**4. Conclusions**

lized as extracellular enzymes such as APase primarily function associated with soil colloids [20]. Compared to the free enzyme, properties and kinetic behavior of such complexed enzymes had a different pH activity dependence and sensitivity to temperature and protein degradation [19]. According to reports in the literature, the kinetics of APase in synthetic enzyme complexes simulating those usually encountered in soil, showed Michaelis-Menten kinetics with a lower Vmax and higher Km values as compared to the free enzyme [13]. Many APase isozymes exist in the root and leaves but only one of them was secreted into the rhizosphere in a large amount [25]. When the enzyme was mixed with aqueous solutions extracted from a P-deficient soil its activity declined to 55% of its original activity after 14 days and to 9% after 21 days. We have performed experiments applying the secreted APase enzyme solution obtained from low P grown plants of *Centrosema rotundifolia* and *Crotalaria juncea* to low P soils [6]; according to the results APase activity in the soils showed significant differences depending on soil type and root secretion but was higher in soils with the secreted APase from *Crotalaria* plants. Under the conditions of a higher Km the enzyme will not efficiently function under P starvation as a higher substrate concentration is needed to achieve half the maximal velocity. Under these circumstances, in order to unbind APase to perform efficiently, besides having a lower Km value, the roots should have the ability to secrete larger amounts of the enzyme into the rhizosphere to compensate for the low Vmax. It has been shown that the APase secreted by white lupin roots is stable in soil solution and shows low substrate specificity which is important to improve their ability to use organic P [12]. According to our results, true saturation Michaelis-Menten kinetics was not observed for all the species, specially for the enzyme from +P plants; we have also found similar results with crude extracts from other wild and cultivated species, and as seen from the shape of the plots of enzyme velocity *versus* substrate concentration, the presence of several isoenzymes should not be discarded. In this context the Hofstee plot (v vs, v/S) is the best alternative in detecting the presence of multiple enzymes that catalyze the same reaction [23]. For agronomic purposes, it is better to assay the crude enzyme secretion or extract, without further purification, as it is the form that it is released from the roots to the environment. Differences in APase activity for *Phaseolus vulgaris* as seen from the Km values apparently indicate the lack of phosphate starvationinducible APase, as it has been found in other crops, for example, see [14]; Vmax values on the other hand were higher in -P plants; however the combination of a high Km with a high Vmax could improve plant behaviour under P deficiency. The opposite was noted with *Vigna unguiculata* where a low Vmax in -P plants may be compensated by a lower Km. As compared to *Phaseoulus* and *Vigna,* the APase secreted from the roots of *Crotalaria juncea* showed consid‐ erably greater kinetic diversity depending on the methods of plotting enzyme kinetics data for the calculation of Km and Vmax values for -P and +P plants. For maximum efficiency it seems reasonable to expect that the enzyme from low P plants under the conditions of this study would have a low Km and a high Vmax; we have found for *Crotalaria* differences in the Km from -P and +P plants, but not for Vmax where the values were similar for the enzyme secreted from low P and high P plants, as found for *Phaseoulus and Vigna*. The less suited combination for the enzyme to perform efficiently under P deficient conditions is to have a high Km and a low Vmax (which means that the substrate concentration must be high and does not compen‐ sate for a low Vmax). From our results, it is seen that the enzyme from -P plants is better suited

92 Plants for the Future

The practical implications of kinetic constants Km and Vmax for the enzyme exuded by the roots of several plant species were analyzed in this study in terms of the potential for Pliberation under limiting condition of Pi bioavailability in soil; an increased Pi uptake is likely to occur if the APase released by the roots has a low Km value (in the neighborhood of the soil P concentration) and a high Vmax, in order to be efficient in liberating Pi from the soil organic-P pool. It has been shown that one of the advantages of the APase secreted from the roots of some leguminous species such as lupinus, was a higher Vmax value as compared to the enzyme from other species. We have found similar results for enzyme from *Desmodium, Phaseoulus* and *Vigna*. The numerical value of the Km for the substrate p-nitrophenyl-P provided a means of comparing the enzyme from high or low-P plants.Our results showed that Km is a reliable physiological tool for assessing plant adaptability to P-deficiency and its is suggested that Km, enzyme activity (Vmax), total leaf area and relative growth rate (RGR) may be used as physiological indicators to differentiate plants grown under P deficiency or sufficiency.
