Involvement of Stromal ATP in the Light Activation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Intact Isolated Chloroplasts

Abstract

Light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and stromal ATP content were measured in intact isolated spinach chloroplasts. Treatments which decreased stromal ATP, such as incubation with the ATP analog #,,'y-methylene adenosine triphosphate or with the energy transfer inhibitor phloridzin inhibited the light activation of rubisco. In the absence of added inorganic phosphate (Pi), light activation of rubisco was inhibited, coincident with low stromal ATP. Addition of methyl viologen restored both stromal ATP and rubisco activity to levels observed in the presence of Pi. Activation of rubisco was inhibited in the presence of 2 millimolar dihydroxyacetone phosphate or 3-phosphoglycerate and stromal ATP was also decreased under these conditions. Both were partialy restored by increasing the Pi concentration. The strong correlation between activation state of rubisco and stromal ATP concentration in intact chloroplasts under a wide variety of experimental conditions indicates that light activation of rubisco is dependent on ATP and proportional to the ATP concentration. These observations can be explained in terms of the rubisco activase protein, which mediates activation of rubisco at physiological concentrations of CO2 and ribulose-1,5-bisphosphate and is dependent upon ATP.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Diurnal Changes in Irradiance

“…10). This result is consistent with previous suggestions that Rubisco activity is coupled to a common factor such as ATP pool size (16,25).…”

Pyruvate,Orthophosphate Dikinase in Leaves and Chloroplasts of C3 Plants Undergoes Light-/Dark-Induced Reversible Phosphorylation

“…6B). This also is consistent with the C 4 RP reaction/ regulatory mechanism because MV has been shown to stimulate C 3 chloroplast ATP production and hence decrease stromal ADP levels via photophosphorylation (Robinson and Portis, 1988). Further, because the site of action of MV prevents photosynthetic reduction of stromal thioredoxin and NADP, reversible phosphorylation of PPDK in C 3 leaves appears neither to be redox regulated, which is consistent with previous studies concerning C 4 RP (Yamamoto et al, 1974;Nakamoto and Young, 1990;Smith et al, 1994b), nor dependent on Calvin cycle activity.…”

“…6A). In contrast, the artificial PSI terminal electron acceptor MV, which stimulates thylakoid ATP synthesis in the presence exogenous Pi (2 mm in these experiments) and conversely lowers stromal [ADP] (Robinson and Portis, 1988), had no effect on the light-induced dephosphorylation of PPDK (Fig. 6B).…”

“…Hence, Pi deprivation during illumination of isolated C 3 chloroplasts may prevent dephosphorylation by limiting substrate for the RPcatalyzed reactivation (dephosphorylation) reaction. Alternatively, decreased stromal [Pi] would impair ATP synthesis in the light (Robinson and Portis, 1988) and thereby result in increased ADP levels. Thus, the inhibitory effect of Pi deprivation on the dephosphorylation of C 3 chloroplast PPDK (Fig.…”

“…Intact spinach and V. faba chloroplasts were isolated using a previously developed protocol, except that before leaf harvest the plants were dark adapted for 1.5 h at room temperature (Robinson and Portis, 1988). The Percollgradient purified, intact chloroplast pellet was resuspended in 0.33 m sorbitol; 50 mm HEPES-KOH, pH 7.6; 2 mm EDTA; 1 mm MgCl 2 ; 1 mm MnCl 2 ; and 0.2% (w/v) bovine serum albumin (BSA) to a final concentration of approximately 150 g chlorophyll mL Ϫ1 and stored on ice until use.…”