1 Rothamsted Research, West Common, Harpenden, Herts, AL5 2JQ, UK
2 Research Centre for Eco-environmental Sciences, The Chinese Academy of
Sciences, Beijing 10085, China
3 Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am
Mühlenberg 1, D-14476 Golm, Germany
* Author for correspondence (e-mail: ruth.gordon-weeks{at}bbsrc.ac.uk)
Accepted 1 April 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Phosphate transporter, Potato root, Immunolocalisation, Plasma membrane, Polar localisation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies of the regulation and tissue and cellular distribution of members
of the Pht1 family are commencing and this has indicated that many of the
genes are expressed in the roots in response to P deprivation. In some cases
transcripts or proteins have been localised to the root epidermis and even
root hairs, strongly suggesting a role in Pi capture and uptake
(Chiou et al., 2001;
Daram et al., 1998
;
Karthikeyan et al., 2002
;
Mudge et al., 2002
;
Liu et al., 1998a
). The
detection of transporters that are more widely distributed throughout plant
tissues and that are less tightly regulated by P depletion provides evidence
that some members of the Pht1 family may be involved in the internal
distribution of Pi (Daram et al.,
1998
; Leggewie et al.,
1997
; Liu et al.,
1998a
; Mudge et al.,
2002
; Rosewarne et al.,
1999
). Such transporters may be responsible for loading or
unloading from the xylem or phloem, deposition into the seed and storage
tissue or remobilisation of Pi from senescing leaves. Two Arabidopsis
mutants, pho1 and pho2, exhibit defects in xylem or phloem
loading that may be due to mutations in the Pi transport system. pho1
lacks the ability to load Pi into the xylem, causing shoot Pi concentrations
to be reduced (Poirier et al.,
1991
). However, the gene involved, PHO1, may be
regulatory, rather than directly involved in Pi efflux from stellar cells
(Hamburger et al., 2002
).
pho2 accumulates high levels of Pi in leaves even during P
deprivation. A phloem loading defect has been proposed
(Dong et al., 1998
), but shoot
cell regulation of internal Pi concentrations may be responsible.
In addition to the high-affinity system, Pht2;1, a member of the Pht2
low-affinity transporter family (Km for Pi, 0.4 mM), has
been identified in Arabidopsis
(Daram et al., 1999). Although
this protein is structurally related to members of a mammalian Na/Pi
transporter family, its activity in a yeast expression system is dependent on
the proton gradient across the yeast plasma membrane but independent of the
sodium gradient. The mammalian counterpart is present in many cell types and
is believed to be responsible for the absorption of Pi for normal cellular
function. Pht2;1 is constitutively expressed in green tissue, suggesting that
it possesses a comparable `house keeping' function, but it has recently been
shown, by green fluorescent protein (GFP) promoter fusion analysis, to be
associated with the chloroplast and to have a very specific transport function
(Versaw and Harrison, 2002
).
The subcellular distribution of members of the Pht1 family has so far
exclusively indicated localisation at the plasma membrane
(Chiou et al., 2001
;
Muchhal and Raghothama,
1999
).
In potato, three high-affinity transporters, StPT1, StPT2 and StPT3, have
been cloned and sequenced (Leggewie et
al., 1997; Rausch et al.,
2001
). Northern blot analysis has shown that StPT1 mRNA is present
not only in root tissue but also to a lesser extent in the source leaves,
flowers and tubers. Although amounts of StPT1 mRNA in the root increase in
response to P depletion, a low level of constitutive expression can be
detected throughout the plant. StPT2 mRNA, however, is only detected by
northern blots in roots after P depletion and StPT3 is only present in roots
colonised by mycorrhiza (Rausch et al.,
2001
).
In the present work, we have used an antibody to a unique sequence from StPT2 that is not present in StPT1 or StPT3, or any other known plant protein, to study the tissue, cellular and subcellular distribution of the transporter in both hydroponically and quartz grown plants. Our findings suggest that StPT2 is expressed most strongly near the root tip in the zone of elongation behind the meristematic region and in the root hairs. Immunolocalisation using confocal microscopy shows that in those cells that express StPT2, the transporter is present only in the apical plasma membrane.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of antisera and immunoglobulin fractions
Two peptides, VEIESEEAKIEQISRDETC, corresponding to a nonconserved region
from the central loop of StPT2, and CLIEEEEGIND, from a conserved region of
the vacuolar pyrophosphatase (VPPase), were synthesised by Sigma Genosys
(Pampisford, UK). The peptides (5 mg) were conjugated to 5 mg of maleimide
activated keyhole limpet haemocyanin or bovine serum albumen (Sigma Genosys)
in 1 ml of deionised water overnight at room temperature and purified on a
sephadex G25 column. Rabbits were immunised with 1 mg of conjugate with 0.5 ml
Freunds complete adjuvant, followed by a boost one month later. After a
further 2 weeks, repeated at 3 weekly intervals, bleeds were collected and IgG
was purified from the sera using an Econo-Pac serum IgG purification kit
(Bio-Rad, Hemel Hempstead, UK) according to the manufacturer's instructions.
IgG was prepared in the same manner from pre-immune sera. The VPPase antibody
detected a protein band on western blots with an apparent molecular weight of
66 kDa in tonoplast vesicle preparations from wheat and mung bean (R.G.-W.,
unpublished), and no bands were detected in plasma membrane preparations
(Theodoulou et al., 2003).
Rat monoclonal antibodies to -tubulin were purchased from Serotec
(Oxford, UK). Rat monoclonal antibodies to plant cell wall methyl esterified
pectin (JM 7) were a kind gift from Maxwell S. Bush, John Innes Centre,
Norwich, UK (Bush and McCann,
1999
) and plasma membrane ATPase antibodies were a gift from Mark
Boutry, Université Catholique de Louvain
(Morsomme et al., 1998
).
Fluorescent labelled secondary antibodies (Alexa FluorTM 568 goat
anti-rabbit and Alexa FluorTM 488 goat anti-rat) were purchased from
Molecular Probes (Cambridge, UK).
Preparation of whole extracts
Whole extracts were prepared from fresh tissue. Segments of root were
excised, weighed and transferred to a chilled glass homogeniser. Gel
electrophoresis loading buffer (10% glycerol, 2% sodium dodecyl sulphate
(SDS), 5% mercaptoethanol, 0.00125% bromophenol blue and 62.5 mM Tris HCl, pH
6.8) was added in the ratio of 10 µl per µg of tissue. The tissue was
homogenised and the homogenate was centrifuged for 2 minutes in a microfuge at
50,000 g to remove the cell debris. Aliquots of the
supernatant (50 µl per lane) were loaded immediately onto SDS
polyacrylamide gel electrophoresis (PAGE).
Preparation of plasma membrane vesicles
Plasma membrane vesicles were purified from whole root tissue and leaves as
described by Larsson et al. (Larsson et
al., 1994) with minor modifications. Briefly, tissue was harvested
on ice and homogenised for in 4x15 minutes in a Waring blender in
homogenisation buffer (500 mM sorbitol, 50 mM HEPES, 5 mM EGTA, 1 mM
dithiothreitol (DTT), 1 mM PMSF, pH 7.5) at a ratio of 2:1 (volume/fresh
weight). The mixture was filtered and centrifuged for 12 minutes at 1000
g. The supernatant was collected and centrifuged for a further
45 minutes at 50,000 g and the pellet was resuspended in 1/25
the original volume of resuspension buffer (330 mM sorbitol, 5 mM KCl, 1 mM
DTT, 5 mM K-phosphate, pH 7.8) and homogenised in a glass homogeniser. 4.5 ml
of the mixture was loaded onto 13.5 g gradients comprising
6.6% dextran T500, 6.6% polyethylene glycol 3350, 330 mM sorbitol, 5 mM KCl, 1
mM DTT, 5 mM K-phosphate, pH 7.8, mixed and incubated on ice for 5 minutes.
The gradients were centrifuged for 10 minutes at 1000 g and
the upper phases were removed to fresh lower phases, prepared by centrifuging
13.5 g gradients and removing the upper phases, which were
then used to wash the lower phases. The phases were centrifuged again and the
phase partitioning was repeated before the upper phases, enriched in plasma
membrane vesicles, were pooled and centrifuged for 60 minutes at 100,000
g. The resulting pellet was resuspended in 330 mM sorbitol, 5
mM Tris-maleic acid, pH 7.8 and the protein concentration assayed by a
modification of the method of Appleroth and Angsten
(Appleroth and Angsten, 1987
)
with bovine serum albumen as a standard. For western blotting 20 µg of
protein was loaded onto each lane.
The purity of the plasma membrane fractions was determined using marker
enzymes (Gordon-Weeks et al.,
1996; Hodges and Leonard,
1974
) and the activities were compared to the activities of the
enzymes in microsomal and tonoplast (Rea
et al., 1992
) fractions. In a typical experiment the specific
activity of the plasma membrane marker (vanadate-sensitive ATPase) in the
plasma membrane fraction was 14.20 µmol Pi released per mg protein per
hour, whereas the specific activity in the microsomal fraction was 5.8 and in
the tonoplast, 1.73. The VPPase activity was 1.34 µmol pyrophosphate
hydrolysed per mg protein per hour, compared with 10.60 (microsomes) and 27.28
(tonoplast). Azide sensitive ATPase activity (mitochondrial contamination) was
0.88 µmol Pi released per mg protein per hour (1.60 in the microsomes) and
NADH-dependent cytochrome c reductase (endoplasmic reticulum contamination)
was 2.61 µmol cytochrome c reduced per mg protein per hour (11.84 in the
microsomes).
SDS PAGE and western blotting
Protein samples were separated on 12.5% SDS PAGE gels and transferred
overnight to nitrocellulose hybond c membranes (Amersham Biosciences, Little
Chalfont, UK) in transfer buffer containing 48 mM Tris, 39 mM glycine, 0.03%
SDS and 10% methanol. Membranes were blocked for 1 hour in Tris buffered
saline (TBS; 20 mM Tris, 2 mM NaCl, pH 7.5) containing 5% dried milk. Blots
were probed with purified IgG antibodies for 4 hours at room temperature at a
1:1000 dilution in TBS containing 1% dried milk. After washing for 1 hour in
TBS with 1% milk, the blots were exposed to secondary antibody (donkey
anti-rabbit purchased from Sigma Genosys) conjugated to horse radish
peroxidase for 45 minutes at a 1:10,000 dilution. The blots were washed three
times in TBS and the immunoreactive bands were visualised using enhanced
chemiluminescence kits (Amersham Biosciences) according to the manufacturer's
instructions.
RNA isolation
For total RNA isolation, root tissue was ground to a powder under liquid
nitrogen. 0.2 g were removed to a 2 ml tube containing 1.5 ml Trizol (Sigma,
Poole, UK) and the contents vortexed for 30 seconds. After a 5 minute
incubation at room temperature the suspension was centrifuged at 12,000
g for 10 minutes and the supernatant removed to a clean 2 ml
tube. An equal volume of chloroform was added and the phases vortexed for 15
seconds and incubated for 5 minutes at room temperature. The phases were
separated by centrifugation at 4°C for 15 minutes at 12,000
g and the aqueous layer removed to a clean 2 ml tube. An equal
volume of chloroform-isoamylalcohol was added and the aqueous layer separated
as before. A one-tenth volume of 3.0 M sodium acetate and one-sixth volume of
isopropanol were added, and after 10 minutes incubation at room temperature
the RNA was pelleted by centrifugation at 12,000 g at 4°C
for 10 minutes. The pellet was washed twice with 70% alcohol and air
dried.
Relative quantitative reverse-transcription polymerase chain reaction
(RQRT-PCR)
PCR reactions used cDNA prepared from total RNA isolated from root tissue
using 18S RNA as a loading control
(Burleigh, 2001). For
amplification of StPT2, 5 pmol of Oligo (dT)12-18 was used as the
antisense primer for first strand cDNA and synthesis of 18S RNA used 5 pmol of
the antisense primer 5'-CAC TTC ACC GGA CCA TTC AAT CG-3'.
Reactions used 0.5 µg RNA and Superscript IITM RT (GibcoBRL, Paisley,
UK), following the manufacturer's recommended protocol. PCR was done initially
using 18S cDNA with one-twentieth volume of the first strand cDNA as template,
2.5U Taq DNA polymerase (Promega, Southampton, UK), PCR buffer
supplemented with 1.5 mM MgCl2 (MBI-Fermentas, St Leon-Rot,
Germany), and nucleotide (Amersham Biosciences) concentrations as recommended
by the supplier in a 50 µl reaction volume. Sense (5'-GAG GGA CTA TGG
CCG TTT AGG-3') and antisense primers were used at a final concentration
of 200 pmol. Reactions were performed on an Omnigene Thermal Cycler with a
heated lid (Hybaid, Ashford, UK) programmed to give a temperature profile of 2
minutes at 94°C followed by 40 cycles of 30 seconds at 94°C, 30
seconds at 55°C, 1 minute 30 seconds at 72°C and a final 5 minute
extension at 72°C. PCR products were analysed on 1% (w/v) Tris acetate
EDTA-agarose gels, using a GeneRulerTM 1 kb DNA ladder (MBI Fermentas).
Gels were visualised using an Eagle-eye II system (Stratagene, La Jolla, CA).
Template amounts were adjusted to achieve equal loading and the corresponding
amounts of oligo (dT)12-18 cDNA were used for StPT2 PCR reactions.
GCT CGC GTC GGC CTC CGT CAC was used as the forward primer and CCA ATA CGG TTG
GCC TCC AAT G as the reverse primer sequence. The PCR reaction conditions were
as described above except that the cycle number was reduced to 30.
Confocal microscopy
Analysis of root tissue for confocal microscopy followed the pre-embedding
staining method of Wick et al. (Wick et
al., 1981). Root tissue was fixed for 3 hours with 2% formaldehyde
in phosphate buffered saline (PBS) comprising 2.7 mM KCl, 1.47 mM
KH2PO4, 0.13 M NaCl and 8 mM
Na2HPO4 at pH 7.3. Tissue was then incubated for 30
minutes at room temperature in PBS containing 0.5% cellulase and washed
repeatedly with PBS. The tissue was blocked overnight in blocking buffer (5%
(v/v) normal horse serum, 5% (v/v) normal goat serum and 50 mM L-lysine in
PBS, pH 7.2) containing 2% Triton X-100 before the addition of primary
antibodies. Rabbit antipeptide IgGs and JM7 were added at a concentration of
1:50 in blocking buffer and rat anti-
-tubulin at a concentration of
1:3. After 12 hours the tissue was taken out of the antibody solution, washed
in PBS and incubated for a further 12 hours with fluorescent labelled
secondary antibody raised in goat (Serotec) at a concentration of 1:50 in
blocking buffer. Alexa Fluor 488 gave green labelling and Alexa Fluor 568,
red. Tissue was stored in PBS with 1 mM sodium azide at 4°C.
Immunolabelled roots for sectioning were embedded in gelatin (0.4% w/v) containing albumen (30% w/v) and sucrose (20% w/v) polymerised by the addition of glutaraldehyde (2.5% v/v). Sections (50 µm) were cut with a vibrating microtome (Camden Instruments, Camden, UK).
Root tissue labelled by immunofluorescence was viewed with a Leica TCS confocal microscope equipped with Argon, Krypton and HeNe lasers. Cells were imaged with 10x/0.3 or 20x/0.75 PL Fluotar objectives or 40x/1.0 or 63x/1.32 PLANAPO oil-immersion objectives and recorded at 1024x1024 pixels per image. Switching off the appropriate laser line using the acousto-optical transmission filter (AOTF) in the confocal microscope showed that there was negligible `bleed-through' between channels. Fluorescent images in TIFF format were manipulated using Adobe PhotoShop and analysed using Leica TCS software.
On request, all novel material described in this publication will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this paper that would limit their use for noncommercial research purposes.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig. 1 shows a typical result from one of three replica experiments using quartz-fed plants. Blots from both fed and depleted roots contained two broad bands of approximately 52 kDa and 30 kDa, and both bands were reduced in intensity in membranes isolated from P-fed root tissue. No bands were present in plasma membrane preparations from leaves. Similar results were obtained when hydroponically grow plants were analysed, except that the 52 kDa and 30 kDa bands were weaker in the P-fed tissue (data not shown).
|
The true molecular mass of StPT2, deduced from the cDNA sequence, is 58
kDa, and although an antibody to the Pi transporter from tomato, LePT1,
detected a protein of the expected size on SDS-PAGE
(Muchhal and Raghothama,
1999), membrane proteins often behave as smaller molecules on such
gel systems. Chiou et al. (Chiou et al.,
2001
) raised an antibody to a C-terminal sequence from MtPT1, a 59
kDa protein, that detected a diffuse protein band with an apparent molecular
mass of 45 kDa on western blots of extracts from Pichia pastoris
transformed with MtPT1. The antibody immunoprecipitated two
35S-methionine-labelled proteins of 45 kDa and 33 kDa (believed to
be an N-terminally truncated product) that had been synthesised in an in vitro
transcription/translation coupled system. We therefore assume that the 52 kDa
band corresponds to the transporter.
Whole tissue extracts were then made from potato roots from plants grown
hydroponically both in the absence and in the presence of P. Separate extracts
were made from three different parts of the root: the tip region (1 cm
segments), the middle region (4 cm segments above the tip section) and 2 cm
segments from the upper root region. The extracts were analysed by western
blotting probed with the StPT2 antibody. The experiment was performed three
times and Fig. 2 (upper panel)
shows a typical result. Two bands (52 kDa and 30 kDa) were detected by the
antibody in the root tip region and both bands were more intense in tissue
from P-depleted plants. A further faint band of approximately 55 kDa was
present in the extracts from P-depleted roots. The intensity of the 52 kDa
band decreased and that of the 30 kDa band increased if the sample was
incubated at 30°C for 2 hours before loading (data not shown), indicating
that the 30 kDa band could be a degradation product. Furthermore, the reduced
intensity of the 30 kDa band relative to the 52 kDa band in whole extracts
compared with that found in plasma membrane extracts suggests that degradation
of the protein could have taken place during the plasma membrane isolation
procedure. We were not able to identify the 55 kDa band, but StPT2 contains
two conserved phosphorylation sites
(Leggewie et al., 1997) and
its molecular mass relative to that of the major immunoreactive band suggests
that it could be a phosphorylated form of the protein.
|
In extracts from the middle root region only the 52 kDa band was detected, and although the band was stronger in P-depleted tissue it was much fainter than in the tip region. In the upper root region the 52 kDa band was only visible in the extract from P-depleted tissue and it was very weak.
RQRT-PCR
Total RNA was extracted from the tip, middle and upper regions of P-fed and
P-depleted hydroponically grown roots using the same segments that were used
for western blot analysis. The experiment was performed three times, and
Fig. 2 (middle panel) shows a
typical result. The RNA was standardised using cDNA generated with an 18S
reverse primer as template amplified with the 18S primers as described in
Materials and Methods (lower panel). Using the corresponding amounts of oligo
(dT) cDNA as template, PCR reactions using the StPT2 primers generated a
product of the expected size (1 kb) in each of the samples (middle panel).
Expression was highest in the root tips and in the middle and upper root
regions the expression was higher in P-depleted than in P-fed roots.
Confocal microscopy
Whole mounts
The results obtained from western blotting and RQRT-PCR indicated that the
expression of the StPT2 in potato roots is predominantly confined to a region
close to the tip. We therefore examined this area of the root using
immunofluorescence and a confocal microscope to study the distribution of the
transporter in more detail.
Root tissue was double labelled with StPT2, VPPase or plasma membrane
ATPase antibodies and -tubulin antibody. Plants grown both in
hydroponic and in quartz media were studied in the presence and absence of P.
Although it is possible to manipulate the nutrient status of hydroponically
grown plants accurately, this method of culture affects the root morphology
in particular, root hair development. Hence, a comparison of StPT2
labelling in roots subjected to both treatments was performed to confirm that
the localisation pattern was not due to growth conditions. In all cases at
least three root tips from three separate experiments were analysed.
Fig. 3A shows a low-power
image of a root tip from a P-depleted plant grown in hydroponic culture
immunolabelled with StPT2 (red) and -tubulin (green). The figure shows
StPT2 labelling confined to a region just behind the root tip, between 250
µm and 1200 µm from the root cap, whereas
-tubulin labelling
extends the full length of the root. Quartz-grown plants showed identical
staining patterns (not shown). In all P-supplied roots examined, labelling for
StPT2 appeared to be confined to a slightly more restricted area, further from
the tip (Fig. 3B). IgG from
pre-immune serum did not label the tissue
(Fig. 3C); similarly, omitting
the primary antibody gave no labelling (not shown). The full length of the
root was immunolabelled with the VPPase antibodies
(Fig. 3D) and the ATPase
antibodies (Fig. 3E). Yellow
indicates colocalisation of the two secondary antibodies, and there was a
higher proportion of yellow labelling in the VPPase-labelled roots and in the
ATPase-labelled roots away from the tip.
|
To examine the transverse distribution of StPT2 at the root tip, an optical
section was taken at 100 µm beneath the root surface, the maximum depth
that the confocal microscope can detect fluorescence
(Fig. 3F). This showed that the
-tubulin antibody labelling extends across the entire root width,
whereas the red StPT2 labelling is confined to the periphery, presumably in
the epidermis.
The expression of Pi transporters in root hairs has been reported in
tomato, Arabidopsis and Medicargo truncatula
(Chiou et al., 2001;
Daram et al., 1998
;
Karthikeyan et al., 2002
;
Mudge et al., 2002
). To study
the expression of StPT2 in root hairs, root parts from P-depleted quartz-grown
potato plants that contained hairs were labelled with the antibody. However,
many of the root hairs appeared to have burst during the cell wall digestion
and blocking procedures used to examine the whole roots, and there was
evidence of bacterial contamination inside the tissue. Milder conditions were
required (cellulase digestion reduced to 10 minutes and blocking buffer Triton
X-100 concentration to 0.5%) to examine the root hairs.
Fig. 3G shows a projected image
of the region above the extension zone that contains root hairs
(Marschner, 1995b
) prepared
under these conditions. StPT2 labelling (red) is present at the ends of the
root hairs but not in the main part of the root beneath.
High-power images
Western blotting detected StPT2 immunoreactive bands in purified plasma
membrane preparations from potato roots, and tissue was examined under high
magnification to further examine the subcellular localisation of the StPT2
labelling. Optical sections from tissue labelled with StPT2, plasma membrane
ATPase, VPPase, -tubulin and cell wall antibodies were compared. The
cell wall provides a barrier to molecules greater than 3-5 nm, which would
impede the passage of antibodies (Carpita
et al., 1979
), and preparation of root material included partial
enzymatic digestion of the tissue in an attempt to permealise the cell wall.
The peptide to which the StPT2 antibody is raised is on the central loop of
the protein, which is believed to be cytoplasmically orientated, and the
ATPase antibody is also raised to a cytoplasmic-facing domain
(Morsomme et al., 1998
).
Therefore, apart from the cell wall antibody, each of the antibodies must pass
through not only the cell wall, but also the plasma membrane to label their
targets. The roots were treated with high concentrations (2%) of Triton X-100
(Dyer and Mullen, 2001
) to
solubilise both the plasma membrane and the internal membranes of the cells.
The complete effectiveness of this procedure could be confirmed by labelling
the tissue with the VPPase because the epitope recognised by this antibody is
on part of the molecule that protrudes into the vacuole
(Kim et al., 1995
).
Tissue 500 µm from the tip at approximately 10 µm depth from roots
from P depleted plants, grown both in hydroponic and quartz media, was
examined to view epidermal cells. All labelling experiments were performed at
least three times on roots excised from different plants, and each gave
similar labelling patterns. Fig.
4A shows a root from a hydroponically grown plant with
cross-sectional views of some cells showing StPT2 labelling (red) peripheral
to the -tubulin (green) (see dotted arrow). This pattern is consistent
with the localisation of StPT2 in the plasma membrane. The cells in this plane
are not uniformly labelled with StPT2, however, and in some cells the
labelling covers the
-tubulin labelling (solid arrow), suggesting a
superficial view of the cells covered with labelled plasma membrane; in others
regions, however, the cells are only labelled with
-tubulin (double
arrow). The plasma membrane ATPase (red) labelled the epidermal cells more
uniformly in this plane than those in Fig.
4A, with all cells showing ATPase labelling peripheral to
-tubulin (Fig. 4B) and
only a few showing red covering the
-tubulin labelling (see solid
arrow).
|
VPPase labelling (red) in double-labelled images with -tubulin
(green) at this magnification also suggests labelling associated with a
membrane (Fig. 4C), with the
higher proportion of yellow indicating overlapping of the labelling. To
confirm that StPT2 antibodies do not label the cell wall, labelling of roots
with cell wall and StPT2 antibodies was compared
(Fig. 4D), and this showed that
StPT2 labelling (red) is inside the green cell wall labelling.
Transverse sections
Optical sections suggest that StPT2 labelling is on the outer surface of
the root tip, in the epidermal cells and on part of the root cap
(Fig. 3F). The width of roots
examined near the tip ranged between 200 µm and 300 µm, which is outside
the range of the confocal microscope. Therefore, to obtain a more complete
picture of the lateral distribution of StPT2 labelling in whole mounted tissue
we examined transverse sections. Sections of approximately 50 µm in width
were taken from between 1000 and 250 µm from the root tip of hydroponically
grown P-depleted plants labelled with StPT2 and -tubulin antibodies
before sectioning. Low-power images of all sections showed labelling with
StPT2 only at the periphery (Fig.
5A,C) but
-tubulin labelling present across each section
(Fig. 5B,C). At 300 µm from
the tip (Fig. 5C) all the cells
were labelled with
-tubulin, but where the root was more differentiated
(700 µm, Fig. 5B), the
-tubulin became weaker in the cortical cells than in the epidermis and
endodermis. StPT2 labelling was most intense at 500 µm from the tip and at
300 µm labelling began to decrease. Cross-sections of roots 700 µm from
the tip labelled with StPT2 (Fig.
5D) and plasma membrane ATPase
(Fig. 5E) (red) both show
labelling of the epidermis in this region, and ATPase labelling is also
detectable in the plasma membrane of the outer cortex. However, the ATPase
labelling is present all around the epidermal cells, whereas the StPT2
labelling is confined to the apical surface and the disto-lateral regions of
the cells, but is virtually absent from the basal surface.
Fig. 5F,G shows dual labelling
of the same sections with
-tubulin (green) (F and G) and StPT2 (F) and
plasma membrane ATPase (G), red. StPT2 labelling is only present on the apical
surface of the cells surrounding the
-tubulin labelling (see double
dotted arrow). A small amount of ATPase labelling appears to be associated
with cytoplasmic components, possibly membrane vesicles, in the epidermis
(arrow head), and a superficial view of other cells has been captured in this
plane (double arrow).
|
This distribution pattern is confirmed by measurement of the pixel density across labelled cells (Fig. 6), which shows similar intensities in the basal and apical plasma membrane in ATPase-labelled cells, but only in the apical membrane in StPT2-labelled cells.
|
In sections taken 250 µm from the tip ATPase labelling is found only in the epidermis and not the cortex. Flattened cells outside the epidermis are present in this region that may be `root boarder' cells from the root cap (Hawes et al., 1990), and these are labelled by both ATPase and StPT2 antibodies (not shown). Although these cells are partly detached from the root they have been shown to be viable and may play a role in plant defence against pathogens.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Localisation of Pht1 transporters in different root parts
Concentration of StPT2 expression at the root tip may reflect the fact that
the most active zone in absorption is the elongation zone near the tip, where
an elevation in metabolic activity is required to sustain the degree of cell
growth that occurs. This must place a high demand on cells for mineral
translocation and could result in a localised upregulation of the expression
of certain plasma membrane transporters. In rice, for example, maximum
NH4 uptake has been shown to occur at a distance of 1 mm behind the
root tip (Colmar et al., 1998). The root tip also plays an important role in
nutrient acquisition because it penetrates into fresh areas of the soil likely
to be richer in nutrients than the soil around the more mature parts of the
root, which forms the depletion zone
(Clarkson, 1991;
Marschner, 1995a
).
In some plant species the whole root has been shown to be capable of
absorbing Pi (Ferguson and Clarkson,
1975) and of responding to P deprivation by upregulation of the
rate of Pi uptake (Clarkson et al.,
1978
). Accordingly, the expression of some members of the Pth1
family in roots is not restricted to the tip. In tomato, LePT1, an StPT1
homologue, was found to be expressed in all parts of the root by western blot
analysis, apart from the apex, and most weakly in younger tissue
(Muchhal and Raghothama,
1999
). StPT1 and LePT1 are members of the subfamily 1
(Bucher et al., 2001
) and both
transporters are constitutively expressed and more widely distributed in plant
tissues than StPT2 (Leggewie et al.,
1997
; Liu et al.,
1998a
), which is a member of the subfamily 2, and is confined to
roots. However, in another study, in situ hybridisation analysis of tomato
roots showed that LePT1 expression is strongest in the differentiation zone
and root cap (Daram et al.,
1998
). The longitudinal distribution of LePT2, also a member of
subfamily 2 and more likely to resemble the pattern of StPT2, has not yet been
investigated. However, MtPT1, a further member of subgroup 2 from
Medicargo truncatula, is present in all root parts
(Chiou et al., 2001
). In
Arabidopsis members of subfamilies 1 and 3 show different expression
patterns within the root, with the expression of some being absent from the
root tip but others being expressed in all root parts including the tip region
(Karthikeyan et al., 2002
;
Mudge et al., 2002
).
We were able to measure a detectable but much reduced level of StPT2
expression in upper root parts compared with the tip by western blotting and
RQRT-PCR. However, no StPT2 labelling was found on the main root under the
confocal microscope away from the tip, possibly as a result of damage during
fixation to the outer surface of the epidermis in the mature tissue, where any
traces of StPT2 labelling would be located. By contrast, VPPase, plasma
membrane ATPase and -tubulin labelling extended up the root.
Tissue and cellular distribution of root Pi transporters
Members of subgroups 1 and 3 of the Pth1 family are found in the epidermis,
cortex and steele (particularly behind the cap in response to P depletion)
(Daram et al., 1998;
Liu et al., 1998a
), whereas
both LePT2 (Liu et al., 1998a
)
and MtPT1 (Chiou et al., 2001
),
members of subgroup 2, are found predominantly in the root epidermis. In
Arabidopsis expression of Pi transporters from groups 1 and 3 has
been detected predominantly in the epidermis, and also in other tissues
(Karthikeyan et al., 2002
;
Mudge et al., 2002
).
Strong expression of Pi transporters has been detected in root hairs
(Chiou et al., 2001;
Daram et al., 1998
;
Karthikeyan et al., 2002
;
Mudge et al., 2002
). Hairs
form above the root tip (Marschner,
1995b
), when plants are grown in solid media or aeroponic culture.
The low levels of the potato Pi transporter protein in upper root parts that
we detected by western blotting, compared with the levels of LePT1 reported
for aeroponically grown tomato roots
(Muchhal and Raghothama,
1999
), may have been due to a greater number of hairs on the upper
roots of the tomato plants. Our quartz-grown plants had relatively few hairs,
partly because of damage to the roots on removal from the solid medium.
Alternatively, StPT2 may not be as highly expressed in hairs as LePT1.
GFP-promoter fusion analysis has suggested that expression of one Pi
transporter in Arabidopsis roots occurs only in the trichoblasts
(Mudge et al., 2002). In this
plant trichoblasts form from cells overlying the cortical cell junctions
(Gilroy and Jones, 2000
), but
we found that all epidermal cells appear to express StPT2 equally
(Fig. 5F), although it may be
significant that these particular roots did not form hairs. In some
hair-bearing regions of Medicago truncatula roots all epidermal cells
are labelled with MtPT1 (Chiou et al.,
2001
), but we were not able to detect labelling in the epidermal
cells of the underlying root in the root hair regions.
Subcellular distribution of Pi transporters
We have shown by immunolocalisation of potato root tissue that StPT2 is
only present in the plasma membrane. All members of the Pth1 family have so
far been shown to be similarly localised, but this has been determined by
western blotting of purified membrane fractions and heterologous transient
expression (Muchhal and Raghothama,
1999; Chiou et al.,
2001
), as opposed to the direct examination of plant tissue that
we have used here.
Cytoplasmic Pi homeostasis is partly regulated at the tonoplast
(Sakano et al., 1995), but no
transporters involved in the bidirection movement of Pi across the vacuolar
membrane have been identified. Members of the Pi transporter families could be
present on the tonoplast, or possibly other endomembranes. The cytoplasmic
volume is 4% of the total cell volume, causing the plasma membrane and
tonoplast to be separated from each other by a narrow band of cytoplasm and
not easily distinguishable under the confocal microscope. StPT2-(plasma
membrane) and VPPase-(tonoplast) labelled tissue did appear different,
however, as both low- and high-power images labelled with the VPPase antibody
contained more yellow (colocalisation) than those labelled with StPT2. This
may be because the tonoplast has more folds or projections into the cytoplasm
than the plasma membrane or it may be less well fixed, causing the
membrane-associated proteins to merge with cytoplasmic components. The
presence of a small amount of VPPase immunoreactivity present in plasma
membrane fractions has been a matter of some dispute, and a low level of
targeting of the VPPase to the plasma membrane could also affect the labelling
pattern (Williams et al.,
1990
).
Immunolocalisation has shown that the Medicago truncatula
transporter MtPT4 is in close proximity to mycorrhizal arbuscules, suggesting
that it is present on the periarbuscular membrane and providing an essential
step towards understanding the complex process of nutrient exchange at the
interface during symbiosis (Harrison et
al., 2002). It would be interesting now to examine immunolabelled
potato root tissue colonised with mycorrhiza using antibodies raised to StPT3
to establish whether the transporters are similarly localised in potato.
Cellular polarity
Our high-power images of transverse sections indicate that StPT2 is
restricted to the apical surface of the plasma membrane, showing cellular
polarity. This also explains the absence of StPT2 label in some cells in the
optical section shown in Fig.
4A, where the section must pass through these cells beneath the
superficial labelling. However, labelling only on the external surface of the
epidermal cells could indicate that the StPT2 antibodies can not penetrate the
cell wall (Carpita et al.,
1979) or that they bind nonspecifically to adhesive components
present in the cell wall or on the cell surface
(Clarke et al., 1979
)
mucigel is present only on the surface of younger unthickened root tissue
(Hay et al., 1986
). However,
pre-immune serum did not label the cell surface, cell wall labelling is
outside that of StPT2 and membrane ATPase antibody is able to label the whole
cell periphery. In all species analysed the plasma membrane ATPase is encoded
by a multigene family that displays complex differential expression patterns
within plant tissues. One isoform has been shown to be asymmetrically
distributed in the plasma membrane of epidermal and cortical cells of the
maize root apex (Jahn et al.,
1998
). The ATPase antibody used here, raised to 110 residues from
a tobacco ATPase sequence (Morsomme et
al., 1998
), is likely to crossreact with more than one potato
isoform.
The concentration of StPT2 at the tips of root hairs also suggests cellular
polarity, and this distribution pattern is also observed for MtPT1
(Chiou et al., 2001). However,
it may reflect the fact that the tip region is more accessible to antibodies.
The use of transformed plants expressing GFP-fusion proteins may provide more
insight, although the question of the correct targeting of the GFP-tagged
proteins remains.
Polar localisation of a membrane protein has recently been observed for an
auxin transporter, AtP1N1. The transporter is only present at the basal end of
auxin-transport competent cells (Palme and
Galweiler, 1999), which facilitates the unidirectional flow of the
hormone. The mechanisms responsible for the targeting of the AtP1N1 to the
restricted area of the cell are currently being investigated and are believed
to involve rapid actin-dependent cycling of the protein between the plasma
membrane and endosomal compartments
(Geldner et al., 2001
). It
would be interesting to establish whether actin filaments are also involved in
the targeting of StPT2 to the apical plasma membrane.
Regulation of Pi transporter expression
Northern blots have shown that StPT2 mRNA was not detectable in roots of
P-fed plants (Leggewie et al.,
1997), and we found significant upregulation in response to P
depletion in the upper parts of the root using western blots and RQRT-PCR. By
contrast, we found that expression at the tip was still detectable, in P-fed
plants. However, not only are northern blots less sensitive than RQRT-PCR but
also localised StPT2 expression could be diluted beyond detectable levels in
whole root extracts because the tip region represents a very small proportion
of the whole root volume. Our findings suggest that it is necessary for the
plant to maintain a high level of transport activity at the root tip under
both circumstances in order to sustain growth. In addition, differences
between transcript and protein levels could reflect a delay in protein
turnover, and the existence of a number of conserved putative phosphorylation
sites present within the Pht1 family suggests that regulation of the
transporters at the post-translational level may also occur.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Appleroth, K. J. and Angsten, H. (1987). An improvement of the protein determination in plant tissues with the dye binding method according to Bradford. Biochem. Physiol. Pflanz. 182,85 -89.
Bucher, M., Rausch, C. and Daram, P. (2001). Molecular and biochemical mechanisms of phosphorus uptake into plants. J. Plant Nutr. Soil Sci. 164,209 -217.[CrossRef]
Burleigh, S. H. (2001). Relative quantitative RT-PCR to study the expression of plant nutrient transporters in Arbuscular mycorrhizas. Plant Sci. 160,899 -904.[CrossRef][Medline]
Bush, M. S. and McCann, M. C. (1999). Pectic epitopes are differentially distributed in the cell walls of potato (Solanum tuberosum) tubers. Physiol. Plant. 107,201 -213.[CrossRef]
Carpita, N., Sabularse, D., Montezinos, D. and Delmer, D. P. (1979). Determination of the pore size of call walls of living plant cells. Science 20,1144 -1147.
Chiou, T. J., Liu, H. and Harrison, M. J. (2001). The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface. Plant J. 25,281 -293.[CrossRef][Medline]
Clarke, A. E., Gleeson, P., Harrison, S. and Knox, R. B. (1979). Pollen-stigma interactions: Identification of surface components with recognition potential. Proc. Natl. Acad. Sci. USA 76,3358 -3362.[Abstract]
Clarkson, D. T. (1991). Root structure and the sites of ion uptake. In Plant Roots: The Hidden Half (ed. Y. Waisel, A. Eshel and U. Kafkafi), pp.417 -454. New York, Basel, Hong Kong: Marcel Dekker.
Clarkson, D. T., Sanderson, J. and Scattergood, C. B. (1978). Influence of phosphate stress on phosphate absorption and translocation by various parts of the root system of Hordeum vulgare L (Barley). Planta 139,47 -53.
Colmer, T. D. and Bloom, A. J. (1998). A comparison of NH4+ and NO3- net fluxes along roots of rice and maize. Plant Cell Environ. 21,240 -246.[CrossRef]
Daram, P., Brunner, S., Persson, B. L., Amrhein, L. and Bucher, M. (1998). Functional analysis and cell specific expression of a phosphate transporter from tomato. Planta 206,225 -233.[CrossRef][Medline]
Daram, P., Brunner, S., Rauch, C., Steiner, C., Amrheim, N. and
Bucher, M. (1999). Pht2;1 encodes a low-affinity phosphate
transporter from Arabidopsis. Plant Cell
11,2153
-2166.
Dong, B., Rengel, Z. and Delhaize, E. (1998). Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana. Planta 205,251 -256.[CrossRef][Medline]
Dyer, J. M. and Mullen, R. T. (2001). Immunocytological localisation of two plant fatty acid desaturases in the endoplasmic reticulum. FEBS Lett. 494, 44-47.[CrossRef][Medline]
Ferguson, I. B. and Clarkson, D. T. (1975). Ion transport and endothermal suberization in the roots of Zea maize. New Phytol. 75,69 -79.
Furihata, T., Suzuki, M. and Sakurai, H. (1992). Kinetic characterisation of two phosphate uptake systems with different affinities in suspension-cultured Catharanthus roseus protoplsts. Plant Cell Physiol. 33,1151 -1157.
Geldner, N., Frimi, J., Stierhof, Y-D., Jurgens, G. and Palme, K. (2001). Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413,425 -428.[CrossRef][Medline]
Gilroy, S. and Jones, D. L. (2000). Through form to function: root hair development and nutrient uptake. Trends Plant Sci. 5,56 -60.[CrossRef][Medline]
Gordon-Weeks, R., Steele, S. H., Leigh, R. A.
(1996). The role of magnesium, pyrophosphate, and their complexes
as substrates and activators of the vacuolar H+ pumping inorganic
pyrophosphatase. Plant Physiol.
111,195
-202.
Hamburger, D., Rezzonico, E., Petetot, J. M. C., Somerville, C.
and Poirier, Y. (2002). Identification and characterization
of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem.
Plant Cell 14,889
-902.
Harrison, M. J., Dewbre, G. R. and Liu, J.
(2002). A phosphate transporter from Medicago truncatula
involved in the acquisition of phosphate released by arbuscular mycorrhizal
fungi. Plant Cell 14,2413
-2429.
Hawes, M. C. and Hao-Jan, L. (1990). Correlation of pectolytic enzyme activity with the programmed release of cells from root caps of pea (Pisum sativum). Plant Physiol. 94,1855 -1859.
Hay, M. J. M., Dunlop, J. and Hopcroft, D. H. (1986). Phosphate uptake and anatomy of unthickened and secondarily thickened adventitious root of field grown white clover (Trifolium repens) L. New Phytol. 103,659 -668.
Hodges, T. K. and Leonard, R. T. (1974). Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol. 32,392 -406.[Medline]
Jahn, T., Baluska, F., Michalke, W., Harper, J. F. and Volkmann, D. (1998). Plasma membrane H+-ATPase in the root apex: Evidence for strong expression in xylem parenchyma and asymmetric localisation within cortical and epidermal cells. Physiol. Plant. 104,311 -316.[CrossRef]
Kai, M., Masuda,Y., Kikuchi,Y., Osaki, M. and Tadano, T. (1997). Isolation and characterisation of a cDNA from Catharanthus roseus which is highly homologous with phosphate transporter. Soil Sci. Plant Nutr. 43,227 -235.
Karthikeyan, A. S., Varadarajan, D. K., Mukatira, U. T., Urzo,
M. P., Damsz, B. and Raghothama, K. G. (2002). Regulated
expression of arabidopsis phosphate transporters. Plant
Physiol. 130,221
-233.
Kim, E. J., Zhen, R.-G. and Rea, P. A. (1995).
Site-directed mutagenesis of vacuolar H+-pyrophosphatase. Necessity
of Cys634 for inhibition by maleimide but not catalysis.
J. Biol. Chem. 270,2630
-2635.
Larsson, C., Sommarin, M. and Widell, S. (1994). Isolation of highly-purified plant plasma membranes and separation of inside-out and right-side-out vesicles. Methods Enzymol. 228,451 -469.
Leggewie, G., Willmitzer, L. and Riesmeier, J. W.
(1997). Two cDNAs from potato are able to compliment a phosphate
uptake-deficient yeast mutant: Identification of phosphate transporters from
higher plants. Plant Cell
9, 381-392.
Liu, C., Muchal, U. S., Mucatira, U., Kononowicz, A. K. and
Raghothama, K. G. (1998a). Tomato phosphate transporter genes
are differentially regulated in plant tissues by phosphorus. Plant
Physiol. 116,91
-99.
Liu, H., Trieu, A. T., Blaylock, L. A. and Harrison, M. J. (1998b). Cloning and characterisation of two phosphate transporters from Medicago truncatular roots: regulation in response to phosphate and to colonisation by arbuscular mycorrhizal (AM) fungi. Mol. Plant Microbe Interact. 11, 14-22.[Medline]
Marschner, H. (1995a). Nutrient availability in soils. In Mineral Nutrition of Higher Plants, pp.483 -507. Academic Press, London Harcourt Brace.
Marschner, H. (1995b). Effect of internal and external factors on root growth and development. In Mineral Nutrition of Higher Plants, pp. 508-536. Academic Press, London Harcourt Brace.
Morsomme, P., Dambly, S., Maudoux, O. and Boutry, M.
(1998). Single point mutations distributed in 10 soluble and
membrane regions on the Nicotiana plumbaginifolia plasma membrane
PMA2 H+-ATPase activate the enzyme and modify the structure of the
C-terminal region. J. Biol. Chem.
273,34837
-34842.
Muchhal, U. S. and Raghothama, K. G. (1999).
Transcriptional regulation of plant phosphate transporters. Proc.
Natl. Acad. Sci. USA 96,5868
-5872.
Muchhal, U. S., Pardo, J. M. and Raghothama, K. G.
(1996). Phosphate transporters from the higher plant.
Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA
93,10519
-10523.
Mudge, R. S., Rae, A. L., Daitloff, E. and Smith, F. W. (2002). Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. Plant J. 31,341 -353.[CrossRef][Medline]
Okumura, S., Mitsukawa, N., Shirano, Y. and Shibata, D. (1998). Phosphate transporter gene family of Arabidopsis thaliana. DNA Res. 5,261 -269.[Medline]
Palme, K. and Galweiler, L. (1999). PIN pointing the molecular basis of auxin transport. Curr. Opin. Plant Biol. 2,375 -381.[CrossRef][Medline]
Poirier, Y., Thoma, S., Sommerville, C. and Schiefelbein, J. (1991). A mutant of Arabidopsis deficient in xylem loading of phosphate. Plant Physiol. 97,1087 -1093.
Rausch, C., Daram, P., Brunner, S., Jansa, J., Laloi, M., Leggewie, G., Amrhein, N. and Bucher, M. (2001). A phosphate transporter expressed in arbuscule-containing cells in potato. Nature 414,462 -466.[CrossRef][Medline]
Rea, P. A., Britten, C. J. and Sarafian, V. (1992). Common identity of substrate-binding subunit of vacuolar H+ translocating inorganic pyrophosphatase of higher plant cells. Plant Physiol. 100,723 -732.
Röhm, M. and Werner, D. (1987). Isolation of root hairs from seedlings of pisum sativum: identification of root hair specific proteins by in situ labelling. Physiol. Plant. 69,129 -136.
Rosewarne, G. M., Barker, S. J., Smith, S. E., Smith, F. A. and Schachtman, D. P. (1999). A Lycopersicon esculentum phosphate transporter (LePT1) involved in phosphorus uptake from a vesicular-Arbuscular mycorrhizal fungus. New Phytol. 144,507 -516.[CrossRef]
Sakano, K., Yazaki, Y., Okihara, K., Mimura, T. and Kiyoa,
S. (1995). Lack of control in inorganic phosphate uptake by
Catharanthus roseus (L.) G. Don cells. Cytoplasmic inorganic
phosphate homeostasis depends on the tonoplast inorganic phosphate transport
system? Plant Physiol.
108,295
-302.
Schachtman, D. P., Reid, R. J. and Ayling, S. M.
(1998). Phosphorus uptake by plants: from soil to cell.
Plant Physiol. 116,447
-453.
Smith, F. W., Ealing, P. M., Dong, B. and Delhaize, E. (1997). The cloning of two Arabidopsis genes belonging to a phosphate transporter family. Plant J. 11, 83-92.[CrossRef][Medline]
Theodoulou, F. L., Clark, I. M., He, X.-L., Pallett, K. E., Cole, D. J. and Hallahan, D. L. (2003). Co-induction of glutathione-S-transferases and multidrug resistance associated protein by Xenobiotics in wheat. Pest Manag. Sci. 59,202 -214.[CrossRef][Medline]
Versaw, W. K. and Harrison, M. J. (2002). A
chloroplast phosphate transporter, PHT2;1, influences allocation of phosphate
within the plant and phosphate-starvation responses. Plant
Cell 14,1751
-1766.
Wick, S. M., Seagull, R. W., Osborn, M., Weber, K. and Gunning, B. E. S. (1981). Immunofluorescence microscopy of organised microtubule arrays in structurally stabilised meristematic plant cells. J. Cell Biol. 89,685 -690.[Abstract]
Williams, L. E., Nelson, S. J. and Hall, J. L. (1990). Characterisation of solute transport in plasma membrane vesicles isolated from cotyledons of Ricinus communis L. I. Adenosine triphosphatase and pyrophosphatase activities associated with a plasma membrane fraction isolated by phase partitioning. Planta 182,532 -539.
|