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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
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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
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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-
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L. C. M. CAPACCIO AND J. A. CALLOW
(a ) ^ . ^ (b)
Fig. 1
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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.
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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
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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
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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.
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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
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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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