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Plant Physiol, September 2000, Vol. 124, pp. 461-474
Regulation of Sulfur Nutrition in Wild-Type and Transgenic Poplar
Over-Expressing
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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This study with poplar (Populus tremula × Populus alba) cuttings was aimed to test the hypothesis
that sulfate uptake is regulated by demand-driven control and that this
regulation is mediated by phloem-transported glutathione as a
shoot-to-root signal. Therefore, sulfur nutrition was investigated at
(a) enhanced sulfate demand in transgenic poplar over-expressing
-glutamylcysteine (-EC) synthetase in the cytosol and (b) reduced
sulfate demand during short-term exposure to H2S.
H2S taken up by the leaves increased cysteine, -EC, and
glutathione concentrations in leaves, xylem sap, phloem exudate, and
roots, both in wild-type and transgenic poplar. The observed reduced
xylem loading of sulfate after H2S exposure of wild-type
poplar could well be explained by a higher glutathione concentration in
the phloem. In transgenic poplar increased concentrations of
glutathione and -EC were found not only in leaves, xylem sap, and
roots but also in phloem exudate irrespective of H2S
exposure. Despite enhanced phloem allocation of glutathione and its
accumulation in the roots, sulfate uptake was strongly enhanced. This
finding is contradictory to the hypothesis that glutathione allocated
in the phloem reduces sulfate uptake and its transport to the shoot.
Correlation analysis provided circumstantial evidence that the sulfate
to glutathione ratio in the phloem may control sulfate uptake and
loading into the xylem, both when the sulfate demand of the shoot is
increased and when it is reduced.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Sulfur ranks fifth or sixth by
quantity of nutrient elements in plants (Cram, 1990
). In its reduced
form, i.e. in the oxidation state-II, sulfur is mainly found as a
structural and functional component of proteins. Sulfur is mostly
available to plants in the oxidized form of sulfate in the soils and
must thus be reduced and assimilated into the sulfur-containing amino
acids Cys and Met to fulfil requirements of protein synthesis. The
processes of both reduction and assimilation are often restricted to
mature leaves (Brunold, 1990), but especially in trees, considerable reduction and assimilation may also occur in the roots. The
supply of stem tissues and young developing leaves with reduced sulfur from assimilatory sulfate reduction in the roots is particularly notable in trees (Herschbach and Rennenberg, 1997).
Sulfate supply at the whole plant level may be controlled by regulation
of sulfate uptake by the roots whereas the supply to the shoot may be
determined by xylem loading of sulfate in the root. In herbaceous
plants, several pieces of evidence indicate that sulfate uptake and
subsequent loading into the xylem are regulated by phloem allocation of
glutathione. When glutathione was supplied to excised tobacco roots,
sulfate uptake and loading into the xylem stream were inhibited
(Herschbach and Rennenberg, 1991). Exposure of the shoot to atmospheric
H2S enhanced the glutathione contents of leaves
and roots and, simultaneously, diminished sulfate uptake and xylem
loading (Herschbach et al., 1995a, 1995b; Rennenberg and Herschbach,
1996; De Kok et al., 1997, 1998). Since H2S can be used as a source of reduced sulfur by the leaves (De Kok, 1990; Rennenberg and Herschbach, 1996; De Kok et al., 1997, 1998), it was
concluded from these experiments that glutathione can signal the sulfur
status of the shoot to the root (Rennenberg, 1995; Rennenberg and
Herschbach, 1995). Consistent with this view Lappartient and Touraine
(1996) found that in canola, glutathione transported via the phloem to
the roots reduced sulfate uptake by the roots. Apparently,
phloem-mediated shoot-to-root allocation of glutathione executes
demand-driven control of sulfate uptake and/or loading into the xylem
stream by the roots in herbaceous plants.
In perennial plants regulation of sulfate nutrition seems to be much
more complex. Storage and mobilization processes require seasonal
changes in the regulation of sulfate uptake and xylem loading of
sulfate that are at least partially independent of the sulfur status of
the shoot (Herschbach and Rennenberg, 1997; Schulte et al., 1998).
These processes are reflected by seasonal changes in the sulfur
composition and contents of xylem sap along the tree axis (Schupp et
al., 1991; Rennenberg et al., 1994; Schneider et al., 1994). Because of
the relatively long distances between root and shoot and the
corresponding time delay, shoot-to-root signaling via phloem transport
of glutathione may not be an exclusive signal in the control of sulfate
uptake and loading into the xylem in adult trees. This conclusion is
supported by a lack of basipetal phloem transport of glutathione fed to
the needles of spruce trees (Schupp et al., 1992; Blaschke et al.,
1996). However, basipetal phloem transport of glutathione has been
observed in seedlings of several deciduous tree species (Herschbach and
Rennenberg, 1995, 1996; Schulte et al., 1998). However, this transport
was not necessarily connected with the regulation of sulfate nutrition. In mycorrhizal and non-mycorrhizal beech, sulfate uptake by excised roots was inhibited by Cys and Met, but not by glutathione; xylem loading of sulfate was enhanced rather than decreased in beech seedlings when glutathione or Cys were fed (Kreuzwieser et al., 1996;
Kreuzwieser and Rennenberg, 1998). In excised poplar (Populus tremula × Populus alba) roots sulfate uptake and
loading into the xylem were inhibited by glutathione (Van der Zalm et
al., 2000). Hence, it has been suggested that the model of
demand-driven control of sulfur nutrition by phloem transport of
glutathione from the shoot to the root cannot easily be transferred
from herbaceous to perennial plant species (Herschbach and Rennenberg,
1997).
In the present study two experimental approaches were used to test the
model of demand-driven control. In a first approach we reduced the
sulfate demand of poplar shoots by H2S
fumigation; under these conditions significant amounts of
H2S are taken up by the leaves and are
incorporated into organic sulfur compounds, whereas assimilatory
sulfate reduction declines (Brunold, 1990; De Kok, 1990). In a second
approach the sulfate demand of poplar shoots was enhanced by
over-expression of -glutamyl-Cys (-EC) synthetase
(-ECS) in the cytosol. In contrast to recent experiments with tobacco plants that over-expressed -ECS in the
chloroplasts (Creissen et al., 1999), transgenic poplar that
over-expressed -ECS in the cytosol displayed no symptoms
of oxidative stress (Noctor et al., 1998). Over-expression of
-ECS in the cytosol may overcome Cys limitation of
glutathione synthesis and enhanced amounts of glutathione and -EC
are synthesized in the leaves (Noctor et al., 1996). The sulfate
requirement for enhanced -EC and glutathione synthesis in poplar
lines over-expressing -ECS must increase the sulfate
demand. In both experimental approaches, sulfate and reduced sulfur
compounds were analyzed in leaves, roots, phloem exudates, and xylem
saps; in addition, sulfate uptake and xylem loading of sulfate of
excised poplar roots were determined.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Developmental Stage of Wild-Type and Transgenic Poplar during H2S Exposure
Both wild-type and transgenic poplar over-expressing the bacterial
gene of -ECS in the cytosol had developed 11 to 17 leaves at the end of the fumigation experiment. Although transgenic and wild-type poplar were in the same age and from the same batch of
micropropagated trees, transgenic plants were slightly greater in shoot
fresh weight and height than wild-type plants irrespective of
H2S fumigation but were similar in root fresh
weight, shoot to root ratio, or shoot and root fresh weight to dry
weight ratio (Table I). Short-term
exposure to H2S did not significantly alter any
of the growth parameters (Table I).
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H2S Uptake, Photosynthesis, and Transpiration
Transpiration and the rate of photosynthesis were similar in both
wild-type and transgenic poplar, respectively (Table
II). Poplar shoots formed a sink for
atmospheric H2S. At 0.25 µL
L
1 the rates of H2S
uptake of wild-type and transgenic poplar were similar (Table II).
H2S fumigation did not significantly influence the rate of photosynthesis or transpiration in either wild-type or
transgenic plants (Table II).
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Sulfur Compounds in Poplar Leaves
Sulfate and reduced soluble sulfur compounds were determined in
the leaves of wild-type and transgenic poplar (Fig.
1). As previously published (Noctor et
al., 1996), Cys concentrations did not differ significantly between the
lines. Similar results were obtained for Met and sulfate. In the leaves
of transgenic poplar, -EC concentrations were 14 times and
glutathione concentrations more than twice those in leaves of the wild
type (Fig. 1) as also observed by Noctor et al. (1996). Exposure to
H2S had differing effects on concentrations of
thiols, Met, and sulfate in leaves of wild-type and transgenic poplar
(Fig. 1). Cys and Met concentrations were enhanced in the leaves of
both poplar lines to a similar extent, whereas -EC concentrations of
the leaves increased more markedly in transgenic lines as compared with
the wild type in response to H2S fumigation (Fig.
1). Similar results were observed for glutathione, but the glutathione
concentration of 1,425 ± 371 nmol g1
fresh weight in the leaves of transgenic poplar exposed
to ambient air was not significantly different from that of 1,642 ± 338 nmol g1 fresh weight in leaves of
transgenic poplar exposed to H2S. Apparently, glutathione concentrations in the leaves of the transgenic poplar were
already close to maximum without H2S exposure
(Rennenberg, 1997). Further increases in glutathione in leaves in
response to H2S may be limited either by
glutathione synthesis (Rennenberg, 1997) or glutathione export
(Herschbach et al., 1998). Sulfate concentrations of the leaves were
not affected by H2S fumigation in either
wild-type or transgenic lines (Fig. 1).
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Sulfur Compounds in Phloem Exudates
Over-expression of -ECS increased the concentrations
of -EC, glutathione, and sulfate in phloem exudates (Fig.
2) as previously found by Herschbach et
al. (1998). -EC increased more than 10-fold, glutathione 3.4-fold,
and sulfate 1.6-fold. Cys and Met concentrations in phloem exudates
were unchanged. Exposure to H2S increased
concentrations of thiols in phloem exudates but not those of Met or
sulfate (Fig. 2). Cys concentrations of phloem exudates
significantly increased in response to H2S
fumigation in both wild-type and transgenic poplar. -EC was only
detected in phloem exudates of transgenic poplar and increased 2-fold
in concentration in response to H2S exposure.
Glutathione concentrations were significantly increased in phloem
exudates of wild-type as well as transgenic poplar plants fumigated
with H2S.
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Sulfur Compounds in Lateral Roots
Compared to poplar leaves, sulfate concentrations in lateral roots
were generally greater and thiol and Met contents generally lower,
independent of the poplar line studied and the treatment applied.
-ECS over-expression increased concentrations of Cys in
roots by approximately 2-fold, of -EC by more than 10-fold, and of
glutathione by approximately 3-fold (Fig.
3). Also, Met concentrations in roots
were significantly increased by -ECS over-expression,
whereas sulfate concentrations were not (Fig. 3). Exposure to
H2S had no effect on Cys, Met, or sulfate
concentrations independent of the poplar line analyzed (Fig. 3),
whereas -EC and glutathione concentrations in the roots of
transgenic poplar were about twice those in control plants. In
wild-type poplar glutathione concentrations in roots increased
slightly, but not significantly by H2S
fumigation; -EC was not detected in wild-type poplar irrespective of
H2S fumigation (Fig. 3).
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Sulfur Compounds in Xylem Sap
Compared to wild-type poplar over-expression of -ECS
enhanced concentrations of Cys in xylem sap more than 2-fold, of -EC more than 10-fold, and of glutathione about 5-fold (Fig.
4). Also, sulfate concentrations in the
xylem sap of transgenic poplar were increased compared with the
wild-type control (Fig. 4). Exposure to H2S
doubled the concentration of Cys in xylem sap in both wild-type and
transgenic poplar (Fig. 4). H2S exposure
increased concentrations of glutathione by more than 2-fold in both
wild-type and transgenic poplar and increased concentrations of -EC
by approximately 4-fold in transgenic poplar (Fig. 4). Sulfate
concentrations in xylem sap were not affected by
H2S exposure irrespective of poplar line (Fig.
4).
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Sulfate Uptake and Xylem Loading of Sulfate
Independent of H2S exposure, over-expression
of -ECS led to a significant increase in sulfate uptake
by excised non-mycorrhizal poplar roots. Xylem loading of sulfate was
only slightly but not significantly enhanced by over-expression of
-ECS (Table III). The
proportion of sulfate taken up that was loaded into the xylem stream
was similar in wild-type and transgenic plants. Exposure of the shoot
to H2S did not change sulfate uptake by the roots either in wild-type or transgenic poplar (Table III). In wild-type but
not in transgenic poplar xylem loading of sulfate was significantly diminished upon H2S exposure (Table III).
Correspondingly, the proportion of the sulfate taken up that was loaded
into the xylem decreased significantly.
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Correlation Analysis
Glutathione concentrations in phloem exudates increased with increasing glutathione concentrations in leaves (Fig. 5). Glutathione concentrations in phloem exudates were also closely related to glutathione concentrations in the roots (Fig. 5). Remarkably, the slope of both regressions was identical. Apparently, glutathione concentrations in roots are largely determined by the production of glutathione in the leaves and transported to the roots via the phloem. For the poplar lines studied and the range of applied treatments, concentrations of glutathione in phloem exudate or roots, and sulfate uptake and loading into xylem were not related (data not shown). However, the sulfate-to-glutathione ratio in phloem exudates declined in a clear-cut, non-linear fashion with both increasing sulfate uptake and loading into xylem (Fig. 6, A and B). Correlation coefficients for exponential models of both sulfate uptake (r = 0.89) and xylem loading (r = 0.84) at P < 0.5 were strong.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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In the present study, the hypothesis of glutathione-mediated,
demand-driven control of sulfur nutrition, previously suggested for
herbaceous plants (Rennenberg, 1995; Rennenberg and Herschbach, 1995;
Lappartient and Touraine, 1996), was tested with young poplar trees.
Rates of sulfate uptake by excised non-mycorrhizal poplar roots were 3 to 4 times greater than those by excised, non-mycorrhizal roots of
beech (Kreuzwieser et al., 1996), twice as great as found for oak
(Seegmüller et al., 1996), but still significantly less than
previously reported for herbaceous plants (Herschbach and Rennenberg,
1991; Herschbach et al., 1995a, 1995b). In comparison to oak and beech,
poplar grows more quickly and may, therefore, have a greater demand for
sulfate. However, rates of loading of sulfate into the xylem were
similar in poplar (this study), oak (Seegmüller et al., 1996),
and beech seedlings (Kreuzwieser et al., 1996), suggesting that
enhanced sulfate reduction in roots may contribute to increased demand
for sulfate by poplar.
Plants can absorb and assimilate atmospheric H2S
in their shoots, a pathway that may compete with pedospheric supply as
sulfur sources for growth (De Kok et al., 1991, 1997; Stuiver and De Kok, 1997, 1998). Poplar foliage was a sink for atmospheric
H2S in the present experiments and rates of
H2S uptake by shoots of both wild-type and
transgenic poplar were quite similar. H2S
exposure generally results in a slight overload of reduced sulfur as is illustrated by increases in size and change in composition of the thiol
pool, particularly in shoots (De Kok, 1990; De Kok et al., 1997;
Stuiver and De Kok, 1997, 1998). Likewise, exposure of poplar to
H2S resulted in enhanced thiol concentrations in both wild-type and transgenic poplar as evident from increased Cys,
-EC, and glutathione concentrations in leaves (Fig. 1). Reduced
amounts of sulfate for reduction and assimilation are thus required in
leaves of poplar exposed to H2S. Since sulfate did not accumulate in poplar leaves upon exposure to
H2S (Fig. 1), as also found for other plant
species (De Kok, 1990), reduced allocation of sulfate to the leaves via
the transpiration stream may be assumed. This view was supported in the
present study by analysis of sulfate transport into and inside roots
(Table IV). Xylem loading of sulfate, but
not uptake was reduced in roots of wild-type and transgenic poplar
exposed to H2S (Table III). Apparently, upon
short-term H2S exposure, xylem loading of sulfate rather than sulfate uptake is down-regulated in response to a reduced
sulfate demand by the leaves. Similar results have been previously
obtained with herbaceous plants (Herschbach et al., 1995a, 1995b)
indicating separate regulation of sulfate uptake and loading into the
xylem stream. Potassium transport to the shoot is also regulated at the
site of loading into the xylem stream (Engels and Marschner, 1992;
Wegner and De Boer, 1997) and reduced K+
concentrations in xylem parenchyma cells are refilled by an increased K+ uptake (Wegner and De Boer, 1997).
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The shoot-derived signal responsible for regulation of sulfate
transport into and inside roots is still a matter of debate. In
herbaceous plants, glutathione or Cys fed to the roots reduced sulfate
uptake and loading into the xylem (Herschbach and Rennenberg, 1991,
1994; Lappartient and Touraine, 1996). Similar results were obtained
for sulfate transporter transcripts. Reduced expression of a
high-affinity sulfate transporter in barley was correlated with large
concentrations of Cys, glutathione, and sulfate in roots (Smith et al.,
1997). Vidmar et al. (1999) found that glutathione reduced the
transcript of the high-affinity sulfate transporter in barley. A
similar effect was observed with maize, whereas Cys rather than
glutathione reduced the transcript of the plant-specific, high-affinity
sulfate transporter (Bolchi et al., 1999). A low-affinity sulfate
transporter was expressed under sulfur deficiency in Arabidopsis in the
central cylinder, but not in the xylem, endodermis, cortex, and
epidermis (Takahashi et al., 1997). This transporter was also down-regulated by glutathione (Lappartient et al., 1999).
In the present study with poplar, glutathione, Cys, and Met
concentrations in roots increased in response to
H2S fumigation (Fig. 3). Also glutathione and Cys
concentrations in phloem sap increased under these conditions (Fig. 2)
seemingly without inhibiting sulfate uptake (Table III). Apparently
neither Cys nor glutathione regulated sulfur nutrition via sulfate
uptake when shoot demand for sulfur was reduced. However, xylem loading
of sulfate was inhibited under these conditions (Table III). Since both
Cys and glutathione concentrations were increased in xylem saps and
roots of plants exposed to H2S, we can neither
definitively identify the reduced sulfur compound responsible for
mediating inhibition of loading of sulfate into the xylem stream nor
the location of the responsible metabolic pool of that reduced sulfur
compound. However, since only glutathione concentrations in leaves and
roots strictly correlated with glutathione concentrations in phloem exudates, this tripeptide seems to be a likely candidate as a shoot-to-root signal for mediating the control of sulfur nutrition at
reduced demand. The strategy is similar for nitrogen. Increasing concentrations of amino acids (Gln, Glu, Asn, and Asp in beech and Arg
and Ala in soybean) in phloem exudates correlate with a reduced nitrate
uptake. These amino compounds are thought to be involved in
adaptation of nitrogen nutrition to the demand (Muller and Touraine,
1992; Gessler et al., 1998).
In early investigations of regulation of sulfate uptake and transport,
sulfate itself was suggested as a shoot-to-root signal (Jensén
and König, 1982; Cram, 1983a, 1983b) as demonstrated for
potassium, which regulates its own demand (Wegner and De Boer, 1997;
White, 1997). Also, in recent studies high-sulfate concentrations in
barley roots were correlated with slow rates of sulfate uptake (Smith
et al., 1997). In the present investigation, reduced concentrations of
sulfate in xylem sap and phloem exudate of plants exposed to H2S were not observed, although rates of xylem
loading were diminished. Because we cannot distinguish between sulfate
and reduced sulfur loaded into the xylem, it is possible that reduced
sulfur rather than sulfate loaded into the xylem was reduced. Also
other sulfate pools were not affected by H2S
fumigation of wild-type plants (Table IV). It may therefore be
concluded that changes in sulfate pools are not responsible for the
regulation of sulfur nutrition under conditions of reduced demand in
poplar plants.
Poplar plants over-expressing the bacterial gene of -ECS in the
cytosol have a greater demand for sulfate in the shoot as compared with
the wild type as evidenced by the observed increase in accumulation of
reduced sulfur compounds. Hence, concentrations of sulfur compounds in
different plant compartments and the rates of sulfate transport
processes were compared between transgenic and wild-type plants to test
the hypothesis of glutathione-mediated and demand-driven control of
sulfate uptake and loading into the xylem. Biomass and its distribution
among components and rates of photosynthesis did not differ between
transgenic and wild-type poplar (Tables I and II). When transgenic
poplar were exposed to ambient air, rates of sulfate uptake were
significantly increased and rates of xylem loading of sulfate were
slightly increased (Table III), reflecting increased sulfate demand of
the shoot. Concentrations of reduced sulfur compounds in phloem
exudates and in roots should be less in transgenic as compared with
wild-type plants (Table IV) if the hypothesis of glutathione-mediated,
demand-driven regulatory control is correct. Counter to this
expectation glutathione concentrations in phloem exudates, roots, and
xylem sap (Figs. 2-4; Table IV) and Cys concentrations in roots
and xylem sap of transgenic poplar were greater than those in the wild
type (Figs. 3 and 4).
We suggest the hypothesis of glutathione-mediated, demand-driven
control of sulfate nutrition cannot be applied under conditions of
enhanced sulfate demand. Since sulfate concentrations in phloem exudates, roots, and xylem sap were greater in transgenic than wild-type poplar, sulfate can also be excluded as the regulatory signal
of enhanced demand (Table IV). These results are consistent with
conclusions drawn from experiments on the regulation of sulfate uptake
with barley (Smith et al., 1997) that include suggestions reduced
sulfur may act as a negative metabolic regulator, in contrast to
positive regulation of sulfate uptake exerted by other compounds than
reduced sulfur. A comparable, but reversed mechanism was suggested for
potassium. Enhanced K+ demand of the shoot seems
to be signaled by a reduced K+ transport in the
phloem (Wegner and De Boer, 1997). As a consequence xylem loading of
K+ by a transporter sensitive to abscisic acid
was increased (De Boer, 1999). Abscisic acid reduced
K+ transport from xylem parenchyma cells into
xylem and stimulates K+ uptake into xylem
parenchyma cells (Roberts, 1998; De Boer, 1999). In studies on the
regulation of sulfur nutrition hormonal compounds were not yet
investigated and therefore cannot be excluded as additional regulatory factors.
Also in the present study, sulfate uptake and loading into xylem seem to be regulated to the needs of the shoot irrespective of demand. Any signal that reduced xylem loading of sulfate at reduced demand, (e.g. as a consequence of H2S exposure of the shoot), was counteracted by another signal that stimulated both sulfate uptake and loading into the xylem at enhanced demand (e.g. in transgenic plants with enhanced thiol synthesis). From the present results it appears that the sulfate-to-glutathione ratio in the phloem rather than the concentration of the individual compounds best reflects both reduced and enhanced sulfate demand of the shoot (Fig. 6). This ratio declined non-linearly with both increasing sulfate uptake and increasing xylem loading of sulfate independent of H2S fumigation and independent of the poplar line studied. While these results provide circumstantial evidence (rather than a casual relationship), further investigations are required to test whether the sulfate-to-glutathione ratio can be considered the dominant shoot-to-root signal controlling sulfate uptake and loading into the xylem.
Finally, since nitrogen and sulfate assimilation may be coordinated by
the Cys precursor O-acetyl-Ser (OAS; Ostrowski and Kredich,
1989; Brunold, 1993) we cannot exclude the possibility that enhanced
sulfate assimilation in transgenic poplar stimulates nitrogen
assimilation and, as a consequence, sulfate uptake. This possibility is
supported by the observation that the concentrations of total free
amino acid, mainly Gln, is generally greater in transgenic poplar as
compared with wild-type plants (Noctor et al., 1997). The expression of
a high-affinity, sulfate transporter is down-regulated at
high-glutathione, -Cys, and -sulfate concentrations but this reduction
is greatly counteracted by increased concentrations of OAS (Smith et
al., 1997). Hence OAS is also a likely candidate for over-ruling the
signal inhibiting xylem loading of sulfate in poplar under conditions
of reduced demand. The finding that OAS stimulates sulfate uptake by
excised mycorrhizal beech roots is consistent with this view
(Kreuzwieser and Rennenberg, 1998) and further studies on the role of
OAS in the regulation of sulfur nutrition are urgently required.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Plant Material
The present experiments were performed with two, independent
lines of wild-type poplar and two transgenic (Populus
tremula × Populus alba ggs28 and ggs11)
that over-expressed the bacterial gene -ECS in the
cytosol (Strohm et al., 1995; Noctor et al., 1996; Arisi et al., 1997).
Since similar results were obtained with different lines, data for one
wild-type and one transgenic line (ggs28) are shown. Transgenic and
wild-type poplar were micropropagated and cultivated under sterile
conditions. After 4 weeks, cuttings were transferred into a soil
mixture and grown in a greenhouse under long-day conditions in pots of
10 cm in height, length, and width (Strohm et al., 1995). The soil
mixture consisted of 1 part of silica sand, particle size 0.06 to 0.2 mm, 1 part of sterilized commercial soil, and 2 parts of perlit
(Agriperl, Perlite-Dämmstoff-GmbH, Dortmund, Germany). Plants
were fertilized every 2 weeks with 200 mL of a 3 g
L1 solution of a commercial fertilizer (Hakaphos blau,
COMPO GmbH, Munster, Germany; as declared by the manufactory the
fertilizer contained: 15% [w/w] N, 10% [w/w]
P2O5, 15% [w/w] K2O, 2%
[w/w] MgO, 0.01% [w/w] B, 0.02% [w/w] Cu,
0.05% [w/w] Fe, 0.05% [w/w] Mn, 0.001%
[w/w] Mo, and 0.015% [w/w] Zn). After 5 to 8 weeks of further growth, six to eight wild-type and transgenic plants were
exposed either to ambient air or to H2S.
H2S Fumigation
Nine- to 12-week-old poplar plants were exposed for 48 h to
0.25 µL L1 H2S or ambient air as described
by Van der Kooij et al. (1997). Transgenic and wild-type poplar from
the same batch were either exposed to H2S or ambient air
during a 3-week period of the experiments. Before treatments were
started the soil of the pots was covered with one layer of parafilm
and, subsequently, with aluminum foil. Poplar plants were fumigated in
0.185-m3 size cylindrical (diameter 0.65 m) stainless
steel cabinets with polycarbonate tops. The air temperature was
controlled by adjusting the cabinet wall temperature. The air flow was
2.28 m3 h1, and the air inside the cabinet
was mixed by two fans placed at the bottom (59 m3 h1 each). To avoid chamber effects, poplar plants from
each treatment, ambient air or H2S fumigation, were
exchanged after 24 h between the fumigation cabinets. Pressurized
H2S (1,000 µL L1 in nitrogen, Hoekloos, The
Netherlands) was injected into the incoming air stream by electronic
mass flow controllers (ASM, type AFC-260, Bilthoven, The Netherlands).
The photoperiod was 16 h at a photon flux density of 380 ± 20 µmol m2 s1 (within the 400-700 nm
range) with a Phillips HPL(R) N 400 W (Phillips, Eindhoven, The
Netherlands) as light source. Day and night temperatures were
approximately 20°C and approximately 18°C, respectively, and the
relative humidity was 60% to 70%.
Determination of H2S Deposition, Transpiration, and Photosynthesis
The rates of H2S uptake, transpiration, and
photosynthesis were derived from measurements of the differences in
H2S, water, and CO2 concentrations between
outlet and the inlet port of a fumigation and reference cabinet
(containing pots without plants), rates of air flow through the
cabinet, and the shoot weight as described previously (De Kok et al.,
1991; Van der Kooij et al., 1997; Van der Kooij and De Kok, 1998).
H2S concentrations were monitored with a SO2
analyzer (model 9850) equipped with a H2S converter (model
8770, Monitor Labs, Lear Siegler Measurement Controls Corporation,
Englewood, CO). Measurements were corrected for controls containing
pots with detached plants. Water and CO2 concentrations
were measured with an infrared gas analyzer (ADC 225 MK2, Hoddesdon, UK).
Collection of Phloem Exudate
Phloem exudates were collected from slices of stem bark from six
to eight wild-type and transgenic poplar plants from each treatment as
described by Herschbach et al. (1998). Bark slices of approximately 150 mg fresh weight (1-2 cm2, 0.5-1.5 mm thick) were
separated from the wood, washed in 2 mM EDTA, and allowed
to equilibrate in different incubation solutions at 4°C. The
incubation solution for thiols, i.e. Cys, -EC, and glutathione,
contained 2 mM EDTA and 1 mM cyanide at pH 5.8. To prevent destruction of thiols by reactions with phenolic compounds, polyvinypolypyrrolidone (PVPP, Sigma, Deisenhoven, Germany) was added
at a PVPP to bark fresh weight ratio of 2. For Met exudation bark
slices were incubated in 2 mM EDTA, pH 6.8. Sulfate was
measured in phloem exudates from bark slices equilibrated in distilled water. Patterns of exudation of Suc were determined as a control and
were independent of the equilibration solution applied (data not
shown). After 5 h, exudation was nearly complete as indicated by
the release of Suc from the bark slices (Herschbach et al., 1998). From
previous experiments, contamination of the phloem sap can be neglected
under the experimental conditions applied (Schneider et al., 1996;
Herschbach et al., 1998).
Xylem Sap Sampling
Xylem sap was collected from poplar shoots by the modification
of the pressure chamber technique of Scholander et al. (1965) described
by Rennenberg et al. (1996). Poplar shoots were cut 2 to 5 cm above the
ground. Bark and cambium were removed at a length of 20 mm from the cut
end. Shoots were fitted into the pressure chamber (Soil Moisture, Santa
Barbara, CA) with 10 mm of the cut end protruding. Subsequently, the
pressure in the chamber was raised slowly and the cut end was observed
with a dissecting microscope. The pressure at which xylem sap first
appeared at the cut end was recorded as the actual shoot water
potential, and the initial exudate was discarded to avoid
contamination. The pressure was then raised to 0.5 MPa over shoot water
potential and kept constant for the following 2 min. The exuding xylem
sap was collected in Eppendorf caps, frozen under liquid nitrogen, and
stored until analysis at 
80°C. Contamination with cellular compounds was measured by ATP analysis as described by Schneider et al.
(1996) and was less than 1%, as previously reported for other plant species.
Collection of Leaves and Roots
For reduced sulfur and sulfate analysis, two young, fully
expanded leaves were selected from each poplar plant. Lateral roots were washed in water to remove sand, soil, and perlite particles. Both
leaves and roots were frozen in liquid nitrogen and stored at 80°C
until analysis.
Analysis of Thiols
Thiols in phloem exudates were analyzed as previously described
by Herschbach et al. (1998). For this purpose, phloem exudates were
centrifuged at 16,000g and 4°C for 10 min. Aliquots of
300 µL of the supernatant were adjusted to pH 8.3 ± 0.2 by
adding 100 µL of 1 M CHES
(2-[cyclohexylamino]-ethansulfonacid), pH 8.4. Reduction of thiols
was initiated by addition of 20 µL of 15 mM dithiothreitol (DTT) and terminated after 60 min by addition of 30 µL
of 30 mM monobromobimane (mBBr) for derivatization. After 15 min, derivatization was stopped by acidification with 50 µL of
30% (v/v) acetic acid to stabilize mBBr-thiol derivatives. Aliquots of
100 µL were used for HPLC analysis. Thiols in xylem sap were analyzed
according to Schupp et al. (1991). For this purpose, 50 µL of
distilled water and 50 µL of 1 M CHES, pH 8.5, was added
to 50 µL of xylem sap. Thiols were reduced by addition of 10 µL of
15 mM DTT, incubated for 60 min, derivatized by addition of
15 µL of 30 mM mBBr, incubated for 15 min, and finally
stabilized by addition of 150 µL of acetic acid (10%, v/v). Aliquots
of 100 to 200 µL were used in HPLC analysis. Thiols in leaves and
roots were homogenized under liquid nitrogen and extracted as described by Strohm et al. (1995). Approximately 50 mg of frozen powder were
transferred into precooled (4°C) vials containing 1.5 mL of 0.1 N HCl and 80 mg of insoluble PVPP. Samples were centrifuged at 16,000g and 4°C for 15 min. Aliquots of 120 µL of
the supernatant were adjusted to pH 8.3 ± 0.2 with 180 µL
of 200 mM CHES, pH 9.3. Oxidized thiols were reduced for 60 min by adding 30 µL of 5 mM DTT. Derivatization was
performed with 20 µL of 30 mM mBBr for 15 min.
Subsequently, thiol derivatives were stabilized with 80 µL of acetic
acid (30%, v/v). Aliquots of 15 to 150 µL were used for to HPLC
analysis. Thiol derivatives were separated and quantified by
fluorescence detection as described by Schupp and Rennenberg (1988).
Peaks were identified and quantified using a standard solution
containing 0.2 mM Cys, 0.1 mM -EC, and 1 mM glutathione in 0.01 M HCl.
Met Analysis
For Met analysis in phloem exudates 1.3-mL aliquots were
freeze-dried (Herschbach et al., 1998). The dried material was
resuspended with 100 µL of 0.2 M sodium citrate buffer,
pH 3.35, and 70-µL aliquots were analyzed using an amino acid
analyzer (Biochrom, Pharmacia LKB, Freiburg, Germany). Aliquots of 50 µL of xylem sap were analyzed directly for Met. Met in tissue samples
was extracted according to Winter et al. (1992). For this purpose, 500-mg aliquots of powdered tissue were homogenized in 0.6 mL of HEPES
(4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid) buffer (20 mM HEPES, 5 mM EGTA, and 10 mM NaF,
pH 7.0) plus 5 mL of methanol:chloroform (3.5:1.5, v/v). The homogenate
was incubated at 4°C for 30 min. Met was extracted twice with 3 mL of
double-distilled water. Aqueous phases were combined, freeze-dried
(Alpha 2-4 and LDC-1 M, Christ, Osterode, Germany) and dissolved in
100 µL of 0.2 M sodium citrate buffer, pH 3.35. A 70-µL
aliquot of each sample was analyzed using the amino acid analyzer. Met
was separated on a cation exchange column (PEEK column, 100 × 4.6 mm, Laborbedarf und Analysetechnik Karin Grüning, Olching,
Germany) using a sodium citrate pH gradient. The flow of 0.2 M sodium citrate was 16.1 mL h1 and the pH
increased from 3.35 to 4.25 within 24 min. Thereafter, the column was
regenerated within 8 min using 0.4 M NaOH, 2.7 mM EDTA, and equilibrated within 19 min with 0.2 M sodium citrate at pH 3.35. Separated Met was derivatized,
post-column, with ninhydrin. The absorption of ninhydrin derivatives
was recorded at 570 nm. Peaks were identified and quantified using a
standard solution containing 500 µM Met.
Sulfate Analysis
Aliquots of 1.5 mL of phloem exudate or xylem sap were incubated
for 60 min with 20 mg of PVPP at 4°C and analyzed for sulfate by
anion-exchange chromatography with an automatic ion analyzer (DX 100, Dionex, Idstein, Germany). Sulfate was extracted from tissue samples
powdered under liquid nitrogen in a mortar. Aliquots of 150 mg were
suspended in 2 mL of twice-distilled water containing 20 mg of
insoluble PVPP to remove phenolic compounds. After shaking for 1 h
at 4°C, samples were boiled for 15 min, and centrifuged for 5 min and
then for 10 min, at 16,000g and 4°C (Centrifuge 5402, Eppendorf, Engelsdorf, Germany). The clear supernatant was used for
sulfate analysis by anion-exchange chromatography. In all samples
anions were separated on a IonPac column (AS9-SC, 250 × 4 mm;
Dionex) eluted with a mixture of 1.8 mM
Na2CO3 and 1.7 mM
NaHCO3 at a flow rate of 1.1 mL min1. Sulfate
was detected by a conductivity detector module (CDM, Dionex).
Uptake and Xylem Loading of Sulfate
Uptake and xylem loading of sulfate were measured as described
by Herschbach and Rennenberg (1991). Poplar roots were washed with
water to remove sand, perlite, and soil particles. Then roots were cut
with a razor blade under transport medium consisting of 5 mM bis-tris-propane buffer pH 7.0, 0.5 mM CaCl2, and 0.1 mM
K2SO4. To measure uptake, xylem loading, and
exudation of 35SO42 with excised
roots, we used the modification of the incubation chamber of Pitman
(1971) described by Herschbach and Rennenberg (1991). Six poplar roots
were placed horizontally in the incubation chamber. The cut ends were
bathed in 10 mL of transport medium in the exudation compartment, and
root tips in 85 mL of transport medium, containing 0.1 mM
K2[35S]O4, in the uptake
compartment. These two compartments were separated by a third (buffer
compartment) filled with 9 mL of transport medium. The roots were fixed
between the compartments with plastibase (Bristol Meyers Squibb GmbH,
Regensburg, Germany). The uptake compartment was stirred, and each
compartment was covered with slides. For equilibration, roots were
pre-incubated with transport medium for 2 h followed by 4 h
of exposure to [35S]sulfate. The transport medium in all
compartments was renewed immediately before labeled sulfate was added
as carrier-free [35S]sulfate (Amersham, Hertogenbosch,
The Netherlands; 0.9 × 104 to 1.6 × 104 kBq). The final concentration of sulfate was 0.1 mM with 1.1 × 106 to 1.9 × 106 kBq mmol1. After a 4-h incubation at room
temperature uptake and xylem loading was terminated. For this purpose,
three 0.5-mL aliquots of transport medium from each compartment were
transferred into scintillation vials. Subsequently, the incubation
compartment was washed 3 times for 20 s each time with 50 mL of
transport medium without
[35S]SO42. Root segments from
each chamber compartment were then cut with a razor blade and were
transferred separately into scintillation vials.
Analysis of 35S
For liquid scintillation counting 4 mL of scintillation fluid (OptiPhase HiSafe 2, Wallac Oy, Turku, Finland) was added to the sampled transport media. Root samples were digested in 3 mL of tissue solubilizer (Soluene 350, Canberra Packard, Frankfurt) for 2 d at 70°C with a maximum of 150 mg fresh weight per sample. Subsequently, samples were bleached with 200 to 400 µL of H2O2 (30%-35%, v/v) after addition of 200 µL of isopropanol. After 1 d at root temperature 10 mL of liquid scintillation fluid (OptiPhase HiSave 3, Canberra Packard) was added. 35S was detected by liquid scintillation counting (2000 CA, Tri-CARB, Packard Instruments, Chicago).
Data Analysis
Net uptake of sulfate into the roots was calculated as the sum
of radioactivity in each root segment plus the radioactivity exported out of the cut end of the roots (Herschbach and Rennenberg, 1991). Radioactivity in the root segments of the exudation and the
buffer compartment plus the radioactivity in the solution of the
exudation compartment was defined as the amount of "sulfate loaded
into the xylem." Experiments were performed with six to eight poplar
plants each. Linear and exponential fits were performed with Microcal
Origin (Microcal Software Version 5.0, Northampton, MA).
Statistical analysis was performed using the Duncan's multi-factorial analysis with SPSS (SPSS for Windows, Release 7.0, Chicago) or Student's t test.
| |
ACKNOWLEDGMENTS |
|---|
We thank Dr. Monika Schulte and Ulrike Heizmann for technical support during the experiments and Prof. Mark Adams for critical reading of the manuscript. The skillful technical assistance of Tanja Hartmann, Ulrike Hanemann, and Tatja Dopatka is gratefully acknowledged.
| |
FOOTNOTES |
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Received February 7, 2000; accepted May 31, 2000.
1 This work was supported by the Deutsche Forschungsgemeinschaft (contract nos. Re 515/6 and He 3003/1).
* Corresponding author; e-mail herschba{at}uni-freiburg.de; fax 49-761-203-8302.
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LITERATURE CITED |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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-glutamylcysteine synthetase, but not of glutathione synthetase elevates glutathione allocation in the phloem of transgenic poplar (Populus tremula × Populus alba) trees.
Plant Cell Physiol
39: 447-451
influx in spring wheat roots.
Physiol Plant
55: 459-464
uptake in intact canola: the role of phloem-translocated glutathione.
Plant Physiol
111: 147-157
[Abstract] uptake by various phloem-translocated amino acids in soybean seedlings.
J Exp Bot
43: 617-623
-glutamylcysteine synthetase.
Planta
202: 357-369
[CrossRef][Web of Science]-glutamylcysteine synthetase.
Plant Physiol
112: 1071-1078
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-Glutamylcysteine Synthetase in the Cytosol as
Affected by Atmospheric H2S
, ambient air;
, H2S) and transgenic poplar (
,
ambient air; 
, H2S) were exposed for 48 h
to ambient air or 0.25 µL L
, ambient
air; 
)
independent of the treatment and of the poplar line used. Linear fits
of the glutathione concentration in phloem exudates against the
glutathione concentration (a) in the leaves (straight line) with
y = 
2 = 6.5 and r2 = 0.795 (A) and y = 1.9 + 10.9e(
). FW, Fresh weight.