Antimycin A-sensitive Pathway Research Articles

Characterization of the Wave Phenomenon in Flash-Induced Fluorescence Relaxation and Its Application to Study Cyclic Electron Pathways in Microalgae.

Photosynthesis is a series of redox reactions, in which several electron transport processes operate to provide the energetic balance of light harvesting. In addition to linear electron flow, which ensures the basic functions of photosynthetic productivity and carbon fixation, alternative electron transport pathways operate, such as the cyclic electron flow (CEF), which play a role in the fine tuning of photosynthesis and balancing the ATP/NADPH ratio under stress conditions. In this work, we characterized the electron transport processes in microalgae species that have high relevance in applied research and industry (e.g., Chlorella sorokiniana, Haematococcus pluvialis, Dunaliella salina, Nannochloropsis sp.) by using flash-induced fluorescence relaxation kinetics. We found that a wave phenomenon appeared in the fluorescence relaxation profiles of microalgae to different extents; it was remarkable in the red cells of H. pluvialis, D. salina and C. sorokiniana, but it was absent in green cells of H. pluvialis and N. limnetica. Furthermore, in microalgae, unlike in cyanobacteria, the appearance of the wave required the partial decrease in the activity of Photosystem II, because the relatively high Photosystem II/Photosystem I ratio in microalgae prevented the enhanced oxidation of the plastoquinone pool. The wave phenomenon was shown to be related to the antimycin A-sensitive pathway of CEF in C. sorokiniana but not in other species. Therefore, the fluorescence wave phenomenon appears to be a species-specific indicator of the redox reactions of the plastoquinone pool and certain pathways of cyclic electron flow.

The antimycin A-sensitive pathway of cyclic electron flow: from 1963 to 2015.

Cyclic electron flow has puzzled and divided the field of photosynthesis researchers for decades. This mainly concerns the proportion of its overall contribution to photosynthesis, as well as its components and molecular mechanism. Yet, it is irrefutable that the absence of cyclic electron flow has severe effects on plant growth. One of the two pathways mediating cyclic electron flow can be inhibited by antimycin A, a chemical that has also widely been used to characterize the mitochondrial respiratory chain. For the characterization of cyclic electron flow, antimycin A has been used since 1963, when ferredoxin was found to be the electron donor of the pathway. In 2013, antimycin A was used to identify the PGRL1/PGR5 complex as the ferredoxin:plastoquinone reductase completing the last puzzle piece of this pathway. The controversy has not ended, and here, we review the history of research on this process using the perspective of antimycin A as a crucial chemical for its characterization.

Redox regulation of the antimycin A sensitive pathway of cyclic electron flow around photosystem I in higher plant thylakoids

The chloroplast must regulate supply of reducing equivalents and ATP to meet rapid changes in downstream metabolic demands. Cyclic electron flow around photosystem I (CEF) is proposed to balance the ATP/NADPH budget by using reducing equivalents to drive plastoquinone reduction, leading to the generation of proton motive force and subsequent ATP synthesis. While high rates of CEF have been observed in vivo, isolated thylakoids show only very slow rates, suggesting that the activity of a key complex is lost or down-regulated upon isolation. We show that isolation of thylakoids while in the continuous presence of reduced thiol reductant dithiothreitol (DTT), but not oxidized DTT, maintains high CEF activity through an antimycin A sensitive ferredoxin:quinone reductase (FQR). Maintaining low concentrations (~2mM) of reduced DTT while modulating the concentration of oxidized DTT leads to reversible activation/inactivation of CEF with an apparent midpoint potential of −306mV (±10mV) and n=2, consistent with redox modulation of a thiol/disulfide couple and thioredoxin-mediated regulation of the plastoquinone reductase involved in the antimycin A-sensitive pathway, possibly at the level of the PGRL1 protein. Based on proposed differences in regulatory modes, we propose that the FQR and NADPH:plastoquinone oxidoreductase (NDH) pathways for CEF are activated under different conditions and fulfill different roles in chloroplast energy balance.

Complexities and protein complexes in the antimycin A-sensitive pathway of cyclic electron flow in plants

Antimycin A-sensitive cyclic electron flow (AA-sensitive CEF) was discovered by Arnon and co-workers more than 50 years ago and serves to recycle electrons from ferredoxin (Fd) to plastoquinone (PQ). A role in AA-sensitive CEF has been attributed to the two thylakoid proteins PGR5 and PGRL1 ever since their identification, but this assignment remains controversial. While current technical limitations have prevented unequivocal clarification of their precise function in CEF in vivo, recent biochemical experiments have implied that PGRL1/PGR5 complexes possess Fd-PQ reductase (FQR) activity in vitro. Consequently, PGRL1-PGR5 complexes in flowering plants appear to shuttle between photosystem I (PSI) and the cytochrome (Cyt) b6f complex, whereas in the green alga Chlamydomonas PGRL1 (but not PGR5) has been detected in a PSI-Cyt b6f supercomplex that has intrinsic CEF activity.

Elevated Expression of PGR5 and NDH-H in Bundle Sheath Chloroplasts in C4Flaveria Species

Cyclic electron transport around PSI has been proposed to supply the additional ATP required for C(4) photosynthesis. To investigate the nature of cyclic electron pathways involved in C(4) photosynthesis, we analyzed tissue-specific expression of PGR5 (PROTON GRADIENT REGULATION 5), which is involved in the antimycin A-sensitive pathway, and NDH-H, a subunit of the plastidial NAD(P)H dehydrogenase complex, in four Flaveria species comprising NADP-malic enzyme (ME)-type C(4), C(3)-C(4) intermediate and C(3) species. PGR5 was highly expressed in the C(4) species and enriched in bundle sheath chloroplasts together with NDH-H, suggesting that electron transport of both PGR5-dependent and NDH-dependent cyclic pathways is promoted to drive C(4) photosynthesis.

An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex.

Cyclic electron flow (CEFI) has been proposed to balance the chloroplast energy budget, but the pathway, mechanism, and physiological role remain unclear. We isolated a new class of mutant in Arabidopsis thaliana, hcef for high CEF1, which shows constitutively elevated CEF1. The first of these, hcef1, was mapped to chloroplast fructose-1,6-bisphosphatase. Crossing hcef1 with pgr5, which is deficient in the antimycin A-sensitive pathway for plastoquinone reduction, resulted in a double mutant that maintained the high CEF1 phenotype, implying that the PGR5-dependent pathway is not involved. By contrast, crossing hcef1 with crr2-2, deficient in thylakoid NADPH dehydrogenase (NDH) complex, results in a double mutant that is highly light sensitive and lacks elevated CEF1, suggesting that NDH plays a direct role in catalyzing or regulating CEF1. Additionally, the NdhI component of the NDH complex was highly expressed in hcef1, whereas other photosynthetic complexes, as well as PGR5, decreased. We propose that (1) NDH is specifically upregulated in hcef1, allowing for increased CEF1; (2) the hcef1 mutation imposes an elevated ATP demand that may trigger CEF1; and (3) alternative mechanisms for augmenting ATP cannot compensate for the loss of CEF1 through NDH.

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.

A Balanced PGR5 Level is Required for Chloroplast Development and Optimum Operation of Cyclic Electron Transport Around Photosystem I

PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of non-photochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis.

Respiration in the filarial nematode Brugia pahangi

d-Glucose-supported O 2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30–40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO 2 by B. pahangi in the presence of d-glucose was stimulated by O 2; the stimulation of CO 2 production was sensitive to antimycin A. The O 2 dependencies of respiration showed that the apparent O 2 affinity for B. pahangi was diminished in the presence of antimycin A; O 2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O 2 concentrations. H 2O 2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H 2O 2 production suggest two sites of H 2O 2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O 2 dependencies of H 2O 2 production and respiration may indicate that H 2O 2 production is involved in O 2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O 2 .− radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H 2O 2 production and O 2 .− production by 30,20 and 25%, respectively, whereas higher concentrations (> 2.5 m m) inhibited respiration by 75% and H 2O 2 and O 2 .− production by up to 85%.

Effects of inhibitors on the oxygen kinetics of Nippostrongylus brasiliensis

Endogenous respiration of the parasitic nematode Nippostrongylus brasiliensis and the succinate oxidase activity of isolated mitochondria were partially inhibited by antimycin A; the remaining respiratory activity was sensitive to salicylhydroxamic acid (SHAM). Sub-millimolar concentrations of SHAM markedly stimulated respiration by 60% in whole N. brasiliensis and isolated mitochondria; stimulation by SHAM was not observed in the presence of antimycin A. Little change in the relative fluxes of electrons through the classical, antimycin A-sensitive pathway and the alternative SHAM sensitive pathway was observed between low and high O 2 concentrations; this may suggest that the O 2 affinities of both pathways are similar. O 2 dependence of respiration showed O 2 thresholds above which respiration decreases; in the absence of inhibitors whole N. brasiliensis and isolated mitochondria had threshold values around 60 μM O 2. Increased O 2 threshold values were observed in the presence of SHAM and antimycin A. The apparent K m values for O 2 of whole N. brasiliensis and isolated mitochondria were 31 ± 2 μM O 2 and 3.5 ± 0.2 μM O 2 respectively; this difference in apparent K m values may reflect the presence of O 2 gradients in the whole worm. The K m and O 2 inhibition threshold values observed for whole N. brasiliensis are in good agreement with the proposed range of O 2 concentrations thought to exist within the worm's natural environment. H 2O 2 production was detected in respiring uncoupled mitochondria, but H 2O 2 could not be detected in the medium surrounding whole N. brasiliensis. SHAM-stimulated respiration was accompanied by increased H 2O 2 production which was prevented by the addition of antimycin A.

Cellular differentiation in the anamniota: I. Initial studies on the aerobic metabolic pathways of differentiation and morphogenesis in teleosts and urodeles

Reasonably precise measurements in embryos of Fundulus heteroclitis and Ambystoma maculatum indicate the existence of aerobic electron transport systems during all stages of embryogenesis. Glycolytic systems are also present. Dinitrophenol and cyanide in appropriate concentration inhibit cellular differentiation without drastically disturbing protoplasmic maintenance processes. It appears highly probable that differentiation and morphogenesis in the anamniota are strictly dependent upon aerobic metabolic processes, but that maintenance and movement may be powered by glycolytic metabolism. The aerobic metabolic pathways exhibit an ontogeny of their own in development of F. heteroclitis. An initial antimycin A-insensitive metabolic pathway is maintained while a new antimycin A-sensitive pathway develops following gastrulation. Both systems are present in the adult teleost but vary with different organs. The enzyme systems studied here exist in the cells of the developing embryo and are absent from the yolk.

The site of electron transfer to various acceptors in sucinoxidase system, with special reference to that of ethylurethane.

A study of succinoxidase system in rat liver mitochondria has been made, with special reference to the site of action of ethylurethane, and the site of electron transfer to several electron acceptors in this system.1. The inhibitory action of ethylurethane on succinoxidase system varied with the variety of acceptors used and it was not observed when brilliant cresyl blue was the acceptor. The inhibition was completely reversible up to the concentration of 0.5 M.2. The existence of two pathways: an “antimycin A-sensitive” and an “antimycin A-resistant, ” was indicated to link ferricyanide, 2, 6-dichlorophenolindophenol, and methylene blue with the succinoxidase system. One of the factors involved in the antimycin A-sensitive pathway is cytochrome c.3. The sensitivity of succinoxidase toward ethylurethane was exaggerated by the treatment with antimycin A and BAL and by the addition of cytochrome c. On the other hand, simultaneous addition of ethylurethane failed to protect the inhibition by BAL. Antimycin A titer of succinoxidase was decreased slightly by the presence of ethylurethane.4. From these results, the mechanism of action of ethylurethane was discussed together with that of antimycin A and BAL, and it was concluded that the site of action of ethylurethane lies at the portion of cytochrome b and Slater factor.