Measurement of Photorespiratory Cycle Enzyme Activities in Leaves Exposed to Abiotic Stress.

Photorespiration, an essential metabolic component, is a classic example of interactions between the intracellular compartments of a plant cell: the chloroplast, peroxisome, mitochondria, and cytoplasm. The photorespiratory pathway is often modulated by abiotic stress and is considered an adaptive response. Monitoring the patterns of key enzymes located in different subcellular components would be an ideal approach to assessing the modulation of the photorespiratory metabolism under abiotic stress. This chapter describes the procedures for assaying several individual enzyme activities of key photorespiratory enzymes and evaluating their response to oxidative/photooxidative stress. It is essential to ascertain the presence of stress in the experimental material. Therefore, procedures for typical abiotic stress induction in leaves by highlighting without or with menadione (an oxidant that targets mitochondria) are also included.

Redox basis of photosynthesis inhibition at supra-optimal bicarbonate in mesophyll protoplasts of Arabidopsis thaliana.

We examined the patterns of photosynthetic O2 evolution at 1 mM (optimal) and 10 mM (supra-optimal) bicarbonate in mesophyll protoplasts of Arabidopsis thaliana. The photosynthetic rate of protoplasts reached the maximum at an optimal concentration of 1 mM bicarbonate and got suppressed at supra-optimal levels of bicarbonate. We examined the basis of such photosynthesis inhibition by mesophyll protoplasts at supra-optimal bicarbonate. The wild-type protoplasts exposed to supra-optimal bicarbonate showed up signs of oxidative stress. Besides the wild-type, two mutants were used: nadp-mdh (deficient in chloroplastic NADP-MDH) and vtc1 (deficient in mitochondrial ascorbate biosynthesis). The protoplasts of the nadp-mdh mutant exhibited a higher photosynthetic rate and greater sensitivity to supra-optimal bicarbonate than the wild-type. The ascorbate-deficient vtc1 mutant had a low photosynthetic rate and no significant inhibition at high bicarbonate. The nadp-mdh mutants had elevated activities, protein, and transcript levels of key antioxidant enzymes. On the other hand, the antioxidant enzyme systems in vtc1 mutants were not much affected at supra-optimal bicarbonate. We propose that the inhibition of photosynthesis at supra-optimal bicarbonate depends on the redox state of mesophyll protoplasts. The robust antioxidant enzyme systems in protoplasts of nadp-mdh mutant might be priming the plants to sustain high photosynthesis at supra-optimal bicarbonate.