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PubMedDate not supplied

Redox regulation and modification of proteins controlling chloroplast gene expression.

Chloroplasts are typical organelles of plant cells and represent the site of photosynthesis. As one very remarkable feature, they possess their own genome and a complete machinery to express the genetic information in it. The plastid gene expression machinery is a unique assembly of prokaryotic-, eukaryotic-, and phage-like components because chloroplasts acquired a great number of regulatory proteins during evolution. Such proteins can be found at all levels of gene expression. They significantly expand the functional and especially the regulatory properties of the "old" gene expression system that chloroplasts inherited from their prokaryotic ancestors. Recent results show that photosynthesis has a strong regulatory effect on plastid gene expression. The redox states of electron transport components, redox-active molecules coupled to photosynthesis, and pools of reactive oxygen species act as redox signals. They provide a functional feedback control, which couples the expression of chloroplast genes to the actual function of photosynthesis and, by this means, helps to acclimate the photosynthetic process to environmental cues. The redox signals are mediated by various specific signaling pathways that involve many of the "new" regulatory proteins. Chloroplasts therefore are an ideal model to study redox-regulated mechanisms in gene expression control. Because of the multiple origins of the expression machinery, these observations are of great relevance for many other biological systems.

Chloroplasts↗
PubMed2006

Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period.

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.

Animals↗
PubMed1996

Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii.

The biosynthesis of the photosynthetic apparatus depends on the concerted action of the nuclear and chloroplast genetic systems. Numerous nuclear and chloroplast mutants of Chlamydomonas deficient in photosynthetic activity have been isolated and characterized. While several of these mutations alter the genes of components of the photosynthetic complexes, a large number of the mutations affect the expression of chloroplast genes involved in photosynthesis. Most of these mutations are nuclear and only affect the expression of a single chloroplast gene. The mutations examined appear to act principally at post-transcriptional steps such as RNA stability, RNA processing, cis- and trans-splicing and translation. Directed chloroplast DNA surgery through biolistic transformation has provided a powerful tool for identifying important cis elements involved in chloroplast gene expression. Insertion of chimeric genes consisting of chloroplast regulatory regions fused to reporter genes into the chloroplast genome has led to the identification of target sites of the nuclear-encoded functions affected in some of the mutants. Biochemical studies have identified a set of RNA-binding proteins that interact with the 5'-untranslated regions of plastid mRNAs. The binding activity of some of these factors appears to be modulated by light and by the growth conditions.

Animals↗
PubMedDate not supplied

Participation of nuclear genes in chloroplast gene expression.

The expression of the plastid genome is dependent on a large number of nucleus-encoded factors. Some of these factors have been identified through biochemical assays, and many others by genetic screens in Arabidopsis, Chlamydomonas and maize. Nucleus-encoded factors function in each step in plastid gene expression, including transcription, RNA editing, RNA splicing, RNA processing, RNA degradation, and translation. Many of the factors discovered via biochemical approaches play general roles as components of the basic gene expression machinery, whereas the majority of those identified by genetic approaches are specifically required for the expression of small subsets of chloroplast genes and are involved in post-transcriptional steps. Some of the nucleus-encoded factors may play regulatory roles and modulate chloroplast gene expression in response to developmental or environmental cues. They may also serve to couple chloroplast gene expression with the assembly of the protein products into the large complexes of the photosynthetic apparatus. The convergence of biochemical approaches with those of classical and reverse genetics, and the contributions from large scale genomic sequencing should result in rapid advances in our understanding of the regulatory interactions that govern plastid gene expression.

Cell Nucleus↗
PubMed1990

Cell cycle-dependent transcriptional and post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii.

The regulated expression of five chloroplast genes in Chlamydomonas reinhardtii during a 24 hour cell cycle (12 hours light, 12 hours dark) was analyzed. Transcription rates of the genes encoding the two reaction center proteins of Photosystem I (psaA, psaB), the subunits alpha and beta (atpA, atpB) of chloroplast ATP synthase and for chloroplast elongation factor tu (EF-tu) were measured during the cell cycle. All genes are maximally transcribed at the beginning of the light period. Transcription was induced before the onset of illumination by a light-independent mechanism. Transcript abundance of the same genes during the cell cycle was determined by quantification of Northern blots hybridized with gene-specific probes. The atpA, atpB and psaB mRNAs were most abundant in the first 6 hours of the light period and decreased to about 15% of maximum in the dark. The abundance of psaA mRNA showed less variation and was maximal around the middle of the cell cycle. The EF-tu mRNA showed a maximum early in the light period, but decreased to almost undetectable levels in the second half of the light period. Because of the similar transcriptional patterns observed, the differential steady state levels of these chloroplast transcripts appeared to be regulated at the post-transcriptional level.

Blotting, Northern↗
PubMed2005

Contribution of 5'- and 3'-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes.

Expression of chloroplast genes is primarily regulated posttranscriptionally, and a number of RNA elements, found in either the 5'- or 3'-untranslated regions (UTRs) of plastid mRNAs, that impact gene expression have been identified. Complex regulatory and feedback mechanisms influence both translation and protein accumulation, making assignment of roles for specific RNA elements difficult. To identify specific contributions made by various UTRs on translation of plastid mRNAs, we used a heterologous gfp reporter gene that is fused combinatorially to chloroplast 5'- and 3'-UTRs. In general, the 5'-UTR, including the promoter, of the plastid atpA and psbD genes produced the highest levels of chimeric mRNA and protein accumulation, while the 5'-UTR of the rbcL and psbA genes produced less mRNA and protein. Varying the 3'-UTR had little impact on mRNA and protein accumulation, as long as a 3'-UTR was present. Overall, accumulation of chimeric mRNAs was proportional to protein accumulation, with a few notable exceptions. Light-regulated translation continues to operate in chimeric mRNAs containing the 5'-UTR of either the psbA or psbD mRNAs, despite translation of these two chimeric mRNAs at very different efficiencies, suggesting that translational efficiency and light-regulated translation are separate events. Translation of some chimeric mRNAs was much more efficient than others, suggesting that interactions between the untranslated and coding sequences can dramatically impact translational efficiency.

3' Untranslated Regions↗
PubMed2002

Translational regulations as specific traits of chloroplast gene expression.

Studies of protein synthesis in the chloroplast compartment have revealed a unique combination of translational autoregulations and trans-regulations due to the delivery of a variety of nuclear factors that act post-transcriptionally. We show how these two characteristics concur to set the major step in the regulation of chloroplast gene expression at the translational level, leading to a surprisingly low sensitivity of chloroplast protein synthesis in response to extensive changes in plastome copy number and transcript concentration.

Animals↗
PubMed1998

A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplast gene expression.

Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane. Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane. A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane. Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role. To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene. Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA. Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA. The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and DeltapH-dependent thylakoid-targeting pathways are disrupted. Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways. CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.

Amino Acid Sequence↗
PubMed2002

Transcript abundance supercedes editing efficiency as a factor in developmental variation of chloroplast gene expression.

In maize plastids, transcripts are known to be modified at 27 C-to-U RNA editing sites, affecting the expression-of 15 different genes. The relative contribution of editing efficiency versus transcript abundance in regulation of chloroplast gene expression has previously been analyzed for only a few genes. We undertook a comprehensive analysis of the editing efficiency of each of the 27 maize editing sites in 10 different maize tissues, which contain a range of plastid types including chloroplasts, etioplasts, and amyloplasts. Using a reproducible poisoned primer extension assay, we detected variation between RNA editing extent of different sites in the same transcript in the same tissue, and between the same site in different tissues. The most striking editing deficiency is in an editing site in ndhB that is edited at only 8% and 1% in roots and callus plastids respectively, whereas green leaf chloroplasts edit this site at 100%. Editing efficiencies of some sites are not affected by the developmental stages we examined and are always edited close to 80-100%. The relative amounts of transcripts of each of the 10 genes that exhibited variable editing extents were determined by real-time PCR. Seven genes exhibited over 100 times lower transcript abundance in either roots or tissue-cultured cells relative to green leaf tissue. The quantitative analysis indicates that a particular editing site can be efficiently edited over a large range of transcript abundance, resulting in no general correlation of transcript abundance and editing extent. The independent variation of editing efficiency of different sites within the same transcript fits with a model that postulates individual trans-acting factors specific to each editing site. Because tissues where editing efficiency at certain sites is low invariably also exhibited greatly decreased abundance of the transcripts carrying those sites, decrease in the amounts of particular RNAs rather than a lack of editing is predicted to have the most significant impact on gene expression under steady-state conditions. Our data is consistent with the hypothesis that the role of editing in angiosperm plastids is to correct otherwise detrimental mutations rather than to generate significant protein diversity.

Amino Acid Sequence↗
PubMed1997

hcf5, a nuclear photosynthetic electron transport mutant of Arabidopsis thaliana with a pleiotropic effect on chloroplast gene expression.

A photosynthetic mutant of Arabidopsis thaliana, hcf5, was isolated by screening M2 seedlings for high chlorophyll fluorescence. Thylakoid morphology was strikingly abnormal, with large grana stacks and almost no stroma lamellae. Fluorescence induction kinetics, activity assays, and immunoblotting showed that photosystem II was absent. Polypeptides of the photosystem I complex, the Cyt b6/f complex, coupling factor, and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase were also severely depleted. However, the nuclear-encoded chlorophyll a/b light-harvesting complex polypeptides were unaffected. The rbcL transcript was present at very low levels, the pattern of transcripts from the polycistronic psbB-psbH-petB-petD operon was abnormal, and the mature psbH message was almost completely lacking. This suggests that the hcf5 locus may encode a product required for the correct expression of several chloroplast genes.

Arabidopsis↗
PubMed2004

PPR motifs of the nucleus-encoded factor, PGR3, function in the selective and distinct steps of chloroplast gene expression in Arabidopsis.

Plastid gene expression is regulated by a variety of nuclear genes. We have isolated Arabidopsis thaliana proton gradient regulation 3 (pgr3) mutants, which display aberrant chlorophyll fluorescence because of defects in chloroplast gene expression. High chlorophyll fluorescence (HCF) because of a reduced level of the cytochrome b6/f complex was observed in two alleles, pgr3-1 and pgr3-2 but not in pgr3-3. In contrast, a transient increase in fluorescence after turning off the actinic light, which was ascribed to chloroplast NADPH dehydrogenase (NDH) activity, was impaired in pgr3-1 and pgr3-3 but not in pgr3-2. Both phenotypes were complemented by the introduction of a single gene, PGR3, encoding a protein containing 27 pentatrico-peptide repeat (PPR) motifs. PPR motifs are present in proteins functioning in the post-transcriptional regulation of organellar gene expression. The conserved threonine in the motif was substituted by isoleucine in the 15th and 12th PPR motifs in pgr3-1 and pgr3-2, respectively, and the conserved leucine by phenylalanine in the final incomplete motif of pgr3-3. We consider that the different domains of the PPR repeats in PGR3 might have different functions in conferring RNA stability and probably allowing translation as well as recognizing at least two distinct target RNAs.

Amino Acid Motifs↗
PubMed2002

Post-transcriptional control of chloroplast gene expression. Accumulation of stable psaC mRNA is due to downstream RNA cleavages in the ndhD gene.

Intergenic cleavages, intron splicing, and editing of primary transcripts of the plastid ndhH-D operon produce multiple overlapping RNAs, of which the most abundant by far is the monocistronic 400-nucleotide mRNA of psaC (encoding the PsaC protein of photosystem I), in contrast with the low level of transcripts of the six ndh genes. Like other plastid operons containing genes for functionally unrelated proteins, the contrasting accumulation of ndh and psaC transcripts provides a model to investigate the mechanisms of the post-transcriptional control of gene expression, a feature of chloroplast genetic machinery, with a minimum of interference by transcriptional control. In leek (Allium porrum L), the ndhD transcript (which follows the psaC gene and ends the ndhH-D operon) requires C --> U editing to restore its start codon and may be used as a marker for the processing of psaC and ndhD transcripts. By determining the editing state and 5' end sequences of specific transcripts, we demonstrated that stable monocistronic psaC mRNA results from downstream cleavages in the ndhD sequence, which renders non-functional ndhD transcripts as by-products. Alternative psaC-ndhD intergenic cleavages produce complete mRNAs for both genes, but only take place in precursors containing editing-restored ndhD start codons. Hence, post-transcriptional control acts by promoting the ndhD cleavage alternative, which allows the accumulation of psaC mRNA at the expense of ndhD mRNA levels.

Base Sequence↗
PubMed1996

Regulation of gene expression in chloroplasts of higher plants.

Chloroplasts contain their own genetic system which has a number of prokaryotic as well as some eukaryotic features. Most chloroplast genes of higher plants are organized in clusters and are cotranscribed as polycistronic pre-RNAs which are generally processes into many shorter overlapping RNA species, each of which accumulates of steady-state RNA levels. This indicates that posttranscriptional RNA processing of primary transcripts is an important step in the control of chloroplast gene expression. Chloroplast RNA processing steps include RNA cleavage/trimming, RNA splicing, ENA editing and RNA stabilization. Several chloroplast genes are interrupted by introns and therefore require processing for gene function. In tobacco chloroplasts, 18 genes contain introns, six for tRNA genes and 12 for protein-encoding genes. A number of specific proteins and RNA factors are believed to be involved in splicing and maturation of pre-RNAs in chloroplasts. Processing enzymes and RNA-binding proteins which could be involved in posttranscriptional steps have been identified in the last several years. Our current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are also considered.

Chloroplasts↗
PubMed2003

Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii.

An exogenous gene, placed between the 5'-upstream regions of the Chlamydomonas reinhardtii chloroplast genes, rbcL or psbA, and the 3'-end of the rbcL gene, do not have the same expression pattern as endogenous genes in the C. reinhardtii chloroplast. Here, we chose four chloroplast genes, rbcL, psbA, psbD and atpA, and examine the effects of chloroplast gene coding regions on gene expression in C. reinhardtii. We constructed chimeric genes composed of the promoter, 5'- and 3'-untranslated regions, varying lengths of protein coding regions of the chloroplast genes, and the bacterial beta-glucuronidase (GUS) gene (uidA) as a reporter gene, and introduced into chloroplast genomes. The transformants, which contained the rbcL-uidA and psbA-uidA chimeric genes fused to the coding region of each gene, showed high expression of uidA mRNA as compared with the previously generated transformants, RG and PG, in which uidA was only fused to the promoter and 5'-UTR of each gene. The difference in the accumulation of uidA transcripts among the transformants was the result of different rates of transcription. This result indicates that the coding region is necessary for sufficient expression of rbcL and psbA. On the other hand, the psbD and atpA coding region portions did not affect chimeric gene expression.

Journal Article↗
PubMed1999

Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression.

The maize nuclear gene crp1 is required for the translation of the chloroplast petA and petD mRNAs and for the processing of the petD mRNA from a polycistronic precursor. In order to understand the biochemical role of the crp1 gene product and the interconnections between chloroplast translation and RNA metabolism, the crp1 gene and cDNA were cloned. The predicted crp1 gene product (CRP1) is related to nuclear genes in fungi that play an analogous role in mitochondrial gene expression, suggesting an underlying mechanistic similarity. Analysis of double mutants that lack both chloroplast ribosomes and crp1 function indicated that CRP1 activates a site-specific endoribonuclease independently of any role it plays in translation. Antibodies prepared to recombinant CRP1 were used to demonstrate that CRP1 is localized to the chloroplast stroma and that it is a component of a multisubunit complex. The CRP1 complex is not associated detectably with either chloroplast membranes or chloroplast ribosomes. Models for CRP1 function and its relationship to other activators of organellar translation are discussed.

Amino Acid Sequence↗