Journal of Experimental Botany, Vol. 48, No. 306, pp. 93-100, January 1997
Journal of
Experimental
Botany
Effect of sulphate deficiency on the plasma membrane
polypeptide composition of Brassica napus
Agnes Massonneau, Nicole Cathala, Claude Grignon and Jean-Claude Davidian1
Laboratoire de Biochimie et Physiologie Moldculaire des Plantes, Ecole Nationale Superieure Agronomique de
Montpellier, INRA, CNRS (URA 2133), 34060 Montpellier Cedex 1, France
Received 21 May 1996; Accepted 14 August 1996
Abstract
Highly purified oilseed rape {Brassica napus) root
plasma membrane fractions were prepared and their
polypeptide patterns analysed by two-dimensional gel
electrophoresis. Sulphur starvation enhances the
sulphate uptake capacity of B. napus roots. The relative abundance of several polypeptides increased significantly and specifically after sulphur starvation.
Several of them (37, 38, 60, and 65 kDa), found in
sulphur-starved plants, were more abundant in a
phase-partitioned membrane fraction treated with
Triton X-100/KBr, indicating that they are intrinsic
polypeptides. One polypeptide (47 kDa) was identified
in the in vitro translation products of the roots mRNAs
as specific for S-starved plants. It was also present
among the intrinsic polypeptides specific for — S
plants. These plasma membrane polypeptides might
be involved in sulphate uptake.
Key words: Sulphate, sulphur-starvation, plasma membrane, polypeptides, root, transport.
Introduction
Sulphur can be acquired by plants as mineral sulphur
(Landry et ai, 1991), sulphur dioxide, hydrogen disulphide (Krouse, 1977), and sulphate. Sulphate absorbed by
roots is the major sulphur source in higher plants. After
its reduction through the assimilatory pathway, sulphur
is incorporated in a variety of organic compounds, including proteins and sulpholipids (Heinz, 1993). Cysteine
rich-proteins are involved in protection against predators
and pathogen microbes (Bohlman, 1993), and in heavy
metal detoxification (Marschner, 1986; Rauser, 1993).
1
Physiological and biochemical studies have shown that
sulphate transport through root plasma membrane is
controlled by a high affinity transport system (Jensen
and Konig, 1982; Cram, 1983; Clarkson et ai, 1993;
Hawkesford et ai, 1993). The first sulphate transporter
gene was cloned from Neurospora crassa by Ketter et ai
(1991). Several cDNAs coding for sulphate transport
systems have been cloned recently from rat (Markovich
et ai, 1993; Bissig et ai, 1994), man (Hastbacka et ai,
1994), yeast (Smith et ai, 1995a), and higher plants
(Smith et ai, 19956).
To identify the plasma membrane polypeptides related
to sulphate transport in plant roots, a treatment was used
which has been shown specifically to increase the root
capacity for sulphate uptake (Clarkson et ai, 1992).
When oilseed rape seedlings were grown in the absence
of a sulphur source, root sulphate uptake capacity was
7-fold greater than in non-sulphur-starved plants
(Massonneau, 1994). Moreover, sulphur starvation did
not interfere with phosphate influx, nor with net proton
and potassium transport. It was found that the sulphate
uptake stimulation observed under sulphur deficiency was
inhibited by cycloheximide, indicating that this stimulation was related to protein synthesis. A greater 35Ssulphate uptake was observed in purified oilseed rape
root plasma membrane vesicles prepared from sulphurdeficient plants in comparison to sulphur-sufficient plants
(Hawkesford et ai, 1993). These results are consistent
with an increase of the amount of plasma membrane
polypeptides involved in sulphate transport in response
to plant sulphur starvation. Two-dimensional (2-D) gel
electrophoresis permits the resolution of around a thousand polypeptides (O'Farrell, 1975). In combination with
silver staining, it allows the detection of as little as 0.02 ng
To whom correspondence should be addressed. Fax: +33 4 67 52 57 37. E-mail: davidian6ensam.inra.fr
Abbreviations: SDS, sodium dodecyl sulphate; TEMED, tetra methyl ethylene diamine.
© Oxford University Press 1997
94
Massonneau et al.
of protein. Such a technique may be expected to detect
membrane polypeptides implicated in transport whose
relative abundance is very low (Sussman and Harper,
1989). Therefore, 2-D gel electrophoresis silver staining
was used to examine changes in the plasma membrane
polypeptide populations in response to sulphur
availability.
Materials and methods
buffer and centrifuged for 60 min at 135 000 xg. All the
membrane fractions were kept in the conservation buffer at
- 8 0 ° C until used.
Enzyme assays
Plasma membrane vanadate-sensitive ATPase activity was
measured as described by Gallagher and Leonard (1982).
Pyrophosphatase and IDPase activities were used respectively
as tonoplast (Rea and Poole, 1985) and Golgi enzyme markers
(Ray et al., 1969). The protein content was estimated as
described by Schaffner and Weissmann (1973) with BSA as a
standard.
Plant culture
Oilseed rape seeds (Brussica napus Metzger var. Drakkar,
Etablissement Ringot, La Chapelle d'Armentieres, 59930,
France) were germinated for 3 d in a dark room as previously
described (Hawkesford et al., 1993). The seedlings were
transferred to an illuminated chamber with a photoperiod of
14/10 h (400 /xmol m " 2 s~' photosynthetically active radiation),
with 70% relative humidity. They were cultivated for 4 d on
+ S or — S nutrient solutions changed daily. The + S solution
contained 1 mM KNO 3 , 0.5 mM Ca(NO 3 ) 2 , 0.1 mM KH 2 PO 4 ,
2 mM MgSO 4 , 0.1 mM EDTA-NH 4 Fe, 0.05 mM KCI, 0.03 mM
H 3 BO 4 , 0.01 mM MnSO 4 , 1 ^M CuSO 4 , 1 ^M ZnSO 4 , and 0.3
/xM Na 2 MoO 4 . In — S solutions, all the sulphate salts were
replaced by their corresponding chloride salts.
Membrane isolation
All the steps of membrane isolation were carried out at 4°C.
Seven-day-old oilseed rape roots were harvested on ice, chopped
with a razor blade and ground for 5 min in a mortar in the
presence of4 ml g" 1 FW of a grinding buffer containing 25 mM
BTP-MES pH 7.8, 250 mM sucrose, 10% (w/v) glycerol, 2 mM
EGTA, 2 m M DTT (dithiothreitol), 2 mM EDTA, and 1 mM
PMSF (phenylmethylsulphonyl fluoride). The extract was then
filtered and centrifuged for 10 min at 13 000 x g. The supernatant
was recovered and centrifuged for 30 min at 80 000 x g. The
'microsomal' pellet was resuspended in 1 ml g" 1 FW of grinding
buffer and centrifuged for 30 min at 80000xg. Fractions
enriched in plasma membrane were obtained from this 'microsomal' pellet either by phase partition or by centrifugation on
a sucrose gradient. Phase partitioning was carried out as
described by Widell et al. (1982) by resuspending the pellet in
250 mM sucrose and 10 mM KH 2 PO 4 . The two-phase partitioning aqueous system was made up to a final composition of
5.8% (w/w) dry dextran T500 (Pharmacia) and 5.8% (w/w)
polyethylene glycol 3350 (Sigma), 250 mM sucrose, 30 mM
NaCl, 1 mM EDTA, and 10 mM KH 2 PO 4 , at pH 7.8. For the
sucrose gradient membrane preparation, the membranes were
resuspended with 2 mM BTP-MES pH 7.2 and 250 mM sucrose
and layered on to a linear 25-48% (w/w) sucrose gradient
containing 2 mM TRIS-MES pH 7.2 and 1 mM DTT. After a
3 h centrifugation at lOOOOOxg with a swinging bucket rotor,
the plasma membrane enriched fractions were recovered between
34% and 36% (w/w) sucrose, diluted with the conservation
buffer (2mM BTP-MES pH 7.2, 20% (v/v) glycerol, 1 mM
DTT, and 250 mM sucrose) and pelleted by a 45 min
centrifugation at 135 000 x g . A n aliquot of the phase-partitioned
plasma membrane fraction was washed with the extraction
buffer containing 250 mM KI, and pelleted (30 min at
80000 x g ) . The pellet was resuspended in 500 mM KBr, 10 mM
MES-TRIS pH6.5, 30% (w/v) glycerol, 150 mM KCI, 1 mM
EDTA, 2 m M DTT, and 0.25% (w/v) Triton X-100. After 10
min on ice, the membranes were centrifuged for 45 min at
135OOOx£ and the pellet was rinsed with the conservation
RNA extraction and poly (A)+ RNA isolation
RNAs were extracted as described elsewhere (Fourcroy, 1986),
using a guanidine thiocyanate extraction buffer. Poly (A) +
RNAs were purified from this extract by an affinity chromatography on oligo dT cellulose as described by Maniatis et al.
(1989). The polypeptides encoded by these RNAs were obtained
after an in vitro translation using a rabbit reticulocyte lysate
system (Amersham Kit N90).
Two-dimensional gel electrophoresis
Membrane proteins were solubilized as described by Hurkman
and Tanaka (1986) by adding to the protein sample (400 ^g/3
IEF gels), 2 ml of extraction buffer (0.7 M sucrose, 0.5 M
TRIS, 30 mM HCI, 50 mM Na-EDTA, 0.1 M KCI, 2.5 mM
DTT, and 2 mM PMSF) and 2 ml of saturated phenol in 0.1 M
TRIS pH 8.0. The two phases were mixed by a 1 min agitation
and centrifuged at room temperature for 10 min at 4000 xg.
The aqueous phase was removed, the phenolic phase was
re-extracted with 1 vol. of extraction buffer, and 5 vols of 0.1 M
ammonium acetate (in methanol) were added to the phenolic
phase. The proteins were precipitated overnight at — 20 °C and
pelleted by a 10 min centrifugation at 4000 xg (4°C). The
pellet was washed three times with the solution of ammonium
acetate, then with a cold (— 20 °C) acetone 80% solution. After
a 5 min centrifugation at 3600 xg (4°C), the pellet was dried
and solubilized with 180 /x\ of lysis buffer (9 M urea, 4% (w/v)
Nonidet P40, 2% (v/v) Resolytes pH 4-8, and 3% (v/v) 0mercaptoethanol). In vitro translation products were solubilized
by adding lysis buffer to the sample.
The 2-D gel electrophoresis was carried out as described by
Ageorges (1992). The IEF gels were made in 2 mm internal
diameter, 15 cm long tubes. The gels were made up with 4%
(w/v) acrylamide, 9.2 M urea, 2% (w/v) Nonidet P40, 2%
ampholytes (Resolytes p H 4 - 8 ) , 0.01% (w/v) ammonium persulphate and 1.4% (v/v) TEMED. The cathodic buffer was a
0.5% (v/v) ethanolamine solution, and the anionic buffer a 0.2%
(v/v) H 2 SO 4 solution. The gels were pre-run for 15 min at 200
V, 30 min at 300 V and 30 min at 400 V. The sample were
loaded at the acidic end and the electrophoresis was run for
15 h at 300 V and 1 h at 400 V. The IEF gels were then
equilibrated by two washes of 15 min in a solution containing
10% (w/v) glycerol, 2.3% (w/v) SDS, 2% (v/v) jS-mercaptoethanol, and 62.5 mM TRIS-HC1 pH 6.8. The gel was then
loaded on a discontinuous SDS-gel, 4.8% (w/v) acrylamide in
the stacking gel at pH 6.8 and 10% (w/v) in the running gel
at pH 8.8.
Non-radioactive samples were silver-stained at room temperature. The gels were treated for 30 min with methanol/acetic
acid/water (50/10/40, by vol.) and 30 min with methanol/acetic
acid/water (5/7/87, by vol.). These treatments were followed by
1 h in 7% (v/v) glutaraldehyde, and six washes in water (total
2 h). The gels were then incubated for 25 min in the staining
Sulphate and plasma membrane polypeptides
solution (0.2% (w/v) AgNO 3 , 0.25% (v/v) NH 4 OH and 0.022
N NaOH) and washed three times for 5 min with water. The
staining was revealed by a solution containing 0.05% (v/v)
formaldehyde (37%) and 0.0025% (w/v) citric acid, and stopped
by 1% (v/v) acetic acid.
In experiments with [35S]-labelled polypeptides, the polyacrylamide gels were fixed by a 2 h treatment in methanol/acetic
acid/water (40/10/50, by vol.), rinsed for 10 min in 100% acetic
acid and treated for 1 h in 22% (w/v) 2,5-diphenyloxazole
dissolved in acetic acid (Hames, 1988).
Electrophoresis image analysis
Polypeptide abundance was estimated by using an image
analysis system (Samba 2005, Alcatel-TITN, Grenoble, France)
fitted with a black and white CCD camera and an image
processing card ( 4 x 5 1 2 x 5 1 2 x 8 bits, Matrox MVP/AT), as
described by Masson and Rossignol (1995). In all the gels
analysed, four polypeptides were identified whose abundance
did not vary in response to the plant sulphur treatments and
which have been chosen as internal references. The relative
abundance of the polypeptides which vary in response to
sulphur treatments is expressed in terms of their absorbance as
a percentage of the sum of the absorbance of the internal
reference.
95
Plasma membrane polypeptides obtained by phase
partition and sucrose gradient
The polypeptide patterns of the plasma membranes purified by phase partition or by sucrose gradient were
different only for polypeptides with a low relative abundance (Plate 1A, B). The comparison of polypeptide patterns from — S and +S roots (Plate 2) was achieved with
an image analyser. An arbitrary identification number
was allocated to each of the variable spots (Table 2).
Three polypeptides appeared specific to the plasma membrane purified by phase partition from — S plants, as
compared to the +S sample (32 kDa, p/6.5; 32 kDa, p/
5.3; and 30 kDa, p/ 6.4) (Table 2). These polypeptides
were, however, found in the plasma membrane purified
on sucrose gradient from +S plants. For 16 other polypeptides, the — S treatment resulted in an increase in
abundance. For one among them, this behaviour was
only observed in phase partition plasma membrane, and
not in the other membrane preparation (spot 55: 44 kDa,
p/ 6.6). The differences between the polypeptides of — S
and +S plants were generally larger in plasma membrane
prepared by phase partition.
Plasma membrane intrinsic polypeptides of — S and +S
plants
Results
Plasma membrane purity in relation to membrane isolation
The relative abundance of plasma membrane associated
with microsomal, sucrose gradient and two phase partition membrane fractions extracted from oilseed rape roots
has been evaluated by comparing the specific activities of
three membrane markers, vanadate-sensitive ATPase,
pyrophosphatase and latent IDPase (Table 1). The
plasma membrane marker specific activity, vanadatesensitive ATPase, measured in the 34-36% sucrose gradient fraction and the phase partition (upper phase) were
identical, and twice that found in the microsomal fraction.
The ATPase activity relative to the pyrophosphatase and
the latent IDPase is higher in the membrane fraction
prepared by two phase partition than in the microsomal
and the sucrose gradient fractions. This indicates that
membranes purified by this technique are less contaminated by vacuole and Golgi membranes.
The plant sulphate transporter, in common with other
membrane transporters, is certainly an intrinsic protein.
In order to eliminate some of the — S specific polypeptides
detected in the plasma membrane fraction and not implicated in the sulphate transport, a detergent treatment to
eliminate extrinsic polypeptides has been used. The plasma
membrane fraction obtained by phase partition was
treated with Triton X-100 and the intrinsic polypeptides
of — S and + S plants were studied by 2-D gel electrophoresis. This treatment reduced by more than 75% the amount
of polypeptides from the plasma membrane fraction
(Galtier et ah, 1987). Several plasma membrane intrinsic
polypeptides affected by S growth conditions can be identified. The relative abundance of 18 among them was much
larger in plasma membrane purified from — S plants than
from +S plants (numbered in Plate 3). In the Triton
X-100 membrane fraction, six polypeptides (spots 70, 73,
Table 1. Enzyme activity of membrane markers in different membrane preparation from oilseed rape roots
The enzyme specific activities were measured on a micTOSomal fraction, a 34-36% sucrose gradient fraction and the upper part of a two phase
partition system. The results are the mean of three independent replicates
Membrane markers activities (^mol h" 1 mg " ' protein)
Microsome
Sucrose gradient
Phase partition
Vanadate-sensitive ATPase
Pyrophosphatase
Latent IDPase
117
242
228
24
8
3
16
21
0.1
96
Massonneau et al.
IEF
5 | kDa pH 5.0
S
'.5
5.0
7.5
66
•
•*
ift
• •
Plate I. Comparison of polypeptide patterns of plasma membrane fractions prepared by phase partition (A) and sucrose gradient ccntrifugatiion
(B). Enriched plasma membrane fractions were purified from roots of + S plants. The polypeptides were separated by two-dimension
gel
electrophoresis, and silver stained.
."s
-a
26
28
30
m
^
•
-
40 -
«31
»34
41
44
49 54
Plate 2. Effect of sulphur starvation on the root plasma membrane polypeptide pattern. Polypeptides from enriched plasma membrane fraction
purified by phase partition were separated by two-dimensional gel electrophoresis, and silver stained. Each number indicates polypeptides whose
relative abundance increases after sulphur starvation (—S) in comparison to control ( + S) plants. The characteristics of these polypeptides are
summarized in Table 2.
Sulphate and plasma membrane polypeptides
97
Table 2. Identification and characterization of oilseed rape root plasma membrane polypeptides
Three ennched plasma membrane fractions were obtained after phase partition with or without detergent treatment (Triton X-100 + KBr), and
sucrose gradient. All the polypeptides whose abundance increased after sulphur starvation are identified by an arbitrary number, and their respective
molecular mass (kDa) and p/ The relative abundance of each spot is expressed as the ratio of its absorbance after sulphur starvation to the one in
the presence of sulphate. ' —S' indicates spots which are detected only after sulphur deprivation. '-' indicates that the spot is absent from the
membrane fraction.
Spot number
Molecular mass
(kDa)
p'
Fractional increase after S starvation
Phase partition
4
9
15
19
20
26
28
30
31
34
39
40
41
42
44
46
49
50
54
55
70
73
83
85
86
90
68
60
47
44
44
38
37
35
35
65
33
32
32
32
30
28
28
27
28
44
27
27
47
55
55
62
5.9
5.8
5.9
6.1
6.2
6.2
6.5
6.5
6.6
6.6
5.7
6.5
6.5
53
6.4
64
5.9
58
6.1
6.6
6.5
6.6
7.0
7.0
6.5
5.5
13.5
3.8
1.4
1.2
2.1
2.7
7.5
11.5
5.4
15.4
1.4
5.7
-S
-S
-S
0.7
2.3
_
0.9
4.1
_
_
_
-
Phase partition
+ TX-100 + KBr
-S
-S
-s
-s
-s
-S
-s
-S
2.8
-S
~S
24.9
_
-S
21.4
-S
-S
-S
11.0
Sucrose gradient
.5
.9
.2
:(.9.8
:!.O
;!.7.7
.7
.1
.2
.1
.3
.4
.3
.0
;>.5
4.9
0.9
83, 85, 86, 90) were observed in the plasma membrane
fraction purified from —S plants (Table 2). These six
membrane intrinsic polypeptides were not detectable in
the absence of detergent probably because of their low
relative abundance in this fraction. Among the 19 — S
polypeptides responsive to S status identified in the plasma
membrane fraction in the absence of detergent treatment,
seven were no longer detected after this treatment (spots
4, 20, 30, 42, 44, 49, 55) (Table 2). Thus, only the other
12 polypeptides are intrinsic (spots 9, 15, 19, 26, 28, 31,
34, 39, 40, 41, 46, and 54) and might be directly involved
in the sulphate transport. However, the possibility cannot
be excluded that the extrinsic polypeptides might play an
important role in the sulphate transport and be necessary
for this function.
tional dependence and a translational regulation are
involved in the response of sulphate uptake to sulphur
starvation in roots.
In vitro translation products of poly(A) + RNA extracts
from — S and + S oilseed rape roots were analysed by 2-D
gel electrophoresis (Fig. 1). Six polypeptides were specific
for the — S fraction (absent from + S fraction). Among
them, only polypeptide 15 (47 kDa, p / 5.9) was present in
the plasma membrane fraction. As described above, it
belongs to the group of polypeptides found specifically in
the plasma membrane of the — S plants, and the intrinsic
polypeptides fraction (Table 2). The other — S specific
translation products did not correspond with any other
polypeptide of the plasma membrane fraction.
In vitro translation products
Discussion
The effects of ribosomal inhibitors suggest that protein
synthesis is necessary to the stimulation of sulphate
uptake in response to S starvation (Rennenberg et cii,
1989; Clarkson et al., 1992). It was found that a-amanitin
(a transcription inhibitor) prevents 78% of the stimulation
of sulphate uptake increase in — S oilseed rape plants
(Massonneau, 1994). These results shows that a transcrip-
In this study, it has been shown that no significant differences were detectable in the plasma membrane polypeptide
patterns prepared with sucrose gradient and phase partition (Plate 1). The purity of the plasma membrane fraction
is, however, higher when prepared by phase partition
(Table 1). Among the 19 polypeptides whose abundance
was increased by sulphur starvation in the phase-
98
Massonneau et al.
IEF
s
D
S
kDa pH 5.0
7.5
-s
5.0
7.5
+s
—
•
-90
-86
85
—_
-
15
83
—
19
•
26
*
28
*
31
34
-
39
*
_
41
40
46
54
73
70
*
Plate 3. Effect of sulphur depnvation on the root plasma membrane intrinsic polypeptide pattern. The root plasma membrane fraction of control
( + S) and sulphur-starved plants ( —S) were purified by phase partition. Membranes were then washed with 0.25% Triton X-100 in the presence of
500 mM KBr. The intrinsic polypeptides were then separated by two-dimensional gel electrophoresis, and silver stained. Each number indicates
polypeptides whose relative abundance increases after sulphur starvation ( —S) in comparison to control ( + S) plants The characteristics of these
polypeptides are summarized in Table 2.
I
Spot number 15
MM (kDa) 47
p/ 5.9
205
42
6.4
201
28
5.7
202
28
5.5
203
27
5.3
204
28
5.9
Fig. 1. In vitro translation product analysis in response to sulphur
starvation. The poly(A) + RNAs were extracted from sulphur-starved
( —S) and control ( + S) plants, and the "S-methionine/ 35 S-cysteine
labelled in vitro translation products analysed by two-dimensional gel
electrophoresis. Each spot number indicate polypeptides whose relative
abundance increases after sulphur starvation (—S) in comparison to
control ( + S) plants Their abundances are expressed in term of their
absorbance as a percentage of the sum of the absorbance of four nonvariant polypeptides which are used as internal references. The results
presented are obtained from two independent experiments. MM and p /
represent, respectively, the polypeptide molecular mass (kDa) and the
isoelectric point.
partitioned membrane fraction, only one (spot 55) differed
in behaviour from the samples from sucrose gradient
centrifugation. In most cases, the increase in the intensity
of the — S polypeptides was lower in the fractions prepared
by sucrose gradient centrifugation. Such a difference may
be the result of a 'dilution' effect on the plasma membrane
peptides by contaminant peptides from other membranes.
Since the gels were loaded with equal amounts of proteins,
these contaminant polypeptides would lower the average
intensity. This effect would be still more marked if some
of the — S peptides of the plasma membranes were present
in a constitutive form in other membranes. Thus, the
relative changes in abundance upon — S treatment would
be enhanced upon using a more highly purified plasma
membrane fraction, as indeed is observed (Table 2). In
the same way, the number of polypeptides responsive to
— S treatment increased from 19 in native phasepartitioned plasma membrane to 40 after washing by
Triton X-100 (data partly shown in Table 2). This result
might be due to the enrichment of the membrane fraction
in minor polypeptides, revealing the variation in relative
abundance between the two S treatments.
As compared to the plasma membrane preparation, the
2-D electrophoresis of in vitro translation products identified only six polypeptides as specific for the sulphur
starvation treatment. Among them, only polypeptide 15
Sulphate and plasma membrane polypeptides
(47 kDa, p / 5.9) was present in the plasma membrane
fraction. As described above, it belongs to the group of
polypeptides found specifically in the plasma membrane
of the — S plants and in the intrinsic fractions. The other
five — S specific translation products, which did not
correspond with any other polypeptides of the plasma
membrane fraction, may either be soluble polypeptides
or membrane polypeptides lacking their post-translational
modifications. Their absence from the membrane polypeptides might also be due to the staining protocol of
membrane polypeptides: some polypeptides cannot be
silver stained (Rochette-Egly and Stussi-Garaud, 1984).
Some sulphur metabolic activities have been reported to
be enhanced by sulphur starvation such as the ATP
sulphurylase (Logan et al., 1996) and the APS (adenosine
phosphosulphate) sulphotransferase (Brunold, 1993).
None of these — S-dependent polypeptides corresponds
to 48 kDa deduced molecular mass of the mature ATP
sulphurylase protein (Logan et al., 1996). The chloroplastic APS-kinase (24 kDa, p / 5.5) (Schiff et al., 1993)
may correspond to one of the four — S specific translation
products whose molecular weight and p / are similar to
those of this enzyme. The enhancement of the abundance
of a soluble polypeptide (28 kDa, p / 5.2) in response to
sulphur deprivation has already been reported in tomato
roots (Hawkesford and Belcher, 1991) and in Anacystis
nidulans (Green and Grossman, 1988).
The increased abundance of six in vitro translation
products in — S plants shows that there is a transcriptional
regulation in the plant in response to sulphur deprivation.
These polypeptides may be involved in the sulphate transport, as are the two mRNAs coding for a high affinity
sulphate transporter in the roots of Stylosanthes hamata
(Smith et al., 19956), which are regulated by the sulphate
status of the plant. Alternatively, they may be involved in
sulphur assimilation, or in any pathway which could be
activated in response to sulphur deficiency (Pedersen et al.,
1978; Malagoli et al., 1994). For instance, the effect of
sulphur deficiency on the nitrogen assimilation pathway is
well documented (Friedrich and Schrader, 1978;
Karmoker et al., 1991; Brunold, 1993; Bell et al., 1995).
Thus, proteins involved in the nitrogen pathway are potential candidates for the — S specific in vitro translation
products. Indeed, the high number of plasma membrane
polypeptides with increased abundance in — S plants suggests that the sulphate transporter(s) are not the sole
system(s) which are responsive to the S nutritional status.
Since more than 400 polypeptides can be detected in
plasma membrane by the 2-D gel electrophoresis method,
the sulphur-responsive polypeptides represent a significant
fraction of the plasma membrane polypeptides. Other
examples of changes in abundance of many polypeptides
upon starvation in various elements are documented in E.
coli (Pedersen et al., 1978).
In summary, three plasma membrane polypeptides
specific for sulphur-starved plants have been identified in
99
tomato roots (Hawkesford and Belcher, 1991): 36 kDa, p /
6.3; 43 kDa and 47 kDa. One of these polypeptides
(36 kDa) may correspond to one of the following polypeptides found in oilseed rape root plasma membrane: spot
28 (37 kDa, p / 6.5) or spot 30 (35 kDa, p / 6.5). Green
and Grossman (1988) have also identified 36 kDa and
43 kDa polypeptides whose abundance increased in relation to sulphur starvation in Anacystis nidulans, a unicellular cyanobacterium. The 43 kDa polypeptide of tomato
may correspond to our polypeptide 20 (44 kDa, p / 6.2).
The 47 kDa polypeptide identified by Hawkesford and
Belcher (1991) fits well with our polypeptide 15 (47 kDa,
pi 5.9). This polypeptide was found to be an intrinsic
polypeptide and was specific for the — S plants in the
plasma membrane fraction, in the intrinsic plasma membrane polypeptides and in the in vitro translation products.
This polypeptide is likely to be involved in the sulphate
transport.
Acknowledgements
We thank Dr Malcolm Hawkesford (BBSRC, Bristol, UK) for
critically reading the manuscript. Dr Agnes Massonneau was
supported by a grant from the French Ministere de la Recherche
et de la Technologic
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