Influence of Antimycin A and Uncouplers on Anaerobic Photosynthesis in Isolated Chloroplasts

Abstract

Anaerobiosis depresses the light-and bicarbonate-saturated rates of 02 evolution in intact spinach (Spinacia oleracea) chloroplasts by as much as 3-fold from those observed under aerobic conditions. These lower rates are accelerated 2-fold or more by the addition of 1 ,UM antimycin A or by low concentrations of the uncouplers 0.3 mM NH4CI or 0.25 jaM carbonyl cyanide m-chlorophenylhydrazone. Oxaloacetate and glycerate 3-phosphate reduction rates are also increased by antimycin A or an uncoupler under anaerobic conditions. At intermediate light intensities, the rate accelerations by either antimycin A or uncoupler are inversely proportional to the adenosine 5'-triphosphate demand of the reduction process for the acceptors HCO3-, glycerate 3-phosphate, and oxaloacetate. The acceleration of bicarbonate-supported 02 evolution may also be produced by adding an adenosine 5'-triphosphate sink (nbose 5-phosphate) to anaerobic chloroplasts. The above results suggest that a proton gradient back pressure resulting from antimycin A-sensitive cyclic electron flow is responsible for the depression of light-saturated photosynthesis under anaerobiosis.Photosynthetic CO2 fixation requires that intact chloroplast electron transport supply 3 moles of ATP and 2 moles of NADPH for each mole of CO2 fixed in the Calvin cycle (4). However, uncertainty in the in vivo coupling ratio (11, 26) and the fact that average quantum requirements of 10 to 12 quanta/ CO2 fixed (10) exceed the expected value of 8, provide doubt that 3 ATP molecules may be formed for every 2 NADP reduced by the linear scheme of photosynthetic electron transport (26). If insufficient ATP is generated during NADP reduction, the extra quanta must be required in some other coupled photoreaction. There is evidence (9) that 02 reduction may be coupled to ATP formation through a portion of the electron carrier sequence normally involved in NADP reduction. A ferredoxin-dependent cycle around PSI has been shown to generate ATP under anaerobic conditions (3) and may also function aerobically in vivo. Several investigators (3,14,25,26) have suggested the existence of a cycle in which electrons from the primary acceptor of PSI reduce Cyt b-563 and are subsequently returned to the oxidized PSI trap, P700+, in a process coupled to the production of ATP. The inhibitor antimycin A of mitochondrial b and c type Cyt interaction (23) has previously been used to study the function of b type Cyt in both bacterial and green plant cyclic electron transport (1,3,(13)(14)(15)19).

Proton Gradient Regulation 5-Mediated Cyclic Electron Flow under ATP- or Redox-Limited Conditions: A Study of ƊATPase pgr5 and ƊrbcL pgr5 Mutants in the Green Alga Chlamydomonas reinhardtii

“…Possible scenarios involve metabolic shuttles between the chloroplast and the mitochondrion. Oxaloacetate and 3-phosphoglycerate reduction rates have been shown to increase in the absence of CEF in isolated chloroplasts under acceptor-limiting conditions (Slovacek and Hind, 1977). Similar in vitro enzymatic assays showed that the malate valve could operate as rapidly as the major (linear) photosynthetic flow rate (Fridlyand et al, 1998).…”

“…It parallels some earlier observations. In isolated chloroplasts, a slowing down of linear electron flow under acceptor-limited conditions was attributed to proton gradient backpressure generated by CEF (Slovacek and Hind, 1977). In tobacco (Nicotiana spp.)…”

Evidence for Cyclic Photophosphorylation during 14CO2 Fixation in Intact Chloroplasts

“…The effect of antimycin A on photosynthetic activities in thylakoids and intact chloroplasts is complex and apparently depends on the experimental conditions involved (7,14,19,20,22,23,25,28). Figure 1 shows that the photoreduction of methyl viologen in intact chloroplasts is insensitive to antimycin A up to 4 Am in the presence and absence of 10 mm NH4CL.…”

“…For example, red light was used in the experiments of Heber et al (14) while the uptake of phosphate had been used as an indicator reaction in the evidence discussed by Simonis and Urbach (24) and Gimmler (1 1). The reported stimulation of photosynthesis in chloroplasts by antimycin A under anaerobic and aerobic conditions (19,22,25,28) indicates the complex nature of the direct and indirect effect of antimycin A on photosynthetic carbon metabolism. However, like Heber et al (14), we have never observed stimulation of photosynthesis by antimycin A in intact chloroplasts.…”

“…In intact chloroplasts, antimycin A stimulated CO2 fixation under anaerobic conditions (19,22,25) and in aerobic conditions which were suboptimal (e.g. high Pi concentration) for CO2 fixation (28).…”