Molecular cloning and nucleotide sequence of the petG gene of the cyanelle genome of Cyanophora paradoxa.
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Amino Acid Sequence↗
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Nucleotide sequence of the 5.2 kbp barley chloroplast DNA fragment, containing psbB-psbH-petB-petD gene cluster.
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Amino Acid Sequence↗
Nucleotide sequence of the chloroplast petD gene of Chlamydomonas eugametos.
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Amino Acid Sequence↗
Endonucleolytic activation directs dark-induced chloroplast mRNA degradation.
Plastid mRNA stability is tightly regulated by external signals such as light. We have investigated the biochemical mechanism responsible for the dark-induced decrease of relative half-lives for mRNAs encoding photosynthetic proteins. Protein fractions isolated from plastids of light-grown and dark-adapted plants correctly reproduced an RNA degradation pathway in the dark that is downregulated in the light. This dark-dependent pathway is initiated by endonucleolytic cleavages in the petD mRNA precursor substrate proximal to a region that can fold into a stem-loop structure. Polynucleotide phosphorylase (PNPase) polyadenylation activity was strongly increased in the protein fraction isolated from plastids in dark-adapted plants, but interestingly PNPase activity was not required for the initiation of dark-induced mRNA degradation. A protein factor present in the protein fraction from plastids of light-grown plants could inactivate the endonuclease activity and thereby stabilize the RNA substrate in the protein fraction from plastids of dark-adapted plants. The results show that plastid mRNA stability is effectively controlled by the regulation of a specific dark-induced RNA degradation pathway.
3' Untranslated Regions↗
Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily.
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.
Base Sequence↗
Photosynthetic electron transport controls expression of the high light inducible gene in the cyanobacterium Synechococcus elongatus strain PCC 7942.
The hliA gene of Synechococcus, encoding a photoprotective high light inducible polypeptide, is up-regulated by high light (HL) or low intensity blue/UV-A light (BL). hliA expression was found to be up-regulated by KCN in low light (LL) (but not in the dark), and up-regulation in HL, BL, and LL (with KCN) was inhibited by 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone. A working hypothesis is proposed whereby up-regulation is in response to the reduced state of cytochrome b(6)f or a carrier beyond in photosynthesis. Modest up-regulation occurs in LL by treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but this is related to effects on hliA mRNA stability rather than on transcription.
Bacterial Proteins↗
Flexibility in photosynthetic electron transport: a newly identified chloroplast oxidase involved in chlororespiration.
Besides electron transfer reactions involved in the 'Z' scheme of photosynthesis, alternative electron transfer pathways have been characterized in chloroplasts. These include cyclic electron flow around photosystem I (PS I) or a respiratory chain called chlororespiration. Recent work has supplied new information concerning the molecular nature of the electron carriers involved in the non-photochemical reduction of the plastoquinone (PQ) pool. However, until now little is known concerning the nature of the electron carriers involved in PQ oxidation. By using mass spectrometric measurement of oxygen exchange performed in the presence of 18O-enriched O2 and Chlamydomonas mutants deficient in PS I, we show that electrons can be directed to a quinol oxidase sensitive to propyl gallate but insensitive to salicyl hydroxamic acid. This oxidase has immunological and pharmacological similarities with a plastid protein involved in carotenoid biosynthesis.
Animals↗
Quantum Yields and Rate Constants of Photochemical and Nonphotochemical Excitation Quenching (Experiment and Model).
Sunflower (Helianthus annuus L.), cotton (Gossypium hirsutum L.), tobacco (Nicotiana tabacum L.), sorghum (Sorghum bicolor Moench.), amaranth (Amaranthus cruentus L.), and cytochrome b6f complex-deficient transgenic tobacco leaves were used to test the response of plants exposed to differnt light intensities and CO2 concentrations before and after photoinhibition at 4000 [mu]mol photons m-2 s-1 and to thermoinhibition up to 45[deg]C. Quantum yields of photochemical and nonphotochemical excitation quenching (YP and YN) and the corresponding relative rate constants for excitation capture from the antenna-primary radical pair equilibrium system (k[prime]P and k[prime]N) were calculated from measured fluorescence parameters. The above treatments resulted in decreases in YP and K[prime]P and in approximately complementary increases in YN and K[prime]N under normal and inhibitory conditions. The results were reproduced by a mathematical model of electron/proton transport and O2 evolution/CO2 assimilation in photosynthesis based on budget equations for the intermediates of photosynthesis. Quantitative differences between model predictions and experiments are explainable, assuming that electron transport is organized into domains that contain relatively complete electron and proton transport chains (e.g. thylakoids). With the complementation that occurs between the photochemical and nonphotochemical excitation quenching, the regulatory system can constantly maintain the shortest lifetime of excitation necessary to avoid the formation of chlorophyll triplet states and singlet oxygen.
Journal Article↗
The light reactions: a guide to recent acquisitions for the picture gallery.
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Cytochrome b6f Complex↗
Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators.
In Chlamydomonas reinhardtii, cytochrome c6 (cyt c6) is synthesized only under conditions of copper deficiency when plastocyanin cannot be synthesized. In previous work, the copper-responsive regulation of cyt c6 synthesis was demonstrated to occur by control of transcription, with no contribution from post-transcriptional processes. To understand the mechanism underlying its regulation, the genomic DNA encoding cyt c6 (Cyc6) was analyzed for the presence of copper-responsive elements. Sequences lying between positions -127 and -7 with respect to the start site of transcription were found to be sufficient to confer copper-responsive expression on either a promoterless or a minimal beta-tubulin promoter-driven (arylsulfatase-encoding) reporter gene. Analysis of this 120-bp fragment indicated that copper-responsive elements lie in two distinct regions (between -110 to -56 and -127 to -109). ATG fusions between copper-insensitive promoters and the coding plus 3' untranslated region of the Cyc6 gene resulted in the accumulation of cyt c6 in copper-supplemented medium; this confirms earlier studies indicating a lack of post-transcriptional control in this copper-responsive pathway. In the context of a constitutive promoter (derived from the beta-tubulin gene), each region was found to function as an activator of transcription in copper-deficient cells, and the metal specificity of the response of reporter genes containing either one or both regions was identical to that of the endogenous Cyc6 gene. The copper-responsive synthesis of cyt c6 is thus attributed to these two 5' upstream sequences.
Animals↗
Estimation of chloroplast macromolecular complex copy numbers and subunit stoichiometries during the Chlamydomonas reinhardtii cell cycle.
An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg cell-1 of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis, and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05 × 106 molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08 × 106 complexes per cell. PSI averages 1.75 × 106 complexes per cell and cytochrome b6f averages 0.77 × 106 complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artifacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.
Chlamydomonas reinhardtii↗
Chlamydomonas reinhardtii in the landscape of pigments.
This review focuses on the biosynthesis of pigments in the unicellular alga Chlamydomonas reinhardtii and their physiological and regulatory functions in the context of information gathered from studies of other photosynthetic organisms. C. reinhardtii is serving as an important model organism for studies of photosynthesis and the pigments associated with the photosynthetic apparatus. Despite extensive information pertaining to the biosynthetic pathways critical for making chlorophylls and carotenoids, we are just beginning to understand the control of these pathways, the coordination between pigment and apoprotein synthesis, and the interactions between the activities of these pathways and those for other important cellular metabolites branching from these pathways. Other exciting areas relating to pigment function are also emerging: the role of intermediates of pigment biosynthesis as messengers that coordinate metabolism in the chloroplast with nuclear gene activity, and the identification of photoreceptors and their participation in critical cellular processes including phototaxis, gametogenesis, and the biogenesis of the photosynthetic machinery. These areas of research have become especially attractive for intensive development with the application of potent molecular and genomic tools currently being applied to studies of C. reinhardtii.
Animals↗
Extensive RNA editing in transcripts from the PsbB operon and RpoA gene of plastids from the enigmatic moss Takakia lepidozioides.
RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.
Base Sequence↗
Topography of the chloroplast cytochrome b6: orientation of the cytochrome and accessibility of the lumen-side interhelix loops.
The topography of chloroplast cytochromes f and b6 was probed with proteases carboxypeptidase A (CpA), trypsin, and Staph, aureus V8. The cytochrome and its proteolytic products were detected by heme stain and, in most experiments, by immunoreaction. In thylakoids, the only protease that significantly affected the intactness of cytochrome f was CpA that caused a small (delta Mr = -1-2000) decrease in the apparent molecular weight. In SDS-treated thylakoids, both trypsin and V8 degraded cytochrome f. The inferred topography of cytochrome f., with the COOH-terminus on the stromal (n) side, one membrane-spanning alpha-elix near the COOH-terminus, and most of the Cyt f mass on the lumen (p) side, is consistent with that previously inferred by others. Cytochrome b6 was not sensitive to CpA, but was more sensitive to trypsin and V8 protease than cytochrome f, cytochrome b-559, or the 17 kDa OEC extrinsic protein. Trypsin caused a small decrease in size of cytochrome b6, which was observed using whole protein antibody as a single smaller band (delta Mr approximately 2000) or two smaller discrete bands (delta Mr = -1000 and 2500, respectively) which, unlike the untreated protein, did not react with antibody generated to a peptide mimicking Asp-5-Gln-14 near the NH2-terminus. These shortened tryptic fragments were attributed to cleavage after R-10 and K-23 near the NH2-terminus, implying an orientation with the NH2-terminus on the stromal side of the membrane. The sensitivity of cytochrome b6 toward this trypsin cleavage was increased if the membranes were first incubated with CpA, showing that the NH2-terminal region of cytochrome b6 is masked by the COOH-terminal domain of one or more thylakoid proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Photoinactivation of photosystem II and degradation of the D 1 protein are reduced in a cytochrome b6/f-less mutant of Chlamydomonas reinhardtii.
The effect of unoccupancy of the QB site by plastoquinone on the photoinactivation of reaction center II in a Cyt b6/f-less mutant of Chlamydomonas reinhardtii, B6, was investigated. In these cells the oxidation of plastoquinol generated by electron flow via RC II to plastoquinone and thus the turnover of PQH2/PQ via the QB site are drastically reduced. Reaction center II of the mutant cells was resistant to photoinactivation relative to the control cells as demonstrated by measurements of light-induced destabilization of S2-QB- charge recombination, rise in intrinsic fluorescence and loss of variable fluorescence. These parameters relate to functions involving the reaction center II D 1 protein. The light-induced degradation of D 1 in the mutant cells was also considerably reduced, with a t1/2 value of 7 h as compared, under similar conditions, to about 1.5 h for the control cells. These results indicate that the photoinactivation of RC II and turnover of the D 1 protein are related and require occupancy of the QB site by PQ and its light-driven reduction.
Chlamydomonas↗
Effects of tamoxifen and levonorgestrel treatment on carbon tetrachloride induced alterations in rats.
Oestrogens cause progression in liver lesion, and thus ovariectomy improves hepatic injury both functionally and histologically. In the present study the efficacy of the antioestrogen tamoxifen (CAS 10540-29-1) was examined in chronical carbon tetrachloride damage. The results were compared with the effect of the progestogen levonorgestrel. Male Wistar rats were treated for 16 days. Blood sampling and autopsy were performed 2 h after last tamoxifen resp. levonorgestrel and 1 h after last CCl4 dosage. Tamoxifen diminished water content of the healthy liver. By fairly the same ratio it decreased high water content of the damaged liver. Levonorgestrel moderated water imbibition in CCl4 impairment. Tamoxifen caused protein synthesis in healthy and in injured liver. Levonorgestrel could not prevent protein loss associated with CCl4 damage. Tamoxifen counteracted, levonorgestrel moderated glycogen loss in liver lesion. Blood glucose was normal in all examined groups. Cytochrome P-450 decrease in CCl4 injury was moderated but not normalised by tamoxifen. Levonorgestrel was less effective. Cytochrome b5 content diminished in CCl4 lesion and both treatments restored it. Aminopyrine-N-demethylase was impaired by liver injury. An improvement was measured correlating with microsomal cytochrome P-450 content. This was significant with tamoxifen but not following levonorgestrel administration. The pathological serum bilirubin level of CCl4 lesion was normalised by tamoxifen as well as levonorgestrel treatment. The progestogen levonorgestrel moderated liver injury in reducing high water content, glycogen loss and normalising serum bilirubin. The antioestrogen tamoxifen seems to be a promising treatment in chronic hepatic impairment.(ABSTRACT TRUNCATED AT 250 WORDS)
Aminopyrine N-Demethylase↗
Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding.
Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work (Stern, D. B., and Gruissem, W. (1987) Cell 51, 1145-1157), we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability.
Base Sequence↗
Polyadenylation accelerates degradation of chloroplast mRNA.
The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation.
Chloroplasts↗