Isolation and Characterization of a Chlamydomonas reinhardtii Mutant Resistant to Photobleaching Herbicides

1993 ◽  
Vol 48 (3-4) ◽  
pp. 339-344 ◽  
Author(s):  
Hiromichi Oshio ◽  
Hideyuki Shibata ◽  
Nobuaki Mito ◽  
Masako Yamamoto ◽  
Elizabeth H. Harris ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Oxidase Activity ◽  
Higher Plants ◽  
Diphenyl Ether ◽  
Protoporphyrin Ix ◽  
Nuclear Genome ◽  
Photosynthetic Electron Transport ◽  
Wild Type ◽  
Protoporphyrinogen Oxidase ◽  
Isolation And Characterization

A group of highly active N -phenylimide photobleaching herbicides have been synthesized. These N -phenylimide herbicides as well as diphenyl ether herbicides induce protoporphyrin IX accumulation and inhibit protoporphyrinogen oxidase activity at extremely low concentrations in higher plants. The binding of a 14C -labeled N -phenylimide herbicide S-23121 [N-[4-chloro- 2-fluoro-5-[(1-m ethyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide] to the solubilized plastid fractions of greening corn seedlings is competed by the diphenyl ether herbicide acifluorfen-ethyl, but not by diuron, an inhibitor of photosynthetic electron transport. These results indicate a similar mode of action for both N -phenylimide and diphenyl ether herbicides.In order to investigate the mechanism of photobleaching herbicides at the molecular level, a strain of Chlamydomonas reinhardtii RS-3 resistant to N -phenylimide S-23142 [N -(4-chloro- 2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide] was isolated by mutagenesis with N -m ethyl-N′-nitro-N -nitrosoguanidine. The 90% inhibition concentration of N -phenylimide S-23142 for growth of RS-3 was 100 times higher than that for wild type. Maximum accumulation of protoporphyrin IX was reached at 0.03 μᴍ of S-23142 for the wild type and 3 μᴍ for RS-3. RS-3 was resistant to oxadiazon, oxyfluorfen and acifluorfen-ethyl which had been shown to have the same mechanism of action as N -phenylimide herbicides, but not to paraquat, diuron or fluridone. Genetic analysis of RS-3 strain showed that the resistance results from a dominant mutation ( rs-3) in the nuclear genome. The magnesium protoporphyrin IX synthesizing activity from 5-am inolevulinic acid in chloroplast fragments isolated from RS-3 was less sensitive to S-23142 than that from wild type (CC-407). Protoporphyrinogen oxidase activity in Percoll™ -purified chloroplasts from RS-3 was also less sensitive to S-23142 than that from wild type. These results indicate that the resistance of RS-3 is specific for photobleaching herbicides, and that the mutation is related to protoporphyrinogen oxidase, the primary site of the photobleaching herbicide action.

Molecular Aspects of Herbicide Action on Protoporphyrinogen Oxidase

1993 ◽  
Vol 48 (3-4) ◽  
pp. 326-333 ◽  
Author(s):  
Beate Nicolaus ◽  
Gerhard Sandmann ◽  
Peter Böger
Keyword(s):  
Diphenyl Ether ◽  
Protoporphyrin Ix ◽  
Protoporphyrinogen Oxidase ◽  
Relative Specificity ◽  
Oxidase Inhibition ◽  
Dilution Experiments ◽  
Membrane Bound ◽  
The Common ◽  
Molecular Aspects ◽  
Derivatives Of

Abstract Protoporphyrinogen oxidase, the last enzyme of the common tetrapyrrole biosynthetic pathway, is inhibited by several peroxidizing compounds resulting in accumulation of photodynamic tetrapyrroles, mainly protoporphyrin IX. The inhibition characteristics of two chemi­cally unrelated compounds were studied using membrane bound protoporphyrinogen oxidase from corn etioplasts. As shown by Lineweaver-Burk-analysis, the inhibition of enzyme activity by the diphenyl ether oxyfluorfen and the cyclic imide MCI 15 are competitive with respect to the substrate. The competitive interaction of protoporphyrinogen and the two chemically un­related inhibitors indicate a relative specificity of the binding site. The reversibility of oxyfluorfen inhibition was evaluated by dilution experiments and was shown to be independent of the presence of DTT. The analysis of structure-activity-relationship on protoporphyrinogen oxidase inhibition was investigated with para-substituted derivatives of phenyl-3,4,5,6-tetrahydro-phthalimides. The results obtained by QSAR -calculation yielded a good correlation of the inhibitory activity determined by the lipophilicity of the para-substituent. These data point to one binding region of the inhibitors within a lipophilic environment associated with the active center of the enzyme.

scholarly journals Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides

1989 ◽  
Vol 260 (1) ◽  
pp. 231-235 ◽  
Author(s):  
M Matringe ◽  
J M Camadro ◽  
P Labbe ◽  
R Scalla
Keyword(s):  
Zea Mays ◽  
Solanum Tuberosum ◽  
Diphenyl Ether ◽  
Protoporphyrin Ix ◽  
Molecular Target ◽  
Plant Tissues ◽  
Mitochondrial Membranes ◽  
Protoporphyrinogen Oxidase ◽  
Cellular Target

Diphenyl ether herbicides induce an accumulation of protoporphyrin IX in plant tissues. By analogy to human porphyria, the accumulation could be attributed to decreased (Mg or Fe)-chelatase or protoporphyrinogen oxidase activities. Possible effects of acifluorfen-methyl on these enzymes were investigated in isolated corn (maize, Zea mays) etioplasts, potato (Solanum tuberosum) and mouse mitochondria, and yeast mitochondrial membranes. Acifluorfen-methyl was strongly inhibitory to protoporphyrinogen oxidase activities whatever their origins [concn. causing 50% inhibition (IC50) = 4 nM for the corn etioplast enzyme]. By contrast, it was roughly 100,000 times less active on (Mg or Fe)-chelatase activities (IC50 = 80-100 microM). Our results lead us to propose protoporphyrinogen oxidase as a cellular target for diphenyl ether herbicides.

Localization of Target-Site of the Protoporphyrinogen Oxidase-Inhibiting Herbicide, S-23142, in Spinacia oleracea L.

1993 ◽  
Vol 48 (3-4) ◽  
pp. 350-355 ◽  
Author(s):  
Fang-Sik Che ◽  
Yoko Takemura ◽  
Naoko Suzuki ◽  
Katsunori Ichinose ◽  
Jim-Ming Wang ◽  
...  
Keyword(s):  
Spinacia Oleracea ◽  
Protoporphyrin Ix ◽  
Target Site ◽  
Synthetic Activity ◽  
Wild Type ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Protoporphyrinogen Oxidase ◽  
Spinacia Oleracea L ◽  
Tolerant Cell

Effects of S-23142 on protoporphyrin IX (Proto IX) biosynthesis in chloroplasts isolated from Spinacia oleracea L. were examined using reverse phase HPLC with fluorescence monitoring. The synthesis of Proto IX was inhibited to a level of 50% by 10-9 ᴍ of S-23142 in this system. The effects of S-23142 was also tested in chloroplasts isolated from two types of photomixotrophic tobacco cells, wild type and S-23142 tolerant cells. The biosynthesis of both the wild type cells and YZI-1 S cells were inhibited at 50% by 10-9 ᴍ and 10-7 ᴍ of S-23142, respectively. It is, therefore evident that the mutation in the tolerant cell is associated with the Protox. To investigate the localization of Proto IX biosynthesis and the target site of S-23142, spinach chloroplasts were osmotically broken and separated into stroma and membrane (thylakoid and envelope) fractions. A very active Proto IX synthesis from ALA was found in the stromal fraction, while no activity of Proto IX synthesis was observed in the membrane fraction. These results suggest that most Proto IX synthetic activity and a target-site of S-23142 exist in the stromal fraction.

scholarly journals An h.p.l.c. assay for protoporphyrinogen oxidase activity in rat liver

1987 ◽  
Vol 243 (3) ◽  
pp. 863-866 ◽  
Author(s):  
F Li ◽  
C K Lim ◽  
T J Peters
Keyword(s):  
Rat Liver ◽  
Oxidase Activity ◽  
Mitochondrial Protein ◽  
Protoporphyrin Ix ◽  
Internal Standard ◽  
Reversed Phase ◽  
Tissue Homogenate ◽  
Protoporphyrinogen Oxidase ◽  
Liver Tissue Homogenate

An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.

Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

2008 ◽  
Vol 411 (2) ◽  
pp. 241-247 ◽  
Author(s):  
María-Jesús García-Murria ◽  
Saeid Karkehabadi ◽  
Julia Marín-Navarro ◽  
Sriram Satagopan ◽  
Inger Andersson ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Large Subunit ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Bisphosphate Carboxylase ◽  
Specificity Factor

Proximal Cys172 and Cys192 in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys172 and Cys192 by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The Vc (Vmax for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the Km for CO2 and O2 was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

scholarly journals Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii.

1989 ◽  
Vol 108 (4) ◽  
pp. 1221-1226 ◽  
Author(s):  
R F Matagne ◽  
M R Michel-Wolwertz ◽  
C Munaut ◽  
C Duyckaerts ◽  
F Sluse
Keyword(s):  
Mitochondrial Dna ◽  
Cytochrome C ◽  
Chlamydomonas Reinhardtii ◽  
Cytochrome C Oxidase ◽  
Oxidase Activity ◽  
Wild Type ◽  
Apocytochrome B ◽  
Cytochrome Pathway ◽  
Apocytochrome B Gene

In addition to lethal minute colony mutations which correspond to loss of mitochondrial DNA, acriflavin induces in Chlamydomonas reinhardtii a low percentage of cells that grow in the light but do not divide under heterotrophic conditions. Two such obligate photoautotrophic mutants were shown to lack the cyanide-sensitive cytochrome pathway of the respiration and to have a reduced cytochrome c oxidase activity. In crosses to wild type, the mutations are transmitted almost exclusively from the mating type minus parent. A same pattern of inheritance is seen for the mitochondrial DNA in crosses between the two interfertile species C. reinhardtii and Chlamydomonas smithii. Both mutants have a deletion in the region of the mitochondrial DNA containing the apocytochrome b gene and possibly the unidentified URFx gene.

Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12

1993 ◽  
Vol 39 (12) ◽  
pp. 1155-1161 ◽  
Author(s):  
Alexandre Sasarman ◽  
Jaroslav Letowski ◽  
Guy Czaika ◽  
Volta Ramirez ◽  
Michael A. Nead ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Oxidase Activity ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Translation System ◽  
Protoporphyrinogen Oxidase ◽  
Escherichia Coli K12 ◽  
Amino Terminal

The hemG gene of Escherichia coli K12 is involved in the activity of protoporphyrinogen oxidase, the enzyme responsible for the conversion of protoporphyrinogen IX into protoporphyrin IX during heme and chlorophyll biosynthesis. The gene is located at min 87 on the genetic map of E. coli K12. The hemG gene was isolated by a mini-Mu in vivo cloning procedure. As expected, the hemG gene is able to restore normal growth to the hemG mutant, and the transformed cells display strong protoporphyrinogen oxidase activity. Sequencing of the hemG gene allowed us to identify an open reading frame of 546 nucleotides (181 amino acids), within the minimal fragment able to complement the mutant. The presumed molecular mass of the HemG protein is 21 202 Da, in agreement with values found by SDS-PAGE, in a DNA-directed coupled transcription–translation system. The identity of the first 18 amino acids at the amino-terminal end of the protein was confirmed by microsequencing. To our knowledge, this is the first cloning of a gene involved in the protoporphyrinogen oxidase activity of E. coli.Key words: protoporphyrinogen oxidase (PROTOX), hemG gene, Escherichia coli, DPE herbicides, heme.

scholarly journals Genome-Based Approaches to Understanding Phosphorus Deprivation Responses and PSR1 Control in Chlamydomonas reinhardtii

2006 ◽  
Vol 5 (1) ◽  
pp. 26-44 ◽  
Author(s):  
Jeffrey L. Moseley ◽  
Chiung-Wen Chang ◽  
Arthur R. Grossman
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Expression Patterns ◽  
Photosynthetic Electron Transport ◽  
Phosphorus Limitation ◽  
Phosphate Transport ◽  
Wild Type ◽  
Sulfur Starvation ◽  
Chloroplast Translation ◽  
Phosphorus Deprivation ◽  
Genes Encoding

ABSTRACT The Chlamydomonas reinhardtii transcription factor PSR1 is required for the control of activities involved in scavenging phosphate from the environment during periods of phosphorus limitation. Increased scavenging activity reflects the development of high-affinity phosphate transport and the expression of extracellular phosphatases that can cleave phosphate from organic compounds in the environment. A comparison of gene expression patterns using microarray analyses and quantitative PCRs with wild-type and psr1 mutant cells deprived of phosphorus has revealed that PSR1 also controls genes encoding proteins with potential “electron valve” functions—these proteins can serve as alternative electron acceptors that help prevent photodamage caused by overexcitation of the photosynthetic electron transport system. In accordance with this finding, phosphorus-starved psr1 mutants die when subjected to elevated light intensities; at these intensities, the wild-type cells still exhibit rapid growth. Acclimation to phosphorus deprivation also involves a reduction in the levels of transcripts encoding proteins involved in photosynthesis and both cytoplasmic and chloroplast translation as well as an increase in the levels of transcripts encoding stress-associated chaperones and proteases. Surprisingly, phosphorus-deficient psr1 cells (but not wild-type cells) also display expression patterns associated with specific responses to sulfur deprivation, suggesting a hitherto unsuspected link between the signal transduction pathways involved in controlling phosphorus and sulfur starvation responses. Together, these results demonstrate that PSR1 is critical for the survival of cells under conditions of suboptimal phosphorus availability and that it plays a key role in controlling both scavenging responses and the ability of the cell to manage excess absorbed excitation energy.

scholarly journals Mg-Protoporphyrin IX and Heme Control HEMA, the Gene Encoding the First Specific Step of Tetrapyrrole Biosynthesis, in Chlamydomonas reinhardtii

2005 ◽  
Vol 4 (10) ◽  
pp. 1620-1628 ◽  
Author(s):  
Zinaida Vasileuskaya ◽  
Ulrike Oster ◽  
Christoph F. Beck
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Posttranscriptional Regulation ◽  
Higher Plants ◽  
Chlorophyll Biosynthesis ◽  
Single Gene ◽  
Protoporphyrin Ix ◽  
Tetrapyrrole Biosynthesis ◽  
Gene Encoding ◽  
Mrna Accumulation ◽  
Specific Step

ABSTRACT HEMA encodes glutamyl-tRNA reductase (GluTR), which catalyzes the first step specific for tetrapyrrole biosynthesis in plants, archaea, and most eubacteria. In higher plants, GluTR is feedback inhibited by heme and intermediates of chlorophyll biosynthesis. It plays a key role in controlling flux through the tetrapyrrole biosynthetic pathway. This enzyme, which in Chlamydomonas reinhardtii is encoded by a single gene (HEMA), exhibits homology to GluTRs of higher plants and cyanobacteria. HEMA mRNA accumulation was inducible not only by light but also by treatment of dark-adapted cells with Mg-protoporphyrin IX (MgProto) or hemin. The specificity of these tetrapyrroles as inducers was demonstrated by the absence of induction observed upon the feeding of protoporphyrin IX, the precursor of both heme and MgProto, or chlorophyllide. The HEMA mRNA accumulation following treatment of cells with light and hemin was accompanied by increased amounts of GluTR. However, the feeding of MgProto did not suggest a role for Mg-tetrapyrroles in posttranscriptional regulation. The induction by light but not that by the tetrapyrroles was prevented by inhibition of cytoplasmic protein synthesis. Since MgProto is synthesized exclusively in plastids and heme is synthesized in plastids and mitochondria, the data suggest a role of these compounds as organellar signals that control expression of the nuclear HEMA gene.

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