Altered expression of nuclear genes encoding chloroplast

Mol Gen Genet (1996) 252:362-370 © Springer-Verlag 1996

Daniela Hahn • Pierre Bennoun • Ulrich Kiick

Altered expression of nuclear genes encoding chloroplast polypeptides in non-photosynthetic mutants of Chlamydomonas reinhardtii: evidence for post-transcriptional regulation

Received: 5 February 1996/Accepted: 11 May 1996

Abstract In photoautotrophic organisms it is well documented that the expression of nuclear genes encoding plastid proteins can be regulated at various levels. We present here the analysis of a non-photosynthetic strain (CC1051) of the green unicellular alga Chlamydomonas reinhar&ii; this strain carries a mutation in the newly identified Cen gene involved in the co-regulated expression of several different nuclear genes encoding plastid proteins. We performed a differential screening strategy to isolate cDNAs corresponding to genes that are differentially expressed in mutant and wild-type strains. Extensive hybridization experiments revealed that the 15 cDNA clones isolated represent five different mRNAs that fail to accumulate in the non-photosynthetic mutant. Comparative analysis of DNA sequencing data showed that they all code for plastid proteins. In particular, we identified genes for the chlorophyll a/b binding protein of the light-harvesting complex II (LHCII), for subunits II and III of photosystem I (PsaD, PsaF), for pentose-5-phosphate 3-epimerase (PPE), an enzyme of the Calvin cycle, and for an unidentified 7 kDa protein with a suggested lumenal location. With the exception of the gene for LHCII, all proteins are encoded by single-copy genes. Evidence from run-on transcription experiments is pre- sented showing that expression of the above mentioned plastid proteins is affected at the post-transcriptional level in the mutant strain CC1051 with a defect in the Cen gene. Our results suggest that the product of the Cen gene is involved in stabilization and/or processing

Communicated by R. G. Herrmann

D. Hahn • U. Kiick ( [~) Lehrstuhl fiir Allgemeine Botanik, Ruhr-Universitiit Bochum, D-44780 Bochum, Germany

P. Bennoun Institut de Biologic Physico-Chimique, 13, Rue Pierre et Marie Curie, 75005 Paris, France

of transcripts from nuclear genes encoding chloroplast proteins.

Key words C h l a m y d o m o n a s • Non-photosynthetic mutant • Differential hybridization • Nuclear gene expression

Introduction

The assembly of fully functional chloroplast proteins requires the coordinated expression of nuclear and plastid genes. In general, gene expression can be regulated at a variety of different steps including transcription, mRNA processing and stability, translation as well as by post-translational events. Developmental and light-dependent regulation of plastid gene expression has recently been intensively studied in both plants and algae. Evidence currently accumulating indicates that changes in plastid-encoded polypeptides can be attributed mainly to post-transcriptional, translational and post-translational control mechanisms (Rochaix 1992a; Gruissem and Schuster 1993). In contrast, molecular details of the mechanisms that control nuclear gene expression in photosynthetic organisms have only been partially resolved. Work to date has mainly been limited to the RbcS and Lhcb gene families, which code for the small subunit of the ribulose-l,5-bisphos- phate carboxylase/oxygenase and the chlorophyll a/b binding proteins, respectively (for review see Thom- pson and White 1991; Herrmann et al. 1992; Kuh- lemeier 1992). It was previously assumed that nuclear gene expression, unlike plastid gene expression, is mainly controlled at the transcriptional level. However, it is now clear that the biosynthesis of nuclear-encoded plastid components is also subject to other control mechanisms. For instance, the photocontrolled accumulation of nuclear-encoded thylakoid polypeptides in tobacco seems to be regulated at both the post-transcriptional and post-translational levels (Herrmann

et al. 1992; Palomares et al. 1993). Reporter gene analyses and run-on assays in pea and Arabidopsis have recently revealed that the gene for chloroplast fer- rodoxin shows light-regulated expression, mediated by transcriptional as well as post-transcriptional events (Dickey et al. 1992; Bovy et al. 1995). So far however, genes encoding factors that control post-transcriptional expression have not been described in photoautotrophic organisms. Here we present the analysis of a ChIamydomonas reinhar&ii mutant that shows a defect in the post-transcriptional regulation of nuclear- encoded chloroplast proteins.

Due to the ability of C. reinhardtii to grow hetero- trophically, many photosynthesis-deficient mutants have been isolated (Harris I989). Most of the mutations characterized so far have a nuclear location, showing a defect in chloroplast gene expression (for review see Rochaix 1992b; Gillham et al. 1994). In the present study, we have investigated a photosynthesis-deficient strain (CC1051) that had previously been shown to affect trans-splicing of chloroplast psaA mRNA and fails to accumulate several nuclear-encoded plastid polypeptides (Girard et al. 1980; Kiick et al. 1987; Herrin and Schmidt 1988; Goldschmidt-Clermont et al. 1990). Using a differential screening method we have isolated five cDNAs representing mRNAs that fail to accumulate in the mutant. Sequence analyses revealed that these cDNAs encode five different plastid proteins.

We provide evidence that the non-photosynthetic strain CC1051 carries a nuclear mutation, 'cen', that specifically affects the post-transcriptional control of expression of nuclear genes encoding plastid polypeptides. To our knowledge this is the first analysis of a mutant from a photoautotrophic organism that is deficient in the post-transcriptional regulation of nuclear genes encoding plastid proteins.

Materials and methods

Strains and culture conditions

C. reinhardtii CC410 (SAG 11-32c, m r ) and the cell wall-deficient CC406 (cwl5, mr-) were used as wild-type control strains. The photosynthesis-deficient strain CC1051 is maintained by the Chlamydomonas Genetic Center, Duke University (Durham, N.C.) and was used throughout the experiments. This strain originally carried only the M18 mutation (Girard et aI. 1980) but has picked up a second, named Cen, which is described in this paper. Cells were grown in Tlis-acetate-phosphate (TAP) medium (Gorman and Levine 1965) or high salt (HS) medium in continuous light at 25 ° C as described by Harris (1989). Genetic crosses were performed as described previously (Bennoun et aI. 1992).

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was isolated either via two cycles of polyU)-Sepharose chromatogra- phy (Pharmacia, Freiburg, Germany) or by using the Poly ATract mRNA isolation system (Promega, Heidelberg, Germany) as indicated by the manufacturer. For RNA hybridizations, RNA was separated on agarose/formaldehyde gels and transferred to Hybond N membranes (Amersham, Braunschweig, Germany). Hybridization was carried out using standard methods as described by Sambrook et aL (1989).

cDNA library construction

First- and second-strand cDNAs were synthesized using the materials and protocols of the cDNA Synthesis System Plus (Amersham, Braunschweig, Germany). The resulting double-stranded cDNA was size-fractionated on a Sephacryl S 500 column and fractions containing cDNA larger than 500 bp were pooled and precipitated with ethanol. The cDNA was cloned into ;~ gtl0 dephospho- rylated arms (Promega, Heidelberg, Germany) as described in Sambrook et al. (1989) and packaged using extracts (Promega) as outlined in the manufacturer's instructions.

Differential screening

Approximately 21 000 recombinant phages were plated (10 ~ plaques per petri dish) and transferred to nitrocellulose filters. Duplicate filters were differentially screened with 32p-labelled cDNA derived from mRNA isolated from cells of either the photoantotrophic (CC406) or the photosynthetically deficient (CC1051) strain.

cDNA probes were synthesized from 5 pg poly(A) + RNA by using an oligo(dT) primer in a first-strand synthesis reaction containing 50ram Tris-HC1 pH 8.3, 10mM MgC12, 150mM KC1, 4mM Na,~P2OT, 10mM DTT, t mM dGTP, 1 mM dTTP, 0.03 mM dATP, 0.03 mM dCTP, 50 gCi [c~-3Zp]dATP (>3000Ci/mmol), 50 gCi [~-32p]dCTP (> 3000 Ci/mmol), 80 units RNAsin (Boehrin- ger, Mannheim, Germany) and 15 ~tg Oligo dT(18) (Pharmacia, Freiburg, Germany). After incubation for 1 h at 42 ° C, the reaction was stopped by adding 2 mM EDTA and labelled cDNA was purified from unincorporated nucleotides by passage through a Sephadex G50 column. The template RNA was degraded by hydrolysis with 0.5 M NaOH for 30 rain at 70 ° C and subsequently neutralized with 1 M Tris-HC1 pH 7.2.

Prehybridizations (6 h) and hybridizations (20 h) were carried out at 37 ° C in 5 x SSC, 5 x Denhardt's solution, 50 % formamide, containing 100 gg/ml of sheared, denatured salmon sperm DNA and 20 gg/ml poly U (Pharmacia, Freiburg, Germany). Following hybridization, the filters were washed twice in 1 x SSC, 0.1% SDS and once in 0.1 x SSC, 0.1% SDS for 30rain at 37°C. The dried filters were exposed to Fuji X-ray films RX at - 20 ° C, intensifying screens (Kodak X-Omatic Regular) were used.

Subcloning and sequencing

In order to subclone the cDNA inserts, recombinant 2 gtl0 DNA was digested using EcoRI, the inserts purified on agarose gels and cloned into the EcoRI sites of pT3T7 (Boehringer, Mannheim, Germany). Both strands of the clones were sequenced using the dideoxy method (Sanger et al. 1977) and the resulting sequences were analyzed using computer software (EMBL, Heidelberg).

RNA isolation and Northern blot analysis

Total RNA was prepared by phenol extraction and selective LiC1 precipitation as already described (Kiick et al. 1987). Poly(A) + RNA

Run-on transcription assays

For in vivo RNA labelling, cells were permeabilized using a freeze- thaw procedure described by Gagn6 and Guertin (1992). Run-on

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transcription was performed in a 60 gl reaction mixture containing 200 mM sucrose, 12 mM MgC12, 10 mM HEPES pH 7.5, 3 mM DTT, 10 mM NaF, i0 mM phosphoenolpyruvate, 0.25 mM GTP, 0.25 mM UTP, 0.5 mM ATP, 250 gCi [~-32p]CTP (> 800 Ci/mmol), 4.5 units pyruvate kinase, 80 units RNAsin (Boehringer) and approximately 5 x l0 T permeabilized cells. The mixture was incu- bated for 15 min at 26 ° C. DNA was hydrolyzed by adding an equal volume of RNase-free DNaseI (Boehringer, Mannheim, Germany) solution (15 units DNaseI in 10 mM MgC12, 1% Nonindet P-40, 10 mM HEPES pH 7.5). Following a 3 min incubation at 26 ° C, DNaseI was inactivated by adding Proteinase K solution (100 gg/ml final concentration in 0.5 % SDS, 10 mM EDTA, 20 mM Tris-HC1 pH 8.0). After 1 h at 37 ° C, total RNA was extracted with phenol/chloroform. The remaining aqueous phase was precipitated with ammonium acetate (2 M final concentration) and ethanol, to remove unincorporated nucleotides. The pellet was washed once with 70 % ethanol, and resuspended in TE (10 mM Tris-HC1 pH 7.4, 1 mM EDTA) for direct use in dot blot hybridizations.

Dot blot hybridizations

Aliquots (10 gg) of cloned cDNAs were immobilized on Hybond N membranes (Amersham) by using a vacuum apparatus (SRC 96 Minifold I, Schleicher and Schiill) as outlined in the manufacturer's protocol. Prehybridization (12 h, 37°C) and hybridization (48 h, 37 ° C) were carried out in a solution containing 5 x SSC, 5 x De- nhardts, 50 % formamide, 0.2 % SDS containing 100 gg/ml sheared, denatured salmon sperm DNA. After hybridization, membranes were washed twice in 2 x SSC, 0.1% SDS and once in 0.2% SSC, 0.1% SDS for 15 rain at 65°C. Membranes were exposed to Fuji X-ray films at - 70 ° C using intensifying screens (Kodak X-Omatic Regular).

Quantitative measurement of the hybridization signals was performed using the Cybertech CS-1 image documentation system together with the corresponding CAM software (Cybertech, Berlin, Germany).

Results

Isolation of cDNA clones corresponding to genes that are differentially expressed in wild-type and non-photosynthetic mutants

C. reinhardtii strain CC1051 is a non-photosynthetic strain carrying a nuclear mutation (M18) that was obtained previously using azide as a mutagen (Girard et al. 1980). Genetic analysis identified the mutant as being defective in the trans-splicing of the transcript of the plastid psaA gene encoding a CP1 apoprotein of the PSI (Kiick et al. 1987; Herrin and Schmidt 1988; Gold- schmidt-Clermont et al. 1990). In addition, this strain also fails to accumulate at least six low molecular weight polypeptides of the photosystem I (PSI) complex (Girard et al. 1980). To isolate genes that are differentially expressed in strain CC1051, a 2 gt l0 cDNA library was constructed using poly(A) ÷ RNA from wild-type C. reinhardtii cells, and approximately 2.1 x 104 recombinant phages were screened with single-stranded, 32p-labelled cDNA, isolated from wild-type and mutant cells. In total, 15 clones were isolated that hybridized to the wild-type cDNA but

Table 1 Summary of clones isolated by differential hybridization

Clone cDNA size mRNA size Protein ~

65-3 1.3 kb 1.3 kb Lhcbl 68-3 1.0 kb 1.1 kb PsaD 44-3 1.3 kb 1.3 kb PsaF 4-3 1.0 kb 1.0 kb 7 kDa 53-1 0.8 kb 1.4 kb PPE

Deduced from the primary cDNA sequence

Fig. 1 Altered transcript accumulation in the non-photosynthetic strain CC105I. Aliquots (25 gg) of total RNA isolated from both photoautotrophic (CC406) and photosynthetically-deficient (CC1051) cells were separated by electrophoresis on formaldehyde- containing agarose gels, transferred to nylon membranes and probed separately with radiolabelled cDNA inserts from each of the clones, pMY60, a rRNA-specific probe (Verbeet et al. 1983), was used as an internal control to quantify the amounts of RNA applied

0 0 0 0

65"3

44-3

68-3

4-3

53-1

pMY60

failed to hybridize with strain CC1051-derived cDNA. Cross-hybridization analysis showed that eleven of the clones represented the same transcript; hence, the isolated cDNA clones derive from five different mRNAs (Table 1). As a control, we carried out a Northern blot analysis with total RNA from photosynthetic and non- photosynthetic cells. Figure 1 shows that each cloned cDNA hybridizes to a wild-type RNA species and none of the corresponding transcripts accumulate in the photosynthesis-deficient strain.

The isolated cDNAs encode plastid polypeptides

The complete nucleotide sequence of both strands of the five cDNAs was determined. The cDNA clones 65-3 and 44-3 encode putative polypeptides of 253 and 235 amino acids, respectively. By comparing the amino acid sequence with databases, the cDNAs were identified as the Lhcbl cDNA (65-3) encoding a chlorophyll a/b binding protein of the light-harvesting complex II (LHCII) (Imbault et al. 1988) and the PsaF cDNA (44-3) that codes for the subunit III of PSI (Franz6n et al. 1989). The protein encoded by cDNA 68-3 contains 196 amino adds, which includes a putative transit peptide of 35 amino acids exhibiting several

characteristic features of C. reinhardtii stroma-targeting chloroplast transit peptides (cTPs) (Franz6n et al. 1990). The deduced 68-3 amino acid sequence was compared with databases and was shown to be 46-48% identical to the PSI subunit II (PsaD) from higher plants. A molecular mass of 18 kDa was also calculated for the 68-3 peptide sequence, a molecular mass similar to that of higher plant PsaD, further indicating that we have cloned a PsaD cDNA from C. reinhardtii. The sequence data reported here were deposited (1993) in the EMBL data library under the accession number X74419. During preparation of this manuscript Farah et al. (1995) reported the isolation of a C. reinhardtii PsaD cDNA which is homologous to cDNA 68-3.

The cDNA 53-1 nucleotide sequence (X96878) seems to be incomplete at its 5' end since the transcript size (1.4 kb) does not correspond to the size of the cDNA (0.8 kb). The cDNA 53-1 contains a single open reading frame (ORF) of 143 amino acids with a restricted codon usage common to C. reinhardtii nuclear genes. To determine potential functions of the cDNA 53-1 encoded protein, we compared the predicted sequence with

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known sequences from protein databases. The ORF shares 41% identity and 73.4% similarity with the pentose-5-phosphate 3-epimerase (PPE) of Alcaligenes eutrophus (Kusian et al. 1992). PPE is a Calvin cycle enzyme catalyzing the epimerization of xylulose-5- phosphate to ribulose-5-phosphate. To date, little in- formation about the properties of PPE is available. Except for A. eutrophus, no PPE sequences from any source have been reported so far. The high degree of similarity between the polypeptide encoded by cDNA 53-1 and the PPE of A. eutrophus indicates that we have cloned the first PPE cDNA from a eukaryotic organism.

The 1022bp cDNA 4-3 can be translated into a protein of 117 amino acid residues with a calculated molecular mass of 7 kDa (Fig. 2). A search of current data libraries revealed no other sequences with signifi- cant homology to the 7 kDa protein. However, the first 52 amino acids exhibit a high degree of similarity to lumenal cTPs from C. reinhardtii (Table 2). Hydro- pathy plots (Kyte and Doolittle 1982) indicate that the 7 kDa protein does not contain hydrophobic domains except for the one in the putative transit peptide that

Fig. 2 Nucleotide and deduced M A A t R e s V A T R A A V P A R amino acid sequences of cDNA -31 AGTCCATAAGCCGTTACGCACATTTTGC/d~ ATG GCC GeT CTG CGC TGC TCT GTT GCC ACT CGC GCG GCC GTC CCG GeT CGC

4-3 (7 kDa lumenal protein) from G $ S V V V R A $ T E O T T I( R A N L G L L A G k C. reinhardtii. Asterisks denote 5z GGC AGe TCC GTC GTG GTG CGC GCC AGe ACT GAG CAG Ace Ace AAG CGC GeT ATG CTG GGC CTG CTG GeT GGT GCC

stop codons, and the V A G A L L V A P A E A I R I P S H E F T G R 14 V polyadenylation signals 127 GTC GeT GGT GCC CTG CTG GTG GeT CCC GeT GAG GCC ATe CGC ATe CCC TCG CAC GAG TTC Ace GGC eGG ATG GTG

(TGTAA) are underlined. The K G G G S S P K S A T A A S 14 E S Y T L E G T K I( sequence data have been 202 AAG GGC GGC GGC TCT TCC CCC AAG TCG GCC Ace GeT GCC TCG ATG GAG AGe TAC Ace CTG GAG GGC Ace AAG AAG

deposited in the EMBL data O G V s L K T K K 1( L L A K V R E N G 0 K S A S S

library under the accession 277 CAG GGT GTC TCC CT6 AAG Ace AAG AAG AAG CTG CTG GCC AAG GTG CGC GAG AAC GGC CAG AAG TCT GCC TCC TCG number X96877

352 TAA GCA~CAACAGCTGAG~GCT~CGAT~CTGTGT~GCG~GCATGGGCACGAGTTCGACGTTTGCT~CGCGCACAAAGCTCGCAGATGCGCATGATCCGG

451 CGACGGCTGCGTCGTGGCTGGTGCCTAGTCGGAGCCAGGAGCAAAGCGGGGTATGCA•GGACGGGAGTCCGCCGAGGTTATTGGTGGATGCTTGAATTGG

551 ATGGATTTAGGCGTTCGGTTTTTTGG•GCTGCCTATGCGACCAACGGTCTTTGTGTAGGCCAGCGTGATGCATGCGAAGGTGAGCGCACT•CTTAGGGCG

651 CGCGCCTGCCTGTATCGATGACTGGT•GTGGT•CCGGCATCCAATGA•GGTT•TAT•T•CGCGTTGGTCCGCTGTTGAGTGGT•CGAGGGCAGCAG•CTC

751 C~CC~CTCACTAC~TCCCAACCACTATAGAAGAGAA~J̀TGATACTCATTTAGGACGAATGCG~GAT~TGGACA~CGATATACGGCTTCACT~CTCTCT~

851 GACCAGTTTTGTGTGATGTGTTTTTTGGT•CT•CATGGACCATTC•••TT•C•GAATGGAAATGAGCGCGGTGTGAAGT•TCAGCTGACACGCGGCATAC

951 ACGCTGTACACTGTAAAGCTGTATGATTCC(A)11

Table 2 Chloroplast lumen-targeting transit peptides from C. reinhar&ii

Protein Sequence Reference

7 kDa OEE1 OEE2 OEE3 cyt~552 PsaF

MAALRCSVATRAAVPARGSSVVVRASTEQTTKRAMLGLLAGAVAGALLVAPA A E [1] MALRAAQSAKAGVRAARPNRATAVVCKAQKVGQAAAAAALATAMVAGSANA A L [2] MATALCNKAFAAAPVARPASRRSAVVVRASGSDVSRRAALAGFAGAAALVSSSPANA A A [3] MALASKVATRPAVASRRGAVVVRASGESRRAVLGGLLASAVAAVAPKAALA A L [4] MLQLANRSVRAKAARASQSSARSVSCAAAKRGADVAPLTSALAVTASILLTTGAASASA A A [3] MALTMRNPAVKASSRVAPSSRRALRVACQAQKNETASKVGTALAASALAAAVSLSAPAAMAA D ~5]

[1] This work; [2] Franz6n et al. (1987); [3] Mayfield et al. (1989); [4] Mayfield et al. (1987); [-5] Merchant and Bogorad (1987). Sites of cleavage of the transit peptide are indicated by A. Boldface type indicates positively charged amino acids. For the 7 kDa protein and the OEE3 protein, the processing sites have not been determined experimentally, but are deduced from the sequence comparisons

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directs the protein into the thylakoid lumen (data not shown). Due to its presumed lumenal localization, we assume that the polypeptide encoded by cDNA 4-3 is a still unidentified protein involved in the photo- kb

synthetic process. 2a.~- The Lhcbl gene is a member of a small gene family,

whereas the subunit III of PSI (PsaF) seems to be 9.4- encoded by a single gene (Imbault et al. 1988; Franz6n 6.7- et al. 1989). As shown in Fig. 3, the other three cDNAs hybridize to only one or two fragments in several re- 4.a- striction digests. These results, together with the North- ern analysis (Fig. 1), indicate that the subunit II of PSI, the pentose-5-phosphate 3-epimerase, and the unidentified 7 kDa lumenal protein are encoded by single- 2 . a -

copy genes. 2 . o -

Strain CC1051 carries at least two distinct nuclear mutations

The photosynthetically deficient CC1051 strain was kept separately by the Duke strain collection for several years and was previously described as the nuclear mutant M18 defective in trans-splicing of the plastid psaA gene (Kiick et al. 1987; Herrin and Schmidt 1988; Goldschmidt-Clermont et al. 1990). To test whether the failure to accumulate the nuclear-encoded plastid polypeptides is due to a pleiotropic effect, we crossed mutant CC1051 with a wild-type strain. If strain CC1051 carries a single mutation (M18), meiotic products should only exhibit either a parental wild-type or a mutant expression pattern. Recombinant phenotypes, however, are expected if CC1051 carries more than one mutation. Meiotic products were analyzed and the in- dividual members were plated on both TAP medium and minimal medium to determine photoautotrophic growth. Only one of the segregants (C4) is capable of growing on minimal medium. Additionally, transcript analyses were carried out using the psaA gene and the five isolated cDNAs as probes. As shown in Fig. 4, two of the progeny display the same expression pattern as their parents. C4 corresponds to the parental photoautotrophic strain and accumulates the nuclear transcripts as well as the mature psaA mRNA, while C1, like CC1051, is unable to accumulate neither the nuclear mRNAs nor the mature psaA transcript. In contrast, C2 and C3 both display a recombinant phenotype, i.e. they are splicing deficient but able to accumulate the nuclear transcripts encoding plastid polypeptides (PsaD, PsaF, PPE, Lhcbl, 7 kDa protein). We conclude that strain CC1051 carries at least two nuclear mutations; one, Mt8, affects the expression of the chloroplast psaA gene and the other affects the coregulated expression of nuclear genes encoding plastid proteins. This was further supported by the finding that two other strains carrying the single M18 mutation (P. Bennoun, unpublished) did not show the mature psaA mRNA, but accumulate the nuclear

I psa° I I B I p l s l

t PPE I [cDNA4"3 1

k b k b

- - 2 3 . 1 - - 2 3 . 1

- - 9 . 4 - - 9.4

- - 6 . 7 - - 6 ~ 7

- - 4 . 3 - - 4 , 3

- - 2 . 3

- - 2 . 0

- - 2 . 3

- - 2 . 0

Fig. 3 Southern blot analysis of genomic DNA from C. reinhardtii. Aliquots (50 lag) of DNA from the wild-type C. reinhardtii strain CC406 were digested with either SalI (S), PstI (P), or BamHI (B) and fractionated in 0.8% agarose gels, transferred to nylon membranes and then hybridized separately with cDNAs 68-3 (PsaD), cDNA 53-1 (PPE) and cDNA 4-3 (TkDa lumenal protein) under stringent conditions. Positions and sizes of DNA marker fragments from HindIII-digested phage Lambda DNA are indicated

icciclclclc cc 406 4 3 2 1 1051

Lhcbl

PsaF

PsaD

cDNA 4- 3

m

PPE

psaA

pMY60

Fig. 4 Northern blot analysis of four randomly selected meiotic products from a cross between CC1051 and CC406. Aliquots (25 lag) of total RNA from CC406, C1051, and each of the four meiotic segregants (C1 to C4) were fractionated on formaldehyde/agarose gels, transferred to nylon membranes and hybridized separately to the radiolabelled cDNA inserts (Lhcbl, PsaF, PsaD, cDNA 4-3, PPE) and radiolabelted chloroplast psaA DNA. pMY60, a rRNA- specific probe (Verbeet et al. 1983), was used as an internal control to quantify the amounts of RNA applied. The hybridization signals detected by the psaA probe correspond to the mature psaA mRNAs; unspliced precursors are not shown

transcripts encoding the above-mentioned plastid proteins (data not shown). The second mutated gene in strain CC1051 was named Cen (coregulated expression of nuclear genes).

Gene expression is effected at the post-transcriptional level in strain CC1051

The inability of strain CC1051 carrying the mutated Cen gene to accumulate mRNAs encoding plastid proteins could be the result of a defect in transcriptional and/or post-transcriptional regulation. To determine whether an alteration in the rate of transcription affects mRNA abundance, we have carried out run-on transcription assays using permeabilized wild-type and mutant cells (Gagn6 and Guertin 1992). In three independent experiments, the radioactively labelled transcripts from both wild-type and mutant cells were used to probe immobilized cDNA fragments specific for PsaD, PsaF, Lhcbl, PPE and for the 7kDa protein (cDNA 4-3). As a control, the synthesis of mRNAs encoding the ribosomal proteins $18 and $27 (Hahn and Kiick 1.995) was monitored. In Northern analyses, equal amounts of Rpsl8 and Rps27 transcripts can be detected in both wild-type and mutant cells (data not shown). As shown in Fig. 5, there was very little difference between the hybridization signals obtained from the wild-type and mutant cell-derived run-on products. This was con- firmed by quantitative measurement of the hybridization signals using an electronic image documentation system (data not shown). Although strain CC1051 fails to accumulate mRNAs encoding PsaD, PsaF, PPE, and the 7 kDa protein (Fig. 1), the corresponding genes seem to be transcribed at wild-type rates (Fig. 5). Only in case of the PsaF gene does the transcription rate seem to be reduced by about 8 to 10% compared to the rates detected for the other genes in strain CC1051.

Fig. 5 Analysis of radiolabelled transcripts produced in permeabilized wild-type and mutant cells. 5 x 107 cells, from both wild-type and mutant cultures, were permeabilized using a freeze-thaw procedure, and used in in vitro transcription assays. The radioactively labelled transcripts isolated were used to probe excess amounts of filter-immobilized cDNAs. Rps18, Rps27 (Hahn and Kfick 1995) and plasmid DNAs (pT3T7BM and M13mpl9) were used as controls

pT3TTBM

M13mp19

Rpsl8

Rps27

PPE

cDNA 4-3

PsaF

PsaD

Lhcbl

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However, this reduction cannot explain the lack of PsaF transcript accumulation in the mutant strain (Fig. 1). Hence, the nuclear factor missing in strain CCt051 is not required for the transcription of the genes investigated, but would seem to be involved in post-transcriptional events, such as RNA stabilization and/or RNA processing.

Discussion

Transcripts encoding a distinct set of plastid proteins do not accumulate in the non-photosynthetic strain CC1051

Several non-photosynthetic mutants of C. reinhardtii have been characterized in order to elucidate mechanisms of chloroplast gene expression (for review see Rochaix 1992a; Gillham et al. 1994; Mayfield et al. 1995).

In the present study we have used a differential screening strategy to investigate nuclear gene expression in photosynthetically deficient cells. We report the isolation of five cDNAs from a photoautotrophic C. reinhardtii strain for genes whose transcripts fail to accumulate in the non-photosynthetic strain CC1051. Besides the two cDNAs ( PsaF and Lhcbt) that have already been isolated from C. reinhar&ii (Imbault et al. 1988; Franz6n et al. 1989) we have identified three previously uncharacterized C. reinhar&ii cDNAs. One represents a completely novel cDNA (4-3) encoding a 7kDa protein with a lumen-targeting transit sequence, whereas the other two cDNAs encode polypeptides sharing similar sequences with other proteins (PsaD, PPE). Hence, four out of five cDNAs code for plastid proteins whose functions are specifically asso- ciated with photosynthesis and carbon assimilation. Although the function of the 7 kDa polypeptide encoded by cDNA 4-3 remains unknown, it is conceivable that it represents a novel small subunit of PSI. The number of non-pigment-binding PSI subunits found in C. reinhardtii and higher plants is still unknown. Recently, Knoetzel and Simpson (1993) reported the isolation of a novel PSI subunit (PsaN), as well as demonstrating that at least two other unknown protein bands are present among lower molecular weight species in a native PSI preparation from barley. Since we have isolated two other cDNAs encoding PSI subunits and in view of the fact that strain CC1051 lacks PSI, the 7kDa protein could represent a low-molecular-mass subunit of PSI that has not yet been isolated from either C. reinhardtii or any higher plant.

A nuclear factor coregulates nuclear genes encoding plastid proteins

Our results suggest that CC1051 carries a mutation in the nuclear Cen gene which is required in trans for the

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expression of at least five different nuclear genes each encoding a different plastid protein (Figs. 1,4). This mutation is unique among non-photosynthetic mutants of C. reinhardtii, for which only defects affecting single chloroplast RNAs have otherwise been described (for review see Rochaix 1992a). In maize, however, photosynthetic mutations are usually more pleiotropic and affect the synthesis of several subunits (Miles et al. 1979; Barkan et al. 1986, 1994). The nuclear mutation crpl, for example, causes the loss of the cyt b6/fcomplex, suggesting that one gene may function to integrate the expression of several genes coding for subunits of the same complex (Barkan et al. 1994). Uniform changes in levels of cytoplasmic mRNAs for thylakoid components have also been observed in spinach, when etiolated seedlings are transferred to high- intensity white light (Flieger et al. 1993). Since nuclear genes are transcribed independently of one another, regulatory mechanisms would seem to be necessary to coordinate expression of genes with related functions. Although the nature of the genetic defect in strain CC1051 remains elusive, our data indicate that we have identified a nuclear gene function that links the expression of several genes encoding proteins involved in both photosynthesis and carbon assimilation.

The nuclear-encoded factor missing in strain CC1051 may stabilize transcripts that code for plastid proteins

Run-on transcription experiments show that wild-type and mutant hybridization signals are more or less identical, demonstrating that the rate of transcription is not altered in the mutant. Thus, the mutation in the Cen gene of strain CC1051 affects the abundance of cytoplasmic transcripts encoding plastid polypeptides at the post-transcriptional level. Post-transcriptional mechanisms seem to play an important role in chloroplast gene expression (Gruissem and Schuster 1993; Rochaix 1992a). Several mutants have been isolated in C. reinhardtii and higher plants which were shown to be affected in chloroplast mRNA stability (Kuchka et al. 1989; Barkan et al. 1994; Monod et al. 1992). Recently, it has become evident that no distinction can be made between chloroplast components of nuclear and plastid origin, and that nuclear genes for plastid proteins can be regulated at various levels. The light-dependent accumulation of psaD and psaF transcripts in spinach, for instance, is regulated both transcriptionally and post- transcriptionally (Flieger et al. 1994). In tobacco, post- transcriptional mechanisms operating both in the cytosol and in the plastids are crucial for the biogenesis of the photosynthetic membrane (Herrmann et al. 1992; Palomares et al. 1993). A similar situation is found in Euglena gracilis, where post-transcriptional and post- translational mechanisms of plastid and nuclear gene control seem to be the main regulatory steps in thylakoid protein synthesis (Weiss et al. 1992). Gagn6

and Guertin (1992) have demonstrated that the L1818 gene, a member of the Lhc gene family, is regulated at the post-transcriptional level in C. reinhardtii. Taken together, it would appear that similar mechanisms regulate the levels ofplastid proteins in both C. reinhardtii and higher plants, irrespective of whether the plastid proteins are encoded in the chloroplast or nucleo-cytoplasmic compartments.

The biochemical basis for the loss of transcripts encoding plastid proteins in strain CC1051 carrying the Cen mutation is not known but is most simply ex- plained by a defect in mRNA stability. We assume that in the wild-type, a specific nuclear gene product stabil- izes the mRNAs which are absent from the mutant, thereby coordinating the expression of genes with related functions.

Changes in mRNA stability are believed to be mediated by interactions among specific cis-acting determinants and trans-acting factors. Sequence determinants that contribute to post-transcriptional control have been identified in transcripts from several eu- karyotes. Examples include the 5' untranslated regions (5' UTRs) found in the chloroplast psbD and rbcL transcripts from C. reinhardtii, as well as the 5' UTR found in the mitochondrial cyt b gene from Sacchar- omyces cerevisiae (Diekmann and Mittelmeier 1987; Salvador et at. 1993; Nickelsen et al. 1994). Sequence- specific motifs, such as the AU-rich elements commonly found in the 3' UTRs from mammalian lymphokine and protooncogene mRNAs, and the DST elements in the 3' UTRs from soybean and Arabidopsis SAUR (small auxin up RNAs) genes, can negatively affect the cytoplasmic stability of mRNA in plant cells (Newman et al. 1993; Ohme-Takagi et al. 1993). Several mechanisms can be envisioned whereby the nuclear-encoded factor missing in strain CC1051 could stabilize transcripts coding for plastid proteins. One possibility is that the nuclear-encoded factor could interact with sequence elements in the 3' UTRs of the transcripts investigated, protecting them from nuclease attack. This hypothesis is further supported by the finding that chimeric genes, containing the 5' UTR from the Lhcbl gene and the arylsulfatase coding region, are expressed in both wild-type and mutant cells (D. Hahn, unpublished results). However, a comparison of the 3' UTRs from the cDNAs isolated indicated that there are no obvious conserved sequence motifs. One could also imagine that the factor physically interacts with sec- ondary structures commonly found in the 3' UTRs thereby stabilizing the mRNA molecules. Most chloroplast transcription units, for instance, contain 3 r inverted repeats and some of these potential stem-loop structures have been identified as stability determinants in vitro and in vivo (Stern and Gruissem 1987; Stern et al. 1989, 1991). With regard to the data obtained with strain CC1051 it would be important to isolate and characterize the missing nuclear-encoded factor. This would help to elucidate its functional role, and will

369

provide insight into the mechanisms of gene expression in general. The recently established transformation system for C. reinhardtii (Debuchy et al. 1989; Kindle 1990) makes it feasible to isolate nuclear factors by complementing appropriate C. reinhardtii mutants.

Acknowledgements We thank Ingeborg Godehardt for excellent technical assistance, Hans Rathke for the artwork, and Dr. Leonard Stevenson for critical reading of the manuscript. DH was a member of the DFG graduate program "Biogenese und Mechanismen komplexer Zellfunktionen". Our work was supported by grants from the Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg, Germany) and by an EC grant to P.B. and U.K. within the Chtamydomonas Network.

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Altered expression of nuclear genes encoding chloroplast