Disappearance of the cyanide-insensitive pathway of oxidation in mitochondria of MI-1 mutant of Neurospora crassa in vitro.

Oxidation of exogenous NADH in mitochondria isolated from wild type and mi-1 mutant of Neurospora crassa decreases rapidly in vitro. In mi-1 mutant mitochondria the inactivation concerns the alternate pathway of oxidation whereas in the wild type it involves an unknown component of the respiratory chain. The activity of the primary NADH dehydrogenase is constant within the time of the experiments (2-4 h). NADH oxidase is not inactivated if oxygen is removed from the incubation medium by nitrogen bubbling. Succinate oxidase does not show any remarkable changes in activity within 2-3 h. In fresh mitochondria of the mi-1 mutant reduced ubiquinone is completely reoxidized by cytochrome oxidase but only 80% reoxidized by the alternate oxidase. In aged mitochondria of the mi-1 mutant in the presence of cyanide, ubiquinone is reduced to the level characteristic for fresh mitochondria in which respiration is completely inhibited by cyanide plus salicylhydroxamic acid. In these mitochondria the reoxidation of the reduced ubiquinone proceeds only via the cytochrome pathway. It is supposed that a labile component(s) of the respiratory chain present in the mi-1 mutant and the wild type mitochondria may, in mi-1 mutant, act as an alternate oxidase.     

Inactivation of ycf33 results in an altered cyclic electron transport pathway around photosystem I in Synechocystis sp. PCC6803

ycf33 encodes a small protein with a molecular mass of 7.5 kDa and is found from cyanobacteria to higher plants. A ycf33 deletion mutant was constructed in Synechocystis sp. PCC6803 and characterized. The mutant showed a higher phycobilisome/chlorophyll ratio than the wild type and a higher photosystem II/photosystem I fluorescence ratio measured at 77 K. Under photoautotrophic conditions, the growth rates were not much different from those of the wild type. Cyclic electron transport activities around photosystem I were not much different between the wild type and the mutant. However, the effects of diphenyleneiodonium, an inhibitor of flavoprotein, on cyclic electron transport in the mutant were different from those in the wild type; it was severely inhibited in the wild type but not much in the mutant. Together with the effects of nitrite, which accepts electrons from ferredoxin via nitrite reductase and those of HgCl2, it was suggested that the pathway of cyclic electron transport is altered in the mutant.     

Expression of a familial amyotrophic lateral sclerosis-associated mutant human superoxide dismutase in yeast leads to decreased mitochondrial electron transport

Strains of Saccharomyces cerevisiae that express either the wild type or the amyotrophic lateral sclerosis-associated mutant human copper-zinc superoxide dismutase (SOD1) proteins A4V and G93A, respectively, in a yeast SOD1-deficient parent strain were used to investigate the hypothesis that expression of a mutant SOD1 protein causes deficient mitochondrial electron transport as a possible mechanism for disease induction. Mitochondria isolated from the wild type SOD1-expressing yeast were identical to mitochondria from the parent strain in heme content and activities of complexes II, III, and IV. Mitochondria isolated from the A4V-expressing yeast had decreased rates of electron transport in complexes II+III, III, and IV and corresponding decreases in hemes b, c-c1, and a-a3 content compared to mitochondria from wild type human SOD1-expressing yeast. Mitochondria isolated from G93A-expressing yeast had decreased rates of electron transport in complex IV and probably in complex II with a corresponding decrease in heme a-a3 content. These results suggest that mutant SOD1-expression causes defective electron transport complex assembly and that the yeast system will provide an excellent model for the study of the mechanism of mutant SOD1-induced mitochondrial electron transport defects.     

Ubiquinone Function in Neurospora crassa

Mitochondria of cytoplasmic respiratory mutants [mi-1] (poky) and [mi-4] contain about a fourfold molar excess of ubiquinone as compared to the wild-type strain of Neurospora crassa. In the wild type and [mi-1] cultures the concentration of ubiquinone remains constant during the exponential and stationary phase of growth. In [mi-4] cultures it markedly decreases in the stationary phase. The reduction of ubiquinone by substrates is approximately the same in the three strains tested and amounts 60 to 70% of total ubiquinone present in mitochondria, independent of its absolute amount. The reduction of ubiquinone on addition of substrates is accompanied by the similar reduction of cytochrome c. These indicate that mitochondrial ubiquinone and cytochrome c are involved in processes of oxidation in Neurospora and that ubiquinone belongs mainly if not entirely to the cytochrome system of electron transport in these strains.     

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b.

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reoxidation is observed in the wild type in the present of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steady-state reduction; reduction in the presence of substrate, cyanide and oxygen; the 'red shift' and lowering of E'-o of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH-2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycin-binding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Studies of Respiratory Components and Oxidative Phosphorylation in Mitochondria of mi-1 Neurospora crassa

Oxidative phosphorylation has been demonstrated with mitochondria of the mi-1 respiratory mutant of Neurospora crassa. The P/O ratios observed with these mitochondria were approximately 0.8 with citrate and 0.4 with either externally added reduced nicotinamide adenine dinucleotide (NADH), succinate, or ascorbate-tetramethyl-p-phenylenediamine (TPD). These P/O ratios suggest that there are only two sites of phosphorylation in mitochondria isolated from young (20 to 24 h) cultures of the mi-1 mutant. The energy-dependent reduction of NAD(+) with succinate and the phosphorylation associated with ascorbate-TPD oxidation indicate that the first and the third sites of energy coupling are present in this mutant. Difference spectra of mitochondria from young cultures of the mi-1 mutant revealed the presence of cytochrome c. Cytochromes b and a + a(3) were not detected. However, in the presence of antimycin A, a small peak in the Soret region at 430 nm was observed. A carbon monoxide difference spectrum revealed the presence of a component of the respiratory chain with a spectrum similar to that of cytochrome o. It is of interest that respiratory inhibitors such as antimycin A, 2-n-nonylhydroxyquinoline N-oxide, and cyanide abolished phosphorylation but only partially inhibited oxidation. It is postulated that the mi-1 respiratory system contains two pathways of electron transport-the first is associated with a phosphorylating pathway, whereas the second is a non-phosphorylating electron transport pathway.     

Photoinhibition and light-induced cyclic electron transport in ndhB(-) and psaE(-) mutants of Synechocystis sp. PCC 6803

The ndhB^– and psaE^– mutants of the cyanobacteriumSynechocystis sp. PCC 6803 are partly deficient in PSI-drivencyclic electron transport. We compared photoinhibition in thesemutants to the wild type to test the hypothesis that PSI cyclicelectron transport protects against photoinhibition. Photoinhibitorytreatment greatly accelerated PSI cyclic electron transportin the wild type and also in both the mutants. The psaE^–mutant showed rates of PSI cyclic electron transport similarto the wild type under all conditions tested. The ndhB^–mutant showed much lower rates of PSI cyclic electron transportthan the wild type following brief dark adaptation but exceededwild type rates after exposure to photoinhibitory light. Thewild type and both mutants showed similar rates of photoinhibitiondamage and photoinhibition repair at PSII. Photoinhibition atPSI was much slower than at PSII and was also similar betweenthe wild type and both mutants, despite the known instabilityof PSI in the psaE^– mutant. We conclude that photoinhibitorylight induces sufficient PSI-driven cyclic electron transportin both the ndhB^– and psaE^– mutants to fulfill anyrole that cyclic electron transport plays in protection againstphotoinhibition. ⁴ Corresponding author: E-mail, sherbert@uwyo.edu; Fax, +1-307-766-2851;Phone, +1-307-766-4353.     

Antimycin-insensitive mutants of Candida utilis. II. The effects of antimycin on cytochrome b

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutant. A similar reoxidation is observed in the wild type in the presence of low concentrations of antimycin.

2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steadystate reduction; reduction in the presence of substrate, cyanide and oxygen; the ‘red shift’ and lowering of E′₀ of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable.

3. The red shift in the mutant is more extensive than in the wild type.

4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes.

5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant.

6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH₂-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycinbinding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Gene mutation eliminating antimycin A-tolerant electron transport in Ustilago maydis

A class of mutants of Ustilago maydis selected on a fungitoxic oxathiin lack of antimycin A-tolerant respiratory system which is present in wild-type cells. This system provides, directly or indirectly, for considerable resistance to antimycin A because growth of mutant cells lacking the system is much more sensitive to the antibiotic than that of the wild type. Antimycin A-sensitive O(2) uptake and growth is found in half of the progeny from crosses of mutant to wild type. All antimycin A-sensitive segregants are somewhat more resistant to oxathiins than the antimycin A-resistant segregants. The respiration of the mutant is strongly inhibited by cyanide and azide at concentrations which stimulate respiration of the wild type. Respiration of both mutant and wild type is about equally inhibited by rotenone. It appears that the mutation alters some component of the respiratory system located between the rotenone inhibition site and the antimycin A inhibition site that permits shift of electron transport to an alternate terminal oxidase when the normal electron transport pathway is blocked.     

Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.

Wild-type Streptomyces clavuligerus NRRL 3585 is unable to utilize glucose. A glucose-utilizing (gut-1) mutant of S. clavuligerus NRRL 3585 has been obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The gut-1 mutant is able to grow on glucose or galactose, while the wild type is unable to catabolize these hexoses. Similar binding affinities of glucose by cells of the wild type and the gut-1 mutant were found, but the wild type was unable to complete glucose transport. A soluble intracellular ATP-dependent (but not phosphoenolpyruvate-dependent) glucokinase activity was found both in the wild type and the gut-1 mutant. The gut-1 mutant has acquired a functional transport system that allows transport of glucose, 2-deoxyglucose, and galactose, as shown by hexose competition experiments. The gut-1 transport system concentrates glucose inside the cell at least 10- to 20-fold and is strongly inhibited by respiratory inhibitors, which prevent the establishment of a proton motive force, and by proton-conducting ionophores, suggesting that it is energized by a proton motive force. The new transport system is not completely sugar specific (transporting galactose and glucose through the same system), as opposed to the hexose-specific system reported in wild-type Streptomyces griseus.     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO(2) concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.     

The maintenance of the energized membrane state and its relation to active transport in Escherichia coli.

1. An ATPase mutant of Escherichia coli and two partial revertants of that mutant were examined for the ability to generate a high energy membrane state with D-lactate or ATP, as measured by the quenching of the fluorescent dye quinacrine. 2. All three strains showed reductions in the aerobically-driven quenching of fluorescence compared to the wild type, but the reduction could be reversed by the addition of eitherN,N'-dicyclohexylcarbodiimide or the crude soluble ATPase of the wild type. 3. The mutant exhibited a decreased ability to accumulate sugars and amino acids and showed an increased permeability to protons. 4. One partial revertant showed a slight increase in active transport and a slight decrease in proton permeability. 5. The other partial revertant showed a large increase in transport ability and a large decrease in proton permeability. 6. A model is proposed in which the conformation of the Mg-2+-ATPase is important in the utilization of energy derived from the electron transport chain and this function is independent of the catalytic activity of the Mg-2+-ATPase.     

Characterization of factors affecting the activity of photosystem I cyclic electron transport in chloroplasts

PSI cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, the antimycin A-sensitive pathway is the main route of electrons in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, its function is still unclear, and there are numerous debates on the rate of electron transport in vivo and its regulation. To assess how PGR5-dependent PSI cyclic electron transport is regulated in vivo, we characterized its activity in ruptured chloroplasts isolated from Arabidopsis thaliana. The activity of ferredoxin (Fd)-dependent plastoquinone (PQ) reduction in the dark is impaired in the pgr5 mutant. Alkalinization of the reaction medium enhanced the activity of Fd-dependent PQ reduction in the wild type. Even weak actinic light (AL) illumination also markedly activated PGR5-dependent PSI cyclic electron transport in ruptured chloroplasts. Even in the presence of linear electron transport [11 mumol O2 (mg Chl)(-1) h(-1)], PGR5-dependent PSI electron transport was detected as a difference in Chl fluorescence levels in ruptured chloroplasts. In the wild type, PGR5-dependent PSI cyclic electron transport competed with NADP+ photoreduction. These results suggest that the rate of PGR5-dependent PSI cyclic electron transport is high enough to balance the production ratio of ATP and NADPH during steady-state photosynthesis, consistently with the pgr5 mutant phenotype. Our results also suggest that the activity of PGR5-dependent PSI cyclic electron transport is regulated by the redox state of the NADPH pool.     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO₂ concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.     

Functional analysis of the PsbX protein by deletion of the corresponding gene in Synechocystis sp. PCC 6803

The psbX gene (sml0002) coding for a 4.1 kDa protein in Photosystem II of plants and cyanobacteria was deleted in both wild type and in a Photosystem I-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Polymerase chain reaction and sequencing analysis showed that the mutants had completely segregated. Deletion of the PsbX protein does not seem to influence growth rate, electron transport or water oxidation ability. Whereas a high light induction of the psbX mRNA could be observed in wild type, deletion of the gene did not lead to high light sensibility. Light saturation measurements and 77K fluorescence measurements indicated a minor disconnection of the antenna in the deletion mutant. Furthermore, fluorescence induction measurements as well as immuno-staining of the D1 protein showed that the amount of Photosystem II complexes in the mutants was reduced by 30%. Therefore, PsbX does not seem to be necessary for the Photosystem II electron transport, but directly or indirectly involved in the regulation of the amount of functionally active Photosystem II centres in Synechocystis sp. PCC 6803.     

Regulation of reductase formation in Proteus mirabilis

Summary The levels of several redox enzymes in a chlorate-resistant mutant of Proteus mirabilis, which is partially affected in the formation of formate hydrogenlyase, thiosulfate reductase and tetrathionate reductase, were compared with those of the wild type. The composition of the electron transport system of both strains was almost the same in cells grown aerobically, but very different in cells grown anaerobically. In the mutant, the cytochrome content increased twofold, whereas the level of the anaerobic enzymes is strongly diminished. The anaerobic formation of electron transport components in the mutant was, in contrast to that of the wild type, not influenced significantly by azide. During anaerobic growth with nitrate low levels of a functional nitrate reductase system were formed in the mutant. Under these conditions the formation of formate dehydrogenase, formate hydrogenlyase, formate oxidase, thiosulfate reductase, tetrathionate reductase, cytochrome b_563,5 and partly that of cytochrome a₂, was repressed. The repressive effect of nitrate, however, was completely abolished by azide. Therefore, it seems likely that a functional nitrate reductase system, rather than nitrate, controls the formation of the enzymes repressible by nitrate.     

Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes

Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90 degrees reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation.     

Defective long-distance auxin transport regulation in the Medicago truncatula super numeric nodules mutant

Long-distance auxin transport was examined in Medicago truncatula and in its supernodulating mutant sunn (super numeric nodules) to investigate the regulation of auxin transport during autoregulation of nodulation (AON). A method was developed to monitor the transport of auxin from the shoot to the root in whole seedlings. Subsequently, the transport was monitored after inoculation of roots with the nodulating symbiont Sinorhizobium meliloti. The sunn mutant showed an increased amount of auxin transported from the shoot to the root compared to the wild type. The auxin transport capacity of excised root segments was similar in wild type and sunn, suggesting that the difference in long-distance auxin transfer between them is due to loading in the shoot. After inoculation, wild-type seedlings showed decreased auxin loading from the shoot to the root; however, the sunn mutant failed to reduce the amount of auxin loaded. The time of reduced auxin loading correlated with the onset of AON. Quantification of endogenous auxin levels at the site of nodule initiation showed that sunn contained three times more auxin than wild type. Inoculation of sunn failed to reduce the level of auxin within 24 h, as was observed in the wild type. We propose a model for the role of auxin during AON of indeterminate legumes: 1) high levels of endogenous auxin are correlated with increased numbers of nodules, 2) inoculation of roots reduces auxin loading from the shoot to the root, and 3) subsequent reduction of auxin levels in the root inhibits further nodule initiation.     

Digalactosyl-diacylglycerol deficiency impairs the capacity for photosynthetic intersystem electron transport and state transitions in Arabidopsis thaliana due to photosystem I acceptor-side limitations

Compared with wild type, the dgd1 mutant of Arabidopsis thaliana exhibited a lower amount of PSI-related Chl-protein complexes and lower abundance of the PSI-associated polypeptides, PsaA, PsaB, PsaC, PsaL and PsaH, with no changes in the levels of Lhca1-4. Functionally, the dgd1 mutant exhibited a significantly lower light-dependent, steady-state oxidation level of P700 (P700(+)) in vivo, a higher intersystem electron pool size, restricted linear electron transport and a higher rate of reduction of P700(+) in the dark, indicating an increased capacity for PSI cyclic electron transfer compared with the wild type. Concomitantly, the dgd1 mutant exhibited a higher sensitivity to and incomplete recovery of photoinhibition of PSI. Furthermore, dgd1 exhibited a lower capacity to undergo state transitions compared with the wild type, which was associated with a higher reduction state of the plastoquinone (PQ) pool. We conclude that digalactosyl-diacylglycerol (DGDG) deficiency results in PSI acceptor-side limitations that alter the flux of electrons through the photosynthetic electron chain and impair the regulation of distribution of excitation energy between the photosystems. These results are discussed in terms of thylakoid membrane domain reorganization in response to DGDG deficiency in A. thaliana.