Transcripts and sequence elements suggest differential ... - CiteSeerX

Ó Springer-Verlag 2000

Curr Genet (2000) 37: 45±52

ORIGINAL PAPER

Holger Summer á Thomas Pfannschmidt á Gerhard Link

Transcripts and sequence elements suggest differential promoter usage within the ycf3 -psaAB gene cluster on mustard (Sinapis alba L.) chloroplast DNA Received: 19 July / 8 October 1999

Abstract The mustard chloroplast DNA region spanning the ycf3 gene and part of the psaAB operon was investigated. The ycf3 gene reveals two class-II introns that are removed during processing to give a mature 0.7-kb transcript, but no RNA editing seems to be involved. RNase protection and RT-PCR experiments suggest cotranscription of ycf3 with the downstream psaA gene, possibly from a NEP promoter upstream of ycf3, whereas distinct ycf3 and psaA transcripts are each initiated from PEP promoters. This situation is reminiscent of that for the trnK-psbA gene region. The implications for light-regulated versus light-independent expression of photosystem core-protein genes are discussed. Key words Chloroplast tetratricopeptide protein gene á Complex transcription unit á Plastid promoter mapping á Ycf3-psaAB gene cluster

Introduction Chloroplasts play an important role in plant cells both as the sites of photosynthesis and as important anabolic pathways. Many of the photosynthesis-related proteins, as well as components of the plastid gene-expression apparatus, are known to be encoded by chloroplast genes (Sugiura 1992) and complete sequences of plastid

This paper is dedicated to Professor Gerhard Richter on the occasion of his 70th birthday Communicated by A. Brennicke H. Summer á T. Pfannschmidt1 á G. Link (&) Arbeitsgruppe P¯anzliche Zellphysiologie und Molekularbiologie, Ruhr-UniversitaÈt Bochum, D-44780 Bochum, Germany e-mail: [email protected] Tel.: +49-234-322 5495 Fax: +49-234-3214 188 Present address: 1 Institut fuÈr Allgemeine Botanik, Abteilung P¯anzenphysiologie, Friedrich-Schiller-UniversitaÈt, D-07743 Jena, Germany

genomes from a number of higher-plant species are now available (Shinozaki et al. 1986; Maier et al. 1995). Nevertheless, the function of a number of conserved open reading frames (ORFs) remains to be established. Most of these enigmatic ORFs termed ycfs (hypothetical chloroplast open reading frames) (Hallick and Bairoch 1994) seem to be restricted to certain groups of organisms, but a few are present in all the chloroplast genomes that have so far been sequenced. For instance, ycf3 was found to be conserved among higher plants (Hiratsuka et al. 1989; McCullough et al. 1991) as well as in algae (Hallick et al. 1993; Stirewalt et al. 1995; Wagasuki et al. 1997) and cyanobacteria (VoÈroÈs et al. 1992). Its gene product was recently shown to be required for the assembly and/or stability of photosystem I (PSI) (Boudreau et al. 1997; Ruf et al. 1997), although the exact role of YCF3 in this processes remains to be elucidated. In all known plastid genomes of higher plants the ycf3 gene is located in front of the psaA-psaB-rps14 gene cluster (encoding the two core apoproteins of PSI and ribosomal protein 14 of the small subunit). Therefore the close physical proximity of ycf3 to the psaA/B genes could mean that common gene regulatory mechanisms might exist, which are involved in the balanced production of functional PSI reaction-center proteins. Studies on the expression characteristics of ycf3 in higher plants have until now been restricted to monocots, i.e. maize (McCullough et al. 1991; Ruf and KoÈssel 1997), rice (Kanno and Hirai 1993) and barley (Hess et al. 1994). In all of these species the ycf3 gene region revealed multiple transcripts ranging in size from 9.5 to 0.58 kb. This highly complex pattern made it dicult to analyse the tissue-speci®c or developmental accumulation of the mature processed transcript(s). For instance, di€erences in ycf3 transcript sizes between leaves and endosperm were initially reported to exist in maize (McCullough et al. 1991). In contrast, in a more recent study on maize only very small di€erences between the transcript patterns of various tissues were detected (Ruf and KoÈssel 1997). Small di€erences in transcript sizes

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were also observed between green and white tissues of the barley ``albostrians'' mutant (Hess et al. 1994). Here we report on the structural organization and expression of the ycf3-psaA region from mustard (Sinapis alba L.), a dicotyledonous crucifer. We have established the mustard ycf3 nucleotide sequence in order to provide a basis for a comparative analysis of the derived amino-acid sequence, exon/intron organization, and the putative transcription start and stop signals. Furthermore, we have investigated ycf3 gene expression at the RNA level during mustard seedling development. Transcript mapping by primer-extension and RNaseprotection techniques provided clues to sequence elements that might serve as signals for transcription and/ or processing events. The results are discussed in relation to ®ndings on the same genes in di€erent plant species, and to other complex transcription units on mustard chloroplast DNA.

Materials and methods Plant material and plasmids Mustard seedlings (cv ``Albatros'') were grown on moist ®lter paper in plastic boxes at 25 °C either in continuous darkness or under continuous white light from ¯uorescent lamps (40 á lmol á m)2 á s)1). Cotyledons were harvested at 24-h intervals after germination, immediately frozen and then stored at )80 °C until further use. Plasmid pSA244 has been described previously (Dietrich and Link 1985). Plasmid pSA244/1375B (Fig. 1, probe b) contains a 1375-bp BamHI subfragment of pSA244. Plasmid pSA244/500 EB (Fig. 1, probe a) is a 0.5-kb EcoRI/BamHI subfragment of pSA244 located upstream of pSA244/1375B. Plasmid pSA244/560BH is a 0.56-kb BamHI/HincII subfragment of pSA244/1375B (Fig. 1, probe c). Plasmid pSAYCF3 contains the processed intron-less ycf3 gene generated by RT-PCR (AMV-RT, Promega) (Ausubel et al. 1990). Primers HS1, 5¢-CTGAGCTCGCCAAGATCGCGTAT-3¢, and HS2, 5¢-GCGTCGACTTCGAAACG CCTTGTG-3¢, are complementary to the 5¢ end of exon 1 (HS1) and the 3¢ end of exon 3 (HS2). Both primers were constructed to contain sequences also recognized by the restriction enzymes SacI (HS1) and SalI (HS2).

DNA sequencing To obtain the ycf3 sequence, the inserts of pSA244/500EB and pSA244/1375B were sequenced by the dideoxy chain-termination method (Sanger et al. 1977). A 32P-labelled RNA probe was synthesized by transcription of the EcoRI-linearized plasmid pSA244/ 500 EB with T7 RNA polymerase (Melton et al. 1984). This probe was used in Southern-hybridization experiments (Southern 1975) of BamHI-restricted mustard chloroplast DNA for detection of the exon 1 region of the ycf3 gene. A 1.8-kb fragment covering exon 1, intron 1 and exon 2 was isolated, cloned and subsequently sequenced. Chloroplast DNA was isolated as previously described (Dietrich et al. 1987). Isolation of RNA, primer extension and Rnase protection Total RNA of cotyledons was isolated as previously described (Hughes et al. 1987). Chloroplast RNA from 5-day old light-grown seedlings was isolated from intact chloroplasts fractionated on sucrose gradients (Reiss and Link 1985) using the same procedure as for total RNA. Oligonucleotides HS4 (5¢-CATTTATACGCGATCTTGGC-3¢) and HS8 (5¢-TCCGGCGAACGAATAATC3¢), which are complementary to the 5¢end of exon 1 of ycf3 (HS4) and the 5¢end of psaA (HS8), were used in primer-extension experiments. The primers were labelled with c-32P-ATP at the 5¢ end using T4 polynucleotide kinase (Sambrook et al. 1989). Ten micrograms of total RNA or chloroplast RNA were incubated in the presence of 5 pmol of labelled primer for 20 min at 56 °C, followed by a standard reverse transcription reaction with AMV-RT at 48 °C according to the manufacturer's protocol (Promega). The reaction was stopped and the products were electrophoresed on 6% denaturing urea sequencing gels at 25 mA. The gels were dried and auto-radiographed. RNase protection mapping of in vivo RNA was performed with 10 lg of chloroplast RNA and 32P-labelled in vitro synthesized cRNA of linearized plasmid pSA244/1375B using T7 RNA polymerase (Sambrook et al. 1989). Hybridization was carried out at 47 °C for 16 h. The plasmid covers exon 3 of ycf3, the intergenic region, and 52 bp of psaA. RT-PCR and PCR Chloroplast RNA was reverse-transcribed with primer HS8 and AMV-RT (Promega). Chloroplast DNAs and cDNAs were ampli®ed by a standard protocol in the presence of 6 mM of MgCl2, using 30 cycles at 94 °C (1 min), 54 °C (1 min) and 72 °C (1.5 min). A 4-min extension at 94 °C was added to the ®rst cycle and a 5-min extension at 72 °C to the last cycle. Ampli®cation products were electrophoretically separated on a 0.7% agarose gel and visualized by ethidium bromide staining. The primers used in PCR were HS7 (5¢-ACTCCTGGTAATTATATTG-3) and HS3 (5¢-GGATGTCGGCTCAATCTGAAGG-3¢). Northern analysis

Fig. 1 Schematic representation of the rps4-ycf3-psaA-psaB-rps14 gene region on mustard chloroplast DNA. Filled boxes coding regions; open boxes introns. Arrows position and orientation of primers used for RT-PCR and primer extension. Positions of probes used for sequencing, Northern-hybridization and RNase-protection mapping are given at the bottom. Numbers below arrows are sizes in bp

RNA (10 lg) of mustard cotyledons was heat-denatured, separated electrophoretically on a 1.7% (w/v) agarose gel containing 19% (v/ v) formaldehyde and transferred to nitrocellulose membranes (Schleicher and SchuÈll, BAS-85) using standard procedures (Ausubel et al. 1990). Blots shown in Fig. 3A were hybridized with 32 P-labelled RNA probes prepared by the transcription of SacIlinearized plasmid pSAYCF3 with T3 RNA polymerase (Melton et al. 1984). All hybridization steps were carried out as previously described (Hughes et al. 1987). Blots in Fig. 3B were hybridized with DIG-labelled antisense RNA synthesized by the transcription of linearized plasmids pSA244/500BE (probe a) and pSA244/ 560BH (probe c) either with T7 or T3 RNA polymerase (Promega) and NTP-labelling mixture (Boehringer Mannheim). Hybridisation at 68 °C, washing, and luminescent detection of DIG-labelled

47 RNA were carried out according to the manufacturer's instructions (Boehringer Mannheim).

Results Features of the mustard ycf3 gene and its derived protein We established the gene structure of chloroplast ycf3 from mustard by sequencing a 3.2-kb region upstream of, and extending into, the psaA-psaB-rps14 operon (EMBL accession no. AJ242660). The ycf3 gene was found to be arranged in three exons (126, 228 and 150 bp respectively) interspersed by two introns (723 bp and 738 bp). The ycf3 coding region within the 3.2-kb fragment is ¯anked by a 455-bp upstream sequence and is followed by an intergenic region of 762 bp and 52 bp of the psaA gene sequence (Fig. 1). These data are in agreement with the conserved organization of chloroplast genes from other higher plants (Shimada and Sugiura 1991). The precise positions of exon/intron borders were identi®ed by sequencing of an intron-less RT-PCR product of ycf3 (504 bp in size). Comparison to the genomic sequences revealed that apparently no RNA editing occurs within this region in the dicot plant mustard (data not shown), in contrast to previous results obtained for ycf3 of the monocot plant maize (Ruf et al. 1994 ; Ruf and KoÈssel 1997). The analysis of the deduced amino-acid sequence (168 residues) shows three tetratricopeptide repeat (TPR) sequence elements (Goebl et al. 1991), which is a conserved YCF3 feature initially noted for Chlamydomonas (Boudreau et al. 1997) (Fig. 2).

Ycf3 is expressed in a light-independent manner during seedling development Accumulation of ycf3 transcripts during mustard seedling development (24±96 h after sowing) was detected in Northern analyses using the intronless RT-PCR product as a probe. Using total RNA (Fig. 3), only one prominent hybridization signal of 0.7 kb was observed with increasing intensity during seedling development and without signi®cant di€erence in intensity between darkand light-grown seedlings. Using the chloroplast RNA speci®c probes ``a'' (mainly exon 2) and ``c'' (mainly exon 3), however, further distinct transcripts in addition to the 0.7-kb signal became visible (Fig. 3B). These larger transcripts were likely to represent larger processing intermediates of ycf3 that contain intron sequences as well as additional 5¢ and/or 3¢ sequences, perhaps extending into the ¯anking rps4 and psaA genes. Fig. 2 Alignment of deduced amino-acid sequences for YCF3. Alignment of the mustard (S.a.) sequence with those from Arabidopsis (A.th., Kaneko and Tabata 1999), tobacco (N.t., Shinozaki et al. 1986), maize (Z.m., Maier et al. 1995), rice (O.s., Hiratsuka et al. 1989), black pine (P.t., Wagasuki et al. 1994), liverwort (M.p., Ohyama et al. 1986), Clamydomonas reinhardtii (C.r., Boudreau et al. 1997), Chlorella vulgaris (C.v., Wagasuki et al. 1997), Synechocystis PCC6803 (Syn., VoroÈs et al. 1992), Porphyra purpurea (P.p., Reith and Munholland 1995), Cyanophora paradoxa (C.p., Stirewalt et al. 1995), Cyanidium caldarium (C.c., AF022186), Guillardia theta (G.t., Douglas and Penny 1999), and Odontella sinensis (O.s., Kowallik et al. 1995). Regions representing TPR domains are indicated by bars above the alignment. The TPR consensus sequence according to Lamb et al. (1995) is given on top. An asterisk indicates identical amino acids for all proteins. The relative position of amino acids is given at the end of each line. In brackets the percent sequence similarity compared to the mustard sequence is given

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Fig. 3 A,B RNA gel-blot hybridization analysis of ycf3 transcripts. A upper panel 10 lg of total RNA were loaded on each lane of a denaturing 1.7% agarose gel containing formaldehyde. Following electrophoresis and Northern transfer to nitrocellulose, the blot was hybridized with an intron-less ycf3 RNA probe prepared by in vitro trancription. Lower panel ethidium bromide-stained agarose gel. Arrow position of the 0.7-kb transcript. B Northern analysis with probes ``a'' and ``c'' (see Fig 1). Left panel hybridization signals. Right panel corresponding ethidium bromide-stained lanes. Probes used for hybridization are indicated below. The arrow marks the 0.7-kb signal as in A. The asterisk marks putative ycf3-psaA cotranscripts. 120 h cpRNA chloroplast RNA from seedlings grown for 120 h under continuous light. 72 h light total RNA from 72-h light-grown seedlings

Prokaryotic-type promoter elements are found upstream of the putative transcription start sites of ycf3 and psaA To locate putative in vivo transcription start sites of mustard ycf3 and psaA, primer-extension experiments were carried out with primers complementary to the 5¢ coding regions of ycf3 exon 1 and of psaA, respectively. As shown in Fig. 4A for ycf3, a major extension product is visible at about 110 bases upstream of the translation start (Fig. 4A, lanes 5±12). Using chloroplast RNA in-

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stead of whole-cell RNA, additional signals are visible, which might re¯ect transcription start sites 189 and 239 bp upstream of the translation start (Fig. 4A, lane 16). Directly in front of these sites are sequence motifs that conform to the consensus sequence for NEP (nuclear-encoded polymerase) class Ia and Ib promoter elements (Liere and Maliga 1999; Weihe and BoÈrner 1999) (Fig. 4C). In the case of psaA, one signal re¯ecting an RNA-end 187 bp upstream of the coding sequence was found (Fig. 4B, lane 5). Sequence motifs resembling the `-10' and `-35' elements of eubacterial sigma-70 promoters (Kung and Lin 1985) were detected in front of the mapped 5¢ RNA ends of both genes, which suggest a role as putative transcription initiation sites for these regions. The motifs TATAAA (-10-like) and TTGGAA (-35-like) are present upstream of ycf3 (Fig. 4A) with similar motifs, TAATAG and TTGAGC, upstream of the psaA gene (Fig. 4B). The changes in relative signal intensity of the ycf3 extension product, as observed with RNA from light- vs dark-grown and old- vs young-seedlings (Fig. 4A, 5±12), are consistent with the results of the Northern analysis (Fig. 3A). Furthermore, when using the mustard ycf3 primer (HS4) and 10 lg of total RNA of the related crucifers Arabidopsis thaliana and Brassica napus (Fig. 4, lanes 13, 14), the primer extension experiments resulted in products showing the same sizes as those found in mustard. Evidence for cotranscription of the mustard ycf3 and psaA sequences The Northern analysis shown in Fig. 3B suggested the possible existence of rps4-ycf3 and psaA-psaB-rps14 cotranscripts. To check for the presence of transcripts that span the ycf3-psaA intergenic region, RNase protection and RT-PCR experiments were carried out. RNase mapping (Fig. 5) revealed several protected fragments with sizes ranging from approximately 50 to 1000 bp (Fig. 5A and B). The major protected fragment ``b'' has b Fig. 4 A,B Determination of 5¢ transcript ends of the ycf3 and psaA genes by primer-extension analyses. A primer extension experiments with dark (lanes 5±8)- and light (lanes 9±12)- grown mustard seedlings and primer HS4. Total RNA (lanes 5±12) and chloroplast (cp) RNA (lane 16). Lanes 1±4 cDNA sequencing products obtained with the same primer. Lanes 13 and 14 products obtained with primer HS4 for total RNA from 96-h B. napus and A. thaliana seedlings, respectively. Lane 15 control without RNA template. Asterisk in lane 4 putative (PEP) transcription start site. Left margin DNA sequence with motifs resembling `-10' and `-35' promoter elements (boxed). Right margin positions of DNA size markers (pBR322/HinfI fragments). Transcripts arising from putative NEP promoters (PNEP-Ia at position 239, PNEP-Ib at position 189) are indicated in lane 16. B primer extension experiments with 10 lg of chloroplast RNA from 5-day old seedlings (lane 5) with primer HS 8. Lanes 1±4 show cDNA sequencing products obtained with the same primer. Left margin `-10' and `-35' sequence region. C ycf3 5¢ end sequence covering the two putative NEP-like promoters. Elements that conform to the consensus sequences of NEP Ia and Ib promoters are marked bold-face; positions of the mapped RNA ends are underlined

Fig. 5 A,B RNase protection mapping of in vivo transcripts within the ycf3 3¢ - psaA 5¢ gene region. A following RNA-RNA hybridization and treatment with RNase, resistant products were separated on a 6% denaturing polyacrylamide gel. A labelled in vitro transcript that covers the region ``b'' (see Figs. 1 and 5B) was used as a probe. Left margin sizes (bp) of the end-labelled DNA size marker (pBR322/HinfI) in lane 1. Protected RNA species are shown in lane 2. Right margin arrows point to the major protected RNA fragments a, b, c, and d, each of which matches one of the regions speci®ed in B. Asterisks denote minor products that may have resulted from incomplete digestion and/or degradation. B schematic drawing of the region represented by hybridization probe ``b'' in A, which depicts the organization of the ycf3 3¢ end and its ¯anking downstream region including the 5¢end of psaA. Open box part of ycf3 intron 2 (452 bp); single-lined intergenic ycf3-psaA region (762 bp); ®lled boxes ycf3 exon 3 (150 bp) and part of psaA coding region (®rst 52 bp). A potential stem-loop structure and prokaryotic-type promoter elements within the intergenic region are also indicated (top portion). Bottom fragments (horizontal arrows) corresponding to the hybridization signals a±d in A, with their sizes given on the right

a length of 310 bp, which might include 150 bp of exon 3 and 160 bp of the 3¢-untranslated region of the ycf3 mRNA. Fragment ``c'' with a size of approximately 230 bp (Fig. 5A and B) matches 52 bp of the 5¢ psaA coding sequence (represented by product ``d'') and 187 bp of its untranslated region as shown in the primer extension analysis (Fig. 4B, lane 5). Product ``a'' (Fig. 5A and B) is approximately 1000 bp in length, which is the size of potential readtrough transcripts of ycf3 and the adjacent psaA region. The less-abundant

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bands marked by asterisks are possibly products generated by incomplete RNase digestion and/or degradation, although they could also represent additional, less frequent, in vivo transcript ends. To con®rm the principal conclusions drawn from the RNase protection data, RT-PCR experiments were carried out (Fig. 6) using total RNA as a template for cDNA synthesis with a primer complementary to the 5¢ coding region of the psaA gene (HS8). Gene-speci®c expression products were then ampli®ed by a combination of HS8 with exon-speci®c ycf3 primers (HS1, HS3 and HS7). The resulting PCR ampli®cation products show sizes expected for read-through transcripts (Fig. 6 A,B lanes 3±8). The usage of chloroplast DNA versus cDNA also showed the correct splicing of the two introns of ycf3 primary transcripts. Taken together, this clearly indicates cotranscription of the ycf3 and psaA sequences (Fig. 7).

Discussion In this work we have focused on the expression of the S. alba chloroplast DNA region containing the ycf3

Fig. 6 A,B Evidence for ycf3-psaA cotranscription by RT-PCR. Products obtained by PCR using di€erent primer pairs. A ampli®cation was carried out either with the cDNA obtained by reverse transcription of mustard chloroplast RNA with HS8 or with chloroplast DNA. Left margin size marker. Top primer combination and used template source. B schematic representation of the ycf3-psaA gene region. Lines intergenic regions, open boxes introns, ®lled boxes exons of ycf3 and coding sequence of psaA-psaB-rps14. Arrows indicate the position and orientation of primers. Products and expected sizes for each primer combination are presented below

Fig. 7 Schematic representation of promoters and transcripts within the ycf3-psaA region on mustard chloroplast DNA. Filled boxes ycf3 and psaA genes. Line intergenic regions. Arrows transcripts

gene upstream of the psaA-psaB-rps14 operon. The organization of the ycf3 gene from mustard is highly conserved with respect to known sequences from other plants. Previous studies on ycf3 in the monocot plants maize, rice and barley (McCullough et al. 1991; Kanno and Hirai 1993; Hess et al. 1994) had shown that the primary transcript undergoes a series of RNA maturation steps. Using various intron- and exon-speci®c probes, about ten di€erent transcripts with sizes ranging from 9.5 to 0.58 kb were reported and the size of the mature RNA was estimated to be 0.95±0.58 kb. In maize, ycf3 RNA editing was detected and found to restore two conserved amino-acid residues (Ruf and KoÈssel 1997). In our analysis of the dicot plant mustard we did not obtain any sequence evidence supporting the possibility that RNA editing might play a role in ycf3 gene expression. Using an intron-less ycf3 probe and total RNA, only one major transcript of 0.7 kb was visible in Northern experiments (Fig. 3A). Larger-sized ycf3 transcripts representing putative processing intermediates were, however, detectable by using chloroplast RNA and probes that included intron sequences (Fig. 3B). The intron-less ycf3 probe hence allowed us to speci®cally investigate the accumulation of the mature ycf3 transcript during seedling development. Northernand primer-extension experiments (Figs. 3 and 4) revealed a light-independent but developmental stagedependent mode of RNA accumulation. Furthermore the results of the RT-PCR experiments (Fig. 6) were consistent with the cotranscription of ycf3 with sequences of the downstream psaA-psaB-rps14 operon. Large transcripts within a size range that would be expected for those that contain extensive downstream sequences were also detected by Northern analysis (Fig. 3B; 120 h cpRNA; see asterisk). In order to locate possible cis-elements that may be involved in ycf3 transcription initiation, we mapped the 5¢- and 3¢-ends of the ycf3 mRNA (Figs. 4 and 5). In front of the putative main transcription start site we found typical prokaryotic-like `-35' and `-10' promoter elements (Kung and Lin 1985; Link 1994), suggesting that the major ycf3 transcript initiates from a typical PEP promoter (Maliga 1998). In addition, we detected minor RNA ends further upstream at distances of 189 and 239 bp from the translation start (Fig. 4A, lane 16). In front of these sites are sequence elements, PNEP-Ia and PNEP-Ib (Fig. 4C), which conform to the consensus architecture of class Ia and Ib NEP promoters (Weihe and BoÈrner 1999). We note, however, that despite the pres-

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ence of putative promoter elements the possibility cannot be excluded that some of the detected 5¢ ends of ycf3 transcripts were created by RNA processing. Maturation of the di€erentially initiated ycf3 transcripts might take place at their 3¢-end at a stable stemloop structure (+83 to +111 from stop codon), which we identi®ed behind the putative 3¢-processing site. This structure could act as an accumulation and processing signal (Rochaix 1996; Rott et al. 1998). The putative transcription start site in front of the psaA gene was found to be located 187 bp upstream of the coding region (Fig. 4B). Like the major RNA end in front of ycf3 (Fig. 4A), it is also preceded by prokaryotic-like `-10' and `-35' elements similar to those described for psaA in maize (Fish et al. 1985), rice (Chen et al. 1993) and barley (Berends et al. 1987). The expression characteristics of ycf3, together with the exon-intron structure and location in front of a photosynthetic core-protein gene (psaA), are very similar to those of the trnK gene from mustard (Hughes et al. 1987). The latter also contains an intron, is light-independently transcribed, and shows cotranscription with the downstream psbA gene (Nickelsen and Link 1991). In addition, cis-elements identi®ed in the ycf3/psaA gene region have structural and functional homologues within the trnK/ psbA gene region, including the conserved NEP Ib promoter that can be identi®ed in front of trnK (at position 210±212/195±197 with regard to the coding region). Both the psaA and the psbA genes possess their own (Escherichia coli-like) promoters, from which transcription is initiated in a light-dependent manner (Dietrich et al. 1987; Hughes et al. 1987, this work). It is tempting to speculate that the presence of a NEP promoter may enable cotranscription of ycf3 and psaA as well as the trnK and psbA genes at a basal level, whereas the major transcripts arise from the prokaryotic-like PEP promoters of ycf3 and psaA. Light-independent cotranscription with a 5¢-upstream non-photosynthetic gene instead of, or in combination with, light-regulated expression from its own promoter could be a possible mechanism for the expression of photosynthetic core proteins under variable environmental in¯uences. In addition such a mechanism would allow the expression of chloroplast genes lacking a NEP promoter by the NEP, at least at a low level, and may help to explain the existence of such transcripts in plant cells containing ribosome-free plastids (Hess et al. 1993). The deduced amino-acid sequence of the mustard ycf3 gene shows three TPR (tetratricopeptide repeat) motifs. TPRs are degenerated 34 amino-acid repeats capable of forming antiparallel folds (Das et al. 1998), resulting in the interaction of two subdomains termed `knob' and `hole' (Goebl and Yanagida 1991). These repeats are thought to play a role in protein-protein interaction, as was shown, e.g., for HSP90 (OwensGrillo et al. 1996). This would be consistent with the proposed function of the YCF3 protein as a factor for the assembly and/or stability of photosystem I (Boudreau et al. 1997; Ruf et al. 1997).

Acknowledgements We thank Sacha Baginsky and Karsten Ogrzewalla for helpful discussion. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, Germany.

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