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The role of acid phosphatases in plant phosphorus

metabolism

Article  in  Physiologia Plantarum · April 1994

DOI: 10.1111/j.1399-3054.1994.tb02539.x

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 PHYSIOLOGIA PLANTARUM 90- 791-800 1994 Cpyngh, © PhyM,,t«gia Plmumm 1994
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The role of acid phosphatases in plant phosphorus metabolism

Stephen M, G. Duff, Gautam Sarath and William C. Plaxton

Duff. S. M. G.. Sarath, G. and Plaxton, W. C. 1994. The role of acid phosphatases in
plant phosphorus metabolism. - Physiol. Plant. 90; 791-800.

Hydrolysis of phosphate esters is a critical process in the energy metabolism and

metabolic regulation of plant ceils. This review summarizes the characteristics and
putative roles of piant acid phosphatase lAPasel. Although immunologicaliy closely
related, plant APases display remarkable heterogeneity w ith regards to their kinetic and
molecular properties, and subcellular location. The secreted APa.ses of roots and cell
cultures are relati\ ely non-specific enzymes that appear to be important in the hydroly-
sis and mobilization of P, from extracellular phosphomonoesters for plant nutrition.
Intracelluiar APases are undoubtedly involved in the routine utilization of P, reserves
or other P, containing compounds. A special class of intracellular APase exists that
demonstrate a clear-cut (but generally nonabsoiute) substrate selectivity. These .^.Pases
are hypothesized to have distinct metabolic functions and include; phytase, phospho-
glycoiate phosphatase. 3-pbosphoglycerate phosphatase. phosphoenolpyruvate phos-
phatase. and phosphotyrosyl-protein phosphatase. APase expression is regulated by a
variety of developmental and environmental factors. P, starvation induces de novo
synthesis of extra- and intracellular APases in cell cultures as well as in whole plants.
Recommendations are made to achieve uniformity in the analyses of the different
.A.Pase isoforms normally encountered within and between different plant tissues.
Key words - .\cid phosphatase. isozymes. phosphate metabolism, phosphoenolpyru-
vate phosphatase. 3-phosphoglycerate phosphatase, phospboglycolate phosphatase,
pho.sphoprotein phosphatase. phytase.

.V. M, G. Duff and G. Santth. Dept of Biochenii.stry, Univ. of Nebraska-Lincoln,

Lincoln. NE 68583-0718. USA: W. C, Plaxton (corresponding author), Dept of Biol-
ogy. Queen s Univ,. Kingston. Ontario K7L 3N6. Canada.

Int od f Phosphatases have been traditionally classified as be-

One of the most important, yet least available mineral
nutrients for plant growth is phosphorus. The orthophos-
phate anion (P,) is the preferentially assimilated form of
phosphorus for organisms such as plants that acquire
their mineral nutrients directly from the environment. P,
not only plays a vital functional role in energy transfer
and metabolic regulation, but is also an important struc-
tural constituent of many bioniolecules. Consequently, P,
assimilation, storage and metabolism are of critical im-
portance to plant growth and development. Efficient ac-
quisition and utilization of phosphorus requires a ubiqui-
tous class of enzymes known as phosphatases which
function to hydrotyse P, from orthophosphate monoesters
in a thermodynamically favourable process (iiG°'< - 9 kj
mol"') (Vincent et al. 1992).
ing alkaline or acid phosphatases according to whether
their optimal pH for catalysis is above or below pH 7.0
(Vincent et al. !992). Plant alkaline phosphatases gener-
ally display an absolute suhstrate specificity. Examples of
this class of phosphatase in plants are the cytosolic fruc-
tose-l,6-bisphosphatase and sucrose-6-P phosphatase,
both of which are involved in sucroneogenesis from
triose-P. By contrast, acid phosphatases (APases) do not
normally exhihit an absolute substrate specificity. Never-
theless, two distinct categories of plant APases can appar-
ently be distinguished on the basis of their relative sub-
strate selectivities. The first type of plant APase are those
truly 'non-specific' enzymes that show little or no sub-
strate specificity. Non-specific APases occur in a wide
variety of species and tissues and feature considerable
diversity with regards to tbeir physical properties. How-

Received 10 September. 1993

52 Physiol. Planl. W. l ? ^ 791

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ever, they all appear to be important in the production,
transport and recycling of P|. Tbe second category of
plant APases are specialized enzymes such as the 3-P-
glycerate (3-PGA) phosphatase from maize leaves (Ran-
dall and Tolbert 1971) and the phosphoenolpyruvate
(PEP) phosphatase of Brassica nigra (black tnustard)
suspension cell cultures (Duff et al. 1989a) wbich display
a clear hut non-absolute substrate specificity. These
APases are hypothesized to have distinct tnetabolic func-
tions.
The diversity and ubiquity of plant APases together
make a consensus on their precise physiological role(s)
difficult to achieve. Nonetheless, a large and growing
body of literature exists which deals with these itnportant
enzymes and their possible functions. Much of this in-
formation, however, is fragtnentary owing to the incon-
sistent manner in which plant APases have been exam-
ined. Studies on plant APases have been performed by
scientists from a variety of fields having a variety of
perspectives. Few complete studies have been reported,
and characteristics of the enzyme or system considered
important by one researcher may not be included by
anotber. The aim of this review is to summarize the
characteristics and possible functions of plant APases.
Our focus will be to highlight areas of study that are
needed to enhance our utiderstanding of these ubiquitous
enzymes. In addition, some suggestions are made to
achieve unifortrtity in the anaiy.ses of the different APase
isoforms normally encountered within and between plant
tissue.';.
Abbreviations - APase. acid phosphatase; EPSP, 5-enolpyruvyl-
shikamate-3-phosphate; pNPP. p-nitrophenyl phosphate; PEP,
phosphoenolpyruvate; 3-PGA. 3-phosphoglycerate; P,. ortho-
phosphate; PP,. pyropbosphate; P-Ser, O-phospho-L-serine; P-
Tyr. (5-phospho-L-tyrosine.
Imnnunologica) properties
One of the first reports on the immunological properties
of plant APases was provided by Kamenan et al. (1985)
who examined three purified APase isoforms of yam
tubers. Two of the yam APases are membrane bound,
with the third localized in the cytoplasm (Tab. 1, Kame-
nan and Diopoh 1983). One of the membrane bound
APase isoforms was fotind to be immunologicaliy related
to the cytoplasmic APase suggesting that the two APases
possess common antigenic determinants (Kamenan et al.
1985). By contrast, polycional antibodies generated
against the other membrane bound isoform failed to rec-
ognize either of the other two yam APases. This is an
interesting finding considering that all three enzymes are
glycosylated, and tbe two membrane bound enzymes
appear to differ only in the nature of tbeir glycosylating
groups (Kamenan and Diopoh 1983). Subsequent work
revealed that monospeciftc polycional antisera raised
against a glycosylated APase from Poa pratensis seed-
lings cross-reacted with purified APases from a number
of other grass species (Lorenc-Kubis 1989). Similar re-
52* Physint Plam. W. 1994
sults were provided by Haraguchi et al. (1990) who
demonstrated the immunological relatedness of several
APase isoforms from several tissues of Vigno mungo
seedlings. Duff et al. (1991b) prepared affinity-purified
polycional rabbit IgG against the vacuolar PEP-specific
APase of B. nigra suspension cells. Preliminary itnmu-
noblotting studie.s demonstt^ted that the anti-(B. nigra
PEP-APase) IgG cross-reacted with numerous plant gly-
coproteins, which is not unexpected since plant glycopro-
teins generally cross-react owing to the presence of iden-
tical or related glycans which are highly immunogenic.
Therefore, in order to abolish carbohydrate epitopes,
without affecting peptide epitopes, antigenic glycosylated
side-chains of the blotted B, nigra glycoproteins were
routinely oxidized by pretreatment of the immunoblots
with sodium m-periodate. Interestingly, the anti-(B. nigra
PEP-APase) IgG strongly cross-reacted with a cell wall-
localized non-specific B. nigra APase (Duff et al. 199ib),
despite the fact that the two APase isoforms show sub-
stantial differences in their respective cyanogen bromide
peptide maps and kinetic properties (Duff et al. 1991a).
Furthermore, the anti-(B. nigro PEP-APase) IgG recog-
nized APases from a wide range of higher plants, but did
not cross-react witb APases from Escherichia coli. yea.st,
Drosophila. and human prostate (Duff et a). 1991b).
Overall, the various studies described ahove suggest that
the major plant APase isoforms tend to be immunolog-
icaliy closely related indicating that they may have
evolved from a common ancestral APase gene.
Genes
The only sequence of a plant APase gene that has ap-
peared to date is that encoding the APase-1 isoform from
nematode resistant tomato (Williamson and Colwel!
1991). The APase-1 protein, a homodimer of about 51
kDa, had previously been purified from tomato cell sus-
pension cultures and sbown to have a very low pH opti-
mum for activity, pH 3.5 (o 4.0, compared to tnost other
plant APases. The cDNA encoding tomato APase-1 was
found to contain an open reading frame predicting a
hydrophobic amino-terminal transit peptide of 38 amino
acids and a mature APase with a molecular mass of
24 981 Da (Williamson and Coiwell 1991). Consistent
with the inability of an anti-(plant APase) IgG to recog-
nize non-plant APases (Duff et al. 1991b), a computer
search of amino acid sequence data banks revealed no
homology hetween the deduced tomato APase-1 se-
quence and any of the various non-plant APase sequences
contained in the data base (Williamson and Coiwell
1991). The sequence most similar to that encoded by the
APase-1 gene was a 29 kDa soybean vegetative storage
protein (Williamson and Coiwell 1991). Tbis correlation
was substantiated by the subsequent demonstration that
soybean vegetative storage proteins are APases active on
polyphosphates (Dewald et al. 1991). However, their
relatively low activity (Tab. I) raises doubts about tbe
793
physiological relevance of the APase activity of soybean
vegetative storage proteins.
Assay, kinetic and physical properties
p-Nitrophenyl-P (pNPP) is the most commonly utilized
substrate for the in vitro estimation of APase activity in
plant extracts. Whereas this substrate is useful for routine
assays, the broad specificity of these enzymes necessi-
tate.s a study of the kinetics of P; release from various
physiologically relevant phosphorylated metabolites. Un-
fortunately, because most APases have been typically
viewed as being completely non-specific enzymes, few
investigators have assessed the V^a^ values, K^ values
and the related specificity constants (i.e. V^,/K™) of their
particular APase for different non-artificiai substrates of
possible physiological relevance. In many instances, pu-
tative physiological suhstrates are assayed only at a sin-
gle saturating concentration which makes meaningful ki-
netic comparisons between the different APases impos-
sible. Kinetic parameters such as pH activity range,
cofactor requirement, effect of metal ions, and substrate
specificity have a direct bearing on the assignment of
enzyme function, especially when the cellular location
and enzymatic responses to physiological modulators are
known. Kinetic parameters for several purified plant
APases are shown in Tab. 1. Many APases show sub-
stantial activity w ith key metabolites of intermediarj' me-
tabolism such as ATP, PP,, 3-PGA, and PEP. The K^
value of the APases for these substrates are frequently in
the ^iM range suggesting tbat these compounds could
serve as substrates for the APase in vivo.
Several metal cations are known to affect AFase activ-
ity in vitro, and the same substances can have markedly
different effects on APases isolated from different
sources. For example, divalent metal cations can be stim-
ulator)' (Christeller and Tolbert 1978, Gibson and Ullah
1988, Duff et a!. 1989a, 1991a, Sugiura et al. 1992),
inhibitory (Duff et al. 19B9a), or without effect (Duff et
al. 1991a). Competitive inhibition by P, and uncompet-
itive, noncompetitive or mixed competitive inbibition by
fluoride are also ob.served (Randall and Tolbert 1971,
Duff et al. 1991a). Potent competitive inhibition of plant
APases by other P, analogues such as arsenate, molyh-
date, ascorbate, tartrate and vanadate have also been
reported (Randail and Tolbert 1971, Duff et al. 1989a,
Duff et al. 1991a). Feedback inhibition of APa.ses by P,
may represent a general form of cellular regulation of
these enzymes.
The purple APases are readily distinguished from other
APases by their purple or red-vioiet color in solution,
which is due to the presence of a manganese or an
iron-zinc center (Beck et al. 1986, Sugiura et al. 1981,
LcBansky et al. 1992, Vincent et al. 1992). These en-
zymes are also distinguised by their immunity to inhib-
ition by tartrate, a potent inhibitor of most other APases.
It is speculated that the purple APases utilize the metal
anion to induce effective binding of the cationic substrate
794
and structural ,stability of tbe enzyme in an acidic envi-
ronment (Sugiura et al. 1981, Vincent et al. 1992).
Flant APases exhibit considerable heterogeneity with
regards to their native molecular mass, subunit structure,
and pi (Tab. 1), However, the majority of APases appear
exist as monomeric or dimeric glycoproteins having sub-
unit molecular masses of approximately 50 to 60 kDa
(Tab. 1). This suggests that there may be some structural
and evolutionary relatedness between the major plant
APases.
AFase isoforms from the same tissue or eel! type often
display substantial differences in their respective physical
and/or kinetic properties. For example, the cytoplasmic
APase from yam storage tubers exhibited maximal activ-
ity with phenyl-P, whereas the two membrane bound
isoforms from this tissue hydrolysed AMP preferentially
(Kamenan and Diopoh 1983). Likewise, the cell wall-
localized APase purified from B. nigra suspension cells
was a heat-stable non-specific enzyme that displayed
substantial pyrophosphatase activity, and was potently
inhibited by P;, molyhdate, or fiuoride (Duff et al. 1991a).
By contrast, the purified vacuolar AFase isoform of B.
nigra was relatively heat-labile, showed a high degree of
specificity for PEP. displayed no pyrophosphatase activ-
ity, and was far less susceptible to inhihition hy Fi, mo-
tybdate, or fluoride (Duff et al. 1989a). These different
properties between APases from the same cell probably
reflect differences in enzyme function.
Overview of the distribution, localization and
function of plant acid phosphatases
Acid phosphatases have been found in all species and
tissues that have been studied, and many electrophoret-
ically distinguishable isoforms are often present in a
given tissue or cell type. However, given the giycosylated
nature of most APases, the detection of several bands of
phosphatase activity upon non-denaturing gel electropho-
resis of a plant extract could reilect differences in the
extent of glycosylation of a parent APase molecule,
rather than the existence of isozymes encoded by distinct
genes. Differential glycosylation might represent a suhtle
form of cellular control, either in targeting phosphatases
to specific compartments, or in increasing their affinity
for specific proteins or enzymes.
Secreted acid phosphatases
Extracellular AFases appear to be ubiquitous in roots and
plant cell cultures. Extracellular APases may be localized
within the cell wall, and/or may be secreted by the root/
suspension cells into tbe surrounding rhizosphere/culture
media (Ueki and Sato J977, Lee 1988, Duff et al. 1991a,
Goldstein et al. 1989, Kaneko et al. 1990, Lefebvre et al.
1990, LeBansky et al. 1992, Miemyk 1992). The precise
physiological roles of plant extracellular AFase activity
remains to be ftrmly established. However, the low level
of soluble Pi in .soils (often ! jM or less) is in direct
Phy.siot Plant. 90. 1994
contrast to the large amounts of soil organic and insoluble
mineral P| (Goldstein et al. 1989). Thus, the extracellular
APa.se of roots, which is localized mainly in apical meris-
tems and outer surface cells, is undoubtedly involved in
hydrolysing and mobilizing F; from organic phosphates
in the soil for plant nutrition (Lee 1988, Lefebvre et al.
1990). Since root surfaces of many plants are known to
be acidified, an AFase (rather than alkaline phosphatase)
is obviously well suited for this region.
A physiological role for the secretory AFase of plant
suspension cell cultures is not evident since the cell wall
or culture medium does not normally contain phosphory-
lated compounds. It has been suggested, however, that
the extema! AFase of F, starved suspension cells may
function as part of a Fj "salvage system' wbich converts
any leaked esterified-F to F, which is readily reabsorbed
(Lefebvre et al. 1990).
Intracellular acid phosphatases
Intracellular plant AFases appear to be ubiquitous since
they have been found in dormant seeds (Park and Van
Etten 1986. Ching et al. 1987, Chung and Folya 1992),
developing seeds (F. E. Podesta and W. C. Plaxton, un-
published data), germinating .seeds (Nishimura and Beev-
ers 1978, Bhargava and Sachar 1987, Gibson and Ullah
1988, Ullah and Gibson 1988, Haraguchi et al. J990,
Biswas and Cundiff 1991), leaves (De Leo and Sacher
1970, Randall and Tolben 1971, Barret-Lennard et al.
1982, Pan 1987, Polya and Hunziker 1987), stems (Lo-
renc-Kubis 1989, Duff et al. 1991b), roots (Panara et al.
1990, Duff et al. 1991b), storage tubers (Stigiura et al.
1981, Kamenan and Diopoh 1983, Polya and Wettenhall
1992, Gellatly et al. 1993), flowers (Lai and Jaiswal
1988), fruit (Kanellis et al. 1989), and cultured cells (Paul
and Williamson 1987. Vogeli-Lange et al. 1989, Duff et
al. 1989a, 1991a). Based on the pH optima for activity of
between pH 5 and 6 for most AFases (Vincent et al.
1992), it has been traditionally assumed that the intracel-
lular APases are present mainly, if not exclusively, in the
cell vacuole (Nishimura and Beevers 1978). Indeed, both
histochemical and subcellular fractionation studies have
clearly confirmed the presence of AFase in the cell vacu-
ole, and there is strong evidence that all plant cell vacu-
oles contain APase activity (Nishimura and Beevers
1978, Boiler and Kende 1979, Vogeli-Lange et al. 1989,
Duff et al. 1991a). The existence of vacuoiar APase is
somewhat enigmatic, however, since nutrient sufficient
plants store 'excess" phosphorus in their vacuole in the
form of Pi (Theodorou and Plaxton 1993). Compart-
mentation of APase within the cell vacuole allows the
enzyme access only to those substrates that are permeable
to the tonoplast, while at the same time removing it from
the cytoplasm where uncontrolled catalysis could poten-
tially cause considerable cellular damage. Decompart-
mentation of the vacuoiar APase has been proposed to
provide a mechanism for P, mohilization from endoge-
nous phosphorylated compounds of senescent plant tis-
Physiol. Plam. W. t
sues (De Leo and Sacher 1970). Although tbe precise
metabolic function(s) of the vacuolar APase of non-se-
nescent, non-stressed, plant tissues remains to be eluci-
dated fully, they are undoubtedly involved with the rou-
tine utilization of Pi reserves or other P,-containing com-
pounds. Vacuolar APases may also play an important role
in the sequestration within the vacuole of compounds tbat
are potentially toxic or accumulate in the cell. For exam-
ple, exposure of plants to the herbicide glyphosate results
in a massive vacuolar accumulation of shikimate (Vogeli-
Lange et al. 1989). Glypbosate inhibits the enzyme 5-
enolpyruvylshikimate-3-P (EPSP) synthase, thus interfer-
ing with aromatic amino acid and phenylpropanoid bio-
synthesis. Studies with glyphosate-treated buckwheat
suspension cell cultures have revealed that shikimate-3-P,
the suhstrate of EPSP-synthase, appears to be transported
into the vacuole and bydrolysed within the vacuole, since
only the vacuolar APases possess the required specificity
(Vogeli-Lange et al. 1989). Suhceliular fractionation
studies have revealed that intracellular APases are not
confined to the vacuole, but may also exist in the cy-
toplasm (Kamenan and Diopoh 1983, Panara et al. 1990),
plastids (Randall and Tolben 1971, Christeller and Tol-
bert 1978, Baldy et al. 1989), and in the symbiosome
membrane of soybean root nodules (Sarath et al. 1993).
These findings indicate that an acidic pH optimum does
not preclude function of a phosphatase in a non-acidic
metaholic compartment.
Regulation of acid phosphatase eiqiression
The great diversity of plant APases in terms of their
biochemical properties, compartmentation, and tissue
specificity suggests a complex regulatory pattern of
APase getie expression. In common with many plant
proteins, regulation of APase expression is modulated by
a variety of developmental and environmental factors.
Developmental factors
Seed germination is one of the hest characterized devel-
opmental cues which results in a significant induction of
AFases including phytase (see section; Specialized acid
phosphatases). Although ungerminated seeds contain a
small amount of constitutive AFase activity, a large in-
crease in AFase activity (up to 30-fold) concomitant with
a decrease in seed organic phosphate reserves (i,e. phytic
acid) usually accompanies germination (Gibson and Ul-
lah 1988, Biswas and Cundiff 1991). Tbis large induction
appears to arise from gibberellic acid-regulated de novo
APase synthesis (Bhargava and Sachar 1987).
Other developmental processes that cause AFase in-
duction include flowering and senescence (De Leo and
Sacher 1970, Lai and Jaiswal 1988), greening of etiolated
seedlings (Christeller and Toblert 1978) and fruit ripening
(Kanellis et al. 1989). Abscisic acid has been reported to
accelerate the on.set and enhance Che magnitude of the
increase in APase activity which accompanies leaf
795
senescence (De Leo and Sacher 1970). Likewise, several
plastidic APases, including F-glycolate pbosphatase and
3-FGA pbosphatase (see section; Specialized acid phos-
phatases) that are associated with the attainment of photo-
synthetic competence are induced during chioroplast bio-
genesis (Randall and Tolbert 1971, Cbristeller and Tol-
bert 1978).
Enviroiunental factors
Environmental determinants that elicit increased expres-
sion of intra- and/or extracellular AFases include; (i) the
exposure of roots to divalent cations such as Ni'*, Ca-*,
and Mg=* (Gabbrielli et al. 1989); (ii) salt stress (Pan
1987); and (iii) water deficit stress (Barret-Lennard et al.
1982). However, since AFases are believed to be impor-
tant in the acquisition and recycling of Fi, APase activity
could be expected to be elevated in plants undergoing
nutritional F, limitation. Indeed, a plethora of reports
have clearfy documented how the intra- and/or extracellu-
lar AFase activities of pbotosynthetic and nonphotosyn-
thetic plant tissues and cell cultures respond dramatically
to cellular F^ status (Ueki and Sato 1977, Barret-Lennard
etal. 1982, Lee 1988, Duff etal. 1989b, 1991 b, Goldstein
et al. 1989, Lefebvre et al. 1990). Ueki and Sato (1977)
found tbat both the synthesis and secretion of an AFase
by cultured tobacco cells were regulated by F,. A F,-
starvation inducible AFase is also secreted by tomato
suspension cells and has been suggested to function ei-
ther as a F, transport agent or as a Fi-scavenging agent
acting on phosphorylated compounds of the culture
media (Goldstein et ai. 1989). Studies with B, nigra
suspension cultures have clearly documented de novo
synthesis of both a vacuolar PEP-specific AFase (Duff et
al. 1989a,b) and a cell wall-localized non-specific APa.se
(Duff etal. 1991a) in response to Pi deprivation. Quantifi-
cation of immunoblots indicated that in B, nigra suspen-
sion cells or intact roots experiencing a transition from P,
sufficiency to deficiency or vice versa, the amount of
total .^Pase protein was closely correlated with total
AFase activity (Duff et al. 199Jb). F; starvation-induced
increases in activities of botb B, nigra AFase isozymes
occurred simultaneously with a substantial induction of
the F, generating enzymes, PFrdependent phosphofructo-
kinase and PEP carboxylase (TTieodorou and Plaxton
1993, 1994), and an enhancement in cellular P-absorp-
tive capacity (Lefebvre et al. 1990). Parallel induction of
these processes suggests the existence of a higher plant
'Pi regulon' (i.e. a set of genes that are coregulated by Pi),
as has been demonstrated to exist in a variety of microor-
ganisms (Goldstein et ai. 1989).
The aforementioned studies underscore three impor-
tant features in the study of plant APases. Firstly, an
apparent common denominator for enhanced phosphatase
activities and expression is the need to reutilize Fi from
stored reserves during germination, growth or senes-
cence, and the need to recycle or scavenge Fi under
conditions of Pi stress. Constitutive intracellular AFases
796
might be involved in the hydrolysis of esterified F, from
specific substrates. Secondly, the numerous effectors of
AFase induction underscores tbe importance that a care-
ful examination of APases in a given cell or tissue type
must be undertaken under clearly defined conditions.
Thirdly, these data demonstrate our lack of knowledge of
the molecular events underlying APase induction.
Specialized acid phosphatases
A special class of intracellular plant AFases exists that
have a clear but non-absolute substrate specificity. These
APases are presumed to have more specialized metabolic
functions than those non-specific AFases that display
absolutely no substrate preference. Until more data is
available on the catalytic specificities of plant AFases it
will be impossible to conclusively determine if all of
these hydrolases serve specialized functions or if 'gen-
eral' non-specific intracellular plant APases actually ex-
ists.
Phytases
Most seeds contain significant amounts of phytic acid
(inositol hexaphosphate) which is hydrolysed to F, and
inositol during germination by one or more phytases.
Fhytic acid thus serves as an Important reserve of inositol
and F, for the growing seedling. Most seed phytases that
have been examined belong to a special class of APase
with optimal activity between pH 4.0 and 5.6 (Yamagata
et al. 1980, Gibson and Ullah 1988). Unlike other special-
ized APases. phytases are relatively non-specific en-
zymes that are able to hydrolyse a variety of natural and
synthetic P, esters with similar efficiencies. Those germi-
nating seed AFases that exhibit significant phytase activ-
ity can be considered to be a phytase on the basis that
pbytic acid is the most probable physiological substrate
in vivo. Gibson and Ullah (1988) purified a phytase from
germinating soybean cotyledons and separated it from a
non-specific APase that had no phytase activity. The
phytase had a relatively low V„,^^ with phytic acid as
substrate (Tab. 1) and was strongly inhibited by P,. The
enzyme was suggested to function as a slow release
mechanism of F; from phytic acid during soybean getini-
nation so that the developing seedling is supplied with a
constant amount of F..
Phosphoglycolate pbosphatase
In contrast to most plant AFases, P-giycolate phosphatase
is unusual in that it displays an absolute substrate speci-
ficity (Christeller and Tolbert 1978, Baldy et al. 1989).
This chloroplast-localized APase (optimal activity at pH
6.3) catalyzes the conversion of F-glycolate to glycolate,
a reaction necessary for photorespiration. F-glycolate
phosphatase activities can easily account for glycoiate
production during photorespiration and it appears to be
indirectly light-regulated by stromal fluctuations in pH,
Ptiysiut. Plain. «>. 1994
energy charge, and levels of Mg-* and NADFH (Baldy et
al. 1989).
3-PGA phosphatase
Randall and co-workers (1971) reported tbe presence of
3-FGA phosphatase in 52 diverse plant taxa, and the
enzyme from sugarcane leaves was extensively character-
ized (Randall and Tolbert 1971). This enzyme catalyzes
the hydrolysis of F; from 3-FGA and has proposed to
provide an alternate metabolic route from glycerate to
serine in place of photorespiration and the glycolate path-
way in leaves (Randall and Tolbert 1971). It has the
general characteristics of an AFase with 3-PGA being the
best, but not the only substrate hydrolysed in vitro. The
enzyme appears to be associated with both the stroma and
the thylakoid membranes of spinach chloroplasts (Ran-
dall et al. 1971). A starch-bound 3-PGA phosphatase is
also present in spinach leaves that has properties very
similar to the soluble form (Randall et al. 1971). The
function of this enzyme was proposed to be related to the
control of starch synthesis, since the pool of 3-PGA could
allosterically modulate the activity of the key regulatory
enzyme of starch biosynthesis, ADF-glucose pyrophos-
phorylase. Tbe 3-FGA phosphatase of C4 leaves appears
to be mainly localized in the cytosol of mesophyll cells
while phosphoglycolate phosphatase is primarily found
in the bundle sheath chloroplasts (Randall et al. 1971).
The role of 3-FGA phosphatase in the metabolism of C4
leaves is uncertain. One suggestion is that it facilitates
shuttling of 3-PGA from bundle sheath to mesophyll
cells. An alternative hypothesis is that, together with
glyoxylate reductase, it leads to serine synthesis in Cj
leaves (Randall et al. 1971).
Regulation of 3-FGA phosphatase may arise from the
enzyme's pH optimum for activity being in the range of
pH 5.6 to 6.0 (Randall and Tolbert 1971, Randall et al.
1971) and the fact that the pH of the chloropiast stroma
increases from about pH 7.0 to pH 8.0 following the dark
to light transition. 3-FGA pbosphatase has been shown to
be active in the hydrolysis of 3-FGA at neutral pH, but is
essentially inactive at pH 8,0 (Randall and Tolbert 1971).
Thus, diurnal fluctuations in pH, which depend on in-
cident light, may result in the regulation of this enzyme in
vivo.
Phosphoenolpyruvate phosphatase
Duff et al. (1989a) reported the purification and charac-
terization of a FEP phosphatase from heterotrophic B,
nigra suspension cells. Although its substrate specificity
was not absolute, tbis APase was designated a 'PEP
phospbatase' owing to a specificity constant (V,n^/K,,,)
for PEP tbat was at least 6-foid greater than tbe value
obtained for any other of 14 non-synthetic substrates that
were identified. Furthermore, the K^ (PEP) of the en-
zyme was well within estimated physiological concentra-
tions of tbis compound and is equivalent to Kn, (PEP)
Phy.siol. Planl. 90. i9<)4
values reported for various plant cytosolic pyruvate ki-
nases. These data suggested that PEP phospbatase could
potentially compete with cytosolic pyruvate kinase for a
common intracellular pool of PEP. Notably, B, nigra PEP
pbosphatase also demonstrated potent feedback inhib-
ition by P, (Duff et ai. 1989a), and its specific activity was
increased more than 10-fold following Pi-deprivation
(Duff et al. 1989b). Similar results have been reported for
the FEF phosphatase of a Fi-iimited green alga, Sele-
nastrum minutum (Theodorou and Flaxton 1993). It was
postulated (Duff et al. 1989a, Tbeodorou and Flaxton
1993) that; (i) the ADF-dependent cytosolic pyruvate
kinase is functionally eliminated from cellular metabo-
lism during severe F, stress (owing to > 10-fold reduc-
tions in intraceilular ADF levels), and (ii) FEF phospha-
tase functions as a F, starvation inducible bypass to the
cytosolic pyruvate kinase of B, nigra and S. minutum.
Although energetically wasteful, this adaptation would
allow Pi-deficient plants and algae to utilize the F, pro-
vided by adenylate catabolism, without impairing the
conversion of FEF to pyruvate. PEP pbosphatase may
also fulfil an additional role as a Fi-recychng system that
converts esterified phosphate to F, that would be rapidly
reassimilated into the metabolism of the F, deficient cells.
Subsequent studies suggested that B, nigra PEF phospha-
tase may function during Fi deprivation as one of a series
of P, starvation inducible 'bypasses' of P, or nucleotide
phosphate-dependent glycolytic enzymes (Duff et al.
1989b, Theodorou and Flaxton 1993, 1994). B, nigra
PEP phosphatase has been localized to the cell vacuole
(Duff et al. 1991a), a location consistent with the en-
zyme's acidic pH optimum (Duff et al. 1989a). The
'glycolytic bypass' theory for PEP phosphatase, there-
fore, requires that Pi-depleted B, nigra suspension cells
transport FEF into, and pyruvate out of, the cell vacuole.
As no data is presently available concerning this possibil-
ity, future studies must evaluate if the tonoplast of F,-
starved plant cells is permeable to FEF and pyruvate.
Phosphoprotein phosphatase
Frotein modification by the reversible covalent incorpo-
ration of Pi is a widespread phenomenon with important
ramifications for metabolic control in vivo. Conse-
quently, phosphoprotein phosphatases have been widely
implicated in the acute and long-term regulation of plant
and non-plant metabolism. Information on tbe properties
and roles of phosphoprotein phosphatases in plants is
relatively scarce. Recent studies have identified protein
phosphatases in higher plants that correspond to the se-
rine/threonine-specific type I and 2 protein pbosphatases
present in animal cells (MacKintosh et al. 1991). The
plant type 1 or 2 protein phosphatases appear to function
in the in vivo dephosphorylatioti/regulation of several
cytosolic enzymes including sucrose-6-P synthase, FEF
carhoxylase, and quinate debydrogenase (MacKintosh et
al. 1991). However, protein phosphatases 1 and 2 cannot
be classified as AFases as they exhibit an alkaline pH
797
 optima for activity. Nevertheless, there have been a va-
riety of recent reports describing the purification of plant
APases displaying a relatively high activity and specifici-
ty for phosphoamino acids and/or pbosphopeptides from
diverse sources including poppy seeds (Chung and Polya
1992), wheat seedlings (Cheng and Tao 1989), maize
seedlings (Jagiello et al. 1992), barely roots (Panara et al.
1990), beet leaves (Polya and Hunziker 1987), and potato
tubers (Folya and Wettenhall 1992, Gellatly et al. 1993).
Few plant APases active against O-phospho-L-serine
(F-Ser) or O-phospbo-L-threonine, and/or pbosphoseryl
or phosphothreonyl peptides have been isolated and char-
acterized (Fanara et al. 1990). However, based upon the
limited studies to date, APases that exhibit high activity
and specificity with O-phospho-L-tyrosine (F-Tyr) appear
to be relatively abundant in plant tissues (Folya and
Hunziker 1987, Cheng and Tao 1989, Fanara et al. 1990,
Chung and Folya 1992, Folya and Wettenhall 1992, Gel-
latly et al. 1993). This agrees with the observation that
phosphotyrosyl proteins are present in plant cells at levels
far greater (relative to phosphoseryl or phosphothreonyl
proteins) thatt are found in animal cells (as cited in Folya
and Wettenhall 1992). Most of the piant F-Tyr APases
that have been characterized thus far share several impor-
tant properties with various mammalian and yeast P-Tyr
phosphatases which appear to be involved in the regu-
lation of the cell cycle and cellular development. Similar
to the plant F-Tyr APases, non-plant P-Tyr phosphatases
frequently display an acidic pH optima, show potent
inhibition by vanadate, molybdate and Zn-*, and will
catalyze the dephosphorylation of pNPF as well as a
variety of other phosphomonoesters (Walton and Dixon
1993).
The major APase isoform from potato tubers was re-
cently purifted to homogeneity and a final P-Tyr hydro-
lysing specific activity of 1917 pmol P, produced (mg
protein)-' min"' (Gellatly et al. 1993) (to the best of our
knowledge this is the highest V^^, yet reported for a plant
APase; see Tab. 1). Although the potato APase also
catalyzed the dephosphorylation of other phosphomo-
noesters including pNPP, P-Ser, PEP, FPi, and ATF, the
enzyme's V^^, and specificity constant were significantly
higher with P-Tyr. Monospeciftc antibodies to P-Tyr were
used to identify polypeptides phosphoryiated on tyrosine
in potato tuber extracts (Gellatly et al. 1993). Although
most of these phosphoproteins could serve as substrates
for the purifted potato APase, a 20 kDa P-Tyr-protein was
preferentially dephosphorylated in vitro. These data sug-
gest that the predominant APase of potato tubers could
function in vivo as a P-Tyr protein phospbatase. Whether
potato APase plays an important role in the regulation of
shoot formation after tuber dormancy remains to be seen.
Although its abundance in tuber tissue might argue
against such a function, it may act in enzyme activation
and mobilization of nutrient reserves during this critical
period of high metabolic activity.
798
Concluding remarks
The most significant obstacle to the elucidation of the
roles of APases in plants has heen the identification of tbe
in vivo substrate(s) for tbe various intracellular AFase
isoforms. Altbough this problem also exists for other
enzymes such as protein kinases, phosphatases have an
immense pool of potential substrates. Theoretically, any
phosphorylated molecule could serve as a substrate, and a
given phospbatase might utilize several different sub-
strates in vivo. Thus, the precise determination of the in
vivo function(s) for plant APases is difficult and is com-
plicated by the following factors; (i) the heterogeneity of
APases within a given cell type rarely permits the puri-
fication and thorough characterization of all of the iso-
forms; (ii) subtle differences may exist in the kinetic
properties of the various APase isoforms for related me-
tabolites; (iii) there is a lack of kinetic data for non-
synthetic substrates for most of the APases that have been
examined; (iv) many APases are inducibie and the condi-
tions and mechanisms for their induction need to be fully
understood; and (v) few studies have localized these
enzymes at the subcellular level (Tab. 1).
Whereas many plant AFases detnonstrate significant
activities against substrates such as ATF or PP,, they
frequently demonstrate much less efficient hydrolysis of
substrates with relatively lower free energies of hydroly-
sis such as glycerol-2-P and phytic acid. It is Jikely that
the use of such compounds will reveal differences in the
specificities and activities of the various AFases. Thus,
we recommend that purified plant AFases be routinely
characterized with regards to their ability to catalyze the
hydrolysis of Fi from the following compounds; ATF. PP,,
glycerol-2-F, PEP, 3-FGA, phytic acid, F-Tyr, and the
synthetic substrate pNPF (in addition to other putative
physiological substrates that the researcher is specifically
interested in). It is also possible that APases may fall into
specific categories based on their inhibition by P, and F,
analogues, or activation by divalent metal cations. Hence,
the characterization of the inhibition and activation of
AFase activity hy F, and divalent metal cations, respec-
tively, should be carefully evaluated. Such an attempt to
catalogue these ubiquitous enzymes will be a ftrst step
towards their classification, and will hopefully serve as a
template for further refinement by other researchers.
Additional investigations on the immunologicaJ and
molecular characteristics of these enzymes as well as
their differential expression within and hetween different
plant tissues are also needed. These studies in conjunc-
tion with appropriate physiological and biochemical anal-
yses should yield additional insights about the cellular
functions of this important class of plant enzymes.
Acknowledgements - Tbis work was supported by grants from
Tbe University Research Council and tbe Center for Biotech-
nology, University of Nebraska-Lincoln to GS, and Tbe Natural
Sciences and Engineering Research Council of Canada
(NSERC) to WCP. SMGD was the recipient of an NSERC
Post-doctoral Fellowship. The critical reading of the manuscript
Physidl. Planl. W. 1994
by Drs. S. Madhavan, J. P. Markwell, and P. E. Staswick is
gratefully acknowledged.
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