Plant Physiol. (1981) 68, 430-4340032-0889/81/68/0430/05/$00.50/0

Nutritional Regulation of Organelie Biogenesis in Euglena12INDUCTION OF MICROBODIES

Received for publication December 9, 1980 and in revised form February 23, 1981

MARK A. HORRUM3 AND STEVEN D. SCHWARTZBACH4School of Life Sciences, University of Nebraska, Lincoln, Nebraska 68588

ABSTRACT

Exposure of dark grown resting Euglena to ethanol produced a transientincrease in the specific activity of the glyoxysomal enzyme malate synthase.Enzyme specific activity increased during the first 24 hours of ethanoltreatment and then declined. Light exposure or malate addition failed toincrease enzyme specific activity. The increase and decrease in enzymespecific activity represented changes in the amount of active enzyme. Inboth wild type cells and the plastidless mutant W3BUL, enzyme levels werealways higher in the dark than in the light.The specific activity of the peroxisomal enzyme glycolate dehydrogenase

began to increase 24 hours after dark grown resting Eugkena were exposedto light. Ethanol, but not malate, prevented the increase and promoted adecrease in glycolate dehydrogenase levels. Cycloheximide produced adecline in enzyme levels similar to the decline produced by ethanol addition.Glycolate dehydrogenase was present in the plastidless mutant W3BULindicating that it is coded in the nucleus and synthesized on cytoplasmicribosomes. Streptomycin, a specific inhibitor of chloroplast protein synthe-sis and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosyn-thetic CO2 fixation, inhibited the photoinduction of glycolate dehydroge-nase while having no effect on the photoinduction of NADP dependentglyceraldehyde-3-phosphate dehydrogenase, another light induced, nuclearcoded, cytoplasmically synthesized enzyme. Taken together, these resultssuggest that microbodies are continuaDly synthesized in resting Euglenaand their enzyme complement is determined through substrate inductionof glyoxysomal and peroxisomal enzymes.

Two types of specialized microbodies are found in higher plants(1). Glyoxysomes are found in the storage tissue of fatty seedlingswhere they are the site of fatty acid breakdown and the glyoxylatecycle. Glyoxysomes are formed during germination and are de-graded when the oxidation of stored fats is completed (17). Per-oxisomes are found in photosynthetic tissue and contain theenzymes for the irreversible conversion of glycolate to glycine andthe reversible conversion of serine to glycerate, key reactions ofthe glycolate cycle (28). Light acting through phytochrome inducesperoxisome formation (10). The synthesis of enzymes of both thereversible (hydroxypyruvate reductase) and irreversible (glycolate

' This research was supported by National Institutes of Health GrantGM26994, Biomedical Support Grant RR-07055, and funds from theResearch Council, University of Nebraska.

2This work was taken from a dissertation submitted to the graduatefaculty of the University of Nebraska by M. A. H. in partial fulfillment ofthe requirements for the PhD degree.

3 Present address: University of Colorado Health Science Center, De-partment of Microbiology, Denver, Col. 80262.

4 To whom reprint requests should be addressed.

oxidase) portions of the glycolate pathway is photoregulated (10,17). The formation of the peroxisome is, however, unrelated tothe synthesis of chloroplast enzymes and the development ofphotosynthetic competence (10, 11, 17). In fatty cotyledons whichbecome functional photosynthetic tissues upon light exposure, thedisappearance of glyoxysomes is hastened by light, the inducer ofperoxisome development (17). The actual role of light in glyoxy-some development is unclear.The control of peroxisome and glyoxysome biogenesis in Eu-

glena differs significantly from the control ofmicrobody biogenesisin higher plants. Glycolate dehydrogenase is found in the mito-chondria and peroxisomes of Euglena (8, 32). Exposure ofEuglenato light under conditions favoring phosphoglycolate synthesis(high 02 to CO2 ratio) induces the synthesis of peroxisomalglycolate dehydrogenase (18, 32). When Euglena are grown in thelight on air supplemented with 5% CO2, peroxisomal glycolatedehydrogenase levels are low and enzyme levels increase upontransfer to unsupplemented air (6, 18, 32). Even though light ispresent, the peroxisomal glycolate dehydrogenase accumulatesonly if phosphoglycolate can be formed. The inducibility ofperoxisomal glycolate dehydrogenase contrasts with the constitu-tive synthesis of hydroxypyruvate reductase (13, 16), anotherperoxisomal enzyme (8). Light exposure has no effect on hydroxy-pyruvate reductase levels (13, 16) suggesting that in contrast tohigher plants (1, 10, 17), in Euglena the reversible and irreversibleportions of the glycolate pathway are independently regulated.

Ethanol and acetate, specific repressors of light-induced chlo-roplast development in Euglena (14), induce the transcription ofthe gene for malate synthase (31), a glyoxysomal marker enzyme(8). Increased enzyme levels, therefore, result from de novo enzymesynthesis (29). Malate synthase levels are, however, lower in cellsgrown in the light (9, 21) suggesting that light, the inducer ofchloroplast development represses glyoxysomal development. Inthis paper, we use resting Euglena to study the interaction betweenlight and ethanol in the regulation of microbody biogenesis. Briefreports of this work were presented at the 1979 (12) and the 1980(13) annual meeting of the American Society of Plant Physiolo-gists.

MATERIALS AND METHODS

Euglena gracilis Klebs var. bacillaris Cori maintained in thedark for many years and the bleached mutant W3BUL derivedfrom this strain (obtained from Dr. J. A. Schiff, Brandeis Univer-sity) were used throughout this work. Conditions for cell growth(15), light-induced chloroplast development (15), the preparationof resting cells (15), the technique for washing cells (26), theconditions for inhibiting protein synthesis (26), the determinationof cell number (14), the determination of Chl content (14) and thecarbon supplementation of resting cells with 84 mm ethanol or 84mM L-malate (14, 15) have been described. DCMU (10 ,M finalconcentration) was dissolved overnight in resting medium, pH 5.0,and filter sterilized.

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MICROBODY BIOGENESIS IN EUGLENA

For the determination of enzyme activity, cells were harvested,washed, and resuspended in 50 mm Tris-HCl (pH 7.5); 10 mmMgCl2 (malate synthase); 50 mM K-phosphate (pH 6.75) (glycolatedehydrogenase) or 10 mm Tris-HCl (pH 8.0); 0.1 mm Na2 EDTA;10 mmi ,f-mercaptoethanol; 30% glycerol (NADP-glyceraldehyde-3-P dehydrogenase). Cells were disrupted by two 30-s bursts(malate synthase), one 40-s burst (glycolate dehydrogenase) orthree 30-s bursts (NADP-glyceraldehyde-3-P dehydrogenase) ofsonication. The sonicate was clarified by centrifugation at 12,000gfor 10 min. The respective supernatants contained all of theenzyme activity. For the determination of malate synthase andglycolate dehydrogenase activity, extracts were chromatographedon Sephadex G-25 columns prior to assay. All manipulations wereperformed at 0-4 C.

Activity of malate synthase (30), glycolate dehydrogenase (32)and NADP-glyceraldehyde-3-P dehydrogenase (14) was deter-mined as described previously. One unit of malate synthase hy-drolyzed 1 nmol of acetyl-CoA per min. One unit of glycolatedehydrogenase reduced 1 nmol of 2,6-dichlorophenolindolphenolper min. One unit of NADP-glyceraldehyde-3-P dehydrogenaseoxidized 1 nmol NADPH per min. Protein was determined by themethod of Lowry (19) using BSA as standard. The data presentedare the results from a typical experiment and each experiment hasbeen repeated at least three times. For all determinations, replicatesamples differed by less than 15%.

RESULTS AND DISCUSSION

Photoinhibition of Malate Synthase Induction. The addition ofethanol to dark-grown resting Euglena maintained in the light orthe dark produced a transient increase in the specific activity ofmalate synthase, a glyoxysomal marker-enzyme (Fig. 1). Enzymeactivity increased during the first 24 h after ethanol addition andthen declined. The extent ofmalate synthase induction in the dark(8-fold) was always significantly higher than in the light (4-fold).Nonsupplemented and malate-supplemented resting cells incu-bated in light or dark maintained a constant low enzyme levelduring the 72-h experimental period (Fig. 1). The changes inenzyme specific activity were not due to the presence in the cell-free extracts of low molecular weight effector molecules since theextracts were chromatographed on Sephadex G-25 columns priorto assay.

WILD TYPE

0 ° ° LIGHToR DARKX8 Q° LIGHTop DARK + MALATEV1 C LIGHT + ETHANOLZ6-ADARK +ETHANOLZ a. 60-

uwE< : 40-

20

00 24 48 72

TIME(HOURS)FIG. 1. Photoinhibition of the ethanol induction of malate synthase in

wild-type Euglena At zero time, the following compounds were added todark grown resting cells, half of each culture was incubated in the lightand half was incubated in the dark: no addition; 84 mM ethanol; 84 mmL-malate. Samples were removed at appropriate times and the specificactivity of malate synthase was determined. When the enzyme specificactivity for cells incubated in the light was not significantly different fromthe specific activity for cells maintained in the dark, the two cultures arerepresented by a single half shaded, half unshaded data point.

Table I. Activity ofMalate Synthase per ml of Culture in Carbon-supplemented, Dark-Grown Resting Euglena

Enzyme Units'/ml culture

Additions to 0 h 24 h 72 hResting Media

Dark Light Dark Light Dark LightNone 1.9 1.9 2.2 1.8 2.2 0.8Ethanol, 84 mM 1.9 1.9 28.3 15.2 10.0 3.7Malate, 84mM 1.9 1.9 3.3 2.3 1.7 1.6

a 1 Unit, 1 nmol of acetyl-CoA consumed/min.

The addition of ethanol to dark grown resting Euglena inducesa net synthesis of protein (14). An increase in total cell proteinwithout a concomitant synthesis of malate synthase would de-crease the specific activity of the enzyme. In this case, the de-creased specific activity of malate synthase seen after 24 h mightnot represent a loss of active enzyme. To see if the decline in thespecific activity of malate synthase represents a true decrease inthe amount of functional enzyme, we have measured the amountof enzyme per ml of culture at 0, 24 and 72 h after ethanoladdition (Table I). Total enzyme activity increased in the light orthe dark during the first 24 h after ethanol-supplementation (TableI). Enzyme levels were higher in the dark than in the light (TableI). Between 24 and 72 h after ethanol addition, malate synthaseactivity per ml of culture decreased. In unsupplemented andmalate supplemented resting cells, there was little change in theamount of active enzyme (Table I). Thus, the changes in enzymespecific activity represent changes in the actual amount of activeenzyme.The ethanol specific induction of malate synthase is regulated

at the transcriptional level (31) and the increase in active enzymeresults from de novo protein synthesis. The decline in the specificactivity of malate synthase occurring 24-72 h after inducer addi-tion probably results from a decreased rate of enzyme synthesisand degradation of existing enzyme. It is unlikely that a decreasein inducer level (ethanol) can account for a decreased rate ofmalate synthase synthesis since ethanol dependent synthesis ofparamylum, the Euglena storage carbohydrate, continues for atleast 48 h after ethanol addition (data not shown), and thesynthesis of chloroplast localized enzymes continues to be re-pressed by ethanol for at least 72 h after ethanol addition (13, 14).

Just as ethanol specifically inhibits the photoinduction of anumber of chloroplast localized enzymes (13, 14), light appears toinhibit the ethanol specific induction of the glyoxysomal enzyme,malate synthase. To see if this inhibition was mediated by anonchloroplast photoreceptor, the induction of malate synthasewas studied in the plastidless mutant W3BUL. This mutant lacksdetectable chloroplast DNA and Pchl(ide), the chloroplast local-ized photoreceptor (22). W3BUL contains a functional nonchlo-roplast photoreceptor as evidenced by the photoinduction ofprotein synthesis (24), paramylum breakdown (25), and cytoplas-mic rRNA turnover (7). The basal level of malate synthase waslower in W3BUL (Fig. 2) than in wild-type cells (Fig. 1). Theaddition of ethanol to dark-grown resting W3BUL induced atransient increase in the specific activity of malate synthase (Fig.2). The enzyme induction kinetics in resting W3BUL cells weresimilar to those seen in wild-type cells. During the first 24 h afterethanol addition, cells induced in the dark synthesized signifi-cantly more enzyme than cells induced in the light. In the absenceof ethanol, enzyme levels were unaltered over a 72 h period (Fig.2). A photoinhibition of malate synthase induction has also beenobserved with cells growing exponentially (9, 21) indicating thatthis photoinhibition is not due to a competition between light-induced and ethanol-induced protein synthesis for a limited poolof amino acids. It remains to be determined whether light acting

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HORRUM AND SCHWARTZBACH

80Lu

Cd,

TI..

60

U E< 40<2C

20

0 24 48TIME (HOURS)

72

FIG. 2. Photoinhibition of the ethanol induction of malate synthase inthe plastidless mutant W3BUL. Experimental conditions are identical toFigure 1. Samples were removed at appropriate times and the specificactivity of malate synthase was determined. When the enzyme specificactivity for cells incubated in the light was not significantly different fromthe specific activity for cells maintained in the dark, the two cultures are

represented by a single half shaded, half unshaded data point.

C,)

z 8ll ._Lu.:

, E

0C]

2

o0 24 48 72

TIME (HOURS)

FIG. 3. Ethanol inhibition of the induction of glycolate dehydrogenasein wild-type Euglena Experimental conditions are identical to Figure 1.Samples were removed at appropriate times and the specific activity ofglycolate dehydrogenase was determined. When the enzyme specific activ-ity for cells incubated in the light was not significantly different from thespecific activity for cells maintained in the dark, the two cultures are

represented by a single half shaded, half unshaded data point.

through the nonchloroplast photoreceptor inhibits at the transcrip-tional, translational, or postranslational level.

Ethanol InhibPdon of Glycolate Dehydrogenase Induction. Per-oxisomes are microbodies associated with photosynthetic metab-olism, and at least one peroxisomal enzyme, glycolate dehydro-genase, is photoinduced in Euglena (4). To better understand theinteraction between light and ethanol in the regulation of micro-body biogenesis, we have studied the photo and nutritional regu-lation of glycolate dehydrogenase synthesis. Exposure of dark-grown resting Euglena to light increased the specific activity ofglycolate dehydrogenase (Fig. 3). After a 24 h lag period, thespecific activity of glycolate dehydrogenase increased in malatesupplemented and unsupplemented cells incubated in the light(Fig. 3). Malate supplementation, however, produced a slight butreproducible inhibition of the photoinduction of glycolate dehy-drogenase. Enzyme levels were unaltered over a 72 h period inunsupplemented and malate supplemented resting cells incubated

in the dark (Fig. 3). The addition of ethanol to cells incubated inthe light or the dark produced an immediate decrease in thespecific activity of glycolate dehydrogenase (Fig. 3). The specificactivity of glycolate dehydrogenase 72 h after ethanol additionwas 50%o of the activity in unsupplemented or malate supple-mented cells maintained in the dark. Similar decreases in thespecific activity of glycolate dehydrogenase were seen when ace-

tate was added to resting cells maintained in the light or the dark(data not shown). Glycolate dehydrogenase was present but notinducible in the plastidless mutant W3BUL (data not shown). Theaddition of ethanol to dark grown resting W3BUL produced a

decline in glycolate dehydrogenase specific activity (data notshown).The ethanol-induced decline in the specific activity of glycolate

dehydrogenase was not due to the presence in the cell-free extractsof low molecular weight effector molecules since the cell-freeextracts were chromatographed on Sephadex G-25 columns priorto assay. Mixing experiments (data not shown) established thatthe decreased glycolate dehydrogenase activity in ethanol-supple-mented cultures was not due to the presence in the cell-freeextracts of high molecular weight inhibitors.The decrease in the specific activity of glycolate dehydrogenase

produced by ethanol addition represented a true loss of activeenzyme rather than an increase in total cell protein in the absenceof concomitant enzyme synthesis. Over a 72 h period, the units ofglycolate dehydrogenase decreased from 0.32 to 0.1 1/ml ofculturefor cells incubated in the light, and from 0.32 to 0.13/ml of culturefor cells incubated in the dark.When cycloheximide, an inhibitor of protein synthesis on cy-

toplasmic ribosomes (3), was added to cells maintained in the lightor the dark, there was a decrease in the specific activity ofglycolatedehydrogenase (Fig. 4). Apparently, this enzyme is unstable inresting cells. In the absence of continued enzyme synthesis on

cytoplasmic ribosomes, enzyme levels decline. The decrease in thespecific activity of glycolate dehydrogenase produced specificallyby ethanol addition (Fig. 3) was similar to the decrease producedby cycloheximide addition (Fig. 4), suggesting that ethanol in-hibited the synthesis of glycolate dehydrogenase with a resultantdecline in enzyme levels due to degradation or inactivation ofexisting enzyme.

o-o LIGHT<t * DARK

a LIGHTop DARK +CYCLOHEXIMIDE

OD

0

6

IN.

80 24-87

LU-J

0

-J

TIME (HOURS)

FIG. 4. Cycloheximide inhibition of glycolate dehydrogenase induc-tion. At zero time, cycloheximide (12 ,ug/ml) was added to dark grownresting Euglena, and cells were incubated in the light or the dark. Sampleswere removed at appropriate times and the specific activity of glycolatedehydrogenase was determined. When the enzyme specific activity forcells incubated in the light was not significantly different from the specificactivity for cells maintained in the dark, the two cultures are representedby a single half shaded, half unshaded data point.

W3BUL° LIGHToF DARK1 LIGHTo; DARK + MALATE-LIGHT + ETHANOL

A-'DARK +ETHANOL

-~~~ 0

_SH =

X, ID

O-o LIGHT0-. DARKm-- LIGHToR DARK + ETHANOL&--. LIGHT + MALATE

*-ADARK + MALATE

or_H~~~~

I I

_ ,d.r_ .432 Plant Physiol. Vol. 68, 1981

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MICROBODY BIOGENESIS IN EUGLENA

X o-o LIGHT<K * DARK

J s o-cH LIGHT. STREPTOMYCINa(D °-- DARK + STREPTOMYCIN

0

CD

g2t ]~~~~~~~~~74 24487

TIME (HOURS)

FIG. 5. Streptomycin inhibition of glycolate dehydrogenase induction.At zero time, streptomycin (0.05%) was added to dark grown restingEuglena and cells were incubated in the light or the dark. Samples wereremoved at appropriate times and the specific activity of glycolate dehy-drogenase was determined. When the enzyme specific activity for cellsincubated in the light was not significantly different from the specificactivity for cells maintained in the dark, the two cultures are representedby a single half shaded, half unshaded data point.

LLC')

z *Lu0 0C: 6J aI E

W-C]4LS'r

21

8 2-JcD)

( L

0 24 48

TIME (HOURS)

72

FIG. 6. DCMU inhibition of glycolate dehydrogenase induction. Atzero time, dark grown resting cells were harvested aseptically by centrif-

ugation and resuspended in either DCMU containing (10 PM) or DCMU-free resting media. Cells were incubated in the light or the dark. Sampleswere removed at appropriate times and the specific activity of glycolatedehydrogenase was determined. When the enzyme specific activity forcells incubated in the light was not significantly different from the specificactivity for cells maintained in the dark, the two cultures are representedby a single half shaded, half unshaded data point.

Dependence of Glycolate Dehydrogenase Synthesis on Photo-synthesis. Glycolate dehydrogenase is repressed when Euglena isgrown phototrophically in air supplemented with 5% CO2 (6, 18,32). When the CO2 concentration is lowered, glycolate dehydro-genase is induced (6, 18, 32). Glycolate levels increase in light-dark synchronized cultures prior to the synthesis of glycolatedehydrogenase (5, 6). Based on these observations, Merrett pro-posed that a product of photosynthesis, rather than light, is theactual inducer of glycolate dehydrogenase (20). The 24-h lagperiod for the photoinduction of glycolate dehydrogenase in rest-ing Euglena (Fig. 4) differs from the lag periods (0-12 h) previouslyreported for other light-induced enzymes (3, 14, 15). In restingcells, glycolate dehydrogenase synthesis appears to begin onlyafter the cells become photosynthetically competent (27) support-

ing Merrett's proposal (20) that glycolate dehydrogenase synthesisis controlled by a product of photosynthesis rather than directlyby light exposure. To distinguish between a direct induction bylight and induction by a product of photosynthesis, we havedetermined glycolate dehydrogenase levels under conditionswhich dissociate the synthesis of light-induced enzymes on cyto-plasmic ribosomes from the development of photosynthetic com-petence.

Streptomycin is a specific inhibitor of protein synthesis onchloroplast ribosomes (2). The photoinduction of cytoplasmicallysynthesized chloroplast localized enzymes is unaffected by thepresence of streptomycin (3). Thus, after 72 h of light exposure inthe presence of streptomycin, Euglena is unable to photosynthet-ically fix CO2 even though the cells contain a normal complementof cytoplasmically synthesized Calvin cycle enzymes such asNADP-dependent glyceraldehyde-3-P dehydrogenase (3). Whendark-grown resting Euglena were exposed to light in the presenceof streptomycin, glycolate dehydrogenase was not induced (Fig.5).

In Euglena, DCMU at a concentration of 10 jIM completelyinhibits photosynthetic CO2 fixation while having little effect onlight-induced chloroplast development (23). Because of its lowsolubility in H20, DCMU is normally prepared as a concentratedstock solution dissolved in ethanol and then added to restingculture for a final ethanol concentration of 17 mi (23). In orderto use DCMU to study the relationship between photosyntheticCO2 fixation and glycolate dehydrogenase induction, DCMUcould not be dissolved in ethanol since ethanol inhibits the syn-thesis of glycolate dehydrogenase. Resting cells were thereforeaseptically harvested by centrifugation, a procedure which has noeffect on subsequent chloroplast development (26), and resus-pended in filter-sterilized resting medium containing 10 t,MDCMU. After 72 h of light exposure, untreated cells contained7.5 pg Chl/cell; cells exposed to light in the presence of 10 /IMDCMU contained 2.9 pg Chl/cell; and cells exposed to light inthe presence of 10 tLM DCMU, 17 mM ethanol contained 1 1.1 pgChl/cell. Small amounts ofethanol reversed the DCMU inhibitionof Chl synthesis. The synthesis of NADP-glyceraldehyde-3-P de-hydrogenase, a light-induced cytoplasmically synthesized chloro-plast enzyme (3) was, however, unaffected by DCMU. Over a 72h period, the specific activity of this enzyme increased from 30units/mg protein to 130 units/mg protein in the absence ofDCMU, and 110 units/mg protein in the presence of 10 tiMDCMU. Since DCMU had little effect on the light-induced syn-thesis of NADP-glyceraldehyde-3-P dehydrogenase, an energyrequiring process, the DCMU inhibition of Chl synthesis was notdue to an inhibition of respiration. It appears that the carbonskeletons required for Chl synthesis are derived from the productsof photosynthetic CO2 fixation and in the absence ofCO2 fixation,the precursors for Chl synthesis can be obtained from exogenouscarbon.The levels of glycolate dehydrogenase in dark grown resting

cells resuspended in medium containing 10 tIM DCMU and ex-posed to light are seen in Figure 6. In untreated cells, glycolatedehydrogenase levels increased between 24-72 h after light expo-sure. Glycolate dehydrogenase levels were unaltered during thistime in cells maintained in the dark or exposed to light in thepresence of 10 tIM DCMU (Fig. 6). The DCMU inhibition ofglycolate dehydrogenase induction was not reversed by 17 mMethanol (data not shown). Thus the inhibition of glycolate dehy-drogenase induction by DCMU is consistent with the proposalthat glycolate dehydrogenase is induced by a product of photo-synthesis.The inducibility of glycolate dehydrogenase and malate syn-

thase contrasts with the constitutive synthesis of the microbodyenzyme hydroxypyruvate reductase (13, 16). Light, ethanol, andmalate addition have no effect on the synthesis of hydroxypyru-

o-0 LIGHT.-. DARK

DCMU (105M) LIGHT&--& DCMU (10-5M) DARK

*QEE~~~~~~~~l~

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HORRUM AND SCHWARTZBACH

vate reductase, an enzyme ofthe reversible portion of the glycolatepathway (13, 16). It appears that a microbody containing theenzymes required for the conversion of glycerate to serine, (thereversible portion of the glycolate pathway) is synthesized regard-less of the source of carbon and energy. In addition to theseenzymes, the microbody contains enzymes which are required forthe utilization of specific carbon sources present in the environ-ment. Based on the results presented in this paper, we proposethat microbodies are continually synthesized in resting Euglenaand that the actual enzyme complement of the microbody isdetermined through substrate regulation of the genes coding forglyoxysomal and peroxisomal enzymes. When substrates of theglyoxylate cycle are present (ethanol and acetate), the synthesis ofenzymes of the irreversible portion of the glycolate cycle is re-pressed while enzymes of the glyoxylate cycle are induced andincorporated into the newly formed microbody resulting in a

glyoxysome which also contains enzymes of the reversible portionof the glycolate pathway. When glycolate is synthesized photosyn-thetically, glycolate dehydrogenase is induced and incorporatedinto the newly formed microbody and a peroxisome is produced.By continuously degrading existing microbodies and by regulatingmicrobody enzyme synthesis through substrate induction, Euglenahas evolved a mechanism for adapting the microbody populationto a constantly changing environment. This level of control con-trasts with microbody biogenesis in obligate phototrophs, higherplants, which is controlled directly by light acting through phy-tochrome (peroxisomes) and by the developmental program of thecotyledon [glyoxysome, (1)].

LITERATURE CITED

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2. BOVARNICK JG, SW CHANG, JA SCHIFF, SD SCHWARTZBACH 1974 Eventssurrounding the early development of Euglena chloroplasts. 3. Experimentswith streptomycin in non-dividing cells. J Gen Microbiol 83: 51-62

3. BOVARNICK JG, JA SCHIFF, Z FREEDMAN, JM EGAN JR 1974 Events surroundingthe early development ofEuglena chloroplasts. 4. Cellular origins ofchloroplastenzymes in Euglena J Gen Microbiol 83: 63-71

4. CODD GA, MJ MEaarr 1970 Enzymes of the glycolate pathway in relation togreening in Euglena gracilis. Planta 95: 127-132

5. CODD GA, MJ MERRBTr 1971 Photosynthetic products of division synchronizedcultures of Euglena. Plant Physiol 47: 635-639

6. CODD GA, MJ MERRErr 1971 The regulation of glycolate metabolism in divisionsynchronized cultures of Euglena. Plant Physiol 47: 640-643

7. COHEN D, JA SCHIFF 1976 Events surrounding the early development ofEuglenachloroplasts. 10. Photoregulation of the transcription of chloroplastic andcytoplasmic ribosomal RNAs. Arch Biochem Biophys 177: 201-216

8. COLLINs N, MJ MERRETr 1975 Microbody marker-enzymes during transitionfrom phototrophic to organotrophic growth in Euglena. Plant Physiol 55: 1018-1022

9. COOK JR, M CARVER 1966 Partial photo-repression of the glyoxylate bypass inEuglena. Plant Cell Physiol 7: 377-383

10. FEIERABEND J 1975 Developmental studies on microbodies in wheat leaves. III.On the photocontrol of microbody development. Planta 123: 63-77

11. FEIERABEND J, U SCHRADER-REiCHHARDT 1976 Biochemical differences of plas-tids and other organelles in rye leaves with high-temperature-induced defi-ciency of plastid ribosomes. Planta 129: 133-145

12. HORRUM MA, SD SCHWARTZBACH 1979 Glyoxysomal and mitochondrial enzymeactivity during light-induced chloroplast development in Euglena. Plant Physiol63: S-160

13. HoRRuM MA, SD SCHWARTZBACH 1980 Photo and nutritional regulation of fourphotorespiratory enzymes in greening Euglena Plant Physiol 65: S-19

14. HORRUM MA, SD SCHWARTZBACH 1980 Nutritional regulation of organellebiogenesis in Euglena. Repression of chlorophyll and NADP-glyceraldehyde-3-phosphate dehydrogenase synthesis. Plant Physiol 65: 382-386

15. HORRUM MA, SD SCHWARTZBACH 1980 Nutritional regulation or organellebiogenesis in Euglena: Photo- and metabolite induction of mitochondria.Planta 149: 376-383

16. HoRRuM MA, SD SCHWARTZBACH 1980 Absence of photo- and nutritionalregulation of two glycolate pathway enzymes in Euglena. Plant Sci Lett 20:133-139

17. KAGAWA T, 01 McGREGOR, H. BEEvERs 1973 Development of enzymes in thecotyledons of watermelon seedlings. Plant Physiol 51: 66-71

18. LoRD JM, MJ MERRETr 1971 The intracellular localization of glycolate oxido-reductase in Euglena gracilis. Biochem J 124: 275-281

19. LowRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measure-ment in the Folin phenol reagents. J Biol Chem 193: 265-275

20. MERRETT MJ, JM LoRD 1973 Glycolate formation and metabolism by algae.New Phytol 72: 751-767

21. MITCHELL JLA 1971 Photoinduced division synchrony in permanently bleachedEuglena gracilis. Planta 100: 244-257

22. SCHIFF JA 1974 The control of chloroplast differentiation in Euglena. In MAvron, ed, Proceedings of the Third International Congress on Photosynthesis,Elsevier, Amsterdam, pp 1691-1717

23. SCHIFF JA, MH ZELDIN, J RUBMAN 1967 Chlorophyll formation and photosyn-thetic competence in the presence of 3,(3,4-dichlorophenyl)l,I-dimethyl urea(DCMU). Plant Physiol 42: 1716-1725

24. SCHWARTZBACH SD, JA SCHIFF 1979 Events surrounding the early developmentof Euglena chloroplasts. 13. Photocontrol of protein synthesis. Plant CellPhysiol 20: 827-838

25. SCHWARTZBACH SD, JA SCHIFF, NH GOLDSTEIN 1975 Events surrounding theearly development of Euglena chloroplasts. V. Control of paramylum degra-dation. Plant Physiol 56: 313-317

26. SCHWARTZBACH SD, JA SCHIFF, S KLEIN 1976 Biosynthetic events required forlag elimination in chlorophyll synthesis in Euglenae Planta 131: 1-9

27. STERN Al, JA SCHIFF, HT EPSTEIN 1964 Studies of chloroplast development inEuglena V. Pigment biosynthesis, photosynthetic oxygen evolution and carbondioxide fixation during chloroplast development. Plant Physiol 39: 220-226

28. TOLBERT NE 1979 Glycolate metabolism by higher plants and algae. In M Gibbs,E Latzko, eds, Encyclopedia of Plant Physiology, N. S., Vol 6, Chap 26.Springer-Verlag, Berlin, pp 338-352

29. WOODWARD J, MJ MERRETT 1975 Induction potential for glyoxylate cycleenzymes during the cell cycle of Euglena gracilis. Eur J Biochem 55: 555-559

30. WOODCOCK E, MJ MERETrr 1978 Purification and immunochemical character-ization of malate synthase from Euglena gracilis. Biochem J 173: 95-101

31. WOODCOCK E, MJ MERnRT 1980 Malate synthase messenger RNA in relationto enzyme derepression in Euglena gracilis. Arch Microbiol 124: 33-38

32. YOKOTA A, Y NAKANO, S KITAOKA 1978 Different effects of some growingconditions on glycolate dehydrogenase in mitochondria and microbodies inEuglena gracilis. Agric Biol Chem 42: 115-120

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