J. Cell Sd. 61, 413-422 (1983) 413Printed in Great Britain © The Company of Biologists Limited 1983

DEGENERATION AND REGENERATION OF

CHLOROPLASTS IN EUGLENA GRACILIS GROWN IN

THE PRESENCE OF ACETATE: ULTRASTRUCTURAL

EVIDENCE

GIAN LUIGI VANNINIInstitute of Botany, University of Ferrara, 1-44100 Ferrara, Italy

SUMMARY

When green cells of Euglena gracilis, strain Z, were light-grown for several months on a solidmedium containing an excess of sodium acetate (1-0% instead of the normal 0 1 %), some 30% ofthe cells were colourless. The 'acetate-bleached' organisms, isolated by plating methods andsubsequently incubated in the light in a liquid medium, regained the capacity to form chlorophyllin a few days in the absence of any organic carbon source, and within 1-2 weeks in the presence of0-1 % acetate. A number of bleached colonies, however, gave rise to populations in which the delayin pigment synthesis initiation was at least 2 months.

Besides numerous paramylum granules and lipid inclusions, the acetate-bleached cells exhibitedvariably shaped and sized plastids, apparently lacking in ribosomes and showing a deeply disor-ganized membrane system. In the alga greened in the presence of 0-1% acetate, the pattern ofplastidome reorganization was altered; the thylakoids were often unpaired and vesiculated in dif-ferent degrees, owing primarily to the swelling of the lumen. A complete recovery of normalchloroplast structure occurred only after several weeks of exponential growth. The entire populationgreened in the absence of acetate constantly showed normal chloroplasts with perfectly reassociatedthylakoids and clear partitions.

INTRODUCTION

Because of its plasticity in nutritional requirements, the protist Euglena graciliscan be placed between the animal and plant kingdoms. For example, when light andreduced organic compounds are supplied together, photosynthesis and heterotrophicnutrition cooperate in cell growth and multiplication of the alga (Cook, 1968; Nigon& Heizmann, 1978). The two metabolisms, however, interfere at some points. WhenEuglena is grown in the presence of acetate, which is its preferred substratum (Bates& Hurlbert, 1970; Droop, 1974), the acetate brings about a reduction in thechlorophyll content and rate of photosynthesis activity (App & Jagendorf, 1963;Buetow, 1967), and it enters lipids and paramylon only. On the other hand, acetatedoes not enter proteins, which are formed almost exclusively by carbon fixed throughphotosynthesis (Cook, 1965).

Increasing amounts of acetate, as well of other nutrients, increasingly inhibitchlorophyll formation (Buetow, 1967). However, the total repression of the latterprocess by an organic source has never been observed. Thus the view that nonutritional condition can induce the total disorganization of the pre-existing

414 G.L. Vannini

chloroplasts in Euglena has obtained widespread credibility (Mego, 1968; Nigon &Heizmann, 1978; Schiff, 1980).

In this paper we provide evidence that, in a defined condition, an excess of acetate( 1 % instead of the normally supplied 0-1%) produces bleached cells in whichchloroplast structures are almost completely degraded. In addition, the process ofregreening of the white organisms is disturbed by acetate at the relatively low con-centration of 0-1 %.

MATERIALS AND METHODS

E. gracilis Klebs, strain Z, was cultivated on agar slants prepared with Starr's medium (Starr,1960) containing 0-1 % or, alternatively, 1 % acetate. The cultures were maintained in laboratoryconditions like those described by Starr (1973).

After 3 months, cells from each culture type were plated in Mego's agar medium (Mego, 1964),and the dishes were kept in continuous fluorescent light (130-50ft-candles; 1 footcandle =10-764 lx) at 24-26 °C. After 1 week, the ratio of green to white colonies was calculated for each dish.

Colourless organisms, which developed only from cells previously grown in the presence of 1 %acetate, were taken for fluorescence and electron microscopic examinations and to inoculate Mego'sliquid medium with or without 0-1 % acetate. The cultures so obtained were constantly stirred andilluminated at 24-26 °C and kept in the logarithmic phase of growth by frequent subculturing. Inaddition, some cultures were allowed to reach the stationary phase of growth or were prepared inthe resting medium of Stern, Epstein & Schiff (1964).

The regreening process in all cultures was followed by determining chlorophyll content in acetone80 extracts (MacKinney, 1941), and by fluorescence and electron microscopic observations. Theexamination under ultraviolet light was made on living cells using a Zeiss Photomicroscope IIequipped with an epi-fluorescence condenser and a high-pressure mercury lamp HBO W/4 (Van-nini, Bonora & Dall'Olio, 1981).

The specimens for the ultrastructural investigation consisted of organisms harvested once a dayfrom the regreening cultures and then routinely fixed, dehydrated and embedded in an Araldite/Epon mixture (Vannini, Fasulo, Bruni & Dall'Olio, 1978). The ultrathin sections, doubly stainedwith uranyl acetate and lead citrate, were observed and photographed with a Siemens Elmiskop 101(Electron Microscopy Center of Ferrara University).

RESULTS

Once plated, the cells harvested from the tubes containing 0-l % acetate raised98-100% green colonies. On the other hand, the algae sampled from the mediumsupplemented with 1 % of the organic source produced about 30 % of white colonies.The latter consisted of organisms, that can be named 'acetate-bleached' cells, in whichchlorophyll was undetectable by both fluorescence examination in vivo and acetoneextraction.

When examined at the ultrastructural level, the bleached microorganisms exhibitedcytoplasm filled with numerous paramylon grains (Fig. 1) and lipid inclusions consist-ing either of fat globules (Fig. 2) or membrane whorls with varying degrees of or-ganization. These membranous components often appeared in close connection with

Figs 1, 2. Sections through acetate-bleached cells of E. gracilis showing abundantparamylon granules (pa) and lipid inclusions (/). Both X18 000.

Acetate and chloroplast regression in Euglena 415

Figs 1 and 2

416 G. L. Vannini

Figs 3 and 4

Acetate and chloroplast regression in Euglena 417

bodies of uncertain nature, possibly lipid drops or organelles resembling themicrobodies described by Osafune, Klein & Schiff (1980) in etiolated Euglena cells(Fig. 3).

Especially noteworthy was the constant presence of variably shaped plastids, whichapparently lacked ribosomes and possessed a deeply disorganized thylakoid system(Fig. 4). These organelles, which often contained a great number of plastoglobules(Fig. 5), were larger than the proplastids of the dark-grown cells.

The acetate-bleached algae kept in a liquid medium depleted of acetate regained theability to form chlorophyll within 2—3 days. After this lag period, the kinetics ofpigment synthesis, as well as that of the structural development of the chloroplasts,were virtually the same as have been described repeatedly for cells first cultivated inthe dark and then exposed to the light (see Nigon & Heizmann, 1978). In particular,the thylakoids appeared perfectly associated in all phases of the developmental process(Fig. 6).

When 0-1 % acetate was present in the medium, the acetate-bleached cells under-went a 1 to 2-week lag period in chlorophyll formation and in plastid structuraldevelopment. Subsequently, the pattern of plastidome organization went through anabnormal phase characterized by the presence of organelles, often possessing a well-structured pyrenoid (Fig. 7). The plastids were unusual in that the thylakoids werevesiculated to varying degrees, due primarily to the swelling of the lumen (Figs 8, 9).This stage of vesiculation lasted approximately 1—2 weeks when the cultures weremaintained in the exponential phase of growth, and longer for organisms in a restingmedium or in the stationary phase of growth. In either case, the algae characterizedby vesiculated plastids contained an amount of chlorophyll virtually the same as thatin the control group, which was formed by etiolated cells exposed for several days tocontinuous light in a medium containing 0-1 % acetate.

Finally, a complete recovery of chloroplast structure occurred, with perfectlyreassociated thylakoids and clear partitions (Fig. 10). All the stages in plastidregeneration were accompanied by a decrease in paramylon and lipid inclusions.

Fig. 3. Section through a cell as in Fig. 1, showing relationship of lipid bodies (/) andmembrane whorls (miv). X55 000.

Fig. 4. Degraded plastids in an acetate-bleached cell. X40 000.

Fig. 5. Plastid of a bleached cell displaying a stroma with several plastoglobuli, fewthylakoids and apparently no ribosome. X48000.Fig. 6. Section through a bleached cell cultivated in the light in a liquid medium deprivedof acetate. The plastids show a normally developed and arranged thylakoid system.X30000.

Figs 7-9. Sections through bleached cells cultivated for 2 weeks in the light in a liquidmedium containing 0-1 % acetate.

Fig. 7. A vesiculated chloroplast with a well-developed pyrenoid (p) surrounded byparamylon granules (pa). X28000.

Fig. 8. An abnormally shaped chloroplast with numerous and large vesicles.X 23 000.

Fig. 9. Highly malformed chloroplast, partly occupied by large vesicles and partlyshowing an almost normal structure. X23 000.

418 G. L. Vannini

Figs 5 and 6. For legend see p. 417.

Acetate and chloroplast regression in Euglena 419

Figs 7-9. For legend see p. 417.

420 G. L. Vannini

Fig. 10. Section through a bleached cell after 3 weeks of regreening in a medium contain-ing 0-1 % acetate. The chloroplast shows no apparent signs of vesiculation and displaysperfectly reassociated thylakoids. X35 000.

DISCUSSION

Contrary to published studies (Cook, 1968; Nigon & Heizmahn, 1978), we haveprovided evidence of nutrition-induced chloroplast degeneration inEuglena grown inthe light.

On the whole, 1 % acetate in defined conditions apparently acts on this alga likeexcess glucose on Chlorellaprotothecoid.es. Excess glucose is commonly used to inves-tigate the conversion of green cells into white organisms and vice versa undercontrolled nutritional conditions (Hase, 1971; Osafune & Hase, 1975).

Caution, however, is required in comparing experiments in which differentnutrients are employed. In addition, Euglena and Chlorella are not equivalent sys-tems, because the latter alga forms chloroplasts both in the light and in the dark.Despite this, it is reasonable to assume that some common event occurs in thenutrition-induced regression of chloroplasts into plastid remnants in facultativephoto-organotrophs. More precisely, in accordance with most studies of the influenceof carbon sources on both the above-mentioned algae (see Harris & Kirk, 1969; Hase,1971; Schuler, Brandt & Wiessner, 1981), we believe that an unknown repressor

Acetate and chloroplast regression in Euglena 421

substance(s) forms during acetate utilization. In particular, over prolonged exposureto the carbon source, the slowly dividing cells oi Euglena could accumulate numerousparamylon grains and lipid inclusions (according to the biochemical data of Cook,1965) as well as repressor intermediate(s). The latter may form in such large amountsthat the internal structures of the pre-existing chloroplasts cannot be maintained.When the acetate-bleached cells are transferred into an acetate-free medium, the toxicsubstance(s) may be readily degraded and/or diluted among the generations. On thecontrary, when 0-1% acetate is present, the amount of bleach-inducing inter-mediate^) decreases more slowly, so that chloroplast morphogenesis starts afterseveral days and follows an abnormal pattern. This observation seems to show that oneor more regulatory interactions are disturbed, presumably until the level of toxicsubstance(s) exceeds a critical value.

The morphological data presented here do not furnish indications as to which cellcompartment concerned with plastid development is more affected by the repressorcompound(s) (see Herrmann, Borner & Hagemann, 1980; Schiff, 1980). However,an initial study with etiolated Euglena cells, first grown in the dark and thenilluminated in a resting medium supplemented with 1 % acetate, shows that chloro-phyll a to b ratio during both the lag period and the major period of chloroplastdevelopment (see Nigon & Heizmann, 1978; Schiff, 1980) is constantly less than 2-5.

Therefore, the repressive effects seem to interact primarily with the synthesizingsystem for the thylakoid particles known to have a high chlorophyll a to b ratio (PS Icomplex), whereas the formation of particles in which this ratio is low (PS II complex)(Brandt, 1980, 1981) is conceivably less affected by acetate. If so, differences in themechanism of action of acetate and glucose exist, because the latter primarily affects PSII when applied in excess to regreenmg Euglena (Schwelitz, Cisneros & Jagielo, 1978;SchulereJa/. 1981). However, it should be remembered that glucose, under the con-ditions used, does not act as a bleach-inducing agent tor Euglena.

Although considerable work remains to be done to determine the mode in whichacetate acts on chloroplasts, this carbon source appears to be a unique tool in helpingto explain the nutritional control of plastid development in Euglena.

I thank Professor Giuseppe Dall'Olio for critical reading of the manuscript. I also thank DrArnaldo Donini for experimental collaboration, Mr Andrea Campana for technical assistance, andMr Giovanni Rompietti for typing the manuscript. This work was supported in part by a grant fromthe Italian Research Council (CNR).

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(Received 19 May 1982 -Accepted 22 November 1982)