Adenylate Levels, Energy Charge, and Phosphorylation Potential during Dark-Light and Light-Dark Transition in Chloroplasts, Mitochondria, and Cytosol of Mesophyll Protoplasts from Avena sativa L.

Abstract

The compartmentation of cellular energy relations during dark-light and light-dark transitions was studied by means of a newly developed technique to fractionate oat (Avena sativa L., var. Arnold) mesophyll protoplasts. Using an improved microgradient system with hydrophobic and hydrophilic layers of increasing density, a pure plastid pellet (up to 90% of total chloroplasts) could be separated from an interphase of only slightly contaminated mitochondria (70 to 80% of total mitochondria), and a cytoplasmic supernatant could be obtained within 60 seconds. Appropriate controls indicate that, under the conditions employed, metabolic interconversions of adenylates can be kept to a minimum and, thus, be determined and corrected for. Cross contamination of the fractions, as well as liberation of organelles to the supernatant, was assessed by specific markers, and the metabolite levels recorded were corrected accordingly. Using this technique, we found that, during dark-light transition, the chloroplastic and cytosolic ATP exhibits a rapid increase, while the mitochondrial ATP level decreases. In all compartments, ADP levels mirror alterations of the ATP pool in the opposite way, at least to some extent. To compensate fully for the rise in ATP, chloroplastic and mitochondrial AMP levels change accordingly, indicating that, due to the more or less unchanged level of total adenylates, there is no net flux of adenylates between the compartments. In contrast to the organelles, no AMP could be detected within the cytosol. When the light is turned off, a decrease of ATP coincides between chloroplast stroma and the cytosol for only about 30 seconds. Under prolonged dark treatment, cytosolic ATP rises again, while stroma ATP levels exhibit a further decrease. After about 60 seconds of darkness, the cytosolic ATP level is back to its initial value. This obviously is due to the immediate rise in mitochondrial ATP upon darkening, which cumulates after about 60 seconds; then, caused by an ATP/ADP exchange with the cytosol, it levels off again at the state before changing the conditions, as soon as the cytosolic ATP is also back to its original level. All of these events are closely mirrored by the change in the ATP/ADP ratio and the energy charge within the compartments. While the values for chloroplasts exhibit considerable differences between dark and light, those calculated for mitochondria and the cytosol exhibit only transient changes. These are limited to about 60 seconds of undershoot or overshoot, with respect to the cytosol, and then return to nearly the levels observed before changing the conditions. Adenylate kinase was found to be exclusively associated with chloroplasts (90% of total activity level) and mitochondria. Isotonic liberation of vacuoles did not point toward a significant association of adenylates with this compartment.The results are discussed with respect to an effective collaboration between photosynthetic and oxidative phosphorylation in order to keep the cytosolic energy state at a constant, preset value.