RNA synthesis in isolated chloroplasts of Euglena gracilis

Plant Science Letters, 16 (1979) 203--210 © Elsevier/North-Holland Scientific Publishers Ltd.

203

RNA SYNTHESIS IN ISOLATED CHLOROPLASTS OF

EUGLENA GRACILI,q R. WOLLGIEHN and B. PARTHIER

Akademie der Wissenschaften der D.D.R., Institut fiir Biochemie der Pflanzen, DDR-401 Halle/Saale, Postfach 250 (G.D.R.) (Received April 11th, 1979) (Revision received and accepted June 2nd, 1979)

SUMMARY

RNA synthesis in isolated chloroplasts of Euglena gracilis was measvred by [ 14C]ATP incorporation into electrophomtically~separable RNA species. The incorporation kinetics reached a plateau after 30 rain incubation, probably due to completing RNA strains which had been initiated previously. We failed to inhibit RNA polymerase activity by addition of 100 ~g/ml rifamycin suggesting a lack of transcription initiation in isolated chloroplasts, in contrast to in vivo experiments. A precursor RNA transcript of mol. wt approx. 1.8 × 106 was found both in mostly intact and broken chloroplasts. In the latter this RNA accumulated but was not processed to immediate precursor or mature rRNAs. The 1.8 X 106 size of the presumed initial common precursor for Euglena chloroplast rRNAs is in good agreement with the length of the respective rRNA gene previously determined.

INTRODUCTION

In vivo and in vitro studies with different plants have shown that the two mature high molecular weight chloroplast ribosomal RNAs (1.05 and 0.56 X 106) result from stable immediate precursors of molecular weights of approx. 1.2 and 0.65 X 106 [ 1 - 1 3 ] . A high molecular weight primary transcription product of the rDNA region containing all kinds of ribosomal RNA was found only occasionally, probably because of the extreme lability of this precursor molecule. From pulse-chase experiments in vivo with Spinacia and Spirodela [4,7] and from labelling experiments with intact isolated Spinacia chloroplasts in a light-driven reaction [ 3,4~14,15] a primary transcription product of 2.7 X 106 was observed. On the other hand, Stempka and Richter [16] reported 1.8 × 106 RNA as a common precursor of chloroplast rRNAs in the two alga species, Chlorella vulgaris

and Cyanidium caldarium.

204

Despite the abundant knowledge of chloroplast RNA synthesis in the phytoflagellate Euglena gracilis a common precursor for the chloroplast rRNAs has not been demonstrated [10--12]. According to the size of each of the rRNA regions in the Euglena chloroplast chromosome [17--19] a precursor smaller than 2 X 10 ~ should be expected. The results on in vitro RNA synthesis with isolated chloroplasts described in this paper confirm this assumption. MATERIALS AND METHODS

Euglena culture and chloroplast isolation. Euglena gracilis, strain Z, was grown autotrophically in Cramer and Myers' medium [20] as described earlier [21 ]. Cells of the late logarithmic phase (3--5 X 10 ~ cells per ml) were used for the experiments. Chloroplasts were isolated according to Manning et al. [22] with the following variations: after washing the cells with medium A (50 mM Tris-HCI, pH 7.8; 10 mM MgCI2; 10 mM KCI; 6 mM mercaptoethanol) containing 10% sucrose, 10 g of cells were suspended in 30 ml of the same medium and disrupted by means of the Aminco French Pressure Cell at 138 Pa. The homc~genate was diluted with 40 ml Honda medium (25 mM Tris--HCl, pH 7.8; 1 mM MgCI2; 5 mM mercaptoethsnol; 0.25 M sucrose; 25 g/l Ficoll; 50 g/l Dextran T 40) and stirred carefully for 5 min. Cells, cell debris and nuclei were separated by two times centrifugation for 20 min at 150 g, and the crude chloroplast fraction was sedimented at 3 000 g for 10 rain. Chloroplasts were suspended in 15 ml medium A plus 20% sucrose (w/v) and mixed carefully with 100 ml medium A containing 2.20 M sucrose for flotation. After centrifugation at 15 000 g for 45 min the chloroplasts were found as a thin layer on the surface of the sucrose buffer. The layers were removed and suspended in medium A (plus 20% sucrose). The chloroplasts were sedimented by centrifugation at 3 000 g and were suspended directly in sucrose-containing buffer for RNA synthesis (see below), in order to maintain organelle integrity. These 'intact' chloroplasts appear swollen in the electron microscopic pictures, but internal membrane and envelope structures seem to be mostly intact. Nevertheless they have lost stroma protein and contain only one third of the ribulose bisphosphate carboxylase activity of the whole cell (on chlorophyll basis). Broken chloroplasts were obtained by suspending 'intact' chloroplasts in 50 mM Tris-HCl (pH 7.8); 10 mM MgCI2; 10 mM KCI; 5 mM mercaptoethanol. After keeping for 10 rain at 0°C, the suspension was centrifuged for 5 min at 3000 g and the green sediment was used for RNA synthesis by preparations containin£ broken chloroplasts.

RNA synthesis in vitro. Chloroplasts at a concentration of I mg chlorophyll per ml were incubated at 25°C in a medium containing 50 mM Tris-HCI (pH 7.8); 10 mM MgCl2; 10 mM KCI; 5 mM mercaptoethanol; 2.5 mM phosphoenol pyruvate, 50 ~g/ml pyruvate kinase, 0.025 mM [~4C]ATP

205

(1.25 pCi/ml), 0.1 mM each of the three other non-labelled nucleoside triphosphates, 1 mg/ml bentonit [ 1,13 ], and in all experiments with 'intact: chloroplasts 0.4 M sucrose. The addition of bentonit was necessary to prevent RNA degradation. Without bentonit no high molecular RNA was obtained. RNA-polymerase reaction product was measured as follows: 75/zl of the incubation mixture was pipetted on a Whatman G F / C glass microfibre filter. The discs were quickly transferred into cold 0.2 N HCIO4 containing 2% pyrophosphate, and then washed three times with the same acid, twice with 70% ethanol, once with 96% ethanol, once with ethanol/ether (3 : 1) and finally with ether. After drying the discs the radioactivity was counted in a Packard scintillation spectrometer. RNA extraction, RNA analysis by gel electrophoresis and the sources of chemicals used were described earlier [13]. RESULTS

In previous studies on RNA synthesis with isolated, broken Euglena

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= e/cctrophoret/c mob///cy Fig. 2. Gel eleetrophoresis of RNA synthesis in isolated chloroplasts. 'Intact' (A and B) and broken chloroplasts (C and D) were incubated 7 min. (A and C) or 35 min (B and D) with ['4C]ATP. (o =) radioactivity, ( ~ ) absorbanee from non-labelled marker chloroplast RNA. The numbers in the figures show the positions of rRNA precursor RNA and mature rRNA in mol. weight x 10 s. RNA separation was performed in 2.4% polyarcylamide with 0.5% agarose at 6 mAlgel for 3 h. Under these conditions the low molecular weight RNAs (4S, 5S) migrate off the gel.

207

chloroplasts we were unable to identify discrete RNA classes, i.e. ribosomal RNA and its precursors. Therefore we compared broken chloroplasts with 'intact' chloroplasts though it is known that uptake of nucleoside triphosphates into intact chloroplasts is limited. For this reason Hartley and Ellis [4] and Bohnert and Schmitt [3] used nucleosides, which in a lightdependent reaction were incorporated into plastid RNA. The kinetics of the [ z4C]ATP incorporation is principally the same in the two chloroplast preparations, though radioactivity is lower in RNA of the 'intact' chloroplasts than in that of broken chloroplasts. Thus, the 'intact' chloroplasts in our preparation are able to take up and incorporate nucleoside triphosphates into RNA (Fig. 1). This seems not to be due to the proportion of broken chloroplasts in that preparation as shown by the qualitative differences in the labelled RNA profiles (Fig. 2B versus 2D). The reaction stops after 45 min. This is not due to substrate deficiency, because after addition of new enzyme (chloroplasts) RNA synthesis increased further, but the addition of new substrate has no influence on the amount of incorporation. The reason may instead be an accumulation of suppressing reaction products (pyrophosphate?) [13], or the absence of initiation of new RNA chains under our in vitro conditions is responsible for the course of incorporation kinetics. The second possibility is supported by the observation that neither in 'intact' nor in broken chloroplasts is the incorporation of ATP influenced by rifamycin (Table I), although chloroplast RNA synthesis in whole Euglena cells is clearly inhibited by the drug [ 13]. All previous analyses of chloroplast RNA synthesised in vitro have shown that ribosomal RNA is preferentially synthesised [ 1,3,4,12,14,15,19]. However, as demonstrated in Fig. 2, heterogeneous profiles of radioactivity are always observed in the electropherograms. Provided that high molecular weight messenger RNA in approximate sizes of rRNA is also synthesised one should expect that besides a weakly labelled 'background' of heterogeneous TABLE I EFFECT OF RIFAMYCIN ON RNA SYNTHESIS IN ISOLATED CHLOROPLASTS Chloroplasts ('intact' or broken)were suspended in incubation medium (see under Materials and Methods) without nucleoside triphosphates in presence or absence of 100 ~g/ml rifamycin SV and preincubated 15 min at 25°C. Nucleoside triphosphates were then added to start the reaction. Radioactivity in epm/75 ~1 incubation suspension.

Without ri famycin Plus rifamycin

'Intact' chloroplasts

Broken chloroplasts

10 min

60 min

10 min

60 min

2790 2880

9140 9080

4300 4160

13500 13800

208

mRNA certain mRNAs may be labelled more pronounced, e.g. the 0.45 X 106 messenger for a chloroplast membrane protein called P protein [ 23--25] or the 0.6--0.7 × 106 messenger for the large subunit of ribulose bisphosphate carboxylase [25]. Bearing in mind these data we can explain the results of our in vitro experiments as following: 1. In accordance with our earlier results [ 13] the lengths of the RNA molecules increase during incubation time (Fig. 2). Assuming a lack of initiation in our chloroplast preparations (Table I) the profiles in Fig. 2 also indicate that the transcription products are accomplished not earlier than 30 min. 2. After short, time incubation (7 rain) the RNA preparations from 'intact' and broken chloroplasts give nearly the same profiles of radioactivity (Fig. 2A and C). The appearance of labelled peaks in the region of the mature rRNAs should not lead to the conclusion of a complete synthesis of immediate precursors and processing to mature rRNAs during the first minutes of incubation. This RNA distribution is probably due to nonfinished polynucleotide chains, which by chance have the same size as mature rRNAs. 3. In both systems RNA up to approx. 1.8 × 106 is synthesised during 30 min of incubation (Fig. 2B and D). We believe that the 1.8 × 106 RNA is the primary transcript of the rRNA region in the chloroplast DNA. Our data [ 13] about in vitro synthesised minor RNA fractions with a higher molecular weight than 1.8 × 106 have to be corrected as they are not reproducible under improved conditions of incubation and RNA analysis. 4. In broken chloroplasts the 1.8 × 106 RNA is accumulated (Fig. 2D). Labelled mature rRNA is scarcely detected. 5. The RNA synthesised in 'intact' chloroplasts is more heterogeneous in size (Fig. 2B) than that of broken chloroplasts (D). In the radioactivity profile we find peaks both in the regions of rRNA precursors (approx. 1.8, 1.2 and 0.65 × 106) and of mature rRNAs (1.05 and 0.56 X 106). 6. Prolongation of the time of incubation or a chase (up to 60 min) after a 30 min incubation period with [ 14C]ATP has no influence on further processing (not shown here). But during long incubation we observed some decrease in the amount of radioactivity and the profile became more heterogeneous, presumably due to fragmentation of newly-synthesised RNA. DISCUSSION Transcription takes place in isolated, mostly intact and broken chloroplasts of Euglena, though rRNA processing is disturbed in the broken organelles. We suppose that factors necessary for processing were removed during osmotic disruption and washing of the chloroplasts while the membran~bound transcription complex remained intact. In spinach chloroplasts high KCI in the medium likewise leads to accumulation of the primary transcription product [ 14,15].

209 For chloroplasts of Spinacia (perhaps all higher plants) a 2.7 × 10 ~ primary rRNA transcription product was claimed [3,4,7,14,15]. It corresponds in size to the rRNA transcription region of the chloroplast DNA, which contains besides the genes for 23S, 16S and low molecular weight rRNAs a spacer of approx. 1400 base pairs between the 23S and 16S RNA genes. The sizes of the intermediate products of various processing steps up to the immediate precursors are still in discussion. The scheme of processing seems to be different in alage: from kinetic and inhibitor experiments m vivo Stempka and Richter [ 16] concluded that the two rRNAs originate from a single precursor having an apparent molecular weight of 1.8 × 106 This corresponds with our data in Euglena. Our in vivo experiments also revealed a labelled very small RNA fraction with a molecular weight of approx. 1.8 × 106, the synthesis of which is inhibited by rifamycin [ 13,26 ]. However, we did not conclusively prove the preribosomal nature of this peak. In vitro, on the other hand, 1.8 × 106 RNA forms a distinguishable fraction, and in broken chloroplasts it is strongly accumulated and represents the major labelled fraction. Each of the three repeated rRNA regions in Euglena chloroplast DNA consists of 5600 base pairs [ 17--19] ; it contains a cluster of genes including the 23S, 16S and 5S rRNAs and likewise tRNA in the short spacer regions. Since the two immediate precursors of rRNA (0.65 plus 1.16--1.20 × 106) require 5.3 thousand base pairs, the theoretical size of the primary transscription product of Euglena rDNA is in the magnitude of 1.8 × 106. It seems improbable but at present not yet excluded that the total rRNA is transcribed in one piece of unstable pre-rRNA giving rise to 1.8 X 106 RNA as a stable cleavage product. The profile of radioactivity (Fig. 2B) is more heterogeneous and contains some other fractions besides the three postulated precursor RNAs and two mature rRNAs. A classification of the non-identified labelled peaks has not yet been done. The 0.9 and 0.45 × 10 ~ RNAs could by fragmentation products of the 23S RNA, although newly synthesised rRNA appears to be very stable [ 27]. It is also possible that additional RNA fractions including mRNAs were synthesised in isolated chloroplasts under our conditions. Hybridisation experiments may give further information. REFERENCES 1 2 3 4 5

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