**2. Results**

(when indicated) as mg total P in plant in dry root biomass per mg P in the nutrient solution (phosphorus absorption efficiency PAE), and as g the total dry biomass per mg total P in the

The extraction experiment for the root surface Na-soluble acid phosphatase from +P and -P grown plants was performed by incubating three intact plants from each P treatment in 250 mL beakers wrapped in aluminum foil with their roots immersed in 100 mL of a 0.1 M NaCl solution inside a well lighted refrigerator at 4 C. After 6 h the solutions in the three flasks per P treatment were separated and filtered using Whatman paper # 1, and as the crystal-clear filtrate was obtained, used for the kinetic studies [5].The solution with the enzyme obtained in this experiment is referred to as extracted APase. The secretion experiment for the acid phosphatase enzyme was performed with the roots of +P and -P intact 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 following the procedures reported [24].. After 24 h the solution inside the dialysis tubes of three plants was collected for kinetic studies when plants in the low P treatment showed moderate P deficiency symptoms as shown by growth inhibi‐ tion. The solution with the enzyme obtained in this experiment is referred to as secreted APase. Root surface Na-soluble acid phosphatase (APase) activity (reaction velocity v) was assayed using aliquots of the extracted or secreted enzyme with the substrate p-nitrophenyl-1 phosphate, buffer Na-acetate 50 mM pH 5.0 in a water bath at 34C. Reaction was stopped after 30 min with a saturated Na2CO3 solution and the yellow p-nitrophenol (PNP) read at 410 nm in an spectrophotometer Varian DMS-90. Kinetic constants Km and Vmax were determined using a range substrate concentration (S) based on the Km value for the purified enzyme [1, 16]. Two different ranges depending on the species (0.2; 0.33; 0.50; 1,00;1,43; 2,00; 2,50; 3,33 and 5,0 or 1.0 ; 1.25; 1.43; 1.67; 2.0; 2.50; 3.30 and 5.50 mM p-NPP) were assayed as indicated in the enzyme activity plots using the root exudation or the secretion from different plants. APase activity (v) was expressed as μmoles PNP/h per g root fresh (FWr) or dry weight DWr. The first step was to examine the v vs. S curve that reflects the hyperbolic Michaelis- Menten to determine the degree of substrate saturation; in theory as the velocity of the enzyme reaction (v) responds in a characteristic way to increasing substrate concentration, but when crude extracts clear substrate saturation is not always observed, and in order to determine the affinity constant Km and maximal velocity of the enzyme reaction Vmax different graphical repre‐ sentations based on linear transformations of the data are used for the determination of the affinity constant (Km) and the maximal velocity of the enzyme reaction (Vmax). The most widely used graphical representations are: 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, where v represents initial enzyme velocity at any given substrate concentration (S). Under the condi‐ tions of this study, the Km refers to the apparent Km for the enzyme activity, or the concen‐ tration of substrate at which activity is one half the maximal velocity and Vmax refers to the apparent maximal velocity for enzyme activity, which is the maximum rate of P- hydrolysis.

plant respectively (phosphorus use efficiency PUE).

82 Plants for the Future

**1.2. Acid phosphatase activity and kinetic constants**

Enzyme kinetics data were analyzed using the Hyper32 free software.

The first visual indication of P deficiency as observed for the plants of this study, was a reduction in leaf area to different degrees in different species, which was reflected in lower biomass values. The large differences among -P and +P plants are explained in part due to soluble Pi concentration in leaves which remained higher in +P plants during the entire growth period. In spite of the large differences in growth and Pi content, P efficiencies were much higher in the low P plants where larger differences were found for PUE (indicating a superior ability of -P plants to convert phosphorus into biomass). The superior acquisition efficiency (PAE) for some plant species has been attributed to a more efficient mycorrhizae and/or root acid phosphatase activity in addition to other factors as superior uptake kinetic. For the plants of this study we focused the strategy on the kinetic constants for the secreted APase under P deficient conditions. Enzyme activity is induced under P deficient conditions which depends on the proper timing for the onset of the P stress; the low Pi content in leaves of -P plants on the other hand was compensated for by a 10 times higher phosphorus use efficiency (PUE) for the plants of this study, which is in agreement with the hypothesis that the efficient recycling of Pi inside the plant is an important mechanism for survival under conditions of P starvation.

#### **2.1. Acid Phosphatase Kinetics of** *Desmodium tortuosum* **(beggar weed)**

After 28 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.01 mM P) were immersed in the collection flasks or in the dialysis membrane and after the extraction period aliquots were taken to perform kinetic studies for (a) the extracted enzyme and (b) the secreted enzyme. For the secreted enzyme saturation kinetics was observed for the enzyme from both high and low grown plants (+P and -P) as substrate concentration was increased from 0.2 to 5.0 mM, as seen from the hyperbolic Michaelis-Menten plot in Figure 1a and Figure 2a Figure +P (a) and -P (a).

The apparent Km and Vmax values determined from Lineweaver-Burk, Hanes and Hofstee plots; Figures +P and -P. For the high P plant enzyme, Km values of 2.81, 1.54, 2.63 and 1.07 mM p-NPP and Vmax of 80.5, 57,3 80.85 and 51.6 μmol PNP/h/g FW roots were calculated from the plots depicted in Figure 1 (b, c and d) in Figure +P For the low-P plant enzyme values were 0.92, 0.56, 1.58 and 0.73 mM p-NPP and 105.1, 87.6, 138.4 and 101.3 μmol PNP/h/g FW roots (Figure 2 (b, c and d) (Figure -P). As seen from the results obtained in this investigation, lower Km a higher Vmax values were found for the enzyme from -P Desmodium plants with any of the plots used to transform the data. For the extracted enzyme Vmax values were similar for the +P and -P plant enzymes but a lower Km was found for -P as compared to +P grown plants: 2.00, 1.48, 1.78,1.61 mM p-NPP *vs* 3.23, 3.27, 2.96, and 2.69 mM p-NPP for +P, as calculated from the plots seen in Figures 3 and 4, for +P and –P treatments using flasks for the extraction of the enzyme. Besides enzyme velocity (Vmax) was lower for the extracted enzyme between 8.85 and 11.63 μmol PNP/h/g FW roots for +P and -P plants) as compared to the secreted enzyme.

Figure 1.Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated. **Figure 1.** Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated.

3 Figure 2. Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated The apparent Km and Vmax values determined from Lineweaver-Burk, Hanes and Hofstee plots; Figures +P and -P . For the high P **Figure 2.** Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated

plant enzyme, Km values of 2.81, 1.54, 2.63 and 1.07 mM p-NPP and Vmax of 80.5, 57,3 80.85 and 51.6 µmol PNP/h/g FW roots were calculated from the plots depicted in Figure 1 (b, c and d) in Figure +P For the low-P plant enzyme values were 0.92, 0.56, 1.58 and 0.73 mM p-NPP and 105.1, 87.6, 138.4 and 101.3 µmol PNP/h/g FW roots (Figure 2 (b, c and d) (Figure -P). As seen from the results obtained in this investigation, lower Km a higher Vmax values were found for the enzyme from -P Desmodium plants with any of the plots used to transform the data. For the extracted enzyme Vmax values were similar for the +P and -P plant enzymes but a lower Km was found for -P as compared to +P grown plants: 2.00, 1.48, 1.78,1.61 mM p-NPP *vs* 3.23, 3.27, 2.96, and 2.69 mM p-NPP for +P, as calculated from the plots seen in Figures 3 and 4, for +P and –P treatments using flasks for the extraction of the enzyme. Besides enzyme velocity (Vmax) was lower for the extracted enzyme between 8.85 and 11.63 µmol PNP/h/g FW roots for +P and -P plants) as compared to the secreted enzyme. Acid Phosphatase Kinetics in *Phaseoulus vulgaris* (common bean) After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were

immersed in the dialysis membrane

3

#### **2.2. Acid Phosphatase Kinetics in** *Phaseoulus vulgaris* **(common bean)** with any of the plots used to transform the data. For the extracted enzyme Vmax values were similar for the +P and -P plant enzymes but a lower Km was found for -P as compared to +P grown plants: 2.00, 1.48, 1.78,1.61 mM p-NPP *vs* 3.23, 3.27, 2.96, and 2.69

immersed in the dialysis membrane

After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were immersed in the dialysis membrane mM p-NPP for +P, as calculated from the plots seen in Figures 3 and 4, for +P and –P treatments using flasks for the extraction of the enzyme. Besides enzyme velocity (Vmax) was lower for the extracted enzyme between 8.85 and 11.63 µmol PNP/h/g FW roots for +P and -P plants) as compared to the secreted enzyme. Acid Phosphatase Kinetics in *Phaseoulus vulgaris* (common bean) After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were

the results obtained in this investigation, lower Km a higher Vmax values were found for the enzyme from -P Desmodium plants

Figure 2. Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated The apparent Km and Vmax values determined from Lineweaver-Burk, Hanes and Hofstee plots; Figures +P and -P . For the high P

Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated **Figure 3.** Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P sufficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated

Figure 3.Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent

For the extracted enzyme the roots of 21 days old plants were immersed in the collection flasks and after the extraction period aliquots were taken to perform kinetic studies for (a) the extracted enzyme and (b) the secreted enzyme. For the secreted enzyme saturation kinetics was observed for the enzyme from both high and low grown plants (+P and -P) as substrate concentration was increased from 0.2 to 5.0 mM, as seen from the hyperbolic Michaelis-Menten plot in Figure 5a and Figure 6a Figure +P (a) and -P (a). The apparent Km and Vmax determined from Lineweaver-Burk, Hanes and Hofstee plots Figures +P and -P. for the high P plant enzyme Figure 5 (a, b, c and d) were Km values of 0.93, 0.73, 1.36, and 0.85 mM p-NPP and Vmax values of 8.5, 7.66, 10.13, and 8.34 μmol PNP/h/ g FW roots. For the low P grown plants Km values were similar to those found for the +P plant enzyme as calculated from plots in Figure 6 (a, b, c, and d) while the Vmax values were higher for the low P plants between 13.36 and 14.28 as calculated from Figure 6 (a, b c and d). For the extracted enzyme Km values were inconsistent and no clear differences were found between +P and -P plants (calculated from plots in Figure 7 and 8 respectively).

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(d) Hofstee plots as indicated.

84 Plants for the Future

immersed in the dialysis membrane

(d) Hofstee plots as indicated

Figure 1.Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated. **Figure 1.** Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and

Figure 2. Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated The apparent Km and Vmax values determined from Lineweaver-Burk, Hanes and Hofstee plots; Figures +P and -P . For the high P plant enzyme, Km values of 2.81, 1.54, 2.63 and 1.07 mM p-NPP and Vmax of 80.5, 57,3 80.85 and 51.6 µmol PNP/h/g FW roots were calculated from the plots depicted in Figure 1 (b, c and d) in Figure +P For the low-P plant enzyme values were 0.92, 0.56, 1.58 and 0.73 mM p-NPP and 105.1, 87.6, 138.4 and 101.3 µmol PNP/h/g FW roots (Figure 2 (b, c and d) (Figure -P). As seen from the results obtained in this investigation, lower Km a higher Vmax values were found for the enzyme from -P Desmodium plants with any of the plots used to transform the data. For the extracted enzyme Vmax values were similar for the +P and -P plant enzymes but a lower Km was found for -P as compared to +P grown plants: 2.00, 1.48, 1.78,1.61 mM p-NPP *vs* 3.23, 3.27, 2.96, and 2.69 mM p-NPP for +P, as calculated from the plots seen in Figures 3 and 4, for +P and –P treatments using flasks for the extraction of the enzyme. Besides enzyme velocity (Vmax) was lower for the extracted enzyme between 8.85 and 11.63 µmol PNP/h/g FW roots for +P and -P plants) as compared to the secreted enzyme. Acid Phosphatase Kinetics in *Phaseoulus vulgaris* (common bean) After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were

**Figure 2.** Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and immersed in the dialysis membrane

Figure 3.Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate p-

Figure 2. Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated The apparent Km and Vmax values determined from Lineweaver-Burk, Hanes and Hofstee plots; Figures +P and -P . For the high P plant enzyme, Km values of 2.81, 1.54, 2.63 and 1.07 mM p-NPP and Vmax of 80.5, 57,3 80.85 and 51.6 µmol PNP/h/g FW roots were calculated from the plots depicted in Figure 1 (b, c and d) in Figure +P For the low-P plant enzyme values were 0.92, 0.56, 1.58 and 0.73 mM p-NPP and 105.1, 87.6, 138.4 and 101.3 µmol PNP/h/g FW roots (Figure 2 (b, c and d) (Figure -P). As seen from the results obtained in this investigation, lower Km a higher Vmax values were found for the enzyme from -P Desmodium plants with any of the plots used to transform the data. For the extracted enzyme Vmax values were similar for the +P and -P plant enzymes but a lower Km was found for -P as compared to +P grown plants: 2.00, 1.48, 1.78,1.61 mM p-NPP *vs* 3.23, 3.27, 2.96, and 2.69 mM p-NPP for +P, as calculated from the plots seen in Figures 3 and 4, for +P and –P treatments using flasks for the extraction of the enzyme. Besides enzyme velocity (Vmax) was lower for the extracted enzyme between 8.85 and 11.63 µmol PNP/h/g FW roots for +P and -P plants) as compared to the secreted enzyme. Acid Phosphatase Kinetics in *Phaseoulus vulgaris* (common bean) After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were

nitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated. For the extracted enzyme the roots of 21 days old plants were immersed in the collection flasks and after the extraction period aliquots were taken to perform kinetic studies for (a) the extracted enzyme and (b) the secreted enzyme. For the secreted enzyme **Figure 4.** Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated.

saturation kinetics was observed for the enzyme from both high and low grown plants (+P and -P) as substrate concentration was

Figure 4. Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate p-

Figure 5. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated. **Figure 5.** Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate p-ni‐ trophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated.

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Acid Phosphatase Kinetics as a Physiological Tool for Assessing Crop Adaptability to Phosphorus Deficiency http://dx.doi.org/10.5772/60975 87

Figure 6. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated. **Figure 6.** Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate p-ni‐ trophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated. Figure 6. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated.

Figure 7. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated **Figure 7.** Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate p-ni‐ trophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P sufficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated

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3

Figure 5. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated. **Figure 5.** Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate p-ni‐ trophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and

Figure 2. Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated The apparent Km and Vmax values determined from Lineweaver-Burk, Hanes and Hofstee plots; Figures +P and -P . For the high P plant enzyme, Km values of 2.81, 1.54, 2.63 and 1.07 mM p-NPP and Vmax of 80.5, 57,3 80.85 and 51.6 µmol PNP/h/g FW roots were calculated from the plots depicted in Figure 1 (b, c and d) in Figure +P For the low-P plant enzyme values were 0.92, 0.56, 1.58 and 0.73 mM p-NPP and 105.1, 87.6, 138.4 and 101.3 µmol PNP/h/g FW roots (Figure 2 (b, c and d) (Figure -P). As seen from the results obtained in this investigation, lower Km a higher Vmax values were found for the enzyme from -P Desmodium plants with any of the plots used to transform the data. For the extracted enzyme Vmax values were similar for the +P and -P plant enzymes but a lower Km was found for -P as compared to +P grown plants: 2.00, 1.48, 1.78,1.61 mM p-NPP *vs* 3.23, 3.27, 2.96, and 2.69 mM p-NPP for +P, as calculated from the plots seen in Figures 3 and 4, for +P and –P treatments using flasks for the extraction of the enzyme. Besides enzyme velocity (Vmax) was lower for the extracted enzyme between 8.85 and 11.63 µmol PNP/h/g FW roots for +P and -P plants) as compared to the secreted enzyme. Acid Phosphatase Kinetics in *Phaseoulus vulgaris* (common bean) After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were

Figure 3.Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated

Figure 4. Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated. For the extracted enzyme the roots of 21 days old plants were immersed in the collection flasks and after the extraction period aliquots were taken to perform kinetic studies for (a) the extracted enzyme and (b) the secreted enzyme. For the secreted enzyme saturation kinetics was observed for the enzyme from both high and low grown plants (+P and -P) as substrate concentration was increased from 0.2 to 5.0 mM, as seen from the hyperbolic Michaelis-Menten plot in Figure 5a and Figure 6a Fig +P (a) y -P (a). The apparent Km and Vmax determined from Lineweaver-Burk, Hanes and Hofstee plots Figures +P and -P . for the high P plant enzyme Figure 5 (a, b, c and d) were Km values of 0.93, 0.73, 1.36, and 0.85 mM p-NPP and Vmax values of 8.5, 7.66, 10.13, and 8.34 µmol PNP/h/ g FW roots. For the low P grown plants Km values were similar to those found for the +P plant enzyme as calculated from plots in Figure 6 (a, b, c, and d) while the Vmax values were higher for the low P plants between 13.36 and 14.28 as

**Figure 4.** Enzyme kinetics plots of the acid phosphatase activity from *Desmodium tortuosum* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and

(d) Hofstee plots as indicated.

(d) Hofstee plots as indicated.

immersed in the dialysis membrane

86 Plants for the Future

Figure 6. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated.

Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated

Figure 8. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated. Vmax values were lower than those for the secreted enzyme although higher for -P plants, as found for the secreted enzyme . **Figure 8.** Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate p-ni‐ trophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated.

However, no clear tendency was found in the Km values for the extracted APase collected from root extracts. Acid Phosphatase Kinetics in *Vigna unguiculata* (cowpea) After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were immersed in the dialysis membrane. and after the extraction period aliquots were taken to perform kinetic studies for the secreted enzyme. Data for the extracted enzyme are not presented as they were very low and similar Vmax values were lower than those for the secreted enzyme although higher for -P plants, as found for the secreted enzyme. However, no clear tendency was found in the Km values for the extracted APase collected from root extracts.

for +P and -P plants and in some cases not detectable. For the secreted enzyme saturation kinetics was observed for the enzyme from

Lineweaver-Burk, Hanes and Hofstee plots Figures +P and -P . for the high P plant enzyme Figure 9 (a, b, c and d) were Km values

#### both high and low grown plants (+P and -P) as substrate concentration was increased from 1.0 to 5.0 mM, as seen from the hyperbolic Michaelis-Menten plot in Figure 9a and Figure 10a Fig +P (a) y -P (a). The apparent Km and Vmax determined from **2.3. Acid Phosphatase Kinetics in** *Vigna unguiculata* **(cowpea)**

of 1.02, 1.31, 1.04 and 0.96 mM p-NPP and Vmax values of 4.11, 4.46, 4.12 and 4.03 µmol/h/ g FW roots. For the low P grown plants Km values were lower (0.72, 0.68, 0.98 and 0.71) to those found for the +P plant enzyme as calculated from plots in Figure 10 (a, b, c, and d) while the Vmax values were higher for the high P plants (4.11, 4.46, 4.12, and 4.03 as compared to 3.23, 3.19, 3,52 and 3.23 µmol PNP/h/g FW roots for -P enzyme as calculated from Figures 9 and 10. Acid Phosphatase Kinetics in *Crotalaria juncea* (sunn hemp). After 20 days of planting the roots from +P (0.86 mM P) and -P (.004 mM P) intact plants were individually immersed in a dialysis membrane and after an extraction period of 24 h, aliquots from the solution inside the dialysis membrane were taken to perform the kinetics studies of root secreted acid phosphatase. Two separate experiments were performed using different plants. The following concentration range for the substrate was used: 1.0, 1.25, 1.43, 1.67, 2.0, 2.50, 3.30 and 5.50 mM p-NPP and as no clear substrate saturation was observed for either +P or -P enzymes using the hyperbolic Michaelis-Menten plot, the apparent Km After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were immersed in the dialysis membrane and after the extraction period aliquots were taken to perform kinetic studies for the secreted enzyme. Data for the extracted enzyme are not presented as they were very low and similar for +P and -P plants and in some cases not detectable. For the secreted enzyme saturation kinetics was observed for the enzyme from both high and low grown plants (+P and -P) as substrate concentration was increased from 1.0 to 5.0 mM, as seen from the hyperbolic Michaelis-Menten plot in Figure 9a and Figure 10a Figure +P (a) and -P (a). The apparent Km and Vmax deter‐ mined from Lineweaver-Burk, Hanes and Hofstee plots Figures +P and -P. for the high P plant enzyme Figure 9 (a, b, c and d) were Km values of 1.02, 1.31, 1.04 and 0.96 mM p-NPP and Vmax values of 4.11, 4.46, 4.12 and 4.03 μmol/h/ g FW roots. For the low P grown plants Km values were lower (0.72, 0.68, 0.98 and 0.71) to those found for the +P plant enzyme as calculated from plots in Figure 10 (a, b, c, and d) while the Vmax values were higher for the high P plants (4.11, 4.46, 4.12, and 4.03 as compared to 3.23, 3.19, 3,52 and 3.23 μmol PNP/h/g FW roots for -P enzyme as calculated from Figures 9 and 10.

4

#### **2.4. Acid Phosphatase Kinetics in** *Crotalaria juncea* **(sunn hemp)**

After 20 days of planting the roots from +P (0.86 mM P) and -P (.004 mM P) intact plants were individually immersed in a dialysis membrane and after an extraction period of 24 h, aliquots from the solution inside the dialysis membrane were taken to perform the kinetics studies of root secreted acid phosphatase. Two separate experiments were performed using different plants. The following concentration range for the substrate was used: 1.0, 1.25, 1.43, 1.67, 2.0, 2.50, 3.30 and 5.50 mM p-NPP and as no clear substrate saturation was observed for either +P or -P enzymes using the hyperbolic Michaelis-Menten plot, the apparent Km and Vmax values for the two experiments were determined from Lineweaver-Burk, Hanes and Hofstee plots and linear transformations were adjusted to the best fit line.

nitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated **Figure 9.** Enzyme kinetics plots of the acid phosphatase activity from *Vigna unguiculata* roots with the substrate p-ni‐ trophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P sufficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated

Figure 9. Enzyme kinetics plots of the acid phosphatase activity from *Vigna unguiculata* roots with the substrate p-

It is important to note that a homogeneous preparation is by no means necessary for kinetic analyses, but the purer the enzyme the less complications from competing reactions that may use up the substrate or the product (23). As shown from the results in Table 1, apparent Km and Vmax values, showed considerable kinetic diversity but the degree of adjustment for the linear equations (r2 ) was always higher than 0.70 except for the Hofstee plot for +P plants in experiment 1, where a low r2 value of 0.50 was obtained. The apparent Km values determined from Lineweaver-Burk plots were, for the first and second experiments, 0.53 and 0.57 mM for -P and 0.82 and 0.76 mM p-NPP for +P plants respectively; from the Hanes plot 1.75 and 0.79 (-P) and 0.62 and 1.13 (+P) and from the Hofstee plot 0.53 and 0.59 (-P) and 0.86 and 0.78 (+P).These results show that the Km from low P plants was lower than that for +P plants (except when calculated from the Hanes plot) and varied from 0.53 to 0.59 mM in -P and 0.76 and 1.13

5

4

Figure 6. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweawer-Burk (c) Hanes and (d) Hofstee plots as indicated.

Figure 7. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P sufficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated

Figure 8. Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate pnitrophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated. Vmax values were lower than those for the secreted enzyme although higher for -P plants, as found for the secreted enzyme . However, no clear tendency was found in the Km values for the extracted APase collected from root extracts. Acid Phosphatase Kinetics in *Vigna unguiculata* (cowpea) After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were immersed in the dialysis membrane. and after the extraction period aliquots were taken to perform kinetic studies for the secreted enzyme. Data for the extracted enzyme are not presented as they were very low and similar for +P and -P plants and in some cases not detectable. For the secreted enzyme saturation kinetics was observed for the enzyme from both high and low grown plants (+P and -P) as substrate concentration was increased from 1.0 to 5.0 mM, as seen from the hyperbolic Michaelis-Menten plot in Figure 9a and Figure 10a Fig +P (a) y -P (a). The apparent Km and Vmax determined from Lineweaver-Burk, Hanes and Hofstee plots Figures +P and -P . for the high P plant enzyme Figure 9 (a, b, c and d) were Km values of 1.02, 1.31, 1.04 and 0.96 mM p-NPP and Vmax values of 4.11, 4.46, 4.12 and 4.03 µmol/h/ g FW roots. For the low P grown plants Km values were lower (0.72, 0.68, 0.98 and 0.71) to those found for the +P plant enzyme as calculated from plots in Figure 10 (a, b, c, and d) while the Vmax values were higher for the high P plants (4.11, 4.46, 4.12, and 4.03 as compared to 3.23, 3.19, 3,52 and 3.23 µmol PNP/h/g FW roots for -P enzyme as calculated from Figures 9 and 10. Acid Phosphatase Kinetics in *Crotalaria juncea* (sunn hemp). After 20 days of planting the roots from +P (0.86 mM P) and -P (.004 mM P) intact plants were individually immersed in a dialysis membrane and after an extraction period of 24 h, aliquots from the solution inside the dialysis membrane were taken to perform the kinetics studies of root secreted acid phosphatase. Two separate experiments were performed using different plants. The following concentration range for the substrate was used: 1.0, 1.25, 1.43, 1.67, 2.0, 2.50, 3.30 and 5.50 mM p-NPP and as no clear substrate saturation was observed for either +P or -P enzymes using the hyperbolic Michaelis-Menten plot, the apparent Km

**Figure 8.** Enzyme kinetics plots of the acid phosphatase activity from *Phaseolus vulgaris* roots with the substrate p-ni‐ trophenylphosphate (p-NPP). The extracted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and

Vmax values were lower than those for the secreted enzyme although higher for -P plants, as found for the secreted enzyme. However, no clear tendency was found in the Km values for

After 14 days of planting the roots of four plants grown under either P sufficiency (+P 1.0 mM P) or deficiency (-P 0.005 mM P) were immersed in the dialysis membrane and after the extraction period aliquots were taken to perform kinetic studies for the secreted enzyme. Data for the extracted enzyme are not presented as they were very low and similar for +P and -P plants and in some cases not detectable. For the secreted enzyme saturation kinetics was observed for the enzyme from both high and low grown plants (+P and -P) as substrate concentration was increased from 1.0 to 5.0 mM, as seen from the hyperbolic Michaelis-Menten plot in Figure 9a and Figure 10a Figure +P (a) and -P (a). The apparent Km and Vmax deter‐ mined from Lineweaver-Burk, Hanes and Hofstee plots Figures +P and -P. for the high P plant enzyme Figure 9 (a, b, c and d) were Km values of 1.02, 1.31, 1.04 and 0.96 mM p-NPP and Vmax values of 4.11, 4.46, 4.12 and 4.03 μmol/h/ g FW roots. For the low P grown plants Km values were lower (0.72, 0.68, 0.98 and 0.71) to those found for the +P plant enzyme as calculated from plots in Figure 10 (a, b, c, and d) while the Vmax values were higher for the high P plants (4.11, 4.46, 4.12, and 4.03 as compared to 3.23, 3.19, 3,52 and 3.23 μmol PNP/h/g FW roots for

(d) Hofstee plots as indicated.

88 Plants for the Future

the extracted APase collected from root extracts.


**2.3. Acid Phosphatase Kinetics in** *Vigna unguiculata* **(cowpea)**

Figure 10. Enzyme kinetics plots of the acid phosphatase activity from *Vigna unguiculata* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated. It is important to note that a homogeneous prearatin is by no means necessary for kinetic analyses, but **Figure 10.** Enzyme kinetics plots of the acid phosphatase activity from *Vigna unguiculata* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated.

the purer the enzyme the less complications from competing reactions that may use up the substrate or

39.90 and 25.97 for -P and 25.44 and 25.38 µmol PNP/g root DW/h for experiments 1 and 2

mM in +P plants. Thus for practical purposes and according to the results presented in this study, the Lineweaver-Burk and Hofstee plots are the best options to fit the data, if the objective of the study is to find out the differences in Km between -P and +P plant enzymes The apparent Vmax values determined from Lineweaver-Burk double reciprocal plot were 39.90 and 25.97 for -P and 25.44 and 25.38 μmol PNP/g root DW/h for experiments 1 and 2 respectively; from the Hanes plot these values were 33.78 and 28.08 (-P) and 38.04 and 28.73 (+P) and from the Hofstee plot 36.94 and 26.12 (-P) and 26.13 and 25.64 (+P).These results show that Vmax values were similar for the secreted enzyme from roots of low P and high P plants. the product (23). As shown from the results in Table 1, apparent Km and Vmax values, showed considerable kinetic diversity but the degree of adjustment for the linear equations (r2 ) was always higher than 0.70 except for the Hofstee plot for +P plants in experiment 1, where a low r2 value of 0.50 was obtained. The apparent Km values determined from Lineweaver-Burk plots were, for the first and second experiments, 0.53 and 0.57 mM for -P and 0.82 and 0.76 mM p-NPP for +P plants respectively; from the Hanes plot 1.75 and 0.79 (-P) and 0.62 and 1.13 (+P) and from the Hofstee plot 0.53 and 0.59 (-P) and 0.86 and 0.78 (+P).These results show that the Km from low P plants was lower than that for +P plants (except when calculated from the Hanes plot) and varied from 0.53 to 0.59 mM in -P and 0.76 and 1.13 mM in +P plants. Thus for practical purposes and according to the results presented in this study, the Lineweaver-Burk and Hofstee plots are the best options to fit the data, if the objective of the study is to find out the differences in Km between -P and +P plant enzymes The apparent Vmax values determined from Lineweaver-Burk double reciprocal plot were


+P Y=0,0393+0,0348x 0,9888 1,13 28,73

II -P Y=0,0282+0,0356x 0,9948 0,79 28,08

Hoffstee I -P Y=36,9396-0,5331x 0,8929 0,53 36,94

5

Acid Phosphatase Kinetics as a Physiological Tool for Assessing Crop Adaptability to Phosphorus Deficiency http://dx.doi.org/10.5772/60975 91


**Table 1.** Apparent Km and Vmax values for the root secreted acid phosphatase in +P and -P grown plants of *Crotalaria juncea*. (Ascencio and Santana, unpublished) -P= 0,004 mM P; +P= 0,86 mM P

#### **3. Discussion**

mM in +P plants. Thus for practical purposes and according to the results presented in this study, the Lineweaver-Burk and Hofstee plots are the best options to fit the data, if the objective of the study is to find out the differences in Km between -P and +P plant enzymes The apparent Vmax values determined from Lineweaver-Burk double reciprocal plot were 39.90 and 25.97 for -P and 25.44 and 25.38 μmol PNP/g root DW/h for experiments 1 and 2 respectively; from the Hanes plot these values were 33.78 and 28.08 (-P) and 38.04 and 28.73 (+P) and from the Hofstee plot 36.94 and 26.12 (-P) and 26.13 and 25.64 (+P).These results show that Vmax values

Figure 10. Enzyme kinetics plots of the acid phosphatase activity from *Vigna unguiculata* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solutions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and (d) Hofstee plots as indicated.

**Figure 10.** Enzyme kinetics plots of the acid phosphatase activity from *Vigna unguiculata* roots with the substrate pnitrophenylphosphae (p-NPP). The secreted enzyme was obtained from plants previously grown in P deficient solu‐ tions. Apparent Km and Vmax values were obtained from: (a) Michaelis Menten (b) Lineweaver-Burk (c) Hanes and

> **Km (mM p-NPP)**

Vmax (µmol PNP/h/g DW r )

) was always


Km (mM p-NPP)


**Vmax (µmol PNP/h/g DW r)**

5

were similar for the secreted enzyme from roots of low P and high P plants.

It is important to note that a homogeneous prearatin is by no means necessary for kinetic analyses, but the purer the enzyme the less complications from competing reactions that may use up the substrate or the product (23). As shown from the results in Table 1, apparent Km and Vmax values, showed

higher than 0.70 except for the Hofstee plot for +P plants in experiment 1, where a low r2 value of 0.50 was obtained. The apparent Km values determined from Lineweaver-Burk plots were, for the first and second experiments, 0.53 and 0.57 mM for -P and 0.82 and 0.76 mM p-NPP for +P plants respectively; from the Hanes plot 1.75 and 0.79 (-P) and 0.62 and 1.13 (+P) and from the Hofstee plot 0.53 and 0.59 (-P) and 0.86 and 0.78 (+P).These results show that the Km from low P plants was lower than that for +P plants (except when calculated from the Hanes plot) and varied from 0.53 to 0.59 mM in -P and 0.76 and 1.13 mM in +P plants. Thus for practical purposes and according to the results presented in this study, the Lineweaver-Burk and Hofstee plots are the best options to fit the data, if the objective of the study is to find out the differences in Km between -P and +P plant enzymes The apparent Vmax values determined from Lineweaver-Burk double reciprocal plot were 39.90 and 25.97 for -P and 25.44 and 25.38 µmol PNP/g root DW/h for experiments 1 and 2 respectively; from the Hanes plot these values were 33.78 and 28.08 (-P) and 38.04 and 28.73 (+P) and from the Hofstee plot 36.94 and 26.12 (-P) and 26.13 and 25.64 (+P).These results show that

considerable kinetic diversity but the degree of adjustment for the linear equations (r2

Vmax values were similar for the secreted enzyme from roots of low P and high P plants.

ent Treatment Equation r2

**Plot Experiment Treatment Equation r2**

Hanes <sup>I</sup> -P Y=0,0520+ 0,0296x 0,9075 1,75 33,78

Hoffstee I -P Y=36,9396-0,5331x 0,8929 0,53 36,94

<sup>I</sup> -P Y =0,0271+0,0144x 0,9209 0,53 36,90

II -P Y=0,0385+0,0221x 0,8869 0,57 25,97

II -P Y=0,0282+0,0356x 0,9948 0,79 28,08

+P Y= 0,0393+0,0321x 0,7144 0,82 25,44

+P Y=0,0394+0,0299x 0,8542 0,76 25,38

+P Y=0,0163+ 0,0262x 0,9980 0,62 38,04

+P Y=0,0393+0,0348x 0,9888 1,13 28,73

Lineweaver

Lineweaver

Plot Experim

(d) Hofstee plots as indicated.

90 Plants for the Future

I

II

Root acid phosphatase activity for the plants was higher in -P plants at the beginning of the growth period (depending on the species) and that the proper timing for the onset of the Pstress is apparently crucial for induction of APase in different species [2, 6]. It has been shown in this and many other reports in the literature, that P deficient conditions in the plant can trigger APase activity [5, 10, 23]; and that new isoenzymes could be activated under P deficiency in roots [12, 16], leaves [29] and seedlings [14].However for bean and cowpea APase activity appears not to be inducible when 0.02 mM P was used to grow the plants under P deficient conditions even though differences in P concentration in the dry matter were large enough to suspect that plants were suffering from mild P stress [9]. In the present study 0.005 mM P was used in the low P treatment and even though large differences were not found for enzyme activity between +P and -P plants larger differences in total dry matter, leaf area and soluble Pi concentration in leaves were found. However from an agronomic point of view and focusing on the APase role to dissolve organic-P in soils with low P bioavailability, the enzyme secreted with the root exudates to the soil is a major concern in plant adaptation, specially to phosphorus-limited tropical soils [21]. Like many other secreted proteins the APase is glycosylated, which protects the enzyme against proteolytic enzymes and contributes to its stability over a wide pH range. Secretory APase can liberate bound P from soil and have shown to deplete organic P in the rhizosphere of several plant species; however, mineral phosphorus is hardly available in tropical soils and for organic P to be used in agricultural soils, increased secretion of APase may be involved as part of the coordinated adaptive strategies to withstand P deficiency. However, under soil conditions where extracellular enzymes such as APase function are associated with soil colloids, a large fraction of the free enzyme may be immobi‐

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 to cope with P deficiency, due to a consistent lower Km. In this context, favorable kinetic properties (low Km and highVmax) as well as the amount of secreted enzyme are important as one may compensate for each other; in this connection the best combination for the enzyme to perform efficiently under natural conditions, where a low P concentration exist in the soil, would be a low Km and a great amount of secreted enzyme to the environment. This might be the better strategy for plant species to perform efficiently under low P soils. We have shown from previous studies that leguminous plants have developed several growth strategies to withstand P limitations imposed by the soil; under P deficiency total leaf area, relative growth rate (RGR) and root length were reduced by 50% in severely stressed *Desmodium tortuosum* and other plant species [4,5], and that for tomato APAse activity was highly correlated to development and recovery from P stress and that total weight and average root diameter decreased under P stress while root surface area per unit dry weight increased, [10]. As large differences were found in Relative Growth Rate (RGR) between the high and low P plants and as these differences were consistent for all the species analyzed, RGR is an adequate physio‐ logical indicator of plant performance under P deficient conditions and a useful tool if used in screening purposes.
