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Chloroplast mRNA maturation is catalyzed by nucleus-encoded processing enzymes. We previously described a recessive nuclear mutation (crp3) that affects 3'-end formation of several chloroplast mRNAs in Chlamydomonas reinhardtii (Levy, H., Kindle, K. L., and Stern, D. B. (1997) Plant Cell 9, 825-836). In the crp3 background, atpB mRNA lacking a 3'-inverted repeat normally required for stability accumulates as a discrete transcript. The mutation also affects the atpA gene cluster; polycistronic mRNAs with psbI or cemA 3'-ends accumulate to a lower level in the crp3 background. Here, we demonstrate that the crp3 mutation also alters 3'-end formation of psbI mRNA and cemA-containing mRNAs. A novel 3'-end is formed in monocistronic psbI transcripts, and this is the only terminus observed when the psbI 3'-untranslated region is fused to an aadA reporter gene. Accumulation of mRNAs with 3'-ends between cemA and atpH, which is immediately downstream, was reduced. However, this sequence was not recognized as a 3'-end formation element in chimeric genes. The crp3 mutation was able to confer stability to three different atpB 3'-stem-loop-disrupting mutations that lack sequence similarity, but are located at a similar distance from the translation termination codon. We propose that the wild-type CRP3 gene product is part of the general 3' --> 5' processing machinery.

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Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

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Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5' and 3' untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5'-->3' and 3'-->5' exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions -20, +25, +165 or +25/+165 relative to the mature petD 5' end, mRNAs accumulate with 5' ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5' end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5'-->3' exoribonucleolytic activity. In the case of the -20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5' end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5' ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) -20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.

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Nucleus-encoded chloroplast proteins of vascular plants are synthesized as precursors and targeted to the chloroplast by stroma-targeting domains in N-terminal transit peptides. Transit peptides in Chlamydomonas reinhardtii are considerably shorter than those in vascular plants, and their stroma-targeting domains have similarities to both mitochondrial and chloroplast targeting sequences. To examine Chlamydomonas transit peptide function in vivo, deletions were introduced into the transit peptide coding region of the petE gene, which encodes the thylakoid lumen protein plastocyanin (PC). The mutant petE genes were introduced into a plastocyanin-deficient Chlamydomonas strain, and transformants that accumulated petE mRNA were analyzed for PC accumulation. The most profound defects were observed with deletions at the N-terminus and those that extended into the hydrophobic region in the C-terminal half of the transit peptide. PC precursors were detected among pulse-labeled proteins in transformants with N-terminal deletions, suggesting that these precursors cannot be imported and are degraded in the cytosol. Intermediate PC species were observed in a transformant deleted for part of the hydrophobic region, suggesting that this protein is defective in lumen translocation and/or processing. Thus, despite its shorter length, the bipartite nature of the Chlamydomonas PC transit peptide appears similar to that of lumen-targeted proteins in vascular plants. Analysis of the synthesis, stability, and accumulation of PC species in transformants bearing deletions in the stroma-targeting domain suggests that specific regions probably have distinct roles in vivo.

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The acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5' untranslated region (UTR). However, when this 5' UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5' end of the petD 5' UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5' UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5'-->3' exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5' UTR protects the mRNA from 5'-->3' degradation in wild-type cells.

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FUD6 is a non-photosynthetic Chlamydomonas mutant that lacks the cytochrome b6/f complex, due to a 236 bp deletion that removes the promoter and part of the 5' untranslated region (UTR) of the chloroplast petD gene, which encodes subunit IV of the complex. Two photosynthetic revertants of FUD6 that synthesized wild-type levels of subunit IV were found to contain related inversions of the chloroplast genome that resulted from recombination between small inverted repeats. These inversions created a functional chimeric petD gene that includes the promoter and part of the 5' UTR of the newly identified ycf9-psbM transciption unit, fused to the petD 5' UTR upstream of the FUD6 deletion. Accumulation of the ycf9-psbM dicistronic transcript was disrupted in the revertants, but monocistronic psbM mRNA accumulated normally. The FUD6 revertants demonstrate the ability of the Chlamydomonas chloroplast genome to undergo a large inversion without a deleterious effect on chloroplast function, reminiscent of events that have led to the evolutionary divergence of chloroplast genomes.

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Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the alpha-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-alpha can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter.

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We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the gamma-subunit of chloroplast ATPase-coupling factor 1 (CF1-gamma, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-gamma accumulation in vivo. The CF1-gamma transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-gamma.

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Nucleus-encoded chloroplast proteins that reside in the thylakoid lumen are synthesized as precursors with bipartite transit peptides that contain information for uptake and intra-chloroplast localization. We have begun to apply the superb molecular and genetic attributes of Chlamydomonas to study chloroplast protein import by creating a series of deletions in the transit peptide of plastocyanin and determining their effects on translocation into isolated Chlamydomonas chloroplasts. Most N-terminal mutations dramatically inhibited in vitro import, whereas replacement with a transit peptide from the gamma-subunit of chloroplast ATPase restored uptake. Thus, the N-terminal region has stroma-targeting function. Deletions within the C-terminal portion of the transit peptide resulted in the appearance of import intermediates, suggesting that this region is required for lumen translocation and processing. Thus, despite its short length and predicted structural differences, the Chlamydomonas plastocyanin transit peptide has functional domains similar to those of vascular plants. Similar mutations have been analyzed in vivo by transforming altered genes into a mutant defective at the plastocyanin locus (K. L. Kindle, manuscript in preparation). Most mutations affected in vitro import more severely than plastocyanin accumulation in vivo. One exception was a deletion that removed residues 2-8, which nearly eliminated in vivo accumulation but had a modest effect in vitro. We suggest that this mutant precursor may not compete successfully with other proteins for the translocation pathway in vivo. Apparently, in vivo and in vitro analyses reveal different aspects of chloroplast protein biogenesis.

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The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.

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We previously created and analyzed a Chlamydomonas reinhardtii strain, [delta]26, in which an inverted repeat in the 3[prime] untranslated region of the chloroplast atpB gene was deleted. In this strain, atpB transcripts are unstable and heterogeneous in size, and growth is poor under conditions in which photosynthesis is required. Spontaneous suppressor mutations that allow rapid photosynthetic growth have been identified. One strain, [delta]26S, retains the atpB deletion yet accumulates a discrete and stable atpB transcript as a consequence of a recessive nuclear mutation. Unlike previously isolated Chlamydomonas nuclear mutations that affect chloroplast mRNA accumulation, the mutation in [delta]26S affects several chloroplast transcripts. For example, in the atpA gene cluster, the relative abundance of several messages was altered in a manner consistent with inefficient mRNA 3[prime] end processing. Furthermore, [delta]26S cells accumulated novel transcripts with 3[prime] termini in the petD-trnR intergenic region. These transcripts are potential intermediates in 3[prime] end processing. In contrast, no alterations were detected for petD, atpA, or atpB mRNA 5[prime] ends; neither were there gross alterations detected for several other mRNAs, including the wild-type atpB transcript. We suggest that the gene identified by the suppressor mutation encodes a product involved in the processing of monocistronic and polycistronic messages.

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Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin (oriA). Transformants were recovered only with the plasmid containing oriA, and all transformants contained an integrated plasmid copy at oriA, suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.

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A suppressor of a translation initiation defect caused by an AUG to AUU mutation in the Chlamydomonas reinhardtii chloroplast petD gene was isolated, defining a nuclear locus that we have named SIM30. A dominant mutant allele at this locus, sim30-1d, was found to increase the translation initiation rate of the mutant petD mRNA. sim30-1d was also able to suppress the translational defect caused by an AUG to AUC mutation in the petD gene, and an AUG to AUU mutation in the chloroplast petA gene. We therefore suggest that the SIM30 gene may encode a general chloroplast translation factor. The ability of sim30-1d to suppress the petD AUG to AUU mutation is diminished in the presence of one or more antibiotic resistance markers located within the 16S and 23S rRNAs, suggesting that the activity of the sim30-1d gene product in translation initiation may involve interaction with ribosomal subunits.

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Nuclear genes play important regulatory roles in the biogenesis of the photosynthetic apparatus of eukaryotic cells by encoding factors that control steps ranging from chloroplast gene transcription to post-translational processes. However, the identities of these genes and the mechanisms by which they govern these processes are largely unknown. By using glass bead-mediated transformation to generate insertional mutations in the nuclear genome of Chlamydomonas reinhardtii, we have generated four mutants that are defective in the accumulation of the cytochrome b6f complex. One of them, strain abf3, also fails to accumulate holocytochrome c6. We have isolated a gene, Ccs1, from a C. reinhardtii genomic library that complements both the cytochrome b6f and cytochrome c6 deficiencies in abf3. The predicted protein product displays significant identity with Ycf44 from the brown alga Odontella sinensis, the red alga Porphyra purpurea, and the cyanobacterium Synechocystis strain PCC 6803 (25-33% identity). In addition, we note limited sequence similarity with ResB of Bacillus subtilis and an open reading frame in a homologous operon in Mycobacterium leprae (11-12% identity). On the basis of the pleiotropic c-type cytochrome deficiency in the ccs1 mutant, the predicted plastid localization of the protein, and its relationship to candidate cytochrome biosynthesis proteins in Gram-positive bacteria, we conclude that Ccs1 encodes a protein that is required for chloroplast c-type holocytochrome formation.

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The small gene family encoding the chlorophyll a/b-binding proteins of photosystem II (CABII or lhcb) is known to exhibit circadian rhythms of mRNA abundance in Chlamydomonas reinhardtii. In this study we investigated the role of transcription in the phenomenon. We used as reporters Chlamydomonas genes that encode nitrate reductase (NITI) and arylsulfatase (ARS2) transcriptionally fused to sequences upstream of one of the CABII genes (called CABII-1). We found that both reporters exhibited the same circadian rhythm of mRNA abundance in phase, period, and amplitude as does the endogenous CABII-1 gene. We also evaluated the efficacy of arylsulfatase enzymatic activity as a reporter and found that its half-life is too long to make it a useful reporter of rhythmic transcription during a circadian or diurnal cycle. The amount of mRNA synthesis from the CABII-1 gene was examined by in vivo labeling experiments and a circadian rhythm in transcription rate was demonstrated. In vivo labeling also revealed a circadian rhythm of mRNA synthesis for the CABII gene family as a whole. The results from the transcriptional reporter assays together with the in vivo labeling experiments strongly support the conclusion that the biological clock regulates the transcriptional activity of the CABII-I gene, and moreover that regulation at the transcriptional level is the predominant mode by which the clock regulates this gene.

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The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5'-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5'-upstream region contains four short stretches of sequence similar to the so-called 'tub box', a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.

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To study translation initiation in Chlamydomonas chloroplasts, we mutated the initiation codon AUG to AUU, ACG, ACC, ACU, and UUC in the chloroplast petA gene, which encodes cytochrome f of the cytochrome b6/f complex. Cytochrome f accumulated to detectable levels in all mutant strains except the one with a UUC codon, but only the mutant with an AUU codon grew well at 24 degrees C under conditions that require photosynthesis. Because no cytochrome f was detectable in the UUC mutant and because each mutant that accumulated cytochrome f did so at a different level, we concluded that any residual translation probably initiates at the mutant codon. As a further demonstration that alternative initiation sites are not used in vivo, we introduced in-frame UAA stop codons immediately downstream or upstream or in place of the initiation codon. Stop codons at or downstream of the initiation codon prevented accumulation of cytochrome f, whereas the one immediately upstream of the initiation codon had no effect on the accumulation of cytochrome f. These results suggest that an AUG codon is not required to specify the site of translation initiation in chloroplasts but that the efficiency of translation initiation depends on the identity of the initiation codon.

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A genomic region from the filamentous, thermophilic non-N2-fixing cyanobacterium Phormidium laminosum was cloned and sequenced. It includes the nitrite reductase gene (nirA) and three other genes (nrtA, B and C) located downstream of nirA, which are related to the nitrate transport system on the basis of a comparison with the homologous system from Synechococcus sp. PCC 7942. No additional nitrate assimilation-related genes were identified in about 5 kb sequenced downstream of nrtC. All four genes are arranged as an operon with a promoter-like region upstream of the nirA gene. Transcripts of these nitrate assimilation genes accumulated after long periods of nitrogen starvation. This operon also contains inverted repeat sequences in the intercistronic regions which might be involved in mRNA processing or stability.

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The nitrite reductase (NiR) gene (nirA) has been isolated and sequenced from the filamentous, thermophilic non-N2-fixing cyanobacterium Phormidium laminosum. Putative promoter-like and Shine-Dalgarno sequences appear at the 5' end of the 1533 bp long nir-coding region. The deduced amino acid sequence of NiR from P. laminosum corresponds to a 56 kDa polypeptide, a size identical to the molecular mass previously determined for the pure enzyme, and shows a high identity with amino acid sequences from ferredoxin-dependent NiR. This cyanobacterial NiR gene has been efficiently expressed in Escherichia coli DH5 alpha from the E. coli lac promoter and probably from the P. laminosum NiR promoter.

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Translational control is an important regulatory mechanism in chloroplasts, and is thought to be mediated by cis-acting elements in the 5' untranslated regions (UTRs) of mRNAs. Chloroplast transformation was used to replace the wild-type Chlamydomonas reinhardtii petD 5' UTR with mutated versions. Transformants containing altered 5' UTRs had either a wild-type photosynthetic phenotype, a leaky non-photosynthetic phenotype, or were unable to grow photosynthetically. Among those transformants with a wild-type phenotype were ones containing mutations in a putative Shine-Dalgarno sequence element. The results indicate that two regions of the 362 nucleotide (nt) 5' UTR may act as positive elements for translation, one located between nt 150 and 200, and the other situated approximately 40 nt upstream of the start codon, at nt 320. In every case where translation was compromised, petD mRNA accumulated to a lower level than in wild-type cells, ranging from 15% to 60% in different strains. It was concluded that specific regions of the petD 5' UTR mediate translational activation, and that mRNA stability may be linked to translatability.

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