Plant Molecular Biology7: 105-113, 1986 © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
105
The expression of chloroplast genes during cotton embryogenesis Katyna E. Borroto & Leon Dure III
Biochemistry Department, University of Georgia, Athens, GA 30602, U.S.A. Keywords: chloroplast genes, cotton, embryogenesis, germination
Summary The expression of chloroplast genes during cotton embryogenesis was followed by measuring hybridization of cloned chloroplast genomic fragments to total RNA immobilized on membrane filters. RNA was extracted from the cotyledons of cotton embryos at different stages of development ranging from very small embryos (5 mg) to the mature dry seed and from the cotyledons of seeds germinated for several days. The clones used in the hybridization reaction were those for 4 known chloroplast genes and one chloroplast sequence whose protein product is yet to be determined; all are from organisms other than cotton. The results of these dot blot hybridizations show that these genes are expressed as RNA constitutively during embryogenesis and that this expression increased between 5 and 20-fold (depending on the genomic sequence) during germination. In some cases it appears that these genes are expressed at a higher level in very young embryos than in more mature embryos. These results are surprising since cotton cotyledons are not exposed to light during embryogenesis and are not green at any embryonic stage. The extent of artifactual hybridization signals due to the presence of ctDNA in the RNA preparations was measured by treating the RNA on the filters with DNase I prior to hybridization with a labeled probe. To determine if the increased expression of these genes observed during germination is correlated with an increase in chloroplast genomes per cell, we followed the levels of ctDNA during embryogenesis and germination by hybridization of the clones to total DNA from different developmental stages. Although ctDNA per cell increases in the cotyledons during early germination in light or dark conditions, this increase is less than the increase observed in the mRNA levels, suggesting that the transcription rate increases or the mRNA degradation rate decreases for these genes during early germination.
Introduction Biochemical and molecular studies of chloroplast DNA (ctDNA) have progressed to the stage where the structure and function of many chloroplast genes from various species have been determined (2, 8). Photoregulation of chloroplast development during the etioplast to chloroplast transition and the effect of light on chloroplast gene expression have also been extensively studied during the past few years (1, 11, 17). By comparison, relatively little is known about the expression of chloroplast genes during embryogenesis. To
study chloroplast gene expression during the embryogenesis of cotton seeds, we have probed cotyledon RNA from 14 stages of embryogenesis and 4 stages of early germination with radioactive cloned chloroplast genomic fragments representing several chloroplast gene sequences. The cloned chloroplast genomic fragments originated from the ctDNA of several organisms, none of which is cotton itself. The fact that strong hybridization signals were obtained with these heterologous probes reiterates that highly conserved sequences of chloroplast genes are found throughout the plant kingdom (22).
106
Experimental procedures Materials
Cotton plants, Gossypium hirsutum vat. COKER413, were greenhouse grown. Embryonic cotyledons were obtained from embryos at different stages of development ranging from very young embryos (5 mg wet weight) to mature dry seeds. This span represents the last two thirds of embryogenesis and a 25 fold increase in embryo size. Etiolated cotyledons were obtained from seedlings germinated in the dark for 24, 48, 72, and 96 hours. Green cotyledons were obtained from seedlings germinated in continuous white light for 96 hours. Pancreatic deoxyribonuclease (E.C. 3.1.5.4) was purchased from Worthington Biochemical Corp. 32p labeled deoxyribonucleotides were purchased from Amersham. R N A Extraction
Total RNA was prepared from cotyledons by solubilization in SDS, deproteinization with .phenol/chloroform and precipitation with LiC1 and sodium acetate. These methods have been previously described (9). All RNA preparations were analyzed for the presence of high molecular mRNAs by in vitro translation in the wheat germ systems (6). Preparations that did not give a high rate of peptide bond formation or show the synthesis of high molecular weight proteins on SDS gels were discarded. Purified RNA was stored in water at - 8 0 ° C until used.
were adjusted to 1.5 M NaC1, 150 mM sodium citrate and applied to nitrocellulose (Schleicher and Schuell, BA85) using a 192 well manifold. 10/~g of RNA were applied to each 3 mm dot except as noted below. A vacuum was maintained on the manifold during sample application to prevent spreading. Following application of the sample, each well was washed with 1.5 M NaCI, 150 mM sodium citrate pH 7.0. Ethidium bromide (5/~g/ml) was included in the wash to allow visualization of the dots is subsequent steps. The dot blots were air dried and baked for 2 hours at 80°C in a vacuum oven. Dot blots of DNA samples were prepared in a similar fashion except that each dot contained 1 #g of DNA. In the case of the dots probed with the chloroplast rDNA sequence, only 1 #g of RNA was applied to the filters and the specific radioactivity of the probe was one half that of the other probes. This allowed the pattern of the chloroplast rDNA gene expression to be plotted (Fig. 2) with that of the other genes. DNAse treatment
For analysis of possible DNA contamination in the RNA preparations, some of the dots were treated with DNAse I prior to hybridization. Filters were incubated for 20 hours at room temperature in 0.9070 NaC1, 5 mM MgC12, 16.7 mM ammonium acetate pH 5.0, and 3/~g/ml DNase I. Filters were rinsed with several changes of water and air dried prior to hybridization with labeled probes. Filter hybridization
DNA Isolation
DNA was obtained by ethanol precipitation of the LiCI supernatant obtained in the preparation of RNA. The DNA was purified by deproteinization with phenol/chloroform followed by two cycles of banding in CsC1/ethidium bromide gradients. Ethidium bromide was removed by extensive dialysis against 2 mM Tris-HCl pH 7.0. Dot blots
Aliquots of RNA were denatured by heating at 60°C for 10 minutes in 5007o formamide, 607o formaldehyde, 10 mM PO4 buffer pH 6.0. Samples
Hybridization of DNA to RNA on the nitrocellulose filters was conducted as described by Galau et al. (10). All hybridizations were at 68°C for 16-20hours in a buffer containing 4x SSC (1 x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 10 mM PO4 buffer pH 6.0, 10 mM EDTA, 0.1070 SDS, l x Denhardt's solution (5), 0.1070 pyrophosphate, 8°7o dextran sulfate (21), 100/~g/ml denatured herring sperm DNA and 32p labeled cloned chloroplast genomic fragments (106 cpm/ml). Following hybridization, filters were washed 2 times for 30 minutes at the hybridization temperature in the hybridization buffer, 2 times for 30 minutes at the same temperature but in
107 2x SSC, 0.1% pyrophosphate and 0.1% SDS and finally, 2 times for 30 minutes at room temperature in 2 x SSC. Filters were air dried and exposed to Kodak XAR film with Dupont Cronex Lightening Plus screens. The levels of radioactivity which hybridized to the developmental dots were also determined by cutting out the dots and measuring the radioactivity on them by scintillation counting. All values were standardized to allow for probe decay.
Chloroplast DNA probes Chloroplast gene specific probes were kindly provided by Dr J.R.Y. Rawson (SOHIO, Cleveland OH, USA). These probes and the individuals who isolated and identified them are listed in Table 1. The rbcL probe is a 647bp internal Pst I fragment of the maize RUBP carboxylase large subunit gene. The chloroplast ribosomal RNA probe contains all of the 16s rDNA and one half of the 23s rDNA from millet chloroplast rDNA. The psbA probe is a 800 bp Pst I fragment of the 'photogene 32' from Spirodela. Probe pMCPS6 is a 2 kb cDNA made to a chloroplast mRNA whose protein product has not been determined. The atpB probe is a 1.98 Kb EcoRI fragment of the ATPase B subunit from spinach. Probes were made radioactive with 32p labeled dCTP by nick translation (15) to specific activities of about 5 x 10a c p m / ~ .
Results
Chloroplast gene expression during embryogenesis Figure 1 shows a series of dot blot experiments in which aliquots of total RNA from cotyledons at 14 stages of embryogenesis and 4 stages in early germination were bound to nitrocellulose and hybri-
dized with radioactive cloned chloroplast genomic fragments representing the several chloroplast gene sequences. This procedure, though not allowing us to determine the absolute amount of a specific mRNA, enables us to follow the changes in the level of RNA transcripts of these different genes throughout embryogenesis. In every instance, the expression of chloroplast genes as RNA was observed at all stages of cotyledon development. It was determined that these signals were not due to non specific hybridization by comparison with RNA dots which had been hybridized with a probe of animal origin. With the filter exposure times used in these experiments, this probe gave no visually detectable hybridization signal (data not shown). Further analysis of RNA transcript level was obtained by cutting out the dots and measuring radioactivity by scintillation counting. This analysis, given in Fig. 2, quantified the patterns of expression among the different chloroplast clotles shown in Fig. 1. The level of RNA transcripts for the atpB, pMCPS6 and rbcL genes appears to be about 2 times more abundant in very young embryos (5 - 10 mg) than during later stages of embryogenesis where the expression of these genes as mRNA remains relatively constant. Furthermore, the transcript levels of these three genes are roughly the same throughout embryogenesis. By contrast, the level of psbA transcript fluctuates throughout embryogenesis and is slightly higher than the RNA levels of the other probes. In order to plot the expression of the chloroplast rDNA gene with that of the other probes, filters were used which contained only 1 #g of total RNA per dot (versus 10/zg of RNA per dot used for the other chloroplast sequences). Furthermore, it was found that at 10/~g of RNA per dot, the concentration of chloroplast rRNA is so high that probe becomes limiting in the hybridization reaction, and
Table 1. Origin of probes used for hybridization to RNA dot blots. Gene
Organism
Source
ATPase (atpB) Chloroplast rDNA Unknown Photo Gene (psbA) RUBP Carboxylase LSU (rbcL)
Spinacia oleracea Panicum miliaceum Panicum miliaceum Spirodela oligorrhiza Zea mays
R. G. Herrmann (Diisseldorf) J. R. Y. Rawson (Ohio) J. R. Y. Rawson (Ohio) M. Edelman (Rehovot) L. Bogorad (Cambridge)
108
Fig. 1. Hybridization of developmental RNA dot blots with chloroplast probes. RNA was extracted from cotyledons during several stages of enabryogenesis and germination, spotted on nitrocellulose and hybridized with labeled chloroplast probes as described in expednaental procedures" All dots contained 10 #g of RNA per dot except for those probed with chloroplast rDNA which contained 1/~g of RNA per dot. The nunabes over the dots represent the developmental stages in nag weight (embryogenesis) or the hours of germination (germination) of the cotyledons from which the RNA was extracted. The final dot on the right represents 96 hours of germination in the light, 48 and 96D are seedlings germinated for 48 and 96 hours in the dark. DS is dry seed.
6000
.~ o o ATPose : : Unknown a.-.--.o Chloroplast rDNA ...... ~ R U B P - L S U ~ - - - - ~ Photo gene
5000 o
~= 4000
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~P /q.o
-/i
~, 3000
";: .~,
-~ 2ooc °
,~-. /,,,~"-,,,_...,,,,
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:30
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Fig. 2. Radioactivity hybridized to RNA developmental dots. The dots shown in Figure 1 were cut out and the amount of radioactivity hybridized to each one was determined by scintillation counting. The bottom axis represents the developmental stages present on the dots as given in Fig. 1.
109 artifactually low values of hybridization are ob- , served. The results of this experiment demonstrate that the level of chloroplast rRNA, although much higher than that of the other probes, remains contant throughout embryogenesis.
Chloroplast gene expression during germination The expression of chloroplast genes during germination of cotton seedlings was followed by hybridization of the ctDNA sequences to RNA extracted from cotyledons of seeds germinated in the dark or under continuous white light. These results are also shown in Figs. 1 and 2. In all cases there was a marked increase in the abundance of chloroplast transcripts following germination of seedlings for 4 days under both dark and light conditions. By comparison to mRNA levels in the dry seed, expression of the rbcL gene showed the greatest difference with a 20-fold increase in transcript level upon germination in the light and a 13-fold increase during germination in the dark. In contrast, transcript levels for the psbA gene only increased by 3 and 5-fold during germination under dark and light conditions respectively. However, psbA RNA levels were higher in the dry seed than those of the other chloroplast genes, and the final level, after 4 days germination in light or dark regimes, of psbA transcripts is comparable to the transcript levels of the other probes. The transcripts for atpB, ctrRNA and pMCPS6 all increased about 9-fold during germination in the light, whereas their expression under dark growth varied; pMCPS6 transcripts increasing 4-fold, atpB transct~ipts increasing 6-fold and ctrRNA transcripts showing 8-fold increase. Previous studies by Merrick and Dure (13) showed that a 4-fold increase in chloroplast tRNA species during early cotyledon germination is independent of induction by light. These results are also in agreement with other studies which have demonstrated a light stimulated, although not light dependent, increase in the transcription of chloroplast genes (3, 18, 20).
Chloroplast DNA levels A major source of error in these measurements is the possible ctDNA contamination of RNA preparations which would result in artifactually
higher hybridization signals on the developmental dots. DNA contamination does not present a problem when RNA dots are probed with a nuclear single copy gene, since even a 10070nuclear DNA contamination would contribute such a small number of copies of a specific nuclear sequence as to go undetected by this hybridization procedure. However, due to the large number of copies of the chloroplast genome per chloroplast (16) and its low complexity, ctDNA contamination could provide enough copies of a chloroplast gene sequence to affect these results. If we assume that the percent of ctDNA in total DNA is 1°70 in embryonic cotyledons (inferred from calculations by Timmis and Scott, 19) and that the complexity of ctDNA is 100, then a 1°70 total DNA contamination in the RNA preparations would provide approximately as many hybridizable chloroplast sequences as are represented by rare class nuclear mRNAs and would be detected by our procedures. To determine if the signals we observed in our RNA dots were due to contaminating ctDNA in our samples, we treated the RNA dot filters with DNase I prior to hybridization with labeled chloroplast rDNA probe. Figure 3 shows the results of these experiments. In this case, 10/~g of RNA per dot were used in order to increase the levels of radioactivity in this experiment. Note that the increase in hybridization during germination is much less than that observed in Fig. 1 for this probe, which illustrates the effect of having too high a concentration of target molecules on the dot, as mentioned previously. On average, DNase treatment removed 5% of the signal observed from the RNA dots, ut the general pattern of hybridization remained ~he same. We can not explain the drop in hybridizaItion observed at 72 hours of germination but suspect that it may be due to degradation of the RNA sample. No such drop is observed in Fig. 1 with this probe. In order to test the effectiveness of the DNase treatment of the dots, we treated DNA dot filters under the same DNase conditions and hybridized the filters with the chloroplast rDNA probe. Figure 4 shows the result of this experiment. The DNase treatment is seen to remove at least 75070 of the DNA available for hybridization. From these two experiments, we calculate that about 33070 of the radioactivity hybridization values obtained with the chloroplast gene probes (Figs. 1 and 2) is due to probe: ctDNA hybridization and should be
~
110
Fig. 3. (a) Hybridization of developmentalRNA dot blots with the labeled chloroplast rDNA done. Experiments were carried out
as describedin Fig. 1. The top filter was treated with DNase I prior to hybridizationas describedin experimentalprocedures. (b) The levels of radioactivity which hybridizedto the dots in Figure 3a and measured by scintillation counting as described in Fig. 2.
subtracted. Nevertheless, the normalized hybridization is still considerable and represents substantial expression of the chloroplast genes. Figure 4 also shows the hybridization of the chloroplast rDNA probe to developmental DNA dots which we used to determine the relative levels of ctDNA found during embryogenesis and germination. The dots represent three stages o f embryogenesis and germination for 4 days under dark or light conditions. The results o f this experiment show that the ctDNA level remains constant during mid-late embryogenesis and is slightly lower in 5 mg embryos. This would indicate that the higher
RNA levels observed in 5 mg embryos are due to increased transcription of chloroplast genes during this embryonic stage. Upon germination in either dark or light, there is a 2-fold increase in the level of chloroplast DNA. This increase would account in part for the higher levels of RNA we observed during germination. However, since in all cases the level of RNA transcripts increased to a greater extent than the ctDNA level, we conclude that the increase in transcript abundance is due to enhanced expression o f the individual chloroplast genes.
111
Fig. 4. (a) Hybridization of DNA dots with the chloroplast rDNA clone. Total DNA from four stages in embryogenesis and two stages in germination was hybridized with the labeled chloroplast rDNA clone as described in experimental procedures. The top filter was treated with DNase I prior to hybridization as described in Fig. 3. The numbers over the dots represent the developmental stages of the cotyledons from which the DNA was extractea as given in Fig. I. (b) The level of radioactivity which hybridized to the DNA dots in Figure 4a was measured by scintillation counting as described in Fig. 2.
Discussion
Previous workers (7, 20) have shown that RNA dot blots can be used as an accurate tool for the measurement of the relative levels of individual RNA species in total RNA preparations. In this
study, we have used this technique to follow the expression of chloroplast genes during cotton seed embryogenesis and germination. The results of our experiments show that the rbcL, psbA, chloroplast rDNA, atpB and pMCPS6 genes are all expressed as mRNA in the cotyledons of developing embryos
112 and that, with the exception of chloroplast rDNA, the abundance of the mRNAs for these genes is roughly the same throughout embryogenesis. The mRNA levels for these genes increase upon germination under both dark and light conditions, although they are more abundant in the cotyledons of light grown seeds. It is obvious in Figs. 1 and 2 that a good deal of fluctuation in signal occurs with some probes during embryogenesis. We are not convinced that these fluctuations are real, but simply underscore the potential artifacts of this technique in determining precise numerical values. However, what is obvious is that strong hybridization signals are found for all embryonic time points and that these signals increase markedly during the first days of germination in both etiolated and green cotyledons. A portion of these signals may be due to chloroplast DNA contaminating the RNA preparations as inferred from the data in Figs. 3 and 4. Nevertheless, it is also obvious that all these chloroplast genes are being expressed in embryogenesis at substantial levels. It is not surprising that the proplastids of embryonic cotyledon are active in the synthesis of chloroplast ribosomal RNA since the protein synthesizing system of the chloroplast must always be in existence in order to perpetuate itself. However, it is interesting to find the expression as mRNA of genes whose protein products are involved in the photosynthetic apparatus since these cotyledons are not green at any stage of embryogenesis. It should be noted that these experiments have only measured the expression of chloroplast genes as RNA. We do not know whether these transcripts are translated into protein. Expression of the rbcL gene as protein, as well as other chloroplast genes, has been observed in the etiolated leaves of plants from three different families (14). Also, studies conducted on the rbcL gene in Phaseolus vulgaris have shown that this gene is expressed as mRNA during embryogenesis and that the mRNA is translated into protein (12). However, the cotyledons of this species are green throughout much of embryogenesis. Furthermore, expression of the protein in these cotyledons was found to be independent of light. In contrast to these results, studies of the rbcL gene in Euglena have demonstrated a translational regulational of plastid gene expression (4). Transcripts of the rbcL gene were found in both the proplastid and the chloroplast but large subunit protein was
only observed in the chloroplast. This uncoupling of transcription and translation has also been observed for the components of the photosynthetic apparatus in spinach leaf cells. These dark grown leaf cells have been shown to express 35 distinct chloroplast genes as mRNAs. However, western blot analysis revealed that only the genes not involved in the photosynthesis complex were expressed as protein under dark conditions (R Herrmann, personal communication).
References 1. Akoyunoglou G, Argyroudi-Akoynnoglou JH: Chloroplast Development Elsevier/North-Holland Press, Amsterdam, 1978. 2. Bohnert H J, Crouse E J, Schmitt JM: Organization and expression of plastid genomes. In: Parthier B, Boulter D (ed) Encyclopedia of Plant Physiology New Series, vol 14B Springer-Verlag Press, Berlin, 1982, pp 475- 530. 3. Coen DM, Bedbrook JR, Link G, Grebanier A, Steinbeck K, Beaton A, Rich A, Bogorad L: Genes and mRNAs for maize chloroplast proteins: Changes during light-induced chloroplast development. In: Akoyunogiou G, ArgyroudiAkoyunoglou JH (ed) Chloroplast Development Elsevier/North-Holland Press, Amsterdam, 1978, pp 553-558.
4. Cushman JC, Barrasso DS, Price CA: Translational regulation in proplastids of Euglena gracilis. In: Gaiau GA (ed) Abstracts, First International Congress of Plant Molecular Biology. University of Georgia Press, Athens, 1985, pp 54. 5. Denhardt DT: A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Comm 23:641-646, 1966. 6. Dure III LS, Gaiau GA: Developmental biochemistry of cottonseed embryogenesis and germination XIII. Regulation of biosynthesis of principal storage proteins. Plant Physiol 68:187-194, 1981. 7. Dure III LS, Pyle JB, Chlan CA, Baker JC, Gaiau GA: Developmental biochemistry of cottonseed embryogenesis and germination XVII. Developmental expression of genes for the principal storage proteins. Plant Molec Biol 2:199- 206, 1983. 8. Dyer TA: The chloroplast genome and its products. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, Vol 2, Oxford University Press, New York, 1985, pp 147- 177. 9. Gaiau GA, Legocki AB, Greenway SC, Dure III LS: Cotton messenger RNA sequences exist in both polyadenylated and nonpolyadenylated forms. J Biol Chem 256:2551-2560, 1981. 10. Galau GA, Chlan CA, Dure III LS: Developmental biochemistry of cottonseed embryogenesis and germination XVI. Analysis of the principal cotton storage protein gene family with cloned cDNA probes. Plant Molec Biol 2:189- 198, 1983.
ll3 11. Jenkins GI, Hartley MR, Bennett J: Photoregulation of chloroplast development: transcriptional, translational and posttranslational controls? Phil Trans R Soc Lond B 303:419- 431, 1983. 12. Medford J, Sussex I: Embryonic expression of RUBPCase in Phaseolus vulgaris. In: Galau GA (ed) Abstracts, First International Congress of Plant Molecular Biology. University of Georgia Press, Athens, 1985, pp 89. 13. Merrick WC, Dure III LS: The developmental biochemistry of cotton seed embryogenesis and germination IV. Levels of cytoplasmic and chlOroplastic transfer ribonucleic acid species. J Biol Chem 247:7988- 7999, 1972. 14. Nechushtai R, Nelson N: Biogenesis of photosystem I reaction center during greening of oat, bean and spinach leaves. Plant Molec Biol 4:337- 384, 1985. 15. Rigby PWJ, Dieckmann M, Rhodes C, Berg P: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237-251, 1977. 16. Smith H, Grierson D: The Molecular Biology of Plant Development. University of California Press, Berkeley, 1982. 17. Smith H, Billett EE, Giles AB: The photocontrol of gene expression in higher plants. In: Smith H (ed) Regulation of
Enzyme Synthesis and Activity in Higher Plants. Academic Press, London, 1977, pp 9 3 - 127. 18. Smith SM, Ellis RJ: Light-induced accumulation of transcripts of nuclear and chloroplast genes for Ribulosebiosphosphate Carboxylase. J Molec Appl Gen 1:127- 137, 1981. 19. Timmis JN, Scott NS: Sequence homology between spinach nuclear and chloroplast genomes. Nature 305:65-67, 1983. 20. Thompson WF, Everett M, Polans NO, Jorgensen RA, Palmer JD: Phytochrome control of RNA levels in developing pea and mung-bean leaves. Planta 158:487- 500, 1983. 21. Wahl GM, Stern M, Stark GR: Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxmymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683 - 3687, 1979. 22. Whitfeld PR, Bottomley W: Organization and structure of chloroplast genes. Ann Rev Plant Physiol 34:279-310,' 1983. Received 4 February 1986; in revised form 29 April 1986; accepted 12 May 1986.
105
The expression of chloroplast genes during cotton embryogenesis Katyna E. Borroto & Leon Dure III
Biochemistry Department, University of Georgia, Athens, GA 30602, U.S.A. Keywords: chloroplast genes, cotton, embryogenesis, germination
Summary The expression of chloroplast genes during cotton embryogenesis was followed by measuring hybridization of cloned chloroplast genomic fragments to total RNA immobilized on membrane filters. RNA was extracted from the cotyledons of cotton embryos at different stages of development ranging from very small embryos (5 mg) to the mature dry seed and from the cotyledons of seeds germinated for several days. The clones used in the hybridization reaction were those for 4 known chloroplast genes and one chloroplast sequence whose protein product is yet to be determined; all are from organisms other than cotton. The results of these dot blot hybridizations show that these genes are expressed as RNA constitutively during embryogenesis and that this expression increased between 5 and 20-fold (depending on the genomic sequence) during germination. In some cases it appears that these genes are expressed at a higher level in very young embryos than in more mature embryos. These results are surprising since cotton cotyledons are not exposed to light during embryogenesis and are not green at any embryonic stage. The extent of artifactual hybridization signals due to the presence of ctDNA in the RNA preparations was measured by treating the RNA on the filters with DNase I prior to hybridization with a labeled probe. To determine if the increased expression of these genes observed during germination is correlated with an increase in chloroplast genomes per cell, we followed the levels of ctDNA during embryogenesis and germination by hybridization of the clones to total DNA from different developmental stages. Although ctDNA per cell increases in the cotyledons during early germination in light or dark conditions, this increase is less than the increase observed in the mRNA levels, suggesting that the transcription rate increases or the mRNA degradation rate decreases for these genes during early germination.
Introduction Biochemical and molecular studies of chloroplast DNA (ctDNA) have progressed to the stage where the structure and function of many chloroplast genes from various species have been determined (2, 8). Photoregulation of chloroplast development during the etioplast to chloroplast transition and the effect of light on chloroplast gene expression have also been extensively studied during the past few years (1, 11, 17). By comparison, relatively little is known about the expression of chloroplast genes during embryogenesis. To
study chloroplast gene expression during the embryogenesis of cotton seeds, we have probed cotyledon RNA from 14 stages of embryogenesis and 4 stages of early germination with radioactive cloned chloroplast genomic fragments representing several chloroplast gene sequences. The cloned chloroplast genomic fragments originated from the ctDNA of several organisms, none of which is cotton itself. The fact that strong hybridization signals were obtained with these heterologous probes reiterates that highly conserved sequences of chloroplast genes are found throughout the plant kingdom (22).
106
Experimental procedures Materials
Cotton plants, Gossypium hirsutum vat. COKER413, were greenhouse grown. Embryonic cotyledons were obtained from embryos at different stages of development ranging from very young embryos (5 mg wet weight) to mature dry seeds. This span represents the last two thirds of embryogenesis and a 25 fold increase in embryo size. Etiolated cotyledons were obtained from seedlings germinated in the dark for 24, 48, 72, and 96 hours. Green cotyledons were obtained from seedlings germinated in continuous white light for 96 hours. Pancreatic deoxyribonuclease (E.C. 3.1.5.4) was purchased from Worthington Biochemical Corp. 32p labeled deoxyribonucleotides were purchased from Amersham. R N A Extraction
Total RNA was prepared from cotyledons by solubilization in SDS, deproteinization with .phenol/chloroform and precipitation with LiC1 and sodium acetate. These methods have been previously described (9). All RNA preparations were analyzed for the presence of high molecular mRNAs by in vitro translation in the wheat germ systems (6). Preparations that did not give a high rate of peptide bond formation or show the synthesis of high molecular weight proteins on SDS gels were discarded. Purified RNA was stored in water at - 8 0 ° C until used.
were adjusted to 1.5 M NaC1, 150 mM sodium citrate and applied to nitrocellulose (Schleicher and Schuell, BA85) using a 192 well manifold. 10/~g of RNA were applied to each 3 mm dot except as noted below. A vacuum was maintained on the manifold during sample application to prevent spreading. Following application of the sample, each well was washed with 1.5 M NaCI, 150 mM sodium citrate pH 7.0. Ethidium bromide (5/~g/ml) was included in the wash to allow visualization of the dots is subsequent steps. The dot blots were air dried and baked for 2 hours at 80°C in a vacuum oven. Dot blots of DNA samples were prepared in a similar fashion except that each dot contained 1 #g of DNA. In the case of the dots probed with the chloroplast rDNA sequence, only 1 #g of RNA was applied to the filters and the specific radioactivity of the probe was one half that of the other probes. This allowed the pattern of the chloroplast rDNA gene expression to be plotted (Fig. 2) with that of the other genes. DNAse treatment
For analysis of possible DNA contamination in the RNA preparations, some of the dots were treated with DNAse I prior to hybridization. Filters were incubated for 20 hours at room temperature in 0.9070 NaC1, 5 mM MgC12, 16.7 mM ammonium acetate pH 5.0, and 3/~g/ml DNase I. Filters were rinsed with several changes of water and air dried prior to hybridization with labeled probes. Filter hybridization
DNA Isolation
DNA was obtained by ethanol precipitation of the LiCI supernatant obtained in the preparation of RNA. The DNA was purified by deproteinization with phenol/chloroform followed by two cycles of banding in CsC1/ethidium bromide gradients. Ethidium bromide was removed by extensive dialysis against 2 mM Tris-HCl pH 7.0. Dot blots
Aliquots of RNA were denatured by heating at 60°C for 10 minutes in 5007o formamide, 607o formaldehyde, 10 mM PO4 buffer pH 6.0. Samples
Hybridization of DNA to RNA on the nitrocellulose filters was conducted as described by Galau et al. (10). All hybridizations were at 68°C for 16-20hours in a buffer containing 4x SSC (1 x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 10 mM PO4 buffer pH 6.0, 10 mM EDTA, 0.1070 SDS, l x Denhardt's solution (5), 0.1070 pyrophosphate, 8°7o dextran sulfate (21), 100/~g/ml denatured herring sperm DNA and 32p labeled cloned chloroplast genomic fragments (106 cpm/ml). Following hybridization, filters were washed 2 times for 30 minutes at the hybridization temperature in the hybridization buffer, 2 times for 30 minutes at the same temperature but in
107 2x SSC, 0.1% pyrophosphate and 0.1% SDS and finally, 2 times for 30 minutes at room temperature in 2 x SSC. Filters were air dried and exposed to Kodak XAR film with Dupont Cronex Lightening Plus screens. The levels of radioactivity which hybridized to the developmental dots were also determined by cutting out the dots and measuring the radioactivity on them by scintillation counting. All values were standardized to allow for probe decay.
Chloroplast DNA probes Chloroplast gene specific probes were kindly provided by Dr J.R.Y. Rawson (SOHIO, Cleveland OH, USA). These probes and the individuals who isolated and identified them are listed in Table 1. The rbcL probe is a 647bp internal Pst I fragment of the maize RUBP carboxylase large subunit gene. The chloroplast ribosomal RNA probe contains all of the 16s rDNA and one half of the 23s rDNA from millet chloroplast rDNA. The psbA probe is a 800 bp Pst I fragment of the 'photogene 32' from Spirodela. Probe pMCPS6 is a 2 kb cDNA made to a chloroplast mRNA whose protein product has not been determined. The atpB probe is a 1.98 Kb EcoRI fragment of the ATPase B subunit from spinach. Probes were made radioactive with 32p labeled dCTP by nick translation (15) to specific activities of about 5 x 10a c p m / ~ .
Results
Chloroplast gene expression during embryogenesis Figure 1 shows a series of dot blot experiments in which aliquots of total RNA from cotyledons at 14 stages of embryogenesis and 4 stages in early germination were bound to nitrocellulose and hybri-
dized with radioactive cloned chloroplast genomic fragments representing the several chloroplast gene sequences. This procedure, though not allowing us to determine the absolute amount of a specific mRNA, enables us to follow the changes in the level of RNA transcripts of these different genes throughout embryogenesis. In every instance, the expression of chloroplast genes as RNA was observed at all stages of cotyledon development. It was determined that these signals were not due to non specific hybridization by comparison with RNA dots which had been hybridized with a probe of animal origin. With the filter exposure times used in these experiments, this probe gave no visually detectable hybridization signal (data not shown). Further analysis of RNA transcript level was obtained by cutting out the dots and measuring radioactivity by scintillation counting. This analysis, given in Fig. 2, quantified the patterns of expression among the different chloroplast clotles shown in Fig. 1. The level of RNA transcripts for the atpB, pMCPS6 and rbcL genes appears to be about 2 times more abundant in very young embryos (5 - 10 mg) than during later stages of embryogenesis where the expression of these genes as mRNA remains relatively constant. Furthermore, the transcript levels of these three genes are roughly the same throughout embryogenesis. By contrast, the level of psbA transcript fluctuates throughout embryogenesis and is slightly higher than the RNA levels of the other probes. In order to plot the expression of the chloroplast rDNA gene with that of the other probes, filters were used which contained only 1 #g of total RNA per dot (versus 10/zg of RNA per dot used for the other chloroplast sequences). Furthermore, it was found that at 10/~g of RNA per dot, the concentration of chloroplast rRNA is so high that probe becomes limiting in the hybridization reaction, and
Table 1. Origin of probes used for hybridization to RNA dot blots. Gene
Organism
Source
ATPase (atpB) Chloroplast rDNA Unknown Photo Gene (psbA) RUBP Carboxylase LSU (rbcL)
Spinacia oleracea Panicum miliaceum Panicum miliaceum Spirodela oligorrhiza Zea mays
R. G. Herrmann (Diisseldorf) J. R. Y. Rawson (Ohio) J. R. Y. Rawson (Ohio) M. Edelman (Rehovot) L. Bogorad (Cambridge)
108
Fig. 1. Hybridization of developmental RNA dot blots with chloroplast probes. RNA was extracted from cotyledons during several stages of enabryogenesis and germination, spotted on nitrocellulose and hybridized with labeled chloroplast probes as described in expednaental procedures" All dots contained 10 #g of RNA per dot except for those probed with chloroplast rDNA which contained 1/~g of RNA per dot. The nunabes over the dots represent the developmental stages in nag weight (embryogenesis) or the hours of germination (germination) of the cotyledons from which the RNA was extracted. The final dot on the right represents 96 hours of germination in the light, 48 and 96D are seedlings germinated for 48 and 96 hours in the dark. DS is dry seed.
6000
.~ o o ATPose : : Unknown a.-.--.o Chloroplast rDNA ...... ~ R U B P - L S U ~ - - - - ~ Photo gene
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40 50 6 0 70 80 Fresh W e i g h t (rag)
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110 1201 DS I i
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Fig. 2. Radioactivity hybridized to RNA developmental dots. The dots shown in Figure 1 were cut out and the amount of radioactivity hybridized to each one was determined by scintillation counting. The bottom axis represents the developmental stages present on the dots as given in Fig. 1.
109 artifactually low values of hybridization are ob- , served. The results of this experiment demonstrate that the level of chloroplast rRNA, although much higher than that of the other probes, remains contant throughout embryogenesis.
Chloroplast gene expression during germination The expression of chloroplast genes during germination of cotton seedlings was followed by hybridization of the ctDNA sequences to RNA extracted from cotyledons of seeds germinated in the dark or under continuous white light. These results are also shown in Figs. 1 and 2. In all cases there was a marked increase in the abundance of chloroplast transcripts following germination of seedlings for 4 days under both dark and light conditions. By comparison to mRNA levels in the dry seed, expression of the rbcL gene showed the greatest difference with a 20-fold increase in transcript level upon germination in the light and a 13-fold increase during germination in the dark. In contrast, transcript levels for the psbA gene only increased by 3 and 5-fold during germination under dark and light conditions respectively. However, psbA RNA levels were higher in the dry seed than those of the other chloroplast genes, and the final level, after 4 days germination in light or dark regimes, of psbA transcripts is comparable to the transcript levels of the other probes. The transcripts for atpB, ctrRNA and pMCPS6 all increased about 9-fold during germination in the light, whereas their expression under dark growth varied; pMCPS6 transcripts increasing 4-fold, atpB transct~ipts increasing 6-fold and ctrRNA transcripts showing 8-fold increase. Previous studies by Merrick and Dure (13) showed that a 4-fold increase in chloroplast tRNA species during early cotyledon germination is independent of induction by light. These results are also in agreement with other studies which have demonstrated a light stimulated, although not light dependent, increase in the transcription of chloroplast genes (3, 18, 20).
Chloroplast DNA levels A major source of error in these measurements is the possible ctDNA contamination of RNA preparations which would result in artifactually
higher hybridization signals on the developmental dots. DNA contamination does not present a problem when RNA dots are probed with a nuclear single copy gene, since even a 10070nuclear DNA contamination would contribute such a small number of copies of a specific nuclear sequence as to go undetected by this hybridization procedure. However, due to the large number of copies of the chloroplast genome per chloroplast (16) and its low complexity, ctDNA contamination could provide enough copies of a chloroplast gene sequence to affect these results. If we assume that the percent of ctDNA in total DNA is 1°70 in embryonic cotyledons (inferred from calculations by Timmis and Scott, 19) and that the complexity of ctDNA is 100, then a 1°70 total DNA contamination in the RNA preparations would provide approximately as many hybridizable chloroplast sequences as are represented by rare class nuclear mRNAs and would be detected by our procedures. To determine if the signals we observed in our RNA dots were due to contaminating ctDNA in our samples, we treated the RNA dot filters with DNase I prior to hybridization with labeled chloroplast rDNA probe. Figure 3 shows the results of these experiments. In this case, 10/~g of RNA per dot were used in order to increase the levels of radioactivity in this experiment. Note that the increase in hybridization during germination is much less than that observed in Fig. 1 for this probe, which illustrates the effect of having too high a concentration of target molecules on the dot, as mentioned previously. On average, DNase treatment removed 5% of the signal observed from the RNA dots, ut the general pattern of hybridization remained ~he same. We can not explain the drop in hybridizaItion observed at 72 hours of germination but suspect that it may be due to degradation of the RNA sample. No such drop is observed in Fig. 1 with this probe. In order to test the effectiveness of the DNase treatment of the dots, we treated DNA dot filters under the same DNase conditions and hybridized the filters with the chloroplast rDNA probe. Figure 4 shows the result of this experiment. The DNase treatment is seen to remove at least 75070 of the DNA available for hybridization. From these two experiments, we calculate that about 33070 of the radioactivity hybridization values obtained with the chloroplast gene probes (Figs. 1 and 2) is due to probe: ctDNA hybridization and should be
~
110
Fig. 3. (a) Hybridization of developmentalRNA dot blots with the labeled chloroplast rDNA done. Experiments were carried out
as describedin Fig. 1. The top filter was treated with DNase I prior to hybridizationas describedin experimentalprocedures. (b) The levels of radioactivity which hybridizedto the dots in Figure 3a and measured by scintillation counting as described in Fig. 2.
subtracted. Nevertheless, the normalized hybridization is still considerable and represents substantial expression of the chloroplast genes. Figure 4 also shows the hybridization of the chloroplast rDNA probe to developmental DNA dots which we used to determine the relative levels of ctDNA found during embryogenesis and germination. The dots represent three stages o f embryogenesis and germination for 4 days under dark or light conditions. The results o f this experiment show that the ctDNA level remains constant during mid-late embryogenesis and is slightly lower in 5 mg embryos. This would indicate that the higher
RNA levels observed in 5 mg embryos are due to increased transcription of chloroplast genes during this embryonic stage. Upon germination in either dark or light, there is a 2-fold increase in the level of chloroplast DNA. This increase would account in part for the higher levels of RNA we observed during germination. However, since in all cases the level of RNA transcripts increased to a greater extent than the ctDNA level, we conclude that the increase in transcript abundance is due to enhanced expression o f the individual chloroplast genes.
111
Fig. 4. (a) Hybridization of DNA dots with the chloroplast rDNA clone. Total DNA from four stages in embryogenesis and two stages in germination was hybridized with the labeled chloroplast rDNA clone as described in experimental procedures. The top filter was treated with DNase I prior to hybridization as described in Fig. 3. The numbers over the dots represent the developmental stages of the cotyledons from which the DNA was extractea as given in Fig. I. (b) The level of radioactivity which hybridized to the DNA dots in Figure 4a was measured by scintillation counting as described in Fig. 2.
Discussion
Previous workers (7, 20) have shown that RNA dot blots can be used as an accurate tool for the measurement of the relative levels of individual RNA species in total RNA preparations. In this
study, we have used this technique to follow the expression of chloroplast genes during cotton seed embryogenesis and germination. The results of our experiments show that the rbcL, psbA, chloroplast rDNA, atpB and pMCPS6 genes are all expressed as mRNA in the cotyledons of developing embryos
112 and that, with the exception of chloroplast rDNA, the abundance of the mRNAs for these genes is roughly the same throughout embryogenesis. The mRNA levels for these genes increase upon germination under both dark and light conditions, although they are more abundant in the cotyledons of light grown seeds. It is obvious in Figs. 1 and 2 that a good deal of fluctuation in signal occurs with some probes during embryogenesis. We are not convinced that these fluctuations are real, but simply underscore the potential artifacts of this technique in determining precise numerical values. However, what is obvious is that strong hybridization signals are found for all embryonic time points and that these signals increase markedly during the first days of germination in both etiolated and green cotyledons. A portion of these signals may be due to chloroplast DNA contaminating the RNA preparations as inferred from the data in Figs. 3 and 4. Nevertheless, it is also obvious that all these chloroplast genes are being expressed in embryogenesis at substantial levels. It is not surprising that the proplastids of embryonic cotyledon are active in the synthesis of chloroplast ribosomal RNA since the protein synthesizing system of the chloroplast must always be in existence in order to perpetuate itself. However, it is interesting to find the expression as mRNA of genes whose protein products are involved in the photosynthetic apparatus since these cotyledons are not green at any stage of embryogenesis. It should be noted that these experiments have only measured the expression of chloroplast genes as RNA. We do not know whether these transcripts are translated into protein. Expression of the rbcL gene as protein, as well as other chloroplast genes, has been observed in the etiolated leaves of plants from three different families (14). Also, studies conducted on the rbcL gene in Phaseolus vulgaris have shown that this gene is expressed as mRNA during embryogenesis and that the mRNA is translated into protein (12). However, the cotyledons of this species are green throughout much of embryogenesis. Furthermore, expression of the protein in these cotyledons was found to be independent of light. In contrast to these results, studies of the rbcL gene in Euglena have demonstrated a translational regulational of plastid gene expression (4). Transcripts of the rbcL gene were found in both the proplastid and the chloroplast but large subunit protein was
only observed in the chloroplast. This uncoupling of transcription and translation has also been observed for the components of the photosynthetic apparatus in spinach leaf cells. These dark grown leaf cells have been shown to express 35 distinct chloroplast genes as mRNAs. However, western blot analysis revealed that only the genes not involved in the photosynthesis complex were expressed as protein under dark conditions (R Herrmann, personal communication).
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4. Cushman JC, Barrasso DS, Price CA: Translational regulation in proplastids of Euglena gracilis. In: Gaiau GA (ed) Abstracts, First International Congress of Plant Molecular Biology. University of Georgia Press, Athens, 1985, pp 54. 5. Denhardt DT: A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Comm 23:641-646, 1966. 6. Dure III LS, Gaiau GA: Developmental biochemistry of cottonseed embryogenesis and germination XIII. Regulation of biosynthesis of principal storage proteins. Plant Physiol 68:187-194, 1981. 7. Dure III LS, Pyle JB, Chlan CA, Baker JC, Gaiau GA: Developmental biochemistry of cottonseed embryogenesis and germination XVII. Developmental expression of genes for the principal storage proteins. Plant Molec Biol 2:199- 206, 1983. 8. Dyer TA: The chloroplast genome and its products. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, Vol 2, Oxford University Press, New York, 1985, pp 147- 177. 9. Gaiau GA, Legocki AB, Greenway SC, Dure III LS: Cotton messenger RNA sequences exist in both polyadenylated and nonpolyadenylated forms. J Biol Chem 256:2551-2560, 1981. 10. Galau GA, Chlan CA, Dure III LS: Developmental biochemistry of cottonseed embryogenesis and germination XVI. Analysis of the principal cotton storage protein gene family with cloned cDNA probes. Plant Molec Biol 2:189- 198, 1983.
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Enzyme Synthesis and Activity in Higher Plants. Academic Press, London, 1977, pp 9 3 - 127. 18. Smith SM, Ellis RJ: Light-induced accumulation of transcripts of nuclear and chloroplast genes for Ribulosebiosphosphate Carboxylase. J Molec Appl Gen 1:127- 137, 1981. 19. Timmis JN, Scott NS: Sequence homology between spinach nuclear and chloroplast genomes. Nature 305:65-67, 1983. 20. Thompson WF, Everett M, Polans NO, Jorgensen RA, Palmer JD: Phytochrome control of RNA levels in developing pea and mung-bean leaves. Planta 158:487- 500, 1983. 21. Wahl GM, Stern M, Stark GR: Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxmymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683 - 3687, 1979. 22. Whitfeld PR, Bottomley W: Organization and structure of chloroplast genes. Ann Rev Plant Physiol 34:279-310,' 1983. Received 4 February 1986; in revised form 29 April 1986; accepted 12 May 1986.
