Acid Phosphatase Kinetics as a Physiological Tool for Assessing Crop Adaptability to Phosphorus Deficiency

Abstract

Acid phosphatases (APase) exuded from the roots is important in mobilizing organic phosphate in the soil. Enzyme kinetics can provide reliable physiological markers to detect the potential for superior plant performance under low P. Kinetic constants for the secreted APase could be used as an early physiological indicator for P stress tolerance in legumes, Desmodium tortuosum , Phaseolus vulgaris,Vigna unguiculata and Crotalaria juncea were grown from seed in +P and-P nutrient solutions and plants were harvested during the early vegetative phase in order to collect the root exudates in vivo and for dry biomass, leaves soluble Pi, and total P in the dry biomass. Root surface Na-soluble APase was extracted from +P and-P grown plants by incubating three intact plants in beakers with their roots immersed in a 0.1 M NaCl solution. Secreted APase was obtained with the roots of three plants individually immersed in a dialysis tube (12 kD) containing NaCl 100 mM and then transferred to a recipient containing 3L of the same solution. Kinetic constants Km and Vmax were determined using a range substrate (p-NPP)concentration (S). Activity (v) was expressed as µmoles PNP/h per g root fresh (FWr) or dry weight DWr. Graphical representations were used for the determination of the Km and Vmax: Linewaver-Burk double reciprocal plot 1/v vs. 1/S plot; Hanes-Wolf plot S/v vs. S and Woolf-Augustinsson-Hofstee plot v vs. v/S. The first visual indication of P deficiency was a reduction in leaf area and dry biomass and a higher soluble Pi in the leaves of +P plants. Activity was higher in-P plants at the beginning of the growth period and the proper timing for the onset of the P-stress was apparently crucial for the induction of APase. For Phaseolus vulgaris Km values apparently indicate the lack of phosphate starvation-inducible APase and a higher Vmax in-P plants; however, with the combination of a high Km with a high Vmax plant behaviour could be improved under P deficiency. In Vigna unguiculata the low Vmax in-P plants may be compensated for by its lower Km. Crotalaria juncea showed considerably greater kinetic diversity, but Km was lower in-P plants. The practical implications of Km and Vmax are explained in terms of the potential for P-liberation under limiting Pi ; to be efficient an increase in Pi uptake is likely to occur if the APase released has a low Km (in the neighborhood of the soil P concentration) and a high Vmax as found for Desmodium, Phaseoulus and Vigna. The Km provided a means of comparing the enzyme from high or low-P plants indicating that Km is a reliable physiological tool for assessing plant adaptability to P-deficiency and it is suggested that Km, Vmax with total leaf area and relative growth rate (RGR).