photoinduced carotenogenesis in chlorotic euglena gracilis1 · win (11) and chromatographed on an...

Plant Physiol. (1970) 46, 685-691

Photoinduced Carotenogenesis in Chlorotic Euglena gracilis1Received for publication June 8, 1970

WARREN D. DOLPHIN2Department of Zoology, University of Maine, Orono, Maine 04473

ABSTRACT

Light induces 1-carotene synthesis in streptomycin-bleached Euglena gracilis Z. Light-adapted, chemostat-grown cells have up to 10-fold as much a-carotene and 25%more protein than similarly grown, dark-adapted cells.Carotenogenesis does not occur under anaerobic conditionsor in the presence of diphenylanmine, cyanide, or cyclo-lieximide. The blue portion of the spectrum (360-560 nm) ismost active in initiating carotenogenesis. The level of cel-lular carotenoids is influenced by the type of carbon sourceand to some degree by pH. Phytofluene and (-carotene arepresent in dark-grown cells but not in cells grown aerobic-ally in white light (360-1120 nm). These pigments, however,were present in cells grown in yellow or green light (above486 nm) or in cells exposed to white light anaerobically. Thecarotenoids are localized in two types of structures at thelight microscope level. A protoporphyrin was isolated fromEuglena, and its role as a possible photoreceptor duringcarotenogenesis is suggested.

Exposure to visible light stimulates carotenoid formation inyeasts, fungi, bacteria, and higher photosynthetic organisms (12,17). In Euglena carotenogenesis parallels chlorophyll synthesis inlight-synchronized cultures (2). However, when chlorophyllsynthesis is blocked by mutagenic agents, carotenogenesis stilloccurs and is stimulated by light (9). Bleached mutants generallylack the xanthophylls found in wild-type Euglena, and thecarotenes (,-carotene, i-carotene, and phytofluene) occur atreduced levels (13). In wild-type cells there are shifts in the relativeamounts of individual carotenoid pigments when they are placedin the light (20), but the effect of light on the carotenoid composi-tion of mutants is unknown except for a brief report by Gross andStroz (15).

Bacterial mutants lacking colored carotenoids (22), similarmutants of Chlorella (18), and white mutants of Chlamnydomonas(16) are killed by visible light under aerobic conditions. Thecarotenoids as a class can act as potent energy traps in photo-sensitized oxidations (8), and possibly the primary function ofthe carotenoids is to protect cells from photosensitizations bytheir own endogenous pigments like the hemes, porphyrins, andcytochromes (21). Krinsky (20) has proposed that the carotenoidsprotect normal Euglena from the photosensitizing action ofchlorophyll by quenching triplet oxygen formed in the light. The

1 This work was supported by United States Public Health ServiceGrant GM-12179 to J. R. Cook and United States Public HealthService Postdoctoral Fellowship GM-35,542 to W. D. Dolphin.

2 Present address: Department of Zoology and Entomology, IowaState University, Ames, Iowa 50010.

function of the carotenoids in the bleached mutants, if any, hasnot been demonstrated.Cook (3) has shown that inhibition of growth occurs in Euglena

and Astasia cultures when they are exposed to visible light.Photoinhibition in wild-type Euglena is transitory while that incultures of Astasia and bleached Euglena is more permanent.While continuing this work, we noticed that dark-grown cells ofa streptomycin-bleached strain changed color from cream toorange when placed in the light. The growth rate of these cellswas unaffected by light. Because of the possibility that caroteno-genesis in these cultures was an adaptive photoprotective mecha-nism, I studied it in detail. This report describes the kinetics ofcarotenoid synthesis with shifts in the relative amounts of caro-tenes and discusses some of the factors which govern cellularcarotenoid levels.

MIATERIALS AND METHODS

Axenic cultures of Euglena gracilis Z, substrain A (strepto-mycin-bleached; stock cultures kindly provided by Dr. F. Child)were grown at 30 C on a defined salt medium (5) at pH 3.5 withvarious carbon sources or on 2'%c multipeptone (Fisher ScientificCo.). The actual experimental conditions are listed in the figurelegends. Cells were cultivated either as batch cultures in Erlen-meyer flasks or continuously in a chemostat as described by Cook(4), with one modification: a vacuum line maintained the desiredlevel in the growth chamber (see Ref. 29) rather than achievingconstant volume by removal with a second pump.Dark conditions were imposed by covering the growth cham-

bers with black cloth. A 500-w Quartzline lamp (GE 500T3/Cl)mounted in a parabolic reflector was the light source unless other-wise noted. Batch cultures were irradiated from below through 10cm of water in a glass-bottomed, constant temperature bath.Chemostat cultures were irradiated laterally through two rec-tangular window glass tanks each 10 cm thick. When filled withwater, these tanks served as infrared filters and were used toprevent heating of the culture chamber in the white light studies.Spectral regions were isolated from the output spectrum of thelamp by filling the tank nearest to the chemostat with a coppersulfate solution or using a third tank 1 cm thick filled with apicric acid solution (modified from Ref. 30). Light intensitieswere measured with a Weston photometer employing a quartzfilter at the surface of the culture vessel.

All gassing and inhibitor studies were done with 100-ml water-jacketed cylinders as culture chambers. A magnetic stirring barand aeration tubes provided adequate mixing. Cell suspensionstaken from the chemostat were used in these comparatively shortstudies.

Cell counts were made with a Coulter electronic cell counter,pore size 140 A. Protein was assayed by a modified biuret tech-nique (10). Oxygen consumption was measured at 30 C at roomintensity light with a Clark electrode and a recorder.

Carotenoid samples were obtained by extracting approximately3 X 106 cells three times with hot 95 c ethanol. The pooled ex-tracts were concentrated in the dark by heating to 50 C while N2

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Plant Physiol. Vol. 46, 1970

was bubbled through the solution. The concentrate was centri-fuged to remove a fine white precipitate, and the absorptionspectrum was read with a Beckman DB scanning spectrophotom-eter with recorder. The amount of (-carotene per cell was esti-mated by the technique of Goodwin (11) with an extinctioncoefficient at 453 nm of 2500.

RESULTS

Pigments Present in Streptomycin-bleached Euglena. To deter-mine the pigment composition of the streptomycin-bleachedstrain, an ethanol extract of dark-adapted Euglena was saponifiedand transferred to hexane according to the techniques of Good-win (11) and chromatographed on an ignited MgO-SiO2 (1:1)column using hexane with acetone as a developer. The bands werecut out of the column and eluted with acetone, dried, and dis-solved in hexane. Table I lists the main absorption peaks of thesepigment bands. Tentative identification of the pigments wasbased on known values (11). The data of Goodwin and Gross(13) on the carotenoid composition of a streptomycin-bleachedstrain of Euglena are included for comparison. As Goodwin and

Table I. Comparisont of the Absorptioni Peaks of Ethaniol-extractedCaroten2oids Dissolved in Hexanie with Putblished

Values for Carotenies

ExperimentalGoodwin (11)Goodwin andGross (13)

Spectral Peaks

Phytoene Phytofluene -Caroteiie s-Carotene

niii

271,283,293 332,348,358 378,400,425'275,285,296 332,348,367 378,400,425

348,3681 380,402,425

DARK1-

Z 3000

E 5.50(I,

a)540.0

E 5.300

0.30

a)- 0.20-

a) 0.1 0-0CZ .i

0)

426,450,477425,451,482425,449,474

Gross found, the xanthophylls, if present, were at such reducedlevels that they were undetected by this technique. In the follow-ing studies only (-carotene, c-carotene, and phytofluene wereroutinely assayed.

Light Adaptation. When cells growing in a chemostat in thedark were irradiated with white light, several changes occurred(Fig. 1). (3-Carotene content per cell increased to 10-fold theamount found in dark-grown cells and remained at that levelthroughout the light period. The amount of protein per cell alsorose rapidly and came to equilibrium at a value 25 % greater thanin dark-adapted cells. The 25 rise in protein was not nearlyenough to account for the large (-carotene increase by assuminga constant ratio between these compounds. Constant cellularlevels of both protein and (3-carotene were reached simultane-ously during the light period. Oxygen consumption per cell didnot change significantly during light adaptation. Shifts in therelative amount of carotenes in the cells occurred. The absorptionpeaks corresponding to phytofluene and A-carotene apparentlydisappeared from the crude ethanolic extracts of the cells, andonly the (-carotene peak remained during the ensuing lightperiod (Fig. 2). Early analysis showed that these changes occurredwithin 30 min of the time the cells were placed in the light.Dark Adaptation. When light-adapted cultures were placed in

the dark, a transition phase occurred during which (-caroteneand protein levels decreased to their original dark-adapted values(Fig. 1). When the transitional phase values of (-carotene andprotein were plotted on semilog paper, the losses were found tobe exponential (Fig. 3). Half-life values of 35 hr for both com-pounds corresponded to the doubling time of the chemostat cellpopulation. A similar kinetic relationship was found in anotherchemostat population with a doubling time of 27 hr. These re-sults suggest that light-adapted cells placed in the dark immedi-ately decreased carotenoid and protein synthesis to a basal rateand lost the high cellular levels of these compounds by simplepartition during cell division. Within 2 hr of entering the dark,

LIGHT DARK

IL

Protein |

Cell No.

.- i.

1100 1200 1300 1400 1500 1600

Culture Age (Hr.)1700 1800

FIG. 1. Effect of incandescent light on ,3-carotene and protein content of chemostat-grown cells. Chemostat culture with 5 mM acetate limitingat pH 3.5. Doubling time at equilibrium was 35 hr. Period between dashed vertical lines represents continuous white light at 5500 ft-c. A: Proteinper cell in ,u,ug; 0: d-carotene per cell in ,u,ug; @: log of cell concentration.

DOLPHIN686

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CAROTENOGENESIS IN EUGLENA

10

-

Q

0.30

0. 20-

0. 15 -

0. 10 -

0.05 -

360 460 440 480 520

Wavelength (nm)560

FIG. 2. Absorption spectra of ethanolic extracts of cells grown in a

chemostat (equal cell numbers) during light regime in Figure 1. Arrowsindicate peaks corresponding to c-carotene. Extracts made from cellsin chemostat population shown in Figure 1 at the following times:prelight, 1280 hr; light, 1550 hr; postlight, 1605 hr.

phytofluene and A-carotene reappeared in ethanolic extracts ofthe cells (Fig. 2).

In summary, light has three effects on dark-adapted cultures.It causes (a) an apparent decrease in the cellular levels of D-carotene and phytofluene, (b) an increase in the cellular levels ofA-carotene, and (c) an increase in cellular protein. Under theseculture conditions it does not affect chemostat population densityor growth rate, nor does it uncouple respiration from the normalgrowth processes.

Protein Synthesis and Carotenogenesis. The simultaneous riseof protein with fl-carotene in chemostat cultures adapting to lightraises the question whether these are coupled or independentevents. Part of the higher protein content per cell could reflectan increase in carotenogenic enzyme levels, the synthesis of a

structural protein involved in carotenoid localization, or theparallel formation of protein not specific for ,B-carotene synthesis.Cycloheximide inhibits protein synthesis in wild-type Euglena(19). When cycloheximide was added to batch cultures ofbleached Euglena and the cultures were exposed to light for 24 hr,carotenogenesis was inhibited (Table II). Thus carotenogenesisis in some manner coordinated with protein synthesis.

Inhibition of Carotenogenesis. Carotenogenesis in Euglena is anaerobic process. When cultures were gassed with air or N2 aftertransfer to light, fl-carotene was synthesized only in the aeratedcultures (Fig. 4A). Two interpretations of the role of 02 in (-

carotene synthesis are possible. Oxygen could induce caroteno-genesis through a photosensitized oxidation, or it may be neededas a terminal acceptor of electrons during the synthesis of (-

carotene. f-Carotene is thought to be synthesized through thesequential dehydrogenation of more saturated polyenes likephytofluene and c-carotene (25). The lack of a terminal acceptorwould cause a malfunction of the dehydrogenase system, stoppingf-carotene synthesis and possibly causing the accumulation ofprecursor compounds. When the absorption spectra of ethanolicextracts of cells from these two treatments were compared, shiftsin carotenoid composition were seen in the aerated cultures

D i1o 150 200

Normalized Culture Time (Hr)

FIG. 3. Derived curves showing loss of 3-carotene and protein fromlight-adapted cells growing in chemostat in Figure 1 and transferredto dark. Concentration of p-carotene or protein per cell during thetransition period minus the dark-adapted concentration is plotted.Half-lives of 35 hr for both compounds in a chemostat with cellshaving a doubling time of 35 hr indicate that the high light-adaptedlevels of these compounds were lost by simple dilution kinetics of celldivision.

Table lI. Iiihibitiont of Caroteniogeniesis by CycloheximideBatch cultures of E. gracilis Z were grown in Cramer-Myers

medium at pH 5 with 0.08 M ethanol and irradiated with coolwhite fluorescent lights at 500 ft-c. Cycloheximide concentrationwas 10lg/ml. Cultures were kept in the dark until stationaryphase growth and then exposed to experimental treatment for24 hr.

Light andTime Dark Light Cycloheximide

hr rnAg 0-carotene/cell0 0.291 0.293 0.2944 0.289 0.372 0.29124 0.300 0.385 0.295

(data not shown). The peaks corresponding to phytofluene andc-carotene disappeared from the aerated culture extract but notfrom the N2-gassed one.

Diphenylamine is a potent inhibitor of carotenogenesis whichblocks the polyene dehydrogenase system (26). A concentrationof 7.4 X 10-5 M allowed growth in dark Euglena cultures but in-hibited fl-carotene synthesis. In cells exposed to light the inhibi-tion of carotenogenesis was reversible, and once DPA3 was

washed away the cells synthesized fl-carotene at the normal rate(Fig. 4B). Ethanol extracts of both the control and DPA-treatedcells, however, lacked the absorption peaks corresponding tophytofluene and ¢-carotene (data not shown) regardless of thestate of f-carotene synthesis. The disappearance of these satu-

3Abbreviation: DPA: diphenylamine.

:. .Post-lightLight *

. , \~~~I

/: /

.*e / ,Pe:lgh

687Plant Physiol. Vol. 46, 1970




rated polyenes from extracts of light-treated cells appears to beindependent of ,B-carotene synthesis.To determine if the role of 02 was to act as a terminal acceptor

of electrons, cells were transferred to aerated cultures in the light,and cyanide was added (Fig. 4C). A-Carotene synthesis was

0)

a)

1-1

a

a)

02

a)

Co

0)

01)

CONUI

0. 35'

0.25/

o. 1, a A A N2A

0 .60-

0.50

0.40

0.30

5 10 15 20 25 30 35

Time in Light (Hrs.)

c100

._

0

FIG. 4. Inhibition of carotenogenesis. Cell suspensions from darkchemostat were placed in 100-ml cylinders and irradiated with coolwhite fluorescent light at 900 ft-c. A: Comparison of carotenogenesisin suspensions gassed with air or N2. B: Carotenogenesis in the pres-ence of 7.4 X 10- M diphenylamine. Diphenylamine washed out at23 hr by resuspending cells twice in fresh Cramer-Myers medium withcarbon source. C: Effect of 2 X 104 M KCN on carotenogenesis.

360 400 44D 480 520

Wavelength (nm)

FIG. 5. Absorption spectra of ethanolic extracts containing phyto-fluene, ¢-carotene, and A-carotene after exposure to 2000 ft-c light for3 hr. Solid line: Solution gassed with N2; dashed line: solution gassedwith air.

0.25

0. 15

0.10

0.351

400 440 480 520

Wavelength (nm)

FIG. 6. Absorption spectra of ethanolic extracts of dark-adaptedcells before and after 30-min exposure of the cells to 2000 ft-c of fluo-rescent light. Solid line: 0 time sample; dashed line: 30-min sample.

inhibited in these cultures, indicating that a functional electrontransport system is necessary for pigment synthesis. This does notpreclude other roles for 02 in carotenogenesis. In addition, theethanolic extracts of control and cyanide-treated cultures lackedthe absorption peaks corresponding to phytofluene and t-caro-tene (data not shown). The disappearance of these pigments fromcells in the light is dependent upon the presence of 02 but not oneither d-carotene synthesis or the presence of a functioning elec-tron transport system.

Proof of the photolability of the saturated polyenes was ob-tained in vitro when ethanolic extracts containing ,B-carotene,c-carotene, and phytofluene were exposed to cool white fluorescentlight while air or N2 was bubbled through the solutions. Nochanges occurred in the absorption spectrum of the N2-gassedsolution, but in the aerated solution the peaks corresponding toc-carotene and phytofluene disappeared and only :-carotene re-mained (Fig. 5). The more saturated polyenes evidently can bephoto-oxidized under the conditions employed in these experi-ments.Pathway of fl-Carotene Synthesis. If the saturated polyenes are

indeed photolabile, this raises the question of the pathway off-carotene synthesis. To determine if part of the saturated polyenepool was converted to fl-carotene as well as some being degradedby light, an aerated culture was exposed to light, and the carot-enoids were sampled. If some of the phytofluene and i-carotenewere converted into fl-carotene, the latter should increase percell. Figure 6 shows the absorption spectra of ethanolic extractsfrom this experiment at 0 and 30 min. The peaks correspondingto c-carotene and phytofluene have disappeared by 30 min, butthere is no corresponding rise in fl-carotene. Since all three of thecarotenoids have extinction coefficients which are nearly thesame, there should have been a rise in absorbance at the fi-carotene peak corresponding to decreasing absorbance at thephytofluene and ¢-carotene peaks. The conclusion drawn fromthis is that f-carotene is not synthesized from the pools of satu-rated polyenes which occur in dark-grown Euglena.

Spectral Regions Active in Carotenogenesis. Solution filterswere used to isolate spectral regions from the output of thequartzline lamp. Table III lists the characteristics of these filtersand the rates of carotenogenesis observed in cells growing in a

)B Control

S ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

A PAtratd Ws

688 DOLPHIN




Table III. Rate of Caroten2oid Synthesis in Chemostat CellPopulations Exposed to Differenit Spectral

Regions of Visible LightChemostat conditions were the same as in Figure 1. Initial

white light intensity was 5500 ft-c, and source intensity was notvaried during use of various filters. Solution filters were stand-ardized in spectrophotometer.

Wave Caroteno-

Light Color Solution Filter Laengths K 1 genesis due to

Lasenghd Wave Length

~~~Interval

nm %White 20 cm H20 360-1120 6.2 100Blue 10 cm H20 + 10 cm 360-560 4.2 67.5

13%o CuS04Yellow 20 cm H20 + 1 cm 486-1120 2.3 37.3

5%l, picric acid

Green 10 cm H20 + 10 cm 486-560 0.6 9.313% CuSO4+ 1cm 5%c picric acid

1 K = initial increase in #3-carotene (Cugg/cell- hr) in chemostatcell populations exposed to light after dark adaptation.

0.20 Yellow light

15

0.10

0

D. °sL - Dar

360 460 440 480 520

Wavelength (nm)

FIG. 7. Comparison of absorption spectra of ethanol extracts ofchemostat cells grown under different light conditions. : Yellowlight;- white light; *---: dark. Peaks corresponding to phyto-fluene and c-carotene are present in dark-grown and yellow light-treated cells but are not present in cells exposed to white light. Chemo-stat conditions as in Figure 1.

chemostat exposed to these spectral regions. The blue end of thespectrum was the most effective in promoting carotenogenesis.The sum of the effects from each area is not equal to 100% andmay be explained either by the synergism of different spectralregions or by partial transmission of light near the extremes of afilter's pass band.The studies with inhibitors inferred that phytofluene and A-

carotene are unstable in light. Corroborative data were obtainedduring the spectral studies. Ethanolic extracts of cells grown inblue or white light always lacked the peaks corresponding tothese polyenes. Cells grown in yellow (Fig. 7) or green light hadthese two pigments present. Yellow and green light consisted ofwave lengths greater than those absorbed by these pigments(Tables I and III).

Extraction of Protoporphyrin. The porphyrins and their deriva-tives have a strong Soret band near 400 nm and secondary peaksabove 560 nm. Dubash and Rege (6) reported that bleached

Table IV. Carotenoid Levels per Cell over the Growth Cycle inCramer-Myers Medium with Variable Carbon Sources or in a

Peptonie Medium

Dark Light (incandescent 4500ft-C)

PA9 A;~~~,&g LIDcells/ml 8-carotene cells/ml $-carotene o-caro-

Icell Icell tene/cell

Acetate (0.03 M;pH 7) Td1= 13 hr 14 hr

Initial 5.0 X 104 0.013 5.0 X 104 0.013 1Late log 3.0 X 105 0.180 3.0 X 105 0.310 1.71Stationary 6.0 X 105 0. 210 5.9 X 105 0.340 1.61

Ethanol (0.03 M;pH 7) Td= 12 hr 12 hr

Initial ... .. ... ... ...

Late log 2.7 X 101 0.238 2.0 X 105 0.640 2.7Stationary 1.1 X 106 0.430 6.0 X 105 1.030 2.4

Glucose (0.03 M;pH 7) Td= 13 hr 20 hi

Initial 4.2 X 104 0.004 4.4 X 104 0.004 1Late log 3.0 X 105 0.068 1. 5 X 105 0.200 2.9Stationary 3.5 X 106 0.150 ... ... ...

Multipeptone (2%c;,pH 7) Td= 10 hr 10 hr

Initial 7.6 X 104 0.016 7.2 X 104 0.016 1Late log 3.0 X 105 0.120 2.5 X 105 0.203 1.7Stationary 6.0 X 105 0.220 5.0 X 105 0.620 2.8

1 Td = Doubling time of cell populations in batch cultures.

Table V. Carotenzoid Levels over the Growth Cycle inCramer-Myers Medium with 0.03 mi Ethanzol as

Carbon Source anid Variable Iznitial pH

Dark Light (incandescent 4500 ft-c)

I ~~~~~~~LIDcells/titl Ppg f-caro- cells?n1 pg -caro- 0-carotenetenelcell el/,l enecelil otn

pH 3Late log 2.3 X 105 0.079 2.1 X 105 0.167 2.1Stationary 8.0 X 105 0.290 7.5 X 105 1.100 3.8

pH 4Late log 1.8 X 105 0.140 1.8 X 105 0.569 4.1Stationary 6.2 X 105 0.340 3.9 X 105 1.300 3.2

pH 6Late log 2.6 X 105 0.216 11.7 X 105 0.574 2.7Stationary 8.9 X 10D 0.530 5.5 X 105 1.120 2.1

pH 7'

1 See Table IV.

Euglena excrete protoporphyrin IX into the culture medium.Gibor and Granick (9) found that some streptomycin-bleachedstrains retained the ability to synthesize protoporphyrins. Acompound was extracted from dark-grown E. gracilis Z withglacial acetic acid and concentrated HC1 which had the absorp-tion spectral properties in 1 N HCI of a protoporphyrin. Thiscompound may be acting as the receptor for the changes detailedhere.Optimal Conditions for Photostimulation of Carotenogenesis.

Table IV illustrates the ,3-carotene content of cells grown as batchcultures in the medium of Cramer and Myers with various carbonsources or in 2%- multipeptone. In all media as the cultures aged,the amount of f-carotene per cell increased. Increasing cellularcarotenoid content with age has been reported previously bySmillie and Rigopoulos (28). Light stimulated carotenogenesis

Plant Physiol. Vol. 46, 1970 689




in all media at all growth stages. Ethanol as a carbon source pro-duced cells with the highest carotenoid concentration whether inthe light or the dark.The effects of the initial pH of the medium on carotenogenesis

were studied with ethanol as a carbon source (Table V). Caro-tenogenesis is favored by an acid pH with the optimum being atpH 6 for dark-grown cells and pH 4 for light-grown cells.

Localization of Carotenoids. As seen in the light microscope,the carotenoids are localized in two types of structures: smallgranules and large discs roughly the size of paramylum granules.The small granules are numerous and found in all cells scatteredthrough the cytoplasm with occasional clusters near the gullet.They are similar to the hematochrome granules of wild-typeEuglena and are usually red-orange in color. The other type ofstructure is found in about half of the cells in the cytoplasm andis pale yellow in color.

DISCUSSION

Light unquestionably stimulates d-carotene formation in strep-tomycin-bleached E. gracilis. Comparison of the chemostatkinetics of the accumulation and loss of :-carotene and proteinand the cycloheximide experiments demonstrate that these proc-esses are coupled. However, whether the requirement for proteinsynthesis involves the induction of an enzymatic pathway or theformation of a structure involved in carotenoid localization can-not be specified at this point. Because of the large increase in pro-tein during light adaptation, other proteins besides carotenogenicenzymes are probably formed. A specific structural protein asso-ciated with 3-carotene has been described in spinach leaves (24),and a similar protein may be found in Euglena, accounting forpart of the rise in protein.The observation of structures containing carotenoids in the

cytoplasm of Euglena indicates that the pigments are in part lo-calized. The occurrence of plastid-like structures in bleachedEuglena has been reported by other authors (9, 23, 27). Thesereports describe concentric whirls of membrane which are thoughtto be plastids. We are studying these cells by electron microscopyto determine the ultrastructure of the cytoplasmic bodies seenwith the light microscope. Preliminary results confirm that largestructures consisting of lamellar whirls of membranes do developin the light-adapted cells, but the localization of d-carotene inthese structures has not been demonstrated. Autoradiographicstudies with selective ,B-carotene precursors should unequivocallydemonstrate the pigment distribution. Speculating at this point,the carotenoids may possibly be dissolved in the lipid componentof the membrane and there be associated with protein.

This work has raised some questions about the sequence of /3-carotene synthesis in Euglena. The Porter-Lincoln series is ac-cepted in some form as the route of 3-carotene synthesis in mostorganisms (25). In this pathway the saturated polyenes, phyto-fluene and D-carotene, are thought to be precursors of 3-carotene.This study has shown several times that the saturated polyenesare photolabile and that they are not converted into /-carotenewhen cells are exposed to light. This does not necessarily mean,however, that /-carotene may not be formed via the Porter-Lincoln series. Dark-grown cells may accumulate large amountsof the saturated polyenes in pools because the synthesis of 3-carotene is essentially blocked at a step between A-carotene and3-carotene. When cells are exposed to light, the block is removed,but the saturated polyenes free in the precursor pools are simul-taneously destroyed. New /-carotene could be synthesized via thePorter-Lincoln series if the saturated polyenes were light-stablewhen they were bound to the dehydrogenase system. When thecells are removed from light, the synthesis of /-carotene is againblocked, and precursor pools of phytofluene and A-carotene ac-cumulate.

Inherent in this postulated scheme is a light-activated enzyme

catalyzing the conversion of the saturated polyenes to /-carotene.Unlike many of the bacterial and fungal systems, in which a pulseof light is sufficient to induce carotenogenesis, the synthesis of 3-carotene in Euglena requires continuous light. Though not shownin the results, when a pulse of light was given to a culture, 3-carotene synthesis was not initiated. This is supported by thechemostat studies in which ,B-carotene synthesis was immediatelyshut off, except for a basal rate, when the cells were placed in thedark. Light apparently not only acts as an agent which inducesprotein (enzyme) formation but also acts as an activating agentof existing enzymes.

In the present study and others (15) blue light stimulated caro-tenogenesis, indicating the presence of a photoreceptor whichabsorbs in that region. Cytochromes and protoporphyrins havebeen postulated as the photoreceptors in Prototheca zopfii (7) andin Myxococcus xanthus (1). The solubility characteristics and theabsorption spectrum of a pigment extracted from the Euglenahave led us to believe that it is a protoporphyrin and it may act asa photoreceptor during carotenogenesis.

Several workers have proposed that carotenoids function assubstrates for photo-oxidation by the excited photosensitizer invarious micro-organisms and that the oxidized carotenoids maythen be cycled back to the reduced form by an enzymatic process(21). I have not as yet noted any oxidation products of /3-carotenein extracts of Euglena, though it is possible that they were not de-tected because they display absorption spectra similar to d-caro-tene or have peaks which occur in the ultraviolet region.Cook (3) found that the blue portion of the spectrum was most

effective in inhibiting the growth of bleached Euglena and Astasia.In these studies there was little photoinhibition of growth. Grossand Jahn (14) showed that strains of Euglena bleached by differ-ent methods varied in their carotenoid composition. Severalworkers feel that streptomycin is not uniform in its bleachingeffect (27). Possibly the lack of growth inhibition in the presentstudies was due to the occurrence of more carotenoids or to anincreased ability to synthesize carotenoids during photostress inthe strain used.

Acknowledgments-The criticism, encouragement, and use of the laboratory facil-ities of J. R. Cook are gratefully acknowledged by the author. I am indebted to C. W.Webb, who worked on the project in partial fulfillment of the requirements for a B.S.with honors from the University of Maine, for many suggestions and technicall assist-ance.

LITERATURE CITEI)

1. BURCHARD, R. P. AND M. DWORKIN. 1966. Light-induced lysis and carotellogene-sis in Mjxococcus oanttltus. J. Bacteriol. 91: 535-545.

2. COOK, J. R. 1961. Euglenia gracilis in synchronous division. II. Biosynthetic ratesover the life cycle. Biol. Bull. 121: 277-289.

3. COOK, J. R. 1968. Photoinhibition of cell division and growth in Euglenoid flagel-lates. J. Cell Physiol. 71: 177-184.

4. COOK, J. R. 1968. A continuous culture device for protozoan cells. J. Protozool.15: 425-455.

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photoinduced carotenogenesis in chlorotic euglena gracilis1 · win (11) and chromatographed on an...

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