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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1.30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5′-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Higher plants use inorganic sulfate as the major source of sulfur for the synthesis of sulfur-containing amino acids such as cysteine and methionine. The assimilation of sulfate is not only essential for the biosynthesis of cysteine and methionine, but also for the production of coenzymes and components of plant membranes. The final step of sulfur assimilation is the formation of cysteine from sulfide and O-acetyl- l-serine. Most sulfur containing bio-molecules present in plants are synthesised directly or indirectly from cysteine. Consequently, the formation of cysteine is the crucial step for assimilation of reduced sulfur into organic compounds ( Giovanelli et al. 1980 ; Schmidt & Jäger 1992).

In cysteine biosynthesis, an acetyl-group from acetyl-CoA is transferred to l-serine, creating the highly reactive compound O-acetyl- l-serine (OAS). This reaction, of which the precise mechanism has been initially investigated in prokaryotes at the molecular level ( Kredich 1987 and references therein), is catalysed by serine acetyltransferase (SAT). The subsequent synthesis of cysteine in plants is accomplished by the sulfhydrylation of O-acetyl- l-serine in the presence of sulfide. This reaction is catalysed by O-acetylserine(thiol)-lyase (OAS-TL, cysteine synthase, CSase; EC 4.2.99.8.). Both SAT and OAS-TL have been reported to be localised in plastids, mitochondria and cytosol of different plants ( Brunold & Rennenberg 1997; Hell 1997; Hesse et al. 1999 ), suggesting that cysteine synthesis is required in all cellular compartments where protein synthesis occurs ( Brunold & Suter 1982; Rolland et al. 1992 ; Ruffet et al. 1995 ).

Plant cDNAs encoding serine acetyltransferases have been cloned recently from Arabidopsis thaliana, Pisum sativum, Citrullus vulgaris and Spinacea oleracea ( Bogdanova et al. 1995 ; Roberts & Wray 1996; Ruffet et al. 1995 ; Saito et al. 1996 ; Saito et al. 1995 , database). In both plants and bacteria ( Kredich 1993), SAT forms a complex with OAS-TL, suggesting an efficient metabolic channeling from serine to cysteine which prevents the diffusion of the intermediate OAS ( Bogdanova & Hell 1997; Ruffet et al. 1994 ). Recently, Droux et al. (1998) investigated the SAT/O-acetylserine (thiol) lyase complex in vitro. Their results suggest that only the excess of free O-acetylserine (thiol) lyase protein is capable of catalysing cysteine formation. This contradicts with the finding that a bi-enzymatic complex is required to channel the intermediate O-acetylserine ( Bogdanova & Hell 1997).

An important feature of cysteine formation in plants is that the activity of SAT is much lower when compared to the activity of OAS-TL ( Nakamura et al. 1987 ; Ruffet et al. 1994 ). In support of this observation, SAT has been shown to be a low-abundance enzyme in comparison to OAS-TL ( Ruffet et al. 1994 ) and the availability of OAS seems to be rate-limiting for cysteine synthesis in vitro ( Neuenschwander et al. 1991 ; Saito et al. 1994a ). Furthermore, SAT is feedback-inhibited by cysteine ( Saito et al. 1995 ). Thus, evidence that SAT plays an important role in regulating cysteine biosynthesis and sulfate assimilation is considerable. Therefore, we decided to directly determine in vivo whether the SAT-catalysed reaction is indeed rate-limiting in plant cysteine biosynthesis. To this end, the wild-type, cys-sensitive, E. coli SAT cysE gene ( Denk & Böck 1987; Nakamori et al. 1998 ) was constitutively expressed in transgenic potato in order to determine the effect on cysteine biosynthesis in these plants. We also studied the levels of the tripeptide glutathione (γ-glutamylcysteinylglycine) in the transgenic lines because this compound is the main storage form of reduced sulfur in the plant kingdom and serves as the major sink for cysteine ( Alscher 1989; Rennenberg 1995).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Selection of transgenic potato plants expressing the E. coli SAT mRNA

To increase SAT activity in transgenic potato plants, a constitutive overexpression approach was used. To this end, a chimeric gene consisting of wild-type cysE from E. coli and a rbcS signal sequence was transformed into potato plants. To screen for plants expressing the E. coli SAT mRNA, leaf samples were taken from 50 independent lines kept in tissue culture. These samples were subjected to RNA gel blot analysis using a radioactively labeled E. coli SAT cDNA as probe. Five independent transformants which accumulated the highest levels of the heterologous SAT mRNA were selected for further analysis and transferred to the greenhouse. Repeated Northern analysis revealed that (also under greenhouse conditions) the transformants accumulated high amounts of transcript encoding the transgene mRNA ( Fig. 1a). The size of the transcript detected in the transgenic potato plants (approximately 1050 basepairs [bp]) correspond to the size reported for the cysE gene (of 819 bp; Denk & Böck 1987), plus that of the signal sequence of rbcS (240 bp). The analysis of SAT activity in crude leaf extracts of the transgenic potato plants showed an up to 20-fold higher activity of SAT than control plants ( Fig. 1b). Despite this strong increase in SAT activity no phenotypic changes in growth and tuber formation of the transgenic plants were visible (data not shown).

image

Figure 1. Expression of the E. coli SAT in transgenic potato plants.

(a) Accumulation of heterologous SAT mRNA was detected by Northern blot analysis of total RNA extracted from leaves of five independent transgenic (SAT-65, 26, 48, 3 and 71) and two wild-type plants (controls a and b). The blot was probed with 32P-labeled cysE DNA from E. coli. The lanes contained 15 μg of total RNA each.

(b) Maximum catalytic activity of SAT in the leaves of five independent transgenic (SAT-65, 26, 48, 3 and 71) and two wild-type plants (controls a and b). The specific activity of crude extracts is given in pmol produced CoA per min and μg total protein. Error bars represent standard deviation (< 10%). n = 4 independent measurements.

(c) Localisation of SAT in the fractions of wild-type (WT) and transgenic plants (SAT-26 and −48). Specific activity of crude extracts (ce), supernatant (s) and organelle (o) fractions are given in relative values related to the activities in crude extracts. ADP-glucose pyrophosphorylase (AGPase) and UDP-glucose pyrophosphorylase (UGPase) are measured as plastidial and cytosolic marker enzymes, respectively.

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To prove the subcellular localisation of the overproduced E. coli SAT protein, organelles were enriched to measure enzyme activity. To this end, leaves of control plants and transgenic lines SAT-26 and SAT-48 were homogenised and fractionated by centrifugation to yield an organelle fraction (pellet) and a cytosolic fraction (supernatant). These two fractions and a crude extract were assayed for serine acetyltransferase activity. The quality of purification was assessed by the measurement of selected subcellular marker enzyme activities ( Fig. 1c). UDP-glucose pyrophosphorylase (UGPase) and ADP-glucose pyrophosphorylase (AGPase) were used as marker enzymes for the cytosolic and organelle fractions, respectively. As shown in Fig. 1(c), most of the chloroplast marker activity was recovered in the pellet, while the supernatant was contaminated by chloroplast-derived enzymatic activities. The contamination of the organelle fractions by cytosolic enzyme activities was rather low. According to this, serine acetyltransferase activity was detected in both the organelle and the cytosolic fraction. However, as the activity of serine acetyltransferase was clearly appointed to the organelle fraction and the remaining activity in the supernatant was in concordance with the contaminating AGPase activity, it can be deduced that serine acetyltransferase was successfully targeted to the plastids. It can still not be excluded, however, that SAT protein is not targeted completely to chloroplasts and that a part of the activity belonged to protein remaining in the cytosol.

Expression of the wild-type cysE gene leads to an increase in the endogenous levels of cysteine and glutathione in leaves

In order to investigate whether the expression of the cysE gene in potato plants influences the endogenous levels of cysteine, the concentration of this sulfur-containing amino acid was determined in non-transformed and transformed plants. Three transformants with the highest levels of SAT activity, i.e. SAT-26, SAT-48 and SAT-65 (see Fig. 1b), were subjected to cysteine analysis. Young, green and fully expanded leaves of 6-week-old plants were harvested and the cysteine content was determined via HPLC. These measurements revealed that all three transgenic lines exhibited increased levels of cysteine ( Fig. 2a). The cysteine content of SAT-26 was 1.5-fold higher (25.19 nmol gfw−1), of SAT-48 was twofold (32.14 nmol gfw−1) and of SAT-65 was 1.3-fold higher (19.93 nmol gfw−1) than that determined for control plants (15.69 nmol gfw−1).

image

Figure 2. Endogenous levels of cysteine and glutathione in leaves and tubers.

(a) The levels of cysteine in leaves of 6-week-old transgenic (SAT-48, SAT-26 and SAT-65) and wild-type plants (control). The amounts of cysteine are given in nmol per gfw; n = 18; six independent plants per transgenic line, three independent measurements per plant. The differences between wild-type and transgenic plants analysed using Student's t-test were statistically significant (*P < 0.05).

(b) The levels of glutathione in leaves of 6-week-old transgenic (SAT-48, SAT-26 and SAT-65) and wild-type plants (control). The amounts of glutathione are given in nmol per gfw; n = 18; six independent plants per transgenic line, three independent measurements per plant. Differences between wild-type and transgenic plants analysed using Student's t-test were statistically significant (*P < 0.05).

(c) The levels of cysteine in tubers of 14-week-old transgenic (SAT-48, SAT-26 and SAT-65) and wild-type plants (control). The amounts of cysteine are given in nmol per gfw; n = 10; five independent plants per transgenic line, three independent measurements per plant.

(d) The levels of glutathione in tubers of 14-week-old transgenic (SAT-48, SAT-26 and SAT-65) and wild-type plants (control). The amounts of glutathione are given in nmol per gfw; n = 10; five independent plants per transgenic line, three independent measurements per plant.

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The increased amount of cysteine in these transgenic potato plants stimulated us to investigate the effect on glutathione biosynthesis, given that glutathione is the main storage compound of cysteine ( Rennenberg 1995). The levels of glutathione in leaves of the transgenic lines SAT-26, SAT-48 and SAT-65 were analysed via HPLC and were up to twofold higher (450–600 nmol gfw−1) compared to wild-type plants (350–400 nmol gfw−1; Fig. 2b).

Interestingly, the thiol content in tubers was not affected by modulation of the biosynthetic pathway. Tubers from control plants and the transgenic lines SAT-26, SAT-48 and SAT-65 were tested for cysteine ( Fig. 2c) and glutathione content ( Fig. 2d). Both compounds were not changed in content compared to tubers derived from wild-type plants.

Increased endogenous levels of cysteine and glutathione do not influence the expression pattern of OAS-TL isoforms

Both SAT and OAS-TL are transcriptionally regulated by cysteine ( Barroso et al. 1995 ; Takahashi & Saito 1996). To investigate whether the enhanced levels of cysteine in the transgenic potato plants expressing the cysE gene from E. coli influenced the expression pattern of the endogenous potato OAS-TL, leaf samples were taken from transgenic and non-transformed plants and subjected to RNA blot analysis. This analysis revealed that despite the increased levels of cysteine, both the cytosolic and chloroplastidic isoforms of potato OAS-TL ( Hesse & Höfgen 1998) were not altered in their steady state mRNA levels compared to the wild type ( Fig. 3a).

image

Figure 3. The expression and catalytic activity of OAS-TL.

(a) Northern blot analysis of total RNA extracted from the leaves of five independent transgenic (SAT-65, 26, 48, 3 and 71) and two wild-type plants (controls a and b). The blot was probed with 32P-labeled plastidic potato OAS-TL cDNA (p) and with a cytosolic isoform (c), respectively. The lanes contained 15 μg of total RNA each.

(b) In vitro OAS-TL activity in leaves of transgenic (SAT-48, 26 and 65) and wild-type plants (control). The specific activity of crude extracts is given in pmol produced cysteine per min and μg total protein; n = 10 independent measurements.

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Increased endogenous levels of cysteine do not influence the activity of OAS-TL

OAS-TL activity is stimulated by sulfur limitation within the cell ( Barroso et al. 1995 ; Bergmann et al. 1980 ; Passera & Ghisi 1982). In contrast, high concentrations of sulfide or cysteine decrease OAS-TL activity ( Kuske et al. 1994 ; León & Vega 1991). Given the significant changes in SAT activity and cysteine and glutathione levels, we decided to assay the activity of OAS-TL in the transgenic lines. These measurements did not show any major difference in the OAS-TL activities between the leaf extracts of wild-type and transgenic plants ( Fig. 3b). The slight differences observed in activities might be due to biological variations of the tested plants.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Several recent studies have strengthened the hypothesis that SAT plays a crucial role in plant cysteine biosynthesis ( Ruffet et al. 1994 ; Saito et al. 1994a ; Saito et al. 1995 ). To analyse the role of SAT in more detail, we chose to overexpress the enzyme from E. coli (wild-type cysE) in transgenic potato plants. Transgenic plants accumulated the E. coli SAT as a catalytically functional protein mainly, if not exclusively, in chloroplasts. Leaves from these plants contained significantly elevated amounts of cysteine and glutathione although the cys-sensitive wild-type SAT has been expressed. This result demonstrates that under normal conditions, i.e. where the sulfur supply is not limiting, the very low endogenous activity of SAT is one of the factors limiting the cysteine biosynthetic pathway, at least in the chloroplast. Interestingly, overexpression of SAT and accumulation of the enzyme in plastids did not overcome a certain threshold which might be due to the cys-sensitivity of the expressed enzyme due to high local concentrations of free cysteine. Saito et al. (1994b) demonstrated that the overexpression of the spinach cysteine synthase A (an O-acetylserine(thiol)-lyase) in transgenic tobacco plants alone had no effect on the cysteine content. Feeding experiments with isolated chloroplasts from the transgenic plants with OAS and sulfide resulted in a large increase in cysteine synthesis. Thus, OAS and sulfide limit cysteine synthesis. Furthermore, the experiment indicated that under normal conditions the level of OAS-TL is sufficient to convert OAS quantitatively to cysteine. This observation is supported by the fact that OAS-TL activity in plants is much higher than SAT activity ( Nakamura et al. 1987 ; Ruffet et al. 1994 ) and that SAT is present in low abundance in comparison to OAS-TL ( Ruffet et al. 1994 ). This study revealed no influence of cysteine and glutathione levels on the steady state mRNA amounts and only a marginal influence on activity of OAS-TL. This adds to the theory that OAS-TL does not play an important role in the regulation of plant cysteine biosynthesis.

Furthermore, these findings support the important role of OAS in the sulfur assimilation pathway. OAS serves not only as substrate for cysteine synthesis, but it is also discussed as the control metabolite for the pathway itself ( Hawkesford 2000) as supported by recent studies. Sulfur transport proteins and APS reductase are both induced in expression by OAS ( Neuenschwander et al. 1991 ; Smith et al. 1995 ; Smith et al. 1997 ; Takahashi et al. 1997 ). The fact that overexpression of SAT in transgenic potato plants results in increased cysteine and glutathione contents further implies that the accumulation of OAS might induce sulfate uptake and reduction which are necessary for cysteine synthesis. This interpretation would support the results obtained by Saito et al. (1994a) that both, reduced sulfur and OAS limit cysteine synthesis. Finally, it is worth noting that the results presented here demonstrate a direct interaction between the biosynthetic pathways of cysteine and glutathione. The increased levels of cysteine in the transgenic potato plants stimulate the biosynthesis of glutathione, leading to levels of the tripeptide, which are up to twofold higher as compared to wild-type plants. Hence, glutathione biosynthesis in potato leaves is limited to some extent by the availability of cysteine. Recent studies showing that the foliar glutathione biosynthesis in poplar and tobacco is restricted by cysteine availability agree with our findings ( Creissen et al. 1999 ; Noctor et al. 1996 ). Remarkably, the tuber system seems to be differently regulated than the leaf system. Although SAT is expressed in tubers (data not shown) no significant changes could be determined for cysteine and glutathione content. On the one hand, SAT might not be efficiently targeted to plastids in tubers and, on the other hand, activation and reduction steps of the sulfate assimilatory pathway might not be sufficient to increase the flux towards cysteine formation in tubers. Expression analysis of cytosolic and plastidial ATP-sulfurylase isoforms ( Klonus et al. 1994 ) and isoforms of OAS-TL (H. Hesse, unpublished results) revealed that these genes are less expressed in tubers, which might be the cause for the unchanged thiol content. Furthermore, nothing is known about the turnover of glutathione in tubers, the transport capacity of potato phloem or the import mechanisms into tuber parenchyma cells. Further experiments to elucidate the physiology of the tuber system in this respect must be performed.

Despite this observation made for the tuber system, the transgenic potato plants expressing the E. coli SAT might be a tool to examine whether increased levels of cysteine can lead to enhanced levels of methionine additionally to the increases in glutathione, given that cysteine is the sulfur donor of methionine ( Giovanelli et al. 1980 ). Methionine is a limiting essential amino acid in various crops. Therefore, increased levels of this amino acid would eventually lead to plants with improved nutritional qualities.

Furthermore, the transgenic potato plants, which express the bacterial SAT, will provide a favourable experimental system to analyse whether increased levels of glutathione are able to confer resistance towards various forms of stresses. In plants, glutathione plays an important role in the defence against active oxygen species, xenobiotics, heavy metals and other forms of stresses including drought and heat ( Alscher 1989; Rennenberg 1995). Additional knowledge in this respect could also be of considerable practical importance in applied biotechnological approaches. Increased resistance of plants against active oxygen species may play an important role in protecting plants against elevated atmospheric concentrations of ozone. Initial evidence in this respect has been provided in the recent publication of Blaszczyk et al. (1999) . They demonstrated for tobacco expressing both cysE isoforms, the wild-type and a cys-insensitive SAT, respectively, that enhanced SAT activities led to increased thiole contents (two- to threefold for cysteine and twofold for glutathione, which is in the same order of magnitude as described in this paper for transgenic potato plants). The higher thiole content conferred increased tolerance when plants were exposed to H2O2. Coincidentally, with results obtained for SAT expression in potato, transgenic tobacco plants expressing both isoforms of E. coli SAT in cytosol and plastids did not show a severe phenotype. Intriguingly, a severe phenotype has been described by Creissen et al. (1999) for transgenic tobacco plants expressing E. coli gshI (γ-glutamyl-cysteine synthetase) in plastids. Therefore, the question is still open as to whether the development of a phenotype can be correlated with changes in thiole content.

Moreover, an increased tolerance against xenobiotics, for example herbicides, as a result of higher glutathione levels in the plant would lead to the improvement of agricultural properties. Finally, this study may give support to approaches for ‘engineering' transgenic plants for bioremediation purposes, which show good growth characteristics on soils contaminated with heavy metals. Likewise, cadmium tolerance was achieved recently in Indian mustard by enhanced glutathione and phytochelatine concentrations due to overexpression and accumulation in plastids of γ-glutamyl-cysteine synthetase or glutathione synthetase from E. coli, respectively (Zhu et al. 1999a ; Zhu et al. 1999b ).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material and growth conditions

Solanum tuberosum cv Désirée was obtained from Saatzucht Fritz Lange KG (Bad Schwartau, Germany). Wild-type and transgenic plants were kept in tissue culture under a 16 h light/8 h dark period on Murashige and Skoog medium ( Murashige & Skoog 1962) supplemented with 2% (w/v) sucrose. In the greenhouse, plants were grown at 20°C during the light period (16 h) and 15°C during the dark period (8 h). If not indicated otherwise, for each experiment five replicates of each line of transgenic and wild-type plants of primary transformants were planted in the greenhouse. Leaf discs were taken from 6- to 8-week-old plants in the greenhouse and stored at −80°C until usage for Northern blot experiments, enzymatic activity measurements and estimation of thiol content. Leaf material was harvested from tissue of similar developmental stages in the morning until noon. Tubers were harvested from 12- to 14-week-old greenhouse plants.

Construction of a chimeric SAT gene and potato transformation

A DNA fragment encoding the mature protein of SAT from E. coli K12 (M15745) was amplified by polymerase chain reaction generating a NcoI site at its 5′-end and a XbaI site at its 3′-end (oligonucleotides for SAT: EcSAT-N: 5′-GAG AGA CCA TGG CGT GTG AAG AAC TGG AAA; EcSAT-C: 5′-GAG AGA TCT AGA TTA GAT CCC ATC CCC ATA; TIB MOLBIOL, Berlin, Germany). Amplified fragments encoding for the mature protein were ligated into a pUC18 derivative containing the rbcS signal sequence from Arabidopsis thaliana in order to provide a plant targeting sequence to chloroplasts. The fused gene product was inserted as an Asp718/XhoI fragment into an Asp718/SalI predigested binary vector ( Höfgen & Willmitzer 1990) under the control of the 35S-CaMV promotor. Transformation of the potato cultivar Désirée was performed via Agrobacterium tumefaciens according to Rocha-Sosa et al. (1989) .

RNA gel blot analysis

For RNA isolation, plant leaf material was frozen in liquid nitrogen immediately after harvest. Total RNA was extracted from the frozen material according to Logemann et al. (1987) . After denaturation at 65°C the total RNA was separated under denaturing conditions by gel electrophoresis ( Lehrach et al. 1977 ) and then transferred to nylon membranes. Northern hybridisation was performed at 65°C as described by Amasino (1986). The Northern blots were washed three times for 30 min at 55°C in 0.5 × SSC; 0.2% (w/v) SDS. For radioactive labeling of the potato OAS-TL cDNAs ( Hesse & Höfgen 1998) the ‘Multiprime DNA-labeling-Kit' (Amersham Buchler, Braunschweig, Germany) was used.

Assay of SAT activity

SAT activity was assayed as described by Toda et al. (1998) by monitoring either the disappearance of the 232 nm absorbance peak of acetyl-CoA or the appearance of the 412 nm absorbance peak of thionitrobenzoic acid according to Kredich & Tomkins (1966). This method is based on a disulfide interchange between CoA, released from acetyl-CoA during the SAT catalysed reaction, and 5,5′-dithio-bis-(2-nitro-benzoic acid). The formation of CoA was assayed in 50 m m Tris–HCl (pH 7.6) containing 1 m m 5,5′-dithio-bis-(2-nitro-benzoic acid), 1 m m EDTA, 20 m m l-serine and 100 μm acetyl-CoA. The reaction was started by the addition of 10 μl of desalted crude leaf extract (1.5 μg μ1−1 total protein in 50 m m Na-phosphate, pH 7.5), and subsequently incubated at 25°C. The production of thionitrobenzoic acid was monitored at 412 nm in a spectrophotometer (Ultraspec 2000, Pharmacia Biotech, Uppsala, Sweden) using a blank control containing all compounds except l-serine. A calibration curve was established with control solutions containing all compounds and different concentrations of CoA (0–200 nmol ml−1). The linearity of the assay was checked with different volumes of crude leaf extract, i.e. 20, 40 and 60 μl.

Assay of OAS-TL activity

O-acetylserine(thiol)-lyase activity was assayed by measuring the production of l-cysteine. Each assay was started by the addition of 5 μl crude leaf extract (1 μg μl−1 total protein in 50 m m K2HPO4/KH2PO4, pH 7.5). Reactions were conducted in 50 m m K2HPO4/KH2PO4 (pH 7.5) in the presence of 5 m m DTT, 10 m mO-acetylserine and 2 m m Na2S (total volume 100 μl) and allowed to proceed for 20 min at 25°C. They were stopped by the addition of 50 μl 20% (w/v) trichloroacetic acid, and then analysed for l-cysteine production by using the Gaitonde reagent ( Gaitonde 1967). The cysteine content was monitored at 560 nm in a spectrophotometer (Ultraspec 2000, Pharmacia Biotech, Uppsala, Sweden) using a blank control containing all compounds except O-acetylserine. The linearity of the assay was checked with 2.5 and 10 μl of added crude leaf extract.

Preparation of enriched chloroplast fractions

Young potato leaves (4-weeks-old) were homogenised in 350 m m sorbitol, 50 m m Tris/HCl (pH 7.5), and 2 m m EDTA using a Waring blendor. Intact chloroplasts were rapidly prepared and enriched by centrifugation at 4°C for 2 min at 1000 g. Chloroplast fractions were assayed for intactness under the microscope and lysed in a buffer containing 50 m m potassium phosphate (pH 7.2), 2 m m EDTA, 0.1% Triton X-100. Enzyme activities in the isolated fractions were determined immediately or extracts were frozen at −80°C until usage.

Enzyme assays

If not described otherwise, enzymes were assayed spectrophotometrically at 340 nm by measuring the coupled oxidation/reduction of NADH or NADPH in 1 ml reaction volumes.

UDP-glucose pyrophosphorylase (UGPase): The UGPase activity was assayed in 0.1 m Tris–HCl (pH 8.0), 2 m m MgCl2, 0.25 m m NADP, 2 m m NaF, 20 μm glucose-1,6-bisphosphate, 2 m m UDPgluc, 3 U ml−1 phosphoglucomutase from yeast, and 1 U ml−1 glucose-6-phosphate dehydrogenase from yeast, at 25°C. The reaction was started with 2 m m Na2P2O7 (final concentration).

ADP-glucose pyrophosphorylase (AGPase): AGPase activity was measured in a reaction assay containing 80 m m HEPES (pH 7.4), 10 m m MgCl2, 1 m m ADP-glucose, 0.6 m m NAD, 10 μm glucose-1,6-bisphosphate 3 m m DTT, 0.02% BSA, 1 U phosphoglucomutase, 2.5 U NAD-linked glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides) and tuber extract at 30°C. The reaction was initiated by the addition of sodium pyrophosphate (2 m m final concentration).

Extraction and analysis of cysteine and glutathione

Thiols were prepared as described by Rüegsegger & Brunold (1992). Separation and quantification were performed by reverse-phase HPLC after derivatisation with monobromobimane according to Newton et al. (1981) . As a modification, the reduction of disulfides was carried out with bis-2-mercaptoethylsulfone and the labeling reaction with mono-bromobimane was stopped with 15% HCl.

Frozen leaf material (120 mg) was homogenised to a fine powder and then extracted for 20 min in 1.2 ml 0.1 N HCl at 4°C. After centrifugation of the mixture at 4°C (20 min, 14 000 g), 120 μl of the supernatant were added to 200 μl of 0.2 m 2-(cyclohexylamino) ethanesulfonic acid (pH 9.3). Reduction of total disulfides was performed by adding 10 μl bis-2-mercaptoethylsulfone in 9 m m Tris–HCl, 5 m m EDTA (pH 8). After 40 min at room temperature, free thiols were labeled with monobromobimane. To achieve this, 20 μl of 15 m m monobromobimane in acetonitrile were added to the mixture and kept for 15 min in the dark at room temperature. The reaction was stopped by adding 250 μl 15% HCl. After being kept on ice and in the dark for 2 h, the reaction mixture was centrifuged again at 4°C (10 min, 14 000 g). For cysteine and glutathione analysis, the supernatant was diluted with 0.1 N HCl. The samples were analysed according to the method of Schupp & Rennenberg (1988) on a reverse phase HPLC column (C18, 250 × 4 mm, 5 μm particle size, Macherey-Nagel, Oensingen, Switzerland). A solvent system consisting of 10% (v/v) methanol; 0.25% (v/v) acetic acid, pH 3.9 (NaOH) and 90% (v/v) methanol with a flow rate of 1.5 ml min−1 was used. Chromatography was followed by fluorescence detection (excitation: 380 nm, emission: 480 nm, SFM 25 fluorescence detector, Kontron, Zürich, Switzerland). Chromatograms were quantified by integration of peak areas.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Astrid Basner for technical assistance, Astrid Blau and Romy Ackermann for the potato transformation, and Frank Huhn for tending of the greenhouse plants. We are very grateful to Josef Bergstein for carrying out the photographic work. We also thank Prof. Lothar Willmitzer and Dr Georg Leggewie for critical reading of the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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