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First published online February 22, 2005; 10.1104/pp.104.057125 Plant Physiology 137:892-900 (2005) © 2005 American Society of Plant Biologists Impact of Reduced O-Acetylserine(thiol)lyase Isoform Contents on Potato Plant Metabolism1Institut für Botanik, Universität Hannover, D–30419 Hannover, Germany (A.R., J.P.); and Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Molecular Physiology, D–14476 Golm, Germany (K.R., R.H., H.H.)
Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H2S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.
Cys is the first committed molecule in plant metabolism that contains both sulfur and nitrogen, and, thus, the regulation of its biosynthesis is of utmost importance for the synthesis of a number of essential metabolites in plant pathways (Hesse et al., 2004  ). Cys is incorporated into proteins and glutathione (GSH) directly or serves as a sulfur donor for the synthesis of S-containing compounds such as Met and its derivatives S-adenosylmethionine and S-methylmethionine, and many secondary compounds such as S-methylcysteine, S-alkylcysteine, glucosinolates, and phytoalexins (Schmidt and Jäger, 1992; Ravanel et al., 1998; Matthews, 1999; Hesse and Hoefgen, 2003). Furthermore, Cys acts as a general catalyst in redox reactions through the nucleophilic properties of its sulfur atom, utilizing dithiol-disulfide interchange, as displayed in the thioredoxin and the glutaredoxin systems (Schürmann and Jacquot, 2000; Jacquot et al., 2002).
Cys is formed from two substrates, sulfide and activated Ser, as a carbon backbone and is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL), which transfers sulfide to O-acetylserine (OAS) to form Cys. The activated Ser, OAS, is synthesized by Ser acetyltransferase (SAT). In plants, OASTL has been shown to be present in 100- to 400-fold excess over SAT (Schmidt and Jäger, 1992
Cys synthesis occurs at several subcellular locations, each of which has its own enzyme isoforms, at least in Arabidopsis with its gene family consisting of seven isoforms (Jost et al., 2000
To promote our understanding of the role of the different OASTL isoforms, we initiated a transgenic approach of producing potato plants with reduced enzymatic activities for OASTL via antisense-mediated inhibition of previously cloned endogenous potato OASTL isoforms (Hesse and Höfgen, 1998 ). Several plants with respectively decreased activity could be identified. Interestingly, these plants showed no gross alterations of growth or yield under normal growth conditions. It could be demonstrated that the reduction of OASTL enzyme activity in transgenic potato plants resulted in increased thiol levels. These data provide evidence that both isoforms are able to substitute for each other in function, probably due to the transfer of S intermediates between cellular compartments, and indicate that excess OASTL is responsible for the regulation of Cys homeostasis.
Engineering and Screening of Plants with Reduced OASTL Enzyme Activity
To decrease the activity of OASTL, potato plants were transformed with the vector pBinAR harboring a cDNA encoding a sequence from either the StOASTL A or the StOASTL B gene (Hesse and Höfgen, 1998
L-Cys desulfhydrase activity was determined in the same extracts of soluble proteins used for the measurement of OASTL activity as described in Figure 2A. The L-Cys desulfhydrase activity was determined by measuring the formation of H2S from Cys (Fig. 2B). In parallel to the reduction of OASTL activity in the OASTL antisense plants, L-Cys desulfhydrase activity was reduced. In the transgenic plants carrying the OASTL antisense construct against the cytosolic OASTL, the effects were more pronounced than in the plastidial antisense plants. It was shown previously in in vitro experiments that the recombinant purified OASTL B-protein catalyzed the formation of Cys from OAS and H2S, but also the formation of H2S from Cys. In a molar ratio, the enzyme formed about 25 times more Cys than H2S per milligram protein during the same incubation time, suggesting H2S release as a side reaction of the Cys synthase reaction (Burandt et al., 2001 ). Thus, in vitro, the reaction of OASTL is a net H2S-consuming reaction. The results shown in Figure 2B indicate that, also in vitro, the OASTL proteins catalyze the release of H2S from Cys. However, in comparison to the reduction in OASTL activity (Fig. 2A), the level of reduction in H2S-releasing activity is not as strong. Therefore, one can assume that H2S-releasing activity resulted not exclusively from the side activity of OASTL proteins but additionally from other enzymes such as true L-Cys desulfhydrase proteins. The results indicate that a L-Cys desulfhydrase protein is also localized in the cytosol. To further test whether the reduced OASTL activity resulted from a decreased endogenous transcript amount, total RNA from the selected plants was isolated and screened for OASTL expression. For all investigated transgenic lines, a substantial reduction in their transcript levels in comparison to wild-type plants was observed, while RNA levels of the second, not antisensed, isoform were not affected by the manipulation (Fig. 3).
Measurements of OASTL Protein Content in Transgenic Potato Lines and OASTL Activity in a Native Gel Protein extracts from leaves of the selected lines of OASTL antisense plants were subjected to protein analysis with an antiserum directed to OASTL protein (Fig. 4A). In the wild-type potato plants, the antiserum detected proteins with sizes of approximately 34 kD and 36 kD, respectively. This is in agreement with the predicted sizes of the cytosolic OASTL and the mature plastidial OASTL proteins, respectively, as deduced from the sequence analysis. Immunoblotting revealed a slight decrease in OASTL protein levels for the plastidial antisense plants (top band) but a significant decrease in the bottom band corresponding to cytosolic OASTL isoform in comparison to control plants. As judged by the immunoblot experiments, the level of OASTL protein was correlated only to the RNA blot for the cytosolic antisense plants, indicating that repression of OASTL transcript led to a reduced availability of the corresponding mRNA for translation. Using a second antibody directed against CAS, it was revealed that the content of this protein is not affected by decreases in either OASTL. Moreover, due to the similarity between OASTL and CAS at the protein level, a cross detection of the OASTL antiserum cannot be excluded and would explain the lower reduction level of OASTL in the plastidial antisense plants.
To further demonstrate that reductions in OASTL protein levels correlate with decreases in specific OASTL activity in leaves, protein extracts were separated on a native gel and analyzed for OASTL activity (Fig. 4B). In agreement with the data obtained from RNA and protein-blot analysis, the transgenic plants showed lower activities. The other activity seen can be assigned as CAS (Fig. 4B, bottom band). Considering these results, we conclude that repression of the OASTL gene by antisense inhibition resulted in alterations of OASTL protein levels, which were in accordance to the corresponding protein quantities and enzyme activities in both transgenic lines.
The effect of a decreased expression of the OASTL gene on the amounts of thiols and Asp-derived amino acid compounds was tested. Following the commonly held assumption that the de novo synthesis of amino acids in higher plants occurs in the chloroplasts, source leaf tissues were analyzed for soluble metabolites using HPLC (Kreft et al., 2003
Cys represents a key compound with several cellular functions being a proteinogenic amino acid, sulfur donor, or part of protective metabolites (Hesse et al., 2004 ). The multitude of implementations in the cellular network makes it necessary to regulate the synthesis and cellular homeostasis tightly. Cys synthesis can be regulated at several steps (Hesse et al., 2004). Most of our understanding on this particular step is based on studies of E. coli (Kredich et al., 1969) and later studies showing that related regulatory networks are present in higher plants (Hesse et al., 2004). Recent efforts have identified the SAT/OASTL complex as a suitable regulator of Cys synthesis. The current model states that SAT is active in the complex, while OASTL is inactive, resulting in OAS formation and disruption of the complex, followed by Cys synthesis (Hesse et al., 2004). On the other hand, excess OASTL in transgenic tobacco (Nicotiana tabacum) plants revealed a more tolerant phenotype when exposed to oxidative stress but with a minimal increase in thiols under normal growth conditions (Noji et al., 2001; Youssefian et al., 2001; Sirko et al., 2004). In this paper, an antisense-mediated down-regulation of OASTL activity was used to misbalance the SAT/OASTL ratio to investigate the particular roles of OASTL isoforms in Cys formation and homeostasis. A set of transgenic plants was investigated expressing antisense RNA for the cytosolic and the plastidial isoform, respectively. Surprisingly, no effect on the growth habit of the transgenic lines could be observed, although OASTL activities were maximally reduced to 5% in crude extracts of transgenic plants. Generally, the reduction obtained with the cytosolic isoform was higher than for the plastidial one. An explanation for this result remains to be found. Constructs were made with the full-length cDNAs and the impact of antisense down-regulation cannot be predicted. However, this finding might indicate that the effectiveness of the cytosolic antisense RNA is higher than the plastidial RNA with its extension of the plastidial-targeting coding sequence, even though they share 68.1% identity at the nucleic acid level. Another explanation could be that the molar ratio between cytosolic and plastidial OASTL activity is displaced in potato toward higher cytosolic activity as occurs in pea (Pisum sativum; Lunn et al., 1990). The down-regulation of OASTL activity is clearly a result of the antisense approach. RNA-blot analysis revealed a specific reduction in OASTL RNA levels of the antisensed isoform in transgenic plants corroborated by the enzyme activity measurements, protein-blot analysis, and the zymogram.
OASTL catalyzes the incorporation of reduced sulfur into organic compounds. Modifying the enzyme activity at such a critical position in a pathway should therefore lead to alterations in metabolic turnover and affect levels of the respective biosynthetic end product. In this respect, changes in OASTL activity levels are likely to result in alterations of soluble Cys contents. Such a possibility has not been investigated in Arabidopsis or other plants. Overexpression of OASTL in Arabidopsis and tobacco has been shown to result in only moderate increases in Cys and GSH levels, though these increases led to augmented tolerance to oxidative stress or cadmium exposure (Harada et al., 2001
The increase in Cys and GSH levels in both types of antisense plants indicate that sulfur-related metabolites are exchanged between compartments, at least between chloroplasts and cytosol. Although the activity of the plastidial isoform is down-regulated, thiol levels were increased. It has to be assumed that sulfide, although very reactive, is able to cross the plastidial membrane, suggesting that both isoforms can substitute for each other (Fig. 6). Other compounds such as GEC must also cross organelle membranes, since GSH synthetase is localized in both cytosol and chloroplasts while GEC synthetase occurs only in plastids (Cobbett et al., 1998 ; The Arabidopsis Genome Initiative, 2000; Noctor et al., 2002; Hartmann et al., 2003). Through this interorganelle transfer, the plant cell is able to keep Cys homeostasis constant, although it is questionable how the cells sense the Cys content in each compartment. In this context, it is of interest why OASTL protein is in such an excess over SAT. Apparently, from the obtained results, it can be assumed that the excess of OASTL is, under normal conditions, responsible for both de novo synthesis and breakdown of Cys to keep Cys or reduced sulfur levels in balance (Fig. 6). The breakdown of Cys, the inverse reaction of OASTL (Burandt et al., 2002), results in pyruvate, ammonia, and H2S production. The latter is volatile and could possibly be used as latent natural pathogen protection. A reduced H2S-releasing capacity in the transgenic potato plants was observed; it would be interesting to investigate if these plants are less resistant to pathogen infections. An H2S-releasing activity of true Cys desulfhydrase proteins is probably induced only in certain physiological states. There are a number of Cys desulfhydrase candidates in the Arabidopsis genome such as NifS-like proteins (Leon et al., 2002). However, the percentage of the turnover of Cys to H2S catalyzed by desulfhydrases and by the side reaction of OASTL proteins is not known. The presented data provide evidence for a more complex regulation of Cys synthesis and homeostasis. The formation of the SAT/OASTL complex is likely part of the regulatory cascade. The obtained data indicate that other players are involved in the regulation of Cys biosynthesis in plants and that the current models require adjustments.
Generation of Transgenic Potato Lines
Potato (Solanum tuberosum; Saatzucht Lange AG, Bad Schwartau, Germany) OASTL isoforms (Hesse and Höfgen, 1998
Transgenic OASTL antisense plants were propagated in tissue culture along with potato wild-type plants and transferred into soil after 2 weeks of cultivation. The rooted shoots were planted in small pots and grown in the phytotron with a light regime of 200 to 250 µmol s–1 m–1 (16 h/8 h) under a hood to retain high air humidity. After 2 weeks, plants were transferred into pots with a diameter of 20 cm and cultivated in a greenhouse providing nearly natural light conditions with an approximately 16-h-light/8-h-dark period plus natural sunlight. Light intensity and temperature were dependent on environmental conditions, but light did not fall below 250 to 300 µmol photons m–2 s–1, and temperature did not sink below 18°C. Leaf material was harvested from greenhouse-grown plants after approximately 8 weeks of cultivation, before the onset of flowering. Leaf discs were excised from tissues of similar developmental stage. Transition to the reproductive stage could usually be observed only in plants older than 10 weeks. All plant material was sampled in the morning and immediately frozen in liquid nitrogen before storage at –80°C.
Total RNA was extracted essentially as described by Chomczynski and Sacchi (1987)
For the determination of protein steady-state levels, 100 mg plant material was mortared to a fine powder in liquid nitrogen. Protein estimation was done according to Bradford (1976)
Extracts of soluble proteins were prepared using 100 mg frozen plant material and 250 µL 20-mM Tris-HCl, pH 8.0. Of each sample, 150 µg protein was separated by native PAGE (8%) at a constant current of 25 mA at 4°C. After electrophoresis, the gel was immersed for 3 min at 30°C in the reaction mix (2.5 mM KCN, 2.5 mM L-Cys, and 25 mM CAPS buffer, pH 10.0), which had been prewarmed at 30°C. The incubation was stopped by adding 0.2 mM lead acetate, pH 4.0. The appearance of brown bands revealed the position of the enzymes in the gel (Akopyan et al., 1975
Extracts of soluble proteins were prepared using 100 mg frozen plant material and 1 mL 20-mM Tris-HCl, pH 8.0. The mixture was further homogenized and centrifuged for 10 min at 13,000g. The assay for OASTL activity contained in a total volume of 1 mL: 5 mM OAS, 5 mM Na2S, 33.4 mM dithiotreitol, 100 mM Tris-HCl, pH 7.5, and 50 µL enzyme extract (Schmidt, 1990
L-Cysteine desulfhydrase activity was measured by the release of sulfide from Cys. The assay for measuring L-Cys desulfhydrase activity contained in a total volume of 1 mL: 100 mM Tris-HCl, pH 8.0, 2.5 mM dithiothreitol, 0.8 mM L-Cys, and 100 µL enzyme extract. After 15 min at 37°C, the reaction was terminated by adding 100 µL of 30 mM FeCl3 dissolved in 1.2 N HCl and 100 µL 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 N HCl (Siegel, 1965
Individual soluble thiols were determined as the sum of their reduced and oxidized forms. One hundred milligrams of fresh ground leaf material was added to 100 mg of polyvinylpolypyrrolidone (previously washed with 0.1 M HCl) and 1 mL of 0.1 M HCl. The samples were shaken for 60 min at room temperature. After centrifugation (15 min at 13,000g; 4°C), the supernatants were frozen at –20°C until reduction/derivatization. Thiols were reduced by incubation with 10 mM dithiothreitol for 40 min at room temperature and derivatized for 15 min in the dark according to Hell and Bergmann (1990)
For GC-MS analysis, polar metabolite fractions were extracted from 60 mg frozen plant material and ground to a fine powder with MeOH/CHCl3. The fraction of polar metabolites was prepared by liquid partitioning into water as described earlier (Roessner et al., 2000
Soluble amino acids were determined following a modified protocol from Scheible et al. (1997)
We thank Romy Ackermann for performing the potato transformations, the gardeners for excellent greenhouse work, and Josef Bergstein for photographical assistance. We thank P. von Trzebiatowski and J. Volker for their excellent technical assistance. We are grateful to Prof. Dr. A. Schmidt, Hannover, for helpful discussions. We thank Dr. P. Burandt, Hannover, for the rescreening of the different transgenic potato lines. We thank Prof. Dr. K. Saito for providing the antibodies for SAT and Megan McKenzie for carefully revising this manuscript. Received November 23, 2004; returned for revision January 3, 2005; accepted January 3, 2005.
1 This work was supported by the Deutsche Forschungsgemeinschaft (project SCHM 307/15–3 to A.R., J.P.), the European Union (grant nos. Bio4CT 97–2182 and QLRT–2000–00103), and the Max-Planck Society (K.R., R.H., H.H.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.057125. * Corresponding author; e-mail hesse{at}mpimp-golm.mpg.de; fax 49–331–567898247.
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ABSTRACT
RESULTS
-cyanoalanine synthase (CAS) substitutes for this function (
-glutamylcysteine (GEC), and GSH increased significantly (
ex 380/
