Synechocystis PCC6803 and PCC6906 dnaK2 expression confers salt and oxidative stress tolerance in Arabidopsis via reduction of hydrogen peroxide accumulation

Abiotic stress slows plant growth and development. Because salt stress, particularly from NaCl, acts as an important limiting factor in agricultural productivity, the identification and manipulation of genes related to salt tolerance could improve crop productivity. Prokaryotic, heat shock protein (Hsp), DnaK from the ubiquitous Hsp70 family is upregulated in cells that are under abiotic stress. Synechocystis spp. cyanobacteria encode at least three potential DnaK proteins in their genome. Here, expressions of dnaK1s and dnaK2s from two Synechocystis spp. PCC6803 (Sy6803) and PCC6906 (Sy6906), enhanced salt tolerance in a dnaK-defective Escherichia coli strain. In contrast, dnaK3s in both strains were ineffective, indicating that dnaK3 is functionally different from dnaK1 and dnaK2 in Synechocystis spp. under salt stress. Ectopic expression of dnaK2s from Sy6803 and Sy6906 conferred salt tolerance in transgenic Arabidopsis plants, which exhibited greater root length, chlorophyll content, fresh weight, and survival rate than wild type plants, all in the presence of NaCl. In transgenic plants, hydrogen peroxide (H₂O₂) accumulation was reduced under NaCl stress and loss of chlorophyll content was reduced under H₂O₂ stress. Overall results suggest that dnaK2s from Sy6803 and Sy6906 confer salt and oxidative tolerance in transgenic plants by reduction of H₂O₂ accumulation.     

Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize.

Transgenic maize (Zea mays L.) plants have been generated by particle gun bombardment that overproduce an Arabidopsis thaliana iron superoxide dismutase (FeSOD). To target this enzyme into chloroplasts, the mature Fesod coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene. Expression of the chimeric gene was driven by the CaMV 35S promoter. Growth characteristics and in vitro oxidative stress tolerance of transgenic lines grown in control and chilling temperatures were evaluated. The transgenic line with the highest transgenic FeSOD activities had enhanced tolerance toward methyl viologen and had increased growth rates.     

Salt-dependent protein phosphorylation in the cyanobacterium Synechocystis PCC 6803

Abstract We have isolated a Bradyrhizobium japonicum USDA 438 (serogroup 123) mutant which has the ability to form nodules on serogroup 123 nodulation-restricting plant introduction genotypes and soybeans containing the Rj4 allele. The identity of the mutant was confirmed by using a serocluster 123-specific DNA probe, restriction fragment length polymorphism analysis, and serogroup-specific fluorescent antibodies. While the mutant contains Tn 5 inserted into a cryptic, non nod gene-containing locus, site-directed mutagenesis and complementation studies indicated that the transposon is not responsible for host-range extension. The mutant and the wild-type parent had the same chromatographic profiles of [¹⁴C]acetate-labelled extracellular B. japonicum nod factors.     

Expression and oxidative stress tolerance studies of glutaredoxin from cyanobacterium Synechocystis sp. PCC 6803 in Escherichia coli

Glutaredoxin (Grx), which has been found widely in bacteria, plant, and mammalian cells, is an electron carrier for ribonucleotide reductase and a general glutathione-disulfide reductase of importance for redox regulation. The open reading frame designated ssr2061 from cyanobacterium Synechocystis sp. PCC 6803 was found as a homologous gene coding for Grx. The amino acid sequence deduced from ssr2061 shares high identity with that of Grxs from other organisms. In the present study, the protein of Grx2061 encoded by ssr2061 was successfully overexpressed as soluble fraction in Escherichia coli BL21 (DE3). The recombinant protein was purified to near homogenity by two steps involving immobilized metal affinity chromatography and gel filtration chromatography with a yield of 22% and a specific activity of 41.5 micromol NADPH oxidized per milligram of protein in the 2-hydroxyethyl disulfide assay. The pET-2061 transformed Escherichia coli cells showed higher Grx activity and tolerance to H(2)O(2) mediated growth inhibition compared to cells transformed with the vector alone. This suggests that overexpression of Grx from Synechocystis sp. PCC 6803 may provide protection to E. coli cells against oxidative stress mediated by H(2)O(2).     

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803

Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium- Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein’s glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST- sll0067.     

The thioredoxin reductase-glutaredoxins-ferredoxin crossroad pathway for selenate tolerance in Synechocystis PCC6803

Most organisms use two systems to maintain the redox homeostasis of cellular thiols. In the thioredoxin (Trx) system, NADPH sequentially reduces thioredoxin reductases (NTR), Trxs and protein disulfides. In the glutaredoxin (Grx) system, NADPH reduces the glutathione reductase enzyme occurring in most organisms, glutathione, Grxs, and protein disulfides or glutathione-protein mixed disulfides. As little is known concerning these enzymes in cyanobacteria, we have undertaken their analysis in the model strain Synechocystis PCC6803. We found that Grx1 and Grx2 are active, and that Grx2 but not Grx1 is crucial to tolerance to hydrogen peroxide and selenate. We also found that Synechocystis has no genuine glutathione reductase and uses NTR as a Grx electron donor, in a novel integrative pathway NADPH-NTR-Grx1-Grx2-Fed7 (ferredoxin 7), which operates in protection against selenate, the predominant form of selenium in the environment. This is the first report on the occurrence of a physical interaction between a Grx and a Fed, and of an electron transfer between two Grxs. These findings are discussed in terms of the (i) selectivity of Grxs and Feds ( Synechocystis possesses nine Feds), (ii) crucial importance of NTR for cell fitness and (iii) resistance to selenate, in absence of a Thauera selenatis -like selenate reductase.     

Role of Slr1045 in environmental stress tolerance and lipid transport in the cyanobacterium Synechocystis sp. PCC6803

ATP-binding cassette (ABC) transporter proteins mediate energy-dependent transport of substrates across cell membranes. Numerous ABC transporter-related genes have been found in the Synechocystis sp. PCC6803 genome by genome sequence analysis including H(+), iron, phosphate, polysaccharide, and CO(2) transport-related genes. The substrates of many other ABC transporters are still unknown. To identify ABC transporters involved in acid tolerance, deletion mutants of ABC transporter genes with unknown substrates were screened for acid stress sensitivities in low pH medium. It was found that cells expressing the deletion mutant of slr1045 were more sensitive to acid stress than the wild-type cells. Moreover, slr1045 expression in the wild-type cells was increased under acid stress. These results indicate that slr1045 is an essential gene for survival under acid stress. The mutant displayed high osmotic stress resistance and high/low temperature stress sensitivity. Considering the temperature-sensitive phenotype and homology to the organic solvent-resistant ABC system, we subsequently compared the lipid profiles of slr1045 mutant and wild-type cells by thin-layer chromatography. In acid stress conditions, the phosphatidylglycerol (PG) content in the slr1045 mutant cells was approximately 40% of that in the wild-type cells. Moreover, the addition of PG to the medium compensated for the growth deficiency of the slr1045 mutant cells under acid stress conditions. These data suggest that slr1045 plays a role in the stabilization of cell membranes in challenging environmental conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803

This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.     

Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli

The superoxide dismutase (SOD) gene (slr 1516) from the cyanobacterium Synechocystis sp. PCC 6803 was cloned and overexpressed in Escherichia coli BL 21 (DE3) using the pET-20b(+) expression vector. E. coli cells transformed with pET-SOD overexpressed the protein in cytosol, upon induction by isopropyl beta-D-thiogalactopyranoside (IPTG). The recombinant protein was purified to near homogeneity by gel filtration and ion-exchange chromatography. The SOD activity of the recombinant protein was sensitive to hydrogen peroxide and sodium azide, confirming it to be FeSOD. The pET-FeSOD transformed E. coli showed significantly higher SOD activity and tolerance to paraquat-mediated growth inhibition compared to the empty vector transformed cells. Based on these results it is suggested that overexpression of FeSOD gene from a heterologous source like Synechocystis sp. PCC 6803 may provide protection to E. coli against superoxide radical-mediated oxidative stress mediated by paraquat.     

Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

An Arabidopsis mitochondrial uncoupling protein confers tolerance to drought and salt stress in transgenic tobacco plants

Background

Plants are challenged by a large number of environmental stresses that reduce productivity and even cause death. Both chloroplasts and mitochondria produce reactive oxygen species under normal conditions; however, stress causes an imbalance in these species that leads to deviations from normal cellular conditions and a variety of toxic effects. Mitochondria have uncoupling proteins (UCPs) that uncouple electron transport from ATP synthesis. There is evidence that UCPs play a role in alleviating stress caused by reactive oxygen species overproduction. However, direct evidence that UCPs protect plants from abiotic stress is lacking.

Methodology/Principal Findings

Tolerances to salt and water deficit were analyzed in transgenic tobacco plants that overexpress a UCP (AtUCP1) from Arabidopsis thaliana. Seeds of AtUCP1 transgenic lines germinated faster, and adult plants showed better responses to drought and salt stress than wild-type (WT) plants. These phenotypes correlated with increased water retention and higher gas exchange parameters in transgenic plants that overexpress AtUCP1. WT plants exhibited increased respiration under stress, while transgenic plants were only slightly affected. Furthermore, the transgenic plants showed reduced accumulation of hydrogen peroxide in stressed leaves compared with WT plants.

Conclusions/Significance

Higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. Our data support previous assumptions that UCPs reduce the imbalance of reactive oxygen species. Our data also suggest that UCPs may play a role in stomatal closure, which agrees with other evidence of a direct relationship between these proteins and photosynthesis. Manipulation of the UCP protein expression in mitochondria is a new avenue for crop improvement and may lead to crops with greater tolerance for challenging environmental conditions.     

A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily from (i) genes of two-component signal transducers of Synechocystis, (ii) Synechocystis genes whose homologues are conserved in the genome of Arabidopsis thaliana, and (iii) genes of unknown function on the Synechocystis chromosome. A total of 3236 independent two-hybrid interactions involving 1920 proteins (52% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure, as well as the general features of interacting partners. The interaction data obtained in this study should provide new insights and novel strategies for functional analyses of genes in Synechocystis, and, additionally, genes in other cyanobacteria and plant genes of cyanobacterial origin.     

Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis

Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes, slr1736 and HPT1, that encode HPT from Synechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystis sp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803 slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1 in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.     

Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants

Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.     

Isolation and Functional Analysis of Homogentisate Phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis

Tocopherols, collectively known as vitamin E, are lipid-soluble antioxidants synthesized exclusively by photosynthetic organisms and are required components of mammalian diets. The committed step in tocopherol biosynthesis involves condensation of homogentisic acid and phytyl diphosphate (PDP) catalyzed by a membrane-bound homogentisate phytyltransferase (HPT). HPTs were identified from Synechocystis sp. PCC 6803 and Arabidopsis based on their sequence similarity to chlorophyll synthases, which utilize PDP in a similar prenylation reaction. HPTs from both organisms used homogentisic acid and PDP as their preferred substrates in vitro but only Synechocystis sp. PCC 6803 HPT was active with geranylgeranyl diphosphate as a substrate. Neither enzyme could utilize solanesyl diphosphate, the prenyl substrate for plastoquinone-9 synthesis. In addition, disruption of Synechocystis sp. PCC 6803 HPT function causes an absence of tocopherols without affecting plastoquinone-9 levels, indicating that separate polyprenyltransferases exist for tocopherol and plastoquinone synthesis in Synechocystis sp. PCC 6803. It is surprising that the absence of tocopherols in this mutant had no discernible effect on cell growth and photosynthesis.     

Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses

l-Ascorbic acid (Vitamin C, AsA) is an important component of human nutrition. Plants and several animals can synthesize their own ascorbic acid, whereas humans lack the gene essential for ascorbic acid biosynthesis and must acquire from their diet. In the present study, we developed transgenic potato (Solanum tuberosum L. cv. Taedong Valley) over-expressing l-gulono-γ-lactone oxidase (GLOase gene; NCBI Acc. No. NM022220), isolated from rat cells driven by CaMV35S constitutive promoter that showed enhanced AsA accumulation. Molecular analyses of four independent transgenic lines performed by PCR, Southern and RT-PCR revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 7.5% and the time required for the generation of transgenic plants was 6–7 weeks. Transgenic tubers showed significantly enhanced AsA content (141%) and GLOase activity as compared to untransformed tubers. These transgenics were also found to withstand various abiotic stresses caused by Methyl Viologen (MV), NaCl or mannitol, respectively. The T₁ transgenic plants exposed to salt stress (100 mM NaCl) survived better with increased shoot and root length when compared to untransformed plants. The elevated level of AsA accumulation in transgenics was directly correlated with their ability to withstand abiotic stresses. These results further demonstrated that the overexpression of GLOase gene enhanced basal levels of AsA in potato tubers and also the transgenics showed better survival under various abiotic stresses.