New Phytol. (1982) 91, 81-91 8 l

THE ENZYMES OF POLYPHOSPHATEMETABOLISM IN VESICULAR-ARBUSCULAR

MYCORRHIZAS

BY L. C. M. C A P A C C I O AND J. A. CALLOW*Department of Plant Sciences, University of Leeds, Leeds LS2 9yT, U.K.

{Accepted 4 November 1981)

SUMMARYThe presence and distribution of enzymes of polyphosphate (polyP) synthesis and degradationwas examined in mycorrhizal and non-mycorrhizal onion roots, and both external and internalhyphae of the endophyte, Glomus mosseae. PolyP kinase activity was detected in extracts ofexternal hyphae and mycorrhizal roots. Activity was dependent on exogenous polyP and Mg''*,the reaction product being a long chain polyP. Exopolyphosphatase activity was detected inboth mycorrhizal and non-mycorrhizal roots but activity was 134",, greater in the former.Endopolyphosphatase activity was also greater in mycorrhizal roots. Polyphosphatases weredetected in extracts of internal hyphae, but not external hyphae. Polyphosphate glucokinaseactivity was demonstrated in extracts of external hyphae and mycorrhizal roots, the immediate.reaction product being glucose-6-phosphate. These results are discussed in relation to potentialmechanisms of P translocation and transfer in vesicular-arbuscular (VA) mycorrhizas. A noveltechnique for isolating the internal hyphae and associated vesicles and arbuscules from mycor-rhizal onion roots is described.

I N T R O D U C T I O N

Little is known of the biochemical mechanisms involved in the active transportof phosphorus in the fungal component of vesicular-arbuscular (VA) mycorrhizasand its subsequent transfer to the host cells. Callow et al. (1978) showed thatsubstantial quantities of orthophosphate taken up by infected roots are convertedinto condensed polyphosphates. Since uninfected roots did not contain polyphos-phate (polyP) and host cells in infected roots did not stain metachromatically forpolyP, it was concluded that the polyP was synthesized in the fungal componentof the mycorrhiza. Calculation showed that there was a sufficiently high concen-tration of polyP in the fungal component to satisfy the experimentally observedhigh mean values for phosphate flux to the root, based on bulk transport ofcondensed polyP through cytoplasmic streaming. It was further suggested that onreaching the arbuscules polyP would be broken down by either an enzyme of thepolyphosphatase (poly Pase) type, liberating Pi, or a polyphosphate kinase, liberatingATP, the products released then being either directly or indirectly used in thetranslocation of Pi across the host/arbuscule interface. An alternative suggestionwas that the polyP might serve in phosphorylation mechanisms for the activetransport of carbon skeletons into the arbuscule from the host.

An initial approach to understanding the role of mechanisms of this type in thehostendophyte relationship, and further confirmation of the significance of polyPin VA mycorrhizas, requires that the appropriate enzymes of polyP synthesis anddegradation be demonstrated. In this paper we present evidence for the existenceof a number of such enzymes. In addition we report on the development of a noveltechnique for the isolation of milligram quantities of internal fungal hyphae andassociated structures.

To whom further correspondence should be addressed.

0O28-646X/82/050081 + 11 $03,00/0 1982 The New Phytologist

82 L. C. M. CAPACCIO AND J. A. CALLOW

MATERIALS AND METHODS

Growth of plantsOnions {Allium cepa L. Fj hybrid Amber Express) were grown in sand or

perlite and inoculated with Glomus mosseae as described previously (Callow et al.,1978). Six- to 12-week-old plants were used for routine extraction.

Enzyme extractionWashed root tissue was extracted by pestle and mortar in 0-05 M Tris-HCl

buffer, pH 7-2 (0-5 to 10 cm^ g"' tissue). Extracts were centrifuged (10000 g for20 min) then desalted on a 150 x 15 mm column of Sephadex G 25. Elution bufferswere varied as appropriate to the particular enzyme to be assayed.

Enzyme assaysExopolyphosphatase (polyphosphatase; polyphosphate phosphohydrolase,

E.C. 3.6.1.11). (polyP) -f H,O -> (polyP)_, -t- Pi.Reaction mixtures contained 0 5 cm^ extract (generally in 01 M acetate pH 5 0),0-25 cm' polyphosphate [1 mg cm'^ of (polyP)2oo, Sigma], 015 cm 004 M MgCl^and 1 cm^ acetate buffer pH 5-0. Aliquots were removed at various times fordetermination of Pi release by the method of Truog and Meyer (1929). Controlsomitted enzyme or substrate. >

Endopolyphosphatase (polyphosphate depolymerase; polyphosphate poly-phosphohydrolase, E.C. 3.6.1.10).

(polyP)n+ + H^O ^ (polyP) + (polyP)^.Incubations contained 10 cm^ 0-1 M citrate buffer pH 5-0, 9 cm^ polyP [1 mg cm^*long-chain Graham's salt prepared as Kornberg (1957), dialysed before use againstcitrate buffer], 3 cm* extract in citrate buffer and 1 cm^ 0-04 M MgClj. Change inviscosity was measured in a U-tube viscometer (B.S. 188) and parallel determi-nations of Pi released were made.

Polyphosphate kinase (ATP: polyphosphate phosphotransferase, E.C.^ ' ^ ^ ' ^ y (polyP) + ATP - (polyP)+, -|- ADP.Although polyphosphate kinase can be measured in either direction, best resultswere obtained in the direction of synthesis, by measuring the incorporation of ^''Pifrom -)/-[*'^ P]ATP into long chain polyphosphates precipitable with trichloroaceticacid (TCA). Reaction mixtures contained 1-25 cm^ ATP (disodium salt, 20 mM),O'Ol cm' phosphoenolpyruvate (monosodium salt, 23 mg cm~'), 0-005 cm' pyruvickinase (Sigma, rabbit skeletal muscle, 2 mg cm ''), 1 cm' polyphosphate (variablechain length from Sigma, w = 5 to w = 200, 2 mg cm '), 2-5 cm' tissue extract,0-025 cm' 7-['^P]ATP (Amersham Radiochemical Centre, triethylammonium salt,0-5 to 3-0 Ci mmol-i), and 005 cm' MgCl.^ (0-1 M). Aliquots (0-3 cm') wereremoved at zero time, 30 and 90 min, and polyphosphate was precipitated with0-6 cm' ice-cold TCA (7-5%, w/v). After a minimum of 2 h at 4 C insolublematerial was pelleted in Eppendorf micro-tubes at 10000^ for 2 min, andtransferred in 5 % TCA to GF/C glass-fibre discs under suction. Each disc waswashed with 10 cm' 5 % TCA, 10 cm' ethanol/ether (1/1, v/v), before drying and

VA polyphosphate metabolism 83counting in 4 cm^ toluene containing 0 4 % PPO and 001 % POPOP. Alternatively,0-03 cm^ aliquots of one-fifth scale reaction mixtures were spotted directly on toWhatman 3MM chromatography paper prior to high voltage electrophoresis insodium borate pH 92, 5 kV for 45 min. Dried paper strips were scanned usinga n Actigraph Radiochromatogram Scanner (Nuclear Chicago).

Polyphosphate glucokinase (polyphosphate-glucose phosphotransferase, poly-phosphate: D-glucose 6-phosphotransferase, E.C. 2 . 7 . 1 . 63).

(polyP)n 4- glucose -> glucose 6-P 4- (polyP)n_i.Polyphosphate glucokinase was demonstrated by the method of Szymona (1962).Reaction mixtures contained 0-15 cm^ extract, 0-15 cm' Tris buffer pH 8 0 (0 3 M),0-02cm3polyP(l mg cm ^ w = 200), 0025 cm^ KCl (2 M), 0 01 cmMgCl2(0lM),0-01 cm' glucose (0 3 M) and 0 02 cm' D-[U>''C]glucose (Radiochemical Centre,Amersham, 230 mCi mmol"'). After incubation for 30 min, 0 05 cm' aliquots werespotted on to Whatman 3MM chromatography paper and subjected to high voltageelectrophoresis (HVE) in borate buffer, pH 90, or pyridine buffer, pH 6S (5 kV).Radioactive components were localized by scanning the strips of paper as describedfor polyphosphate kinase.

Non-specific acid phosphatase. Phosphatase assays were conducted as describedby Gianinazzi-Pearson and Gianinazzi (1978) using /)-nitrophenol phosphate assubstrate at various pHs.

Isolation of internal hyphae.Roots from perlite or sand culture were washed, cut into 10 mm segments and

incubated for 16 h in an aqueous solution of 01 % cellulase and 1 % pectinase(Sigma). Under a dissecting microscope, hyphal masses could be easily removedfrom root cylinders and collected.

RESULTS

Maceration of onion root segments for 16 h resulted in the hydrolysis of corticaltissue leaving intact fungal masses within cylinders of unhydrolyzed, suberizedhypodermis (F"ig. 1). The fungal masses were readily recovered from the rootcylinders under a dissecting microscope and were usually washed thoroughly inwater before any further treatment. Under the microscope the fungal massesconsisted of granular hyphae, with intact arbuscules and terminal vesicles (Fig. 1).Since a hypotonic maceration medium was employed no contamination with hostprotoplasts was detected. Internal hyphae isolated in this way contained 0-02 %by fresh wt of polyphosphate (assayed by the methods of Callow et al. 1978) andpolyP accounted for 16% of total P. The fresh wt:dry wt ratio was 0 2.

Extracts from both uninfected and infected onion roots were able to release Pifrom long chain polyP, but on a fresh wt basis the activity in infected roots was,on average, 134% greater (Table 1). In addition, there was a qualitative differencein the pattern of polyphosphatase activity detected. For a 10% release of Pi(exo-activity) extracts from infected plants caused a 27 % reduction in viscosityof long chain polyP (endo-activity) compared with 6 % for extracts from uninfectedplants. Extracts from external hyphae collected from sand cultures contained low,or undetectable polyphosphatase but did contain substantial activities of non-

L. C. M. CAPACCIO AND J. A. CALLOW

(a ) ^ . ^ (b)

Fig. 1

VA polyphosphate metabolism 85

Table 1. Exopolyphosphatase and p-nitrophenol phosphatase activities. All assayswere conducted tn triplicate and means are shown

MS Pi released h ' g

Mycorrhizal Non-mycorrhizalExperiment roots

(A)IIIIIIIV(B)III

from polvP9486

102

from /)-nitrophenol phosphate121255

roots

435744

104232

' fresh wt

Externalhyphae

000

1871150

Internalhyphae

107

specific acid phosphatase (115mg Pi released from />-nitrophenol phosphateh^^g"^ fresh wt at pH 5-0 in extracts of external hyphae; 0-231 mg h~'g~' fromuninfected root tissue). However, extracts from internal hyphae isolated fromplants grown in the same way did contain substantial polyphosphatase activity(0-1 mg Pi released from polyP h ' g ' fresh wt). All polyphosphatases examinedshowed a sharp pH optimum at 50 and activities were doubled by 1 mM Mg^^.

Extracts from both external hyphae and infected roots were able to catalyse theincorporation of ''Pi from 7-[''^P]ATP into a high molecular weight, TCA-insoluble product (Pigs 2 and 3), although the assay method was not entirelysatisfactory since zero-time controls gave high background counts due to bindingof labelled ATP to the filter discs. A variety of treatments was employed to reducebackground from this source, including silanizing filter discs, use of Teflon discs,discs pretreated with unlabelled Pi and ATP, but zero-time counts could not bereduced below 5000 d min ^ Activity in extracts from external hyphae andinfected roots was dependent on the addition of exogenous polyP primer suggestingthe presence of an enzyme of the polyphosphate kinase type and activity wasrnaximal at pH 7-2 and dependent on Mg'-'+. Highest activities were obtained 2 to6 h after providing roots with lO*' M orthophosphate. Extracts from uninfectedroots were less able to catalyse incorporation when compared on a fresh wt basis(Fig. 3) and the low activity was independent of exogenous polyP primer.

The products of incorporation were examined by HVE without prior precipi-tation with TCA (Fig. 4). Hyphal extracts produced a clear peak of incorporationof '^P near the origin, in a region staining positively for long chain polyP withtoluidine blue. Low molecular weight compounds, not precipitable with TCA inthe quantitative assay were detected, including Pi and unincorporated ATP. Onelution and hydrolysis in 1 N HCl for 7 min at 100 C, activity in the polyP regionwas totally converted to Pi as detected by re-electrophoresis. Flxtracts frommycorrhizal roots gave similar results.

PolyP kinase from Esherichia coli readily catalyses the reverse reaction, i.e.

Fig. 1. (a) Segments of mycorrhizal onion root after enzyme maceration for 16 h. Complexes ofinternal hyphae can be seen within the unhydrolyzed hypodermal cylinders, x 40, stained withtoluidine blue, (b) Isolated internal hyphae with vesicles, x 600, stained with toluidine blue.

(c) Isolated internal hyphae with arbuscules. x 600, stained with toluidine blue.

86 L. C. M. CAPACCIO AND J. A. CALLOW

20 30 40Time (min)

6 0

Fig. 2. Polyphosphatekinaseactivity in extracts of external hyphae, in the presence (#) and absence(O) of exogenous polyP primer. Activity is expressed per mg fresh wt of hyphae extracted, withbackground, zero-time controls subtracted (equivalent to 1-2 x 10' counts min"' mg"'). Each point

is the mean of triplicate determinations.

60 90Time (mm)

Fig. 3. Polyphosphate kinaseactivity in extracts from mycorrhizal and non-mycorrhizal onion rootsin the presence and absence of exogenous polyP primer. Activity is expressed per g fresh wt oftissue extracted with background, zero-time controls subtracted. Each point is the mean oftriplicate assays. 9 , Mycorrhizal plus polyP; O. mycorrhizal minus polyP; A, non-mycorrhizal

plus polyP; A> non-mycorrhizal minus polyP.

ADP-dependent ATP synthesis frotn polyP (Kornberg, 1957). This activity wastested for in extracts of external hypbae and mycorrhizal roots, using ^^P-labelledpolyP, prepared as described by Kornberg (1957), as substrate. However, inneither case could ATP synthesis be convincingly demonstrated implying that themycorrhizal enzyme is predominantly synthetic.

Polyphosphate glucokinase activity was detected by HVE and radioscanningfollowing incubation of long chain polyP with ['''C]glucose. Extracts of bothexternal hyphae and infected roots, but not uninfected roots, were able to phos-phorylate glucose using polyP as P donor (Fig. 5). The principal low molecular

VA polyphosphate metabolism

2000 - 1

10

/ ~ \/ \/ \

/ \/ \polyp \ ^

'1 11/1/1

V11, V ATP

n,1 \V \

Electrophoretic mobility >

Fig. 4. Products of polyP kinase activity from extracts of external hyphae, as shown by HVE andstrip-scanning. The area of activity shown was eluted and hydrolysed in 1 N HCl, for 7 min at100 "C hefore re-electrophoresis. The product was entirely Pi. , Zero-time incubation;

, 2 h incubation.

1000

1000 -

EI/)

Fig. 5. Products of polyP glucokinase activity in extracts of non-mycorrhizal roots (a), mycorrhizalroots (b) and external hyphae (c), as detected by HVE and strip-scanning. GP, Glucose-6-phosphate

tnarker.

weight product synthesized by extracts of infected roots co-electrophoresed withhexose-6-phosphates and was identified as follows.

The peak of activity was insensitive to acid hydrolysis but sensitive to alkali (F'ig.6) (aldose 1-phosphates are acid labile but alkali stable, Leloir and Cardini, 1957).Secondly, after incubation of the peak with acid phosphatase at pH 5 the labelled

88 L. C. M. CAPACCIO AND J. A. CALLOW

1000 -

1000

Electrophoretic mobility

Fig. 6. Products of polyphosphate glucokinase activity in cell-free extracts of mycorrhizalroots (a), and following treatment of the incubation mixture with 1 N HCl for 10 min at 100 C (b),

and 1 N KOH for 3 min, 100 C (c), immediately prior to separation by HVE.

products co-electrophoresed primarily with glucose. F"inally, after heat denaturationthe total incubation mixture was treated with glucose-6-phosphate dehydrogenase.On subsequent HVE a peak of 6-phosphogluconate was detected rather thanhexose-phosphate. The product of the reaction between polyP and glucose thusappears to be glucose-6-phosphate suggesting the presence of polyphosphateglucokinase. Since all extracts were thoroughly desalted to remove low molecularweight compounds including ADP it is unlikely that the activity observedrepresents a coupled reaction between ATP-generating activity of polyphosphatekinase and a separate hexokinase. P'urthermore, activity was not enhanced in thepresence of ADP and as previously stated, polyphosphate kinase activity in thedirection of ADP-dependent ATP synthesis was undetectable in extracts ofinfected roots or fungus.

Polyphosphate glucokinase activity of infected roots was dependent on 4 mMMg^ "*", inhibited by 20 mM Mg''"'", and showed a pH optimum of 8-0. The reactionproducts from extracts of external hyphae contained a second component inaddition to glucose-6-phosphate, running with hexose-l,6-diphosphates. Thiswas not analyzed further.

VA polyphosphate metabolism 89

D I S C U S S I O N

T h e novel technique described in this paper for isolating the internal hyphae ofV A mycorrhizas permits, for the first time, detailed study of the properties of thatcomponent of the mycorrhizal symbiosis in intimate contact with host cells.Previous studies on hyphae in these associations have been limited to externalhyphae collected from the root surface or rooting medium and which therefore existin a totally different environment and physiological state. The technique has beenused in two ways in the present paper. Firstly, previous studies in this laboratoryshowed that polyP may be present in the fungal endophyte at a concentration of0-05 % by fresh wt (0-15 % by dry wt) and that the proportion of total P as polyPcould be as high as 40% (Callow et al., 1978). Considering the approximationsa n d assumptions made in the latter study, the corresponding figures of 0-02%,0-1 % and 16%, respectively, determined by direct extraction of isolated internalhyphae, are in surprisingly good agreement and emphasize the quantitativeimportance of polyP in the P metabolism of VA mycorrhizas.

Secondly, isolated internal hyphae have been used to study enzyme distributions.T h u s , in the present paper, exopolyphosphatase was shown to be absent fromexternal hyphae, but present in internal hyphae, and Gianinazzi-Pearson, Giani-nazzi and Callow (unpublished results) have shown that a mycorrhiza-specificalkaline phosphatase is present in extracts from internal hyphae. However, whilstpreparations of internal hyphae were well washed, possible adsorption of hostcytoplasm from lysed host cells cannot be completely excluded. Hence conclusionson enzyme localization, for example, must be treated with caution. Internal hyphaeisolated by this technique may prove useful in a variety of studies on VArnycorrhizal physiology including carbohydrate uptake and metabolism.

Callow et al. (1978) suggested that uptake of soil P by external hyphae is coupledto the endergonic synthesis of polyP. Micro-organisms synthesize long chain polyPb y a single pathway catalysed by polyP kinase (Harold, 1966). In the presentexperiments extracts of external hyphae and infected roots catalysed the transferof ternninal phosphate from ATP to a high molecular weight form with thecharacteristics of polyP. The activity detected was rather weak and consequentlydiflficulty was experienced with the quantitative assay due to relatively highnon-specific binding of labelled ATP to the membrane filters. The weak activitydetected may be attributed to the use of a synthetic polyP primer (Nishi, 1960).

Little is known of the control of polyP synthesis in VA mycorrhizal fungi. Yeastcells subjected to phosphate starvation contain enhanced levels of polyP kinase andare thus capable of rapid polyP synthesis when Pi is provided, this being the basisof the 'overplus' phenomenon (Harold, 1966). In VA mycorrhizas, although thereis a rapid accumulation of polyP when infected roots are transferred from low(10~^ M) to high (10~^ M) Pi analogous to the 'overplus' effect (unpublished datafrom this laboratory), the control may be quite different since polyP kinase wasvirtually undetectable in infected roots grown on 10"^ M Pi, and was greatlyenhanced within 2 to 6 h of providing 10"^ M Pi, suggesting that this enzyme maybe inducible or activated in the presence of excess Pi uptake.

A number of enzymes are implicated in polyphosphate degradation in micro-organisms (Harold, 1966). Reversibility of polyP kinase has been demonstrated forsome organisms but not others, and Langen (1965) was unable to detect directconversion of polyP to ATP by yeast cells in vivo. Stepwise hydrolysis to lowmolecular weight polyP and ultimately Pi by polyphosphatases appears to be a

go L. C. M. CAPACCIO AND J. A. CALLOW

more likely degradative pathway (Harold, 1966). On the other hand, polyphos-phatases have been detected in organisms that do not contain polyP (Harold, 1966),as shown here for non-mycorrhizal roots. Nevertheless, a significant role forpolyphosphatase in mycorrhizal P metabolism is suggested since exopolyphos-phatase activity was more than doubled in infected roots and activity was qualita-tively different to that in uninfected roots, with a greater proportion of endopoly-phosphatase activity. Furthermore, extracts of internal hyphae but not externalhyphae contained polyphosphatase, a distribution which might be anticipated ifthere is vectorial translocation of polyP from its site of synthesis in external hyphaeto the site of utilization in internal hyphae and arbuscules (Callow et al., 1978).Tbe polyphosphatase activity in uninfected roots presumably reflects non-specificphosphomonoesterase activity but more study is required to separate and charac-terize the various activities.

Another enzyme that has been well-characterized from various micro-organismsand implicated in polyP breakdown is polyP glucokinase (Harold, 1966). Fungiappear to contain both active and passive sugar transport systems (Rothstein andVan Steveninck, 1966). Woolhouse (1975) suggested that the transport of C-skeletons from host source to fungal sink in VA mycorrhizas may be linked tophosphorylation mechanisms and, in the case of yeast, polyP appears to serve asP donor for the uptake of glucose in the phosphorylated state (Van Steveninck andBooij, 1964). In the present case, the polyP glucokinase activity detected in infectedroots, assuming that this is of fungal origin, is potentially relevant to the transferof glucose from host root to fungal endophyte. However, polyP glucokinase activitywas also detected in extracts of external hyphae where such a role is less obviouslysignificant.

While the demonstration of these enzymes of polyP synthesis and degradationfurther implicates polyP in the P metabolism of VA mycorrhizas, it does little toclarify the precise mechanisms of P uptake, translocation and transfer. The roleof these enzymes and other mycorrhiza-specific enzymes of P metabolism such asthe alkaline phosphatase of Gianinazzi-Pearson and Gianinazzi (1978) mustremain largely speculative in the absence of critical experimentation on thebiochemical control of P metabolism in the fungi of VA mycorrhizas. Thetechnique described in this paper for isolating the endophyte may therefore permitmore detailed studies on this aspect of mycorrhizal physiology.

ACKNOWLEDGEMENTS

The authors wish to thank SERC for financial support and Dr P. B. Tinker forhis collaboration in the initiation of this work.

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