Plant Molecular Biology5: 175-182, 1985 © 1985 Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
Red light inhibits blue light-induced chloroplast development in cultured plant cells at the mRNA level Gerhard Richter & Klaus Wessel Institut ff~'r Botanik, Universitdt Hannover, Herrenhiiuser Strafle 2, D-3000 Hannover 1, F.R.G.
Keywords: blue light-control, chloroplast development, dot-hybridization, nuclear mRNAs, plastid mRNAs, red light-inhibition
Summary During the blue light-dependent chloroplast differentiation in suspension cultured cells of tobacco (Nicotiana tabacum var. 'Samsun') chlorophylls and other pigments as well as specific membrane and stroma proteins are synthesized de-novo: the 32-kD membrane protein of photosystem II, the small subunit (SSU) and the large subunit (LSU) of ribulose-bisphosphate carboxylase/oxygenase (EC. 4.1.1.39; RuBPCase). Parallel with their accumulation the steady-state concentration of the corresponding mRNAs increases rapidly and coordinately as was detected by employing cloned DNA sequences complementary to these plastid and nuclear transcripts as hybridization probes. The blue light-induced change in the concentration of the mRNAs analyzed here is abolished by red light when applied to cells at an early or late stage of chloroplast differentiation. The results indicate that blue light exerts a positive and coordinate influence on both genomes, nuclear and plastid, in chloroplast development of tobacco cells.
Introduction In suspension cultured cells of tobacco (Nicotiana tabacum var. 'Samsun') the development of chloroplasts from leucoplasts is induced and maintained solely by blue light while red light is inefficient in this respect (2). One prominent feature of this major cellular differentiation is the de-novo synthesis of membrane and stroma proteins which is reflected in the activity level of the corresponding mRNAs (10). Concomitantly, the steady-state concentration of two plastid mRNAs coding for LSU and a 35-kD precursor polypeptide (PP-35) of the 32-kD membrane protein increases rapidly in response to blue light irradiation as has been detected by dot-hybridization using the complementary DNA sequences as probes (8). These results support the notion that the actual sequence levels of plastid mRNAs are under the control of blue light. Moreover, they suggest that the latter causes the induction of mRNA, i.e. a transient increase in the rate of transcription though the possibility exists that the
stability of transcripts is affected. However, results of preliminary experiments employing a transcriptionally active 'chromosome' from chloroplasts seem to exclude this alternative (9). While the implication of blue light in affecting transcription of plastid genes is now documented nothing had been known so far about a similar photocontrol of the activity of nuclear genes encoding plastid proteins. Therefore, in the present study the steady-state level of a nuclear transcript, SSU mRNA, in dark-grown tobacco cells exposed to blue light was determined by using a cloned cDNA probe for SSU mRNA of tobacco (7). We present evidence that the expression of nuclear genes is likewise regulated by blue light. We have continued these studies to evaluate to what extent this positive and coordinate control by blue light of the two genomes is counteracted or perhaps taken over by red light given subsequent to a short (24 h) or longer inductive (8 d) exposure to blue light of dark-grown tobacco cells. The results obtained clearly demonstrate that red light cannot adequately replace blue
176 light neither in raising up the levels of plastid and nuclear transcripts nor in maintaining chlorophyll synthesis in tobacco cells.
nick-translation using [a-32p]dCTP (Amersham). About 15 ng/ml (3 × 106cpm/ml) of labeled plasmid DNA was used for hybridization.
Quantitation of m R N A s Materials and methods
Cell culture Freely suspended callus cells from Nicotiana tabacum var. 'Samsun' (2) were grown as reported before (3). The conditions for the blue and red light treatment were those as described (3) with the exception that the dark-grown cells after the transfer to the greening medium were kept for another 4 d in darkness prior to blue light exposure.
Chlorophyll measurement The determination of chlorophylls was carried out as reported elsewhere (3).
RNA preparation The isolation and purification of total RNA were done as described elsewhere (6). Poly(A)-containing and poly(A)-minus RNA were prepared according to the methods of Apel & Kloppstech (1). Before binding to nitrocellulose filter RNAs were denatured by treatment with formaldehyde at 60°C for 15 min (16).
Plasmid preparation ?
Clones pSA 204 and pSA 452 containing coding sequences for the large subunit (LSU) of RuBPCase and the 35-kD precursor polypeptide (PP-35) of the 32-kD membrane protein of photosystem II, respectively, were kindly provided by Dr Link (Freiburg, FRG); they were isolated from mustard leaves (Sinapis alba L.) and cloned in E. coli with pBR 322 as the vehicle (5). Clone pST V 34, which contains a cDNA encoding SSU of Nicotiana silvestris, was constructed by Pinck et al. (7) and given to us by Dr J. Fleck (Strasbourg, France). Plasmids containing these specific inserts were isolated after amplification with chloramphenicol by standard procedures and purified by CsCI/ethidium bromide gradients. They were labeled to a specific activity of approximate 2 × 108 c p m / # g by
The relative concentrations of plastid and nuclear transcripts were determined by dot-hybridization. Dilution series of RNA were fixed to a nitrocellulose filter (GeneScreen, New England Nuclear) using a HYBRI-DOT system (Bethesda Research Laboratories). The hybridization technique has been reported before (8). After autoradiography the labeled spots were cut out and counted in a scintillation counter using Cerenkov radiation. To describe the relative amounts of hybridizable mRNA the values for fully greened cells, i.e. day 15, were set at 100% and all other values refered to it.
Northern blot analysis Poly(A)-minus RNA was glyoxylated (16), samples of 2 #g separated on 1% agarose gel and transfered to nitrocellulose (14). Hybridization conditions were the same as used for the RNA dot-hybridization.
Results and discussion
Two sets of experiments were designed to study to what extent blue light-induced chloroplast formation in cultured tobacco cells is affected by subsequent red light irradiation: dark-grown cells were pre-illuminated with blue light for either 24 hours or 8 days, then treated with continuous red light of equal energy fluence rate (EFR; W. m-2).
Chlorophyll synthesis As shown previously the blue light-dependent transition of leucoplasts to chloroplasts in tobacco cells can be roughly estimated by following the synthesis of chlorophylls (l l). Accordingly, we have used this parameter to evaluate the influence of the two irradiation programs on the plastid transformation. In dark-grown cells illuminated with blue light for 24 h, then with red light no chlorophyll formation was observed up to day 7 (Fig. 1). In control cells which were kept in blue
177 light over the same period of time chlorophyll synthesis proceeded with a constant rate. Prolongation of the initial blue light treatment to 8 d gave rise to a massive chlorophyll accumulation which, however, ceased when these cells were exposed to red light for another 15 d (Fig. 2). Moreover, the chlorophyll a to b ratio changed from about 3.1 to 2.7 within 10 d under these conditions. These data establish that blue light is mandatory for chlorophyll synthesis in the cultured tobacco cells and cannot be replaced by red light of equal EFR even at an advanced stage of chloroplast differentiation, i.e. after several days of greening under appropriate conditions. Apparently, the induction as well as the maintenance of the plastid transition are strictly blue light-dependent processes. This notion is supported by the outcome of a similar experimental approach where dark-grown tobacco cells had been pre-illuminated with blue light for 4 d, then exposed to red light of equal EFR for 20 d: Chlorophyll accumulation ended with the change from blue to red light irradiation. A slight
increase of 5-9% at the end of the stationary growth phase registered in a few instances was due to a certain clonal variation, and was also observed in the control cells kept in continuous red light (11). This phenomenon is often encountered when clonal cell lines had been isolated from cultured plants (4). Plastid m R N A s
The observed ineffectiveness in maintaining induced chlorophyll synthesis of red light provoked the question whether this negative photocontrol is also exerted on the mRNA level. To obtain an answer the steady-state concentration of the plastid mRNAs coding for PP-35 and LSU of RuBPCase was assessed by hybridization employing the structural sequences. The two mustard clones (pSA 204, pSA 452) which served as probes share sequence homology with the corresponding genes of Nicotiana tabacum; thus their sensitivity is sufficient to allow at least a semiquantitative determination of the corresponding mRNA sequences (8). 50.
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Fig. 1. Time course of chlorophyll synthesis in suspension cultured tobacco cells pre-illuminated for 24 h with blue light, then exposed to either red light (R) or blue light (BL) of equal energy fluence rate (EFR; 8 W • m 2) for 7 d.
d BL
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Fig. 2. As described for Fig. 1, except: pre-illumination with blue light was for 8 d; cells were subsequently exposed to red light (R) or blue light (BL) for 15 d.
178 In a first a t t e m p t to estimate the m R N A level for PP-35 equal a m o u n t s of poly(A)-minus R N A f r o m cultured t o b a c c o cells which had been irradiated with red light for different lengths of time after a pretreatment with blue light according to the two irradiation p r o g r a m s were fractionated by agarose gel electrophoresis, transfered by blotting to nitrocellulose and probed for transcripts encoding P P 35. The different R N A samples yielded one single hybridization band at the same position which corresponded with the expected position of the specific m R N A . The different intensity of the radioactive signals suggested that the level of the specific transcript varied: In cells kept in continuous blue light a drastic increase occurred concomitantly with illumination time; in those transfered to red light after an initial blue light exposure for 8 d no such change took place - on the contrary, a decrease with time was observed. The m e t h o d or R N A blotting and hybridization allows only a r o u g h estimate of changes in the steady-state c o n c e n t r a t i o n of a given m R N A sequence. These could be measured more reliably by the technique of dot-hybridization (12). The following a p p r o a c h was used to analyze the effect of red light on the level of PP-35 m R N A in an early stage of induced chloroplast differentiation: D a r k - g r o w n t o b a c c o cells were exposed to blue light for 24 h, then transfered to red light. Poly(A)m i n u s - R N A was isolated after different lengths of time. F o r each preparation identical series of increasing a m o u n t s were dotted on nitrocellulose and hybridized with the nick-translated 32p-DNA of p S A 452. First the filters were a u t o r a d i o g r a p h e d to obtain a visual record on the degree of hybridization (Fig. 3A); afterwards the labeled spots were cut out and counted. The a m o u n t s of 32p-probe hybridized to different quantities of filter-bound poly(A)m i n u s - R N A increases linearly with 2-10 #g (Fig. 3B). The slope of each straight line was the basis for calculating the relative concentration of specific transcript within the p o l y ( A ) - m i n u s - R N A fraction. When the a m o u n t of PP-35 m R N A sequences is plotted against the time of red light-irradiation a steady decrease is observed (Fig. 4). In the control cells, however, a roughly 5 fold increase occurred over the same period of time, a result which is in g o o d agreement with earlier findings (8). Since pre-illumination for 24 h with blue light failed to enhance the level of PP-35 m R N A during the subsequent red light period, the latter was ex-
Fig. 3. Changes in the amount of hybridizable PP-35 mRNA in tobacco cells exposed to a combination of red and blue light or to continuous blue light. Poly(A)-minus RNA applied to the nitrocellulose filter in increasing amounts was isolated from cells pre-illuminated with blue light for 24 h, then with red light for 3, 5 and 7 d, or from cells kept in blue light for 3 and 7 d. Hybridization to the 32p-labeled DNA of pSA 452. A: autoradiogram of 32p-labeled hybrids. B: dots of part A were cut out and counted. The amounts of 32p-label in hybrids as a function of poly(A)-minus RNA applied to the dots. PL: hybridization to increasing amounts of poly(A)-minus RNA from fully greened cells of labeled DNA of pBR 322. dBL: days in blue'light; dR: days in red light.
179 tended to 8 d in order to establish an advanced stage of chloroplast differentiation indicated by the appearance of grana stacks and n u m e r o u s s t r o m a thylakoids (10). These cells f r o m the logarithmic g r o w t h phase contain levels of the m R N A of PP-35 and L S U a b o u t 70% of that f o u n d after 12 d g r o w t h in blue light, i.e. in the fully greened stage (8). The possibility that they m a y mask a specific red light effect on the transcription of these m R N A s could be o v e r c o m e by transfering blue light-treated cells to fresh culture m e d i u m prior to the treatment with red light. As an immediate response the steady-state concentration of the plastid m R N A s drops to a level 2 0 - 3 0 % of that in fully greened cells. Nevertheless, normal growth and chloroplast differentiation continue in blue light (unpublished results). P o l y ( A ) - m i n u s R N A was isolated f r o m the cells at various times after the onset of the red light irradiation and analyzed by dot-hybridization as described above. N o w a slightly different situation was encountered which is reflected in the radioactivity signals as well as in the quantitation of the specific m R N A . In a n u m b e r of experiments the steady-state c o n c e n t r a t i o n of PP-35 m R N A increased slightly during the subsequent red light treatment of the ceils a m o u n t i n g to about 15-20% of the controls on day 10; afterwards it began to decrease (Fig. 5). This enhancing effect t h o u g h near the margin of error was reproducible. With regard to the inhibitory effect of red light on PP-35 m R N A synthesis it is of special interest to investigate whether the transcription of other plastid m R N A species is likewise impaired by red q u a n ta. Therefore, we have focused on the sequence which codes for L S U of R u B P C a s e , representing a n o t h e r m a j o r plastid m R N A . 32p-DNA of p S A 204 was used as probe in the d o t - h y b r i d i z a t i o n assay with poly(A)-minus R N A . The intensity of the radioactive dots correlated well with the extent of D N A / R N A hybridization. A kinetic similar to that of PP-35 m R N A was obtained when d a r k - g r o w n t o b a c c o cells were exposed to red light after a 24 h blue light induction period: A decline in the level of the specific transcript contrasted by a drastic rise in the control cells kept in blue light (Fig. 6). The day-1 value indicates that with the onset of blue light irradiation the transcription of LSU m R N A sequences had been initiated without a lag phase due to the modified culture conditions (s. Material a. Methods). When
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Fig. 4. Time course of irradiation-induced changes in the amount of PP-35 mRNA as determined by hybridization to 32p-labeled pSA 452. Samples were taken from cell cultures subjected either to a combination of blue light (24 h) and red light (R) or to continuous blue light (BL) for the extraction of poly(A)-minus RNA at the times indicated. The data were obtained by counting the radioactivity bound to 8 #g of RNA. The corresponding value for the RNA preparation from fully greened cells (day 15) was set a 100%and all other values referred to it.
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180 the inductive blue light treatment came to 8 days a small, but temporarily increase in the level of LSU mRNA up to day l0 occurred during the subsequent red light period (Fig. 7) which compares fairly well with that of PP-35 mRNA under the conditions of the same irradiation program (s. Fig. 5). Analysis by dot-hybridization of the total RNA instead of poly(A)-minus RNA from tobacco cells similarly treated with both irradiation programs
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yielded the same results in respect to the relative concentration of the two plastid mRNAs (not shown).
Transcripts of nuclear genes Results described in the previous section suggest that in cultured tobacco cells red light fails to provoke a positive response on the level of plastid mRNAs the extent of which depending upon the stage of chloroplast differentiation. They raise the question as to whether the steady-state level of RNAs transcribed from nuclear genes is likewise photoregulated. For this purpose we examined the expression of SSU mRNA in cells which had been exposed to the combined treatment of the two light qualities employing the same technique of dot-hybridization (see above). Poly(A)-containing RNA was isolated from the cells at various times after the onset of red light irradiation, samples were applied to nitrocellulose and probed with the 32p-DNA of pST V 34 whose cDNA insert encodes SSU of tobacco. After autoradiography the dots exhibited radioactive signals the intensity of which depended on the irradiation program applied to the cells. The plot of 32p-labeled DNA probe hybridized versus the amount of poly(A)-containing RNA was linear from 0.125 to 1/~g per dot thus making a quantitation feasible. After treatment of dark-grown cells for 24 h the concentration of SSU mRNA remained practically unchanged during the following red light period (Fig. 8). Pre-illumination with blue light for 8 d generated the same response as observed for LSU and PP-35 mRNA: After the onset of red light irradiation the relatively high level of the transcript as registered at the end of the blue light induction period continued to increase for a limited period of time, i.e. up to day 10 (Fig. 9). This positive effect of red light, however, is modest as compared with that of blue light. The data obtained establish that the level of SSU mRNA parallels that of the two plastid mRNAs under the conditions of both irradiation programs. In order to exclude the possibility that SSU mRNA may be polyadenylated to different extents in blue light and red light irradiated tobacco cells total RNA instead of the polyadenylated RNA
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Fig. 8. Times course of irradiation-induced changes in the amount of SSU mRNA as determined by hybridization to 32p_ labeled pST V 34. Cells were taken from cultures treated either with a combination of blue light (24 h) and red light (R) or with continuous blue light (BL)for the extraction of poly(A)-containing RNA at the times indicated. The other conditions are the same as in Fig. 4,
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light was 8 d followed by exposure to red light for 15 d; samples were analyzed at the times indicated. fraction was analyzed. Even though larger quantities had to be dotted onto nitrocellulose similar effects on the steady-state concentration of SSU m R N A by red light exposure following blue light induction could be demonstrated (not shown).
The results presented here not only confirm previous results (8), but provide first evidence that blue light is also implicated in affecting the concentration of the nuclear transcript coding for SSU. The data suggest that blue quanta cause a true induction of m R N A s in the plastids as well as in the nucleus. Photoregulation of the steady-state level o f m R N A s coding for three major plastid proteins seems to be a major feature of the action of blue light. Effects on m R N A stability seem less likely (see Introduction). From the fact that the corresponding m R N A s follow the same pattern of accumulation respective decrease in the course of the two radiation programs it is conceivable that their transcription is coordinately regulated by blue quanta. Indications are that the concentration of another nuclear-encoded transcript, of the light-harvesting chlorophyll a, b protein, is likewise photocontrolled in the tobacco cells (unpublished results). Thus a parallel exists to the action of red light in pea seedlings (13) and Lemna (15) where nuclear and plastid genome are under phytochrome control. For red light, on the other hand, the transition of leucoplasts to functional chloroplasts seems to be the prerequisite to bring about at least a small, but temporary increase in the level of the three m R N A s analyzed here. Whether this enhancement corresponds to a change in the activity of these transcripts as has been found for tobacco cells continuously irradiated with blue light (10) remains to be clarified. Moreover, the time course of this red light-induced change needs a more detailed analysis. The establishment of a blue light-dependency on the concentration of the m R N A s of LSU, SSU and PP-35 raises the question as to whether the specific photoreceptor acts to affect primarily transcription or messenger stability. To find an answer will be the aim of further studies.
Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft (Ri 73 / 23-1). The authors are grateful to Professor L. Bergmann for supplying callus cultures. We thank Dr G. Link for the generous gift of the plasmids pSA 204 and pSA 452, and Dr J. Fleck for giving us the SSU e D N A clone.
182
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cal systems. Springer, Berlin, Heidelberg, New York, 1984, pp 253-263. 10. Richter G, Beckmann J, GroB M, Hundrieser J, Schneider Ch: Blue light-induced synthesis of chloroplast proteins in cultured plant cells. In: Progr Clinical a. Biol Res, Vo1102B Cell function and differentiation, part B (FEBS Vol 65). Alan R Liss, New York, 1982, pp 267-276. I I. Richter G, Hundrieser J, Grofl M, Schultz S, Bottl~inder K, Schneider Ch: Blue light effects in cell cultures. In: Senger H (ed) Blue light effects in biological systems. Springer, Berlin, Heidelberg, New York, 1984, pp 387-396. 12. Rodland KD, Russell P J: Simplified techniques for hybridization of DNA immobilized on GeneScreen and GeneScreen-Plus,using 32p-labeled or ass-labeled DNA probes. New Prod News 2:1-6, 1983. 13. Sasaki Y, Sakihama T, Kamikubo T, Shinozaki K: Phytochrome-mediated regulation of two mRNAs, encoded by nuclei and chloroplasts, of ribulosebisphosphate carboxylase/oxygenase. Eur J Biochem 257:8569-8572, 1983. 14. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517, 1975. 15. Stiekema WJ, Wimpee ChF, Silverthorne, J, Tobin EM: Phytochrome control of the expression of two nuclear genes encoding chloroplast proteins in Lemna gibba L. G-3. Plant Physiol 72:717-724, 1983. 16. White BA, Bancroft FC: Cytoplasmic dot hybridization. J Biol Chem 257:8569-8572, 1982. Received 8 March 1985; in revised form 5 June 1985; accepted 27 June 1985.
