Planta

Planta (1982)156:274-281

9 Springer-Verlag 1982

Dark-induced chloroplast dedifferentiation in Euglena graciIis A. Scheer and B. Parthier* Institut fiir Biochemie der Pflanzen, Akademie der Wissenschaften der DDR, Weinberg 3, DDR-4020 Halle, German Democratic Republic

Abstract.

Transfer of light-grown autotrophic

Euglena gracilis cells to darkness and carbon (glucose) containing heterotrophic media causes structural and functional decomposition of the photosynthetic apparatus. The process can be ascribed to a strict diluting-out mechanism of stroma constituents among the progeny, as shown for ribulose-l,5-bisphosphate carboxylase (RuBPCase, EC4.1.1.39), and aminoacyl-tRNA synthetases (Aa-RS; especially Leu-RS, EC 6.1.1.4) activities. The diluting-out effect of thylakoid membranes and chlorophyll seems to be superimposed by additional degradations, beginning soon after the transfer of cells to darkness. Cultivation of cells in darkness in 0.03 M KC1 or without utilizable organic carbon (resting media) preserves chloroplast structure and function over a long period, indicating negligible turnover in these cells. Thus, under both growing and resting conditions, darkness induces the arrest of synthesis of plastid constituents. Experiments with the inhibitors cycloheximide, chloramphenicol, and nalidixic acid demonstrate that chloroplast dedifferentiation does not require organelle gene expression, but it is more strictly dependent on biosynthetic events in the nucleo-cytoplasmic compartment than the reverse process, light-induced chloroplast formation. Since cycloheximide at low concentrations in growth medium causes a marked suppression of precursor uptake or re-utilization similar to that in cells of resting media, intracellular precursor deficiency is suggested to control the observed blockade in cytoplasmic synthesis of plastid proteins. On the other hand, darkness might signalize the stop of gene expression in the organelles. * To whom correspondence should be addressed Abbreviations: Aa=aminoacid; CH=cyCloheximide; C M =

chloramphenicol; Leu-RS=leucyl-tRNA synthetase; RuBP= ribulose-1,5-bisphospbate; TCA = trichloroacetic acid

0032-0935/82/0156/0274/$ 01.60

Key words: Amino-acid uptake - Chloroplast dedifferentiation (dark-induced) - Euglena- Plastid stroma enzymes.

Introduction

Euglena gracilis is an unicellular organism with a high degree of metabolic flexibility. It is able to multiply in light, in the absence or in the presence of organic carbon substrates, or under heterotrophic conditions in complete darkness. Transition of cells from darkness to light, or vice versa, must involve drastic changes in metabolic and energetic pathways. Such biochemical changes are connected with alterations in size, ultrastructure, and gene expression of the energy-generating organelles, chloroplasts and mitochondria. Whereas light-induced chloroplast formation from proplastids is well studied (cf. reviews by Schiff 1978; Nigon and Heizmann 1978; Parthier 1981), the reverse process, dark-dependent dedifferentiation of mature chloroplasts to proplastids, has found much less attention. Green Euglena cells transferred to organic media in the dark reduced their chloroplasts both in ultrastructural and functional (photosynthetic) aspects, indicating a diluting-out mechanism of plastid constituents due to cell and organelle divisions (Ben-Shaul et al. 1965; Ophir etal. 1975; Ehara et al. 1975). However, Ophir et al. (1975) compared the rates of decrease of chlorophyll content, thylakoid number, and photosystem I and II activities and found no strict parallelity. Obviously certain constituents of the photosynthetic apparatus are more stable than others under such conditions. Since the components of the photosynthetic membranes (lamellae) are at least partially synthesized on plastid ribosomes, it would be interesting to analyze stroma

A. Scheer and B. Parthier: Chloroplast dedifferentiation in Euglena

enzymes, which originate from the cytoplasm, during chloroplast dedifferentiation in dark-adapting cells. This paper demonstrates changes in activity of ribulose-l,5-bisphosphate carboxylase (EC 4.1.1.39) and some aminoacyl-tRNA synthetases (EC 6.1.1) of autotrophic E. gracilis cells during prolonged dark periods in growth and resting media. The results are correlated with changes in plastid ultrastructure, pigment content, and amino acid uptake. It is suggested that the decrease in stroma enzyme activities are caused by diluting-out of the enzyme proteins upon the dark-induced stop of their synthesis.

275 30 min by the addition of 0.05 m120% cold trichloroacetic acid (TCA). After the protein had been precipitated over night, the supernatant was cleared by centrifugation and the radioactivity of the formed p4C]phosphoglyceric acid was measured on paper filter disks in a scintillation counter.

Aminoacyl-tRNA synthetase activities were assayed in a reaction volume of 0.11 ml containing 50 I.tl aliquots of the hydroxyapatite eluate fractions, 45 gg of deacylated tRNA, 0.1 gM U-14C labeled amino acid (UVVVR Prag, Czechoslovakia; spec. act. 8 GBq retool-l), and the reaction cocktail (Parthier and Krauspe 1974). After 5 and 10 min incubation at 28 ~ C, 50 I~l-aliquots were transferred to paper filter disks which were immediately tipped into cold TCA, washed with cold TCA, ethanol and ether, and were then counted for tRNA-bound radioactivity. Transfer R N A preparation from Anaeystis niduIans and from the W~BUL mutant of E. gracilis has been described earlier (Parthier and Krauspe 1974). E. co# t R N A was a gift from Dr. J. H. Well, Strasbourg, France.

Material and methods Media and growth conditions. All experiments were performed with autotrophic cells of Euglena gracilis, Z strain (No. 1224-5/ 25, Algensammlung G6ttingen). The cells were grown axenically in l-liter cylindric flasks in the autotrophic acid medium of Hutner et al. (1956), supplemented with an air: CO2 (97:3,1/ v) mixture at 5,000 lx and 24 ~ C. The heterotrophic growth medium was the same, but contained 10 g D-glucose per liter. The resting media were composed of 0.03 M KC1 or 0.054 M mannit, 0.01 M KHzPO ~, 0.01 M MgCI2 (mannit medium; Stern et al. 1964). The transfer of cells from the autotrophic medium to other mediums was done under sterile conditions. Aliquots were collected by low-speed (300 g) centrifugation at room temperature, washed with growth or resting medium, and resuspended for a chosen cell concentration. After incubation periods as indicated in the legends of the Figures, the aliquots were harvested by centrifugation, washed twice with 0.01 M potassium phosphate buffer (pH 7.5) containing 1 mM MgClz, 5 mM fl-mercaptoethanol and 15% glycerol, and were then frozen at - 3 0 ~ C.

Preparation of enzyme extracts. After resuspension of usually 5.108 cells (1 g fresh weight) in 3 ml buffer, the cells were completely broken by ultrasonication (Branson Sonifier, 80 W) for 60 s, centrifuged at 6,000 g for 10 rain, and the supernatant at 100,000 g for 60 min. One ml portion of this supernatant was dialyzed against Tris/MgZ+/K+/mercaptoethanol buffer (pH 8.0) and thereafter used for the determination of RuBPCase activity and protein content; another ml-portion was chromatographed on small columns, each containing 5 ml hydroxyapatite, in order to separate organelle and cytoplasmic Aat R N A synthetases from each other by stepwise elution with 0.05 M potassium phosphate and 0.2 M potassium phosphate, respectively (Krauspe and Parthier 1974; Parthier and Neumann 1977). Fractions of 6 ml were collected and then estimated for Aa-RS activities with t R N A preparations from Anacystis nidulans or E. coli for the plastid-specific enzyme and cytoplasmic t R N A of the aplastidic Euglena mutant W3BUL for the cytoplasmic enzyme (Parthier and Krauspe 1974).

Enzyme assays. RuBPCase activity was determined in a reaction mixture of 0.1 ml volume containing 50 m M Tris-HC1, pH 8.0; 1 5 m M MgCtz; 60raM KC1; 1 0 m M 2-SH; 6 . 6 m M NaH14CO3 (spec. act. 155MBq mmol-*); 1.5mM RuBP (Sigma, 98.3 % purity); dialyzed supernatant of 0.2 mg protein. The incubation was done at 28 ~ C and stopped at 10, 20, and

Other determinations. Cell numbers were counted in 1% NaC1 in an electronic particle counter T U R ZG1 (VEB Transformatoren- und R6ntgenwerk, Dresden, GDR) using 140 gm aperture. Chlorophyll was determined according to Arnon (1949) in 80% acetone, carotenoids according to Jensen and Jensen (1971). Protein determination (Lowry et al. 1951) was done with bovine serum albumin as a standard. Aminoacid uptake and incorporation in vivo. This was done according to our earlier experiments (Parthier 1974). In brief, cells were collected by low-speed centrifugation (1,500g), washed, and resuspended in fresh media to a concentration of 107 cells ml-1. The cells were adapted for 30 min before the addition of 14C-amino acids. Incubations were carried out at 26 ~ C in darkness under gentle agitation. 100 gl-aliquots were taken at various time periods, rapidly transferred into 5 ml icecold water, poured onto paper filters, and washed three times with 5 ml ice-cold water on these filters. Then they were dried and counted for radioactivity after removal of the pigments by UV irradiation of the disks in toluol. Corrections were made for the quenching effects. Incorporation into acid-insoluble cell material was similarly done with the variation that ice-cold 10% TCA was used instead of water; TCA-soluble radioactivity was extracted for 3-4 h at 0 ~ C.

Electron microscopy. The cells were treated with 3% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.4, for 2 h, at room temperature and fixed with 1% OsO4 in 0.05 M cacodylate buffer. Contrasting was done with 1.5% uranyl acetate in 70% aceton for 2 h, likewise with lead acetate.

Results Growth curves

Transfer of autotrophic cells to organotrophic (Hutner's) medium and darkness is accompanied with a lag period of 15-20 h followed by a logarithmic growth of the culture (Fig. 1 a). The generation time is 17 h under our conditions. No cell division has been observed in autotrophic cells resuspended in fresh autotrophic medium which contains the

A. Scheer and B. Parthier: Chloroplast dedifferentiation in Euglena

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Fig. 1. Cell numbers of autotrophic E. gracilis after transfer to darkness in different growth media, a, Hutner's growth medium containing 10 g 1-1 D-glucose; b, autotrophic (mineral) medium, the same as in a but lacking glucose; e, 0.03 M KC1 only

same constituents, but lacks glucose (Fig. I b). Likewise, cell division is suppressed in resting media, either consisting of 0.03 M KC1 only (Fig. I c) or 0.054 M mannit and the vitamins. Plastid ultrastructure and pigment contents E. gracilis chloroplast lamellae are extended stacks of two or three thylakoids. The number per organelle varies dependent on the nutritional condition of the cell We find 20-30 lamellae per chloroplast under autotrophic conditions (Fig. 2a), whereas in photoheterotrophic cells only 5-10 lamellae have been observed (Neumann and Parthier 1973). When autotrophic cells are transformed to organotrophic cells in darkness, they reduce continuously both size of chloroplasts and number of lamellae per organelles (Fig. 2 d-g, Fig. 3). One hundred forty-four hours growth (about six generations) in darkness results in bleached heterotrophic cells containing proplastids which cannot be discriminated from those of continuously darkgrown heterotrophic cells (cf. Ophir et al. 1975; Ehara et al. 1975). They are free of photosynthetic membranes, but contain a girdle-lamellae near the plastid envelope (Fig. 1 g). Plastid length and number of lamellae remain constant for at least 96 h in cells of autotrophic or resting media under dark conditions (Fig. 2b, c, Fig. 3). These cells show a certain reduction in the size of chloroplasts (Fig. 3 A) probably due to

Fig. 2a-g. Ultrastructural changes of chloroplasts in autotrophic E. graeilis cells after transfer to darkness in different growth media, a Original autotrophic cells; b after 94 h darkness in autotrophic (mineral) medium; e after 94 h darkness in mannit medium; d after 22 h darkness in glucose containing (Hutner's) medium; e--g the same medium as in d but after 46, 94 and 144 h darkness, m, mitochondria; n, nucleus; o, osmiophilic globuli; pa, paramylon grains; pe, pellicula; pp, proplastid; v, vacuole, a-c, 12,000 x ; fl-g, 24,000 x

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A. Scheer and B. Parthier: Chloroplast dedifferentiation in Euglena

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a broadening of the organelles, but there is no loss in the number of lamellae (Fig. 3 B). The dark-dependent and glucose-induced decrease in size of chloroplasts and number of photosynthetic lamellae is connected with a loss of chlorophyll content per cell. As can be seen in Fig. 4A, the chlorophyll content per cell drops without a lag period soon after transfer of the cells to darkness. Thus, the exponential decrease caused by cell and chloroplast division seemed to be superimposed by chlorophyll destruction before cell division. This picture is consistent with an immediate cease of chlorophyll de nova synthesis and thylakaid formation in darkened cells. After division the preexisting structures are redistributed among the progeny cells. The course of total carotenoid decrease (Fig. 4B) differs slightly from that of the chlorophylls, indicating a partial distribution of carotenoids in cytoplasmic granula (Dolphin 1970). Stroma enzyme activities RuBPCase. Changes in activity of the major stroma enzyme, ribulose-l,5-bisphosphate carboxylase, have been determined in the crude postribosomal supernatants of the cells. In Fig. 5A, curve a illustrates that the dark-dependent reduction in activity on a cell basis strictly follows the cell division curve, thus confirming a pattern which is consistent with a diluting-out effect of the enzyme among the progeny cells. The lag period of decrease in the first 20 h is consistent with a slow turnover of RuBPCase in darkness (cf. Lord et al. 1975). Enzyme activities were likewise compared

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Dark-induced chloroplast dedifferentiation in Euglena gracilis.

Transfer of light-grown autotrophic Euglena gracilis cells to darkness and carbon (glucose) containing heterotrophic media causes structural and funct...
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