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Ribosomal RNA precursor synthesis in tobacco tissue culture
503
BIOCHIMICA ET BIOPHYSICA ACTA
BBA %710
RIBOSOMAL RNA PRECURSOR SYNTHESIS IN TOBACCO TISSUE CULTURE T S A I - Y I N G C H E N G AND G. L. H A G E N
The Institute [or Cancer Research, 77oz Burholme Avenue, Fox Chase, Philadelphia, Pa. x 9 I z I
(u.s.A .)
(Received A u g u s t i z t h , 197 o)
SUMMARY
This study, using an aseptic suspension culture of undifferentiated meristematic parenchyma cells of tumor-forming tobacco hybrid of Nicotiana glauca and Nicotiana langsdor]]ii, has shown an enhancement of RNA synthesis by the phytohormone indole-acetic acid. Analyses by sucrose density-gradient sedimentation and polyacrylamide gel electrophoresis show a rapidly labeled rRNA precursor with a sedimentation coefficient of 3 8 S and a molecular weight of 2.8.IO e. Actinomycin D-chase experiments, further, indicate that the heavy rapidly labeled RNA is indeed the rRNA precursor. An intermediate component which is slightly heavier than large rRNA with a molecular weight of 1.5" lO6 is also detected.
INTRODUCTION
Phytohormones are known to influence plant nucleic acid metabolism x. Numerous studies z-~ further indicate that auxins both enhance the synthesis of all RNA species and preferentially increase the synthesis of rRNA when compared to tRNA and mRNA. In this study, aseptic suspension cultures of white friable undifferentiated callus cells such as those derived from pith explants of the tumor-forming tobacco hybrid of Nicotiana glauca and Nicotiana langsdor]]ii were used. These cells have several unique advantages including: (I) They are potentially tumorous tissueS; (2) they are free from microorganism contamination which has frequently interfered with plant nucleic acid analysis; (3) they are an essentially homogeneous mass of parenchyma cells with meristematic activity; (4) they can be manipulated in a manner comparable to a bacterial system, thus facilitating the experimental scheme; and (5) their uptake of radioactive compounds is rapid and uniform. Further, the techniques of sucrose density-gradient sedimentation and polyacrylamide gel electrophoresis were used instead of methylated albumin coated kieselguhr column chromatography, which has been widely used in the plant nucleic acid research. MATERIALS AND METHODS
Plant and growth conditions The tumor forming hybrid of N. glauca and N. langsdorlfii was used as the source material for these experiments. The pith tissue from non-flowering plants was Biochim. Biophys. Acta, 228 (1971) 503-508
504
T.-Y. CHENG, G. L. HAGEN
aseptically removed and cultured on a modified MURASHIGE AND SKOOG9 medium containing, per 1: (I) inorganic salts, 1.65 g NH,NOs, 1.9o g KNO 3, 0.44 g CaC12' 2H20, 0.37 g MgSO4"7HzO, o.17 g KH2PO4, o.12 g FeSO,.7H20, o.145 g disodium EDTA, 6.20 mg H3BO4, 22.3 mg MnSO,'4H20 , lO.51 mg ZnSOa.7H~O, 0.83 nag KI, 0.25 nag NaMoO~'2H20, 0.025 mg C u S Q ' 5 H 2 0 , and 0.025 mg CoC1~.6H20; and (2) organic substances, IOO mg myoinositol, o.I mg thiamine HC1, I.O mg indole-acetic acid, 20 g sucrose, and 0.8 °/o agar (pH 5.4). Pith cultures were grown in the dark at 25 ° for IO or more days. Under these culture conditions the tissue grows as a friable undifferentiated callus mass.
Radioactive labeling Radioactive labeling experiments were performed b y dispersing logarithmically growing pith cultures in an indole-acetic acid-less modified MURASHIGE AND SKOOG9 liquid medium, harvesting them and resuspending them in a modified MURASHIGE AND SKOOG medium with or without indole-acetic acid. 3H and 14C compounds were added as required followed by incubation with shaking at 25 ° for the lengths of time specified in the figure captions. Reactions were terminated by chilling the cells to o °, after which the cells were centrifuged, washed once with p H 5.0 buffer (o.I M sodium acetate, o.I M NaC1 and o.oi M EDTA) and stored at --20 ° until extraction. Measurement of incorporation of ~3H1- or ~14Cluridine into RNA of whole cells is described in the caption to Fig. i.
Preparation o/RNA The frozen cells were ground with a small amount of purified sand for IO min in a mortar which was surrounded by chipped dry ice. RNA was extracted by adding an equal volume of p H 5.0 buffer, i.o mg per ml bentonite, 0. 5 ~o sodium dodecyl sulfate and 2 vol. of water-saturated phenol to the minced tissue. This was shaken at 4 ° for 5 min. The aqueous layer was separated by centrifugation with a Lourdes 9RA rotor at 12 ooo rev./min for 15 min. The interphase was re-extracted with p H 5.0 buffer, and 0.5 % sodium dodecy! sulfate at 55 ° for 5 rain. The aqueous layer was again centrifuged as before. The RNA's from both extractions were precipitated separately by the addition of 2 vol. of cold ethanol. The resulting solutions were kept for at least 2 h at --20 °. The precipitate was collected by centrifugation, washed once with 95 % ethanol and dissolved in either the pH 5.0 buffer or 3 ° °/o sucrose in electrophoresis buffer.
Analyses o/ RNA Sucrose density-gradient sedimentation was used to identify the RNA as described elsewhere 1° except that a 6-30 °/o sucrose gradient in p H 5.0 buffer was used. The gradients were centrifuged as indicated in the figure captions. Following centrifugation, the gradients were fractionated, the absorbance at 260 nm was measured for each tube and samples assayed for radioactivity. For the polyacrylamide gel electrophoresis, the procedure of LOENING11 was followed. The mixture of 14C-steady state labeled RNA and 3H-pulse labeled RNA were applied to 5 cm 2.8 % polyacrylamide gels. At the end of 3.5 h electrophoresis at 5 mA per tube at room temperature, the gels were frozen in hexane with dry ice and sliced with razor blades into about 60 pieces. Two sliced gels were placed in each scintillation vial and digested with 0.5 ml concentrated NHaOH for at least 3 h before the addition of counting fluid. Biochim. Biophys. Acta, 228 (1971) 5o3-5o8
rRNA
SYNTHESIS IN PLANT TISSUE
505
The radioactivity was determined in a Packard Tricarb liquid scintillation counter. The molecular weight of RNA was calculated according to BISHOP et al. TM by using rRNA from Escherichia coli B as a reference. RESULTS AND DISCUSSION
The rate of RNA synthesis as measured by the incorporation of [3H]uridine into RNA is compared for the suspension culture of 2N hybrid pith of N. glauca and 8000
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Fig. i. The R N A synthesis in tobacco tissue culture. (A) The effect of indole-acetic acid on R N A synthesis. The p i t h tissue of the 2N h y b r i d from N. glauca and N. langsdorHii was grown to a logarithmic phase in a modified MORASHIGE AND SKOOG agar m e d i u m with i.o /~g/ml of indoleacetic acid. The cells were dispersed into liquid m e d i u m w i t h o u t indole-acetic acid, washed once a n d resuspended in the same m e d i u m with ( Q - O ) indole-acetic acid at the concentration of 2.o ffg/ml or w i t h o u t ( × - x ) indole-acetic acid. The culture was incubated at 25 ° with shaking in the dark. The R N A synthesis was measured b y the incorporation of all-labeled uridine into trichloroacetic acid insoluble material. [sH]Uridine (2.o ffC/ml) was added to the culture. Samples of 0.5 m] were w i t h d r a w n at various time intervals and were added to o.5 illl cold Io % trichloroacetic acid. The radioactivity was assayed. (B) and (C) The effect o£ actinomycin D on R i g a synthesis. The experimental details were similar to those of (A) with the following exceptions: (B) Actinomycin D at IO ffg/ml ( O - O ) and 3o/zg/ml ( × - - - × ) was added to the cultures, respectively, at zero time w i t h 0.2 #C/ml of [laC]uridine. (C) The cultures were incubated with o.2/,C]ml of HaC]uridine for 4o rain, t h e n actinomycin D at 15/zg/ml was added to the culture ( O - - - O ) . The samples of 0. 5 ml from (B) and (C) were w i t h d r a w n at various times and assayed for radioactivity. Fig. 2. Sucrose density-gradient analysis of 1RNA from cells grown with indole-acetic acid. The cell cultures were prepared as in Fig. I. The concentration of [SH]uridine in each culture was as follows: A-I, B-I, 16 ffC/ml; A-2, B-2, 8/~C/ml; A- 3, B- 3, 4 ffC/ml. The cultures were incubated for: A-I, B-I, I5 rain; A-2, 13-2, 3 ° rain; A- 3, 13-3, 60 rain at 25 ° in the d a r k with shaking. The R N A was prepared from phenol extraction at 4 ° (A) and re-extraction of the interphase at 55 ° (B). T h e centrifugation was performed at 5 ° ooo r e v . / m i n for 15 ° rain in a SW 5o.1 rotor.
Biochim. Biophys. Acta, 228 (i97 I) 503-508
506
T.-Y. CHENG, G. L. HAGEN
N. langsdor][ii grown in the presence or absence of the plant growth hormone, indoleacetic acid. As shown in Fig. IA, the immediate effect of indole-acetic acid is a stinmlation of RNA synthesis. Therefore, all the experiments were performed in the presence of 2.0 #g per ml of indole-acetic acid. RNA's made during the incubation of cell cultures as measured by [SH]uridine incorporation were analyzed by sucrose density-gradient sedimentation as shown in Fig. 2. Cell cultures were incubated with [3H]uridine for 15 (A-I, B-I), 30 (A-2, B-2) and 60 (A-3, B-3) rain. In Part A, RNA was prepared from the phenol extraction at 4 °, while in Part B the RNA was re-extracted from the interphase with phenol at 55 °. After 15 min of labeling with [3H]uridine (B-I), a heavy rapidly labeled RNA is predominantly seen, about 7o % of the radioactivity being sedimented in this region. This heavy RNA has a sedimentation coefficient of 38 S using E. coli rRNA as a reference. With longer incubation, 3o and 6o rain, the relative amount of radioactivity at the 38-S region was decreased to 59 % and 33 % as compared to the radioactivity at the I8-S plus 25-S rRNA regions. The results of the kinetics 3H
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Fig. 3. Analysis of R N A b y polyacrylamide gel electrophoresis. (A) Time course of R N A synthesis. The cell cultures were prepared as in Fig. I. The concentration of [SH]uridine and t h e incubation time were as follows: A-I, 25.oFC/ml, 3 o m i n ; A-2, 2o.ouC/ml, 60 rain; and A-3, 15 FC/ml, 7° min. The bulk r R N A was removed b y phenol extraction at 4 °. The R N A was t h e n re-extracted from the interphase with phenol at 55 °. The m i x t u r e of ~4C-labeled s t e a d y state R N A ( O - O ) and the 8H-labeled pulse R N A ( Q ) - O ) was applied to 5 cm 2.8 % polyacrylamide gels. At the end of 3.5 h of electrophoresis at 5 mA per t u b e at room temperature, the gels were frozen in hexane with dry ice and sliced with razor blades into a b o u t 60 pieces. Two gel slices were placed in each scintillation vial and digested with o. 5 ml conc. N H a O H for at least 3 h before the addition of counting fluid. Radioactivity was determined in a Packard Tricarb liquid scintillation counter. (B) Actinomycin D pulse-chase experiments. The cultures were prepared as in Fig. i, 25.oFC/ml of [SH]nridine was added to the cultures. After 4 ° min incubation a t 25 ° with shaking, 15/zg/ml of actinomycin D was added to the cultures of 13-2 and B-3. The cultures were incubated further, for B-2, 15 min; and B-3, 3o rain. Culture t3-1 was t e r m i n a t e d a t the end of 4 ° min incubation w i t h o u t actinomycin D t r e a t m e n t . The 8H-labeled R N A ( O - - -C)) was prepared and subjected to co-electrophoresis with x4C-labeled steady state R N A ( 0 - 0 ) The procedures for electrophoresis and radioactive assay are described in Fig. 3 A.
Biochim. Biophys. Acta, 228 (I971) 503-508
rRNA SYNTHESIS IN PLANT TISSUE
507
of the incorporation of E3Hjuridine into the regions of 38-S RNA and rRNA (Fig. 2) and actinomycin D pulse-chase experiments (Fig. 3B) suggest that the 38-S RNA is indeed the rRNA precursor. Polyacrylamide gel electrophoresis is known to be more sensitive in revealing differences in RNA's. Thus, the pulse labeled RNA comparable to the RNA in sucrose-density gradient analysis was subjected to electrophoresis. In this case, ~4Clabeled steady state RNA was mixed with 3H-labeled pulse RNA for direct comparison in the gels, Fig. 3A shows that the heavy rapidly labeled RNA is again seen and the molecular weight is 2.8. lO6 using E. coli rRNA as a reference. The results indicate that rRNA precursor has been converted to rRNA as the time of incubation increases to 60 and 7° min. An intermediate component which is slightly heavier than large rRNA with a molecular weight of 1.5' lO6 is resolved distinctly in all the gel electrophoresis profiles. The high molecular weight rapidly labeled rRNA precursor also has been identified in the carrot 13, pea root tip and artichoke-tuber tissue 14. This intermediate component was also observed in some sedimentation profiles although with less resolution. In this system it is not feasible to chase the radioactive RNA with cold uridine because of the large pool size of the RNA precursor. Actinomycin D was used therefore to inhibit RNA synthesis in order to follow the conversion of rRNA precursor to rRNA. The kinetics of actinomycin D action on the inhibition of the RNA synthesis are shown in Fig. i (B and C). In Part B, actinomycin D at concentrations of io and 30 #g/ml was added to the cultures at zero time with [aHluridine, while in Part C, RNA was allowed to synthesize for 40 rain in the presence of E3H]uridine before the addition of 15 #g/ml of actinomycin D. In both experiments, actinomycin D immediately inhibited RNA synthesis, IO/zg/ml being sufficient to inhibit, Turn-over of radioactive RNA, however, is not detectable for at least 60 rain. The results of actinomycin D pulse-chase experiments are shown in Fig. 3B. About 5 ° °/o of the radioactivity is associated with the rRNA precursor from the culture grown with ~SHluridine for 40 rain. The cultures were subsequently chased with 15 #g/ml of actinomycin D. Approx. 25 o~ of the precursor has been converted into rRNA regions after 15 rain, and after 30 min the majority of the precursor is shifted to rRNA regions. The intermediate component is still detectable in these experiments. In this plant suspension culture the response to auxin with respect to RNA synthesis is earlier and more pronounced than other systems where larger pieces were used. Analysis of RNA synthesis in the presence of indole-acetic acid showed a heavy rapidly labeled RNA component. The kinetic study of the synthesis of the heavy component and its conversion to rRNA as well as actinomycin D pulse-chase experiments indicate that the heavy component is the rRNA precursor. The precursor has a sedimentation coefficient and molecular weight of 38 S and 2.8. lO6, while the molecular weights of the I8-S and 25-S rRNA were calculated to be o.66. lO6 and 1.2. lO6, respectively. An intermediate component with a molecular weight of 1.5" lO6 is also well resolved by gel electrophoresis. This may be an intermediate component for the synthesis of 25-S rRNA. This tissue culture system shows growth enhancement in the presence of indole-acetic acid. It seems reasonable, therefore, that indole-acetic acid may enhance the synthesis of ribosomes.
Biochim. Biophys. Acta, 228 (1971) 503-508
508
T.-Y. CHENG, G. L. HAGEN
ACKNOWLEDGMENTS
These investigations were supported by U.S. Public Health Service grants CA04890, CA-o6927 and RR-o5539 from the National Institutes of Health, grant IN-49 from the American Cancer Society and by an appropriation from the Commonwealth of Pennsylvania. We thank Drs. J. Schultz and R. P. Perry for comments and suggestions. We also thank J. Cadbury for technical assistance. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14
J. SILBERGER AND F. SKOOG, Science, 118 (1953) 443. J. L. KEy, Ann. Rev. Plant Physiol., 20 (1969) 449J. F. FREDRICK, Ann. N . Y . Acad. Sci., 144 (1967) 3-153. J. L. KEY AND J. C. SHANNON, Plant Physiol., 39 (1964) 360. Y. MASUDA AND E. TANIMOTO, Plant Cell Physiol., 8 (1966) 458. C. F. TESTER AND L. S. DURE, 111, Biochemistry, 6 (1967) 2532. U. E. LOENING AND J. INGLE, Nature, 215 (1967) 363 . D. KOSTOFF, Zentr. Bakteriol. Parasitenk. Abt. II, 81 (193o) 244. T. MURASHIGE AND F. SKOOG, Physiol. Plantatum, 15 (1963) 473. T. Y. CHENG AND K. A. HARTMAN, JR., J. Mol. Biol., 31 (1968) 191. U. E. LOENING, Biochem. J., lO2 (1967) 251, D. H. L. BISHOp, J. R. CLAVBROOK AND S. SPIEGELMAN, J. Mol. Biol., 26 (1967) 373. C. J. LEAVER AND J. L. KEY, J . Mol. Biol., 49 (197 o) 671. M. E. ROGERS, U. E. LOENING AND R. S. S. FRASER, J . Mol. Biol., 49 (197 ° ) 681.
Biochim. Biophys. Acta, 228 (1971) 503-508
BIOCHIMICA ET BIOPHYSICA ACTA
BBA %710
RIBOSOMAL RNA PRECURSOR SYNTHESIS IN TOBACCO TISSUE CULTURE T S A I - Y I N G C H E N G AND G. L. H A G E N
The Institute [or Cancer Research, 77oz Burholme Avenue, Fox Chase, Philadelphia, Pa. x 9 I z I
(u.s.A .)
(Received A u g u s t i z t h , 197 o)
SUMMARY
This study, using an aseptic suspension culture of undifferentiated meristematic parenchyma cells of tumor-forming tobacco hybrid of Nicotiana glauca and Nicotiana langsdor]]ii, has shown an enhancement of RNA synthesis by the phytohormone indole-acetic acid. Analyses by sucrose density-gradient sedimentation and polyacrylamide gel electrophoresis show a rapidly labeled rRNA precursor with a sedimentation coefficient of 3 8 S and a molecular weight of 2.8.IO e. Actinomycin D-chase experiments, further, indicate that the heavy rapidly labeled RNA is indeed the rRNA precursor. An intermediate component which is slightly heavier than large rRNA with a molecular weight of 1.5" lO6 is also detected.
INTRODUCTION
Phytohormones are known to influence plant nucleic acid metabolism x. Numerous studies z-~ further indicate that auxins both enhance the synthesis of all RNA species and preferentially increase the synthesis of rRNA when compared to tRNA and mRNA. In this study, aseptic suspension cultures of white friable undifferentiated callus cells such as those derived from pith explants of the tumor-forming tobacco hybrid of Nicotiana glauca and Nicotiana langsdor]]ii were used. These cells have several unique advantages including: (I) They are potentially tumorous tissueS; (2) they are free from microorganism contamination which has frequently interfered with plant nucleic acid analysis; (3) they are an essentially homogeneous mass of parenchyma cells with meristematic activity; (4) they can be manipulated in a manner comparable to a bacterial system, thus facilitating the experimental scheme; and (5) their uptake of radioactive compounds is rapid and uniform. Further, the techniques of sucrose density-gradient sedimentation and polyacrylamide gel electrophoresis were used instead of methylated albumin coated kieselguhr column chromatography, which has been widely used in the plant nucleic acid research. MATERIALS AND METHODS
Plant and growth conditions The tumor forming hybrid of N. glauca and N. langsdorlfii was used as the source material for these experiments. The pith tissue from non-flowering plants was Biochim. Biophys. Acta, 228 (1971) 503-508
504
T.-Y. CHENG, G. L. HAGEN
aseptically removed and cultured on a modified MURASHIGE AND SKOOG9 medium containing, per 1: (I) inorganic salts, 1.65 g NH,NOs, 1.9o g KNO 3, 0.44 g CaC12' 2H20, 0.37 g MgSO4"7HzO, o.17 g KH2PO4, o.12 g FeSO,.7H20, o.145 g disodium EDTA, 6.20 mg H3BO4, 22.3 mg MnSO,'4H20 , lO.51 mg ZnSOa.7H~O, 0.83 nag KI, 0.25 nag NaMoO~'2H20, 0.025 mg C u S Q ' 5 H 2 0 , and 0.025 mg CoC1~.6H20; and (2) organic substances, IOO mg myoinositol, o.I mg thiamine HC1, I.O mg indole-acetic acid, 20 g sucrose, and 0.8 °/o agar (pH 5.4). Pith cultures were grown in the dark at 25 ° for IO or more days. Under these culture conditions the tissue grows as a friable undifferentiated callus mass.
Radioactive labeling Radioactive labeling experiments were performed b y dispersing logarithmically growing pith cultures in an indole-acetic acid-less modified MURASHIGE AND SKOOG9 liquid medium, harvesting them and resuspending them in a modified MURASHIGE AND SKOOG medium with or without indole-acetic acid. 3H and 14C compounds were added as required followed by incubation with shaking at 25 ° for the lengths of time specified in the figure captions. Reactions were terminated by chilling the cells to o °, after which the cells were centrifuged, washed once with p H 5.0 buffer (o.I M sodium acetate, o.I M NaC1 and o.oi M EDTA) and stored at --20 ° until extraction. Measurement of incorporation of ~3H1- or ~14Cluridine into RNA of whole cells is described in the caption to Fig. i.
Preparation o/RNA The frozen cells were ground with a small amount of purified sand for IO min in a mortar which was surrounded by chipped dry ice. RNA was extracted by adding an equal volume of p H 5.0 buffer, i.o mg per ml bentonite, 0. 5 ~o sodium dodecyl sulfate and 2 vol. of water-saturated phenol to the minced tissue. This was shaken at 4 ° for 5 min. The aqueous layer was separated by centrifugation with a Lourdes 9RA rotor at 12 ooo rev./min for 15 min. The interphase was re-extracted with p H 5.0 buffer, and 0.5 % sodium dodecy! sulfate at 55 ° for 5 rain. The aqueous layer was again centrifuged as before. The RNA's from both extractions were precipitated separately by the addition of 2 vol. of cold ethanol. The resulting solutions were kept for at least 2 h at --20 °. The precipitate was collected by centrifugation, washed once with 95 % ethanol and dissolved in either the pH 5.0 buffer or 3 ° °/o sucrose in electrophoresis buffer.
Analyses o/ RNA Sucrose density-gradient sedimentation was used to identify the RNA as described elsewhere 1° except that a 6-30 °/o sucrose gradient in p H 5.0 buffer was used. The gradients were centrifuged as indicated in the figure captions. Following centrifugation, the gradients were fractionated, the absorbance at 260 nm was measured for each tube and samples assayed for radioactivity. For the polyacrylamide gel electrophoresis, the procedure of LOENING11 was followed. The mixture of 14C-steady state labeled RNA and 3H-pulse labeled RNA were applied to 5 cm 2.8 % polyacrylamide gels. At the end of 3.5 h electrophoresis at 5 mA per tube at room temperature, the gels were frozen in hexane with dry ice and sliced with razor blades into about 60 pieces. Two sliced gels were placed in each scintillation vial and digested with 0.5 ml concentrated NHaOH for at least 3 h before the addition of counting fluid. Biochim. Biophys. Acta, 228 (1971) 5o3-5o8
rRNA
SYNTHESIS IN PLANT TISSUE
505
The radioactivity was determined in a Packard Tricarb liquid scintillation counter. The molecular weight of RNA was calculated according to BISHOP et al. TM by using rRNA from Escherichia coli B as a reference. RESULTS AND DISCUSSION
The rate of RNA synthesis as measured by the incorporation of [3H]uridine into RNA is compared for the suspension culture of 2N hybrid pith of N. glauca and 8000
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Fig. i. The R N A synthesis in tobacco tissue culture. (A) The effect of indole-acetic acid on R N A synthesis. The p i t h tissue of the 2N h y b r i d from N. glauca and N. langsdorHii was grown to a logarithmic phase in a modified MORASHIGE AND SKOOG agar m e d i u m with i.o /~g/ml of indoleacetic acid. The cells were dispersed into liquid m e d i u m w i t h o u t indole-acetic acid, washed once a n d resuspended in the same m e d i u m with ( Q - O ) indole-acetic acid at the concentration of 2.o ffg/ml or w i t h o u t ( × - x ) indole-acetic acid. The culture was incubated at 25 ° with shaking in the dark. The R N A synthesis was measured b y the incorporation of all-labeled uridine into trichloroacetic acid insoluble material. [sH]Uridine (2.o ffC/ml) was added to the culture. Samples of 0.5 m] were w i t h d r a w n at various time intervals and were added to o.5 illl cold Io % trichloroacetic acid. The radioactivity was assayed. (B) and (C) The effect o£ actinomycin D on R i g a synthesis. The experimental details were similar to those of (A) with the following exceptions: (B) Actinomycin D at IO ffg/ml ( O - O ) and 3o/zg/ml ( × - - - × ) was added to the cultures, respectively, at zero time w i t h 0.2 #C/ml of [laC]uridine. (C) The cultures were incubated with o.2/,C]ml of HaC]uridine for 4o rain, t h e n actinomycin D at 15/zg/ml was added to the culture ( O - - - O ) . The samples of 0. 5 ml from (B) and (C) were w i t h d r a w n at various times and assayed for radioactivity. Fig. 2. Sucrose density-gradient analysis of 1RNA from cells grown with indole-acetic acid. The cell cultures were prepared as in Fig. I. The concentration of [SH]uridine in each culture was as follows: A-I, B-I, 16 ffC/ml; A-2, B-2, 8/~C/ml; A- 3, B- 3, 4 ffC/ml. The cultures were incubated for: A-I, B-I, I5 rain; A-2, 13-2, 3 ° rain; A- 3, 13-3, 60 rain at 25 ° in the d a r k with shaking. The R N A was prepared from phenol extraction at 4 ° (A) and re-extraction of the interphase at 55 ° (B). T h e centrifugation was performed at 5 ° ooo r e v . / m i n for 15 ° rain in a SW 5o.1 rotor.
Biochim. Biophys. Acta, 228 (i97 I) 503-508
506
T.-Y. CHENG, G. L. HAGEN
N. langsdor][ii grown in the presence or absence of the plant growth hormone, indoleacetic acid. As shown in Fig. IA, the immediate effect of indole-acetic acid is a stinmlation of RNA synthesis. Therefore, all the experiments were performed in the presence of 2.0 #g per ml of indole-acetic acid. RNA's made during the incubation of cell cultures as measured by [SH]uridine incorporation were analyzed by sucrose density-gradient sedimentation as shown in Fig. 2. Cell cultures were incubated with [3H]uridine for 15 (A-I, B-I), 30 (A-2, B-2) and 60 (A-3, B-3) rain. In Part A, RNA was prepared from the phenol extraction at 4 °, while in Part B the RNA was re-extracted from the interphase with phenol at 55 °. After 15 min of labeling with [3H]uridine (B-I), a heavy rapidly labeled RNA is predominantly seen, about 7o % of the radioactivity being sedimented in this region. This heavy RNA has a sedimentation coefficient of 38 S using E. coli rRNA as a reference. With longer incubation, 3o and 6o rain, the relative amount of radioactivity at the 38-S region was decreased to 59 % and 33 % as compared to the radioactivity at the I8-S plus 25-S rRNA regions. The results of the kinetics 3H
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Fig. 3. Analysis of R N A b y polyacrylamide gel electrophoresis. (A) Time course of R N A synthesis. The cell cultures were prepared as in Fig. I. The concentration of [SH]uridine and t h e incubation time were as follows: A-I, 25.oFC/ml, 3 o m i n ; A-2, 2o.ouC/ml, 60 rain; and A-3, 15 FC/ml, 7° min. The bulk r R N A was removed b y phenol extraction at 4 °. The R N A was t h e n re-extracted from the interphase with phenol at 55 °. The m i x t u r e of ~4C-labeled s t e a d y state R N A ( O - O ) and the 8H-labeled pulse R N A ( Q ) - O ) was applied to 5 cm 2.8 % polyacrylamide gels. At the end of 3.5 h of electrophoresis at 5 mA per t u b e at room temperature, the gels were frozen in hexane with dry ice and sliced with razor blades into a b o u t 60 pieces. Two gel slices were placed in each scintillation vial and digested with o. 5 ml conc. N H a O H for at least 3 h before the addition of counting fluid. Radioactivity was determined in a Packard Tricarb liquid scintillation counter. (B) Actinomycin D pulse-chase experiments. The cultures were prepared as in Fig. i, 25.oFC/ml of [SH]nridine was added to the cultures. After 4 ° min incubation a t 25 ° with shaking, 15/zg/ml of actinomycin D was added to the cultures of 13-2 and B-3. The cultures were incubated further, for B-2, 15 min; and B-3, 3o rain. Culture t3-1 was t e r m i n a t e d a t the end of 4 ° min incubation w i t h o u t actinomycin D t r e a t m e n t . The 8H-labeled R N A ( O - - -C)) was prepared and subjected to co-electrophoresis with x4C-labeled steady state R N A ( 0 - 0 ) The procedures for electrophoresis and radioactive assay are described in Fig. 3 A.
Biochim. Biophys. Acta, 228 (I971) 503-508
rRNA SYNTHESIS IN PLANT TISSUE
507
of the incorporation of E3Hjuridine into the regions of 38-S RNA and rRNA (Fig. 2) and actinomycin D pulse-chase experiments (Fig. 3B) suggest that the 38-S RNA is indeed the rRNA precursor. Polyacrylamide gel electrophoresis is known to be more sensitive in revealing differences in RNA's. Thus, the pulse labeled RNA comparable to the RNA in sucrose-density gradient analysis was subjected to electrophoresis. In this case, ~4Clabeled steady state RNA was mixed with 3H-labeled pulse RNA for direct comparison in the gels, Fig. 3A shows that the heavy rapidly labeled RNA is again seen and the molecular weight is 2.8. lO6 using E. coli rRNA as a reference. The results indicate that rRNA precursor has been converted to rRNA as the time of incubation increases to 60 and 7° min. An intermediate component which is slightly heavier than large rRNA with a molecular weight of 1.5' lO6 is resolved distinctly in all the gel electrophoresis profiles. The high molecular weight rapidly labeled rRNA precursor also has been identified in the carrot 13, pea root tip and artichoke-tuber tissue 14. This intermediate component was also observed in some sedimentation profiles although with less resolution. In this system it is not feasible to chase the radioactive RNA with cold uridine because of the large pool size of the RNA precursor. Actinomycin D was used therefore to inhibit RNA synthesis in order to follow the conversion of rRNA precursor to rRNA. The kinetics of actinomycin D action on the inhibition of the RNA synthesis are shown in Fig. i (B and C). In Part B, actinomycin D at concentrations of io and 30 #g/ml was added to the cultures at zero time with [aHluridine, while in Part C, RNA was allowed to synthesize for 40 rain in the presence of E3H]uridine before the addition of 15 #g/ml of actinomycin D. In both experiments, actinomycin D immediately inhibited RNA synthesis, IO/zg/ml being sufficient to inhibit, Turn-over of radioactive RNA, however, is not detectable for at least 60 rain. The results of actinomycin D pulse-chase experiments are shown in Fig. 3B. About 5 ° °/o of the radioactivity is associated with the rRNA precursor from the culture grown with ~SHluridine for 40 rain. The cultures were subsequently chased with 15 #g/ml of actinomycin D. Approx. 25 o~ of the precursor has been converted into rRNA regions after 15 rain, and after 30 min the majority of the precursor is shifted to rRNA regions. The intermediate component is still detectable in these experiments. In this plant suspension culture the response to auxin with respect to RNA synthesis is earlier and more pronounced than other systems where larger pieces were used. Analysis of RNA synthesis in the presence of indole-acetic acid showed a heavy rapidly labeled RNA component. The kinetic study of the synthesis of the heavy component and its conversion to rRNA as well as actinomycin D pulse-chase experiments indicate that the heavy component is the rRNA precursor. The precursor has a sedimentation coefficient and molecular weight of 38 S and 2.8. lO6, while the molecular weights of the I8-S and 25-S rRNA were calculated to be o.66. lO6 and 1.2. lO6, respectively. An intermediate component with a molecular weight of 1.5" lO6 is also well resolved by gel electrophoresis. This may be an intermediate component for the synthesis of 25-S rRNA. This tissue culture system shows growth enhancement in the presence of indole-acetic acid. It seems reasonable, therefore, that indole-acetic acid may enhance the synthesis of ribosomes.
Biochim. Biophys. Acta, 228 (1971) 503-508
508
T.-Y. CHENG, G. L. HAGEN
ACKNOWLEDGMENTS
These investigations were supported by U.S. Public Health Service grants CA04890, CA-o6927 and RR-o5539 from the National Institutes of Health, grant IN-49 from the American Cancer Society and by an appropriation from the Commonwealth of Pennsylvania. We thank Drs. J. Schultz and R. P. Perry for comments and suggestions. We also thank J. Cadbury for technical assistance. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14
J. SILBERGER AND F. SKOOG, Science, 118 (1953) 443. J. L. KEy, Ann. Rev. Plant Physiol., 20 (1969) 449J. F. FREDRICK, Ann. N . Y . Acad. Sci., 144 (1967) 3-153. J. L. KEY AND J. C. SHANNON, Plant Physiol., 39 (1964) 360. Y. MASUDA AND E. TANIMOTO, Plant Cell Physiol., 8 (1966) 458. C. F. TESTER AND L. S. DURE, 111, Biochemistry, 6 (1967) 2532. U. E. LOENING AND J. INGLE, Nature, 215 (1967) 363 . D. KOSTOFF, Zentr. Bakteriol. Parasitenk. Abt. II, 81 (193o) 244. T. MURASHIGE AND F. SKOOG, Physiol. Plantatum, 15 (1963) 473. T. Y. CHENG AND K. A. HARTMAN, JR., J. Mol. Biol., 31 (1968) 191. U. E. LOENING, Biochem. J., lO2 (1967) 251, D. H. L. BISHOp, J. R. CLAVBROOK AND S. SPIEGELMAN, J. Mol. Biol., 26 (1967) 373. C. J. LEAVER AND J. L. KEY, J . Mol. Biol., 49 (197 o) 671. M. E. ROGERS, U. E. LOENING AND R. S. S. FRASER, J . Mol. Biol., 49 (197 ° ) 681.
Biochim. Biophys. Acta, 228 (1971) 503-508