Microbodies (Glyoxysomes and Peroxisomes) in Cucunber ... · Glyoxysomes contain not onlyenzymes directly involved in lipid metabolism, but also catalase. In their possession ofcata-lase

Plant Physiol. (1971) 48, 461-475

Microbodies (Glyoxysomes and Peroxisomes) inCucunber CotyledonsCORRELATIVE BIOCHEMICAL AND ULTRASTRUCTURAL STUDY IN LIGHT- ANDDARK-GROWN SEEDLINGS'

Received for publication January 12, 1971

RICHAiD TRELEASE,2 BECKER, PEER GRUBER3, ELDON NEWCOMB

Department of Botany and the Institute of Plant Development, University ofWisconsin, Madison, Wisconsin 53706

ABSTRACT

The changes in activities of glyoxysomal and peroxisomalenzymes have been correlated with the fine structure of micro.bodies in cotyledons of the cucumber (Cucumis sativus L.)during the transition from fat degradation to photosynthesisin light-grown plants, and in plants grown in the dark andthen exposed to light. During early periods of development inthe light (days 2 through 4), the microbodies (glyoxysomes)are interspersed among lipid bodies and contain relativelyhigh activities of glyoxylate cycle enzymes involved in lipiddegradation. Thereafter, these activities decrease rapidly asthe cotyledons expand and become photosynthetic, and theactivity of glycolate oxidase rises to a peak (day 7); con-comitantly the microbodies (peroxisomes) become preferen.tially associated with chloroplasts.

In seedlings grown in the dark for 10 days, the reservelipid and the glyoxylate cycle enzyme activities persist for alonger time than in the light; correlated with this, there is acontinued association of the microbodies with the lipid bodies.When these dark-grown seedlings are then exposed to 51 hoursof the light-dark cycle, peroxisomal marker enzymes increaserapidly in activity, and the microbodies become appressed tochloroplasts. We conclude that the characteristic associationobserved between glyoxysomes and lipid bodies reflects theirmutual involvement in net gluconeogenesis through the con-version of fatty acids to carbohydrate, while the close spatialrelationship observed between peroxisomes and chloroplastsat later stages of development reflects their mutual involve-ment in glycolate metabolism.

Although glyoxysomal enzyme activities are dropping rap-idly while peroxisomal enzyme activities are increasing rapidlyduring the transition period in the light, the electron micro-scopic evidence does not indicate that glyoxysomes are beingdegraded or peroxisomes are being formed. Since in the dark-grown seedlings the activities of peroxisomal enzymes remainlow and do not increase as they do in the light, an opportunityis afforded to compare quantitatively any changes in numbersof microbodies per cell with the changes in activities of glyoxy-somal enzymes. It is found that the magnitude of the decreasein numbers of microbodies is considerably less than that of

'This research was supported in part by Grant GB-15246 toE.H.N. and W.M.B. from the National Science Foundation.2 National Institutes of Health Postdoctoral Fellow. Present ad-dress: Department of Botany, Arizona State University, Tempe,Ariz. 85281.

'National Science Foundation Predoctoral Fellow.

the decrease in glyoxysomal enzyme activities between days 4and 10. When the cotyledons are exposed to light, peroxisomalenzyme activities increase greatly, but again there is no ultra-structural evidence for the synthesis of a new population ofmicrobodies to accommodate this increase. These results allowus to conclude that the developmental transition from gly-oxysomal to peroxisomal function almost certainly does notinvolve the actual replacement of one population of micro-bodies by another. Rather, the transition probably occurswithin existing particles, either by a sequential functioningof two different kinds of microbodies or by a change in en-zyme complement within a single population. Our findingswith both light- and dark-grown cotyledons favor the latterpossibility. The cytoplasmic invaginations into microbodiesseen during greening of both light-grown cotyledons and etio-lated cotyledons exposed to light may be morphological mani-festations of the mechanism by which the microbodies lose orgain enzymes.

Plants which store lipid in the endosperm or cotyledons ofthe seed convert this lipid into carbohydrate via the glyoxylatecycle during early postgerminative growth (8). Isocitrate lyaseand malate synthetase, key enzymes of the glyoxylate pathway,are present in such fatty seedlings when lipid stores are beingmetabolized in the endosperm (3, 30, 45) or the cotyledons (8,43, 57) and in fact appear to be restricted in higher plants tothis one stage of growth (3). Using castor bean endosperm,Beevers and co-workers (6, 7) localized these enzymes in asubcellular particle, the glyoxysome, which is now also knownto be the site of fatty acid oxidation (12, 26). Since their dis-covery in endosperm, it has been reported that glyoxysomesare the site of the glyoxylate pathway in a number of fattycotyledons, including those of watermelon (11, 28, 39), peanut(11, 35), cucumber (53), and sunflower (22).

Glyoxysomes contain not only enzymes directly involved inlipid metabolism, but also catalase. In their possession of cata-lase and in several other respects, glyoxysomes are similar toleaf peroxisomes. The latter were first isolated from spinachand are known to be involved in the metabolism of glycolate,a product of photosynthesis (50). Both kinds of particles havethe same buoyant density in sucrose and both are bounded bya single membrane, as revealed by electron microscope exami-

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Plant Physiol. Vol. 48, 1971

nation of the isolated organelles (7, 50). Single membrane lim-ited organelles, defined morphologically as microbodies (18,40), have been observed with the electron microscope in bothleaves (15) and castor bean endosperm (55). These observa-tions, coupled with cytochemical detection of catalase in themicrobodies of castor bean endosperm (55, 56) and leaves (16,17), indicate that microbodies are the morphological equiva-lents of glyoxysomes and peroxisomes and that the latter twobiochemically defined particles have certain distinctive simi-larities.

Studies with a variety of species have established a character-istic developmental pattern for the key enzymes of the gly-oxylate cycle during the early postgerminative growth of light-grown fatty seedlings. Little or no isocitrate lyase or malatesynthetase is detectable in dry seeds (38), but both enzymesincrease markedly in activity shortly after germination, reacha peak within a few days, and then decline rapidly as lipid re-serves are depleted (8, 9, 13, 29-31, 35, 38, 39, 53, 54). Thepostgerminative increase in activity is known to result from denovo enzyme synthesis both in endosperm (30) and in cotyle-dons (21, 24, 34). The subsequent decline in activity is cor-related, in the case of endosperm, with loss of the organelle,probably due to proteolytic activity in an organ which is of nofurther use to the plant (20, 56). The microbodies of somefatty cotyledons, however, are of further developmental inter-est, since the cotyledons of species such as cucumber whengrown in the light expand and differentiate into photosyntheticorgans after depletion of lipid stores. In such cotyledons, thedecline in isocitrate lyase and malate synthetase activities isaccompanied by a rapid rise in glycolate oxidase, an enzymecharacteristic of leaf peroxisomes. In green, photosyntheticcotyledons, glycolate oxidase has been localized in catalase-containing particles similar to leaf peroxisomes (22, 28, 39,53, 54). Thus within the same organ, some succession mustexist between glyoxysomes present during early stages andperoxisomes found at later stages.

Developmental changes in enzyme activities which are nowknown to be compartmentalized in glyoxysomes and peroxi-somes have also been studied in dark-grown seedlings in anumber of laboratories. It was shown in pumpkin cotyledonsthat the typical rise and fall in isocitrate lyase (glyoxysomal)activity found during postgerminative development of light-grown seedlings occurs similarly in dark-grown plants, but thepeak activity is then sustained for about 3 days before declining(8). Similar patterns of a slow but eventual decline in activityof glyoxylate cycle enzymes in the dark were also shown inpine (13) and watermelon seedlings (23, 28, 39). Kagawa andBeevers (28) investigated changing activities of glyoxylate cycleenzymes, the peroxisomal enzymes, glycolate oxidase and hy-droxypyruvate reductase, and protein in microbodies isolatedat various stages from watermelon cotyledons grown in thedark and then exposed to light. They concluded from the lossof protein in these fractions that the slow decline in activitiesof the glyoxylate cycle enzymes was due to degradation of theglyoxysomes, and that the peroxisomes which are present afterexposure to light are not derived from pre-existing glyoxy-somes. In the case of etiolated leaves, glycolate oxidase activityhas not yet been localized in peroxisomes, but this activity isdetectable in dark-grown leaves of several species and increasesmarkedly after exposure to light (47, 48).The question has been asked whether the transition from

glyoxysomal to peroxisomal metabolism represents a changein enzyme complement within a single type of particle orwhether it involves two distinct entities, glyoxysomes and per-oxisomes, which arise in the cotyledons as separate populations

of organelles (22, 28, 39, 53). We have reported previously (22)the results of a correlative ultrastructural and enzymologicalstudy of sunflower, cucumber, and tomato cotyledons grownin the light, which established that at a time of relatively highglyoxysomal activity microbodies are intimately associated withlipid bodies, whereas at a time of relatively high peroxisomalenzyme activity they are associated primarily with chloroplasts.These results were obtained from three different stages of post-germinative growth. We now report in more detail the changesin enzyme complement and fine structure of microbodies incucumber cotyledons during the transition from fat degrada-tion to photosynthesis in light-grown plants, and in seedlingsgrown in the dark, then exposed to light. Correlations aremade between the daily changes in enzyme activities and thefine structure of the microbodies seen at the correspondingperiods. The evidence suggests that at least some of the gly-oxysomes may acquire peroxisomal function during the courseof development through a change in enzyme complement.

MATERIALS AND METHODS

Cucumber seeds (Cucumis sativus L., cv. "Improved LongGreen") were planted in moistened vermiculite overlying soiland grown under a 12-12 hr light-dark cycle with the tem-perature maintained at 22 C in the dark and 28 C in the light,or within the range of 22 to 24 C in complete darkness. Lightintensity of approximately 800 ft-c was provided by fluorescentand incandescent light. Exposure of dark-grown seedlings tolight was accomplished by transferring the seedlings to the12-12 hr light-dark cycle. Germination was considered to oc-cur immediately upon planting.Homogenate Assays. Cucumber cotyledons were diced by

hand with razor blades, ground vigorously at 4 C in a mortarcontaining three volumes of grinding medium consisting of 0.3M sucrose in 50 mM sodium cacodylate, pH 7.2, and strainedthrough three layers of cheesecloth. The dark-grown cotyle-dons were ground in a mortar and strained through cheese-cloth under a dim green light. Homogenates obtained in thismanner were assayed for catalase (37), glycolate oxidase (46),glyoxylate (hydroxypyruvate) reductase (46, with glycolate assubstrate), malate synthetase (11), and isocitrate lyase (11) ac-tivities, and for protein (36). Dithiothreitol was added to afinal concentration of 10 mm to the homogenate samples usedfor isocitrate lyase assays.Homogenate Fractionation. Homogenates were differentially

centrifuged twice, first at 5OOg for 10 min to remove cellulardebris, starch, and nuclei, then at 10,800g for 30 min to pro-vide pellets enriched in mitochondria, glyoxysomes, and per-oxisomes. The above mentioned assays were conducted onthese pellets to obtain recovery data and specific activities.

Zonal Rotor Centrifugation. Cucumber seedlings for theseexperiments were grown in the University of Wisconsin Bio-tron programed for the same growth conditions as describedabove. The seeds were planted in moistened vermiculite with-out underlying soil; the seedlings were therefore fed daily withone-half strength Hoagland's solution.Homogenates were prepared without the use of mortar

and pestle by dicing the cotyledons thoroughly with razorblades attached to an electric knife handle (R. G. Jensen, Uni-versity of Arizona, personal communication) in 1.5 volumes ofgrinding medium (0.5 M sucrose in 50 mm sodium cacodylate,pH 7.2), then were strained through three layers of cheese-cloth. This homogenate was differentially centrifuged twice,first at 500g for 10 min to remove starch, nuclei, and cellulardebris, then at 1,000g for 15 min to remove chloroplasts. The

462 TRELEASE ET AL.

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MICROBODIES IN CUCUMBER COTYLEDONS

supernatant from the last spin was used as the applied samplefor isolation of glyoxysomes and peroxisomes on a continuoussucrose gradient.A Beckman zonal rotor (Ti-14) with 650-ml capacity was

used in these experiments. A sucrose gradient, linear withrotor radius, was made using a modification of the apparatusdescribed by Birnie and Harvey (5). The gradients (35-59%w/w sucrose, buffered with 50 mm sodium cacodylate, pH 7.2)were generated with buffered 35% w/w sucrose as the lowdensity solution and buffered 66.8% w/w sucrose as the highdensity solution. The 500-ml continuous gradients were sup-ported on a cushion of approximately 60% w/w sucrose, and20 ml of buffered 10% w/w sucrose was used as the overlayon the 50-ml sample (1,OOOg supernatant). The rotor was ac-celerated to 44,000 rpm and spun for 2.5 hr at 4 C. After de-celeration to 3,000 rpm, the contents were displaced from thecenter by pumping heavy sucrose into the edge of the rotor.Twenty-milliliter fractions were collected into tubes at ice-bathtemperature; approximately 1 ml was removed from each frac-tion for determination of sucrose densities by measurement ofrefractive index at 20 C. The activities of the enzymes men-tioned above were assayed in 0.5-ml aliquots of each 20-mlfraction.

Electron Microscopy. Segments of cotyledons obtained ateach developmental stage were fixed and embedded for elec-tron microscopy as described by Gruber et al. (22) in 3% glu-taraldehyde buffered in 50 mm potassium phosphate buffer,pH 6.8, and postfixed in buffered 2% osmium tetroxide. Re-moval of the cotyledons from the dark-grown seedlings andfixation of tissue in glutaraldehyde during the first 30 minwere done under a dim green light. Pellets obtained by dif-ferential centrifugation (10,800g) were fixed and embeddedusing a modification of the method recommended by Frankeet al. (14). After encasing the pellets in 2% agar, they werefixed at 4 C for 1.5 hr in a solution containing 3% glutaralde-hyde, 1.5% osmium tetroxide, 0.8 M sucrose, and 50 mm so-dium cacodylate, pH 7.2. The material was then transferreddirectly to 30% acetone and dehydrated and embedded inthe usual manner.The method of incubating tissue in DAB' described by

Frederick and Newcomb (16) was used to detect catalasecytochemically in small segments of light-grown cucumbercotyledons, but without removal of the epidermis of the coty-ledons before incubation. Controls were preincubated in ami-notriazole and then incubated in the standard DAB mediumto which aminotriazole had been added.

Profiles of microbodies were counted in sections of cotyle-dons obtained from seedlings grown in the dark for 4, 7, and10 days. Counts were made while viewing the sections in theelectron microscope rather than from electron micrographs.The sections, taken from 3 to 4 separate cotyledon segmentsat each stage of development, were of a uniform thickness ofabout 80 nm. The mean number of microbody profiles at eachstage was determined from counts in sectioned palisade andspongy cells completely visible between grid bars (75 x 300mesh). In order to avoid scoring microbodies in serial sectionson the same grid, large sections were cut, and only one oroccasionally two sections on the grid were used for the counts.The sizes of the palisade and spongy cells were measured

with a light microscope from plastic sections approximately0.5 am in thickness. Measurements of length and width weremade on a large number of cells of each type (see Table III).

'Abbreviations: DAB: 3,3'.-iaminobenzidine; ER: endoplasmicreticulum.

thih Yellow Yellow Green Greenhreon Grween

qz~

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FIG. 1. Diagrammatic representation of the appearance of cu-cumber seedlings grown under the 12-12 hr light-dark cycle.

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DAYSFIG. 2. Changes in glyoxysomal and peroxisomal enzyme activ-

ities in homogenates of cucumber cotyledons grown under the light-dark cycle. Enzyme activity. glycolate oxidase, nmoles substrateconsumed/min-cotyledon; isocitrate lyase, 0.1 X nmoles substrateconsumed/min-cotyledon; malate synthetase, 0.04 X nmoles sub-strate consumed/min cotyledon; and catalase, 0.2 X units/cotyledon(a unit of catalase is that amount of enzyme required to catalyzethe decomposition of 50% of the HO2 present per 100 sec at 25 C).Values given at days 3, 4, 5, and 7 are averages of two independentexperiments.

Vascular tissue was purposely avoided for determinations ofcell size and numbers of microbody proffles, since it containsfew microbodies.

RESULTS

LIGHT-GROWN SEEDLINGS

Cucumber cotyledons were assayed for characteristic glyoxy-somal and peroxisomal enzymes and fixed for electron micros-copy at 24-hr intervals from the 2nd to the 10th day of post-germinative growth in the 12-12 hr light-dark cycle. Thecharacteristic appearance of the developing seedlings at sev-eral daily intervals is shown in Figure 1. From day 7 throughday 10, the cotyledons continue to enlarge and remain green.The developmental stages were separated by the same timeintervals whether the seedlings were germinated and grown inthe Biotron or in growth chambers. To ensure maximum uni-formity among cotyledons used for enzymatic and ultrastruc-tural analysis, plants were carefully screened and selected ateach daily interval to correspond as closely as possible to themorphological stages shown in Figure 1.Microbody Enzyme Actities during Development. Enzyme

profiles for cotyledon homogenates are shown in Figure 2.Isocitrate lyase and malate synthetase (specific for glyoxy-somes) show the characteristic rise and fall in activity reportedto occur in other fatty seedlings. Of particular interest here istheir sharp parallel decline within 24 hr from peak activitiesat day 4 to very low levels at day 5. Thereafter, the activities

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A463Plant Physiol. Vol. 48, 1971



TRELEASE ET AL. Plant Physiol. Vol. 48, 1971

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DAYS AFE EM3DAYS AFTER GERMINATIONFIG. 3. Specific activities of glyoxysomal and peroxisomal en-

zymes in cotyledon homogenates (solid bars) and 10,800g precipi-tates (open bars) during postgerminative growth of cucumber seed-lings under the light-dark cycle. Specific activity: isocitrate lyase,malate synthetase, glycolate oxidase, nmoles substrate consumed/min-mg protein; catalase, units/mg protein (see Fig. 2 for definitionof catalase unit).

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BUOYANT DENSITY g-cm 3

FIG. 4. Distribution of isocitrate lyase, glyoxylate reductase, andglycolate oxidase activities after sedimentation to equilibrium on asucrose gradient in a zonal rotor. The l,OOOg supernatant fractionsfrom 3-, 4-, 41/2-, and 6-day-old cucumber cotyledons were used asthe applied samples. Enzyme activity (per 20 ml fraction): glycolateoxidase, 30 X nmoles substrate consumed/min cotyledon; isocitratelyase, 0.8 X nmoles substrate consumed/min-cotyledon; glyoxylatereductase, 1.7 X umoles NADH oxidized/min cotyledon.

Note added in proof: Reduction of the Figure for publicationresulted in a lack of distinction between the symbols for isocitratelyase and glyoxylate reductase. Isocitrate lyase is the uppermostcurve in the 3- and 4-day proffles and the lowermost curve in the41o- and 6-day plots.

1.81

1.4

remain low; beyond day 7, these enzymes are essentially un-detectable.A marked rise in activity of glycolate oxidase (characteristic

of leaf peroxisomes) is seen between days 2 and 5; after day 5a relatively constant level is maintained. The initial rise inglycolate oxidase activity occurs as isocitrate lyase and malatesynthetase activities are approaching their peak. However, thegreatest rate of increase in glycolate oxidase activity occursconcomitantly with the rapid decline in glyoxysomal enzymeactivities; consequently, for a short period of time relativelyhigh activities of both peroxisomal and glyoxysomal enzymesare demonstrable within the cotyledons.

Catalase, an enzyme common to both particles, closely re-sembles isocitrate lyase and malate synthetase in activity pro-file during early periods of development. These latter two en-zyme activities drop precipitously after reaching their peaks,but catalase activity decreases only to approximately 40% ofits peak and maintains a plateau level for several days (Fig. 2)as the others disappear. The activities of catalase and glycolateoxidase reach plateau levels simultaneously and maintain themfor at least 4 days thereafter.To establish the particulate nature of the enzymes under

study, the homogenates were fractionated by differential cen-trifugation. Approximately 20 to 40% of total homogenate ac-tivity was recovered in pellet form; the recoveries, althoughlow, compare well with values reported by others (11, 49).The specific activities of the enzymes from homogenates and10,800g pellets are compared in Figure 3. In almost all casesthe specific activities are substantially higher in the resus-pended pellets than in the original homogenates. That the10,800g pellet is a rich source of microbodies is shown in Fig-ure 12, a representative electron micrograph of such a pelletfrom 4 day old cotyledons.

Microbodies in situ were found to contain catalase activityat days 3, 5, and 7 by incubation of cotyledonary segments inDAB. Osmium black reaction product attributable to catalaseactivity was specifically localized in microbodies as it had beenin previous work in this laboratory (16, 17, 55, 56). Tissueincubated in both DAB and aminotriazole showed no reactionproduct in the microbodies.

Confirmation of the localization of these enzymes in micro-bodies was provided by equilibrium centrifugation on con-tinuous sucrose gradients, a technique widely used to charac-terize glyoxysomes and both leaf and animal peroxisomes.Shown in Figure 4 are the results of such experiments inwhich 1,000g supernatants obtained from 3-, 4-, 41/2-, and6-day-old cotyledons were used as the applied sample in thezonal rotor. Marker microbody enzymes banded at a buoyantdensity of approximately 1.26 g/cm'. The fractions werenearly free of contamination with mitchondrial marker en-zymes and chlorophyll. Electron microscopic examination ofthe peak fractions from day 4 showed that microbodies werethe only intact organelles, although some fragments of chloro-plasts and mitochondria were evident.

That the change in enzyme activities observed in homog-enates during postgerminative growth is attributable toenzymes localized in microbodies can be determined by com-paring appropriate enzyme activities associated with micro-bodies isolated on sucrose gradients in a zonal rotor. The ac-tivities of microbody enzymes isolated in particulate form atvarious stages of greening are presented in Figure 4. In TableI are summarized the total and percentage activities of theseenzymes recovered in the density range 1.230 to 1.275 g/cm'.From these data it is apparent that the particulate isocitratelyase activity rises to a peak at day 4, then drops precipitously,whereas the particulate glyoxylate reductase and glycolate oxi-dase activities increase rapidly from days 3 through 6. Hencethe enzyme activities recovered from a region of the sucrose

464

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gradient containing microbodies as the only intact organelle,although only a fraction of the total applied activities, faith-fully reproduce the enzyme profiles obtained by assaying ho-mogenates at the same stages (Fig. 2). The amount of pro-

tein recoverable in the range 1.230 to 1.275 g/ cm: increasessharply between days 2 and 4 (Table I). Thereafter, theamount of protein in the microbody region of the gradientremains relatively constant over the time interval studied,whether expressed as ,ug protein per cotyledon or as a per-

centage of the applied protein. It should be pointed out,however, that these values probably do not accurately repre-

sent only microbody protein since electron microscopic ob-servation of material from this region of the gradient generallyreveals some contaminating fragments of other cellular com-

ponents.Ultrastructural Observations of Light-grown Cotyledons.

Cotyledons similar to those selected for enzyme assays were

examined in the electron microscope to establish the changingin situ relationships between microbodies and other cellularcomponents and to aid in the interpretation of their metabolicfunctions. The morphological term "microbody" is used todescribe the glyoxysomal and peroxisomal particles since it isimpossible to distinguish ultrastructurally between them.The cells of the cotyledons observed 2 days after germina-

tion are packed with droplets of storage lipid (Fig. 6). Identi-fication of the microbodies in the early stages of cotyledonarydevelopment is based on the criteria of size and granularityused by Gruber et al. (22). Microbodies (presumably glyoxy-somes) are squeezed among lipid bodies, as are proplastids,promitochondria, and storage protein bodies. The membranesystems of the various organelles, including those of the endo-plasmic reticulum and Golgi, as well as the mitochondrialcristae and plastid thylakoids, are difficult to detect at thisstage.

Three days after germination, the cotyledons have justemerged above ground and are beginning to green (Fig. 1).Activities of the glyoxylate cycle enzymes are relatively highand are increasing, whereas that of glycolate oxidase is justbeginning to rise. The cells are still packed with storage lipid,but are now more hydrated and their membrane systems are

more obvious (see mitochondrial cristae and Golgi mem-

branes, Fig. 7). The microbodies interspersed among the lipidbodies can be positively identified at this stage since theirsingle limiting membrane can be clearly seen. They fall intoa narrow range of sizes and have a matrix homogeneous inappearance. A few of the microbodies show a close associa-tion with short profiles of granular ER.The greening cotyledons at day 4 still contain considerable

storage lipid at the time of peak activity of the glyoxylatecycle enzymes (Fig. 8). The microbodies are localized pre-

dominantly among lipid bodies toward the cell center and are

not found among the plastids which are located close to thecell wall. The plastids exhibit internal thylakoid differentia-tion indicative of an intermediate stage of chloroplast devel-opment (Fig. 8). Most of these 4-day microbodies show insectional view what appear to be invaginations of cytoplasmicmaterial, including ribosomes (Figs. 8, 11). The invaginationsare observed only infrequently in 3-, 5-. and 6-day-old ma-

terial and are not seen at all in 2- and 7-day-old cotyledons(see Fig. 5). It is not certain that the ribosomes within the in-vaginations are grouped into polysomes. although they appear

clustered in most cases. In some of the microbodies the invagi-nations may be enclosed so that they are no longer continuouswith the general cytoplasm. Support for this view is providedby the observation that microbodies pelleted from homoge-nates of cotyledons at day 4 still contain inclusions of ribo-somes (Fig. 12).By day 5. the green cotyledons have expanded, and the cells

465

Table I. Chaniges in Glyoxysomal anld Peroxisomal EnizymeActivities anzd Proteini of Isolated Microbodies from

Cucuimber CotyledonsCucumber seedlings were grown under a 12-12 hr light-dark

cycle. Microbodies were isolated by centrifugation to equilibriumon a sucrose gradient in a zonal rotor. Enzyme activities and pro-tein contents represent totals recovered in the density range 1.230to 1.275 g,cm3. The numbers in parentheses represent the per-centages of the total applied samples recovered in this densityrange.

Enzyme Activities and Protein Content

Day

2

3

4

41 *v6

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Specific Activities

Is- Gly- ,GyIsocitrate Glyoxylate. Glycolate Protein citrate oxylate colate

lyase reductase oxidase Iyase e- soxidase

nmoles suibstrate consumned,' cotyledon nmoles sbstrate con-

min edon Ag. c otYld szumned/min-Ag protein

0.2 (4) 0.21.1 (13) 0.61.3 (15) 3.10.2 (7) 4.30.2 (3) 9.2

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(10) 0.30 8.9

(6) 112.1

70

60

50

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DAYS

FIG. 5. Percentage of total microbody profiles containing invagi-nations or inclusions at various stages of postgerminative growth in

cucumber cotyledons grown under the light-dark cycle. Values were

determined from counts with electron micrographs and sections ob-

served under the electron microscope.

have lost most of the storage lipid seen earlier (Fig. 9). These

changes have been accompanied by a sharp decline in the ac-

tivity of the glyoxylate cycle enzymes. By this time the plastidshave developed much of the internal structure characteristic of

chloroplasts, and a large central vacuole is undergoing for-

mation in the cells. The microbodies are now peroxisomal in

enzyme complement and are seen near both plastids and

lipid bodies, but do not seem to be specifically associatedwith either. They are similar in size to the microbodies seen

at earlier stages and do not appear to include a populationof small forms. Some of the microbodies are observed inclose association with ER cisternae, however.The cotyledons between 6 and 10 days after germination

contain typical photosynthetic parenchymatous cells. A largecentral vacuole occupies most of the cell volume. while thecytoplasm forms a layer next to the cell wall. The micro-bodies are nearly always appressed to the chloroplasts (Fig.10). Reserve lipid droplets are no longer found. As mightbe expected from the micrographs, there is substantial glyco-

Plant Physiol. Vol. 48, 1971 MICROBODIES IN CUCUMBER COTYLEDONS



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- o-:, ,+.._Bg.;s> S.

.-

ji. I ...:-

(.E , '

FIGS. 6, 7, and 8. Electron micrographs of cucumber cotyledonary cells 2, 3, and 4 days after germination in the light. Fig. 6. Lipid bodies (L)

occupy a large part of the cell volume and are packed around microbodies (Mb). Membranes are poorly defined, presumably because of the poor

cellular hydration. X 25,600. Fig. 7. Microbodies (Mb) interspersed among numerous lipid bodies (L) have obvious single limiting membranes.Mitochondria (M) and Golgi stack (G) membranes have become better defined as the cells have become more hydrated. X 20,900. Fig. 8. Micro-bodies (Mb) are located in the central portion of the cell, where vacuoles (V) are forming, and are distributed among the lipid bodies (L). Char-acteristic cytoplasmic invaginations into microbodies (arrows) are seen at this stage of growth. Plastids (P) exhibiting internal membrane differen-tiation are found toward the periphery of the cells next to the cell wall (W). X 8,600.

466

*7

.

9 .-l

1% " I'A

,

i . --R;e ..w w *

.tI5;

X. s.

s w i

c. w

.'

r.44a.

Iqli.I, z44 IL-/ >

w )-11

1. I

*.

I;

a% *,.

.20.-A?r,-77

I"t4

p ,



Plant Physiol. Vol. 48, 1971 MICROBODIES IN CUCUMBER COTYLEDONS 467

V¶.1' _~~~~~~~~~~19iia~~~~~~~~~~~~~~~~~~~'-0P~~~~~~~~~~~~~~~~~~~*I. I,~~~~~~~~~~~~~~~~~.j

Mb:-~~~~~~aIt -, Ln'>~~~~~~~~'"5

t F~~~~~~~~~~~~~~~~~~~~~~J.I' 4-

v~~~~~~~~~~~~~~~~~r

/ U~~~~~~~~

V~~~~~~~~~~~A~~~~~~~' 4~~~~~ *~~~~~~~~*bl'~~~~~~II.44JI KWp~~~~~~~~~~~W

P/x%G.I

I.--4~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ -fk.&~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

1.~~~~~ .. Mix P~~~~~~~~~tc ¼%~~~~~~~~~~~~~~~~~~ 4XS\ C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4.J~ ~ a -'V ' r fI f<.Z~~~~ ~ ~ ~ ~ ~ ~ ~ ~~~Ae

N ~ ~~~~ (

FIGS. 9 and 10. Electron micrographs of cucumber cotyledonary cells 5 and 7 days after germ-ination in the light. Fig. 9. The cells contain en-larging central vacuoles (V) and a peripheral layer of cytoplasm in which there are chloroplasts (P), microbodies (Mb), and residual lipid bodies(L). Microbodies are in contact with both chioroplasts and lipid bodies (L). X 18,900. Fig. 10. Microbodies and a mitochondrion adjacent tochloroplasts mn the penipheral cytoplasm. The center of the cell is occupied by a large vacuole (V). X 33,700.

FIo. 11I. Microbodies with cytoplasmic -invaginations in a different 4-day, light-grown cucumber cotyledonary cell from that shown in Figure 8.The cytoplasmic pockets in the microbodies contain clustered ribosomes in a matrix less electron opaque than that of the cytoplasm. X 20,200(

FIG. 12. Electron micrograph of a portion of 10,800g pellet enriched with microbodies obtained from 4-day, light-grown cucumber cotyledons.A single membrane limits the microbodies (Mb), whereas an envelope consisting of two membranes bounds the mitochondria (M). Arrows indicateportions of cytoplasm containing ribosomes included within microbodies. X 21,600.




z IU.0g 91LLI.8

7006

5-

> 4

03

w 2I

N

Zl1

2 4 6

DAYS

8

FIG. 13. Changes in glyoxysomal and peroxisomal enzyme ac-tivities in homogenates of cucumber cotyledons germinated andgrown in complete darkness. See Figure 2 for units of enzyme ac-

tivity.

0Iw

I-0

w

N

ZIw

10.

9'LIGHT

Bt t ISOCITRATE,X LYASE

7

6 l LYGLYCOLATE

5-

4-

MALATE---'i:

SYNTHETASE ! |

02

4 6 8 '0

FIG. 14. Changes in glyxoysomal and peroxisomal enzyme ac-tivities in homogenates of cucumber cotyledons exposed to a 12-12hr light-dark cycle after being germinated and grown in completedarkness for 5 days. Assays were done at 12-hr intervals. See Figure2 for units of enzyme activities.

Table lI. Chaniges in Gljyoxysomal anid Peroxisomal En-zymeActi ivilies of Honiogenates from Caciunmber Cotyledons

Cucumber seedlings were grown in the dark for 10 days and thenexposed to light. For catalase, I unit is that amount of enzyme re-

quired to catalyze the decomposition of 50(, of the H5O., presentper tOO sec at 25 C.

Isocitrate AMalate Glycolate Glyoxylate ('atalaseLyase Synthetase Oxidase Reductase

nmii.oles szubstrate contstu

Dark (10 days)Dark + 12 hrDark + 27 hrDark + 51 hr

4.6

4.3

2.0

0.6

79

47

nuled m1ti csotyledon 8 cotyledon

0.7 31 11.4

1.4 55 7.24.3 64 6.9

6.0 120 8.4

late oxidase activity in these cotyledons, but no activity ofthe glyoxylate cycle enzymes.

DARK-GROWN SEEDLINGS

Cotyledons from etiolated cucumber seedlings were ho-mogenized at 24-hr intervals from the 2nd through 10thday of postgerminative growth to determine changing ac-

tivities of glyoxysomal and peroxisomal enzymes. These ac-

tivities were correlated with ultrastructural observations madeon the same cotyledons, or in a few cases, on others grownunder the same conditions. At two time periods (days 5 and10), seedlings were exposed to light for various lengths oftime, and the ultrastructural and enzymological changes inthe microbodies were analyzed in a similar manner.The dark-grown cotyledons emerged above ground level

on the same day as light-grown seedlings, but from days 3through 10 remained yellow as they were hooked and pressedtogether. After exposure to light for 12 hr, the cotyledonsspread apart, expanded, and turned yellow-green. During thenext 39 hr. they continued slowly to expand and becamedeep green, except for a thin yellow margin.Enzyme Activities. The enzyme profiles of cotyledons

grown in the dark for 10 days (Fig. 13) show several differ-ences from those determined for light-grown seedlings overthe same time period (cf. Fig. 2). Isocitrate lyase activityexhibits the typical rise and fall, but declines only to approxi-mately 40% of its peak activity, then undergoes a secondincrease, and finally a gradual decrease to about one-sixthof its peak activity. Malate synthetase and catalase activitieshave profiles similar to one another throughout the dark pe-riod, whereas in the light-grown plants the profiles weresimilar only during the first 4 days. The profiles show twoapparent peaks for both enzymes; these differ slightly in timefrom the two apparent peaks of isocitrate lyase activity.Both malate synthetase and catalase decrease rapidly in ac-tivity after day 7, with malate synthetase falling to about40% of its peak activity. By day 10, catalase has decreasedto approximately 60% of its peak activity, i.e., to nearly thesame activity found after 10 days of growth in the light. Asexpected, glycolate oxidase activity was quite low through-out the dark period, but significant activity was always de-tected after day 3.

In the experiment shown in Figure 14 the seedlings weregrown in the dark for 5 days before exposure to 60 hr of thenormal 12-12 hr light-dark cycle. This length of dark periodprior to illumination was chosen because it was shown inan earlier experiment (Fig. 13) that isocitrate lyase activitywas at a peak at day 5 and declined thereafter; it was an-ticipated that exposure of the plants to light at this timewould cause a rapid disappearance of this activity. The pro-files show, however, that isocitrate lyase and malate syn-thetase activity actually increased during the first 12 hr oflight exposure and then decreased rapidly. During the first36 hr of light, glycolate oxidase activity also increased veryrapidly, but from an extremely low level of activity in thedark. The slight decrease shown between 36 and 48 hr ap-parently was in response to the dark half of the 12-12 hrlight-dark cycle. Thus the profiles obtained in this way werevery similar to those seen in light-grown plants, with the over-lap of relatively high glyoxysomal and peroxisomal activitiessimply delayed by approximately 2 days.

Significantly different results were obtained when the plantswere grown in the dark for 10 days before being exposed tolight, since activities of the glyoxylate cycle enzymes did notincrease during the first 12 hr of light under these conditions(Table II). Instead, isocitrate lyase activity decreased slightlyduring the first 12 hr and nearly one-half of the dark malatesynthetase activity was lost. The activities of both glyoxylatereductase and glycolate oxidase nearly doubled from the levelsdetected in the dark. Both activities continued to increaserapidly during the 51-hr period of the light-dark cycle; glyco-late oxidase activity increased 9-fold, whereas glyoxylate re-ductase activity rose approximately 4-fold. Catalase activitydropped sharply at first, then leveled off. The activity of iso-

,SOCITRATE MALATELYASE--X' .-JKSYNTHETASE

I/ . *W*e l i CATALASE

2" GLYCOLATEOXIDASE

[A.1

l I = "I

468 TRELEASE ET AL.




citrate lyase, on the other hand, had become essentially un-detectable by the end of this period.

Ultrastructural Observations of Dark-grown Cotyledons.Cotyledons similar to those selected for enzyme assays at days4, 7, and 10 in the dark, and at 12 and 51 hr light exposureafter growth in the dark for 10 days, were examined in theelectron microscope to determine the extent of changes in(a) the in situ relationships between microbodies and othercellular components, (b) microbody fine structure and size,and (c) the numbers of microbodies per cell.The cells of cotyledons observed after 4 days of growth

contained numerous microbodies and were packed with stor-age protein and lipid bodies (Fig. 15A). Of common occur-rence were pockets with finger-like projections in the lipidbodies containing what appear to be ribosomes (Figs. 15, Aand B). In contrast to plants of the same age grown in thelight, fusion of small vacuoles seemed to be at a minimum,since most of the storage protein was still contained withinthe protein vacuoles (Fig. 1 5A). During this period whenthe activities of the glyoxylate cycle enzymes were relativelyhigh (Fig. 13), the microbodies were located primarily amongand closely appressed to lipid bodies. The microbodies wererelatively large and their matrices uniformly dense. Invagi-nations of cytoplasm containing ribosomes were frequentlyseen in the microbodies, usually as several pockets (Figs. 15,A and C). Only rarely were microbodies seen in contact withendoplasmic reticulum. Plastids were located mostly near thecell walls; they did not exhibit any granal membrane forma-tion but contained conspicuous prolamellar bodies.The cells at day 7 were much more vacuolate (Fig. 16A).

The cytoplasm contained numerous microbodies and sub-stantial storage lipid, the microbodies being dispersed amonglipid bodies rather than in association with plastids, as wouldhave been the case for light-grown plants of the same age.Although the microbodies remained unchanged in generalsize range, their matrix appeared less homogeneous and moreflocculent in texture than at day 4. Cytoplasmic invaginationsinto microbodies were present but were less frequent thanat day 4 and seemed to occur as single pockets and to occupymore space (Fig. 16, B and C).A large single vacuole containing remnants of storage pro-

tein occupied most of the cell volume at day 10. Microbodieswere still commonly observed within the cells and occasion-ally contained invaginated pockets of cytoplasm (Fig. 17).Lipid bodies were much less numerous than at day 7. Someof the microbodies in the peripheral cytoplasm did not ap-pear to be appressed to either plastids or lipid bodies (Fig.18), whereas in other instances some microbodies were seennear both cellular components (Fig. 17). The matrix of themicrobodies was not homogeneous, but was characteristi-cally flocculent in texture. Only rarely were microbodies seenassociated with endoplasmic reticulum, the profiles of whichwere noticeably lacking in the cells throughout the dark pe-riod.

Using the electron microscope, the number of microbodyprofiles per cell section was counted in both palisade andspongy mesophyll cells at days 4. 7. and 10. In addition, celldimensions were measured in sections 0.5 ,um thick withthe light microscope to determine changes in cell size duringthis period. The data presented in Table III indicate thatwith the possible exception of a decrease in mean width ofthe palisade cells, little change in cell size occurred fromdays 4 through 10. On the average, the number of micro-bodies seen per cell section decreased from days 4 through10 from about 8.1 to 6.2 in the palisade cells and from 6.3to 4.1 in the spongy cells. Since the cell size did not changeappreciably, the number of microbodies can be directly com-

pared; the data show that the majority (about 65-75%) of themicrobodies present at day 4 apparently persist at leastthrough day 10 under our conditions.

Transformation of plastid thylakoid membranes into granalstacks was characteristic of the vacuolated cells of cotyledonsgrown in the dark for 10 days and then exposed to 12 hrof light (Figs. 19, 20). The activity of the peroxisomal en-zymes, glycolate oxidase and glyoxylate reductase, nearlydoubled during the 12 hr of light (Table II), and the micro-bodies seemed to show a more preferential relationship withthe developing plastids (Figs. 19, 20). The matrix of themicrobodies was more homogeneous than it was in 7 and10 day dark-grown plants. In some of the microbodies cyto-plasmic invaginations were still present as single pockets(Fig. 19). It is perhaps noteworthy that a significant numberof microbodies observed were more elongated than thoseseen before exposure to light (Fig. 20); this apparently in-volved the transformation of large microbodies already in ex-istence, since small ones were not observed. Profiles of endo-plasmic reticulum appeared to be more numerous, but theywere infrequently associated with microbodies.At the time when the activity of glyoxylate cycle enzymes

had become nearly undetectable and that of the peroxisomalenzymes had become relatively high (51-hr exposure to light),the close association between the microbodies and the well-developed chloroplasts was the most striking feature in thecells (Figs. 21, 22). The relatively large microbodies withmostly homogeneous matrices were nearly always appressedto the surfaces of chloroplasts. In some instances, the shapeof the microbody was irregular and conformed to chloro-plast shape, whereas in others the microbody retained itsrounded outline and the chloroplast was indented to ac-commodate it (Figs. 21, 22). These associations apparentlydid not arise solely because of spatial restrictions in the nar-row peripheral cytoplasm, since some microbodies were ob-served closely appressed to chloroplasts even when there wasconsiderable free cytoplasm nearby (Fig. 21), and micro-bodies were not seen in regions of cytoplasm not occupiedby chloroplasts. Extensive examination of the cells at thisstage failed to reveal any microbodies with cytoplasmic in-vaginations or inclusions.

DISCUSSION

We have reported previously (22) the results of a correlativeultrastructural and enzymological study of microbody changesat three stages of postgerminative growth in sunflower coty-ledons. Microbodies progressed from catalase-containing parti-cles located among lipid and protein bodies (day 1), to gly-oxysomes closely associated with lipid bodies (day 4), toperoxisomes frequently appressed to chloroplasts (day 7).Using a similar correlative approach with cucumber cotyle-Ions grown both in the light and dark, we have now studiedthe timing of the events on a daily basis, with emphasis onthe transition from glyoxysomal to peroxisomal metabolism.

It was found that microbodies were prominent in the cellsof cucumber cotyledons at all stages examined. The follow-ing three criteria were used to establish that these micro-bodies contained enzymes characteristic of glyoxysomes andperoxisomes: (a) the observation of increased specific activ-ities of these enzymes in microbody-enriched pellets ob-tained by differential centrifugation, (b) the detection of theseenzymes at a density of about 1.26 g/cm' when clarified ho-mogenates from different stages of development were cen-trifuged to equilibrium in a zonal rotor, and (c) the localiza-tion of catalase in microbodies cytochemically.The glyoxylate cycle functions only for a few days in coty-

469Plant Physiol. Vol. 48, 1971



1h4 4A~~~~~~~~

-n't~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

VW . Vt

~~ir~~~A~~F. 4-~~~4

*~~~1r4'~~~"AV.. vs~~~~~~~~~~~~~~~~~~~~1

W;V~~~~A

~~=½,- Mb~~~61

a 7~~4r0'r-34 A~~v..A

-(-4- '..*

= .4.*1..*'.. 9-

6 .- '½

<4 7,;1. --....

AV t.YW

C

.t..ww&pt.hr. N

N;?

B

n-i

..t~~M.7

..'t.e_ ~ ~ ~

i. ': :

FIGS. 15, 16, 17, and 18. Electron micrographs of cucumber cotyledonary cells at 4, 7, and 10 days after germination in the dark. Fig. 15A:microbodies (Mb) with homogeneous matrices at day 4 are distributed among lipid (L) and protein bodies (PB). Several small pockets of cyto-plasm have invaginated into some of the microbodies (arrow). X 16,600. Fig. 15B: a pocket of cytoplasm within a lipid body. X 26,600. Fig. 15C:enlarged view of a microbody exhibiting a cytoplasmic invagination containing ribosomes. X 29,400. Fig. 16A: microbodies (Mb), at day 7 withflocculent textured matrices, distributed near lipid bodies (L). X 37,800. Figs. 16, B and C: views of cytoplasmic invaginations into microbodies atday 7. X 36,400 and 29,700. Fig. 17. Microbodies (Mb), at day 10 with flocculent textured matrices, located near both lipid bodies (L) and plastids(P). Conspicuous prolamellar bodies are visible in the plastids. X 25,600. Fig. 18. Microbodies (Mb), at day 10 exhibiting nonhomogeneous mat-rices, situated in the peripheral cytoplasm not associated with other organelles. Vacuole (V). X 23,000.

470 www.plantphysiol.orgon August 20, 2020 - Published by Downloaded from

Copyright © 1971 American Society of Plant Biologists. All rights reserved.



Table III. Changes in Cell Dimenisionis anid Microbody Numbers inEtiolated Cucumber Cotyledons

The greatest length and width of palisade and spongy mesophyllcells in etiolated cucumber cotyledons were measured with a cali-brated ocular micrometer; the values given are means i standarddeviations. The numbers in parentheses indicate total numbers ofcells measured. The number of microbody profiles seen per cellsection was obtained by examination of cotyledon sections withthe electron microscope (see "Materials and Methods"). Valuespresented are means i standard deviations; numbers in paren-theses indicate total numbers of cells in which microbodies werecounted.

Day 4 Day 7 Day 10

Palisade cellsCell length, A 77 i 9 (112) 66 i 3 (210) 75 ± 7 (279)Cell width, MA 14 i 3 (96) 11 i 1 (140) 9 ± 2 (305)Microbodies 8 i 4 (76) 8 i 4 (75) 6 i 4 (75)

Spongy cellsCell length, A 49 + 12 (144) 39 + 13 (166)144 ± 13 (336)Cell width, 1919 ± 8 (185) 21 ± 5 (179) i20 + 10 (326)Microbodies 6 + 4 (81) 6 i 4 (76) 4 ± 2 (78)

ledons of light-grown cucumber seedlings, but remains op-erative longer when the plants are kept in the dark. The risein activities of isocitrate lyase and malate synthetase in light-grown seedlings occur at a time when microbodies (glyoxy-somes) are interspersed among lipid bodies, and the persistenceof these activities in dark-grown plants correlates well withthe prolonged presence of lipid reserves. It has been shownthat in corn embryos and peanut cotyledons the lipid bodiesconsist mainly of triglycerides (27, 52). Glyoxysomes canactivate fatty acids (10), degrade them to the 2-carbon level(12, 26), and convert the 2-carbon fragments to succinate,thereby effecting net gluconeogenesis. The characteristic as-sociation between glyoxysomes and lipid bodies reported hereand elsewhere (22, 56) probably reflects their mutual involve-ment in a net gluconeogenic process. The catalase profileparallels the changes in the glyoxylate cycle enzymes both inlight- and dark-grown plants, suggesting that H202 generationmay accompany S-oxidation in the cucumber cotyledon as hasbeen postulated for castor bean endosperm (12). The pro-jections of the cytoplasmic pockets seen within lipid bodies(Fig. 1SB) in dark-grown seedlings resemble the "channels"described in isolated lipid bodies of castor bean endospermsubjected to degradation by lipase (42). It may be that thesepockets are the corresponding in situ manifestations of lipidbody degradation.The profiles of isocitrate lyase and malate synthetase ac-

tivities show two apparent peaks in the dark. Hock and Beev-ers (24) showed that these enzymes are synthesized de novoin watermelon cotyledons during the first three days of germi-nation, presumably on a relatively stable messenger RNA.The eventual decline in the activities appeared to be a conse-

quence of limited enzyme half-life (2 to 3 days). Hock latershowed (23) that isocitrate lyase activity is inhibited by whitelight in the same tissue, presumably because continued for-mation of the enzyme is suppressed by the availability ofcarbohydrate provided by photosynthesis. It has also beenshown that isocitrate lyase activity is suppressed when Cu-curbita cotyledons are floated on glucose (30). The secondarypeaks of isocitrate lyase and malate synthetase activitiesfound in our etiolated cotyledons may therefore result fromrenewed synthesis of these enzymes occasioned by a needfor further lipid catabolism, the lack of suppression of this

synthesis by products of photosynthesis, and the turnover ofthe original enzyme molecules.A preferential association of microbodies with plastids be-

came evident in light-grown plants only after reserve lipidhad been mobilized and glycolate oxidase had reached near-peak activity. In dark-grown plants, this association oc-curred only upon exposure of the cotyledons to light, afterthe etioplasts had differentiated into chloroplasts and glyco-late oxidase activity had increased 9-fold. This observedassociation with chloroplasts, also reported in a variety ofleaves (15. 16) and in sunflower cotyledons (22), seems tobe more than fortuitous, since peroxisomes are rarely seenin regions of the cytoplasm not occupied by the chloroplasts.It appears, therefore, that both in light-grown cotyledonsand in etiolated cotyledons exposed to light, the preferentialassociation of microbodies with plastids is related to increasedglycolate oxidase activity, which in turn is presumably de-pendent upon the production of glycolate by photosynthesiz-ing chloroplasts (50). Hence the close spatial relationship be-tween the two organelles seen here under these conditionslends support to the conjecture of Frederick and Newcomb(15) that there is an interaction between the two organellesthat probably reflects their mutual involvement in glycolatemetabolism.One of the principal aims of the studies described in this

and a previous paper (22) has been to examine the relation-ship which exists between the glyoxysomes present in fat-storing cotyledons during early lipid-degrading stages and theperoxisomes found later on at photosynthetic stages. Severaldifferent models can be postulated to explain this succession.An obvious possibility (model 1) assumes glyoxysomes andperoxisomes to be separate populations of microbodies, withone population replacing the other during growth and devel-opment. According to this model, the decline in glyoxysomalenzyme activities in both light- and dark-grown cotyledonswould represent the degradation and disappearance of onepopulation of microbodies, and the rise in peroxisomal en-zyme activities would be due to de novo synthesis and re-placement by a different population of particles.Our studies with light-grown cucumber seedlings provide

preliminary evidence bearing on this point. As the cotyle-dons spread and become green between days 4 and 5 in thelight, the rapid depletion of reserve lipid is accompanied bya rapid decline in activities of glyoxylate cycle enzymes,similar to that seen in castor bean endosperm. Gerhardt andBeevers (20) reported that the specific activities of the glyoxy-somal enzymes isolated from sucrose gradients remained con-stant as the enzyme activities declined in castor bean endo-sperm and concluded that a net destruction of glyoxysomesoccurs in this tissue. In contrast, the specific activity of par-ticulate isocitrate lyase isolated from our light-grown coty-ledons decreased as lipid reserves were depleted. because thedecline in enzyme activity was not accompanied by a com-parable decrease in the amount of protein recoverable in themicrobody region of the sucrose gradient (Table I). Thus,these data do not suggest a net destruction of glyoxysomes aspostulated for castor bean endosperm. Unlike castor beanendosperm, however. the cucumber cotyledon is not devoidof function after depletion of lipid reserves, since it expands,becomes green, and persists as a photosynthetic organ. Theincrease in peroxisomal enzyme activities which accompaniesthe decrease of glyoxysomal activities in light-grown cotyle-dons sugrests that a possible net loss of glyoxysomal proteinmight well be obscured by a concomitant increase in peroxi-somes. Because of this comnlication, the relationship betweenglyoxysomes and peroxisomes probably cannot be exploredadequately by measuring changes in enzyme activities and





5'./~ f*' +\

# | ,j :..>'t

19 rI8 'i

-S ,Cffitt' _*Rr Y o'jta 8 <

a.

t A91*f-

3 ~ ~ ~ ~ . 4-.m44 ,

r0' J,

; ~ ~ 1^'a'L?s

;.6,-1Mt

'a

,''A

"'t am.

.44~~~~~~~

tI

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rM bIt

a- ! ' ::;. < .

: _ ''-Mb L

4 k . *.i ,{ .? -s,. . -

FIGS. 19 and 20. Electron micrographs of cucumber cotyledonary cells from plants grown in the dark for 10 days and then exposed to 12 hr oflight. Fig. 19. A microbody situated near both a developing plastid (P) exhibiting granal membrane formation from the prolamellar body, andlipid bodies (L). A pocket of cytoplasm is visible within the microbody. X 32,409. Fig. 20. Elongated microbodies (Mb) located near the surfaceof a plastid (P). Vacuole (V). X 28,300.

FIGS. 21 and 22. Electron micrographs of cucumber cotyledonary cells from plants grown in the dark for 10 days and then exposed to 51 hr ofan alternating 12-12 hr light-dark cycle. Fig. 21. A microbody (Mb) closely appressed and indented into the surface of a well-developed chloro-plast (P). X 33,700. Fig. 22. One microbody (Mb) indented into surface of the chloroplast with the other squeezed between adjacent chloroplasts(P). X 48,600.

472 TRELEASE ET AL.




protein content of gradient fractions obtained light-

grown cotyledons.On the other hand, observations of the cells light-grown

seedlings with the electron microscope during

period should have provided some evidence

of peroxisomes or degradation of glyoxysomes,

both events in fact occur. One possible mechanism

degradation of glyoxysomes would involve lysosome-like

tivity. In castor bean endosperm, Vigil (56)

at least some microbodies disappear in toto

into autophagic vacuoles as the cells age.

ledons, a large central vacuole forms in the

with the decline in glyoxysomal enzyme

apparently a result of the hydrolysis of

fusion of the residual vacuoles (1). However,

tural evidence suggesting the incorporation

any other organelles into vacuoles has been

present study.The possibility that glyoxysomes were not

down in vacuoles but were undergoing autolysis

depletion was also examined, but no electron

evidence for partially degraded or disintegrating

in the cytoplasm was obtained, even though

were made at 24-hr intervals. Of possible

quent invagination of cytoplasm into microbodies

and in other cotyledonary microbodies (54),

of ribosomes within these invaginations

synthesize the lytic enzymes required to degrade

However, no evidence for a sequential

struction of invaginated microbodies was

the invaginated microbodies always appeared

intact, both in situ and in gradient fractions.

acid phosphatase, a lysosomal marker enzyme,

tected in the microbody gradient fractions

lease and Becker, unpublished observations).

possible that proteolytic activity in the microbodies

responsible for the loss of specific enzymes

the glyoxylate cycle and that the invaginations

festations of this activity, it appears unlikely

structural observations that this could involve

of a major fraction of the microbody population.

The electron microscope should also have

detecting the possible de novo synthesis

which is the other principal aspect of model 1.

newly-formed particles should be observed

menting from pre-existing microbodies,

clofibrate-treated rats (32) and in regenerating

forming from endoplasmic reticulum, which

ferred as the site of origin of microbodies

to support either possibility was seen at any

Although our fine-structural observations

that cotyledonary microbodies were being

thesized de novo in light-grown seedlings,

could not be obtained to evaluate model Ireplacement of microbody populations

scured by the substantial overlap in glyoxysomal and peroxi-

somal function and by the similar morphological appearance

of the two kinds of microbodies. Using etiolated cotyledons,

however, the model could be tested more rigorously, since we

were able to obtain an eventual decline in glyoxysomal en-

zyme activities without the usual complicating rise in peroxi-

somal enzyme activities. Under these conditions, we did not

see a decrease in microbody numbers comparable either to

the decline in glyoxvlate cycle enzymes (Fig. 13) or to the

decrease reported by Kagawa and Beevers (28) in the protein

of microbodies isolated from etiolated watermelon cotyle-

dons. The average number of microbody profiles per cellsection of dark-grown cotyledons decreased by only about25% (palisade cells) to 35% (spongy cells) between days 4and 10. These values for cell section profiles are probably a

good indication of the changes in total microbodies per cell,since our measurements of cell dimensions indicate thatneither the palisade nor the spongy cells underwent appreci-able changes in size during the dark period.

In addition, no evidence was found to suggest a de novosynthesis of peroxisomes upon exposure of the etiolated seed-lings to light. The presence of low but consistently detectablelevels of glycolate oxidase in etiolated cotyledons and therapidity with which the activities of both glycolate oxidaseand glyoxylate reductase increase upon exposure to light in-dicate that the enzyme activities increase without an accom-panying synthesis of the requisite particles. Furthermore, nosmall, presumably newly-synthesized microbodies were seenfragmenting or budding from ER upon exposure of the dark-grown cotyledons to light. These observations indicate that atleast in cucumber cotyledons the transition from glyoxysomalto peroxisomal function does not involve the actual replace-ment of one population of microbodies by another.An alternative explanation (model 2), also based on the con-

cept of two distinct kinds of particles, presumes the coex-istence in the cotyledonary cells of both populations of micro-bodies, each active in cellular metabolism at a different stageof development. The transition in function from lipid degra-dation to primarily glycolate metabolism would in this caseinvolve the acquisition or activation of enzymes in presump-tive peroxisomes and the inactivation or degradation of en-

zymes in glyoxysomes, possibly accompanied by the break-down of the glyoxysomes themselves.

Still another mechanism (model 3) by which the successionfrom glyoxysomal to peroxisomal activity might occur in-volves a change in enzyme complement within a single on-

going population of microbodies. In this model, particleswould not need to be either synthesized or degraded during thedevelopment stages under consideration, but would simply un-dergo changes in enzyme content and metabolic capability.

That microbodies are capable of changing their enzymecomplement is indicated in one instance by the experimentsof Longo and Longo (35) with peanut cotyledons. Theyfound that the level of isocitrate lyase can be strongly de-pressed with glucose, but that the total protein in isolatedglyoxysomes did not change. In another instance, Legg andWood (33) found that catalase was inhibited in liver tissuewhen rats were injected with allylisopropylacetamide, butthere was no apparent change in the number and distributionof microbodies or in their relationship to other organelles, as

compared with the controls. A decrease in the microbodymatrix was observed under these conditions; perhaps a similarphenomenon occurs in etiolated cotyledons, where the lesselectron opaque appearance of the microbodies after day 4may be due to the progressive loss of glyoxysomal enzymes

under conditions not conducive to synthesis of peroxisomalenzymes.

It is crucial to the choice between models 2 and 3 as pos-

sible explanations of the transition from glyoxysomal to perox-

isomal function to determine whether the peroxisomalenzymes which undergo striking increases in activity in re-

sponse to light are located in virgin particles which haveserved no previous metabolic function (model 2) or in thesame pprticles which formerly housed glyoxysomal enzymes

(model 3). No definitive data bearing on this question are yetavailable. However, detailed fine-structural observations ofboth light- and dark-grown cotyledons have consistently pro-





duced no evidence suggesting a dichotomy in the microbodypopulation, either in morphological appearance or in asso-ciations with other organelles. We cannot, for example, dis-tinguish presumptive peroxisomes from functional glyoxy-somes during early stages, nor can we differentiate betweenenzyme-depleted glyoxysomes and active peroxisomes afterlipid depletion and exposure to light. We also find no evi-dence to suggest a category of microbodies which are ob-viously unassociated with lipid bodies during early stages, nordo significant numbers of microbodies fail to take up asso-ciations with chloroplasts during later stages. Our data aretherefore most consistent with the concept of a single popu-lation of particles which can undergo changes in enzyme com-plement and metabolic function.

Whether the transition from glyoxysomal to peroxisomalfunction involves a single kind of microbody capable ofchanging its enzyme complement or two kinds of particleswith different and nonoverlapping metabolic roles, some sortof mechanism must exist to degrade or inactivate glyoxysomalenzymes and to synthesize or activate peroxisomal enzymeswithin existing particles. There is some evidence to indicatethat RNA (9, 19) and DNA (9) occur in certain glyoxysomes.Ching (9) suggested from her data that the glyoxysomes inmegagametophyte tissue of pine seedlings can synthesizefunctional enzymes during germination. We have alreadypointed out that the frequent invagination of cytoplasm intomicrobodies seen in light-grown cotyledons during the periodof metabolic transition may represent a mechanism for gly-oxysomal degradation. It is, however, also possible that theinvaginations are involved in the acquisition of peroxisomalfunction through the synthesis of enzymes on the includedclusters of ribosomes. The invaginations were first seen whenglycolate oxidase activity became detectable, and their fre-quency was correlated strikingly with the increase in the ac-tivity of this enzyme (Fig. 5). No invaginations were observedafter the enzyme activity reached a plateau level. Similarinvaginations and inclusions were frequently observed in themicrobodies of the etiolated cotyledons and persisted untilshortly after exposure to light. There appears to be no prece-dent for synthesis of enzymes or other proteins on ribosomeswithin invaginations into organelles, but invaginations of thissort are not uncommon, and have been reported at certainstages in plastids (41) and mitochondria (2). Moreover, in theepidermal cells of spadix appendices in Sauroinatluin, micro-bodies increased in numbers during flowering, at which timeinvaginations into them were commonly observed (4), al-though no attempt was made to correlate the phenomenonwith any enzyme activity.The actual role, if any, of such invaginations in microbody

function is at present unknown, but their presence duringthe transition from glyoxysomal to peroxisomal function inlight-grown cotyledons and their persistence in etiolated coty-ledons until after exposure to light and attainment of highlevels of peroxisomal enzymes strongly suggest that they mayparticipate in some way in the acquisition or conversion ofmicrobody function. We are, however, still exploring ourearlier proposals that the characteristic association of micro-bodies with endoplasmic reticulum may represent a functionalrelationship involving the transfer of enzymes (22).

In summary, correlative fine-structural and enzymologicalstudies of light-grown cucumber cotyledons and of etiolatedseedlings before and after exposure to light allow us to con-clude that the developmental transition in microbody functionfrom glyoxysomal to peroxisomal metabolism almost certainlydoes not involve the actual replacement of one population ofmicrobodies by another. Rather, the transition probably oc-curs within at least some of the existing microbodies, either

by sequential functioning of two different kinds of microbodiesor by a change in enzyme complement within a single popu-lation of organelles. Neither of these possibilities can be ex-cluded at present, although our data appear to favor the lat-ter. In either case, the cytoplasmic invaginations seenconsistently in both light- and dark-grown cotyledons may bea possible mechanism whereby the loss, gain, or change inenzyme content is effected.

Ackiioirledyilqntt-The atitliot.s express their, sincere appreciation to 'MissLinda Crouse for her excellenit technical assistance.

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Documents

Microbodies (Glyoxysomes and Peroxisomes) in Cucunber ... · Glyoxysomes contain not onlyenzymes directly involved in lipid metabolism, but also catalase. In their possession ofcata-lase