Impact of reduced O-acetylserine(thiol)lyase isoform contents on potato plant metabolism

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, H(2)S-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.     

Overproduction of SAT and/or OASTL in transgenic plants: a survey of effects

The last steps of cysteine biosynthesis are catalysed by a bi-enzyme complex composed of serine acetyltransferase (SAT) and cysteine synthase, also called O-acetyl-serine (thiol) lyase (OASTL). SAT is responsible for the production of O-acetyl-serine (OAS) from serine and acetyl-coenzyme A, while OASTL catalyses the formation of cysteine from OAS and hydrogen sulphide. Several distinct nuclear genes for SAT and OASTL enzymes exist in plants. Products of these genes are targeted into at least three cellular compartments: cytosol, chloroplasts, and mitochondria. The SAT and OASTL enzymes are strongly evolutionary conserved, both structurally and functionally. Therefore, isoenzymes from various cellular compartments can be substituted, not only by their plant counterparts from the other cellular compartments but also by their bacterial homologues. During the last decade transgenic plants overproducing SAT, OASTL or both enzymes simultaneously were obtained independently by several research groups. These manipulations led not only to the elevated levels of the respective products, namely OAS and cysteine, but also to increased amounts of glutathione and changes in the levels of other metabolites and enzymatic activities. In several cases, the transgenic plants were also shown to be less susceptible to applied abiotic stresses. In this review, all published and some unpublished results from this laboratory related to heterologous overproduction of SAT and OASTL in transgenic plants are discussed and summarized.     

Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation

Reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide and hydroxyl radicals are generated through normal biochemical processes, but their production is increased by abiotic stresses. The prospects for enhancing ROS scavenging, and hence stress tolerance, by direct gene expression in a vulnerable cell compartment, the chloroplast, have been explored in tobacco. Several plastid transformants were generated which contained either a Nicotiana mitochondrial superoxide dismutase (MnSOD) or an Escherichia coli glutathione reductase (gor) gene. MnSOD lines had a three-fold increase in MnSOD activity, but interestingly a five to nine-fold increase in total chloroplast SOD activity. Gor transgenic lines had up to 6 times higher GR activity and up to 8 times total glutathione levels compared to wild type tobacco. Photosynthetic capacity of transplastomic plants, as measured by chlorophyll content and variable fluorescence of PSII was equivalent to non-transformed plants. The response of these transplastomic lines to several applied stresses was examined. In a number of cases improved stress tolerance was observed. Examples include enhanced methyl viologen (Paraquat)-induced oxidative stress tolerance in Mn-superoxidase dismutase over-expressing plants, improved heavy metal tolerance in glutathione reductase expressing lines, and improved tolerance to UV-B radiation in both sets of plants.     

Reactive Oxygen Species and Antioxidants in Plants: An Overview

Plants exposed to biotic and abiotic stresses generate more reactive oxygen species (ROS) than their capacity to scavenge them. Biological molecules are susceptible to attack by ROS, including several proteins, polyunsaturated fatty acids and nucleic acids. The cellular arsenal for scavenging ROS and toxic organic radicals include ascorbate, glutathione, tocopherol, carotenoids, polyphenols, alkaloids and other compounds. Enzymatic antioxidants including superoxide dismutase, peroxidase, catalase and glutathione reductase detoxify either by quenching toxic compounds or regenerating antioxidants involving reducing power. Various aspects relating to sensors for ROS and signaling role of ROS in plants, improvement of antioxidant systems in transgenic plants and functional genomics approaches used to unravel the reactive oxygen gene network has been discussed.     

Mutational analysis of O-acetylserine (thiol) lyase conducted in yeast two-hybrid system

Cysteine biosynthesis, achieved by the sequential reaction of two enzymes, serine acetyltransferase and O-acetylserine (thiol) lyase (OASTL), represents the final step of sulfur assimilation pathway in plants and bacteria. The two enzymes form a bi-enzymatic cysteine synthase complex through specific protein-protein interactions. To identify the amino acids important for cysteine synthase complex formation, several mutations in bacterial OASTL were designed. Effects of mutagenesis were verified in a yeast two-hybrid model that allowed monitoring both, protein-protein interactions and the enzymatic activity of OASTL.     

Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase

We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.     

An Ipomoea batatas Iron-Sulfur Cluster Scaffold Protein Gene,IbNFU1, Is Involved in Salt Tolerance

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.     

Dominant-negative modification reveals the regulatory function of the multimeric cysteine synthase protein complex in transgenic tobacco

Cys synthesis in plants constitutes the entry of reduced sulfur from assimilatory sulfate reduction into metabolism. The catalyzing enzymes serine acetyltransferase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL) reversibly form the heterooligomeric Cys synthase complex (CSC). Dominant-negative mutation of the CSC showed the crucial function for the regulation of Cys biosynthesis in vivo. An Arabidopsis thaliana SAT was overexpressed in the cytosol of transgenic tobacco (Nicotiana tabacum) plants in either enzymatically active or inactive forms that were both shown to interact efficiently with endogenous tobacco OAS-TL proteins. Active SAT expression resulted in a 40-fold increase in SAT activity and strong increases in the reaction intermediate OAS as well as Cys, glutathione, Met, and total sulfur contents. However, inactive SAT expression produced much greater enhancing effects, including 30-fold increased Cys levels, attributable, apparently, to the competition of inactive transgenic SAT with endogenous tobacco SAT for binding to OAS-TL. Expression levels of tobacco SAT and OAS-TL remained unaffected. Flux control coefficients suggested that the accumulation of OAS and Cys in both types of transgenic plants was accomplished by different mechanisms. These data provide evidence that the CSC and its subcellular compartmentation play a crucial role in the control of Cys biosynthesis, a unique function for a plant metabolic protein complex.     

Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants.

Cysteine (Cys) synthase [O-acetyl-L-Ser(thiol)-lyase, EC 4.2.99.8; CSase] is responsible for the final step in biosynthesis of Cys. Transgenic tobacco (Nicotiana tabacum; F(1)) plants with enhanced CSase activities in the cytosol and in the chloroplasts were generated by cross-fertilization of two transformants expressing cytosolic CSase or chloroplastic CSase. The F(1) transgenic plants were highly tolerant to toxic sulfur dioxide and sulfite. Upon fumigation with 0.1 microL L(-1) sulfur dioxide, the Cys and glutathione contents in leaves of F(1) plants were increased significantly, but not in leaves of non-transformed control plants. Furthermore, the leaves of F(1) plants exhibited the increased resistance to paraquat, a herbicide generating active oxygen species.     

Changes in cysteine and O-acetyl-L-serine levels in the microalga Chlorella sorokiniana in response to the S-nutritional status

We analyzed the effects of deprivation and subsequent restoration of sulphate (S) in the nutrient solution on cysteine (Cys) and O-acetyl-l-serine (OAS) levels in Chlorella sorokiniana (211/8k). The removal of S from the culture medium caused a time-dependent increase in O-acetyl-l-serine(thiol)lyase (OASTL) activity and a decrease in soluble proteins content. The protein gel blot analysis was used to show that OASTL isoforms are located in the chloroplast and in the cytoplasm of S-starved cells. S-deprivation caused a decrease in the intracellular levels of Cys and glutathione (GSH) and an increase in serine (Ser) and OAS, reflecting an imbalance between sulphur and nitrogen assimilation. Re-supplying of sulphate to S-starved cells produced a decrease in OAS levels and concomitant rapid increase in Cys and GSH concentrations. The simultaneous addition of OAS and sulphate to S-starved cells did not further increase the concentration of Cys, suggesting the existence of a threshold level of intracellular Cys that is independent of the cellular concentration of OAS. Our findings that OAS is stored during S-starvation and that its quick decrease appears to be coupled with the increase of Cys levels upon re-supply of sulphate, imply that the central role that these two compounds play is in the regulation of sulphur-assimilating enzymes in response to the S status of the cell.     

Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.     

Signaling in the plant cytosol: cysteine or sulfide?

Cysteine (Cys) is the first organic compound containing reduced sulfur that is synthesized in the last stage of plant photosynthetic assimilation of sulfate. It is a very important metabolite not only because it is crucial for the structure, function and regulation of proteins but also because it is the precursor molecule of an enormous number of sulfur-containing metabolites essential for plant health and development. The biosynthesis of Cys is accomplished by the sequential reaction of serine acetyltransferase (SAT) and O-acetylserine(thiol)synthase (OASTL). In Arabidopsis thaliana, the analysis of specific mutants of members of the SAT and OASTL families has demonstrated that the cytosol is the compartment where the bulk of Cys synthesis takes place and that the cytosolic OASTL enzyme OAS-A1 is the responsible enzyme. Another member of the OASTL family is DES1, a novel l-cysteine desulfhydrase that catalyzes the desulfuration of Cys to produce sulfide, thus acting in a manner opposite to that of OAS-A1. Detailed studies of the oas-a1 and des1 null mutants have revealed the involvement of the DES1 and OAS-A1 proteins in coordinate regulation of Cys homeostasis and the generation of sulfide in the cytosol for signaling purposes. Thus, the levels of Cys in the cytosol strongly affect plant responses to both abiotic and biotic stress conditions, while sulfide specifically generated from the degradation of Cys negatively regulates autophagy induced in different situations. In conclusion, modulation of the levels of Cys and sulfide is likely critical for plant performance.     

Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO₂. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components—ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate–glutathione (Asc–GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways.     

Superoxide Dismutase Transgenes in Sugarbeets Confer Resistance to Oxidative Agents and the Fungus C. beticola

Sugarbeets carrying superoxide dismutase transgenes were developed in order to investigate the possibility of enhancing their resistance to oxidative stress. Binary T-DNA vectors carrying the chloroplastic and cytosolic superoxide dismutase genes from tomato, were used for Agrobacterium-mediated transformation of sugarbeet petioles. The transgenic plants were subjected to treatments known to cause oxidative stress, such as the herbicide methyl viologen and a natural photosensitizer toxin produced by the fungus Cercospora beticola, namely cercosporin. The transgenic plants exhibited increased tolerance to methyl viologen, to pure cercosporin, as well as to leaf infection with the fungus C. beticola.