Aerobic and anaerobic respiration in the intact spinach chloroplast.

Aerobic and anaerobic chloroplastic respiration was monitored by measuring (14)CO(2) evolution from [(14)C]glucose in the darkened spinach (Spinacia oleracea) chloroplast and by estimating the conversion of fructose 1,6-bisphosphate to glycerate 3-phosphate in the darkened spinach chloroplast in air with O(2) or in N(2) with nitrite or oxaloacetate as electron acceptors. The pathway of (14)CO(2) evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide and glycolate 2-phosphate under air or N(2) were those expected from the oxidative pentose phosphate cycle and glycolysis. Of the electron acceptors, O(2) was the best (2.4 nanomoles CO(2) per milligram chlorophyll per hour), followed by nitrite and oxaloacetate. With respect to glycerate 3-phosphate formation from fructose 1,6-bisphosphate, methylene blue increased the aerobic rate from 3.7 to 5.4 micromoles per milligram chlorophyll per hour. A rate of 4.8 micromoles per milligram chlorophyll per hour was observed under N(2) with nitrite and oxaloacetate.

Inhibition of chloroplastic respiration by osmotic dehydration.

The respiratory capacity of isolated spinach (Spinacia oleracea L.) chloroplasts, measured as the rate of (14)CO(2) evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with [(14)C]glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO(2) evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84% respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH(4)Cl (a stromal alkylating agent), iodoacetamide) an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6-bisphosphatase/phosphofructokinase junction.