JXB Advance Access originally published online on March 12, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 837-845, April 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Regulation of sulphate assimilation by glutathione in poplars (Populus tremulaxP. alba) of wild type and overexpressing 
-glutamylcysteine synthetase in the cytosol
Received 17 October 2003; Accepted 26 December 2003
1 Albert-Ludwigs-University of Freiburg, Institute of Forest Botany and Tree Physiology, Georges-Köhler-Allee 053, D-79110 Freiburg, Germany
2 Heidelberger Institute of Plant Sciences (HIP), Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany
* To whom the correspondence should be addressed. Fax: +49 761 2038302. E-mail: Stanislav.Kopriva{at}ctp.uni-freiburg.de
Abbreviations: APR, adenosine 5'-phosphosulphate reductase; APS, adenosine 5'-phosphosulphate; ATPS, ATP sulphurylase; GSH, glutathione; GSHS, glutathione synthetase; 
-ECS, -glutamylcysteine synthetase; OAS, O-acetylserine; OASTL, O-acetylserine (thiol)lyase; SAT, serine acetyltransferase; SIR, sulphite reductase.
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Abstract |
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Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremulaxP. alba) overexpressing
-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3–4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars. Key words: Adenosine 5'-phosphosulphate reductase, cysteine synthesis, glutathione, poplar, sulphate assimilation.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The majority of the essential element, sulphur, in living organisms is in the reduced form of organic thiols, such as the amino acids cysteine and methionine. Plants, yeast, and bacteria are primary producers of these organic thiols since they are able to reduce sulphate, the major form of sulphur in nature, to sulphide and incorporate it in an activated amino acid acceptor to build cysteine or homocysteine (Brunold, 1990; Leustek et al., 2000). The first enzyme involved in this pathway of assimilatory sulphate reduction (Fig. 1) is ATP sulphurylase (ATPS) which catalyses the activation of sulphate by ATP to adenosine 5'-phosphosulphate (APS). In plants and the majority of bacteria, APS is reduced to sulphite by APS reductase (APR) (Suter et al., 2000; Kopriva et al., 2002), whereas in fungi and some enteric bacteria APS must be phosphorylated to phosphoadenosine 5'-phosphosulphate and only this compound can be reduced to sulphite (Jones-Mortimer, 1973; Marzluf, 1996). Sulphite is further reduced by sulphite reductase (SIR). The resulting sulphide is used for cysteine biosynthesis by O-acetylserine (thiol)lyase (OASTL) using O-acetylserine, which is synthesized from serine by serine acetyltransferase (SAT). Cysteine can be used directly for protein synthesis, further metabolized to methionine, or serve as a sulphur donor for the synthesis of coenzymes (thiamine, CoenzymeA, molybdopterine) and glutathione (
-glutamyl-cysteinyl-glycine, GSH). GSH is the most abundant low molecular weight thiol in plants and plays an important role in the defence against various biotic and abiotic stress conditions and in redox buffering of the cell (Noctor et al., 1998a; May et al., 1998). Moreover, GSH is, beside S-methylmethionine, the main storage and transport form of reduced sulphur and is involved in the regulation of sulphate assimilation (Foyer and Rennenberg, 2000).
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GSH is synthesized in the cytosol and in plastids of plant cells from its constituent amino acids via the consecutive action of
-glutamylcysteine synthetase (-ECS), synthesizing -glutamylcysteine (-EC) from glutamate and cysteine, and glutathione synthetase (GSHS), adding glycine to the C-terminal end of -EC (Foyer and Rennenberg, 2000). At the cellular level, glutathione biosynthesis is limited by the availability of cysteine (Strohm et al., 1995; Noctor et al., 1996), which itself is dependent on the supply of both reduced sulphur and carbon precursor (Blaszczyk et al., 1999; Harms et al., 2000). Furthermore, GSH synthesis is regulated by the activity of -ECS which, in vitro, is frequently feedback-inhibited by glutathione (Hell and Bergmann, 1990; Schneider and Bergmann, 1995; Strohm et al., 1995). Indeed, overexpression of -ECS, but not GSHS, in hybrid poplars (Populus tremulaxP.alba) led to an approximately 3-fold increase in foliar GSH concentrations compared with wild-type plants, but did not affect cysteine levels (Noctor et al., 1996; Arisi et al., 1997). The increased GSH synthesis is independent from localization of the overexpressed protein in the chloroplast or the cytosol; all these transgenic plants share similar basic metabolism, biochemistry, and physiology with wild-type poplars (Arisi et al., 1997; Noctor et al., 1998b).
Key points of regulation of assimilatory sulphate reduction by GSH are the enzymes ATPS and APR. In experiments with Brassica napus and Arabidopsis thaliana low GSH levels in the phloem due to sulphur deprivation correlated with increased ATPS activities and mRNA levels (Lappartient and Touraine, 1996; Lappartient et al., 1999). Supplying external cysteine or GSH inhibited ATPS activity and mRNA accumulation. Buthionine sulphoximine (BSO), an inhibitor of -ECS (Fig. 1), reversed the inhibitory effect of cysteine on ATPS leading to the conclusion that GSH and not cysteine is responsible for the regulation of ATPS in response to sulphur availability. On the other hand, Bolchi et al. (1999) showed that cysteine was able to down-regulate the ATPS transcript level when supplied to sulphur-deficient Zea mays seedlings under conditions when GSH synthesis was blocked with BSO. However, different authors found APR more susceptible to a range of regulatory signals than ATPS (Lee and Leustek, 1999; Koprivova et al., 2000; Westerman et al., 2001). Significantly, H2S fumigation of Brassica oleracea led to an increase in cysteine and GSH levels in the shoot, accompanied by a decrease in APR but not in ATPS activity (Westerman et al., 2001). Indeed, externally applied cysteine and GSH to Arabidopsis root cultures decreased APR mRNA, protein, and activity while ATP sulphurylase was much less affected by the treatments. Flux control analysis revealed that APR possess more than 50% control over sulphate assimilation (Vauclare et al., 2002). When BSO was added simultaneously, cysteine had no effect on APR activity, whereas GSH decreased APR mRNA, indicating that also in Arabidopsis GSH and not cysteine is the regulatory substance of the sulphate assimilation pathway (Vauclare et al., 2002).
Investigations with transgenic poplar lines overexpressing -ECS in the cytosol, aimed at comparing the effects of increased internal GSH synthesis and externally supplied GSH on sulphate assimilation, are reported here.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant material
Experiments were performed with young wild-type and transformed hybrid poplars (Populus tremulaxP.alba, INRA clone 717 1B4) overexpressing the bacterial gene for
-ECS in the cytosol (lines ggs5, ggs11, ggs28: Noctor et al., 1996; Arisi et al., 1997). The plants were micropropagated in vitro as previously described (Strohm et al., 1995) and transferred to pots with moistened balls (2–6 mm in diameter) of burned clay (Leca Ton, Leca, Lamstedt, Germany). They were further grown in the greenhouse with a 16/8 h light/dark regime in hydroponic culture (0.3 mM Ca(NO3)2, 0.4 mM KNO3, 0.025 mM CaCl2, 0.3 mM MgSO4, 0.05 mM KCl, 0.26 mM NaH2PO4, 0.1 mM FeSO4, 0.1 mM Na-EDTA, 2 µM MnSO4, 10 µM H3BO3, 0.2 µM CuSO4, 0.2 µM ZnSO4, 0.2 µM Na2MoO4, and 40 nM CoSO4) for 6–10 weeks. For feeding of intact plants the nutrient solution also contained 1 mM glutathione pH 6.0, 1 mM buthionine sulphoximine (BSO), 1 mM O-acetylserine (OAS) or 1 mM GSH and 1 mM OAS, simultaneously.
Analysis of low molecular weight thiols
Low molecular weight thiols were analysed in leaf and root extracts as described before (Herschbach et al., 2000). About 30 mg frozen powder was transferred into precooled (4 °C) vials containing 750 µl 0.1 M HCl and 50 mg insoluble PVPP and were subsequently centrifuged for 10 min at 12 000 g and 4 °C. 120 µl aliquots of the supernatants were adjusted to pH 8.3±0.2 with 180 µl 200 mM CHES pH 9.3. Oxidized thiols were reduced by adding 30 µl 15 mM DTT for 60 min. Derivatization was performed with 45 µl 30 mM monobromobimane (Thiolyte®MB, Calbiochem, Bad Soden, Germany) for 15 min at room temperature and subsequently the bimane derivatives were stabilized with 240 µl 10% acetic acid (v/v). Bimane 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 0.1 mM Cys, 0.1 mM -EC, and 1 mM GSH in 0.1 M HCl.
Enzyme assays
Activities of ATP sulphurylase, APS reductase, sulphite reductase, and O-acetylserine(thiol)lyase were measured in the tenth leaf from the apex in transgenic and wild-type poplars. For determination of serine acetyltransferase leaves 9–11 from the apex were pooled. All leaf samples were taken between 08.00 h and 10.00 h. Extraction of enzymes, enzyme assays, and protein determination were carried out as described previously (Hartmann et al., 2000).
ATPS activity was measured using the luciferin–luciferase system to quantify the ATP produced (Schmutz and Brunold, 1982). The reaction vial contained 20 µl 0.1 mM APS, 100 µl luciferin–luciferase mixture, 5 µl 1:20 diluted extract, and 100 µl 165 µM PPi. The initial rate of ATP production was followed for 30 s with a luminometer (Lumat LB 9501, Berthold, Germany).
APR activity in extracts was measured as the production of [35S]sulphite, assayed as acid volatile radioactivity formed in the presence of 75 µM [35S]APS (15–30 kBq µmol–1) and 4 mM DTE (Brunold and Suter, 1990).
SIR activity was measured by coupling the production of sulphide with synthesis of cysteine by OASTL present in the extracts in excess (von Arb and Brunold, 1983). The enzyme assay contained 0.18 M Tricine–NaOH pH 7.4, 0.7 mM methyl viologene, 6 mM OAS pH 6.0, and 6 mM sodium dithionite (dissolved in 150 mM NaHCO3) in a volume of 1 ml. The cysteine formed was derivatized with monobromobimane, detected, and quantified by HPLC analysis.
OASTL activity was measured in an assay mixture containing 0.2 M TRIS-HCl pH 7.5, 10 mM DTE, 8 mM OAS, and 8 mM Na2S. After 10 min at 30 °C the cysteine produced was determined photometrically at 546 nm as a red ninhydrin complex.
Measurement of SAT activity was performed by a coupled assay, using the excess OASTL present in the protein extract instead of addition of purified enzyme (Nakamura et al., 1987). The reaction mixture (200 µl) contained 140 µl leaf extract (corresponding to c. 1 mg protein), 75 mM TRIS pH 8.0, 0.5 mM DTT, 10 mM serine, 2.5 mM Na2S, and 1 mM acetyl-CoA. Reactions were started by the addition of acetyl-CoA and the samples were incubated at 30 °C for 20 min. The cysteine produced was detected and quantified by HPLC analysis as described above.
cDNA cloning
The cDNA clones for ATPS (GenBank accession number AY353090
[GenBank]
), APR (AY353089
[GenBank]
), and cytosolic isoforms of OASTL (AY353092
[GenBank]
) and SAT (AY353091
[GenBank]
), were obtained by screening a cDNA library from poplar leaf RNA, constructed in Lambda ZAP phages with the ZAP-cDNA synthesis kit (Stratagene, Amsterdam, The Netherlands) with corresponding cDNA clones from Arabidopsis. SIR (AY353094
[GenBank]
) and tubulin (AY353093
[GenBank]
) cDNAs were obtained by RT-PCR amplification of total RNA from poplar leaves with oligonucleotide primers derived from conserved domains. The ATPS cDNA probe, corresponding to accession number U05218
[GenBank]
, was derived from Arabidopsis total RNA by RT-PCR amplification. The identity of the cDNA fragments was verified by sequencing. Cytosolic isoforms of SAT and OASTL were chosen for the expression analysis because, in these transgenic poplars, the cytosolic GSH pool was increased (Hartmann et al., 2003).
RNA blot analysis
The tenth leaves from the top of poplar plants were pulverized in a mortar and pestle in liquid nitrogen. Total RNA was isolated from five poplars per treatment with the RNeasy® extraction kit (Plant Mini Kit, Quiagen, Hilden, Germany) according to the manufacturer’s instruction. Electrophoresis of RNA was performed on 1% formaldehyde–agarose gels at 120 V. RNA was transferred onto Hybond-N and hybridized with 32P-labelled cDNA probes. The membranes were washed three times at different concentrations of SSC in 0.1% SDS for 20 min, the final washing step being 1x SSC, 0.1% SDS at 65 °C for 1 h, and exposed to an X-ray film (Kodak BioMax MS, Integra Biosciences, Fernwald, Germany) for 1–3 d. The autoradiograms were quantified with a densitometer GS-670 (BioRad, Munich, Germany) using the software Molecular Analyst (BioRad) and Adobe PhotoshopTM.
Determination of O-acetylserine (OAS)
100 mg tissue material was ground in liquid nitrogen to a fine powder and extracted with 1 ml 0.1 M HCl for 15 min at 4 °C while shaking. Homogenates were centrifuged twice at 15 400 g, 4 °C for 5 min. The resulting supernatants were used for further analysis. Determination of OAS was based upon derivatization with the fluorescence dye AccQ-Tag (Waters, USA). An appropriate volume of supernatant (5–20 µl) was derivatized according to the manufacturer (Manual WAT052874TP). The resulting OAS derivative was separated form other amino acid derivatives by reverse phase HPLC as described in Rolletschek et al. (2002), with the exception that the pH of buffer A was optimized to pH 6.3. Data acquisition and processing was performed with Millenium32 software (Waters, USA). Recovery rates for OAS were higher than 90% in all tested tissues as determined by spiking of samples with internal standards. All samples were analysed in triplicate.
Statistical analysis
Statistical analysis was performed using the Tukey Test (P 
0.05) or T-test (P 0.05) with SPSS for Windows, Release 9.0.
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Results |
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Effect of enhanced GSH synthesis on sulphate assimilation in transgenic poplar
The effect of enhanced GSH synthesis on the sulphate assimilation pathway was investigated using transgenic poplars overexpressing
-glutamylcysteine synthetase in the cytosol (Noctor et al., 1996; Arisi et al., 1997). Enzymatic activities and mRNA levels of ATPS, APR, SIR, OASTL, and SAT were measured in the tenth leaves of transgenic lines ggs5, ggs11, and ggs28 (Fig. 2). All investigated plant lines have about 2–3-times higher foliar GSH concentration than the wild-type plants, whereas cysteine levels are only slightly enhanced (Arisi et al., 1997). As shown in Fig. 2, the higher thiol contents did not affect the enzyme activities of APR, SIR, OASTL, or SAT; ATPS was increased in two transgenic lines. Steady-state mRNA levels for APR and SIR were not affected in any line. The mRNA level of the cytosolic isoform of OASTL was reduced in line ggs5, but comparable to the wild type in the other lines, whereas the transcript level of cytosolic SAT slightly increased in ggs11 and ggs28, but were not affected in ggs5. The increased activity of ATPS was not accompanied by a corresponding increase in mRNA levels.
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In addition, roots of the transgenic lines ggs5 and ggs28 contained GSH concentrations 2-fold higher than wild-type roots (Fig. 3A). Cysteine and
-EC contents did not differ between the transgenic and wild-type roots. As already shown for leaves, APR mRNA was accumulated in roots to the same level in the wild type and both transgenic lines and was not affected by the increased GSH concentration (Fig. 3B).
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Effect of exogenously applied GSH or BSO on sulphate assimilation in wild-type poplar
Since the well-described feedback inhibition of ATPS and APR by an increased concentration of GSH was not observed in the experiments with transgenic poplars (Fig. 2, 3), it was tested whether an increased GSH content by exogenously applied GSH or its decrease by inhibition of
-ECS with BSO (Griffith, 1982) affects the mRNA level and activity of APR. APR was chosen for these experiments since Vauclare et al. (2002) showed that APR possess a more than 50% control over the flux through the sulphate assimilation pathway. Preliminary experiments revealed that, in the leaves, the effects of GSH and BSO feeding via the roots are much smaller than in the roots, therefore further experiments were performed with the roots. Figure 4 illustrates the effects of the addition of 1 mM GSH or 1 mM BSO to the roots of wild-type poplars for 3 d. Feeding of exogenous GSH increased the GSH contents in the roots approximately 2-fold and also led to a 5-fold increase in cysteine contents (Fig. 4A). This resulted in a 50% decrease in APR activity and mRNA accumulation (Fig. 4B, C, D). The addition of BSO strongly decreased GSH levels in the roots by about 95% and also led to about a 2-fold increase in cysteine. The decrease in GSH content resulted in a big increase in APR transcript abundance (Fig. 4C, D). The changes in mRNA levels were accompanied by an approximately 4-fold increase in APR activity (Fig. 4B). These results also show, that in poplars, APR is regulated by GSH at the transcriptional level.
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OAS attenuates the feedback inhibition of APR by GSH
The simplest explanation for the contradictory results presented in Figs 3 and 4 is that, in the transgenic plants, feedback regulation of APR by GSH was overcome by another signal. O-acetylserine (OAS), a substrate for Cys synthesis, is known to regulate sulphate uptake and APR positively and, thus, would be a good candidate for such a signal. Therefore, OAS was measured in leaves and roots of wild-type and transgenic poplars and in poplars treated with GSH. OAS levels were increased 2-fold in the leaves of the transgenic line ggs5 and slightly, but not significantly, increased in lines ggs11 and ggs28 compared with wild-type levels (Table 1). Roots of the line ggs5 contained lower OAS levels compared with the wild type, whereas in the other two lines OAS contents were not significantly affected.
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In addition, wild-type poplar roots were treated with GSH and OAS alone or in combination for 72 h. These treatments doubled the concentration of GSH and elevated OAS levels 5–8-fold (Fig. 5A, B). Cysteine contents were increased 6–7-fold by all three treatments. APR activity was not significantly altered by the different treatments (Fig. 5C). As expected, the APR mRNA level was increased 4-fold by OAS treatment and decreased by 50% upon addition of GSH (Fig. 5D). Simultaneous addition of OAS and GSH resulted in APR mRNA levels between those of controls and treatments with OAS alone. However, since the roots of transgenic poplars have no feedback inhibition of APR despite high GSH concentration and constant OAS and OAS levels were not affected by the GSH treatment, OAS cannot be the only signal to overcome the feedback inhibition of APR by GSH.
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Discussion |
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Poplars overexpressing bacterial genes for
-ECS and GSHS in the cytosol or in the chloroplast are the best characterized plants with manipulated GSH content. These plants have contributed significantly to the understanding of the regulation of GSH synthesis by its constituent amino acids (Strohm et al., 1995; Noctor et al., 1996, 1997, 1998b). Due to the increased GSH concentration in the plants overexpressing -ECS (Noctor et al., 1996, 1997; Arisi et al., 1997) an additional sink for reduced sulphur was formed. However, the effects of enhanced demand for cysteine on sulphate assimilation have not been addressed in previous reports. Therefore, the enzyme activities and steady-state mRNA levels of enzymes involved in sulphate assimilation in poplars of wild type and those overexpressing the -ECS in the cytosol (Noctor et al., 1996; Arisi et al., 1997) were compared. As demonstrated in Fig. 2, neither the activities nor the mRNA levels of enzymes of the sulphate assimilation pathway were affected in the leaves of the transgenic poplars. This result shows that the capacity of sulphate reduction in poplars is sufficient to increase cysteine biosynthesis to the extent necessary to accommodate enhanced GSH synthesis. On the other hand, it was expected that the increased GSH level might feedback–regulate ATPS and APR, as described for several herbaceous plant species (Lappartient et al., 1999; Vauclare et al., 2002). Inhibition of ATPS by thiols is mediated by GSH in Brassica (Lappartient et al., 1999); however, in maize, the mechanism is different and the molecular signal is most probably cysteine (Bolchi et al., 1999). Poplar, as a tree species with a long life span and different needs for the regulation of nutrient supply than the faster growing herbaceous plants, might easily possess different regulatory elements (Herschbach, 2003) and lack this kind of feedback regulation at all. It is, therefore, significant that exogenous feeding of GSH to the roots caused the APR activity and mRNA accumulation to decrease (Fig. 4). Thus, in poplar as well, GSH can exert feedback regulation on APR and modulate the flux through the sulphate reduction pathway as described in the model of demand-driven control of sulphate assimilation (Lappartient and Touraine, 1996; Lappartient et al., 1999).
Why then were APR and ATPS not down-regulated by even higher GSH concentration in the transgenic poplar lines? The poplar lines used in the study are primary transformants continuously propagated in vitro. One could hypothesize that the plants adapted to the increased GSH levels. Such adaptation would be possible only by mutation in the promoter region of APR. However, the probability that such mutation would occur in three independent lines is extremely low. Also the subcellular compartmentation of GSH most probably cannot explain the contradictory results. In poplars overexpressing -ECS in the cytosol, the increase in total GSH could be ascribed to the increase in the cytosolic GSH pool (Hartmann et al., 2003); exogenous feeding with GSH would, primarily, increase cytosolic GSH levels.
The simplest explanation for the contradictory data would be the action of a second signal that positively influences APR mRNA accumulation and activity and overrules the negative signal of GSH. A good candidate for such a signal is OAS. OAS accumulates during sulphur starvation and was thus proposed to act as a signal of the sulphur status in seeds (Kim et al., 1999). APR activity was induced in Lemna minor by feeding OAS both in the light and in the dark (Neuenschwander et al., 1991). The addition of OAS to nitrogen-deprived Arabidopsis led to an increase in mRNA levels for APR, SIR, OASTL, and SAT-A and to higher incorporation of 35So42– into thiols and proteins (Koprivova et al., 2000). Increased synthesis of OAS due to overexpression of SAT resulted in increased cysteine and glutathione synthesis (Blaszczyk et al., 1999; Harms et al., 2000). Most importantly, OAS de-repressed sulphate transport and accumulation of mRNA for sulphate transporters and, therefore, overrode a negative transcriptional control of the sulphate transporter (Smith et al., 1997). Indeed, simultaneous feeding of GSH and OAS abolished the feedback regulation of APR by GSH and resulted in APR mRNA levels higher than in control plants (Fig. 5). Accordingly, OAS levels were increased in leaves of the transgenic line ggs5 and slightly, but not significantly, elevated in ggs11 and ggs28 (Table 1). These increased OAS contents might reflect sulphur depletion caused by the increased GSH synthesis. However, Herschbach et al. showed that, in the line ggs28, the sulphate contents in leaves and roots and sulphate uptake by the roots did not differ from the wild-type plants (Herschbach et al., 2000) and, thus, there is no indication of sulphur deficiency causing the increase in OAS content. On the other hand, OAS contents in the roots of transgenic lines, did not follow an identical pattern. While in the ggs5 line the OAS level was lower than in wild-type plants, in ggs11 and ggs28 plants the OAS concentration was not different from the wild type; nevertheless, the APR activity and mRNA level were not reduced by a highly elevated GSH content (Fig. 3). Thus, although OAS contents increased above physiological levels prevented feedback regulation of APR by GSH, OAS cannot be the only molecular signal in transgenic poplars responsible for maintaining normal APR mRNA levels at elevated GSH concentrations.
Apparently, poplars are able to distinguish between GSH synthesized in the cell and GSH transported from outside. In analogy to sugar-sensing by hexokinase, a signalling sucrose transporter, and hexose-transport-associated sensor (reviewed in Smeekens and Rook, 1997) two hypotheses may be formed. Firstly, similar to hexokinase, in addition to catalytic activity -ECS initiates a signalling cascade that activates sulphate assimilation and/or prevents feedback inhibition by GSH. When GSH levels are high enough to inhibit -ECS (Hell and Bergmann, 1990), the positive signal disappears and the feedback regulation by GSH takes place. This hypothesis is unlikely since the enzyme overexpressed in poplars is of bacterial origin and its structure is completely different from its plant counterpart (May et al., 1998), so that such a mechanism would have to be extremely well conserved evolutionarily. In the second hypothesis, GSH transport across the plasma membrane is a possible mechanism of the feedback regulation of sulphate assimilation by GSH. The postulation of a signalling GSH transporter, similar to signalling hexose and sucrose transporters in sugar sensing (reviewed in Smeekens and Rook, 1997), would explain why APR was down-regulated only by exogenously added GSH and not in the transgenic poplars. Such a mechanism would also be consistent with GSH transported from the leaves to the roots via phloem acting as a signal of the sulphur status (Lappartient and Touraine, 1996; Herschbach et al., 2000). This hypothesis is compatible with all the data presented in this report, taking into account the previous discussion of OAS as a positive signal in APR regulation. However, this hypothesis does not explain the inhibition of sulphate uptake and assimilation by feeding cysteine, which must be metabolized to GSH for this regulation (Lappartient and Touraine, 1996; Vauclare et al., 2002). Clearly, these data demonstrate that knowledge on the regulation of sulphate assimilation is still partial. The exact nature of the molecular mechanisms of the regulation of APR and other enzymes of sulphate assimilation remains to be elucidated.
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Acknowledgements |
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This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG contract No. Re 515/11) as a part of a research group FOR 383 ‘Sulphur metabolism in plants: Junction of basic metabolic pathways and molecular mechanisms of stress resistance’.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Arisi AC, Noctor G, Foyer CH, Jouanin L. 1997. Modification of thiol contents in poplars (Populus tremulaxP. alba) overexpressing enzymes involved in glutathione synthesis. Planta 203, 362–372.[CrossRef][Web of Science][Medline]
Blaszczyk A, Brodzik R, Sirko A. 1999. Increased resistance to oxidative stress in transgenic tobacco plants overexpressing bacterial serine acetyltransferase. The Plant Journal 20, 237–243.[CrossRef][Web of Science][Medline]
Bolchi A, Petrucco S, Tenca PL, Foroni C, Ottonello S. 1999. Coordinate modulation of maize sulphate permease and ATP sulphurylase mRNAs in response to variations in sulphur nutritional status: stereospecific down-regulation by L-cysteine. Plant Molecular Biology 39, 527–537.[CrossRef][Web of Science][Medline]
Brunold C. 1990. Reduction of sulphate to sulphide. In: Rennenberg H, Brunold C, De Kok LJ, Stulen I, eds. Sulphur nutrition and sulphur assimilation in higher plants. The Hague, The Netherlands: SPB Academic Publishing, 13–31.
Brunold C, Suter M. 1990. Adenosine 5'-phosphosulphate sulphotransferase. In: Lea P, ed. Methods in plant biochemistry, Vol. 3. London: Academic Press, 339–343.
Foyer CH, Rennenberg H. 2000. Regulation of glutathione synthesis and its role in abiotic and biotic stress defence. In: Brunold C, Rennenberg H, De Kok LJ, Stulen I, Davidian JC, eds. Sulphur nutrition and sulphur assimilation in higher plants: molecular, biochemical and physiological aspects. Bern, Switzerland: Paul Haupt, 127–153.
Griffith OW. 1982. Mechanism of action, metabolism, and toxicity of buthionine sulphoximine and its higher homologs, potent inhibitors of glutathione synthesis. Journal of Biological Chemistry 257, 13704–13712.
Harms K, von Ballmoos P, Brunold C, Hofgen R, Hesse H. 2000. Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione. The Plant Journal 22, 335–343.[CrossRef][Web of Science][Medline]
Hartmann T, Mult S, Suter M, Rennenberg H, Herschbach C. 2000. Leaf age-dependent differences in sulphur assimilation and allocation in poplar (Populus tremulaxP. alba) leaves. Journal of Experimental Botany 51, 1077–1088.
Hartmann TN, Fricker MD, Rennenberg H, Meyer AJ. 2003. Cell-specific measurement of cytosolic glutathione in poplar leaves. Plant, Cell and Environment 26, 965–975.[CrossRef][Medline]
Hell R, Bergmann L. 1990. -Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization. Planta 180, 603–612.[CrossRef][Web of Science]
Herschbach C. 2003. Whole plant regulation of sulphur nutrition of deciduous trees – Influences of the environment. Plant Biology 5, 215–340.[CrossRef]
Herschbach C, van Der Zalm E, Schneider A, Jouanin L, De Kok LJ, Rennenberg H. 2000. Regulation of sulphur nutrition in wild-type and transgenic poplar over-expressing gamma-glutamylcysteine synthetase in the cytosol as affected by atmospheric H2S. Plant Physiology 124, 461–473.
Jones-Mortimer MC. 1973. Mapping of structural genes for the enzymes of cysteine biosynthesis in Escherichia coli K12 and Salmonella typhimurium LT2. Heredity 31, 213–221.[Web of Science][Medline]
Kim H, Hirai MY, Hayashi H, Chino M, Naito S, Fujiwara T. 1999. Role of O-acetyl-L-serine in the coordinated regulation of the expression of a soybean seed storage-protein gene by sulphur and nitrogen nutrition. Planta 209, 282–289.[CrossRef][Web of Science][Medline]
Kopriva S, Büchert T, Fritz G, et al. 2002. The presence of an iron-sulphur cluster in adenosine 5'-phosphosulphate reductase separates organisms utilizing adenosine 5'-phosphosulphate and phosphoadenosine 5'-phosphosulphate for sulphate assimilation. Journal of Biological Chemistry 277, 21786–21791.
Koprivova A, Suter M, Op den Camp R, Brunold C, Kopriva S. 2000. Regulation of sulphate assimilation by nitrogen in Arabidopsis. Plant Physiology 122, 737–746.
Lappartient AG, Touraine B. 1996. Demand-driven control of root ATP sulphurylase activity and So42– uptake in intact canola. The role of phloem-translocated glutathione. Plant Physiology 111, 147–157.[Abstract]
Lappartient AG, Vidmar JJ, Leustek T, Glass ADM, Touraine B. 1999. Inter-organ signalling in plants: regulation of ATP sulphurylase and sulphate transporter genes expression in roots mediated by phloem-translocated compound. The Plant Journal 18, 89–95.[CrossRef][Web of Science][Medline]
Lee S, Leustek T. 1999. The affect of cadmium on sulphate assimilation enzymes in Brassica juncea. Plant Science 141, 201–207.[CrossRef]
Leustek T, Martin MN, Bick JA, Davies JP. 2000. Pathways and regulation of sulphur metabolism revealed through molecular and genetic studies. Annual Review of Plant Physiology and Plant Molecular Biology 51, 141–165.[CrossRef][Web of Science]
Marzluf GA. 1996. Molecular genetics of sulphur assimilation in filamentous fungi and yeast. Annual Review of Microbiology 51, 73–96.[CrossRef][Web of Science]
May MJ, Vernoux T, Leaver C, Van Montagu M, Inzé D. 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. Journal of Experimental Botany 49, 649–667.
Nakamura K, Hayama A, Masada M, Fukushima K, Tamura G. 1987. Measurement of serine acetyltransferase activity in crude plant extracts by a coupled assay system using cysteine synthase. Plant and Cell Physiology 28, 885–891.
Neuenschwander U, Suter M, Brunold C. 1991. Regulation of sulphate assimilation by light and O-acetyl-L-serine in Lemna minor L. Plant Physiology 97, 253–258.
Noctor G, Arisi A-CM, Jouanin L, Foyer CH. 1998b. Manipulation of glutathione and amino acid biosynthesis in the chloroplast. Plant Physiology 118, 471–482.
Noctor G, Arisi A-CM, Jouanin L, Kunert KJ, Rennenberg H, Foyer CH. 1998a. Glutathione: biosynthesis, metabolism, and relationship to stress tolerance explored in transformed plants. Journal of Experimental Botany 49, 623–647.
Noctor G, Arisi A-CM, Jouanin L, Valadier M-H, Roux Y, Foyer CH. 1997. The role of glycine in determining the rate of glutathione synthesis in poplar. Possible implications for glutathione production during stress. Physiologia Plantarum 100, 255–263.[CrossRef]
Noctor G, Strohm M, Jouanin L, Kunert K-J, Foyer CH, Rennenberg H. 1996. Synthesis of glutathione in leaves of transgenic poplar overexpressing -glutamylcysteine synthetase. Plant Physiology 112, 1071–1078.[Abstract]
Rolletschek H, Hajirezaei MR, Wobus U, Weber H. 2002. Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake. Planta 214, 954–964.[CrossRef][Web of Science][Medline]
Schmutz D, Brunold C. 1982. Rapid and simple measurement of ATP-sulphurylase activity in crude plant extracts using an ATP meter for bioluminescence determination. Analytical Biochemistry 121, 151–155.[CrossRef][Web of Science][Medline]
Schneider S, Bergmann L. 1995. Regulation of glutathione synthesis in suspension cultures of parsley and tobacco. Botanical Acta 108, 34–40.
Schupp R, Rennenberg H. 1988. Diurnal changes in glutathione content of spruce needles (Picea abies L.). Plant Science 57, 113–117.[CrossRef]
Smeekens S, Rook F. 1997. Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiology 115, 7–13.[Web of Science][Medline]
Smith FW, Hawkesford MJ, Ealing PM, Clarkson DT, van den Berg PJ, Belcher AR, Warrilow AG. 1997. Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter. The Plant Journal 12, 875–884.[CrossRef][Web of Science][Medline]
Strohm M, Jouanin L, Kunert KJ, Pruvost C, Polle A, Foyer CH, Rennenberg H. 1995. Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremulaxP. alba) overexpressing glutathione synthetase. The Plant Journal 7, 141–145.[CrossRef]
Suter M, von Ballmoos P, Kopriva S, Op den Camp R, Schaller J, Kuhlemeier C, Schürmann P, Brunold C. 2000. Adenosine 5'-phosphosulphate sulphotransferase and adenosine 5'-phosphosulphate reductase are identical enzymes. Journal of Biological Chemistry 275, 930–936.
Vauclare P, Kopriva S, Fell D, Suter M, Sticher L, von Ballmoos P, Krähenbühl U, Op den Camp R, Brunold C. 2002. Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5'-phosphosulphate reductase is more susceptible to negative control by thiols than ATP sulphurylase. The Plant Journal 31, 729–740.[CrossRef][Web of Science][Medline]
von Arb C, Brunold C. 1983. Measurement of ferredoxin-dependent sulphite reductase activity in crude extracts from leaves using O-acetyl-L-serine sulphhydrylase in a coupled assay to measure the sulphide formed. Analytical Biochemistry 131, 198–204.[CrossRef][Web of Science][Medline]
Westerman S, Stulen I, Suter M, Brunold C, De Kok JL. 2001. Atmospheric H2S as sulphur source for Brassica oleracea: consequences for the activity of the enzymes of the assimilatory sulphate reduction pathway. Plant Physiology and Biochemistry 39, 425–432.[CrossRef][Web of Science]
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