J. Photo&em.

Photobiol.

B: Bid.,

II

173

(1991) 173-187

Expression of early genes in light-induced chloroplast differentiation of cultured plant cells U. Specht and G. Richter+ Institut fiir Botanik,

Universit&t

Hannmer,

W-3ooO Hannover

21 (F.R.G.)

(Received February 28, 1991; accepted April 11, 1991)

Keywords. Blue light, cDNA library, chloroplast differentiation, gene expression, transcription (nuclear and plastid genes).

Abstract The objective of this study was to identify genes, preferentially of the plastid, which are rapidly expressed during the initial phase of blue-light-induced chloroplast development in suspension-cultured cells (Ch.enopodiunz mm L.) and to analyse the encoded proteins. A cDNA library (A gt IO) was constructed using total BNA from plastid preparations of dark-grown cells exposed to blue light for 3, 6 and 12 h. By differential screening, at least three clones were identified which correspond to rapidly light-induced plastid genes. For these and a number of nuclear genes represented by other clones, a temporary accumulation of the specific mFtNA was observed between 12 and 48 h of blue-light exposure. with regard to their nucleotide sequence and derived ammo-acid sequence they seem to represent a novel group of genes distinctive in structure and encoded product.

1. Introduction Recent studies have shown that the differentiation of chloroplasts from leucoplast-like precursors in suspension-culturedcells of Chenopodium &rum is induced and maintained preferentially by blue light, while red light has little or no effect [ 1, 21. During this developmental process, the activity and steady state level of nuclear and plastid transcripts encoding prominent chloroplast proteins significantly increase. These light-induced changes are also reflected in the activity of cell-free transcription systems such as a plastid DNA-protein complex or isolated nuclei prepared from blue-lightirradiated cell suspensions [ 3, 41. The transcription rates of several genes of the endogenous DNA increase substantiallywith irradiation time, indicating that blue light strongly stimulates the transcription of nuclear and plastid genes which are involved in chloroplast differentiation. In a first attempt to evaluate early events which may play a crucial role in the initial phase of light-induced plastid transformation, i.e. in light +Author to whom correspondence should be addressed.

loll-1344/91/$3.50

0 1991 - Elsevier Sequoia, Lauaanne

174

perception, signal transduction and response, as well as in the coordination of light-inducible genes, we have identified, via their complementary DNAs (cDNAs), a number of nuclear genes which are rapidly induced by blue light in dark-grown cell suspensions of Chenopodium rubrum [ 51. For most of these, a temporary accumulation of the specific mRNA is registered between 30 min and 72 h of blue-light irradiation. With regard to nucleotide sequence and derived amino-acid sequence, a glycine-rich protein, a /3-tubulin-like protein and one species resembling a regulatory acidic ribosomal protein (RLAO) are among the products of these early light-induced genes. The aim of the present work was to search for genes of the plastid which are also rapidly activated during the exposure of dark-grown suspensioncultured cells to blue light and code for proteins exhibiting similar features to the nuclear-encoded counterparts. To this purpose, we followed an alternative experimental strategy designed to facilitate the detection of these genes. The plastid RNAs of short-term-irradiated cells were transcribed in cDNAs which were used for the construction of a clone library. In this paper, we present the first evidence for the existence of plastid genes whose prominent feature is rapid and transient expression during blue-light exposure of darkgrown cell suspensions of Chenopodium rub-urn. Theirnucleotide sequence and derived amino-acid sequence indicate that they differ in structure, and probably also in function, from any plastid proteins observed so far.

2. Experimental

details

2.1. Plant materials Chloroplast-free callus cells of Cherwpodiumrubrum L. (kindly provided by Professor Dr. W. Htisemann, Miinster, F.R.G.) were grown as reported previously [2]. Prior to illumination, cells from the logarithmic growth phase (day 6) were suspended in fresh medium and kept for 48 h in darkness. Irradiation experiments were carried out with cell populations of identical physiological properties. Seeds of Cherwpodiuq rub-urn L. (ecotype 184; supplied by Professor E. Wagner, Freiburg, F.R.G.) were induced to germinate by application of a 12 h temperature cycle (10 “C and 32 “C) in weak white light. Six days after sowing, the seedlings were transferred to the greenhouse and cultivated in standard soil under a light-dark regime of 14 h light and 10 h dark. At the end of 10-12 days of growth, the young plants were between 25 and 30 cm tall. The upper leaves were harvested and used for chloroplast isolation. 2.2. Preparation Plastids from cells were isolated in ref. 3. For the Logemann et al.

and an&@

of plustid RNA

dark-grown and blue-light-irradiated suspension-cultured and purified by sucrose gradient centrifugation as described extraction and purification of their RNA, the protocol of [ 61 was followed. Portions were denatured in formam-

175

ide-formaldehyde and subjected to denaturing gel electrophoresis in 1% agarose [ 71. Transfer by capillary blotting to a nylon membrane for Northern hybridization was carried out as described in ref. 8. For dot hybridization, denatured RNA was directly dotted onto a nitrocellulose membrane in a dilution series as reported in ref. 2 and then treated with UV light for 5 min. Total plastid RNA from different preparations was standardized using ethidium bromide fluorescence of plastid ribosomal RNA species or the amount of LSU-mRNA probed with the specific insertion of clone pSA 204 (see later). Hybridization to 32P-labelledprobes in dot and Northern hybridization was performed at 42 “C as described by Richter et al. [2] with slight modifications in the washing procedure [ 51. The air-dried membranes were exposed to Kodak XAR films with an intensifying screen at - 80 “C. Results were quantified by counting the radioactivity of excised spots using Cerenkov radiation. 2.3. Putifiation of DNA DNA of chloroplasts from cell suspensions or leaves of Cherwpodium rubrum was prepared using the protocol of Whitfeld et al. [9]. In a few instances the DNA was additionally purified by caesium chloride gradient centrifugation (1.33 g ml- ’ plus ethidiumbromide); centrifugation was carried out at 42 000 rev min- ’ and 20 “C for at least 48 h (Beckman Ti 80 rotor). Alternatively, the DNase treatment was replaced by sucrose gradient centrifugation according to Palmer [lo]; the chloroplasts collected were directly subjected to lysis. For the preparation of total DNA from suspension-culturedcells, a portion of 20 g (fresh weight) was washed once with distilled water, quickly chilled in liquid nitrogen and ground in a mortar. The resulting powder was suspended in 10 ml extraction buffer (10 mM Tris-HCl, pH 7.4, 25 mM ethylenediaminetetraacetic acid (EDTA)); 1 ml of 10% sodium dodecylsulphate (SDS) and 11 mg proteinase K were added; the mixture was incubated at 37 “C for l-2 h; another 11 mg of the enzyme were added and the incubation was repeated. After addition of 1 ml 5 M NaCl, extraction with 1 vol. of phenol-chloroform-isoamyl alcohol (10: 10:1) was performed; the aqueous phase was separated by centrifugation (Sorvall HB4 rotor; 10 000 rev ruin- ’ for 15 min) and treated with 2 vol. of diethylether; 2.5 vol. of ethanol were added and the mixture was left overnight at -20 “C. The precipitate was collected by centrifugation, resuspended in 10 ml 0.1 X standard saline citrate (SSC), supplied with 20 ~1 DNase-free RNase (10 mg ml-‘) and incubated at 37 “C for 30 min. DNA was recovered as described above, dissolved in bidistilled water and quantified spectrophotometrically. For Southern blot analysis, the DNA was digested with appropriate restriction enzymes; the fragments were separated by gel electrophoresis on 1% agarose; blotting and hybridization, including the washing procedure, were performed under stringent conditions [5].

176

2.4. Cloniw of complementary DNA (cDNA) Total plastid RNA was isolated as described above, and cDNA was synthesized with materials from a cDNA synthesis kit (Amersham-Buchler, Braunschweig, F.R.G.) according to the supplier’s protocol. First strand cDNA was synthesized with random hexanucleotide primers. cDNA libraries were established in A gt 10 from plastid RNAs of darkgrown cells after 3,6 and 1.2h in blue light using materials from a commercial kit (Amersham-Buchler); these RNA preparations were used individually or a common fraction was employed. Single-strand cDNAs synthesized from plastid RNAs of cells grown in darkness or in blue light for 3, 6 and 12 h or 9 days served as probes for differential screening; they were labelled by random priming [ 111. Plaque hybridization, selection of relevant clones and isolation of plasmid DNA were performed as reported in ref. 5. The cDNA insertions were subcloned into the EcoRI site of plasmid vector Bluescript KS (Stratagene, Heidelberg, F.R.G. [ 121). 2.5. Sequencing of DNA Both strands of cDNA insertions previously subcloned in the vector Bluescript (see above) were analysed by the dideoxy-chain-termination technique with T7 polymerase [ 131 using materials from a commercial T7 sequencing kit (Pharmacia, Freiburg, F.R.G.) and following the supplier’s protocol. Fragments of DNA longer than 400 base pairs (bp), which could not be directly sequenced from both directions, were cut with exonuclease III; blunt ends were obtained by digestion with mung-bean nuclease (Stratagene), and the fragments were subcloned in Bluescript vector; they were subsequently analysed. Sequence data analyses and derivation of protein sequences were performed using the PC Gene program (Genofit). Searches for protein sequences similar to the derived amino-acid sequences of the various cDNA clones were conducted with the Fast Scan Program using a Dayhoff MDM-78 matrix.

3. Results 3.1. Transcripts of early light-induced genes With dark-grown suspension-cultured cells of Chewpodium rub-urn a pronounced effect of blue light on the steady state level of nuclear and plastid mRNAs encoding chloroplast proteins has been observed [ 1,2]. Several accumulate rapidly and often temporarily with the onset of blue-light irradiation. The corresponding nuclear genes can be identified via their cDNAs using cDNA libraries based on polyadenylated RNAs (poly[AJ-RNAs) from cells irradiated for 6-24 h [5]. In order to detect plastid genes with similar expression kinetics in the initial phase of blue-light-induced chloroplast differentiation, plastid RNA was isolated from cells exposed to blue light for 3, 6 and 12 h. These individual preparations were combined and used to construct cDNA libraries with the vector A gt 10. For differential screening,

177

cDNAs synthesized from plastid RNAs of cells, irradiated with blue light for 3, 6 and 12 h or 9 days or kept in darkness, were used as probes. Thus a number of non-cross-hybridizing clones were identified. Several of these hybridized to RNAs which accumulated rapidly in dark-grown cells after exposure to blue light. Clones representative of this group include U 1.1, U 1.2, U 2.2, U 4.1, U 5.2 and U 6.2. 3.2. Temporal pattern of speci$c transcripts during blue-light irradiation The effect of irradiationtime on the abundanceof RNAs rapidly transcribed after exposure to blue light was studied by dot hybridization analysis. Plastid RNA was isolated from dark-grown cells exposed to blue light for various lengths of time (between 3 h and 9 days). For each preparation, identical series of increasing amounts (1 B-2.0 pg) were dotted. They were challenged with the radiolabelled insertions of various clones. Control experiments were conducted with LSU-mRNA as standard. Previous studies have shown that it increases linearly with irradiation time [2]; accordingly, the signal intensities of hybrids formed between the specific probe and RNA of different origin were used for calibration (see Pig. l(a)). In the case of the mRNA specific for clone U 1.2 (U 1.2~n-RNA), hybridization signals of highest intensity were monitored between 9 and 24 h (Fig. l(b)). These visual impressions were quantified by counting the radioactivity of each spot (Fig. 2). A similar temporal accumulation was observed for the mRNAs of clones U 2.2, U 4.1 and U 6.2. As expected the genes specified by these four clones exhibited a rapid and transient activation by blue light. The time courses of temporary accumulation, and thus the positions of the maxima, differed only slightly (Pig. 2). For U 1.2- and U 4.1~mRNA, the most rapid accumulation took place after about 12 h of blue-light irradiation, whereas U 2.2~mRNAdid not reach its highest expression level before 24 h. For the mRNA of clone U 6.5,

Oh 3h

(a)

Oh

6h 12h 20h 48h 9d

3h

6h 12h 20h 48h 9d

@I

Fig. 1. Changes in the amounts of hybridizable LSU-mRNA (a) and U 1.2-mRNA (b) in darkgrown cell suspensions of Chenopodium n&rum exposed to blue light. Assessment by dot hybridization. Total plastid RNA was isolated from cell samples after the onset of irradiation at the times indicated; 1.0, 1.5 and 2.0 pg of each preparation were directly blotted onto a nitrocellulose membrane and hybridized to the 32P-labelIed insertions of clone pSA 204 and U 1.2 respectively. 0 h, dark-grown cells; BL, blue light.

178

representing a second group of transcripts, a steep increase in the steady state concentration was observed until about 12 h after light induction, followed by a more or less constant level until day 9 (not shown).

0

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Fig. 2. Temporal changes in the amounts of RNAs homologous to clones U 1.2 (a), U 2.2 (b), U 4.1 (c) and U 6.2 (d) on irradiation of suspension-cultured cells with blue light. Isolation and handling of RNA samples were as described in Fig. 1. Hybridization was to the radiolabelled cDNA insertions of the four clones. The relative signal intensities were obtained by counting the 32Pradioactivity of hybrids bound to the dots. LSU-mRNA was used as an internal standard (see text). BL, blue light.

180

3.3. Analysis of nucleic-acid sequences 3.3.1. clone u 1.1 By following the strategy outlined in Section 2, we determined the complete nucleotide sequence of the 778 bp cDNA insertion of this clone. A plausible open reading frame (ORF) was ascertained by the method of Shepherd [ 141 which could encode a polypeptide of 138 amino-acid residues with a predicted molecular mass of 16 437. Figure 3 shows that the ORF begins with an ATG initiation codon at position 81 (A) and ends with a stop codon at position 495 (T). The hydropathy plot of the deduced amino-acid sequence (not shown) indicates regions which could form an a! helix. An interesting feature is the presence of three putative phosphorylation sites at positions 18, 45 and 90. The deduced protein does not resemble any species so far present in nucleic-acid and/or protein databases.

1 56

TACTCCTACAAATTCCCCTCCTITITTGATTAAATTCTPC TTAAATTTCATTTCCCTTAATCGAA

ATG GAT CTC GAT CAT AAT CCA MDLDHNP

102

CAT TTT CAC CGA AAA GGA GAA AGG GTA CGG TAC GTT AGC CGA H F H R K G E R V R Y V S R

144

GGG Tl-A TTA GGG GAG GAG AAT ATA GAC GAG AAC AGG GAG CAA G L L G E E N I D E N R E Q

186

ATC GAG AGA ACA AGT TTT TTA TTG AGT TCG ATT CGG TTT TTT I E R T S F L L S S I R F F

220

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CTT AGT GAA lTT TTC GGA TTG AAA CAA ATT ATA AAC TCT CAA L S E F F G L K Q I I N S Q

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AAA ATA TAT CTG TTT TTA GCC TCT CAA ACC TCG TCA TTG TTC K I Y L F L A S Q T S S L F

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AAACCCTCAAAACACGATACmAACCCTAACCCTACCTGCCCCACGAAATTCACCGAACG

692

AGTTGAACTTTTAGTACTT!~'TAAACTGCTTCACACM~XTI?AAAC!'~TAATACACT?JI

747

CTAACTTGTACAAATTCGTACTATAACCACTA

AGATTAC!lTAAATCAACTAACTTCACm

Fig. 3. Nucleotide sequence (non-coding strand) of the cDNA insertion of clone U 1.1 and the derived amino-acid sequence (one letter code).

181

3.3.2. Clone U 1.2 On sequencing the 699 bp insertion, this clone was found to contain an ORF beginning with a feasible initiation codon at position 122 (A) and ending with a stop codon at position 663 Q, which could encode a polypeptide of 180 amino acids in length and a calculated molecular mass of 20 391 (Fig. 4). The position of the translation initiation codon as the first possible in the putative ORF was determined by the fact that three stop codons existed at positions 38, 65 and 119 upstream of the ATG. A predominance of positively charged ammo-acid residues near the amino terminus was registered. Four potential phosphorylation sites existed along the entire amino-acid sequence (positions 5, 89, 171, 178). Comparison with the sequences of published proteins did not reveal any homology.

1 56

GGTMTC~CAGGTGCCTTGTCGCGCGAGAAATXTA'lTGAACTGTCCC~C c!mAAAAAA

GGAACTGC'ITCCTGCTATA~TTACCCCCGTCGCGT

111

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ATG GCA TAT GCA AGT CTC CGT GTJ! GTC GAT AGG MAYASLRVVDR

156

GCT TTC AGG TTC COG CAC GTC GGT TGT AAT TCT AAA ACC CGG A F R F R H V G C N S I( T R

198

GGT GGT GAG GTC TTT 'ITT ATG ACT CGT Am G G E V F F M T R I

ACC GAT CGC GTA T D G V

240

GAG TCA GGT ATC AGA ATA CC0 CCC AAG CM E S G I R I P P K Q

AGG CAC =A R H L

282

GAG GCT GGA GTA CTT CCT AGG TAC CTT CGA ACA ATT ATG TM' EAGVLPRYLRTIMF

324

CGA AGG GCC CCC CAG ACC TCA AGT GGC CTA ATT TTA ATT GTA R R A P Q T S S G L I L I V

366

TAT TAT TGC CCT CTT CCG CCG TCA ACA AAG CTG CCG TGG T'IT Y Y C P L P P S T K L P W F

408

AGT TTG ACT AAA GTA GTA AAC CTC CAC CAA CAA CCG ATC GGT S L T K V V N L H Q Q P I G

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TAC AAT TCT CAG GCA TCT GCC CTA TTT ATA GAT TAC l?l?CCTT Y N S Q A S A L F I D Y F L

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TGT CTT TTT CCC TTT TTT !lTT CTC GGT AAC ACG Tl'T AGT CCG C L F P F F F L G N T F S P

576

GGT TAC TTC ATT TTG CTA AAC CCG AAC AGT AGA ACT TGC ACG GYFILLNPNSRTCT

618

CGT GCT CCT TCG TCA AGT AGC CGA GCC TCG GAA AAG TTC 'XT R A P S S S S R A S E K F L

660

AGC TAG s STOP

CTTCGGTAAAGTACCTTAGCTAATGGCTATl?CTA

Fig. 4. Nucleotide sequence (non-coding strand) of the cDNA the derived amino-acid sequence (one lettercode).

insertionof clone U 1.2 and

182

3.3.3. Clone U 4.1 Sequence analysis of the 534 bp insertion indicates a complete ORF; as shown in Fig. 5 it begins with the first possible initiation codon at position 140 (A), determined by the presence of a stop codon at position 59 upstream of the ATG, and ends at position 512 (T). The protein specified encompasses 124 amino acids with ,a calculated molecular mass of 14 034. A prominent feature of the predicted secondary structure is a hydrophilic region near the amino terminus (residues 24-34) from which an cr helix is deducible. The sequence data indicate that the gene corresponding to clone U 4.1 encodes a protein as yet unidentified in plant cells. 3.4. Location of early light-induced genes In an attempt to clarify the affiliation of the early light-induced genes represented by the various cDNA clones to the nuclear or plastid genome, Southern analyses were carried out. Total DNA and purified chloroplast DNA were digested with the restriction enzymes EcoRI or PstI; the resulting fragments were separated, blotted and hybridized to the 32P-labelled cDNA insertions of clones U 1.2, U 2.2, U 4.1 and U 6.2. As depicted in Fig. 6 two patterns of autoradiographic bands can be distinguished. The first pattern, generated by the insertion of clones U 1.2 and U 2.2 as probes, has detectable hybridization signals exclusively with

1 56

CTTAAGAACAAATCCGTCAAGTATGGTAGGTATGTATCACAMACTAGGTTCTAA AGTl'AAGAAGGTACAAAGTGGTCATCGTATAACAAGGTACCTCGATTCCAGGC!lT

111

TATACCTTTTTGTTCACAAAGGTGCTGAG

158

CAA GGT GAA TTA GGG GGA AAG TAC CGG TGT ATA GAA AGG CCG Q G E L G G K Y R C I E R P

200

ATT CCT TAC CTT !ITA GAA GAG GAG AAC GTA CTT AGG Tl'A MG I P Y L L E E E N V L R L K

242

TTA GTA GGC CTT TTC GGT AGA AAA AAA GTT GTT ACA GAA ACA L V G L F G R K K V V T E T

284

GTA ACT AGG TTA TCA CAA GGC AAT CTA TCC TTG TCT MA V T R L S Q G N L S L S K

326

TTT ATG ACT ATT GAG AGC CTA TCT CAT AAT CTT GCC !l?lTCTA FMTIESLSHNLAFL

368

GGT AAA CTA TTA CTT GAT AAC CAA GAT TCG GTA GAG ACC GCT G K L L L D N Q D S V E T A

410

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TAC

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TGG GTC GCA ACT TTA TCT CTA TAT CCT CCT ATA AAC GCC CTT W V A T L S L Y P P I N A L

494

CAT TCT TCG GGG AAA CTA TAG H S S G K L STOP

AGAAGTAGACGTTTCTTAAG

Fig. 5. Nucleotide sequence (non-coding strand) of the cDNA insertion of clone U 4.1 and the derived amino-acid sequence (one letter code).

183

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Fig. 6. Southern an&y& of puriiied chloroplast DNA and total DNA cut with EdoR (larks a, b) or PstI (lanes c, d); 30 pg of total DNA (lanes a, c) and 10 pg of chloroplast DNA (l&tes b, d) were digested and the fragments were separated on 1% agarose gel, blotted onto a nitrocellulose membrane and challenged with the 32P-labelled insertions of clones U 1.2 (a), U 2.2 (b), U 4.1 (c) and U 6.2 (d). Size markers (M) are EcoRI and Hind III fragqentq of lambda DNA.

184

fragments of total DNA (lanes a, c), even after extremely long exposure times. The second pattern, obtained with the clone-specific probes of U 4.1 and U 6.2, differs insofar as it exhibits strong signals with chloroplast DNA fragments (lanes b, d), but weaker signals with those of total DNA (lanes a, c). The positions of the strong and weak signals are the same when both sorts of DNA are cut by the same enzyme, i.e. with EcoRI (lanes a, b) or Pst I (lanes c, d). Evidently, hybrids formed by the insertion of clones U 4.1 and U 6.2 and restriction fragments of total DNA originate from the portion of chloroplast DNA present, although in small amounts, in the DNA preparations. This conclusion is confirmed by the results of two control experiments (not shown). With the coding sequence of a cab gene (lightharvesting chlorophyll a&protein II) employed as probe in the same Southern hybridization assay, the signals are conllned to fragments of total DNA in both restriction patterns; however, probing with the sequence of the psbA gene (clone pSA 452; courtesy of Professor Dr. G. Link, Bochum, F.R.G.) gives rise to a strong signal with a distinct chloroplast DNA fragment created by either EcoRI or PstI, and a weaker signal of identical position with a total DNA fragment, its size depending on the enzyme used for restriction. These results indicate that the two genes represented by the clones U 4.1 and U 6.2 are located on the plastid DNA, whereas those specified by clones U 1.2 and U 2.2 are part of the nuclear DNA. However, to substantiatethese conclusions, further studies are needed. The results of the Southern blot analyses performed with various DNAs suggest that the genes corresponding to the four clones examined are single copy genes (U 1.2, U 4.1 and U 6.2) or belong to small gene families (U 2.2). 4. Discussion The resultsreported here provide further evidence that blue light activates a number of genes in suspension-culturedcells of Chenopodium rubrum, the products of which are not typical membrane and stroma proteins of the chloroplast. Thus earlier findings [5] have been coniirmed and extended. In ref. 5, the experimental strategy was based on a selection step, preceding the cloning procedure, by which poly [A]-RNAs representative of dark-grown and fully-green cells were removed. Thus the cDNA library established was enriched in clones specifying nuclear mRNAs which were rapidly transcribed within the first 24 h of blue-light treatment. In the present investigation, an alternative approach has been chosen designed to increase the chance of identification of plastid genes with similar rapid expression on blue-light irradiation of the cultured cells. Nuclear contaminations were not removed from the plastid fractions prepared from cell samples irradiated with blue light for 6, 12 and 24 h. This explains the presence of clones in the library which harbour coding sequences of nuclear origin in their insertions. By deliberate omission of additional purification

and selection steps, the conditions for the recovery of very rare mRNA species, transcribed from nuclear and plastid genes, were substantially improved. As a consequence, the characterization of early light-induced genes in terms of their location depends on the demonstration of homologous sequences in the nuclear or plastid DNA. Therefore, the results reported here are preliminary since the hybridization approach can only provide limited information on the precise location of a gene. This emphasizes the importance of studies investigating the complete nucleotide sequence, the exact mapping on either genome and the identification of the encoded product for each relevant gene. These analyses can also be expected to provide clues to the regulatory mechanism(s) involved in the light-dependent expression of the genes detected here. A disadvantage of the strategy followed in this study is the restriction of the search to those species of genes which are transcriptionally activated on blue-light irradiation of the cells. We cannot rule out the possibility that regulation of gene expression also takes place without de ru)~o synthesis of specific proteins, i.e. by post-transcriptional and post-translational modifications of available precursor molecules, by interaction with other regulators on the protein level, by compartmentation of protein factors and by DNAbinding of available protein factors. As expected, several clones were isolated with early transcription activation of the corresponding genes by light. They add to the novel group of genes identified recently in suspension-cultured cells of Chenopodium rubrum by a different experimental approach (see above), which exhibit very similar features, i.e., rapid and mostly transient accumulation of their transcripts in the initial phase of blue-light-inducedchloroplast development, and lack of homology with known plant genes. The genes examined in this paper are novel in the sense that they encode prospective proteins which have not been found in plants so far. Indications are that at least three clones identified here correspond to genes of the plastid. These findings, although preliminary, demonstrate that the experimental strategy chosen was successful with regard to the detection of early light-induced plastid genes via their cloned mRNAs. Therefore, it is probable that more clones of this type, derived from other plastid mRNA species, could be identified in the established library. It is tempting to speculate that the proteins encoded by these early light-induced nuclear and plastid genes may play a role as regulatory factors. Since typical DNA-binding motifs, such as the zinc-binding finger or helixturn-helix, are absent in the amino-acid sequence deduced from the cDNA insertion of the relevant clones, an involvement in other regulatory mechanism(s) must be taken into consideration. Prior to this report the only information concerning plastid-encoded regulatory proteins was the detection of a protein containing a zinc-binding finger [ 15 1. The observed blue-light-induced changes in the expression of nuclear and plastid genes may be interpreted either as a general adaptation of cellular activities to the impact of sudden blue-light irradiation or as a sequence of

186

events in close connection with the induced developmental process of chloroplast differentiation. At least two different sets of genes could participate in the two fields. Our data are too preliminary to allow us to allocate single genes to one or the other category. It is the aim of further studies to elucidate the importance of early bluelight-induced genes and the biological role of their products. Further analysis of the causal relationship between their expression and the differentiation of chloroplasts will aid in the understanding of the molecular mechanisms which determine this elementary process.

Acknowledgments This work was supported by the Ministerium fXir Forschung und Technologie, Bonn (F.R.G.). We thank Mrs. I. Liebscher and P. von Trzebiatowski for expert technical assistance.

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