A New Electrochemical Gradient Generator in Thylakoid Membranes of Green Algae

Abstract

Using a new method of delayed luminescence digital imaging, mutants of Chlorella sorokiniana lacking the chloroplast CF 0 CF 1 ATP synthase were isolated for the first time. Biochemical characterization of these strains indicates a lack of detectable synthesis and accumulation of the ATP synthase subunits R-CF 1 and -CF 1 . Functional characterization indicates the presence of a permanent electrochemical gradient (∆μ ) across the thylakoid membrane in the dark-adapted state, which is not suppressed under anaerobic conditions. Contrary to what is observed in the presence of the CF 0 CF 1 ATP synthase, this gradient is essentially due to an electric field component ∆Ψ with no detectable ∆pH component, under both aerobic and anaerobic conditions. Neither the CF 0 CF 1 ATP synthase nor a respiratory process can thus be responsible for a permanent gradient detected under these conditions. The previous proposal of a new ATP-dependent electrogenic pump in thylakoid membranes is supported by these results that, in addition, indicate a specificity of this new pump for ions other than protons.Using delayed luminescence measurements, Joliot and Joliot (1) showed the existence of a permanent electrochemical gradient across thylakoid membranes in dark-adapted algae under aerobic conditions. This phenomenon was ascribed to the ATP hydrolysis by the chloroplast CF 0 CF 1 ATP synthase. However, a permanent gradient was observed as well in the Chlamydomonas reinhardtii FUD50 mutant lacking this protein (2). The persistence of such a gradient in the absence of the chloroplast CF 0 CF 1 ATP synthase was a strong argument favoring the model of chlororespiration, which could also account for the pattern of dark oxidation and reduction of plastoquinone (2). In this model, the permanent gradient present in the absence of CF 0 CF 1 ATP synthase results from a coupled electron transfer through a chloroplast respiratory pathway, in which plastoquinone is reduced by a chloroplast NAD(P)H dehydrogenase and reoxidized at the expense of oxygen by a chloroplast oxidase. This hypothetical pathway is similar to that prevailing in photosynthetic prokaryotes which associate photosynthesis and respiration in the same membranes (see ref 3 for a review). More recently, however, Bennoun (4) analyzed in detail the effects of myxothiazol, a mitochondrial cytochrome bc 1 inhibitor, on the redox state of plastoquinone in darkness and on the permanent gradient. He concluded that the permanent electrochemical gradient present in the absence of CF 0 CF 1 ATP synthase could not result from the coupling of the chlororespiratory chain, but rather from a new ATPdriven gradient generator present in the thylakoid membranes.We have studied the permanent electrochemical gradient (∆μ) 1 under strict anaerobic conditions to investigate the role of respiration processes in its formation. In wild-type Chlorella sorokiniana, a ∆μ has even been observed under anaerobic conditions (5). In that case, it was assumed that the CF 0 CF 1 ATP synthase was responsible ...

Time-resolution of the Antheraxanthin- and ΔpH-dependent Chlorophyll a Fluorescence Components Associated with Photosystem II Energy Dissipation in Mantoniella squamata¶

“…We show that the high levels of light-induced A and ⌬pH-dependent energy dissipation observed in Mantoniella are independent of Z and lutein. We also confirm that Mantoniella cells, like other algae, are capable of sustaining ⌬pH-dependent energy dissipation in the dark (16,17,28), especially at low temperatures, similar to the chloroplast adenosine triphosphatase (ATPase) reactions with isolated thylakoids reported by Gilmore and Yamamoto (20) and Gilmore and Björkman (29). Our data indicate that the de-epoxidase reaction in Mantoniella is significantly altered, when compared to higher plant chloroplasts, and that the conversion of V to A is more sensitive to the sulfhydryl reagent dithiothreitol (DTT).…”

“…Jakob et al (16) proposed that chlororespiration involves proton translocation across the thylakoid membrane which could directly involve maintenance of an active pH-dependent conformational state for the chloroplast CF 0 CF 1 ATPase. In contrast Rappaport et al (28) recently concluded that the dark-sustained aerobic ⌬pH in wild-type chlorella is not coupled to chlororespiratory electron flow but to ATP hydrolysis. The dark-sustained ⌬pH was absent in chlorella mutants lacking the chloroplast CF 0 CF 1 ATPase and ATP hydrolysis proton translocation activity.…”

Electron Flow between Photosystem II and Oxygen in Chloroplasts of Photosystem I-deficient Algae Is Mediated by a Quinol Oxidase Involved in Chlororespiration

“…This is in apparent contradiction with conclusions of Bennoun (6) who proposed that previous effects of inhibitors on fluorescence transients could be explained by the existence of metabolic interactions between mitochondria and chloroplasts, therefore questioning both the existence of a chloroplast terminal oxidase and of chlororespiration. According to this author, the electrochemical gradient measured in the dark would not be due to chlororespiration, but rather to an ATP-dependent proton pump different from the CF1F0 ATPase (6,44). These apparent contradictions can be alleviated if we consider that, according to its probable composition, the chlororespiratory electron transport chain is likely nonelectrogenic.…”

Nonphotochemical Quenching of Chlorophyll Fluorescence inChlamydomonas reinhardtii

“…While the overall amplitude of the ∆µ H + is the same, the relative contribution of ∆pH is larger in the RubisCO mutant than in the WT. An additional ATPase activity has been identified in the thylakoid membranes, the "X" pump, which shows a selectivity for ions other than protons (60). Its contribution to the generation and dissipation of the ∆µ H + is almost negligible in physiological conditions (see ref 60 for a further discussion).…”