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Plant Physiol. (1998) 118: 1337-1344
Distribution of Sulfur within Oilseed Rape Leaves in Response to
Sulfur Deficiency during Vegetative Growth1
Mechteld M.A. Blake-Kalff*,
Kevin R. Harrison,
Malcolm J. Hawkesford,
Fangjie J. Zhao, and
Steve P. McGrath
IACR-Rothamsted, Soil Science Department (M.M.A.B.-K., K.R.H.,
F.J.Z., S.P.M.), and Biochemistry and Physiology Department (M.J.H.),
Harpenden, Hertfordshire AL5 2JQ, United Kingdom
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ABSTRACT |
The distribution of S to sulfate,
glucosinolates, glutathione, and the insoluble fraction within oilseed
rape (Brassica napus L.) leaves of different ages was
investigated during vegetative growth. The concentrations of
glutathione and glucosinolates increased from the oldest to the
youngest leaves, whereas the opposite was observed for
SO42 . The concentration of insoluble S was
similar among all of the leaves. At sufficient S supply and in the
youngest leaves, 2% of total S was allocated to glutathione, 6% to
glucosinolates, 50% to the insoluble fraction, and the remainder
accumulated as SO42. In the middle and oldest
leaves, 70% to 90% of total S accumulated as
SO42, whereas glutathione and glucosinolates
together accounted for less than 1% of S. When the S supply was
withdrawn (minus S), the concentrations of all S-containing compounds,
particularly SO42, decreased in the youngest
and middle leaves. Neither glucosinolates nor glutathione were major
sources of S during S deficiency. Plants grown on nutrient solution
containing minus S and low N were less deficient than plants
grown on solution containing minus S and high N. The effect of N was
explained by differences in growth rate. The different responses of
leaves of different ages to S deficiency have to be taken into account
for the development of field diagnostic tests to determine whether
plants are S deficient.
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INTRODUCTION |
In higher plants, S is taken up by the roots as
SO42, transported via the
xylem to the leaves, reduced to Cys, and either converted to Met or
incorporated into proteins and Cys-containing peptides such as
glutathione. The uptake and subsequent distribution of SO42 to the leaves is closely
regulated in response to demand. For instance, developing leaves are
strong S sinks, but show a net loss of S after full expansion (Sunarpi
and Anderson, 1996 ). Similarly, the uptake of
SO42 by the roots is
down-regulated when the external S supply is sufficient, but increases
in plants during S deficiency (Clarkson and Saker, 1989; Hawkesford et
al., 1993). It has been suggested that the signal for down-regulation
is either SO42 (Datko and
Mudd, 1984) or a reduced-S-containing compound such as glutathione
(Herschbach et al., 1995; Lappartient and Touraine, 1996). Both
SO42 and glutathione are
mobile in the phloem (Rennenberg et al., 1979; Lappartient and
Touraine, 1996).
It has been suggested that
SO42 stored in the vacuoles of
mesophyll cells is only released under conditions of prolonged S stress
and that this release is too slow to support new growth (Clarkson et
al., 1983; Bell et al., 1995). As a result, the developing leaves are
the first ones to show symptoms of S deficiency. When the youngest
leaves are S deficient, the major fraction of S is in protein. However,
remobilization of S from proteins does not take place under conditions
of S starvation unless N is also deficient (Sunarpi and Anderson, 1996,
1997a). Additionally, low N has been shown to promote the export of
SO42 from mature leaves
(Sunarpi and Anderson, 1997b).
Oilseed rape (Brassica napus L.) is particularly sensitive
to S deficiency because it has a high demand for S (Holmes, 1980); for
example, oilseed rape produces seeds with a high yield of protein with
relatively large quantities of S-containing amino acids (Zhao et al.,
1997), and the plants require S for the synthesis of glucosinolates, a
group of thioglucoside compounds reported to be part of the plant's
defense mechanism against fungi and insects (Chew, 1988). In addition,
glucosinolates may play a role as a S storage source, which can be used
in the event of S starvation (Schnug and Haneklaus, 1993). It is not
yet known if this contribution of S from glucosinolates is sufficient
to counter the effect of S starvation, because the concentration in
vegetative growth is less than 8% of total S (Fieldsend and Milford,
1994) and decreases during leaf expansion (Porter et al.,
1991).
The decrease in atmospheric deposition of S has increased the incidence
of S deficiency in oilseed rape (McGrath and Zhao, 1995). Studies on
the physiological and molecular effects of S nutrition in oilseed rape
will facilitate the prediction of responses to decreased S inputs and
may provide useful diagnostic indicators of S status. In this study the
distribution of S to glutathione, glucosinolates,
SO42, and insoluble S in
leaves of different ages and the contributions of these compounds as a
S source under conditions of S deficiency were investigated.
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MATERIALS AND METHODS |
Plant Material, Growth Conditions, and Experimental Design
Seeds of oilseed rape (Brassica napus L. cv Apex) were
sown in pots with moist vermiculite (medium grade) and germinated at a
constant temperature of 20°C, 75% RH, and a 16-h light period (280-300 µmol m2
s1). After 7 d the seedlings were
transferred to a hydroponic system consisting of 40-L tanks (25-40
plants per tank) filled with a continuously aerated nutrient solution
containing 3 mM KNO3, 2 mM Ca(NO3)2, 1 mM
NH4H2PO4,
50 µM KCl, 25 µM
H3BO3, 2 µM
MnCl2, 2 µM
ZnCl2, 0.5 µM
CuCl2, 0.5 µM
(NH4)6Mo7O24,
and 20 µM NaFeEDTA. The pH of the solution was adjusted
to 5.5 with KOH. MgSO4 was added as indicated in
the experiments and Mg2+ was maintained at 1 mM in all treatments by the addition of
MgCl2 when appropriate.
Two experimental systems were used. In the first, after transfer to the
hydroponic system, seedlings were exposed to three different sulfate
concentrations (20, 100, and 1000 µM
SO42). The nutrient solution
was replaced weekly. Depending on the size, between 4 and 10 plants per
treatment were harvested at regular intervals and weighed. Leaf length
was determined by measuring the leaf along the main vein from the base
to the apex. At each harvest the plants were dissected, and
corresponding leaves of plants were pooled together in muslin cloth and
immediately frozen in liquid N2. L1 corresponded to the
first fully exposed leaf, L2 to the second, etc. The frozen leaves were
lyophilized for 72 h and kept under a vacuum at room temperature
until further analysis.
In the second experimental system, plants were precultured
hydroponically, as described above, for 23 d on nutrient solution containing 1 mM
SO42 and 7 mM
NO3. After this preculture,
the plants were transferred to nutrient solutions containing: (a) 1 mM SO42 and 7 mM NO3 (plus S,
high N); (b) 0 mM
SO42 and 7 mM
NO3 (minus S, high N); (c) 1 mM SO42 and 250 µM NO3 (plus
S, low N); or (d) 0 mM
SO42 and 250 µM
NO3 (minus S, low N). These
nutrient solutions were replaced every 3 d. Three plants per
treatment were harvested at d 0 (the day when treatments were started),
2, 3, 6, 8, and 13. Each plant was dissected into the oldest leaves (L1
and L2), the middle leaves (usually L3, L4, L5, L6, and L7, depending
on the size of the plant), and the youngest leaves (usually L8 and L9).
The three leaf fractions of each plant were frozen separately in liquid N2 and lyophilized for 72 h. The dried leaves were
ground into a fine powder and stored under vacuum at room temperature
until further analysis.
Measurements
Total S was determined by digesting 50 mg of lyophilized plant
material in a mixture of concentrated HNO3 and
HClO4 (85:15, v/v). The digested material was
resuspended in 5% (v/v) HCl and S determined by inductively coupled
plasma-atomic emission spectroscopy (Applied Research Laboratories,
Accuris, Ecublens, Switzerland) at 182 nm. Sulfate and nitrate
were measured by extracting 20 mg of lyophilized plant material in 20 mL of deionized water at 90°C for 2 h, after which the extract
was filtered through filter paper (Whatman no. 42).
SO42 and
NO3 concentrations in the
extracts were determined by ion chromatography (Dionex 2000i/sp) using
an AS9SC separation column fitted with an AS9G guard column (Dionex,
Sunnyvale, CA). The eluent solution consisted of 1.8 mM Na2CO3, 1.7 mM NaHCO3, and the regenerant of
0.025 N H2SO4.
Glutathione was extracted by grinding 10 to 15 mg of lyophilized plant
material (with a mortar and pestle and quartz sand) in 2 mL of a
solution containing 5% (w/v) 5-sulfosalicylic acid and 6.3 mM diethylenetriaminepenta-acetic acid. Total glutathione (GSH plus one-half GSSG, expressed as GSH equivalents) was determined using the 5,5 -dithiobis(2-nitrobenzoic acid) recycling assay as
described by Anderson (1985), with GSSG as a standard. The final
concentration of GSH reductase (type III, Sigma) in the cuvette was 0.5 unit mL1. Glucosinolates were extracted from 50 mg of lyophilized leaf material, and the concentrations of individual
compounds were measured by HPLC according to the protocols of Heaney et
al. (1986). Insoluble S, representing mainly protein S, was determined
by subtracting the concentrations of sulfate, glutathione, and
glucosinolates from the concentration of total S. Chlorophyll was
measured using a SPAD 502 meter (Minolta, Tokyo, Japan). Analysis of
variance was performed on all data.
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RESULTS |
Effects of External S Supply on Leaf Growth and Concentrations of
SO42 and Glutathione
When plants were grown at three different sulfate concentrations,
as described for the first experimental system in "Materials and
Methods," there was a profound effect on the growth and appearance of
oilseed rape plants. The RGRs of the shoots were 0.15, 0.18, and 0.21 d1 for plants grown on 20, 100, and 1000 µM sulfate, respectively. The average leaf length
increased with increasing SO42
concentration (Fig. 1), as well as the
number of leaves developing per plant: at the highest
SO42 concentration (1000 µM) two to four more leaves developed per plant compared
with the lowest concentration (20 µM) (data not shown).
Chlorosis was observed in L6 to L12 in the 20 µM
SO42 treatment after 26 d
and in the 100 µM
SO42 treatment after 34 d. In contrast, plants grown on 1000 µM
SO42 remained green during the
experiment (data not shown).
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| Figure 1.
Effect of external S supply on leaf development in
oilseed rape. Plants were grown continuously in nutrient solutions
containing 20, 100, or 1000 µM
SO42. The effect of increasing
SO42 concentrations was similar in all of the
leaves, but for clarity only the length of the longest leaf is
presented. Data represent the means ± SE of four
separate plants. The LSD (P < 0.05) for each time
point is shown by vertical bars.  , 20 µM
SO42;  , 100 µM
SO42;  , 1000 µM
SO42.
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Initially, L1 and L2 contained similar concentrations of sulfate,
irrespective of the supplied S concentration (Fig.
2). At the lower S treatments (20 and 100 µM), the SO42
concentration decreased gradually throughout the experiment, and little
or no SO42 was observed in the
subsequently developing leaves. At the highest S treatment (1000 µM), SO42
accumulated in all of the leaves, but particularly in the older ones.
In all treatments, total S concentrations in leaves closely paralleled
the SO42 concentrations (data
not shown).
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| Figure 2.
SO42 concentrations in
leaves of different ages grown in nutrient solutions containing 20, 100, or 1000 µM SO42 throughout
the experimental period. The data shown are a representative example of
two different experiments. DW, Dry weight.
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In all of the treatments the concentration of glutathione decreased
during the course of the experiment (Fig.
3). This decrease closely paralleled the
increase in leaf length and was probably caused by growth dilution. In
plants grown on either 20 or 100 µM
SO42, glutathione decreased
more rapidly over time, reaching a minimum concentration after 26 d. The concentration of glutathione in the young leaves showed the
greatest decrease, and only a low concentration was measured in those
leaves.
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| Figure 3.
Total glutathione concentrations (GSH plus GSSH)
in leaves of different ages in nutrient solutions containing 20, 100, or 1000 µM SO42 throughout the
experimental period. The data shown are a representative example of two
different experiments. DW, Dry weight.
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Effects of S and N on Chlorophyll and Growth Rate
We examined the responses of plants grown for 3 weeks on 1 mM SO42 and 7 mM NO3, followed
by a transfer to nutrient solutions containing plus or minus S
in combination with high (7 mM) or low (0.25 mM) NO3
concentrations, as described for the second experimental system in
``Materials and Methods''. The chlorophyll readings decreased rapidly
in the middle and particularly the youngest leaves of plants grown on
solution containing minus S and high N, but not in leaves of plants
grown on minus S and low N (Fig. 4). No
significant changes in chlorophyll over time or between treatments were
observed in the oldest leaves.
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| Figure 4.
Chlorophyll readings in the oldest, middle, and
youngest leaves of plants grown in the presence or absence of
SO42 at either high (7 mM) or low
(0.25 mM) NO3. Plants were grown
for 3 weeks on 1 mM SO42 and 7 mM NO3 before transfer to the
four different treatments at d 0. Data are the means ± SE of three separate plant samples. The LSD
(P < 0.05) for each time point is shown by vertical bars. There
was no significant difference between treatments in the oldest leaves.
Open symbols, minus S; closed symbols, plus S; circles, high N;
triangles, low N.
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The RGRs for the different plant parts are shown in Table
I. Little growth was observed in the
oldest leaves regardless of the treatment, probably because these
leaves were already at full expansion at the start of the experiment.
The growth rates of the middle and youngest leaves were much lower in
the low- than in the high-N treatment. In the middle leaves, the
removal of S did not decrease the growth rate any further in the low-N
treatment, but decreased the growth rate by approximately 50% in the
high-N treatment.
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Table I.
RGRs (d1) of the oldest, middle, and
youngest leaves of oilseed rape plants grown in the presence or absence
of SO42 at either high (7 mM) or
low (0.25 mM) NO3
Data represent the means ± SE.
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Effects of S and N on Glutathione and Glucosinolates
When sulfate was removed from the nutrient solution on d 0, the
glutathione concentration decreased rapidly after 2 d in the middle and youngest leaves, but not in the oldest leaves (Fig. 5). In the youngest leaves, this decrease
in glutathione concentration was faster in plants grown on high N than
in plants grown on low N; the glutathione concentration in plants grown
on high N reached a minimum after approximately 8 d, whereas in
plants grown on low N the decrease was slower. A similar trend in the
decrease of glutathione between plants grown on low and high N after S removal was observed in the middle leaves. The glutathione
concentration in the youngest leaves was approximately 2.5 times that
in the oldest leaves.
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| Figure 5.
Total glutathione concentrations (GSH plus GSSG)
in the oldest, middle, and youngest leaves of plants grown in the
presence or absence of SO42 at either high (7 mM) or low (0.25 mM)
NO3. Plants were grown for 3 weeks on 1 mM SO42 and 7 mM
NO3 before transfer to the four different
treatments at d 0. Data are the means ± SE of three
separate plant samples. The LSD (P < 0.05) for each
time point is shown by vertical bars. There was no significant
difference between treatments in the oldest leaves. Open symbols, minus
S; closed symbols, plus S; circles, high N; triangles, low N. DW, Dry
weight.
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At d 0 in the youngest and middle leaves, the distribution of
glucosinolates between the aliphatic, aromatic, and indolyl types was
16%, 23%, and 61%, respectively. The oldest leaves contained only
aromatic and indolyl glucosinolates. There was a clear difference in
the concentrations of glucosinolates in the different leaf tissues: at
d 0, the youngest leaves contained 8.14 ± 0.54 µmol g1 dry weight (6.4% of total S), whereas the
middle and oldest leaves contained 1.12 ± 0.07 and 0.43 ± 0.08 µmol g1 dry weight (0.6% and 0.1% of
total S), respectively. When the external S supply was withdrawn, the
aliphatic glucosinolates in the youngest leaves of plants grown on high
N decreased at a rate of 0.17 µmol g1
d1 (i.e. 12.8% d1 of
the initial concentration), reaching 0 at d 8, whereas the aromatic and
indolyl glucosinolates decreased at rates of 0.14 and 0.51 µmol
g1 d1 (i.e. 7.6 and
11.3% d1), respectively (Fig.
6). In plants grown on minus S and low N, the rates of decrease in the youngest leaves were 0.11 and 0.22 µmol
g1 d1 for aliphatic and
indolyl glucosinolates, respectively, whereas aromatic glucosinolates
were not affected. In plus-S plants the concentration of glucosinolates
increased in the youngest leaves of plants grown on low N, which was
mainly attributable to a 15-fold increase in the concentration of
aliphatic glucosinolates during the experiment. In the middle leaves of
minus-S plants grown on high N, the concentrations of the aliphatic and
aromatic glucosinolates decreased at rates of 0.035 and 0.033 µmol
g1 d1 (i.e. 13.5% and
9.4% d1 of the initial concentration),
respectively, whereas no significant change was observed in the
concentration of the indolyl glucosinolates. In the middle leaves of
minus-S plants grown on low N, no significant changes were observed in
the glucosinolate concentration for the duration of the experiment.
Like the youngest leaves, in the middle leaves of plus-S plants grown
on low N, the concentration of the aliphatic glucosinolates increased
almost 20-fold between d 0 and 13. In the oldest leaves, no significant
differences were found between the different treatments.
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| Figure 6.
Glucosinolate concentrations in the oldest,
middle, and youngest leaves of plants grown in the presence or absence
of SO42 at either high (7 mM) or
low (0.25 mM) NO3. Plants were
grown for 3 weeks on 1 mM SO42
and 7 mM NO3 before transfer to
the four different treatments at d 0. Data are the means ± SE of three separate plant samples. For clarity, the
y axes for the oldest, middle, and youngest leaves are
presented at different scales. The LSD (P < 0.05) for
each time point is shown by vertical bars. There was no significant
difference between treatments in the oldest leaves. Open symbols, minus
S; closed symbols, plus S; circles, high N; triangles, low N. DW, Dry
weight.
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Effects of S and N on SO42 and Insoluble
S
At d 0, SO42 accounted
for 83%, 68%, and 42% of total S in the oldest, middle, and youngest
leaves, respectively. The SO42
concentration of all of the leaves decreased sharply after the removal
of the external S supply (Fig. 7). No
difference in the decline of the
SO42 concentration between the
high- and low-N treatments could be observed in the youngest and middle
leaves of plants after S withdrawal; in the youngest leaves, the
SO42 concentration decreased
at a rate of 7.0 and 7.4 µmol g1
d1 between d 2 and 8 in plants grown on high
and low N, respectively; in the middle leaves, the
SO42 concentration decreased
at a rate of 26.0 and 25.3 µmol g1
d1, respectively, during the same period. In
both the youngest and middle leaves this decrease in
SO42 concentration was
equivalent to a rate of decrease of approximately 10%
d1 of the initial
SO42 concentration. However,
the rate of decrease was not constant, and once the
SO42 concentration was less
than 20% of the initial concentration the rate of decrease was reduced
considerably. In the oldest leaves there was initially no significant
decline in the SO42
concentration in plants grown on high N, but after d 6 it decreased rapidly at a rate of 34.4 µmol g1
d1 (equivalent to 9.3%
d1). In plants grown on low N, the
SO42 concentration in the
oldest leaves decreased from d 3 onward at a rate of 29.4 µmol
g1 d1 (7.8%
d1).
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| Figure 7.
Concentrations of SO42
and insoluble S in the oldest, middle, and youngest leaves of plants
grown in the presence or absence of SO42 at
either high (7 mM) or low (0.25 mM)
NO3. Plants were grown for 3 weeks on 1 mM SO42 and 7 mM
NO3 before transfer to the four different
treatments at d 0. Data are the means ± SE of three
separate plant samples. The LSD (P < 0.05) for each
time point is shown by vertical bars. For clarity, the
SO42 concentrations of the youngest leaves
are presented on a smaller scale. Open symbols, minus S; closed
symbols, plus S; circles, high N; triangles, low N. DW, Dry weight.
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The concentrations of insoluble S, defined as the difference between
the total S concentration and the sum of the concentrations of
SO42, glutathione, and
glucosinolates, were similar (approximately 100 µmol
g1 dry weight) at the start of the experiment
in the oldest, middle, and youngest leaves, and were less affected by
the withdrawal of the external S supply than the
SO42 concentration (Fig. 7).
In the oldest leaves no significant differences were observed between
the four different treatments: the insoluble S concentration remained
relatively constant during the course of the experiment. In the middle
leaves the insoluble S concentration in the minus-S plants was
significantly decreased compared with the control from d 6 onward at a
rate of 5.3 and 5.2 µmol g1
d1 (5% d1) for plants
grown on high and low N, respectively. In the youngest leaves the
insoluble S concentration decreased at a rate of 13.6 µmol
g1 d1 (12.5%
d1) in plants grown on minus S and high N, but
at a rate of 6.1 µmol g1
d1 (5% d1) in plants
grown on minus S and low N between d 0 and 8, respectively. By d 13, plants grown on minus S and low N contained 55% of the initial
insoluble S concentration, whereas plants grown on high N contained
only 33%.
To determine whether S was relocated from old to young leaves, a
comparison was made between the total S measurements in plants grown on
minus S and high N, and the predicted values for total S for each leaf
type based on the growth rate and assuming that no S was transported
into or out of each plant part (Fig. 8). The predicted values represent a dilution curve attributable to growth.
A measured value higher than the predicted value implies a net gain of
S, and vice versa. The measurements of total S in the youngest leaves
followed the prediction very closely, indicating no net gain or loss of
S. In the middle leaves, the measured total S was consistently lower
than the predicted value for total S during the course of the
experiment, indicating net export. In the oldest leaves, the
measurements of total S were higher than the predicted values up to d
8, indicating net import; however, by d 13 the measured total S in the
oldest leaves was lower than the predicted value, indicating net
export. It is possible that the exported S from the middle and oldest
leaves went to the roots or stems, but this was not determined in the
present study. The S budget in the youngest leaves of plants grown on
minus S and high N was examined more closely by using similar
predictions for the dilution of the concentrations of
SO42, glutathione,
glucosinolates, and insoluble S, together with the difference between
the measured and predicted values by d 13 (Table
II). The data show that the concentration
of insoluble S decreased more slowly than predicted, whereas the
concentrations of SO42,
glutathione, and glucosinolates decreased faster than predicted. A
comparison of the data in Table II with those in Figure 8 suggests that
the net gain of insoluble S in the youngest leaves comes from an
internal redistribution of different S pools, specifically the
conversion of soluble pools of S to insoluble S.
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| Figure 8.
Concentrations of total S in the oldest, middle,
and youngest leaves of plants grown in the absence of S and at high (7 mM) NO3 compared with the
predicted values for each leaf type. Predictions were based on the
growth rate, assuming that no S was transported into or out of each
plant part, and therefore predicted values represent a dilution curve
attributable to growth. Closed circles, Measured values; dashed lines,
predicted values. DW, Dry weight.
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Table II.
Comparison between measured and predicted values of
S-containing compounds in the youngest leaves of plants grown at high
(7 mM) NO3 for 13 d after
removal of the S supply
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DISCUSSION |
The results obtained showed a clear difference in the distribution
of S between leaves of different ages. At sufficient S supply,
approximately 50% of total S in the youngest leaves was incorporated
into insoluble S, 2% into glutathione, 6% into glucosinolates, and
42% accumulated as SO42. In
contrast, in the middle and oldest leaves, 70% to 90% of S
accumulated as SO42, whereas
glutathione and glucosinolates together accounted for less than 1% of
total S. It has been reported that in soybean plants,
SO42 in the transpiration
stream was predominantly delivered to developing leaves, despite the
fact that mature and young leaves transpired at an equal rate (Smith
and Lang, 1988). These results were explained by a very efficient
xylem-to-phloem transfer before any
SO42 delivered to mature
leaves could mix with the intracellular
SO42 pool in the mesophyll
cells and become immobilized. In oilseed rape plants
SO42 accumulated mainly in the
mature leaves, even for days after the S supply was withdrawn. This
indicated that the xylem-to-phloem transfer of
SO42 was not completely
effective and could contribute to the high S demand of oilseed rape
plants.
When the external S supply was removed, the youngest leaves were able
to convert SO42, glutathione,
and glucosinolates to insoluble S. SO42 was the most important
net contributor of S; the contributions by glutathione and
glucosinolates were almost negligible in comparison. These results were
in contrast with the suggestion that glucosinolates play a vital role
as a S source in the event of S deficiency (Schnug and Haneklaus,
1993), but were in agreement with the results obtained from several
oilseed rape varieties grown in the field showing that glucosinolates
in vegetative tissues accounted for only 2% to 8% of total S
(Fieldsend and Milford, 1994).
Little is known about the regulatory aspects of the remobilization of S
from mature leaves. It has been proposed that glutathione acts as a
regulatory signal to decrease the
SO42 uptake in the roots
(Rennenberg et al., 1989; Lappartient and Touraine, 1996), but it is
unknown whether it also acts as a signal for the leaf-to-leaf
translocation of S. Export of S from the oldest leaves was observed
after 6 d of S starvation, predominantly as
SO42 (Figs. 7 and 8), but
during that period no changes in the glutathione concentration were
observed (Fig. 5). It may have been possible that S from these leaves
was exported as glutathione, as suggested by Rennenberg (1984), with
the glutathione pool being replenished at a constant level using the
SO42 pool. However, no
increases in S were observed in the middle and youngest leaves,
although it was unknown whether S increased in the roots or stems.
Furthermore, in soybean plants exposed to 35S
more than 90% of S transported out of the mature leaves was recovered
as SO42, and the export of
glutathione from mature leaves was concluded to be quantitatively
negligible (Smith and Lang, 1988).
The occurrence of S deficiency was not determined by the external S
concentration alone, but also by the external N concentration. Previously, it has been suggested that S stress is reduced in plants
grown on low N, because N stress promotes an increase in the
redistribution of SO42 from
older leaves (Sunarpi and Anderson, 1997a) and stimulates the
hydrolysis of proteins and the subsequent export of insoluble S
(Sunarpi and Anderson, 1997b). In the present study there was no
evidence for the export of insoluble S from the oldest leaves during
the experiment, or for a faster rate of
SO42 export in plants grown on
low N compared with plants grown on high N. Instead, plants grown on
minus S and low N showed less S-deficiency stress symptoms, as measured
by chlorophyll, because of the reduced growth rate; high-N plants grew
faster and therefore had an increased demand for S, which was reflected
by a faster decrease in the concentration of insoluble S compared with
the youngest leaves of plants also grown on minus S but with low N. At
low N and minus S the internal concentrations of S and N were more
balanced. This balance between N and S is closely regulated and has
been reported for several plant species (Friedrich and Schrader, 1978;
Barney and Bush, 1985; Karmoker et al., 1991). The importance of the
balance between N and S is also shown in plants grown on plus S
and low N. In these plants the surplus of S resulted in the
accumulation of aliphatic glucosinolates, which contain two S atoms for
one N atom.
In summary, we conclude that neither glucosinolates nor glutathione
were the major sources of S during S deficiency in oilseed rape, and
that SO42 was by far the most
important source. The increased S-deficiency symptoms at a high
external N concentration compared with a low N concentration were
explained by higher growth rates of the youngest and middle leaves,
rather than by differences in the export of SO42 or in the remobilization
of insoluble S in the old leaves. Finally, the results reported here
show that the various S pools in different leaves responded differently
to S deficiency. These differences have to be taken into account for
the development of field diagnostic tests to determine whether plants
are S-deficient.
| |
FOOTNOTES |
1
This research was supported by the Home-Grown
Cereals Authority (grant no. 015/1/96/OS08/1/96). IACR receives
grant-aided support from the Biotechnology and Biological Science
Research Council of the United Kingdom.
*
Corresponding author; e-mail kalff{at}bbsrc.ac.uk; fax
44-1582-760981.
Received April 14, 1998;
accepted September 2, 1998.
| |
ABBREVIATIONS |
Abbreviation:
RGR, relative growth rate.
| |
ACKNOWLEDGMENTS |
The authors thank Mr. Adrian Crosland for
conducting the total-S measurements using inductively coupled
plasma-atomic emission spectroscopy, and Mr. Guy Kiddle for measuring
glucosinolates.
| |
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Top
Abstract
Introduction