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Modification of non-protein thiols contents in transgenic tobacco plants producing bacterial enzymes of cysteine biosynthesis pathway.

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Liszewska F., Błaszczyk A., Sirko A.

2001

vol. 48

issue 3

647-656

Conditions of achieving the maximal accumulation of sulfhydryl metabolites in the leaves of tobacco were explored. Simultaneous production of bacterial O-acetylserine (thiol)-lyase and serine acetyltransferase resulted in the increased thiols contents as compared to single transformants and controls. However, leaf discs feeding experiments differently affected thiols concentration in different plant groups and suggested that the most promising strategy to obtain plants with a high level of non-protein thiol-containing compounds might be sulfate feeding to plants overproducing serine acetyltransferase.Formation of cysteine from sulfide and O-acetyl-L-serine (OAS) is catalyzed by O-acetylserine (thiol)-lyase (OAS-TL) (EC 4.2.99.8) while OAS is synthesized by serine acetyltransferase (SAT) from acetyl-coenzyme A and serine. Molecular interactions between SAT and OAS-TL are involved in the regulation of the enzymatic activities of these proteins in bacteria (Mino et al., 2000) and in plants (Bogdanova & Hell, 1997; Droux et al., 1998). Experiments with Escherichia coli enzymes clearly indicated that OAS-TL activity was reduced to 30% in a complex (Mino et al., 2000). Similarly, a very dramatic decrease of the catalytic activity of OAS-TL bound in a complex has been reported for the plant enzymes (Droux et al., 1998). On the other hand, the bienzyme complex formation stabilizes bacterial SAT (Mino et al., 2000) and increases the apparent affinity for the substrates of plant SAT (Droux et al., 1998). The stability of the complex is negatively affected by OAS and positively by sulfide (Droux et al., 1998). Bacterial SAT is feedback regulated by cysteine, however, no relationship seems to exist between the complex formation and SAT sensitivity to this inhibition (Mino et al., 2000).Both enzymes, SAT and OAS-TL, are located in three compartments of the plant cell: the cytosol, chloroplasts and mitochondria. Different isoforms of these enzymes are in a different way regulated by sulfur nutrition (Nakamura et al., 1999; Takahashi et al., 1997; Warrilow & Hawkesford, 1998). The feedback regulation by L-cysteine of various isoforms of plant SAT has recently been studied (Inoue et al., 1999; Noji et al., 1998). According to the model proposed, the only role of the chloroplastic and mitochondrial isoforms (that are insensitive to the feedback inhibition) would be the production of OAS for cysteine biosynthesis. The cysteine-sensitive cystosolic isoform of SAT would, according to this model, have two roles: (i) OAS production for cysteine biosynthesis in the cystosol and (ii) control of OAS pool for regulatory purposes. The second postulated function is tightly connected with the fact that OAS acts as a positive regulator of genes whose expression is affected by sulfur status (Saito, 2000).Glutathione, the main low-molecular-mass thiol-containing compound in the plant cell, has multiple functions, including involvement in responses to various environmental stresses and maintenance of the redox homeostasis (Foyer & Rennenberg, 2000). Under non-stressing conditions the majority of glutathione is maintained in the reduced form (GSH) and its concentration is determined mainly by the rate of biosynthesis. GSH is produced from cysteine, glutamate and glycine in two steps catalyzed by γ-glutamylcysteinyl synthetase (γ-ECS) and glutathione synthetase (GS), respectively (Noctor et al., 1998). The biosynthesis and accumulation of GSH has been shown to depend on (i) the activity of γ-ECS, (ii) the availability of cysteine, and (iii) the light-dependent formation of glycine through the photorespiratory pathway (Foyer & Rennenberg, 2000).The main aim of this study was to identify the optimal conditions for the maximal accumulation of non-protein sulfhydryl metabolites in plant leaves. The transgenic tobacco plants with cytosolic production of bacterial enzymes of the cysteine biosynthesis pathway, SAT and OAS-TL, were obtained and analyzed for the transgenes expression. Additionally, leaf discs of either single or double transformants, as well as of control plants, were assayed for the thiol contents upon incubation in solutions of various compounds expected to have an influence on sulfur metabolism.

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

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Liszewska F., Gaganidze D., Sirko A.

2005

vol. 52

issue 1

117-128

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol) lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

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2 Acta Biochimica Polonica

1 2005

1 2001

2 Liszewska F.

2 Sirko A.

1 Błaszczyk A.

1 Gaganidze D.

Search results

Modification of non-protein thiols contents in transgenic tobacco plants producing bacterial enzymes of cysteine biosynthesis pathway.

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.