues are computed from the scanned figures shown in Figure 6.

25s 23s 18s 16s Species

Whole leaf 36.2 18.5 25.7 13.5 6.1Chloroplastic fraction 5.6 36.4 3.2 28.0 26.8

(tubes 14-18)Soluble cytoplasmic 35.2 2.8 22.0 3.3 26.5

fraction (tubes 27-31)

several investigators attempted to enhance the CO2 fixationcapacity of chloroplast preparations by supplementing some Pesters, e.g. fructose-diP, ribose-5-P, and 3-PGA (5). Further-more, one can assume that the contact of cytoplasmic and othercellular inclusions with chloroplasts during the preparative stepof chloroplasts may result in both enzymic and nonenzymicdegeneration of the membranous structure of the latter. Re-cently reported procedures for the isolation of active chloroplastpreparations are all based on very rapid separation using a

medium such as silica gel (19) or two phase dextran-polyethyl-ene glycol (14), but the homogeneity of separated organellefractions has not been well documented in these studies. Al-though the present procedure appears to be far superior toothers to the extent of isolating enzymically and structurally purechloroplasts, further technical improvements are needed in or-

der to obtain fully functional Class A chloroplasts (9).It is known that single membranous organelles such as peroxi-

somes are particularly fragile and are difficult to isolate in theintact form. Lips (15) reported that enzyme content of plantmicrobodies is liable to be affected by experimental proceduressuch as homogenization or sedimentation. It must be pointed outthat there are no satisfactory criteria for the intactness of peroxi-somes. The present method is considered to be useful for sepa-rating them free from other cellular constituents. From thespecific localization of seven major component enzymes with thefractions of d = 1.25 the separation of intact peroxisomes freefrom other organelles can be achieved by the present technique.However, some enzymic activities are leached out into the solu-ble fractions located at the top of the gradient, whereas they mayrepresent specific location in the cytosol in vivo.

Note. After completion of this manuscript our attention was

drawn to the paper by Rathnam and Edwards which describedthe isolation of chloroplasts from various grasses (Rathnam, C.K. M. and G. E. Edwards. 1976. Protoplasts as a tool forisolating functional chloroplasts from leaves. Plant Cell Physiol.,17: 177-186).

Acknowledgments -We wish to record our hearty thanks to H. Beevers for his invaluablediscussion and advice during the course of this investigation and to A. Watanabe for the guidanceof polyacrylamide gel electrophoresis technique.

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M, AND AKAZAWA Plant Physiol. Vol. 58, 1976

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