Mature chloroplasts of Arabidopsis thaliana do not import ATP efficiently

Abstract

The degree at which ATP transport across the chloroplast envelope occurs in vivo is not completely clear. By using AT1.03 fluorescent protein sensing, we compared the MgATP2- concentrations in cytosol and plastid stroma in living Arabidopsis thaliana. In the cotyledon, hypocotyl and roots of 10-day-old seedlings, the stromal MgATP2- concentrations were all significantly lower than the cytosolic MgATP2- concentrations. Only in the cotyledons of very young seedlings (3 and 4-day-old), the stromal MgATP2- levels are as high as that of the cytosol. Addition of exogenous MgATP2- to isolated chloroplasts from 4- and 5-day-old AT1.03 seedlings increased the FRET ratio of the stromal sensor, but not in chloroplasts isolated from 10-day-old cotyledons or 20-day-old true leaves, suggesting that mature chloroplasts do not import ATP efficiently. Transient expression of AtNTT1 and AtNTT2 in mesophyll protoplasts greatly lowered the cytosolic MgATP2- concentrations, indicative of an additional ATP sink being created by increasing the entry of ATP into the chloroplast. Employing a combination of photosynthetic and respiratory inhibitors we corroborate the dependency of photosynthetic ATP production on mitochondrial dissipation of photosynthetic reductant. We propose that during chloroplast maturation, the flux of ATP between the cytosolic and stromal compartments of mature chloroplasts is restricted by the downregulation of the expression of nucleotide transporters. Restricting energy consumption by the chloroplast in source cells at night may allow higher plants to conserve resources to support plant growth. Hence, unlike in diatoms, in Arabidopsis thaliana cytosolic ATP does not enter the mature chloroplasts to support the Calvin-Benson cycle. Instead, chloroplasts adjust the ATP:NADPH ratio by exporting reducing equivalents, which are dissipated by the mitochondria to generate cytosolic ATP.