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The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni2+-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes. 相似文献
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Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability. 总被引:1,自引:0,他引:1
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A saturable and temperature-dependent copper uptake pathway has been identified in Chlamydomonas reinhardtii. The uptake system has a high affinity for copper ions (Km approximately 0.2 microM) and is more active in cells that are adapted to copper deficiency than to cells grown in a medium containing physiological (submicromolar to micromolar) copper ion concentrations. The maximum velocity of copper uptake by copper-deficient cells (169 pmol h-1 10(6) cells-1 or 62 ng min-1 mg-1 chlorophyll) is up to 20-fold greater than that of fully copper-supplemented cells, and the Km (approximately 2 x 10(2) nM) is unaffected. Thus, the same uptake system appears to operate in both copper-replete and copper-deficient cells, but its expression or activity must be induced under copper-deficient conditions. A cupric reductase activity is also increased in copper-deficient compared with copper-sufficient cells. The physiological characteristics of the regulation of this cupric reductase are compatible with its involvement in the uptake pathway. Despite the operation of the uptake pathway under both copper-replete and copper-deficient conditions, C. reinhardtii cells maintained in fully copper-supplemented cells do not accumulate copper in excess of their metabolic need. These results provide evidence for a homeostatic mechanism for copper metabolism in C. reinhardtii. 相似文献
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Alejandro Torrado Carmen Ramírez-Moncayo José A. Navarro Vicente Mariscal Fernando P. Molina-Heredia 《BBA》2019,1860(1):60-68
Cytochrome c6 is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b6-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c6 and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c6 is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c6 has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c6 deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c6 plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin. 相似文献
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Scenedesmus acutus, pregrown autotrophically, forms high levels of plastocyanin during an initiation period of 24 hr in the light, after cupric ions have been added to depleted cells. In the dark, no plastocyanin formation is observed. This is in contrast to heterotrophic cells which, over the same period and under identical conditions, yield about half the plastocyanin level regardless of whether they are incubated in the dark or in the light. The ATP level is high in all cases when substantial formation of plastocyanin takes place, while the 35S-sulfolipid level, the marker of thylakoid formation, is at variance. After the 24-hr plastocyanin formation period, the 35S-sulfolipid level is high in illuminated autotrophic cells as well as in dark-incubated heterotrophic ones, but low in illuminated heterotrophic cells, all of them having substantial plastocyanin contents. Additional experiments on light dependence of copper uptake, influence of light quality, occurrence of apoplastocyanins, provide a set of data to assume that the role of light in plastocyanin formation is primarily through photophosphorylation, the ATP presumably being necessary for processing of plastocyanin precursors to holoprotein. 相似文献
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Transit Peptide Mutations That Impair in Vitro and in Vivo
Chloroplast Protein Import Do Not Affect Accumulation of the
γ-Subunit of Chloroplast ATPase
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We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ. 相似文献
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The Crd1 gene encodes a putative di-iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii
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Chlamydomonas reinhardtii adapts to copper deficiency by degrading apoplastocyanin and inducing Cyc6 and Cpx1 encoding cytochrome c(6) and coproporphyrinogen oxidase, respectively. To identify other components in this pathway, colonies resulting from insertional mutagenesis were screened for copper- conditional phenotypes. Twelve crd (copper response defect) strains were identified. In copper-deficient conditions, the crd strains fail to accumulate photosystem I and light-harvesting complex I, and they contain reduced amounts of light-harvesting complex II. Cyc6, Cpx1 expression and plastocyanin accumulation remain copper responsive. The crd phenotype is rescued by a similar amount of copper as is required for repression of Cyc6 and Cpx1 and for maintenance of plastocyanin at its usual stoichiometry, suggesting that the affected gene is a target of the same signal transduction pathway. The crd strains represent alleles at a single locus, CRD1, which encodes a 47 kDa, hydrophilic protein with a consensus carboxylate-bridged di-iron binding site. Crd1 homologs are present in the genomes of photosynthetic organisms. In Chlamydomonas, Crd1 expression is activated in copper- or oxygen-deficient cells, and Crd1 function is required for adaptation to these conditions. 相似文献
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The cell wall–lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the Tn5 transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (106 mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 103 HygR transformants per 106 recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation ofCh. reinhardtii with heterologous genes are discussed. 相似文献
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《Process Biochemistry》2004,39(11):1685-1691
Cells of CW-15 mutant of Chlamydomonas reinhardtii without a cell wall were transformed by electroporation. The hpt gene of hygromycin phosphotransferase was used as a selective marker. Optimal conditions of transformation were observed in the middle of the logarithmic growth phase at the density of suspension 106 cells/ml, electric field intensity 1 kV/cm, and pulse duration 2 ms. Under these conditions up to 103 hygromycin-resistant clones of trasformants per 106 recipient cells were obtained that was 100 times higher than at the usage of wild-type cells. Exogenic DNA integrated into the genome of the nucleus C. reinhardtii was constantly inherited for more than 350 generations. The use of mutants without a cell wall and certain selective systems enable the efficiency of transformant yield to be doubled problems on unstable expression of geterologous genes to be investigated, and ways of obtaining super producers of foreign proteins using the alga C. reinhardtii investigated. 相似文献
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Most organisms performing oxygenic photosynthesis contain either cytochrome c
6 or plastocyanin, or both, to transfer electrons from cytochrome b
6-f to photosystem I. Even though plastocyanin has superseded cytochrome c
6 along evolution, plants contain a modified cytochrome c
6, the so called cytochrome c
6A, whose function still remains unknown. In this article, we describe a second cytochrome c
6 (the so called cytochrome c
6-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c
6A than to cyanobacterial cytochrome c
6. In this article, we conclude that the cytochrome c
6-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c
6 and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically
in the absence of both cytochrome c
6 and plastocyanin. In any way, the Cyt c
6-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles. 相似文献
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1. Chloroplast fragments from either Chlamydomonas reinhardi or spinach, which lack plastocyanin, or from Euglena gracilis depleted of cytochrome c552, require a large excess of exogenously added plastocyanin or cytochrome c552 to restore Photosystem I activity.2. In the presence of a small amount of polylysine, Photosystem I activity of chloroplast fragments is stimulated greatly by plastocyanin or cytochrome c552, and the reaction is saturated at a lower concentration of these proteins. Higher concentrations of polylysine inhibit Photosystem I activity; the inhibition is not reversed by plastocyanin or cytochrome c552.3. Salt protects chloroplast fragments from stimulation by polylysine plus plastocyanin or cytochrome c552, and also reverses this stimulation.4. The data suggest that polylysine, at low concentration, enhances binding of plastocyanin or cytochrome c552 to chloroplast membranes, thereby increasing the effective concentration at their site of function. The total inhibition of Photosystem I activity, independent of the presence of plastocyanin or cytochrome c552, at higher polylysine concentrations is similar probably to that observed previously in chloroplasts which retain their plastocyanin. 相似文献
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Harold G. Weger 《Planta》1999,207(3):377-384
Cells of the green alga Chlamydomonas reinhardtii Dangeard were grown in Fe-limited chemostat culture over a range of growth rates (0.15–1.5 d−1). Greater cell densities and culture chlorophyll levels were achieved using an excess of chelator [ethylenediamine di-(o-hydroxyphenylacetic acid)] relative to FeCl3 (80:1), compared to growth using a 1:1 chelator:FeCl3 ratio. The C. reinhardtii cells reduced extracellular ferric chelates, and ferric chelate reductase activity increased with increasing Fe-limited growth
rates. However Fe-sufficient cells exhibited a low rate of ferric chelate reductase activity, similar to severely Fe-limited
cells. Iron-limited cells were capable of reducing a wide variety of ferric chelates, representing a wide range of stability
constants, at similar rates, suggesting that the stability constants of ferric complexes are not important determinants of
ferric reducing activity. Cupric reductase activity also increased with increasing Fe-limited growth rates, and Cu(II) was
preferentially reduced compared to Fe(III). These results suggest that both reductase activities may represent the same plasma-membrane
enzyme. The rate of cupric reduction was a function of the free [Cu2+], not the total [Cu(II)], suggesting that free Cu2+ is the actual substrate for cupric reductase activity.
Received: 8 July 1998 / Accepted: 5 August 1998 相似文献
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Christensen Rowan Dave Rajvi Mukherjee Ananya Moroney James V. Machingura Marylou C. 《Plant Molecular Biology Reporter》2020,38(3):503-512
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In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAXoligo) and by a combination of monomeric BAX (BAXmono) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, tcBID). We also examined whether the effects induced by BAXoligo or by BAXmono activated with tcBID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that tcBID plus BAXmono produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology. On the contrary, treatment of mitochondria with BAXoligo resulted in BAX insertion and Cyt c release, which were accompanied by gross distortion of mitochondrial morphology. The effects of BAXoligo could be at least partially suppressed by mitochondrial depolarization. The effects of tcBID plus BAXmono were insensitive to depolarization. BAXoligo produced similar BAX insertion, mitochondrial remodeling, and Cyt c release in KCl- and in N-methyl-d-glucamine-based incubation media indicating a non-essential role for K+ influx into mitochondria in these processes. A combination of cyclosporin A and ADP, inhibitors of the mitochondrial permeability transition, attenuated Cyt c release, mitochondrial remodeling, and depolarization induced by BAXoligo, but failed to influence the effects produced by tcBID plus BAXmono. Thus, our results suggest a significant difference in the mechanisms of the outer mitochondrial membrane permeabilization and Cyt c release induced by detergent-oligomerized BAXoligo and by BAX activated with tcBID. 相似文献
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Chi Zhu Chuanhong Chen Liangyuan Zhao Yihui Zhang Jiaoyan Yang Lirong Song Shao Yang 《Journal of applied phycology》2012,24(5):1245-1251
Bioflocculants of Chlamydomonas reinhardtii were investigated under axenic conditions. C. reinhardtii was found to produce significant amounts of bioflocculants. Flocculating activity by C. reinhardtii began in the linear phase of growth and continued until the end of the stationary phase. The highest flocculating efficiency of the culture broth was 97.06%. The purified C. reinhardtii bioflocculant was composed of 42.1% (w/w) proteins, 48.3% carbohydrates, 8.7% lipids, and 0.01% nucleic acid. The optimum condition for bioflocculant production of C. reinhardtii was as follows: under temperature of 15°C to 25°C, pH 6–10 and illumination of 40–60 μmol photons m?2 s?1. The bioflocculants produced by C. reinhardtii showed maximum activity in pH ranges from 2 to 10. The flocculating activity was significantly enhanced by the addition of CaCl2 as a co-flocculant at an optimal concentration of 4.5 mM. 相似文献
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