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Plant Physiol. (1999) 119: 543-552
Apyrase Functions in Plant Phosphate Nutrition and Mobilizes
Phosphate from Extracellular ATP1
Collin Thomas,
Yu Sun,
Katie Naus,
Alan Lloyd, and
Stanley Roux*
Botany Department and the Institute for Cellular and Molecular
Biology, University of Texas, Austin, Texas 78713
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ABSTRACT |
ATP, which is present in the
extracellular matrix of multicellular organisms and in the
extracellular fluid of unicellular organisms, has been shown to
function as a signaling molecule in animals. The concentration of
extracellular ATP (xATP) is known to be functionally modulated in part
by ectoapyrases, membrane-associated proteins that cleave the  - and
 -phosphates on xATP. We present data showing a previously unreported
(to our knowledge) linkage between apyrase and phosphate transport. An
apyrase from pea (Pisum sativum) complements a yeast
(Saccharomyces cerevisiae) phosphate-transport mutant
and significantly increases the amount of phosphate taken up by
transgenic plants overexpressing the gene. The transgenic plants show
enhanced growth and augmented phosphate transport when the additional
phosphate is supplied as inorganic phosphate or as ATP. When scavenging
phosphate from xATP, apyrase mobilizes the -phosphate without
promoting the transport of the purine or the ribose.
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INTRODUCTION |
Apyrases are enzymes with the unifying characteristic of being
able to hydrolyze both the - and the -phosphate on ATP or ADP
(Plesner, 1995 ). Most apyrases are expressed as plasma
membrane-associated proteins with their hydrolytic activity facing into
the ECM (Wang and Guidotti, 1996). The pea (Pisum sativum)
apyrase used in this investigation, psNTP9, was originally
characterized as a 47-kD nuclear NTPase because it was initially
purified from nuclei (Chen et al., 1987), because it was shown to be
localized in the nucleus by immunocytochemistry (Tong et al., 1993),
and because it had potential nuclear-localization and DNA-binding
sequences (Hsieh et al., 1996). However, the fact that it also had a
signal peptide (Hsieh et al., 1996) led us to investigate whether some
fraction of the pea apyrase is extracellular, and the results shown
here indicate that it is.
Recent reports of ATP in the ECM of multicellular organisms (Sedaa et
al., 1990) and in the extracellular fluid of unicellular organisms (Boyum and Guidotti, 1997) have prompted investigations into
the fate of ATP outside of the cell. One conclusion is that xATP is
hydrolyzed by apyrases. The physiological relevance of xATP
degradation has been demonstrated in at least two systems, synaptic
xATP hydrolysis following nerve stimulation to inactivate xATP as
a neurotransmitter (Todorov et al., 1997), and xATP degradation during
thrombosis (Marcus et al., 1997). The products of ATP hydrolysis do not
accumulate in the extracellular fluid but are presumed to be recouped
by purine transporters. Work in animal systems has shown that adenosine
derived from xATP by the joint action of extracellular apyrases and
ecto-5 -nucleotidases may be transported into cells by a
sodium/adenosine cotransporter (Che et al., 1992). We present evidence
from a plant system that shows that extracellular apyrases have a
biological function in addition to xATP catabolism. We also
demonstrate that apyrase from pea augments the uptake of Pi, as well as
the Pi derived from xATP, without promoting the transport of the purine
or ribose.
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MATERIALS AND METHODS |
Expression, Growth, and Transport in Yeast
The yeast (Saccharomyces cerevisiae) mutant NS219 was
provided by Satoshi Harashima (Osaka University, Japan). The coding region of psNTP9, the pea (Pisum sativum) apyrase cDNA, was
subcloned into pYES2 (Invitrogen, Carlsbad, CA) downstream of the GAL1
promoter and was transformed into the mutant by a PEG lithium acetate
method (Elble, 1992). For time-course growth assays, single colonies of
transformants were grown in a low-Pi (100 µM)
synthetic-defined induction medium containing 2% Gal, 0.1% Glu, and
5% glycerol, and growth was monitored spectrophotometrically. Western
analysis was performed on 30 µg of total protein isolated from
saturated cultures grown in synthetic-defined induction medium using a
polyclonal antiapyrase antibody raised against the purified pea protein
(Tong et al., 1993). For growth assays in which the concentration of Pi
was varied, 10,000 cells from a log-phase culture were added
to an induction medium in which the Pi concentration had been adjusted.
After 48 h of growth, the turbidity of the cultures was analyzed
spectrophotometrically.
The acid-phosphatase assay was performed on cells grown in a high-Pi
(10 mM) synthetic-defined induction medium. Cells were grown to an A660 of 1.0, and 1 mL was
pelleted. The pellet was resuspended in an acetate buffer (pH 4.0)
containing the chromogenic substrate complex nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl-phosphate (KPL, Gaithersburg, MD).
Acid-phosphatase activity was monitored by the development of a blue
color. Time-course transport assays using 32Pi
(Amersham) were performed on cells grown to an
A660 of 1.0 in synthetic-defined induction
medium containing 100 µM Pi. For each sample, 1 mL of cells grown in synthetic-defined induction medium was pelleted
and resuspended in 250 µL of fresh synthetic-defined induction medium
containing 1 µCi 32Pi and 100 µM Pi. The transport reaction was stopped at
various times by the addition of 1 mL of ice-cold water, followed by
four washes in ice-cold water. Kinetic assays were performed in the same manner as the time course of transport, but the concentration of
Pi in the synthetic-defined induction medium was varied, and all
reactions were stopped 8 min after the addition of radioactivity. In
separate experiments transport reactions were performed in the presence
of 2% Glu for the duration of the reaction. Cells used for these
experiments were grown in the low-Glu induction medium until the time
of the experiment, because Glu was found to repress expression of
the psNTP9 gene under the control of the Gal promoter in pYES2.
The kinetic data were fitted using regression analysis.
Transgenic Plant Construction
psNTP9 was subcloned as a SalI to XbaI
fragment into pKYLX71 (Schardl et al., 1987) cut with XhoI
and XbaI. This plasmid was transformed into
Agrobacterium tumefaciens GV3101 [pMP90] (Knocz and
Schell, 1986), which was used to infect root calli from the Wassilewskija ecotype of Arabidopsis under kanamycin
selection (Valvekens et al., 1992). Four individual lines obtained from separate calli were propagated to the T3
generation.
Subcellular Apyrase Distribution in Pea
Etiolated pea plumules served as the tissue source for nuclei and
cytoplasm isolation, as described previously (Chen and Roux, 1986).
Plasma membrane was prepared from 30 g of root tissue. Western
analysis was performed on 15 to 30 µg of protein from the cytoplasm,
plasma membrane, and nuclei using a polyclonal antiapyrase antibody
raised against the purified pea protein (Tong et al., 1993). To
determine the orientation of the pea apyrase in the pea plasma
membrane, outside-out vesicles were prepared, and the accessibility of
the enzyme was determined by selective trypsin proteolysis or membrane
shaving, followed by activity assays and western blotting.
Apyrase Activity Measurement and Immunochemistry in Transgenic
Arabidopsis
Approximately 0.5 g of the total tissue from 3-week-old
plants was frozen and powdered. ECM material was then extracted by the
method of Barcelo et al. (1987). Apyrase activity was determined using
the phosphomolybdate assay (Chen and Roux, 1986). Western analysis was
performed on 20 µg of the total ECM protein using the pea apyrase
antibody. Immunoblots were developed with an alkaline phosphatase
substrate system.
Pi-Uptake Experiments and Growth Assays
In all experiments the growth medium contained no sugar, and
plants were grown in sterile culture at 22°C under 150 to 200 µE of
continuous light. Unless otherwise noted, a standard 0.8% agar medium
containing 100 µM Pi was used for uptake assays
(Somerville and Ogren, 1982). Plants used for the Pi-uptake experiments
were grown singly in 1 mL of the standard agar medium for 15 d
prior to the experiment. On the day of the experiment, 10 µCi
of Pi was applied to the side of the
culture dish and allowed to diffuse through the agar. In kinetic
studies additional Pi was added with the 32P to
the final concentration specified. The lids of the tissue-culture dishes were removed to facilitate transpiration. After 18 h the plants were removed from the medium. The aerial portions of the plant
not in contact with the agar were weighed and counted by liquid
scintillation. For each plant the entire root system was carefully
pulled from the agar and washed in ice-cold water prior to
scintillation counting. For kinetic analysis the data were fitted using
linear regression. In experiments involving uptake from radioactive
adenyl phosphates, 0.8% agar Murashige and Skoog basal salt medium
(Sigma) was used. The procedures were the same as those used for Pi
uptake; however, only the aerial portions of the plants were counted.
In growth assays involving the response of plants to Pi, the standard
0.8% agar medium was used (Somerville and Ogren, 1982), with
appropriate modifications made to the potassium phosphate concentration. Wild-type Arabidopsis (ecotype Wassilewskija) and transgenics were plated on 10 mL of this medium. In growth experiments involving nucleotides, Murashige and Skoog agar medium was used. adenosine nucleotide phosphates were spread onto the medium to a final
concentration of 300 µM; for Pi treatments, the final concentration was 1.3 mM (1 mM in the Murashige
and Skoog basal salt mixture plus 300 µM in the
supplement). Leaf-area assays were performed on an automated imager
(Alpha Imager 2000, Alpha Innotech, San Leandro, CA) and were
calculated as the sum of pixel areas for 400 to 600 plants per
treatment.
Measurement of ATP in a Defined Soil
For measurements of soil ATP, we used uncharged Sunshine Mix 2 potting soil (Hummert, Earth City, MO). The soil was hydrated to 4 times its dry weight and then autoclaved for 0.5 h. When the soil
had cooled to room temperature, it was divided into pots packed with
30 g of sterile soil. One-half of the pots were inoculated with
10,000 colony-forming units of a stock soil flora derived from a single
field sample at the University of Texas (Austin), and the other half
were not inoculated. The pots were then wrapped in plastic, sealed, and
placed in darkness at 37°C for 7 d. After 7 d a 15-g sample
was removed from each pot and pressed in a syringe until 1 mL of soil
fluid was collected. A dilution of this fluid was plated on
Luria-Bertani medium and grown for 12 h at 37°C, and the
colonies were then counted. The remaining fluid sample was centrifuged
for 60 s to pellet soil debris and was then filtered through a
0.2-µm filter. This filtrate was used as the source for the firefly
luciferase assay.
Luminometry was performed in triplicate on 30 µL of each sample
reconstituted in 70 µL of firefly luciferase buffer (Firelight, Analytical Luminescence Laboratory, Cockeysville, MD) After the buffer
was added, all samples were kept on ice. ATP standards (Sigma) were
reconstituted in firefly luciferase buffer with 30% soil water from an
uninoculated sample to account for the inhibitory effects of humic acid
on the luciferase enzyme. Standards and sample were loaded into a
96-well plate and read on an automated luminometer (model MLX, Dynex
Technologies, Chantilly, VA). Samples were processed with the addition
of 50 µL of firefly luciferase, followed by a reading delay of
1.0 s and an integration time of 5 s. Output was taken as an
average of the integration time and was then averaged for the
triplicate. The sample handling time was less than 2 h.
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RESULTS |
Pea Apyrase Complements a Yeast Pi-Transport Mutant
Initial observations of heightened Pi transport by transgenic
plants expressing apyrase suggested that apyrase might be involved in
Pi uptake. To test the ability of an apyrase to function in Pi
nutrition, we expressed a previously characterized pea NTPase gene
psNTP9 (Hsieh et al., 1996), a member of the apyrase family (Handa and
Guidotti, 1996), in a yeast Pho84 mutant (NS219) deficient in a Pi transporter (Bun-ya et al., 1991). The mutant showed reduced Pi
acquisition and a decreased growth rate, and it constitutively expressed the Pi-repressible acid phosphatase because it could not
accumulate Pi. Complementation with a functionally homologous gene
resulted in increased growth, increased Pi transport, and repression of
the acid phosphatase (Harrison and van Buuren, 1995). Based on a survey
of the yeast genomic sequences, yeast lacks membrane-associated
apyrases resembling psNTP9. Since the yeast system is naive to this
apyrase, complementation of the NS219 mutant could be used to determine
whether psNTP9 could function in Pi transport.
Three independent tests indicated that the apyrase gene successfully
complemented the PHO84 mutant phenotype: growth assays (Fig.
1, A and B), repression of acid
phosphatase activity (Fig. 1C), and labeled Pi transport (Fig.
2). The psNTP9 gene significantly improved the growth of NS219 cells (Fig. 1A) that overexpressed the
apyrase (Fig. 1A, inset). Furthermore, the growth benefits conferred to
the mutant by apyrase extended to Pi concentrations as high as 2 mM (Fig. 1B). When cells were incubated under
acid conditions in nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate, mutant cells with
high acid phosphatase activity stained dark, and wild-type cells and
complemented cells stained light. The NS219 cells expressing the pea
apyrase gene had less acid phosphatase activity than a mock-transformed
control (Fig. 1C).
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| Figure 1.
psNTP9 complements a Pi-uptake mutant. A, pYES2
vector alone and pYES2/psNTP9 were transformed into NS219. Single
colonies were picked and grown in low-Pi medium. Values represent the
averages of two independent transformants. Inset shows a western blot
of the psNTP9 transformant and a control (vector alone). B, Growth of
the mutant transformed with the pea apyrase and a mock transformant at
five Pi concentrations. Values are the averages of two independent
transformants for two separate experiments. Cultures were inoculated
with 104 yeast cells from a log-phase culture. C,
Acid-phosphatase activity in NS219 yeast: two independent NS219
transformants harboring the vector alone show the dark staining of the
mutant, whereas two independent NS219 transformants carrying the pea
apyrase gene psNTP9 show the light staining of the wild type.
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| Figure 2.
Pi uptake by NS219 in the presence and absence of
the psNTP9 gene. A, Two independent pYES2/psNTP9 transformants were
tested. Values reported are in nanomoles per milliliter and are the
averages of duplicate samples. B, Lineweaver-Burk plot of Pi uptake
versus the Pi concentration of the culture medium. Linear-regression
analysis was used to fit the data and a first-order polynomial
describing the line of best fit was used to approximate the
Km.
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Although the pea gene partially restored many wild-type properties to
the PHO84 mutant, pleiotropic effects of apyrases, such as
increased turnover of cellular phosphate pools or changes in Pi
metabolism resulting from feedback effects, could also explain the
reversion. Complementation was also confirmed directly by testing Pi
uptake in NS219 cells expressing psNTP9 (Fig. 2A). Cells expressing the
pea apyrase gene followed Michaelis-Menten kinetics and had an
estimated Km of 24 µM in the induction medium containing Gal (Fig.
2B). When 2% Glu was substituted for Gal for the duration of the
transport assay, the Km was estimated at 14 µM.
Detection of the Pea Apyrase in Nuclei and in Purified Plasma
Membrane
An immunoblot assay showed that pea apyrase was associated with
nuclei and with purified plasma membranes but not with the cytoplasm
(Fig. 3A). Protease treatment destroyed
both apyrase activity and antigenicity in outside-out plasma membrane
vesicles. After trypsin treatment the exterior face of the vesicle
showed 30% of the ecto-ATPase activity of the untreated sample.
Endo-ATPase activities were retained after trypsin treatment,
indicating that the digestion occurred exclusively on the exterior face
of the membrane.
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| Figure 3.
Expression of apyrase in pea and in transgenic
lines. A, Immunoblot analysis of subcellular fractions from etiolated
pea plants. Lane 1, Cytoplasm; lane 2, purified plasma membrane; lane
3, purified nuclei; and lane 4, preimmune control of nuclei. B, Top,
The total Pi accumulated in the shoots of the wild type (Wt) and three
independent transformants in an 18-h 32P-uptake assay
tested at 2 mM Pi. Bottom, A corresponding immunoblot
performed on equal amounts of protein isolated from the ECM of
3-week-old wild-type Arabidopsis and the psNTP9 transgenics. C, Assay
of ATPase activity in the ECM fraction of OE1.
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Enhanced Growth of Plants Overexpressing Apyrase Correlates with
Increased Pi Uptake
Our findings in yeast corroborated the observations made with
transgenic Arabidopsis plants. Three of the four transgenic plant lines
constitutively expressed psNTP9 under the control of the cauliflower
mosaic virus 35S promoter, and over an 18-h period showed 2 to 5 times
as much Pi accumulation in shoots as the wild type (Fig. 3B).
Apyrase-expressing plants also showed 4 times as much ATPase activity
in the ECM as the wild type (Fig. 3C). Some percentage of the apyrase
can be found in crude fractions of proteins ionically extracted from
wall fragments in pea epicotyls (data not shown) and in Arabidopsis
seedlings. Because this fraction could be more conveniently prepared
than a plasma membrane fraction from Arabidopsis, it was used to assay
the level of ECM apyrase expression in the transgenic Arabidopsis
plants. Using an antibody raised against the pea apyrase (Tong et al.,
1993), we detected a high level of the 47-kD gene product in the
extracellular fraction of three transgenic lines compared with that
detectable in immunoblots in the lane loaded with the same protein
quantity from wild-type plants (Fig. 3B, bottom).
Kinetic studies of the transgenic and wild-type plants showed large
differences in the relative rate of Pi accumulation in the low-affinity
range. On a per-root basis the average Vmax
was 77.51 pmol Pi h 1 for wild-type plants and
932 pmol Pi h1 for the transgenic plants.
Whereas the roots of transgenic plants accumulated Pi at 12 times the
rate of the wild type (Fig. 4A), these
differences applied to only plants grown in high-Pi medium, because the
affinity for Pi in the transgenic plants was substantially reduced. The
Km of wild-type and transgenic plants was
625 and 3770 µM, respectively. In contrast, the
shoots of both transgenic and wild-type plants retained the same
relative affinity for Pi at high concentrations, but the transgenics
accumulated Pi at about 3 times the rate of the wild type, with a
Vmax of 181 and 50 pmol Pi
mg1 h1 (Fig. 4B).
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| Figure 4.
Kinetic analysis of Pi uptake into wild-type (Wt)
and transgenic Arabidopsis plants. A, Lineweaver-Burk plot of Pi uptake
into roots. Values on the x axis are the averages of Pi
accumulated per minute for three transgenic and three wild-type roots
at each of the five Pi concentrations tested. Linear-regression
analysis was used to fit the data, and a first-order polynomial
describing the line of best fit was used to approximate the
Km. B, Lineweaver-Burk plot of Pi uptake
into shoots. The data were acquired and analyzed in the same
manner as for the root kinetics but were
normalized for weight because of the differences in shoot size. C, To
permit comparisons between the uptake of shoots and roots, the total Pi
accumulation during the 18-h experiment is presented for each tissue.
For each concentration the average uptake of the three plants is
presented.
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A comparison of the total Pi transported into the entire root or shoot
over a range of high Pi concentrations revealed that in both roots and
shoots the transgenic plant accumulated an average of 3 times as much
Pi as the wild type (Fig. 4C). The most dramatic difference in
accumulation between transgenic and wild-type plants was at 2 mM Pi. At this concentration the apyrase-expressing plants accumulated 9 times as much Pi in the root and 3.6 times as much in the
shoot compared with the wild type. When partitioning of Pi between root
and shoot in the transgenic and wild type was compared, both wild-type
and transgenic plants tended to accumulate more Pi in the shoot at
higher concentrations of Pi. At 100 µM external Pi, 63%
of the total Pi transported into the transgenic plants was in the
shoot. In wild-type plants given the same concentration of exogenous
Pi, 53% of the total Pi transported into the plant was in the shoot.
At 10 mM Pi, 87% of the total Pi was found in the shoots
of transgenic plants and 92% in the shoots of wild-type plants.
We examined the transgenic plants for phenotypes that might relate to
the presence of the apyrase gene and found that those grown in high Pi
were significantly larger than wild-type plants (Fig.
5A). Differences in growth became
statistically significant when the Pi concentration was approximately 2 mM (Fig. 5B), at which point the average leaf area of the
transgenic plants was 3 times that of the wild type. The growth
differences between transgenic and wild-type plants at 2 mM
Pi was correlated with the increased Pi uptake of transgenics at this
same concentration (Fig. 4C). The growth and transport phenotypes were
visible only in media depleted of sugar.
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| Figure 5.
A, Representative 20-d-old wild-type (Wt) and
transgenic Arabidopsis plants grown in 2 mM Pi. B, Leaf
area of transgenics compared with wild type. Although there was a trend
for increased growth with increased Pi, the difference was only
statistically significant at 2 mM. Data were analyzed for
significance in a Student's t test
(n = 18; *P < 0.05; error bars = SE).
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Transgenic Plants Can Utilize Pi Supplied as ATP
Since apyrases seem to function in the breakdown of xATP, we
decided to test the ability of root-fed adenosine nucleotide phosphates
to substitute for additional Pi. The basal Pi concentration, a
growth-sustaining concentration at which there was no statistically significant difference between the growth of wild-type and transgenic plants, was 1 mM. Transgenic and wild-type plants were fed
an additional 0.3 mM ATP, ADP, or AMP, and growth was
measured using total mass as an indicator. Transgenic plants on ATP
showed enhanced growth (Fig. 6A). The
xATP-fed transgenic plants had an average wet weight double that of the
wild type, a growth stimulation nearly equivalent to that observed from
the addition of free Pi (Fig. 6B). Whereas xATP had dramatic effects on
growth, xADP and xAMP did not. In contrast to the transgenic plants,
none of the treatments, including additional Pi, significantly affected
wild-type growth, as determined by Student's t test.
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| Figure 6.
Growth of wild-type (Wt) versus transgenic plants
expressing psNTP9 in the presence of exogenous nucleoside phosphates.
A, Representative 3-week-old Arabidopsis plants grown with three
different treatments compared with Murashige and Skoog medium (MS)
alone. B, Plants from the treatments were grown for 17 d and then
weighed. All treatments were cross-analyzed for significance in a
Student's t test. Bars marked by an asterisk differ
significantly from those without an asterisk (n > 20 for all groups; P < 0.05; error bars = SE).
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Transgenic Plants Preferentially Transport the -Phosphate of ATP
The inability of apyrase to translocate xAMP was demonstrated by
the low level of radiolabel accumulated in transgenic plants fed
[2,8-3H]ATP and
[ -32P]ATP (Fig.
7). However, in feeding experiments in
which the -phosphate was labeled, transgenic plants accumulated 3 times as much as the wild type. In separate experiments nonradioactive
xATP was able to competitively inhibit the uptake of
-32P from xATP.
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| Figure 7.
Transport of the products of ATP hydrolysis by
transgenic and wild-type (Wt) plants. [2,8-3H]ATP,
[-32P]ATP, and [-32P]ATP were fed to
15-d-old plants in separate treatments. All treatments were analyzed
for significance in a Student's t test
(n > 4-6 for all groups; *P < 0.05; error
bars = SE).
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Soil ATP
Soil treated with microbes showed increased microbial growth and
an accumulation of ATP after 7 d, whereas those that were not
inoculated accumulated far less ATP. In both instances the presence of
ATP was correlated positively with the number of colony-forming units
(Fig. 8).
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| Figure 8.
Soil ATP measurement with respect to microbial
count (C.F.U. = colony-forming units). Bars are average ATP
concentrations in soil water taken from three to five pots.
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DISCUSSION |
The growing evidence for xATP in animal systems has cast
ectoapyrases as likely regulators of extracellular energy charge. Although this role has functional significance for purinergic receptor
signaling, it may not be the exclusive function of apyrases. Experiments using pea apyrase in both yeast and transgenic Arabidopsis have revealed a new function for these enzymes. Our evidence from the
use of this gene shows that plant apyrases, like their animal counterparts, are located in the ECM and hydrolyze xATP. More importantly, these experiments suggest that apyrases play a role in the
mobilization of Pi and phosphate derived from xATP.
Many apyrases are ectoapyrases, which means that they are anchored in
the plasma membrane with their active site facing into the ECM
(Komoszynski and Wojtczak, 1996; Wang and Guidotti, 1996). Our results
indicate that a significant fraction of the protein encoded by psNTP9
can also be classified as ectoapyrase. As judged by immunoblots, highly
purified plasma membranes from pea roots contain the apyrase oriented
with its activity accessible to proteolytic removal. These results are
consistent with the interpretation that, like most of the animal
apyrases characterized thus far, the hydrophobic signal peptide at the
N terminus of the pea apyrase specifies a transmembrane domain and
orients the enzyme in the plasma membrane so that its activity is in
the ECM. Although animal ectoapyrases are considered to be localized
primarily in the plasma membrane, in some systems a fraction of the
total apyrase population is found free in the ECM (Todorov et al.,
1997). This observation led us to determine whether a similar situation
exists in plants. The results shown in Figure 3B indicate that
transgenic Arabidopsis plants had the 47-kD apyrase in the ECM, whereas
the wild type did not. Although it is possible that the extracellular
apyrase may be secreted, an alternative explanation could be that some fraction of the plasma membrane-anchored protein may be proteolytically removed from the plasma membrane and released into the wall.
Our data indicate that the psNTP9 gene product can function in Pi
nutrition in two heterologous systems. In yeast the apyrase promotes
growth of the NS219 mutant at Pi concentrations up to 2 mM,
above which point the low-affinity system probably masks the apyrase.
This finding is consistent with PHO84 gene-transport activities and growth assays originally reported. In the initial characterization of the yeast PHO mutants (Toh-E and Oshima, 1974; Bun-ya et al., 1991), experiments were performed in two standard Pi-containing media: one at 10 mM (low-affinity)
and one at 0.1 mM (high-affinity). Although pea
apyrase does not have the multiple membrane-spanning regions of
plant Pi transporters recently cloned (Muchal et al., 1996;
Leggecie et al., 1997), the pea gene for psNTP9 complements NS219,
giving the mutant a Km in the high-affinity range.
It is possible that apyrase itself may oligomerize to form a pore in
the membrane. Recent work with the mammalian homolog of psNTP9
indicated that this ectoapyrase tetramerizes in the membrane to form a
structure reminiscent of several channels (Wang et al., 1998).
Alternatively, apyrase could interact with a transport system that
functions as a complex. A similar scenario has been suggested by others
to explain the partial complementation of high-affinity mutants with
low-affinity transporters (Leggecie et al., 1997). Indeed,
extracellular proteins are postulated to bind Pi as part of a
Pi-transport system (Mass et al., 1979).
Although the complementation of NS219 is for a high-affinity
transporter, growth differences in the complemented mutant persist at
external Pi concentrations as high as 2 mM. Similarly,
significant differential growth is seen at Pi levels near or at 2 mM in plants, the concentration at which apyrase-expressing
plants accumulated 9 times as much Pi in the root and 3.6 times as much
in the shoot compared with wild type. However, the accumulation of Pi
in the transgenic Arabidopsis plants is unlike that seen in
pho2, a shoot Pi-hyperaccumulator mutant (Delhaize and
Randall, 1995), because Pi intoxication is not seen. The phenotype of
the transgenics is also different from what might be expected of a
plant overexpressing the gene at the pho1 locus. The pho1
mutant is defective in the ability to transfer Pi from the roots to the
xylem (Poirier et al., 1991).
Plants ectopically expressing the pea apyrase show increased Pi
accumulation in roots and shoots. Apyrase functions in a transport system that is common to the root and xylem. It also seems likely that
elements of this shared system are present in yeast, given the ability
of apyrase to complement NS219. The differences in the kinetics of
transport may be a function of the transport complex stoichiometry. In
such a scenario, increased apyrase levels may modulate the affinity of
a pre-existing low-affinity system by channeling Pi or phosphate
derived from ATP. In the transgenics the dependency of growth and
transport phenotypes on Suc-depleted medium suggests that the Pi
complex in which apyrase participates is tightly linked to
photosynthate availability.
Physiologically, the expression of an extracellular enzyme that can
hydrolyze only - and -phosphates on nucleotides is difficult to
explain, given the abundance of other substrate-versatile phosphatases. Indeed, both plants and yeast have evolved highly effective means of
scavenging organophosphate using acid and alkaline phosphatases (Reid
and Bieleski, 1970; Yoshida et al., 1989). Why are apyrases needed at
all? The immediate answer may be that apyrases, with an ATPase
kcatKm of
108 (Handa and Guidotti, 1996), simply hydrolyze
xATP more quickly than any other enzyme in the ECM.
In animals ATP is released into the ECM in a regulated manner by
ATP-binding cassette transporters (Abraham et al., 1993; Reisin
et al., 1994). The efflux of ATP is thought to be a way of regulating
the intracellular adenylate pool involved in signaling, and this xATP
would need to be hydrolyzed in part because xATP itself is a powerful
elicitor of intracellular calcium signaling (Zheng et al., 1991; Suko
et al., 1997). If xATP is also an elicitor in plants, apyrases may
provide a governable way of inactivating xATP as a signal, an
inactivation analogous to that discovered in the nervous system
(Todorov et al., 1997).
We propose a role for apyrase in addition to quenching xATP signals.
Apyrase may function constitutively to recoup Pi from xATP whether the
xATP accumulates in the soil as it is released from various cellular
sources or, as has been shown in animals, it accumulates in the ECM
when cells release ATP through multidrug resistance proteins or the
cystic fibrosis transmembrane conductance regulator (Abraham et al.,
1993; Reisin et al., 1994). The mdr1 gene is a member of the
ATP-binding cassette family and has been reported to promote the
electrogenic transport of ATP across the plasma membrane into the ECM.
Since a homolog of mdr1 has been found in plants (Dudler and
Hertig, 1992), we hypothesize that it might have ATP-transport
properties similar to channels found in animals.
Given the high catalytic efficiency of apyrases, and the fact that
their ability to generate Pi is limited only by the diffusion of ATP,
we speculate that apyrases could create a microenvironment of
recoverable Pi in the area proximal to MDR1. Estimations of the
kinetics of ATP efflux from cells overexpressing MDR1 are approximately
4 × 106 molecules
cell1 s 1 (Abraham et
al., 1993), but this xATP does not accumulate. In animals a
steady-state level (in femtomoles/cell) of xATP is achieved rapidly
through the action of apyrase and other phosphatases. Although there is
a functional linkage between this apyrase activity and nucleoside
transport in eukaryotes (Che et al., 1992), the fate of the hydrolyzed
Pi has yet to be reported. We believe that apyrase itself may provide a
means of immediately and directly recovering Pi from xATP.
The pea apyrase seems to mobilize the -phosphate of ATP
preferentially. This is probably because the ADPase activity of the pea
apyrase is only 15% of that of the ATPase activity (Chen and Roux,
1986). The lack of responsiveness of the transgenics treated with xAMP
is most likely due to the inability of apyrases to hydrolyze nucleotide
monophosphates (Plesner, 1995).
Although pea apyrase does play a role in the transport of Pi, it does
not facilitate the transport of the other products of ATP hydrolysis
(Fig. 7). Increased extracellular apyrase activity does not result in a
concomitant increase in adenylate transport, indicating that the
regulation of purine uptake is independent of xATPases and Pi uptake.
This exclusion could be physiologically relevant to regulating the size
of the adenylate pool.
In plants considerations of scavenging xATP could also include
environmental ATP. As much as 80% of the phosphorus in soil is in
organic form (Schachtman et al., 1998), and much of this is thought to
be unavailable to plants. Although soil flora may be in competition
with plants for Pi, it seems more likely that soil flora would be the
point of origin for most Pi bound in extracellular adenylate
nucleotides. It has long been known that Escherichia coli
secretes 99.9% of its cAMP into the extracellular fluid (Matin and
Matin, 1982). Yeasts also release significant amounts of
ATP into the extracellular fluid (Boyum and Guidotti, 1997). We
speculate that apyrases could make the organic phosphate released by
microbes in the rhizosphere available to plants.
Measurements of ATP in soil have largely focused on using the
nucleotide as an indicator for soil biomass (Brookes and Jenkinson, 1989). These measurements place priority on the ratio of ATP to carbon
biomass, a ratio that is typically 10 µmol of ATP
g1 carbon biomass (De Nobili et al., 1996). The
procedures required for such measurements require sonication of samples
prior to processing. Furthermore, most samples used for such
measurements are collected from the field and have a clay content
capable of chelating ATP. Although we acknowledge the benefits of
current methods of correlating biomass with ATP, we would like to
suggest that our estimation of aqueous ATP, the ATP fraction in soil
water, may be representative of the amount of ATP available to roots in
a soil rich in organic matter (Fig. 8; even though this is only a very
conservative approximation of the ATP that might actually be present in
the mucilaginous root-soil interface). The ATP in the soil water may
originate from dead cells, efflux, or phage activity. However,
regardless of the source, this ATP could be used as a Pi source by
plants, in which case apyrase may function to mobilize it. Future
experiments will help to delineate which xATP pools suggested in this
report are physiologically relevant to apyrase and Pi transport.
| |
FOOTNOTES |
1
This work was supported by grants from the
National Science Foundation and the National Aeronautics and
Space Administration and by a National Science Foundation graduate
fellowship to C.T.
*
Corresponding author; e-mail sroux{at}uts.cc.utexas.edu; fax
1-512-471-3878.
Received August 28, 1998;
accepted October 26, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ECM, extracellular matrix.
NTP, nucleotide
triphosphate.
xATP, extracellular ATP.
| |
ACKNOWLEDGMENTS |
We thank Dr. G. Thompson, A. Rajagopal, and B. Windsor for
valuable suggestions that aided in the preparation of this manuscript, and Dr. M. Harrison, who helped us to obtain the yeast strains.
| |
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Top
Abstract
Introduction