THE ACTIVITY AND CHARACTERIZATION OF ACID PHOSPHATASES IN ENDOMYCORRHIZAL FUNGI OF THE ERICACEAE

Transcription

1 New Phytol. (1986) 14, THE ACTIVITY AND CHARACTERIZATION OF ACID PHOSPHATASES IN ENDOMYCORRHIZAL FUNGI OF THE ERICACEAE BY C. J. STRAKER* AND D. T. MITCHELL Department of Botany, Unversty of Cape Town, Rondebosch 77, South Afrca {Accepted 5 June 1986) SUMMARY Endomycorrhzal fung were solated from the root systems of Vaccnum macrocarpon Aton, Rhododendron pontcum L., Calluna vulgars (L.) Hull (Ercaceae) and also from a South Afrcan erca, Erca hspdula L. They were grown on a lqud medum contanng nostol hexaksphosphate, fractonated nto cytoplasmc, extracellular and wall- and nembrane-bound fractons and assayed for acd phosphatase. Extracellular phosphatase was demonstrated n all cultures but ts actvty was hghest n the endophyte of E. hspdula. The wall- and membranebound acd phosphatase was the domnant fracton n the European endophytes. The extracellular acd phosphatase of hgh P-fed (323 mm P) mycela of the endophyte of E. hspdula had the greatest actvty (76% of total actvty), whereas n low P-fed (-1 nm P) mycela both wall and extracellular fractons contrbuted almost equally to form 8 % of total actvty. All the fractons contaned an acd phosphatase of MW wth a broad ph optmum of 2- to 6- but a smaller enzyme of 6828 MW wth a ph optmum of 65 also occurred n tbe wall fracton from low P-fed mycela. All the enzymes were nhbted by fluorde, molybdenum, arsenate, cyande, mercury and pbospbate but stmulated by EDTA, ct'ate and tbe ferrc on at low concentratons. A wde substrate specfcty was demonstrated wth maxnum affnty for norganc pyropbospbate, hgh affntes for OL- and y?-napbtbyl phosphate, pbenyl phosphate and a-glyceropbosphate and a low affnty for pbytc acd. Tbe low tnolecular weght enzyme was the only one wbcb bydrolysed organc anhydrdes (ATP, ADP and AMP). Key words: Acd phosphatase, ercod mycorrhzal fung, Ercaceae. INTRODUCTION Acd phosphatases (E.C ) are enzymes of wde specfcty whch cleave phosphate ester bonds and thus play an mportant role n the mneralzaton of organc phosphate (P) n sols, where ther actvty may be correlated wth hgh amounts of organc carbon and organc P and low levels of free norganc ons (P^) (Spers & McGll, 1979; Appah & Thomas, 1982). Hgh acd phosphatase actvty Was demonstrated n ectomycorrhzal fung n pure culture (Theodorou, 1971; Ho & Zak, 1979; Calleja et al., 198), excsed ectomycorrhzal roots (Woolhouse, 1969; Bartlett & Lews, 1973) and onon roots nfected wth vescular-arbuscular (VA) mycorrhzas (Gannazz, Gannazz-Pearson & Dexhemer, 1979). Calleja et al. (198) observed an ncrease n total phosphatase actvty (localzed manly n the cell wall) and the appearance of an extracellular acd phosphatase when three ectomycorrhzal fung were grown under P-defcent condtons. Saprotrophc fung are also known to secrete extracellular phosphatases (San Bas & Cunnngham, 1974; Arnold & Garrson, 1979; Beever & Burns, 198; Bojovc-cvetc & Vujcc, 1982). The only report of acd phosphatase actvty n ercod mycorrhzas * Present address: Staton d'ameloraton des Plantes, lnra BV 154, 2134 Djon-ccdex, France X/86/ / 1986 The New Phytologst

2 244 C. J. STRAKER AND D. T. MITCHELL s that of Pearson & Read (1975). Ths paper nvestgates acd phosphatases n mycela of a South Afrcan and European ercod endophytes. The hgh actvty of an acd phosphatase secreted nto the external medum of cultures of the South Afrcan endophyte prompted a more detaled characterzaton of the enzyme n cytoplasmc, wall- and membrane-bound and extracellular fractons. The results are dscussed n relaton to the phosphatases of VA and ectomycorrhzas and the P nutrton of members of the Ercaceae n heathlands. MATERIALS AND METHODS Cultural procedures Mycorrhzal endophytes of Vaccnum macrocarpon Aton., Rhododendron pontcum L., Calluna vulgars (L.) Hull from the Unted Kngdom and Erca hspdula L. from South Afrca were the same as those used by Straker & Mtchell (1985). The preparaton of lqud cultures and the basal lqud medum were as descrbed by Straker & Mtchell (1985). Fractonaton of extracellular, wall- and membrane-bound, and cytoplasmc fractons of acd phosphatase Mycela of the endophytes of V. macrocarpon, C. vulgars, R. pontcum and E. hspdula, growng on a medum contanng 3^23 mm P as sodum nostol hexaksphosphate (phytate), were harvested at 7, 11, 14, 17, 22 and 25 d after noculaton. The mycelum was fltered under sucton through Whatman No. 4 flter paper and washed wth ce-cold, 5 M acetate buflfer (ph 4^5). The bulked fltrate was passed through a sterle 'Mllpore' flter (pore sze -45/m) and consttuted the extracellular fracton. The mycelum was homogenzed n 1 cm^ of acetate buffer (^5 M, ph 4^5) for 1 mn usng an Ultra-Turrax homogenzer and centrfuged twce at 36^ for 15 mn at C. The combned supernatants consttuted the cytoplasmc fracton and the resdue was resuspended n 1 cm^ ^5 M acetate buflfer (ph 4-5) to form the wall- and membrane-bound fracton. Fve cultures were fractonated separately to gve fve replcates of each fracton whch were assayed for phosphatase actvty and total proten. Preparaton of mycela of the endophyte of E. hspdula pror to partal purflcaton and characterzaton of acd phosphatase The extracton and fractonaton procedure was based upon that of Hasegawa, Lynn & Brockbank (1976). Cultures of the endophyte of Z?. hspdula, growng on meda contanng 323 mm P or 1 mm P as sodum nostol hexaksphosphate were harvested at 13 d,.e. durng exponental growth (Straker, 1986). Each mycelum, fltered under sucton, was rnsed n 1 cm''' ce-cold, dstlled water. The fltered lqud meda of 2 cultures together wth the washngs were passed through a 'Mllpore' flter (pore sze ^45/ym), dalyzed aganst dstlled water at 1 C, lyophlzed and dssolved n 2 cm'* ^1 M acetate buflfer (ph 4-5) to form the crude extracellular fracton. Mycela were bulked, thoroughly washed n ce-cold, dstlled water, homogenzed n dstlled water and centrfuged twce at 12 ^'for 15 mn. The supernatant was dalyzed aganst dstlled water, concentrated by lyophlzaton and dssolved n 2 cm^ ^1 M acetate buflfer (ph 4-5) to form a crude cytoplasmc fracton. The resdue was suspended n cold ^2 % Trton X-1 soluton for 2 h, straned through mult-layered musln cloth and washed repeatedly wth ^2 % Trton X-1 soluton untl neglgble phosphatase actvty was detected n the washng

3 Phosphatases of endomycorrhzal fung 245 medum. The Trton X-1 fltrate was dalyzed aganst dstlled water, concentrated by lyophlzaton and dssolved n -1 M acetate buffer (ph 4-5) to form a membrane-bound fracton. The resdue was rnsed wth dstlled water and ncubated n 1 M NaCl at C overnght to release wall-bound protens. Prelmnary studes sbowed 1 M NaCl was tbe optmal concentraton for the release of wall-bound phosphatase (Straker, 1986). The suspenson was centrfuged at 12 j^ for 15 mn, the supernatant dalyzed aganst dstlled water, lyophlzed and dssolved n 2 cm^ -1 M acetate buffer (ph 4-5) to gve a soluble, wall-bound fracton. The resdue formed the nsoluble, wall-bound fracton. Gel fltraton The acd phosphatase actvty of the crude enzyme fractons was determned mmedately and then stored at 2 C for a maxmum of 2 weeks. The crude enzymes were further purfed by passng 3 cm^ volumes through a Sephacryl S-4 gel column (2-6 x 5 cm) pre-equlbrated wth -5 M acetate buffer (ph4-5); eluton of 4 cm^ fractons occurred at 1 C. Actve fractons were pooled, dalyzed aganst dstlled water and when necessary concentrated by lyophlzaton before scannng for ph optma and testng for substrate specfcty and nhbton characterstcs. The vod volume of the column was determned wth Blue Dextran 2 eluted wth a -1 M phosphate buffer (ph 7-). The molecular weghts of the acd phosphatases were determned by the method of Basha (1984). Tbe protens (3 mg cm"^), e.g. catalase (232), bovne albumn (662) and ovalbumn (45) and cytochrome C (1396) were eluted through the Sephacryl S-4 column wth -5 M phosphate buffer (ph 7-). Proten n tbe collected fractons was assayed by the method of Lowry et al. (1951) and the absorbance of cytocbrome C was measured at 42 nm. Acd phosphatase assay /)-Ntrophenyl phosphate (PNPP) was prepared by dssolvng a 4 mg prewegbed capsule (Sgma) n 3 cm^ dstlled water. A mxture of -1 cm^ enzyme extract, -9 cm^ -1 M acetate buffer (ph 4-5) and 1 cm^ PNPP as the substrate was ncubated for 3 mn n a shakng water-bath at 25 C and the reacton stopped wth 5 cm^ -1 M NaOH (Bartlett & Lews, 1973). Absorbance was measured at 41 nm. A control wthout the enzyme was always ncluded to measure nonenzymc hydrolyss of the substrate. The ncubaton medum used to measure the effects of dfferent compounds on phosphatase actvty contaned -1 cm^ enzyme extract, -9 cm^ of the approprate buffer (determned from ph scans) n whch was dssolved the effector compound and 1 cm' PNPP as substrate. Controls wthout the enzymes were run to adjust the absorbance readng at 41 nm for colour contamnaton. Proten determnatons Total proten was determned by the method of Lowry et al (1951) usng bovne serum albumn as the standard. Phosphorus assay The affnty of the enzyme for dfferent substrates was determned by measurng the level of P released nto the ncubaton medum (-1 cm'' enzyme extract, -9 cm^ buffer, 1 cm^ 5 tnm substrate) after 3 mn ncubaton at 25 C n a shakng water-batb. When sodum phytate was the substrate, ncubaton occurred for 24 h. The fnal substrate concentraton of 2-5 mm was equvalent to

4 246 C. J. STRAKER AND D. T. MITCHELL the concentraton of PNPP under standard condtons. Phosphate was assayed by the method of Murphy & Rley (1962) and controls wthout the enzymes were run to measure non-enzymc hydrolyss or free P contamnaton of the substrates. Gel electrophoress Electrophoress was ntally performed accordng to Davs (1964) wth the use of vertcal, flat-bed gels of 5, 7-5 or 1 % polyacrylamde. No mgraton of the protens occurred ether under basc (^5 M trs-glycne runnng buffer, ph 8-3) or acdc (-5 M /?-alanne-acetc acd runnng buffer, ph 4^5) condtons durng 24 h ncubaton at 3 ma constant current. The enzymes fnally mgrated after more than 24 h at 3 ma constant current n horzontal fat beds contanng 5 % agarose wth a -1 M trs-acetate-edta runnng buffer adjusted to ph 4-4 wth acetc acd. Acd phosphatase was dentfed by ncubatng the gels for 2 h at room temperature n 25 cm^ -2 M acetate buffer (ph 4^), contanng 25 mg a-naphthyl phosphate and 25 mg fast garnet GBC salt. RESULTS Acd phosphatase actvty and proten content of mycelal fractons Proten contents of the fractons of all four endophytes showed an ntal lag phase of 7 d and then ncreased rapdly wth maxmum levels beng attaned 15 to 2 d after noculaton (Eg. 1). Wall- and membrane-bound proten was consderably hgher than extracellular and cytoplasmc proten n the four endophytes durng all the growth phases (Eg. 1). In the endophytes of C. vulgars, R. pontcum and V. macrocarpon, amounts of cytoplasmc and extracellular proten were smlar, whereas cytoplasmc proten was very low n the endophyte of E. hspdula (Eg. 1). In the endophytes of C. vulgars, R. pontcum and V. macrocarpon, wall- and membrane-bound phosphatase was the most actve fracton after an ntal lag phase of 7 d and maxmum actvty was attaned 14 d after noculaton (Eg. 2). Wall- and membrane-bound phosphatase actvty n the endophyte of V. macrocarpon was mantaned durng the fnal phases of growth despte the fact that total proten content declned (Egs 1 and 2). The presence of extracellular phosphatase was demonstrated n all cultures 7 d after noculaton, although the actvty of ths enzyme was hghest n the endophyte of E. hspdula (Eg. 2). At maxmum actvty, the ratos of extracellular, cytoplasmc, wall- and membranebound phosphatase of the endophyte of E. hspdula to those of V. macrocarpon were 2-5, 1^6 and 2 respectvely. Extracellular phosphatase n the endophyte of E. hspdula was more actve than the cytoplasmc and wall- and membrane-bound enzymes 18 d after noculaton. Phosphatase actvty n fractons of mycela of the endophyte of Erca hspdula In the prevous secton, the endophyte of E. hspdula had the hghest total acd phosphatase actvty when compared wth the three European endophytes and was thus selected for further studes. To determne the nfuence of growth under dfferent organc P condtons on phosphatase actvty, fractons were extracted from both hgh P (3-23 mm P as sodum phytate) and low P (-1 mm P as sodum phytate)-fed mycela. In hgh P-fed mycela, the actvty of the extracellular fracton was 5^5 tmes greater than that of the total wall and 19 and 13 tmes greater than that of the cytoplasmc and membrane-bound fractons

5 Phosphatases of endomycorrhzal fung Incubaton perod (d) Eg. I. Proten content of wall- and membrane-bound ( - I), cytoplasmc - ) and extracellular (A A) fractons of endophytes: (a) Rhododendron pontcum; (b) Calluna vulgars; (c) Vaccnum macrocarpon; (d) Erca hspdula. Results are the means of fve replcates (each replcate representng fractons from dfferent cultures). respectvely (Table 1). In hgh P-fed mycela, the actvty of the extracellular fracton was 76 % of total actvty. The actvty of the extracellular and total wall fractons of low P-fed mycela were more smlar and together formed 8 % of total actvty. The low P, extracellular fracton was 4^5 and 3^3 tmes more actve than the low P, cytoplasmc and membrane fractons respectvely (Table 1). It appears that 92 % of the wall-bound phosphatase enzymes of hgh P-fed mycela were solublzed by 1 M NaCl, whereas for low P-fed mycela t was 77 % (Table 1). Gel fltraton Sngle peaks were eluted from all extracts (Fg. 3) except the low P, soluble wall fracton whch produced a major peak concdng wth the sngle peak of the other fractons and a mnor one of a smaller molecular weght [Fg. 3 (b)]. The acd phosphatase actvty of the membrane-bound and the low P, cytoplasmc fractons could not be detected after eluton through the column even after further concentraton by lyophlzaton. The domnant peak, obtaned from all the extracts, corresponded to a molecular weght of , whereas the mnor and lower molecular weght peak only eluted from the low P, soluble wall extract had a molecular weght of (Fg. 4).

6 248 C. J. STRAKER AND D. T. MITCHELL ncubaton perod (d) 2 25 Fg. 2. Acd phosphatase actvty of wall- and memhrane-hound ( ), cytoplasmc ( ) and extracellular (A ) fractons of endophytes: (a) Rhododendron pontcum; (h) Calluna vulgars; (c) Vaccnum macrocarpon and (d) Erca hspdula. Results are the means of fve replcates (each replcate representng fractons from dfferent cultures). ph scans The buffer routnely used for the assay of acd phosphatase was ph 4-5 (Pearson & Read, 1975) but ths mght not have been the optmum ph for all the enzymes extracted from the endophyte of E. hspdula. Moreover, ph serves as a bass for dstngushng the propertes of the extracellular, wall and cytoplasmc fractons. All the fractons, except the low P, soluble wall enzyme (peak II) hydrolyzed PNPP optmally between ph 2- and 6- wth maxmum actvty at ph 2- (Fg. 5). The actvty of these enzymes declned above ph 5-5. The low P, soluble wall enzyme (peak II) showed an optmum actvty at ph 6-5 wth a rapd declne to ph 8- (Fg. 5). Ejfectors Although varatons between enzymes n the degree of response to dfferent agents was observed, the clearest dstncton was between the low molecular weght enzyme and the group of hgh molecular weght enzymes. The agents whch produced the greatest actvty of the hgh molecular weght enzymes were ferrc ons n low concentratons, EDTA and ctrate, whereas ferrc and cuprc

7 Phosphatases of endomycorrhzal fung 249 Table 1. Acd phosphatase actvty n fractons of mycela of the endophyte of Erca hspdula at dfferent stages of purfcaton Actvty" (pmol PNP g ' fresh mass mn ') + SE (nmol PNP mg~^ proten mm"')+ SE Fracton Hgh P mycela Low P mycela Extracellular Crude extract Dalyss + lyophlzaton Sephacryl S (76)" (75) (36) 135±3 (36) 849 ± ±-2 Cytoplasmc Crude extract Dalyss -I- lyophlzaton Sephacryl S (6) 39+1 (6) ± (8) (8) Membrane Crude extract Dalyss -I- lyophlzaton 334+1(4) 28+1 (5) 51± (11) 43+1 (11) Wall (NaCl-soluble) Crude extract Dalyss + lyophlzaton Sephacryl S (13) (13) ± (35) (35) ±l-4 89±4 (peak I) 6 ± (peak II) Wall (NaCl-nsoluble) Crude extract Total (1) 4-4±-8(l) 7888 (1) 621 (1) (1) 39±2(1) 5539 (1) 316 (1) Results are the means of three or four replcates. Fgures n parentheses represent percentage contrbuton of each fracton to total actvty. ons n low concentratons stmulated the actvty of the low molecular weght enzyme (Table 2). Eluorde and molybdenum nhbted all the enzymes, whereas the degree of nhbton by phosphate, cyande, arsenate, mercury, cobalt, copper and ferrc ons was dependent on concentraton (Table 2). Magnesum, calcum and nckel ons caused lttle varaton n the actvty of the hgh molecular weght enzymes but n low concentratons nhbted the actvty of the low molecular weght enzyme (Table 2). Substrate specfcty Acd phosphatases generally hydrolyze a broad range of substrates but may dffer substantally n ther affntes. The substrate most effcently hydrolyzed by

8 25 C. J. STRAKER AND D. T. MITCHELL Fracton number Fg. 3. The eluton profles of proten (O) and acd phosphatase actvty ( ) followng gel fltraton of fractons of cultures of the endophyte of Erca hspdula on a Sephacryl S-4 column: (a) low P, extracellular; (b) low P, soluble wall; (c) hgh P, extracellular; (d) hgh P, cytoplasmc; (e) hgh P, soluble wall. The actvty of D s /mol PNP mg"' proten mn"'. Proten s mg (3 cm-')"' alquot eluted. The symbol V denotes the vod volume of the column. all the fractons was norganc pyrophosphate, wth a-glycerophosphate, cc- and y?-naphthyl phosphate, phenyl phosphate, PNPP and y?-glycerophosphate also showng hgh rates of hydrolyss but wth sodum phytate havng a low rate of hydrolyss (Table 3). All the fractons except the low P, soluble wall (peak II) fracton showed a hgh aflnty for glucose-1-phosphate but not for glucose- 6-phosphate or fructose 6-phosphate whch proved successful substrates for the low P, soluble wall (peak II) enzyme (Table 3). Fructose-1, 6-bsphosphate and the organc anhydrdes, especally ATP, proved sutable substrates for the low P, soluble wall (peak II) fracton but were not hydrolyzed by the other enzymes (Table 3). Gel electrophoress The zymograms showed only a slght dstncton on the bass of electrophoretc moblty between the low P phosphatases and the hgh P phosphatases but Rf values could not be determned because after 24 h the fast green marker had mgrated out of the gel.

9 Phosphatases of endomycorrhzal fung " Volume of eluton from Sephacryl S-4 column (cm^) Fg. 4. Estmaton of molecular weght of acd phosphatase peaks eluted from Sephacryl S-4 column. Molecular weghts of 1 (domnant peak, representatve of all fractons) and II (subsdary peak, present only n low P-fed mycela) were estmated from a standard curve (logy = -1 x +log 7-17, r = -91) prepared usng the followng standards: (l)catalase(232ooo); (2) bovne albumn (662); (3) ovalbumn (45 ); (4) cytochrome C (13 96). Volumes of standards were the means of duplcate elutons and molecular weght estmates are the means of at least three replcates. DISCUSSION The endophyte of E. hspdula, a South Afrcan erca, showed a hgher total acd phosphatase actvty, attrbutable to a more actve extracellular fracton, than the European endophytes (Fg. 2). Ths study was extended by determnng the acd phosphatase actvty n extracellular, wall, cytoplasmc and membrane-bound fractons of the endophyte of E. hspdula grown on a basal medum contanng etber hgh or low concentratons of organc P (sodum phytate). In hgh P-fed mycela, the extracellular enzyme was domnant (76 % of total actvty), whereas n low P-fed mycela the extracellular and soluble wall enzyme contrbuted almost equally to form 8 % of total phosphatase actvty. The total actvty of hgh P-fed mycela was 15 tmes greater than that of low P-fed mycela whch can be attrbuted entrely to the greater actvty of the hgh P extracellular enzyme. In many plants, Pj defcency has been found to nvoke an ncrease n acd phosphatase actvty, part of whch s due to the appearance of a cell wall-bound enzyme (Belesk, 1973; Znk & Velky, 1979). The studes of Calleja et al. (198) and Calleja & d'auzac (1983) have confrmed ths for solated ectomycorrhzal fung. Under condtons of low Pj avalablty, these organsms showed a large ncrease (75 %) n the total accessble phosphatase actvty whch was due to an ncreased contrbuton by tbe cell wall and extracellular enzymes. When the acd phosphatases of mycorrhzal and non-mycorrhzal onon root systems were compared after beng grown wth and wthout the addton of Pj to the sol, no sgnfcant dfferences n actvty were found (Gannazz-Pearson & Gannazz, 1976). A surfet of organc P n the growth medum dd not repress the acd phosphatase actvty of the endophyte of E. hspdula but stmulated the further synthess of an already actve, externally-released enzyme. In Canadan and Ghanaan sols, hgh sol phosphatase actvty has been correlated wth hgh organc P contents n the sol (Appah & Thomas, 1982). In ths study, all the enzymes of the endophyte of E. hspdula except the low P, soluble wall (peak II) enzyme sbowed a wde acd ph tolerance (2- to 6-) wth

10 252 C. J. STRAKER AND D. T. MITCHELL (b) - _ ^ ph - O (g) ph Fg. 5. ph scans of phosphatase actvty n fractons of mycela of the endophyte o Erca hspdula-. (a) hgh P, extracellular; (b) hgh P, soluble wall; (c) hgh P, cytoplasmc; (d) low P, extracellular; (e) low P, soluble wall (peak I); (f) low P, soluble wall (peak II); (g) hgh P. membrane-bound; (h) low P, membrane-bound, (a) ( scanned after eluton through a Sephacryl S-4 column; (g) and (h) were not eluted through the gel fltraton column. Data ponts are the means of four replcates. an optmum at ph 2^ to 2^5 and a molecular weght of The low P soluble wall (peak II) fracton possessed a lower molecular weght phosphatase of 6828 and a ph optmum of 6^5. All the enzymes were nhbted by hgh concentratons of cuprc and ferrc ons but not by low concentratons of these metals, nor dd they show a requrement for magnesum or calcum ons. Moreover, EDTA exerted a stmulatory effect on ther actvty, possbly through bndng nhbtory ons present from the extracton and purfcaton procedures (Schmdt, 1961). These features suggest that the enzymes are not metalloprotens, although NaCN dd exert a strong nhbtory effect on ther actvty. Possbly, the nhbtory effect of ths compound was due to propertes other than ts metalbndng ablty. Lke most phosphomonoesterases, the phosphatases of the endophyte of E. hspdula hydrolyzed a wde range of phosphate esters wth common affntes for /)-ntrophenyl phosphate, norganc pyrophosphate, naphthyl phosphates, phenyl phosphate and a-glycerophosphate but a low phytase ablty. The surface acd phosphatases of beech mycorrhzas hydrolyzed /?-glycerophos-

11 Phosphatases of endomycorrhzal fung 253 Table 2. Influence of varous reagents on the acd phosphatase actvty of fractons of the endophyte of Erca hspdula grown for 12 d on basal medum and ether 3^23 mm P {hgh P) or -1 mm P {low P) as sodum phytate Relatve actvty of fractons (%)" Reagent Concn (mm) Cytoplasmc: hgh P Extracellular Hgh P Low P A Wall (soluble) A Hgh P Low PI Low PI I EDTA Potassum ctrate Magnesum chlorde Calcum chlorde Ferrc chlorde Copper sulphate Znc sulphate Nckel sulphate Cobalt sulphate Mercurc chlorde Sodum arsenate Sodum cyande Sodum fluorde Sodum molybdate Sodum phosphate Sodum ntrate " Actvty expressed as a percentage of the control. Actvty assessed under standard condtons usng PNPP as substrate. Reagents were dssolved n 1 M maleate bufter (ph 6-5) for assay of low P, wall-bound enzyme (peak II) or -1 M glycne/hcl buffc-r (ph 2-2) for assay of other enzymes. Low PI and low PI I refer to the hgh and low molecular weght peaks respectvely. phate, glucose-6-phosphate, pyrophosphate and phytates successfully (Bartlett & Lews, 1973). The low molecular weght phosphatase of the endophyte of E. hspdula represents an soenzyme dstnct from the hgher molecular weght form. Although ts ph optmum s close to neutralty, t does not represent an alkalne phosphatase snce t s nhbted by fluorde and molybdenum, cbaracterstc nhbtors of acd phosphatases (Schmdt, 1961), and t shows neglgble actvty n the alkalne ph range. In onon roots nfected wth VA mycorrhzas, acd phosphatases were

12 254 C. J. STRAKER AND D. T. MITCHELL ^r--^mo-^^..- s r- _ vo rs - I--,.r-t QN \C (N ' ' o^ o^ oo o 1 I "*) - CA 5 c-l T-H ro - CO -,_ r^,_, Th m 1 I ' OTj-corp-^or^ppop^foc^'y'r^ O O O ' - - ' O T ^ O O O O O O O O O (^OOOOO^DO^OOOO(NO^O^OO(N^-' r.)rooot-,-r.\oo^^'^r^fot c^c^'t". (N ro, (N (N OOOOOOOOOOOOOOO Ttr^OOOrofN»n ooooooooooooooo ^ O M 1 I T ' o ^ rt T-H y ^_^ TJ cn rs r tn (N ^ r- fs rs ^ OOOOOOOOOOOOOOO O^OOr^fNrOfNOOOO vprpncor-jcn^orp'osp ^fs):rl«obf^fn(^rvr-' o"oo"^r^f^on~r^ ^-^,. OT-r^ooor--roo-+c7' fn cn ' ' T-fNpT-.T--.T--.-^T--.,-,,-H (N O O O f N O O O O O O O O O O O \D(N'r-'<NOr^c-Tj-cso^ os'd-t^'ocnr^rpor^t I oa'o.n'^t^t^t^oo cn r^ O "3. M) 2 -S u 1 (NOccC-p<NnT^(N cn ooooooooooooooo M u 3 C I % X. X. 1 o a a I X^ cn cn 1. a O O -. c h - 2 I SI' z?5 z; OO X. ~ &!& I o o c c cn C o a c.3

13 Phosphatases of endomycorrhzal fung 255 present only n very young, termnal parts of arbuscule branches but ther actvty dsappeared n older arbuscular branches to be replaced wth a hgh alkalne phosphatase actvty specfc to the mycorrhzal endophyte (Gannazz et al., 1979). The ph optmum for actvty of the low molecular weght enzyme suggests a functonal mportance more closely assocated wth the host (see Pearson & Read, 1975) than wth the external soluton, snce after 12 d growth the ph of the growth medum was between 2-5 and 3- (Mtchell & Read, 1981; Straker, 1986). The hgh ATPase, ADPase and AMPase actvtes of the lower molecular weght enzyme and ts possble assocaton wth the cell wall suggests that t may be mportant n the actve transport of P across rhzosphere/endophyte or endophyte/host nterfaces. The prmary ecologcal sgnfcance of ths nvestgaton s n the release of an actve acd phosphatase nto the external medum by the South Afrcan endophyte grown on an organc P source. The possblty that the extracellular enzyme dentfed n ths study was a product of cell lyss leadng to leakage of cytoplasmc or wall- and membrane-bound protens cannot be supported by the dfferences n propertes shown between enzyme fractons n eft'ector and substrate specfcty studes (Table 2 and 3). Mycela were fractonated durng exponental growth when cell lyss would be expected to be mnmal. In the fynbos vegetaton there s a seasonal pattern n ntrogen mneralzaton (Stock & Lews, 1986; Stock, Lews & AUsopp, 1986), sol mosture levels and total P levels n the acdc sandy sols (Mtchell, Brown & Jongens-Roberts, 1984). Wth the bulk of the P beng organcally bound, the phosphatases secreted by mcro-organsms would be mportant n the catalytc release of P from bound complexes. The acd phosphatases of the endophyte of E. hspdula were partcularly assocated wth the cell wall or released nto the external medum, showed a wde substrate specfcty and a ph range for actvty wthn that of a fynbos sandy sol (Mtchell et al., 1984). However, the formaton and release of extracellular phosphatases by sol saprotrophs s dependent on cultural condtons (Yamamoto, Mnoda & Yamada, 1969; Sakura & Shota, 1977; Bojovc-cvetc & Vujcc, 1982) and the sgnfcance of the extracellular acd phosphatase of the endophyte of E. hspdula n the P nutrton of the host needs to be substantated by further studes on mycorrhzal and non-mycorrhzal root systems. ACKNOWLEDGEMENTS We gratefully acknowledge the FRD, CSIR, South Afrca and the Unversty of Cape Town for fnancal assstance and Dr D. J. Read of Sheffeld Unversty for the use of the European endophytes. REFERENCES APPIAH, M. R. & THOMAS, R. L. (1982). Inostal phosphate and organc phosphorus contents and phosphatase actvty of some Canadan and Ghanaan sols. Canadan Journal of Sol Scence, 62, ARNOLD, W. N. & GARRISON, R. G. (1979). An Fe'+-actvated acd phosphatase n Saccharomyces roux. Journal of Bologcal Chemstry, 254, 4919^925. BARTLETT, E. M. & LEWIS, D. H. (1973). Surface phosphatase actvty of mycorrhzal roots of beech. Sol Bology and Bochemstry, 5, BASHA, SHEIK MEHBOOB (1984). Purfcaton and characterzaton of an acd phosphatase from pesnut (Arachs hypogaea) seed. Canadan Journal of Botany, 62, BEEVER, R. E. & BURNS, D. J. W. (198). Phosphorus uptake, storage and utlzaton by fung. In: Advances n Botancal Research, vol. 8 (Ed. by H. W. Woolhouse), pp Academc Press, London.

14 256 C. J. STRAKER AND D. T. MITCHELL BIELESKI, R. L. (1973). Phosphate pools, phosphate transport and phosphate avalablty. Annual Revew of Plant Physology, 24, Bojoyc-CVETIC, D. & VujCc, R. (1982). Acd phosphatase localsaton and dstrbuton n Aspergllus flavus. Transactons of the Brtsh Mycologcal Socety, 79, CALLEJA, M., MOUSAIN, D., LECOUVREUR, B. & D'AUZAC, J. (198). Influence de la carence phosphatee sur les actvtes phosphatases acdcs de tros champgnons mycorhzcns. Hebeloma edurum Metrod., Sullus granulatus (L. ex FR.) O. Kuntze et Psolthus tnctorus (Pers.) Coker et Couch. Physologe Vegetale, 18, CALLEJA, M. & D'AUZAC, J. (1983). Actvtes phosphatases et carence phosphatee chez des champgnons supereurs. Canadan Journal of Botany, 61, DAVIS, B. J. (1964). Dsc electrophoress. II. Method and applcaton to human serum protens. Annals of New York Academy of Scence, 121, GIANINAZZI-PEARSON, V. & GIANINAZZI, S. (1976). Enzymatc studes on the metabolsm of vesculararbuscular mycorrhza. 1. Effect of mycorrhza formaton and phosphorus nutrton on soluble phosphatase actvtes n onon roots. Physologe Vegetale, 14, GIANINAZZI, S., GIANINAZZI-PEARSON, V. & DEXHEIMER, J. (1979). Enzymatc studes on the metabolsm of vescular-arbuscular mycorrhza. III. Ultrastructural localzaton of acd and alkalne phosphatases n onon roots nfected hy Glomus mosseae (Ncol. and Gerd.). New Phytologst, 82, HASEGAWA, Y., LYNN, K. R. & BROCKIIANK, W. J. (1976). Isolaton and partal characterzaton of cytoplasmc and wall-hound acd phosphatases from wheat roots. Canadan Journal of Botany, 54, Ho, I. & ZAK, B. (1979). Acd phosphatase actvty of sx ectomycorrhzal fung. Canadan Journal of Botany, 57, LowRY, O. H., Ro.sEBROTjGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Proten measurement wth the Foln phenol reagent. Journal of Bologcal Chemstry, 193, MITCHELL, D. T. & READ, D. J. (1981). Utlzaton of norganc and organc phosphates by the mycorrhzal endophytes of Vaccnum macrocarpon and Rhododendron pontcum. Transactons of the Brtsh Mycologcal Socety, 76, MITCHELL, D. T., BROWN, G. & JONGENS-RODERTS, S. M. (1984). Varaton of forms of phosphorus n the sandy sols of coastal fynbos, south-western Cape. Journal of Ecology, 72, MURPHY, J. & RH.EY, J. P. (1962). A modfed sngle soluton method for the determnaton of phosphate n natural waters. Analytca Chmca Acta, 27, PEAR.SON, V. & READ, D. J. (1975). The physology of the mycorrhzal endophyte of Calluna vulgars. Transactons of the Brtsh Mycologcal Socety, 64, 1-7. SAKURAI, Y. & SHIOTA, H. (1977). Two dstnct types of acd phosphatase formatons n Aspergllus sp. Journal of General and Appled Mcrobology, 23, SAN BLAS, G. & CUNNINGHAM, W. L. (1974). Producton of extracellular acd phosphatase hy the yeast Hamenula holst NCYC 56. Bochmca et Bophysca Acta, 343, SCHMIDT, G. (1961). Nonspectc acd phosphomonoesterases. In: The Enzymes, vol. 5, 2nd Edn. (Ed. hy P. D. Boyer, H. Lardy & K. Myrback), pp Academc Press, New York. SPIERS, G. A. & MCGILL, W. B. (1979). Effects of phosphorus addton and energy supply on acd phosphatase producton actvty n sols. Sol Bology and Bochemstry, 11, 3 8. STOCK, W. D. & LEWIS, O. A. M. (1986). Sol ntrogen and the role of fre as a mneralzng agent n a South Afrcan coastal fynhos ecosystem. Journal of Ecology. (In press.) STOCK, W. D., LEWIS, O. A. M. & ALLSOPP, N. (1986). Sol ntrogen mneralzaton n a post-fre coastal fynhos successon at Pella, south-western Cape, South Afrca. Oecologa. (In press.) STRAKER, C. J. (1986). Aspects of phosphorus nutrton n endomycorrhzal fung of the Ercaceae. Ph.D. thess. Unversty of Cape Town. STRAKER, C. J. & MITCHELL, D. T. (1985). The characterzaton and estmaton of polyphosphates n endomycorrhzas of the Ercaceae. New Phytotogst, 99, THEODOROU, C. (1971). The phytase actvty of the mycorrhzal fungus Rhzopogon roseolus. Sol Bology and Bochemstry, 3, WOOLHOUSE, H. W. (1969). Dfferences n the propertes of the acd phosphatases of plant roots and ther sgnfcance n the evoluton of adaptve ecotypes. In: Ecologcal Aspects of Mneral Nutrton of Plants (Ed. hy 1. H. Rorson). Symposum of the Brtsh Ecologcal Socety, 9, YAMAMOTO, S., MINODA, Y. & YAMADA, K. (1969). Localzaton of acd phosphatase actvty n mycela of Aspergllus terreus at the electron mcroscope level. Agrcultural and Bologcal Chemstry, 33, ZNK, M. W. & VELIKY, I. A. (1979). Acd phosphatases of Ipomoea sp. cultured n vtro. I. Influence of ph and norganc phosphate on the formaton of phosphatases. Canadan Journal of Botany, 57,

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