Cytochemical localization of acid phosphatase in Stigeoclonium

Nonspecific acid phosphatases are a group of enzymes whose activity increases the availability of exogenous and endogenous orthophosphate either through extra- or intracellular hydrolysis of phosphate compounds. Our study demonstrates the activity of acid phosphatases in the filamentous freshwater alga Stigeoclonium tenue. These enzymes were detected following a cerium-based method in which cerium was used as an orthophosphate-capture reagent. In thalli from S. tenue from the natural environment, acid phosphatases were found in the longitudinal cell wall, plasmalemma, and vacuole. In thalli from Bold's Basal Medium culture, these enzymes were found mainly in the plasmalemma; they were scarce in the cell wall. In the thalli grown in phosphate-enriched culture medium, enzymes were found only in the plasmalemma. The low availability of orthophosphate in the medium seems to induce the transport of these enzymes to the cell wall. Its abundance, on the contrary, seems to attenuate this response without affecting the localization of acid phosphatases in the plasmalemma.


Introduction
Phosphorus (P) is one of the most important mineral nutrients required by algae for growth and development (Lee et al., 1999).Orthophosphate anion (P i ) is preferentially assimilated by organisms such as algae which obtain their mineral nutrients directly from the environment (Moraes, 1991).
Natural water contains different organic P compounds which degradation is low.Enzymatic hydrolysis is, therefore, the only mechanism by which biologically active orthophosphate is released from organic P (Gilbert et al., 1999;Wasaki et al., 1999).
Acid phosphatases (APases, orthophosphoric-monoester phosphohydrolases) are a group of enzymes that catalyze the hydrolysis of a wide spectrum of orthophosphate monoesters and anhydrides with an optimum pH ranging between 5.0 and 6.0 to finally release phosphate ions (Duff et al., 1994;Gilbert et al., 1999;Ferreira et al., 2000).APases exhibit broad and overlapping substrate specificities (Gellatly et al., 1994).There are two different categories of plant APases that are differentiated from each other according to their relative substrate selectivities.One of such categories includes specialized enzymes such as 3-P glycerate (3-Pga) phosphatase and phosphoenolpiruvate (PEP) phosphatase which display a clear though non-absolute substrate specificity.The second category includes those truly nonspecific enzymes that show either low or no substrate specificity (Duff et al., 1994).These nonspecific enzymes are the focus of attention of the present research.APases are responsible for extra-and intracellular phosphate cleavage and they are apparently ubiquitous in nature, occurring in many animal tissues, plants, and microorganisms (Törnqvist, 1989;Duff et al., 1994;González et al., 1994;Ferreira et al., 2000).Intracellular forms occur in all plant tissues in vacuolar as well as cytoplasmic locations (Gilbert et al., 1999;Turner and Plaxton, 2001).APases might be of relevance mainly in habitats with low concentrations of inorganic phosphorus as they may enable cells to hydrolyze extracellular phosphate esters, increasing the amount of available orthophosphate (Beißner and Römer, 1999).
The genus Stigeoclonium Kütz includes attached, branched, uniseriate, filamentous green algae.The thallus is heterotrichous and is composed of a prostrate and an erect system of filaments.The terminal cells of the erect filaments may produce multicellular, hyaline hairs particularly under nutrient deficiency conditions (Whitton and Harding, 1978).
There have been few studies of APases localization on freshwater algae.Thus far, there have been no studies on filamentous algae.The aim of the current investigation, therefore, was to detect the occurrence and location of APases in a Stigeoclonium species which is very common in the periphyton community all over the world in a wide range of habitats in eu-and hypertrophic aquatic environments (de Vries, 1986) and to ascertain whether hyaline hairs were related with nutrient capture.
To our knowledge, this is the first report on the fine structural distribution of APase activity in the green alga Stigeoclonium tenue (C.Agardh) Kütz.

Material and Methods
The alga Stigeoclonium tenue was collected from Paso de las Piedras dam (Buenos Aires province, 38º 22' S, 62º 12' O).Cultures were grown in Bold's Basal Medium-BBM (Stein, 1973) with both normal phosphate content (7.5 g/l K 2 HPO 4 and 17.5 g/l KH 2 PO 4 )  and containing a 3-fold increase in phosphates (22.5 g/ l K 2 HPO 4 and 52.5 g/l KH 2 PO 4 ) at 10ºC in a 12-12 lightdark regime provided by cool-white fluorescent lamps.S. tenue was grown in P-plus BBM during 15 days.Nonspecific APase detection was carried out according to Robinson and Karnovsky (1983) using sodium-ß glycerophosphate as substrate and cerium as capture reagent.Portions of thalli were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4ºC for 1 h and then washed briefly in the same buffer four times at 4ºC.The material was incubated in medium containing 1mM sodium-ß-glycerophosphate (Sigma Chemical Co) and 2 mM CeCl 3 (Alfa Products) in 0.1 M acetate buffer (pH 5) and Triton X-100 0.0001%.Incubation was then carried out at 37ºC for 60 min with a medium change after 30 min, the medium was filtered through a 0.45 µm pore size Millipore filter just prior to use.Control specimens were incubated in a similar medium without substrate.After incubation, the material was washed twice in 0.1 M acetate buffer at room temperature and twice in 0.1 M cacodylate buffer at 4ºC.Samples were subsequently refixed in 3% glutaraldehyde in 0.1 M cacodylate buffer for 1 h at 4ºC and rinsed over night in the same buffer at 4ºC and postfixed in 2% OsO 4 in cacodylate buffer for 1 h at room temperature.Samples were dehydrated in a graded acetone series (50%, 70%, 90%, 96% and 100%), infiltrated and embedded in Spurr's (1969) resin.Samples were sectioned with a diamond knife, collected on Formvar-coated cooper grids and examined in a Jeol 100 CX-II electron microscope without further staining.

Results
The cytochemical experiments carried out for the localization of APases in vegetative Stigeoclonium tenue cells demonstrated a very fine electron-opaque reaction product which corresponds to cerium phosphate.
Thalli from Paso de las Piedras dam showed a high and uniform density of precipitates along the longitudinal cell wall (Figs.1-2, arrowheads).The reaction product was irregularly distributed in the plasmalemma (Figs.1-3, arrows) although no reaction product was observed in the plasmodesmata (Fig. 3, arrowhead).Labeled vesicles were observed in contact with the plasmalemma in the outer region of the cytoplasm (Fig. 4).Fine and coarse precipitates were found in the lumen of vacuoles and associated with vesicular bodies present within the vacuoles; the tonoplast showed no reaction product (Figs.1-5).Likewise, no reaction product was observed in the dictyosomes, nuclei, mitochondria or chloroplasts (Fig. 2).Hyaline hairs showed a strong APase activity at the cell wall and associated with intravacuolar vesicular bodies next to the tonoplast (Fig. 5).
Thalli grown in BBM showed abundant precipitates in the plasmalemma (Fig. 6, arrow).Precipitates were scarce in the cell wall (Fig. 6, arrowhead).In P-plus BBM, filaments exhibited the reaction product only in the plasmalemma (Fig. 7).Controls from the natural medium as well as from BBM and enriched BBM showed no precipitates (Fig. 8).

Discussion
The method that is traditionally used for localization of APases employs lead as a capture agent of the orthophosphate released in the enzymatic site as an insoluble precipitate phosphate (Gomori, 1952).This method has been criticized by several researchers because it may yield false positive results, partially due to that some enzymes are sensitive to lead.However, it is still used to date for the histochemistry of plants and animals (Tsekos and Schnepf, 1991;Yamaguchi and Anderson, 1994;Weber et al., 2001;Vori√ek and Kalachová, 2003).On the other hand, it has been demonstrated that cerium-based methods are more specific (Robinson and Karnovsky, 1983).The latter have been widely used for ultrastructural localization of several phosphatases and ATPases in animal tissues while they have been used with minor frequency for localization of such enzymes in plant cells (Olmos and Hellín, 1997).The cerium-based localization method was found to be appropriate for the present research as a result of the apparent absence of nonspecific reaction product deposits.
Our study reports the first lines of evidence of activity of nonspecific APases in vegetative cells of Stigeoclonium tenue.S. tenue from its natural environment has been found to evidence an intense enzymatic activity in the longitudinal cell walls.This suggests that S. tenue is able to hydrolyze organic P compounds available in the environment on the cell surface to obtain the necessary orthophosphate.
A significant accumulation of reaction product was observed in hyaline hairs, particularly in the longitudinal walls and in the vesicular bodies next to the tonoplast.S. tenue does not form hairs under regular BBM culture conditions while it does under conditions of N and P deficiency in both culture and the natural environment (Abbas and Godward, 1963).In S. tenue, the hairs formed under P deficiency are more numerous and longer than those formed under N deficiency (Vanaja et al., 1999).Several researchers agree that the hair formation mechanism involves a marked increase in the area-volume relation of thalli which contributes to increasing the capture efficiency of certain nutrients under limiting conditions (Tupa, 1974;Whitton and Harding, 1978;Francke, 1982;Gibson and Whitton, 1987a,b;Vanaja et al., 1999).Our results validate the role of hairs in the absorption of nutrients.
A positive correlation between alcaline phosphatase activity and hair formation has been observed in Stigeoclonium, Chaetophora Schrank, and Draparnaldia Bory (Gibson and Whitton, 1987a,b).Gibson and Whitton (1987a) observed, at light microscope level, alcaline phosphatase activity in hairs of Chaetophora and Draparnaldia and in basal filaments of Stigeoclonium, while at ultrastructural level they observed the product reaction in the cell wall of a hair of Chaetophora.
In filaments of S. tenue growing in BBM, low precipitates were registered in the cell wall while there were no precipitates in P-plus BBM.The regulatory action of orthophosphate has been observed in unicellular Chlorophyta, Euglenophyta, bacteria, fungi and higher plants (Price, 1962;Knutsen, 1968;Moraes, 1991;Lubián et al., 1992;Duff et al., 1994;Wasaki et al., 1999;Ferreira et al., 2000).Our results suggest, therefore, that in S. tenue either the synthesis or the activation of APases might be induced by P deficiency.Worthy of note is that phosphates concentration registered in the natural environment was much lower (ranging from 0.077 to 0.089 mg/l) than that under culture conditions which might explain the intense reaction observed in cell walls of thalli growing in its natural environment.Phosphatase activity has been used as an indicator of P deficiency in algal populations from freshwater habitats (Jansson et al., 1988).
APases were registered in the plasmalemma under the three above-mentioned assayed conditions, suggesting that, at this level, they are not affected by concentration of phosphates in the medium.In contrast, these enzymes may participate in the active transportation of nutrients as it has been demonstrated in some higher plants (Mogensen and Rossiter, 1982;Mogensen, 1985).
Taking into account the hydrolytic activity of vacuoles in any metabolic state of the cell, APases are expected to be found in these organelles in S. tenue in accordance with previous observations in other algal species (Noguchi, 1994;Zhou and Fritz, 1994;Yamaguchi and Anderson, 1994).In S. tenue, the precipitate was found mainly in the membranes of vesicu-lar bodies.These results are consistent with observations in the unicellular green alga Asteromonas gracilis Artari (Swanson and Floyd, 1979).In higher plants, digestion processes of these membranes might be related to its subsequent label (Weber, 2002).
The presence of APases in cytoplasmic vesicles next to the plasmalemma suggests enzyme transport towards the cell wall where they will perform their hydrolytic function at the extracellular level.A similar effect was observed in the mycelium of the Ascomycete Claviceps purpurea Tul., in which the reaction product was accumulated in the cell wall (Vori√ek and Kalachová, 2003).
Under the three assayed experimental conditions, no precipitate was found in the cisterns of dictyosomes.The absence of precipitates in the dictyosomes of other organisms has been interpreted either as a different sensitivity of organelles to fixatives and substrates (Lin et al., 1977) or as an indicator of the metabolic state of the organism (Cooper et al., 1974).
In conclusion, APases in S. tenue might be -in accordance with observations in higher plants by Plaxton (1999)-, part of an extracellular system responsible for generating P i from phosphate esters.Our study reveals the first lines of evidence regarding the localization of APases in S. tenue.It represent, therefore, the starting point for further studies about the dynamics of the activity of these enzymes at ultrastructural level in relation to the level of nutrients in the medium, interesting and useful information from an environmental point of view.