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Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Interspecies RNA-Seq datasets are increasingly common, and have the potential to answer new questions about the evolution of gene expression. Single-species differential expression analysis is now a well-studied problem that benefits from sound statistical methods. Extensive reviews on biological or synthetic datasets have provided the community with a clear picture on the relative performances of the available methods in various settings. However, synthetic dataset simulation tools are still missing in the interspecies gene expression context. In this work, we develop and implement a new simulation framework. This tool builds on both the RNA-Seq and the phylogenetic comparative methods literatures to generate realistic count datasets, while taking into account the phylogenetic relationships between the samples. We illustrate the usefulness of this new framework through a targeted simulation study, that reproduces the features of a recently published dataset, containing gene expression data in adult eye tissue across blind and sighted freshwater crayfish species. Using our simulated datasets, we perform a fair comparison of several approaches used for differential expression analysis. This benchmark reveals some of the strengths and weaknesses of both the classical and phylogenetic approaches for interspecies differential expression analysis, and allows for a reanalysis of the crayfish dataset. The tool has been integrated in the R package compcodeR, freely available on Bioconductor., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

C4 plants, such as maize, strictly compartmentalize Rubisco to bundle sheath chloroplasts. The molecular basis for the restriction of Rubisco from the more abundant mesophyll chloroplasts is not fully understood. Mesophyll chloroplasts transcribe the Rubisco large subunit gene and, when normally quiescent transcription of the nuclear Rubisco small subunit gene family is overcome by ectopic expression, mesophyll chloroplasts still do not accumulate measurable Rubisco. Here we show that a combination of five ubiquitin promoter-driven nuclear transgenes expressed in maize leads to mesophyll accumulation of assembled Rubisco. These encode the Rubisco large and small subunits, Rubisco assembly factors 1 and 2, and the assembly factor Bundle sheath defective 2. In these plants, Rubisco large subunit accumulates in mesophyll cells, and appears to be assembled into a holoenzyme capable of binding the substrate analog CABP (carboxyarabinitol bisphosphate). Isotope discrimination assays suggest, however, that mesophyll Rubisco is not participating in carbon assimilation in these plants, most probably due to a lack of the substrate ribulose 1,5-bisphosphate and/or Rubisco activase. Overall, this work defines a minimal set of Rubisco assembly factors in planta and may help lead to methods of regulating the C4 pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-seq. Using Arabidopsis thaliana as a model, we catalogued >215 primary 5' ends corresponding to transcription start sites (TSS), as well as 1628 processed 5' ends and 1299 3' ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5' and 3' ends, contrasting with the prevailing description of discrete 5' termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-seq was also implemented for pnp1-1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2000 termini were altered in pnp1-1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the absence of light in caves, animals have repeatedly evolved reduced eyes and visual systems. Whether the underlying genetic components remain intact in blind species remains unanswered across taxa. The freshwater crayfish have evolved to live in caves multiple times throughout their history; therefore, this system provides an opportunity to probe the genetic patterns and processes underlying repeated vision loss. Using transcriptomic data from the eyes of 14 species of cave and surface crayfishes, we identify the expression of 17 genes putatively related to visual phototransduction. We find a similarly complete repertoire of phototransduction gene families expressed in cave and surface species, but that the expression levels of those transcripts are consistently lower in cave species. We find statistical support for episodic positive selection, increased and decreased selection strength in caves, depending on the gene family. Analyses of gene expression evolution suggest convergent and possibly adaptive downregulation of these genes across eye-reduction events. Our results reveal a combination of evolutionary processes acting on the sequences and gene expression levels of vision-related genes underlying the loss of vision in caves.

Dissecting the evolutionary genetic processes underlying eye reduction and vision loss in obligate cave-dwelling organisms has been a long-standing challenge in evolutionary biology. Independent vision loss events in related subterranean organisms can provide critical insight into these processes as well as into the nature of convergent loss of complex traits. Advances in evolutionary developmental biology have illuminated the significant role of heritable gene expression variation in the evolution of new forms. Here, we analyze gene expression variation in adult eye tissue across the freshwater crayfish, representing four independent vision-loss events in caves. Species and individual expression patterns cluster by eye function rather than phylogeny, suggesting convergence in transcriptome evolution in independently blind animals. However, this clustering is not greater than what is observed in surface species with conserved eye function after accounting for phylogenetic expectations. Modeling expression evolution suggests that there is a common increase in evolutionary rates in the blind lineages, consistent with a relaxation of selective constraint maintaining optimal expression levels. This is evidence for a repeated loss of expression constraint in the transcriptomes of blind animals and that convergence occurs via a similar trajectory through genetic drift.

Caves are perceived as isolated, extreme habitats with a uniquely specialized biota, which long ago led to the idea that caves are "evolutionary dead-ends." This implies that cave-adapted taxa may be doomed for extinction before they can diversify or transition to a more stable state. However, this hypothesis has not been explicitly tested in a phylogenetic framework with multiple independently evolved cave-dwelling groups. Here, we use the freshwater crayfish, a group with dozens of cave-dwelling species in multiple lineages, as a system to test this hypothesis. We consider historical patterns of lineage diversification and habitat transition as well as current patterns of geographic range size. We find that while cave-dwelling lineages have small relative range sizes and rarely transition back to the surface, they exhibit remarkably similar diversification patterns to those of other habitat types and appear to be able to maintain a diversity of lineages through time. This suggests that cave adaptation is not a "dead-end" for freshwater crayfish, which has positive implications for our understanding of biodiversity and conservation in cave habitats., (© 2017 The Author(s). Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.)

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsis thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators., (Copyright © 2016 Castandet et al.)

RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3' end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3' extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation., (© 2015 American Society of Plant Biologists. All rights reserved.)

Nucleus-encoded ribonucleases and RNA-binding proteins influence chloroplast gene expression through their roles in RNA maturation and stability. One mechanism for mRNA 5' end maturation posits that sequence-specific pentatricopeptide repeat (PPR) proteins define termini by blocking the 5'→3' exonucleolytic activity of ribonuclease J (RNase J). To test this hypothesis in vivo, virus-induced gene silencing was used to reduce the expression of three PPR proteins and RNase J, both individually and jointly, in Nicotiana benthamiana. In accordance with the stability-conferring function of the PPR proteins PPR10, HCF152 and MRL1, accumulation of the cognate RNA species atpH, petB and rbcL was reduced when the PPR-encoding genes were silenced. In contrast, RNase J reduction alone or combined with PPR deficiency resulted in reduced abundance of polycistronic precursor transcripts and mature counterparts, which were replaced by intermediately sized species with heterogeneous 5' ends. We conclude that RNase J deficiency can partially mask the absence of PPR proteins, and that RNase J is capable of processing chloroplast mRNAs up to PPR protein-binding sites. These findings support the hypothesis that RNase J is the major ribonuclease responsible for maturing chloroplast mRNA 5' termini, with RNA-binding proteins acting as barriers to its activity.

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.

Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18-25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5' end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.

Two mechanisms for chloroplast DNA replication have been revealed through the study of an unusual heteroplasmic strain of the green alga Chlamydomonas reinhardtii. Heteroplasmy is a state in which more than one genome type occurs in a mitochondrion or chloroplast. The Chlamydomonas strain spa19 bears two distinct chloroplast genomes, termed PS+ and PS-. PS+ genomes predominate and are stably maintained in vegetative cells, despite their lack of known replication origins. In sexual crosses with spa19 as the mating type plus parent, however, PS+ genomes are transmitted in only approximately 25% of tetrads, whereas the PS- genomes are faithfully inherited in all progeny. In this research, we have explored the mechanism underlying this biased uniparental inheritance. We show that the relative reduction and dilution of PS+ vs. PS- genomes takes place during gametogenesis. Bromodeoxyuridine labeling, followed by immunoprecipitation and PCR, was used to compare replication activities of PS+ and PS- genomes. We found that the replication of PS+ genomes is specifically suppressed during gametogenesis and germination of zygospores, a phenomenon that also was observed when spa19 cells were treated with rifampicin, an inhibitor of the chloroplast RNA polymerase. Furthermore, when bromodeoxyuridine incorporation was compared at 11 sites within the chloroplast genome between vegetative cells, gametes, and rifampicin-treated cells by quantitative PCR, we found that incorporation was often reduced at the same sites in gametes that were also sensitive to rifampicin treatment. We conclude that a transcription-mediated form of DNA replication priming, which may be downregulated during gametogenesis, is indispensable for robust maintenance of PS+ genomes. These results highlight the potential for chloroplast genome copy number regulation through alternative replication strategies.

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.

We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ' untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ' >3 ' degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.

Enzymes from several gene families modify RNA molecules at their extremities. These reactions occur in several cellular compartments and affect every class of RNA. To assess the diversity of a subclass of these enzymes, we searched Chlamydomonas for open reading frames (ORFs) potentially encoding exoribonucleases, poly(A) polymerases, and proteins known to associate with and/or regulate them. The ORFs were further analyzed for indications of protein localization to the nucleus, cytosol, mitochondrion, and/or chloroplast. By comparing predicted proteins with homologs in Arabidopsis and yeast, we derived several tentative conclusions regarding RNA 5'- and 3'-end metabolism in Chlamydomonas. First, the alga possesses only one each of the following likely organellar enzymes: polynucleotide phosphorylase, hydrolytic exoribonuclease, poly(A) polymerase, and CCA transferase, a surprisingly small complement. Second, although the core of the nuclear/cytosolic exosome decay complex is well conserved, neither nucleus-specific activators nor the cytosolic exosome activators are present. Finally, our discovery of nine noncanonical poly(A) polymerases, a divergent family retaining the catalytic domains of conventional poly(A) polymerases, leads to the hypothesis that polyadenylation may play an especially important regulatory role throughout the Chlamydomonas cell, stabilizing some transcripts and targeting degradation machinery to others.

Cell survival depends on the cell's ability to acclimate to phosphorus (P) limitation. We studied the chloroplast ribonuclease polynucleotide phosphorylase (PNPase), which consumes and generates phosphate, by comparing wild-type Chlamydomonas reinhardtii cells with strains with reduced PNPase expression. In the wild type, chloroplast RNA (cpRNA) accumulates under P limitation, correlating with reduced PNPase expression. PNPase-deficient strains do not exhibit cpRNA variation under these conditions, suggesting that in the wild type PNPase limits cpRNA accumulation under P stress. PNPase levels appear to be mediated by the P response regulator PHOSPHORUS STARVATION RESPONSE1 (PSR1), because in psr1 mutant cells, cpRNA declines under P limitation and PNPase expression is not reduced. PNPase-deficient cells begin to lose viability after 24 h of P depletion, suggesting that PNPase is important for cellular acclimation. PNPase-deficient strains do not have enhanced sensitivity to other physiological or nutrient stresses, and their RNA and cell growth phenotypes are not observed under P stress with phosphite, a phosphate analog that blocks the stress signal. In contrast with RNA metabolism, chloroplast DNA (cpDNA) levels declined under P deprivation, suggesting that P mobilization occurs from DNA rather than RNA. This unusual phenomenon, which is phosphite- and PSR1-insensitive, may have evolved as a result of the polyploid nature of cpDNA and the requirement of P for cpRNA degradation by PNPase.

Numerous nuclear gene products are required for the correct expression of organellar genes. One such gene in the green alga Chlamydomonas reinhardtii is MCD1, whose product is required for stability of the chloroplast-encoded petD mRNA. In mcd1 mutants, which are non-photosynthetic, petD mRNA is degraded by a 5'-3' exonuclease activity, resulting in a failure to synthesize its product, subunit IV of the cytochrome b (6)/f complex. Here, we report the sequence of the wild-type MCD1 gene, which encodes a large and novel putative protein. Analysis of three mutant alleles showed that two harbored large deletions, but that one allele, mcd1-2, had a single base change resulting in a nonsense codon near the N-terminus. This same mutant allele can be suppressed by a second-site mutation in the nuclear MCD2 gene, whereas mcd2-1 cannot suppress the deletion in mcd1-1 (Esposito,D. Higgs,D.C. Drager,R.G. Stern, D.B. and Girard-Bascou,J. (2001) Curr. Genet., 39, 40-48). We report the cloning of mcd2-1, and show that the mutation lies in a tRNA(Ser)(CGA), which has been modified to translate the nonsense codon in mcd1-2. We discuss how the existence of a large tRNA(Ser) gene family may permit this suppression without pleiotropic consequences.

Arabidopsis thaliana chloroplasts contain at least two 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3' processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5' and 3' ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3' extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Delta26pAtE 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 Delta26pAtE, 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 Delta26, 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.

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5' to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold-containing proteins from the SDR superfamily, including several RNA-binding proteins and endonucleases. CSP41 is active only in the presence of MgCl2 and CaCl2. Although Mg2+- and Ca2+-activated CSP41 cleave at identical sites in the single-stranded regions of a stem-loop-containing substrate, Mg2+-activated CSP41 was also able to cleave within the double-stranded region of the stem-loop. Mixed metal experiments with Mg2+ and Ca2+ suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Mn2+, Co2+ and Zn2+ compete with Mg2+ for binding, although there is no activity in their presence. Using site-directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal-binding pocket. The activation constant for Mg2+ (K(A,Mg) = 2.1 +/- 0.4 mM) is of the same order of magnitude as the stromal Mg2+ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg2+ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.

The plastid gene clpP is widely regarded as essential for chloroplast function and general plant cell survival. In this note we provide evidence that certain lines of non-photosynthetic maize (Zea mays) Black Mexican Sweet (BMS) suspension cells do not carry clpP in their plastid genomes. We also discuss several incidences in the literature where clpP is either missing or not expressed in other non-green cell lines and plants. We conclude that clpP is not required for general plant cell survival but instead may only be essential for the development and/or function of plastids with active gene expression.

Chlamydomonas reinhardtii is a unicellular eukaryotic alga possessing a single chloroplast that is widely used as a model system for the study of photosynthetic processes. This report analyzes the surprising structural and evolutionary features of the completely sequenced 203,395-bp plastid chromosome. The genome is divided by 21.2-kb inverted repeats into two single-copy regions of approximately 80 kb and contains only 99 genes, including a full complement of tRNAs and atypical genes encoding the RNA polymerase. A remarkable feature is that >20% of the genome is repetitive DNA: the majority of intergenic regions consist of numerous classes of short dispersed repeats (SDRs), which may have structural or evolutionary significance. Among other sequenced chlorophyte plastid genomes, only that of the green alga Chlorella vulgaris appears to share this feature. The program MultiPipMaker was used to compare the genic complement of Chlamydomonas with those of other chloroplast genomes and to scan the genomes for sequence similarities and repetitive DNAs. Among the results was evidence that the SDRs were not derived from extant coding sequences, although some SDRs may have arisen from other genomic fragments. Phylogenetic reconstruction of changes in plastid genome content revealed that an accelerated rate of gene loss also characterized the Chlamydomonas/Chlorella lineage, a phenomenon that might be independent of the proliferation of SDRs. Together, our results reveal a dynamic and unusual plastid genome whose existence in a model organism will allow its features to be tested functionally.

The Chlamydomonas reinhardtii plastid and mitochondrial transcriptomes were surveyed for changes in RNA profiles resulting from growth in 12 culture conditions representing 8 abiotic stimuli. Organellar RNA abundance exhibited marked changes during nutrient stress and exposure to UV light, as revealed by both RNA gel blot and DNA microarray analyses. Of particular note were large increases in tufA and clpP transcript abundance during nutrient limitation. Phosphate and sulfur limitation resulted in the most global, yet opposite, effects on organellar RNA abundance, changes that were dissected further using run-on transcription assays. Removal of sulfate from the culture medium, which is known to reduce photosynthesis, resulted in 2-fold to 10-fold decreases in transcription rates, which were reflected in lower RNA abundance. The decrease in transcriptional activity was completely reversible and recovered to twice the control level after sulfate replenishment. Conversely, phosphate limitation resulted in a twofold to threefold increase in RNA abundance that was found to be a post-transcriptional effect, because it could be accounted for by increased RNA stability. This finding is consistent with the known metabolic slowdown under phosphate stress. Additionally, inhibitor studies suggested that unlike those in higher plants, Chlamydomonas chloroplasts lack a nucleus-encoded plastid RNA polymerase. The apparently single type of polymerase could contribute to the rapid and genome-wide transcriptional responses observed within the chloroplast.

To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the -1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the -1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Polyadenylation of nucleus-encoded transcripts has a well-defined role in gene expression. The extent and function of polyadenylation in organelles and prokaryotic systems, however, are less well documented. Recent reports of polyadenylation-mediated RNA destabilization in Escherichia coli and in vascular plant chloroplasts prompted us to look for polyadenylation in plant mitochondria. Here, we report the use of reverse transcription-polymerase chain reaction to map multiple polyadenylate addition sites in maize mitochondrial cox2 transcripts. The lack of sequence conservation surrounding these sites suggests that polyadenylation may occur at many 3' termini created by endoribonucleolytic and/or exoribonucleolytic activities, including those activities involved in 3' end maturation. Endogenous transcripts could be efficiently polyadenylated in vitro by using maize mitochondrial lysates with an activity that added AMP more efficiently than GMP. Polyadenylated substrates were tested for stability in maize mitochondrial S100 extracts, and we found that, compared with nonpolyadenylated RNAs, the polyadenylated substrates were less stable. Taken together with the low abundance of polyadenylated RNAs in maize mitochondria, our results are consistent with a degradation-related process. The fact that polyadenylation does not dramatically destabilize plant mitochondrial transcripts, at least in vitro, is in agreement with results obtained for animal mitochondria but differs from those obtained for chloroplasts and E. coli. Because fully edited, partially edited, and unedited transcripts were found among the cloned polyadenylated cox2 cDNAs, we conclude that RNA editing and polyadenylation are independent processes in maize mitochondria.

We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

We have previously used a homologous in vitro transcription system to define functional elements of the maize mitochondrial atpA promoter. These elements comprise a central domain extending from -7 to +5, relative to the transcription start site, and an upstream domain of 1-3 bp that is purine rich and centered around positions -11 to -12. Within the central domain lies an essential 5 bp core element. These elements are conserved in many mitochondrial promoters, but their functionality has only been tested for atpA. In this study we have introduced mutations into the corresponding elements of two cox3 promoters and show that while the core element is essential for cox3 promoter activity, upstream element mutations have little or no effect. To define the minimal sequence required for in vitro promoter activity a series of short cloned oligonucleotides corresponding to the atpA promoter was used. While some activity was seen with a 14 bp sequence, full activity required 26 bp, suggesting that elements other than the core and upstream region can influence promoter strength. Another series of clones showed that altered spacing between the upstream and core elements of atpA had a significant effect on promoter activity. These results further define important features of the plant mitochondrial transcriptional machinery.

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.

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.

Correct 3' processing of chloroplast precursor mRNAs (pre-mRNAs) requires a stem-loop structure within the 3' untranslated region. In spinach, a stable 3' stem-loop-protein complex has been shown to form in vitro between petD pre-mRNA, encoding subunit IV of the cytochrome b6/f complex, and chloroplast proteins. This complex contains three chloroplast stem-loop binding proteins (CSPs), namely, CSP29, CSP41, and CSP55. Here, we report the purification of CSP41 and cloning of the csp41 gene and show that CSP41 is encoded by a single nuclear gene. Characterization of bacterially expressed CSP41 demonstrates that this protein binds specifically to the 3' stem-loop structure and a downstream AU-rich element of petD pre-mRNA and that its binding affinity is enhanced by associating with CSP55. Our data also show that CSP41 has substantial nonspecific endoribonuclease activity. These data suggest that CSP41 could be involved in 3' processing of petD pre-mRNA and/or in RNA degradation. The fact that different reaction conditions favor RNA binding over ribonuclease activities suggests a possible mode of in vivo regulation.

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.

Spinach chloroplast petD gene encodes subunit IV of the cytochrome b6/f complex. Like many chloroplast mRNAs, the spinach petD mRNA contains a 3'UTR stem-loop structure that determines correct 3' processing of the pre-mRNA and stability of the mature mRNA. RNA-protein interactions with this structure may be involved in the regulation of petD mRNA processing and/or stability. In this report, a spinach chloroplast 41 kD protein has been identified as a component of the petD mRNA 3'stem-loop:protein complex. The 41 kD protein has been purified and four internal peptide sequences were obtained. Based on the peptide sequences, a 585 bp fragment was amplified from a spinach cDNA library by PCR. And, a 1404 bp cDNA clone was then isolated by using the initial PCR fragment as probe. Nucleic acid sequence analysis of the cDNA clone identified an ORF encoding a predicted 36 kD mature protein. An apparent N-terminal transit peptide in the coding region and a 3' poly(A) tail exist in the cDNA clone indicated that this chloroplast protein as nuclear encoded. The mature protein was expressed in E. coli, and the expressed product with a molecular mass of 41 kD was purified. A gel mobility shift assay demonstrated that the expressed 41 kD protein interacts with the petD mRNA 3' UTR to form an RNA-protein complex.

Chlamydomonas reinhardtii chloroplast transformants that lack an inverted repeat normally found at the 3[prime] end of the chloroplast atpB gene have a slow phototrophic growth phenotype due to reduced accumulation of atpB mRNA and the chloroplast ATPase [beta] subunit. We have recovered transformants exhibiting more robust phototrophic growth at a moderate frequency (~1% relative to slow-growing transformants). Quantitative DNA blot analysis indicated that in one class of these robust photosynthetic transformants, the introduced plasmid DNA is maintained at high copy number-~25 copies per chloroplast genome or 2000 copies per cell. Partial restriction digests resulted in a ladder with at least 15 visible fragments, indicating that most of the transforming DNA is organized as a long head-to-tail tandem repeat. Total atpB transcription and accumulation of atpB mRNA and the ATPase [beta] subunit were increased approximately fivefold relative to transformants that carry a single copy of the truncated atpB gene. The amplified DNA was stably maintained at high copy number under mixotrophic growth conditions. It was inherited uniparentally from the mt+ parent, and its synthesis was sensitive to 5-fluoro-2[prime]-deoxyuridine, an inhibitor of chloroplast DNA synthesis. Therefore, we conclude that the tandem repeat is maintained in the chloroplast. Restriction enzymes that fail to digest the transforming plasmid but have recognition sites in chloroplast DNA did not alter the electrophoretic mobility of the tandem repeat, suggesting that it is not integrated in the chloroplast genome. We conclude that the tandem repeat is probably episomal and hypothesize that its replication is independent of the chloroplast genome.

The Chlamydomonas reinhardtii chloroplast atpB mRNA contains sequences at its 3' end that can form a complex stem/loop structure. Deletions of part or all of this sequence in transformed C. reinhardtii cells led to decreased atpB mRNA accumulation, whereas transcription rates were unaffected. The reduction of mRNA to 20% to 35% of wild-type levels in transformants without 3' stem/loops was correlated with the accumulation of atpB mRNA that was heterogeneous in size. These results indicated that RNA secondary structures function both in mRNA stabilization and in 3' end formation in C. reinhardtii chloroplasts. Furthermore, deletion of the stem/loop resulted in a decrease in the steady-state level of the ATPase beta-subunit to approximately 60% of wild-type levels, suggesting that translational and/or post-translational mechanisms may influence the steady-state level of the atpB gene product.

To investigate the role of mRNA 3' inverted repeats (IRs) in stabilizing plant chloroplast mRNAs, we have measured the processing and stability of wild-type and mutant RNAs corresponding to the 3' end of the spinach chloroplast psbA mRNA. wild-type and mutant 3' IR-RNA precursors were processed at similar rates in a homologous in vitro system, but RNAs with either a mutant loop sequence CUUCGG or a specific base substitution in the IR exhibited an enhanced accumulation of mature product. Incubation of mature products in the in vitro system demonstrated that this was due to an increased stability of the product. These mutant RNAs displayed the same order of stabilities when their decay was measured following electroporation into intact chloroplasts. We found that the in vitro system contains an endonuclease activity that cleaves the wild-type 3' IR-RNA within the loop and also in single-stranded regions, suggesting a possible role for the loop sequence in determining RNA longevity in vitro. Interestingly, the altered loop sequence CUUCGG, which enhances RNA stability in bacteria (1), prolonged the half-life of psbA 3' IR-RNA in vitro and also resulted in an altered endonuclease cleavage pattern. Such nucleases could potentially play an important role in plastid mRNA decay in vivo.

The organisation of the mitochondrial ribosomal RNA genes in maize is described. Each of the rRNAs is encoded by a single gene. The 5S and 18S rRNA genes are close together, and separated from the 26S rRNA gene by 16 kb of DNA. There is no evidence of heterogeneity in this gene arrangement.

A complete physical map of the spinach mitochondrial genome has been established. The entire sequence content of 327 kilobase pairs (kb) is postulated to occur as a single circular molecule. Two directly repeated elements of approximately 6 kb, located on this "master chromosome", are proposed to participate in an intragenomic recombination event that reversibly generates two "subgenomic" circles of 93 kb and 234 kb. The positions of protein and ribosomal RNA-encoding genes, determined by heterologous filter hybridizations, are scattered throughout the genome, with duplicate 26S rRNA genes located partially or entirely within the 6 kb repeat elements. Filter hybridizations between spinach mitochondrial DNA and cloned segments of spinach chloroplast DNA reveal at least twelve dispersed regions of inter-organellar sequence homology.

Several plant mitochondrial genomes contain repeated sequences that are postulated to be sites of homologous intragenomic recombination (1-3). In this report, we have used filter hybridizations to investigate sequence relationships between the cloned mitochondrial DNA (mtDNA) recombination repeats from turnip, spinach and maize and total mtDNA isolated from thirteen species of angiosperms. We find that strong sequence homologies exist between the spinach and turnip recombination repeats and essentially all other mitochondrial genomes tested, whereas a major maize recombination repeat does not hybridize to any other mtDNA. The sequences homologous to the turnip repeat do not appear to function in recombination in any other genome, whereas the spinach repeat hybridizes to reiterated sequences within the mitochondrial genomes of wheat and two species of pokeweed that do appear to be sites of recombination. Thus, although intragenomic recombination is a widespread phenomenon in plant mitochondria, it appears that different sequences either serve as substrates for this function in different species, or else surround a relatively short common recombination site which does not cross-hybridize under our experimental conditions. Identified gene sequences from maize mtDNA were used in heterologous hybridizations to show that the repeated sequences implicated in recombination in turnip and spinach/pokeweed/wheat mitochondria include, or are closely linked to genes for subunit II of cytochrome c oxidase and 26S rRNA, respectively. Together with previous studies indicating that the 18S rRNA gene in wheat mtDNA is contained within a recombination repeat (3), these results imply an unexpectedly frequent association between recombination repeats and plant mitochondrial genes.