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

Jocelyne Ascencio

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60975

#### **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 starvationinducible 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

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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).

**Keywords:** plant enzymes, root secretion, abiotic stress

#### **1. Introduction**

Phosphorus (P) is an essential element in the mineral nutrition of plants and under P starvation a reduction in biomass and leaf area among other physiological traits, is observed. Phosphorus occurs in soils not only as mineral phosphates but also as organic compounds that in order to be available to the roots they must be first hydrolyzed and several studies now indicate that acid phosphatase may have a role in mobilizing organic phosphate in the soil that in infertile tropical soils may contribute significantly to ameliorate P bioavailability from sparingly soluble P forms. Acid phosphatase (APase) from several plant species and genotypes has been shown to liberate P from soil thus decreasing organic phosphorus in the rhizosphere [8, 26, 27] and a large body of evidence now exists showing that P deficiency can trigger an increase in activity of root secreted APase in a variety of wild and cultivated plants and that the activity of the enzyme can vary among species along with the severity of P starvation [4, 24, 25]; however [28] showed that some tropical forage such as *Brachiaria* hybrids and *Arachis pintoi* have the ability to tolerate low-phosphorus stress without showing any increase in APase activity in root exudates. According to [29], the role of secreted APase in plant adaptation to low phosphorus availability is unclear. Enzyme kinetics can used to identify simple and reliable physiological markers, for screening purposes to detect the potential for superior plant performance under low P concentrations. Ascencio [4, 5] calculated the kinetics constants (Km and Vmax) for the exuded APase from different plants species grown under P- sufficient and P-deficient conditions. The numerical values of the Km for the substrate p-nitrophenyl-Phosphate (p-NPP) provided a means of comparing the enzyme from high or low P plants, so it is suggested that Km and enzyme activity (Vmax) may be used as physiological indicators to differentiate plants grown under P deficiency or sufficiency. Km values are related to the substrate concentration at which enzyme velocity is half of the maximum (Vmax), they are not strictly constants as those that are found with the purified enzyme as the activity is assayed using the crude root exudates as they might be released to the soil. Acid phosphatase enzymes are released to the soil as part of the pool of extracellular enzymes along with other products of root exudation; however, under laboratory conditions different forms of the enzyme are present depending on the procedure that is used to collect the enzyme. Collection of the enzyme *in vivo* can be performed directly from the intact plant by immersing their roots in the extracting solution which is assayed without purification (root surface Na-soluble APase) or by separating the enzyme from other exuded compounds by enclosing the roots of the intact plant inside a tube or a dialysis membrane (secreted APase). Collection of the enzyme *in vitro* from the root and other plant tissues (mostly leaves or seedlings) is achieved by grinding and collecting the resulting solution (extractable APase). Further purification of the enzyme in order to assess whether new APase isoenzymes are induced by P deficiency can be achieved using any of the extraction methods described above. The properties of the purified enzyme secreted in vivo by plant roots was first reported by Tadano [23], for the secreted enzyme from Lupinus roots under P-deficient conditions. Under P-deficient conditions new APase isoforms, which are different forms for the enzyme, are released. It has been shown [1,27] that APase isoforms were inducible under P deficient conditions but that for Lupinus, the major activity in the rhizosphere soil and in roots grown under hydroponic conditions corresponded to a previously purified APase secreted by the roots; thus in order to compare the potential of APase in the root exudates released by different plant species and genotypes that may increase plant performance under phosphorus deficient conditions, root secretions can be obtained *in vivo* and acid phosphatase activity measured in the secreted or exuded solution without further purification.

The objective of the present chapter is to give a broader picture, from an agronomical point of view, as to how kinetic constants for the APase secreted "in vivo" by the roots of leguminous plants grown under P-deficiency or sufficiency, could be used as an early physiological indicator for P stress tolerance.

#### **1.1. Research methods**

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

Phosphorus (P) is an essential element in the mineral nutrition of plants and under P starvation a reduction in biomass and leaf area among other physiological traits, is observed. Phosphorus occurs in soils not only as mineral phosphates but also as organic compounds that in order to be available to the roots they must be first hydrolyzed and several studies now indicate that acid phosphatase may have a role in mobilizing organic phosphate in the soil that in infertile tropical soils may contribute significantly to ameliorate P bioavailability from sparingly soluble P forms. Acid phosphatase (APase) from several plant species and genotypes has been shown to liberate P from soil thus decreasing organic phosphorus in the rhizosphere [8, 26, 27] and a large body of evidence now exists showing that P deficiency can trigger an increase in activity of root secreted APase in a variety of wild and cultivated plants and that the activity of the enzyme can vary among species along with the severity of P starvation [4, 24, 25]; however [28] showed that some tropical forage such as *Brachiaria* hybrids and *Arachis pintoi* have the ability to tolerate low-phosphorus stress without showing any increase in APase activity in root exudates. According to [29], the role of secreted APase in plant adaptation to low phosphorus availability is unclear. Enzyme kinetics can used to identify simple and reliable physiological markers, for screening purposes to detect the potential for superior plant performance under low P concentrations. Ascencio [4, 5] calculated the kinetics constants (Km and Vmax) for the exuded APase from different plants species grown under P- sufficient and P-deficient conditions. The numerical values of the Km for the substrate p-nitrophenyl-Phosphate (p-NPP) provided a means of comparing the enzyme from high or low P plants, so it is suggested that Km and enzyme activity (Vmax) may be used as physiological indicators to differentiate plants grown under P deficiency or sufficiency. Km values are related to the substrate concentration at which enzyme velocity is half of the maximum (Vmax), they are not strictly constants as those that are found with the purified enzyme as the activity is assayed using the crude root exudates as they might be released to the soil. Acid phosphatase enzymes are released to the soil as part of the pool of extracellular enzymes along with other products of root exudation; however, under laboratory conditions different forms of the enzyme are present depending on the procedure that is used to collect the enzyme. Collection of the

**Keywords:** plant enzymes, root secretion, abiotic stress

rate (RGR).

80 Plants for the Future

**1. Introduction**

The leguminous plants species (*Desmodium tortuosum* (Sw.) DC, *Phaseolus vulgaris* L var Manuare, *Vigna unguiculata* (L) Walp cv Tuy and *Crotalaria juncea* L, were used in this study. *Desmodium tortuosum* (beggarweed) Fabaceae, is a slow growing tropical non-grain legume that grows wild, and on small farms, in association with other crop species as a nitrogen source, as a forage legume and green manure. *Phaseolus vulgaris* (common bean) and *Vigna unguicu‐ lata* (cowpea), -Fabaceae are very important grain crops used as a protein source worldwide; *Crotalaria juncea* (sunn hemp) Fabaceae is widely grown throughout the tropics and subtropics as a source of green manure, fodder and lignified fiber and has been recently looked at as a possible bio-fuel. Plants were grown from sterilized seeds and water culture experiments were performed in a highly ventilated greenhouse in plastic 950 ml pots with aerated Hoagland solutions containing either sufficient (+P) or deficient (-P) (mM P as KH2PO4 depending of the species). After establishment, plants were harvested during the early vegetative phase for each species and separated into groups in order to perform kinetic studies for the APase in the root exudates collected in vivo, for dry biomass determinations and to measure soluble Pi content in fresh leaves and total P in the dry biomass. The Soluble Pi content in leaves was measured using leaf discs (0.5-10 g fresh weight) macerated in cold 2% acetic acid a the extract diluted and centrifuged at 4 o C and the clear supernatant used for Pi concentration determinations using the colorimetric phosphomolybdate reaction. Total P in the dry biomass was measured using previously digested material with H2SO4:H2O2. Phosphorus efficiencies were calculated (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 plant respectively (phosphorus use efficiency PUE).

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

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. Enzyme kinetics data were analyzed using the Hyper32 free software.
