ACID PHOSPHATASE ACTIVITY AND LEAF PHOSPHORUS CONTENT IN SOYBEAN CULTIVARS

The phosphate fertilization represents the most costly fraction of soybean crop production. Efficient soybean cultivars for P absorption and utilization in soils of medium available P are highly desirable and might contribute for increasing crop production potential. Thirty two soybean [Glycine max (L.) Merr.] cultivars recommended for ‘Cerrado’ and differing in growth cycle (early, semi-early, semi-late, and late) were grown in a dystrophic Typic Haplustox Cerrado soil to evaluate the acid phosphatase activity, P content in the diagnostic leaf, and shoot biomass. There were differences among the soybean cultivars within all maturation groups in acid phosphatase activity and shoot biomass. The diagnostic-leaf P-content showed significant differences on semi-late and late maturation groups’ cultivars. The acid phosphatase activity correlated positively with the plant shoot biomass from semi-early (r = 0.46) and late (r = 0.47) cultivars, and negatively (r = -0.40) with the P content in the diagnostic leaf of late maturation cultivars. The occurrence of soybean cultivars with high and low acid phosphatase activity within the same maturation groups indicates the existence of different mechanisms involving P mobilization in the soil and internal plant P remobilization.


INTRODUCTION
Soybean cultivation in Brazil presents high fertilizer demand, which represents 25% of all Brazilian fertilizer consumption in agriculture.In addition, phosphorus (P) is the most expensive nutrient as compared to the other nutrients (Câmara, 2000).The majority of Brazilian soils are acid and present high P retention capacity, with con-sequent low P availability for the plants (Sanchez & Salinas, 1981).Growers should know the alternatives to reduce production costs, mostly with fertilizers, which correspond to 38% of the total farm input used in the 'Cerrado' cultivation for a five-year cycle estimation (FNP Consultoria e Comércio, 2003).The utilization of more adapted and efficient soybean cultivars in phosphorus uptake and utilization would certainly contribute for yield increase.Phosphorus uptake and utilization efficiency requires a vast and well-distributed class of enzymes known as phosphatases (Duff et al., 1994).The acid phosphatase is a hydrolase which promotes the monoester phosphate hydrolysis, transforming organic phosphate in an inorganic form (Breseghelo et al., 1992).The acid phosphatase activity determination is a fast and sensible test for the P status in the plant, and for this reason, Besford (1979a;1979b) suggested its utilization as a plant P-deficiency indicator.This author observed in tomatoes, in which the unbalance of 15 nutrients was induced, that only P-deficiency caused an increase in leaf acid phosphatase, hence the suggestion that this enzyme might be used to detect P deficiency in these plants (Besford, 1979c).Several reports in the literature indicate that the acid phosphatase activity is correlated to P-concentrations in tissues of several plant species: Cucumis sativus L (Besford, 1978), Licopersicon esculentum Mill (Besford, 1979a;1979b), Triticum aestivum L (McLachlan, 1982), Oryza sativa L (Zaini & Mercado, 1985), Phaseolus vulgaris L. (Breseghelo et al., 1992), Saccharum officinarum L (Silva & Basso, 1993), Phaseolus vulgaris L and Vigna unguiculata L Walp (Fernandez & Ascencio, 1994), Cajanus cajan L and Gossypium hirsutum L (Ascencio, 1994), and Bactris gasipaes Kunth (Bovi et al., 1998).However, all of these works tested P doses, without taking into account the acid phosphatase activity, P-uptake and use efficiency for comparison of cultivars and or species, in only one soil fertility situation in terms of soil P availability.
More efficient P genotypes usually show lower acid phosphatase activity under P stress situation, reflecting better nutritional condition when compared to inefficient genotypes.Such observations were made in pure lines of sorghum, corn and sorghum hybrids, being inconsistent for other species or among heterozygote genotypes of the same species, probably because of many acid phosphatases types can either be or not be released as exudates by the roots, with specific functions (Furlani & Machado, 2002).Recently, genes and proteins of the acid phosphatase synthesis in Arabidopsis, tomatoes and lupine were isolated and completely characterized (Vance et al., 2003).Both the Arabidopsis AtACP5 and tomato LePS2 genes are chiefly related to internal Premobilization than in P-uptake, and were both quickly induced, under P deficiency conditions, thus suggesting that the plant molecular responses to external P concentrations have strict control (Vance et al., 2003).Soybean plants are known to have low or no-secretion roots, that is, are not able to release exudates or present little organic acid exudation in the rhizosphere (Hocking & Randall, 2001), depending on other agents that induce P mobilization to grow in P limiting environments.
The hypothesis in the present study is that, under medium soil-P availability condition, soybean culti-vars grouped according maturation cycle, would present variation in the leaf acid phosphatase activity, and that this enzyme activity might be used as indicator for soybean breeding programs, attempting to increase the plant efficiency in P absorption and utilization.In a preliminary research with soybean cultivars cropped in a 'Cerrado's' typical dystrophic Red Yellow Latosol, with low available P (6 to 8 mg dm -3 -resin method), there was no success in obtaining enough plant material for analytical determinations, since plants showed poor growth under such severe P stress (data not published), indicating the need for a less limiting soil P level to grow soybean plants.The present work evaluated the leaf acid phosphatase activity, the diagnostic-leaf P concentration and the canopy shoot biomass, in soybean cultivars of different maturation groups, cultivated in a 'Cerrado's' typical dystrophic Red Yellow Latosol, at medium P soil level.

MATERIAL AND METHODS
The experiment was carried out in a randomized complete block design, with 32 treatments and three replications, under greenhouse conditions, at Piracicaba, State of São Paulo, Brazil.Seeds of 32 soybean (Glycine max (L.) Merril) cultivars were inoculated with 1 mL per pot of Bradyrhizobium japonicum SEMIA 587 and 5019, at sowing.The chosen soybean cultivars were among those most used in the Brazilian 'Cerrado' producing area, representing the early, semi-early, semi-late and late maturation groups (Table 1).
Non-perforated plastic pots were filled with 200 g of soil and each one received four soybean seeds.Seedlings emerged three days after sowing (DAS).Thining at seven days after emergence (DAE) left, one V1-stage (first trifoliolate leaf) plant per pot.Fifteen DAE, 0.4 mg of potassium as KCl was applied per pot.Water was supplied twice a day, determining the necessary quantity by weighing the pots individually (at 9:00 a.m. and 3:00 p.m.).At 46 DAE the in vivo activity evaluation of the acid phosphatase enzyme (E.C.3.1.3.2) was performed according to Besford (1979c).Leaf sampling criterion followed recommendations of Ambrosano et al. (1996), so the third and the fourth recently mature leaf from the apical bud were collected.
Fragments of recently collected leaves (2-mm leaf blades; 100 mg) were incubated with 8 mL of 0.25 mmol L -1 p-nitrofenilphosphate (p-NNP) in a 0.1 mol L -1 (pH 4.0) sodium acetate buffer.After 20 minutes of incubation in water-bath at 30ºC in the dark, 5 mL of the mixture reaction was interrupted with 2 mL of a 2 mol L -1 NaOH solution.During the incubation period, the buff-ered p-nitrofenilphosphate substrate (p-NPP) is hydrolyzed by the acid phosphatase resulting in p-nitrophenol (p-NP, yellow in base).Readings were made against a blank sample through optic density in a Klett-Summerson photocolorimeter with a blue filter (nº 42) and the p-NP quantity was estimated using a p-NP standard curve (the blank sample was also incubated with all reagents except the leaf fragments).The enzyme activity was expressed as µmoles of hydrolized substrate (p-NPP) per hour, per fresh tissue weight (µmol h -1 g -1 ).
After the acid phosphatase activity evaluation, leaves remnants were dried in a forced air oven at 65°C for 72 hours, weighed, ground and submitted to the chemical analysis for P concentration determination in the diagnostic-leaf, according to Sarruge & Haag (1974).The plant shoots were oven dried to obtain the dry weights, which were added to the leaf weights (leaves used in the enzymatic evaluation and P content determination) to get the total biomass production.
Cultivars were evaluated regarding acid phosphatase activity, diagnostic-leaf P-concentration and biomass production of shoots.Results were submitted to statistical analyses using the SAS -System for Windows 6.11 (SAS Inc., 1996) program, through the GLM procedure.Analysis of variance and the orthogonal decomposition of the treatment degrees of freedom were performed, allowing comparisons among and within each maturation group.When test F was significant, within each group, the Tukey's test (α = 0.05) for multiple mean comparisons was applied to identify differences among cultivars.Correlations between acid phosphatase activity, biomass production and leaf P-concentration were calculated using SAS, through the procedure CORR, calculating the Pearson's correlation coefficients within each maturation group.

RESULTS AND DISCUSSION
There were differences (P < 0.05) among soybean maturation groups, only for the acid phosphatase activity, evidencing different strategies among them for the acid phosphatase synthesis under the tested conditions.Within each maturation group, cultivars differed significantly regarding acid phosphatase activity and biomass production (P < 0.05).Differences (P < 0.05) were observed in leaf tissue P concentration only among the semilate and late cultivars (Table 2).Although the substrate P concentration was considered of medium level (Souza et al., 2002), P concentration in the diagnostic-leaf varied from 0.98 to 1.50 g kg -1 among cultivars, what is considered below the adequate critical level for soybean that is 2.5 to 5.0 g kg -1 (Ambrosano et al., 1996).
No differences were observed (P < 0.05) regarding leaf-diagnostic P-concentration among cultivars in the early maturation group, as well as for the ones of the semi-early group (Table 2).However, differences (P < 0.05) were observed within the semi-late group; IAC PL-1 and DM Vitória showed lower leaf P. The other cultivars did not differ from each other, presenting higher leaf P, but always below the critical level for soybeans (2.5 g kg -1 ) according to Ambrosano et al. (1996).Late soybean cultivars differed in diagnostic-leaf P: DM 309 and BRSGO Jataí presented the highest values (1.50 and 1.49 g kg -1 respectively) and BRAS 97-7371 and DM Nobre cultivars, the lowest values (0.98 and 1.10 g kg -1 , respectively) (Table 2).It was also observed that all late cultivars presented leaf P concentrations below the critical level previously cited.Although correlation coefficients between biomass and leaf P were negative for early (r = -0.38)and semi-early cultivars (r = -0.18),they were nonsignificant.A negative relationship between biomass and leaf P concentration was observed within the semi-late (r = -0.49)and late (r = -0.74)maturation groups.As plant biomass increased there was a decrease in leaf P concentration, characterizing a nutrient dilution effect in the tissues (Jarrel & Beverly, 1981).
Differences in the values for root acid phosphatase activity do not always evidence plant differences regarding P uptake and utilization efficiency (Furlani & Machado, 2002).Studies with genetic markers in bean genotypes, one efficient and another inefficient, proved that the gene locus for root acid phosphatase was not associated to the loci for P uptake and utilization efficiency (Yan et al., 2001).High root enzymatic activity, however, can result in plant stress signals for the need of higher external P supplies for plant development (Furlani & Machado, 2002).
Taking as example the data on the late cultivars BRAS 7371 and DM Nobre, it was observed higher leaf acid phosphatase activity in BRAS 7371 than in DM Nobre, despite their similar biomass and leaf tissue P concentration.I can be thus inferred that BRAS 7371 cultivar should probably be more stressed than DM Nobre (Table 2).On the other hand, the early cultivar M-Soy 8001 presented low leaf acid phosphatase, correspondent to about one third of the value reached by DM-118, but no differences (P < 0.05) were found between the two cultivars regarding biomass production and leaf P-concentration.These are evidences that other mechanisms than acid phosphatase activity, such as root exudation of organic acids, enhance P uptake and utilization efficiency (Hocking & Randall, 2001;Vance et al., 2003), and that should be studied.
Soybeans are well known as non-secreting or poor organic acid root-secreting plants (Hocking & Randall, 2001), depending on other factors that enhance P mobilization under P-limiting environments.Such considerations lead to speculations about the existence of genetically different cultivars regarding the organic acid secretion, what would explain the variation regarding acid phosphatase activity within the same maturation group.
The simple linear correlation analysis between the acid phosphatase activity and shoot biomass within each soybean maturation group showed that the enzyme activity was directly proportional (P < 0.05) to the produced shoot biomass of semi-early (r = 0.46; N = 21) and late (r = 0.47; N = 24), indicating that semi-early and late cultivars with higher biomass yield also presented higher phosphatase activity at the same P level condition (Table 3).At any rate, results of several studies show that the acid phosphatase activity is inversely proportional to the dry matter production under P deficiency (McLachlan, 1982;Breseghelo et al., 1992;Kummerová & Buresová 1990 cited by Furlani & Machado, 2002).There is no reference in the literature concerning the relationship between the acid phosphatase activity and biomass production among soybean cultivars grouped by maturation cycles and evaluated at only one soil P level, but reports about these relations with sorghum and corn are found (Camacho & Malavolta, 2000;Furlani et al., 1984;Kummerová & Buresová, 1990;cited by Furlani & Machado, 2002).
The acid phosphatase activity presented negative correlation (P < 0.05) with leaf P concentration (r = -0.40;N = 24) within the late soybean cultivars group (Table 3), indicating that a decrease in the diagnostic-leaf P-Sci.Agric.(Piracicaba, Braz.), v.61, n.4, p.439-445, July/August 2004 concentration induced increase in the enzyme activity.No significant correlation in the other soybean maturation groups was observed (Table 3).Young developing plants and P-stressed plants usually show higher acid phosphatase activity, which guarantees P supply from older tissues by hydrolyzing P from organic forms to inorganic P, and remobilizing it to the apical growing parts (Duff et al., 1994;Furlani & Machado, 2002).Soybean cultivars with higher biomass production, reduce acid phosphatase activity but keep the same leaf tissue P concentration.On the other hand, cultivars with higher biomass production, increase acid phosphatase activity, and maintain the same leaf tissue P concentration within the same maturation group.Results of this work are evidence that differentiated soybean plant mechanisms are involved in the soil P acquisition and in the internal P-remobilization, and being the need for further biochemical and molecular studies to characterize the genes responsible for these mechanism controls.

Table 2 -
Acid phosphatase activity, shoot biomass and diagnostic-leaf P-concentration in soybean cultivars, according to maturation groups, cultivated in a dystrophic Red Yellow Latosol under 'Cerrado' (means of three replications).

Table 3 -
Simple linear correlation coefficient estimates between leaf acid phosphatase activity, and shoot biomass and diagnostic-leaf P concentration of soybean cultivars according to maturation group.ns = non significant; *, **, significant at 5 and 1%, respectively.