Journal of Experimental Botany vol:52 issue:362 pages:1769-1777
The influence of the O-2 and CO2 concentration and the temperature on the O-2 uptake rate of cool-stored intact pears and pear cell protoplasts in suspension was compared. Protocols to isolate pear cell protoplasts from pear tissue and two methods to measure protoplast respiration have been developed. Modified Michaelis-Menten kinetics were applied to describe the effect of the O-2 and the CO2 concentration on the O-2 uptake rate and temperature dependence was analysed with an Arrhenius equation. Both systems were described with a non-competitive type Of CO2 inhibition. Due to the inclusion of gas diffusion properties, the Michaelis-Menten constant for intact pears (2.5 mM) was significantly larger than the one for protoplasts in suspension (3 muM), which was in turn larger than the Michaelis-Menten constant obtained in mitochondrial respiration measurements described in the literature. It was calculated that only 3.6% of the total diffusion effect absorbed in the Michaelis-Menten constant for intact pears, could be attributed to intracellular gas diffusion. The number of cells per volume of tissue was counted microscopically to establish a relationship between the pear cell protoplast and intact pear O-2 uptake rate. A remarkable similarity was observed: values of 61.8 nmol kg(-1) s(-1) for protoplasts and 87.1 nmol kg(-1) s(-1) for intact pears were obtained. Also, the inhibitory effect Of CO2 on the respiration rate was almost identical for protoplasts and intact pears, suggesting that protoplast suspensions are useful for the study of other aspects of the respiration metabolism.