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First published online October 14, 2004; 10.1105/tpc.104.026203 © 2004 American Society of Plant Biologists Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas ChloroplastsBoyce Thompson Institute for Plant Research, Ithaca, New York 14853 3 To whom correspondence should be addressed. E-mail ds28{at}cornell.edu; fax 607-255-6695.
In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain  26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from 26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3' poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS–, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain 26, in which atpB mRNA is unstable because of the lack of a 3' stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3' 5' exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.
Chloroplasts are photosynthetic organelles that arose from prokaryotic endosymbionts during eukaryotic evolution (reviewed in Gray, 1992  ). Chloroplasts contain their own DNA and gene expression machinery, which have combined features of their progenitors with evolved traits. For example, as in bacteria, chloroplast genes are often organized into operons and clusters; however, once these are transcribed into precursor transcripts, they undergo complex posttranscriptional processing events, including splicing and intercistronic cleavages (reviewed in Rochaix, 1996; Sugita and Sugiura, 1996; Monde et al., 2000). Chloroplasts also tend to use modulation of mRNA stability rather than transcriptional control as a mode of regulating mRNA accumulation, which has led since its initial discovery (Deng and Gruissem, 1987; Mullet and Klein, 1987) to intensive investigations of posttranscriptional control mechanisms.
Considerable information is available on cis-acting elements that can act as positive or negative regulators of chloroplast RNA (cpRNA) stability, some of which resemble those of their bacterial ancestors (reviewed in Hayes et al., 1999
Chloroplast mRNAs can also carry signals for instability, the best characterized of which are poly(A) or A-rich tails that can be added posttranscriptionally at a variety of sites, including the coding region or 3' end (Kudla et al., 1996
In vivo studies of chloroplast RNA stability determinants have been mainly performed using the unicellular green alga Chlamydomonas reinhardtii, a model system for molecular genetic studies of photosynthesis (Grossman et al., 2003
Subsequently, a derivative of atpB
Here, we report on an extensive analysis of such strains, which we have designated as suppressor of polyadenylation (spa). Genetic analysis showed that all the spa strains had chloroplast mutations and that two unusual strains had stably heteroplasmic genomes. This led to the observations that double-stranded RNA (dsRNA) formation between sense and antisense transcripts might overcome poly(A)-mediated instability and that PNPase appears to be required for rapid degradation of polyadenylated Chlamydomonas cpRNA in vivo.
Processing Alterations Prevent the Exposure of the atpB Poly(A) Tail in Some spa Strains The structure of the chloroplast genome in the region surrounding atpB is shown in Figure 1A for wild-type, 26, and 26pAtE cells. In wild-type cells, atpB transcription reads through a downstream IR, followed by a two-step processing mechanism to yield the mature 3' end, which is coincident with the stem-loop (Stern and Kindle, 1993). In 26pAtE, the 3' IR, which is absent in 26, has been replaced by a sequence of 25 adenosines, followed by an ectopic copy of the tRNAGlu (trnE1) gene and flanking sequences and a selectable marker cassette, including the aadA coding region, which confers resistance to spectinomycin and streptomycin (Goldschmidt-Clermont, 1991).
In
This screen identified 38 spontaneous phenotypic revertants of
To test for the failure to expose the poly(A) tail, additional probes were used in RNA gel blot analysis. Two probes downstream of atpB were used: trnE1 and aadA (Figure 2A). The results showed that atpB transcripts of six strains (spa1, spa2, and spa6-8) hybridized with the trnE1 probe, indicating that the pre-mRNA was abnormally processed, avoiding poly(A) tail exposure. The aadA probe revealed a monocistronic mRNA emanating from the selectable marker cassette in pA, spa1, and spa6-8, corresponding to those strains in which the cassette had not been disrupted (see below). The atpB-containing transcripts in spa2 and spa6 also hybridized with aadA, confirming their cotranscription.
In spa3-5, however, the atpB transcripts hybridized with neither trnE1 nor aadA despite their lengths (
When PCR was performed with primer sets YK03/12 + (aadA2/atpA5'/BM3'tE), spa1 and spa6-8 yielded the same sized PCR product as On the other hand, the atpB mRNAs of spa1, spa7, spa8, spa19, and spa23 were close to the size of the wild-type transcript, consistent with a 3' end near the poly(A) tract. However, only the transcripts of spa19 and spa23 failed to hybridize with trnE1 (Figure 2A). In the other cases (spa1, spa7, and spa8), an intercistronic processing defect prevents exposure of the poly(A) tail, explaining the suppression phenotype. Sequence analysis of spa1 and spa7 revealed two and one point mutations in trnE1, respectively, which might compromise RNase P site function. Taken together, these results lent support to the possibility that uniquely in spa19 and spa23, the atpB mRNA might accumulate with an exposed poly(A) tail. Therefore, we focused on how polyadenylated mRNA might be stabilized in these two suppressors. We note that because of the way in which they were recovered, spa19 and spa23 are likely to be clones of a single initial suppressor; in fact, data obtained from them were indistinguishable in every case examined.
Genetic Analysis Revealed an Exceptional Segregation Pattern for spa19/23
To our surprise, the cross (spa19/23 mt+ x CC-124 mt–) yielded two classes of tetrads, one in which the four members were all photosynthetic (PS+), and the other in which all were nonphotosynthetic (PS–). Of 42 complete or partial tetrads, nine were PS+ and 33 were PS–. In all tetrads obtained, Mendelian 2:2 segregation of nuclear alleles was confirmed by PCR analysis of the nuclear mating type loci, as described in Methods (and shown in Figure 3B).
Although several models could be developed to explain this exceptional segregation pattern, the most likely possibility seemed to be that spa19 and spa23 had a mixture of chloroplast genomes and that a poorly transmitted version conferred the PS+ phenotype, whereas a better transmitted one conferred the PS– phenotype. This hypothesis was confirmed by molecular analysis, as described below.
Spa19/23 Maintain Heteroplasmic cpDNA When tetrad progeny were examined, two patterns were found, which correlated with the photosynthetic growth phenotype. In spa19/23 and in PS+ progeny resulting from the cross to CC-124, both 7.8- and 1.95-kb fragments were found (data for spa23 are shown in Figure 3B). Thus, the PS+ progeny resembled the original suppressor. In PS– tetrads, however, only the 7.8-kb fragment was found, leading to the conclusion that the 1.95-kb fragment cosegregated with a PS+ phenotype. We will therefore refer to cpDNA containing the 1.95-kb fragment as the PS+ genome and that containing the 7.8-kb fragment as the PS– genome. We further inferred that the PS+ genome is required for accumulation of atpB transcripts, whereas the PS– genome is insufficient for this purpose. However, because the PS– genome is invariably present, it seemed probable that it contributed essential genes absent from the PS+ genome. Taking the genetic and molecular data together, it was immediately obvious that although the PS– genome was inherited alone more frequently than the PS+/PS– genome combination, the PS– genome is much less abundant than the PS+ genome in vegetative spa19/23 cells (Figure 3A). Indeed, a quantitative comparison of the signal intensities between 1.95-kb/7.8-kb fragments revealed an approximately fourfold molar excess of the PS+ genome (data not shown; see Methods and Figure 5). Thus, the more abundant genome is inherited poorly in genetic crosses, suggesting a defect in transmission and/or replication during zygote maturation.
Inverse PCR Reveals a Large Deletion in the spa19/23 PS+ Genome To clarify the presumed chloroplast mutation underlying the PS+ phenotype of spa19/23, an inverse PCR approach was taken to clone the sequence downstream of atpB in the PS+ genome. PCR was conducted after PstI digestion and subsequent self-ligation of spa19 DNA. Sequencing of the cloned 1.95-kb PstI fragment revealed that in addition to the aadA selectable marker cassette being deleted (Figure 2C), deletion and/or rearrangement had resulted in upstream portions of the endogenous trnE1 gene (i.e., that corresponding to the ectopic one used in 26pAtE) being positioned immediately downstream of atpB, but in an inverted orientation, as shown in Figure 4A.
The presence of both PS+ and PS– genomes in phenotypically PS+, but not PS– strains, was confirmed by PCR. Primer sets specific for the PS+ (YK12+spatE) and PS– (YK12+BM3'tE) genomes were designed, as shown in Figure 4A. DNA was extracted from wild-type, photosynthetic (PS+), and nonphotosynthetic (PS–) progeny from the cross (spa19/23 mt+ x CC-124 mt–) and used as a template for PCR. The PS+ specific primer pair yielded a single PCR product with the predicted size (437 bp) only from photosynthetic progeny (Figure 4B). On the other hand, PS– specific primers yielded the 376-bp product from both types of progeny. These results confirmed that the sequence obtained by inverse PCR from spa19/23 exists in vivo and is maintained heteroplasmically with the PS– genome, consistent with results of the DNA gel blot described above. The fact that the PS+ PCR product was undetectable with DNA isolated from nonphotosynthetic cells further suggests that this configuration is not maintained at a substoichiometric level, as is often seen in plant mitochondria (reviewed in Mackenzie and McIntosh, 1999 ).
Because in wild-type and These signals were quantified, normalized to the signal for 28S rDNA on the same membrane, and plotted against the amount of PCR product present in each dot (Figure 5C). Average relative signals per nanogram of PCR product were calculated, which allowed the seven chloroplast genes tested to be categorized into two clear groups, atpB-petD-tufA and clpP-psbB-rbcL (signal from the short psbN gene was too weak to be used). The slopes for the atpB-petD-tufA group and 28S rDNA were almost identical between the wild type and spa19, with the average ratio of spa19/wild type being 1.1 ± 0.06. On the other hand, the ratio for clpP-psbB-rbcL was 4.5 ± 0.56, indicating that these genes are three to four times more abundant in spa19 than in the wild type. This stoichiometry can be most simply explained by positing that the PS+ genome arose through a deletion of 145 kb and is maintained at three to four times the copy number of the PS– genome in vegetative cells.
Transcriptional Activity of the PS+ Genome
Close inspection of the novel PS+ genome sequence revealed a potential explanation for the accumulation of atpB mRNA. As shown in the bottom panel of Figure 6A, the apposition of two identical copies of trnE1-derived sequences surrounding the RNase P site, in an inverted orientation, could form a stem-loop if present in the atpB mRNA. This near-perfect 24-nucleotide IR is indicated with a line above it in the sequence and if formed would likely protect the 3' end of the RNA with a classical secondary structure. Furthermore, if this stem-loop were present in vivo, both primers spatE3 and spatE2 should yield RT-PCR products with YK12 as a second primer. However, the results obtained by RT-PCR were unexpected. As shown in the middle panel of Figure 6B (+RT), we found that a product was obtained when primers common to PS+ and PS– genomes were used (spatE3-YK12) but not when spatE2, which anneals only 20 nucleotides downstream, was used. This result indicates that an atpB mRNA species accumulated in which the poly(A) tail was in fact not exposed, but was followed by a sequence of 5 to 20 nucleotides, which would be required to stably anneal the 3' end of the spatE3 primer. Although this short sequence would internalize the poly(A) tract, it is not predicted to form a strong secondary structure, and it is known that atpB mRNA lacking an IR (e.g., strain 26) is unstable (Stern et al., 1991). To map the 3'end of atpB mRNA more precisely, S1 nuclease protection analysis was performed, as shown in Figure 7. To generate probes, PCR products were amplified from the PS+ and PS– genome using primers YK14 (within atpB) and spatE and BM3'tE, respectively (Figure 7A). The 3' end of the antisense strand of the PCR product was labeled by taking advantage of a SexAI site that is present 142 bp upstream of the atpB translation termination codon. PCR products were also prepared with three different reverse primers, spatE3, spatE4, and spatE5, and labeled in the same way. These served as precise size markers for the end of the atpB coding sequence (spatE5), the end of the poly(A) sequence (spatE4), and the end of the sequence shared between the PS+ and PS– genomes (spatE3; see Figure 7B for sequence detail).
The results of S1 protection with the PS– probe are shown in Figure 7C. When wild-type RNA was used, the size of protected product was calculated to be 161 nucleotides, very close to the predicted 159 nucleotides, which corresponds to the discontinuity between the engineered sequence in 26pAtE and the wild-type atpB gene. Using spa19 mRNA, a single protected product was also observed. Based on its migration between the markers generated using primers spatE4 and spatE3, its size was 197 nucleotides, very close to the 200 nucleotides that would place it at the boundary of sequences shared between the PS+ and PS– genomes (arrowhead in Figure 7B). Using the PS+ probe instead of the PS– probe, the same  161- and 197-nucleotide protected products were observed for the wild type and spa19 mRNA, respectively, as shown in Figure 7D. This demonstrates that sequences derived uniquely from the PS+ genome are not present in spa19 atpB mRNA at the resolution possible with this analysis, consistent with the RT-PCR experiments shown above. To eliminate the possibility that S1 nuclease protection had generated an artifact because of, for example, strong RNA secondary structure, we performed a control experiment with two in vitro–generated atpB transcripts, as shown in Figure 7E. A transcript having its 3' end coincident with primer spatE3 represented the mRNA accumulating in vivo based on the experiments described above, whereas a transcript with its end coincident with primer spatE included PS+ genome-specific sequences, including the fortuitous IR created during the generation of the PS+ genome. When the transcript with its 3'end at spatE3 was used with a PS+ probe, a single protected product with the same size as the 210-nucleotide marker was observed, as expected. When the transcript with its 3' end at spatE was used, the entire probe was protected, without any evidence of artifactual cleavage products (e.g., within the loop of the putative IR). Thus, the poly(A) sequence of atpB mRNA in spa19 is not exposed, but rather is capped with a short sequence common to the PS+ and PS– genomes. This left the open questions of how such a short and apparently unstructured sequence might stabilize the mRNA, and why atpB transcripts are detected apparently only corresponding to sequences shared between the PS+ and PS– genomes.
Antisense Transcripts for atpB Accumulate and Form Duplexes in spa19/23
Two testable predictions arise from this hypothesis, namely, that antisense atpB mRNA should accumulate in spa19 and that duplexes should form between antisense and sense atpB transcripts. To test the first of these predictions, we performed RT-PCR so as to distinguish sense and antisense transcripts, as shown in Figure 9. Total RNA was extracted from wild-type and spa19 cells, and cDNA was synthesized using forward or reverse primers to detect antisense or sense transcripts, respectively. cDNA was then amplified by PCR with appropriate primer pairs, as shown in Figure 9A. The atpB coding region was analyzed with the primer pair (YK11+KY09). In the wild type, sense atpB transcript was clearly detected, but almost no signal was obtained representing antisense transcripts, as expected (Figure 9B). In spa19, however, PCR products corresponding to not only sense transcripts, but also to antisense transcripts, were detected. A similar result was obtained for the region between the 3' end of atpB and tufA (YK12+spatE3), consistent with antisense transcripts spanning the PS+ genome-specific region into atpB.
To assess the possibility that atpB forms dsRNA in spa19, S1 nuclease protection was used, as shown in Figure 9C. S1 nuclease digests single-stranded DNA or RNA nonspecifically but will not digest fully paired duplexes. Total RNA was extracted from wild-type and spa19 cells, and equal amounts were treated with increasing concentrations of S1 nuclease. Subsequently, RT-PCR was performed using a one-step assay with primers YK11+KY09 to detect the atpB coding region or for another chloroplast gene, petD, as a control. For both genes, clear signals were obtained from the wild type and spa19 when the RNAs were not treated with S1 nuclease. But as higher concentrations of S1 nuclease were used, we found that atpB RNA from spa19 was more resistant to the action of S1 nuclease than that of the wild type. On the other hand, no such difference was observed when petD mRNA was examined using the same RNA samples. These results support the possible formation of double-stranded atpB mRNA in spa19 chloroplasts.
Ectopically Expressed Antisense RNA Suppresses the Instability of atpB mRNA Lacking a 3' IR
The experimental strategy is shown in Figure 10A. A 121-bp segment of the atpB coding region was amplified by PCR, with the reverse primer designed not to include any 3' UTR/IR sequences. This 121-bp sequence was inserted at the end of the aadA coding region in an inverted orientation. The SphI site used for this insertion was 5 bp downstream of the aadA translation termination codon, allowing the expression of chimeric aadA sense–atpB antisense RNA without affecting aadA translation. This engineered aadA cassette was targeted to a site near petD, which is
The antisense-expressing plasmid, along with a control plasmid lacking the antisense segment, were transformed into atpB 26 cells, and colonies were selected on Tris-acetate-phosphate (TAP) medium containing 100 µg/mL of spectinomycin. DNA of several individual transformants was analyzed to confirm correct integration of the aadA cassette and homoplasmicity of chloroplast genomes (data not shown). atpB26 grows slowly on minimal medium because of the unstable atpB mRNA and a dramatic reduction of its gene product (Stern et al., 1991). We therefore compared the growth of two representative control (26AD) and antisense (26ADaB) strains using a dilution series of cells, as shown in Figure 10B. On TAP (acetate-containing) plates, robust and equivalent growth was observed for all strains. On minimal medium, however, the 26ADaB strains showed much more robust growth than the controls, as can be clearly seen by comparing the numbers of cells in the respective strains after plating of equal numbers and incubation for 9 d. This suggested that expression of the antisense RNA increased the expression of the unlinked atpB gene.
Next, the expression of the chimeric aadA-atpB mRNA was analyzed by RT-PCR, as shown in Figure 10C, with actin mRNA as a control. Products of the expected size (897 bp) were detected only from the antisense (
More robust photosynthetic growth, as shown in Figure 10B, would be expected to be accompanied by an increase of the atpB gene product and would be consistent with the greater accumulation of the cognizant mRNA, as shown in Figure 10D. We therefore performed immunoblot analysis, using an antibody raised against the ATPase
PNPase May Degrade atpB mRNA Synthesized from the PS– Genome
In work to be published elsewhere, we have described the generation of Chlamydomonas strains depleted for PNPase (our unpublished data). This depletion was achieved via antisense technology (Figure 11A) to partially silence the nucleus-encoded PNPase gene (note that antisense RNA in the nucleus downregulates gene expression, whereas the model proposed in Figure 8 of this article evokes a stabilizing role for antisense RNA in the chloroplast). One of the antisense strains obtained was Ap6, which, based on immunoblot analysis, retains
Zygotes were dissected on acetate-containing medium containing 2 µg/mL of zeocin. Zeocin was included to select for expression of the antisense RNA, which is cotranscribed with the ble gene. Expression of ble confers zeocin resistance (Stevens et al., 1996 ), and in the absence of constant selection, Chlamydomonas nuclear transgenes are often silenced by methylation or other mechanisms (Cerutti et al., 1997). Consequently, in the 51 tetrads that were obtained, only two of the four progeny survived (i.e., those that received the ble-pnp cassette), a supposition that was confirmed by PCR analysis (data not shown). When replica-plated on minimal medium lacking acetate but containing zeocin, we found that the surviving progeny were all photosynthetic, suggesting a correlation between antisense expression and a PS+ phenotype. RT-PCR was used to confirm expression of the dicistronic ble-pnp antisense transcript (Figure 11B, top panel) and a reduction in the level of Pnp sense transcripts, with actin as a positive control (second and third panels). Subsequently, the presence or absence of PS+ and PS– genomes was assayed by PCR (fourth and fifth panels). No positive signal was obtained for the PS+ genome in any of the 24 zeocin-resistant progeny examined, whereas the PS– genome was clearly detected in all of them. This result is consistent with the interpretation that in the Ap6 background the PS+ genome is no longer required for photosynthesis, presumably because the reduced expression of PNPase results in the stability of otherwise unstable atpB mRNA.
Next, the 3' termini of atpB transcripts in the progeny were estimated by preparing cDNAs with three different reverse primers [KY09, oligo(dT), or spatE3; Figure 12, top]. KY09 was used as a positive control because it anneals to the atpB coding region and was expected to yield a product with RNA from wild-type cells. As shown in Figure 12, bottom, clear signals were obtained from the wild type, spa19, and all the progeny when the atpB mRNA coding region was amplified after cDNA synthesis directed by KY09. When an oligo(dT) primer was used for cDNA synthesis, no signal was obtained from wild-type cells, which is consistent with previous reports showing the exceedingly low abundance of polyadenylated transcripts in chloroplasts (Lisitsky et al., 1996
In this study, we have performed reversion analysis of a strain in which the chloroplast was engineered to generate an mRNA rendered unstable because of exposure of a poly(A) tail. The strain, 26pAtE, gave rise to spontaneous photosynthetic revertants at a remarkably high frequency, all of which turned out to have chloroplast mutations. Deliberately decreasing expression of the nuclear PNPase gene allowed photosynthesis in spite of the accumulation of polyadenylated atpB mRNA, demonstrating that nuclear factors play a role in the polyadenylation-mediated decay pathway. By intensively examining the chloroplast suppressors, we discovered unexpected features of RNA stabilization, including duplex formation between sense and antisense transcripts.
Polyadenylated mRNA Can Be Stabilized by Capping the 3' UTR
Polyadenylated mRNA Is Likely Stabilized by an Antisense Transcript in spa19/23 Upon close inspection of the PS+ genome, we found a fortuitous IR sequence downstream of atpB, so our initial hypothesis was that atpB mRNA transcribed from the PS+ genome would be stabilized by this IR (Figure 6). Indeed, RT-PCR showed that in at least some atpB transcripts the poly(A) tail was not exposed, but rather was flanked by a very short sequence (Figures 6 and 7). However, the 3' end of atpB mRNA we detected was shorter than the end of this IR sequence and therefore was not predicted to form a 3' end secondary structure. Because the primer spatE3 terminates in six G/C residues, this is probably the minimum overlap with the transcript. A maximum length would be defined by the transcript's inability to anneal to the downstream primer spatE2. Taken together, we estimated that 6 to 20 nucleotides flank the poly(A) tract in the accumulating spa19/23 atpB mRNA detected by RT-PCR (Figure 6B).
We also tried to detect this IR in atpB mRNA using S1 protection. The probes were generated from both the PS+ and PS– genomes, which, if the IR were present, would generate longer protected product(s) when the PS+ probe was used. However, such products were not observed (Figure 7C), and indeed, the sizes of the protected products were indistinguishable whether the PS+ or PS– probe was used (Figure 7D). At the resolution of this experiment, we cannot exclude that some atpB transcripts contain a few nucleotides derived from the PS+ genome; however, clearly no transcripts are long enough to contain the full IR and, thus, a 3' stabilizing element. Thus, these observations disagreed with a classical model of IR-mediated stabilization and led us to an alternative and unexpected model presented in Figure 8. In this alternative model (Figure 8), the PS+ genome provides antisense transcripts for the atpB gene, driven by the tufA and/or trnE1 promoters, and these anneal to sense transcripts from the PS– genome. After trimming of the single-stranded 3' regions by ribonucleases, the remaining dsRNA structure would stabilize the 3' end and thus permit translation of the message. The presence of antisense atpB transcripts and dsRNA, shown by RT-PCR (Figure 9), both support the antisense hypothesis. However, we felt it prudent to obtain independent evidence for such an unexpected mechanism and therefore created chloroplast transformants that express artificial antisense transcripts. This experiment showed that the introduction of antisense transcripts could partially stabilize otherwise unstable atpB mRNA in the strain atpB
Another perplexing finding is the apparent unidirectional (antisense) transcription of the PS+ genome atpB region. In principle, the sense transcripts could be generated from the PS+ as well as PS– genome because both contain the atpB-poly(A)-trnE1 (upstream) sequences present in the accumulating mRNA. However, if sense–antisense pairing occurred between transcripts both generated from the PS+ genome, we would expect the duplex—and, hence, the accumulating transcript—to extend considerably further downstream, perhaps as far as the 3' end of tufA, where the incipient antisense transcript presumably would be processed as part of tufA 3' end maturation. However, such a 3' end was not observed using S1 protection analysis (Figure 7). And because we detected only one class of transcript in spa19/23 by RNA filter blot, it is likely that sense transcripts are generated only from the PS– genome and the antisense transcripts solely from the PS+ genome. The basis for this apparent exclusivity remains an open question. One possible explanation might be the predominance of tufA/trnE promoter activity over that of the atpB promoter. Transcriptional interference between convergent genes is well known (e.g., Spencer et al., 1986
On the other hand, the PS+ genome was invariably lost when spa19/23 were crossed to strains expressing nuclear antisense RNA to reduce PNPase expression (Figures 11 and 12). This indicates that the dsRNA structure imparts stability by impeding the action of PNPase and that the rapid degradation of atpB mRNA would be the selective pressure that resulted in at least some progeny from spa19/23 x CC-124 crosses retaining PS+ genomes, although the zygotes were not plated on minimal medium. It is also possible that the reduction in PNPase had indirect effects on cpDNA transmission, for example, by altering expression of some chloroplast genes. In any case, our results suggest that PNPase is the major 3'
Natural Occurrence of Antisense RNA
Apart from the special case of psbN, antisense transcripts do not appear to accumulate generally in chloroplasts. Although many such RNAs may be synthesized as a result of the compact nature of chloroplast genomes, because of the lack of efficient termination, they are likely to be unstable. For example, Chlamydomonas chloroplasts have a highly efficient mechanism for degrading atpB mRNA downstream of its eventual maturation site (Hicks et al., 2002
Because dsRNA-directed gene silencing has been receiving great attention (Cerutti, 2003
Dynamic Alteration of cpDNA in spa Strains
The spa19/23 strains, however, are novel. Stable heteroplasmicity is unknown in chloroplasts except under conditions where both genomes are being selected (Goldschmidt-Clermont, 1991
Our tentative scenario for the spa19/23 suppression event initiates with loss of the aadA cassette, generating the spa19/23 PS– genome, which is nearly identical to the
Heteroplasmicity in spa19/23 has two other features of interest. The first is the imbalance between the PS– and PS+ genomes, with the latter being in threefold to fourfold excess in vegetative cells. The closest precedent for an overabundant genome type in Chlamydomonas also resulted from suppression of a photosynthetic growth phenotype. Strain
The second feature is the unusually poor transmission of the PS+ genome in genetic crosses, despite its high copy number. Presently, we do not have clear mechanistic insights into this phenomenon. However, it is known that a DNA replication step during zygote germination is required for normal inheritance of Chlamydomonas cpDNA (Umen and Goodenough, 2001
Selection and Genetic Analysis of Suppressors The progenitor strain for the suppressor screen was 26pAtE mt+ (Komine et al., 2002). Cells were grown under constant light in TAP medium (Harris, 1989) and harvested at mid-log phase (1 to 2 x 106 cells/mL). Cells (2 x 108) were plated on minimal medium (lacking a reduced carbon source) to select for photosynthetic growth. Resulting colonies were screened by PCR with primers YK03 and B-M-3'-tE (Table 2) to confirm the presence of the atpB-trnE1 fusion in the chloroplast genome. Because the original 26pAtE strain was lost as a result of reversion before a rate of reversion could be calculated, the 10–5 rate reported in Results was derived from a PS– progeny of a cross of spa19 x CC-124, which differs from 26pAtE by its loss of the aadA cassette.
The wild-type strain used in backcrosses was CC-124 mt–. Mating type plus suppressor strains were crossed to CC-124 according to Harris (1989
DNA/RNA/Protein Analysis
For inverse PCR, total DNA was isolated from spa19, and a procedure adapted from Ochman et al. (1990) For DNA dot blot analysis, PCR products for atpB, petD, tufA, clpP, psbN, psbB, rbcL, and 28S rDNA were prepared using primer sets shown in Table 2. PCR products (64, 32, 16, 8, and 4 ng) were blotted on GeneScreen nylon filters using the Bio-Dot microfiltration apparatus (Bio-Rad, Hercules, CA). The DNA filter blots were hybridized with radiolabeled total DNA extracted from wild-type or spa19 cells, which were labeled by random priming. The radioactive dots were visualized using the Storm scanner and quantified using ImageQuant software (Amersham Pharmacia Biotech, Uppsala, Sweden). Microsoft Excel (version X) was used to calculate a least-squares fit line and slope for the data shown in Figure 5C and to generate an exponential curve using the migration of size markers in Figure 7; the derived equation was used to calculate the size of the protected products.
Total protein was isolated as previously described (Komine et al., 2002
RT-PCR Sequencing was performed to ensure that PCR products were derived from the targeted genes. DNA fragments were excised from gels, purified using QiaQuick resin (Qiagen, Valencia, CA), and cloned using the TOPO-TA cloning vector (Invitrogen). DNA from positive colonies was end sequenced using vector primers.
Plasmid Construction and Chloroplast Transformation
We gratefully acknowledge Shinya Murakami and Gadi Schuster for thoughtful insights into this work, which was supported by Japan Society for the Promotion of Science, Francis Goelet postdoctoral fellowships to Y.N., and awards from the Binational Agricultural Research and Development Fund (US-3177-99C) and the National Science Foundation (MCB 0091020) to D.B.S, including a National Science Foundation Research Experience for Undergraduates award to E.A.K.
1 These authors contributed equally this work. 
2 Current address: Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720. The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Yoshiki Nishimura (yn37{at}cornell.edu) and David B. Stern (ds28{at}cornell.edu). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.026203. Received July 18, 2004; accepted August 26, 2004.
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INTRODUCTION
-subunit. To show relative loading, a Ponceau-S stained image is shown at the bottom.
