Transformed Tobacco Plants Research Articles

Chinese cherry CpMYB44-CpSPDS2 module regulates spermidine content and florescence in tobacco.

The flower bud differentiation plays a crucial role in cherry yield and quality. In a preliminary study, we revealed the promotion of spermidine (Spd) in bud differentiation and quality. However, the molecular mechanism underlying Spd regulating cherry bud differentiation remains unclear. To address this research gap, we cloned CpSPDS2, a gene that encodes Spd synthase and is highly expressed in whole flowers and pistils of the Chinese cherry (cv. 'Manaohong'). Furthermore, an overexpression vector with this gene was constructed to transform tobacco plants. The findings demonstrated that transgenic lines exhibited higher Spd content, an earlier flowering time by 6 d, and more lateral buds and flowers than wild-type lines. Additionally, yeast one-hybrid assays and two-luciferase experiments confirmed that the R2R3-MYB transcription factor (CpMYB44) directly binds to and activates the CpSPDS2 promoter transcription. It is indicated that CpMYB44 promotes Spd accumulation via regulating CpSPDS2 expression, thus accelerating the flower growth. This research provides a basis for resolving the molecular mechanism of CpSPDS2 involved in cherry bud differentiation.

Effect of the overexpression of external alternative NADH dehydrogenase gene in Arabidopsis on the resistance of transformed tobacco plants to negative temperatures

The study aims to confirm the functional activity and localization of the At_NDB2 transgenic protein of Arabidopsis in tobacco cells and to evaluate the effect produced by the permanent expression of external alternative NADH dehydrogenase on the resistance of a heat-loving plant to low temperatures. Proteins and mitochondria were isolated from the leaves of tobacco plants grown at 25 °С (day/night) at the 7-leaf stage. At_NDB2 protein localization in mitochondria was determined via electrophoresis and immunoblotting. The functional activity of At_NDB2 was confirmed through the polarography of isolated mitochondria and the specific mitochondrial complex I inhibitor (rotenone). It was also found that the cyanide-resistant respiration rate and the activity of an alternative oxidase enzyme were significantly higher in transgenic plants than in wild-type plants. In order to determine the resistance to low temperatures, the parent and transgenic tobacco plants were grown in soil until the 2–3 and 6–7 leaf stages, after which they were kept at 3 to -3 °С for a day in the dark and left to regrow at 25 °С for seven days. Although it was previously shown that oxidative stress is reduced in transgenic plants at low temperatures as compared to wild-type plants, it was established that the tolerance of transgenic and nontransformed plants does not differ. Thus, alternative NADH dehydrogenase activity was found to reduce oxidative stress and increase alternative oxidase activity, without enhancing the resistance of Nicotiana tabacum to negative temperatures.

Functional Characterization of a Novel Synthetic Herbicide Resistance Gene in a Model Plant

As crop losses can occur owing to the abrupt growth of uncontrollable weeds in the field, research is currently being conducted in Pakistan to eradicate herbs. To carry out our proposed research domain, we have designed a novel synthetically modified EPSPS gene that has a potent role in herbicide-resistance development. The novel codon-optimized synthesized mEPSPS sequence was inserted into the pXCSG-mYFP plant expression vector. Results of colony PCR (1400 bp) confirmed the integration of genes into bacteria. For functional validation of pXCSG-mYFP-EPSPS, transient expression in tobacco (Nicotiana benthamiana) in comparison with PBSF-16 was done. Benth infiltration results showed that transient expression was successfully confirmed through ELISA and western blot analysis via anti-YFP antibody in tobacco leaves. For further validation of transient expression, the stable transformation results of the pXCSG-mYFP-EPSPS vector showed that successful transformation was done via two days of co-cultivation followed by selection and regeneration of transformed tobacco plants. The regenerated tobacco plants were then confirmed through gene-specific-based PCR. After PCR-based confirmation, western blot further validates the 26 kDa anti-YFP antibodies expression in transformed tobacco plants. Another important finding of the study was the bar and PAT gene real-time expression elucidating that the bar gene was 4.9-fold more expressive under the 35S promoter than the PAT gene under the nos promoter.

Efficiency of Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L.) with rice OsNAC6 gene

Crop improvement by genetic engineering requires efficient plant regeneration derived from cultured cells or tissues. The success of cell or tissue culture regeneration depends on reliable explant source. In this study, three different types of explant (callus, stem, and leaf) of tobacco (Nicotiana tabacum L.) were evaluated to determine the most effective explant source for Osnac6 gene transformation. OsNAC6 encodes a transcription factor belonged to NAC transcription factor family in rice (Oryza sativa L.). OsNAC6 transcription factor has been reported in some studies to confer tolerance to drought stress. Agrobacterium tumefaciens strain EHA 105 harboring pC1305 plasmid containing the OsNAC6 gene driven by CaMV 35S promoter (pARNAC6) was used for transformation. The T-DNA contains hygromycin phosphotransferase (hpt) gene as a selectable marker. PCR confirmed the presence of the hpt gene in the T0 generation of the transformed tobacco plants. The PCR result showed that a total of 50 tobacco plants were positive for hpt gene which 18, 20, and 12 of them derived from callus, stem, and leaf explants respectively. The highest transformation efficiency was obtained by using the stem as explant material, i.e. 17%. Meanwhile, the obtained transformation efficiency by using callus and leaf was 15% and 10% respectively.

The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants.

Most angiosperms bear hermaphroditic flowers, but a few species have evolved outcrossing strategies, such as dioecy, the presence of separate male and female individuals. We previously investigated the mechanisms underlying dioecy in diploid persimmon (D. lotus) and found that male flowers are specified by repression of the autosomal gene MeGI by its paralog, the Y-encoded pseudo-gene OGI. This mechanism is thought to be lineage-specific, but its evolutionary path remains unknown. Here, we developed a full draft of the diploid persimmon genome (D. lotus), which revealed a lineage-specific whole-genome duplication event and provided information on the architecture of the Y chromosome. We also identified three paralogs, MeGI, OGI and newly identified Sister of MeGI (SiMeGI). Evolutionary analysis suggested that MeGI underwent adaptive evolution after the whole-genome duplication event. Transformation of tobacco plants with MeGI and SiMeGI revealed that MeGI specifically acquired a new function as a repressor of male organ development, while SiMeGI presumably maintained the original function. Later, a segmental duplication event spawned MeGI’s regulator OGI on the Y-chromosome, completing the path leading to dioecy, and probably initiating the formation of the Y-chromosome. These findings exemplify how duplication events can provide flexible genetic material available to help respond to varying environments and provide interesting parallels for our understanding of the mechanisms underlying the transition into dieocy in plants.

Identification and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth.

Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.

Mitigating Root Knot Nematode Propagation on Transgenic Tobacco via <i>in Planta</i> Hairpin RNA Expression of <i>Meloidogyne incognita</i>—Specific PolA1 Sequence

Root knot nematodes are top priority nematode pests that significantly constrain agricultural productivity globally especially in developing countries. However, expressing double stranded RNA (dsRNA) of essential nematode genes in susceptible plants is known to confer protection against these pests via RNA silencing. This molecular-based strategy is called host induced gene silencing (HIGS) and the selection of appropriate target nematode gene is critical to its success. In this study, therefore, we focused on root knot nematode PolA1, an essential single copy nuclear gene encoding the largest subunit of RNA polymerase I enzyme and evaluated its effectiveness as a target in conferring nematode resistance on Agrobacterium-mediated transformed tobacco plants. Transgenic tobacco expressing Meloidogyne incognita-specific (MiS) dsRNA of PolA1 gene showed significant reduction in nematode fecundity and multiplication compared to wild type plants in both T0 and T1 generations. T0 plants showed varying degrees of agronomic vigorover WT plants possibly due to varying levels of processed siRNA. However, production of MiS siRNAs in the transgenic plants coupled with significant reduction of PolA1 transcript expression in nematodes feeding on roots of transgenic plants provided evidence of HIGS. Taken together, our results show that PolA1 is a potentially effective target for HIGS-mediated reduction of root knot nematode damage on transgenic tobacco. Given the homology of our target sequence among Meloidogyne species, this protection could be broad range against other root knot nematodes aside M. incognita.

An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide.

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be “self-cleaved” into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd-values of 90.13–149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants.

Agrobacterium- Mediated Transformation of Tobacco Plants using Walnut Polyphenol Oxidase Gene

Polyphenol oxidase (PPO) is a well-recognized copper-containing enzyme which can catalyze oxidation of a great variety of phenolic compounds. PPO is found in diverse microorganisms, plants, and animals. To examine the anti-pathogenic role of PPO in walnut (Juglans regia L.), Agrobacterium tumefaciens strain LBA4404 was used to transform tobacco (Nicotiana tobacum L.) explants. Recombinant binary vector pBI121, containing the walnut JrPPO gene and the nptII gene as a selectable marker, was incorporated into A. tumefaciens. MS medium supplemented with 50 mg/l of kanamycin and 200 mg/l of cefotaxime was used as a selection medium. Plantlets were regenerated from putatively transgenic calli and polymerase chain reaction and Southern blot analysis were performed to confirm the integration of JrPPO into the genome. To evaluate the function of PPO in pathogen resistance, transgenic tobacco plants were exposed to Pseudomonas syringae pv. tabaci. According to symptom progress and quantitative analyses, although there was no significant difference in transgenic tobacco, but mean comparison showed that disease severity of transgenic tobacco was less than wild plants. Finally, it may be concluded that PPO has a notable function in the resistance process in walnut, but tobacco transgenic plants might not be a suitable test plant to examine the resistance role of PPO in walnut.

Osmotic stress on genetically transformed tobacco plant seeds

Abstract: Salinity and water deficit limit the productivity of several crops; thus, studies related to the genetic transformation of seeds in a model plant, such as tobacco, can be an alternative to minimize negative impacts caused by environmental conditions. The purpose of this work was to evaluate the tolerance to osmotic stress of seeds from genetically transformed tobacco plants, with the introduction of the proline-synthesizer gene (p5csf129a), under salinity and water deficit conditions. To do so, five events with differences in proline content were selected, ranging from 0.70 to 10.47 µmoles.g-1 of fresh mass. The used saline concentrations were: zero (distilled water); 50; 100; 150 and 200 mmol.L-1 of NaCl, whereas for the water deficit, simulated with PEG 6000, the following osmotic potentials were used: zero (distilled water); -0.2; -0.4; -0.6 and -0.8 MPa. Each tested treatment was evaluated through germination, first germination count and germination speed index tests. It is possible to conclude that seeds from genetically transformed tobacco plants with overexpression of the gene p5csf129a, a proline synthesizer, are more tolerant to osmotic stresses. Tabacco seeds with a proline content of 10.47 µmol.g-1 showed a better perfomance, revealing higher physiological potential.

Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA.

Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA‐MRS and CinH‐RS2, to precisely excise a marker gene from the plastid genome of tobacco. Transplastomic plants transformed with the pTCH‐MRS and pTCH‐RS2 vectors, containing the visual reporter gene DsRed flanked by directly oriented MRS and RS2 recognition sites, respectively, were crossed with nuclear‐genome transformed tobacco plants expressing plastid‐targeted ParA and CinH recombinases, respectively. One hundred per cent of both types of F1 hybrids exhibited excision of the DsRed marker gene. PCR and Southern blot analyses of DNA from F2 plants showed that approximately 30% (CinH‐RS2) or 40% (ParA‐MRS) had lost the recombinase genes by segregation. The postexcision transformed plastid genomes were stable and the excision events heritable. The ParA‐MRS and CinH‐RS2 recombination systems will be useful tools for site‐specific manipulation of the plastid genome and for generating marker‐free plants, an essential step for reuse of SMG and for addressing concerns about the presence of antibiotic resistance genes in transgenic plants.

Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide.

Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.

Transgenic Tobacco Expressing the TAT-Helicokinin I-CpTI Fusion Protein Show Increased Resistance and Toxicity to Helicoverpa armigera (Lepidoptera: Noctuidae).

Insect kinins were shown to have diuretic activity, inhibit weight gain, and have antifeedant activity in insects. In order to study the potential of the TAT-fusion approach to deliver diuretic peptides per os to pest insects, the HezK I peptide from Helicoverpa zea, as a representative of the kinin family, was selected. The fusion gene TAT-HezK I was designed and was used to transform tobacco plants. As a means to further improve the stability of TAT-HezK I, a fusion protein incorporating HezK I, transactivator of transcription (TAT), and the cowpea trypsin inhibitor (CpTI) was also designed. Finally, the toxicity of the different tobacco transgenic strains toward Helicoverpa armigera was compared. The results demonstrated that TAT-HezK I had high toxicity against insects via transgenic expression of the peptide in planta and intake through larval feeding. The toxicity of the fusion TAT-HezK I and CpTI was higher than the CpTI single gene in transgenic tobacco, and the fusion TAT-HezK I and CpTI further enhanced the stability and bioavailability of agents in oral administration. Our research helps in targeting new genes for improving herbivore tolerance in transgenic plant breeding.

Effect of Temperature and ABA Exposure on LlaNAC Expression in Transformed Tobacco Plants

Effect of Temperature and ABA Exposure on LlaNAC Expression in Transformed Tobacco Plants

A novel constitutive promoter and its downstream 5′ UTR derived from cotton (Gossypium spp.) drive high-level gene expression in stem and leaf tissues

The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene expression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton (Gossypium hirsutum) translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter (GhEF1A1.7) and the 5′ untranslated region (5′ UTR), comprising a regulatory region named PGhEF1A8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the β-glucuronidase (GUS) reporter gene to characterize its expression pattern in tobacco. Histochemical and fluorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus (CaMV) 35S promoter::GUS plants, but as same as CaMV 35S promoter::GUS plants in flower and root tissues. GUS expression levels decreased 2–10-fold when the 5′ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEF1A8 sequence showed that the region −647 to −323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEF1A8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.

A Fusion between Domains of the Human Bone Morphogenetic Protein-2 and Maize 27 kD γ-Zein Accumulates to High Levels in the Endoplasmic Reticulum without Forming Protein Bodies in Transgenic Tobacco.

Human Bone Morphogenetic Protein-2 (hBMP2) is an osteoinductive agent physiologically involved in bone remodeling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad) in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs.

Evaluation of antigens stability of tobacco seeds as edible vaccine against VTEC strains

Plants have represent a promising alternative for biopharmaceutical proteins (Ma et al., 2003; Rossi et al., 2014). Many plant based edible vaccines have been shown to be effective in inducing local immune responses ( Rossi et al., 2013). Edible vaccines can activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining. This dual effect would provide first-line defense against pathogens invading through the mucosa. The antigens are released in the intestines are taken up by M cells that are present over the Payer’s patches (in the ileum) and the gut associated lymphoid tissue (GALT). Edible vaccines represent an important worldwide goal for the prevention of the enteric diseases, also in livestock. In particular, the enteric infections are a significant clinical problem in pigs. Verocytotoxic Escherichia (E.) coli strains are responsible for serious enterotoxaemia that causes important economic losses in the pig industry. The production of a vaccine for oral administration of transgenic seeds could be a practical and efficient system to prevent the infection and to reduce the antibiotic use. This study was focused on tobacco plants, previously transformed by agroinfection for the seed-specific expression of antigenic proteins (F18 adhesive fimbriae and the B subunit of the Vt2e toxin) as model of edible vaccines against verocytotoxic E. coli strains. The dietary administration of transgenic tobacco seeds promotes a significant increase in the number of mucosal IgA-producing cells of the tunica propria in both small and large intestine in mice ( Rossi et al., 2013). A protective effect of oral administration of transgenic tobacco seeds was also observed against verocytotoxic Escherichia coli infection in piglets (Rossi et al., 2014). The aim of this study was to assess the seed-expression stability, that is a important requirement in the vaccine production, of F 18 and Vt2e-B heterologous genes into the progeny of transformed tobacco plants.

Co-expression of the tobacco anthranilate synthase β subunit with its feedback-insensitive α subunit as a selectable marker that also markedly increases the free tryptophan content

Anthranilate synthase (AS), a tetramer consisting of two α subunits and two β subunits, is the key control enzyme in the tryptophan (Trp) biosynthesis pathway. A naturally occurring α subunit of AS called ASA2 that is insensitive to Trp feedback inhibition was isolated from a tobacco suspension cell culture and has been extensively studied and used for both nuclear and plastid transformation. However, the obligate β subunit of AS had not been studied in tobacco. Therefore, the tobacco AS β subunit-encoding cDNA was cloned and its encoded protein was verified. Agrobacterium-mediated transformation of tobacco plants was performed, under the selection of the toxic Trp analogs, 7-methyltryptophan or α-methyltryptophan, with a construct containing both ASA2 and AS β subunit genes. Many transgenic plants overexpressing both subunits were identified and examined. Compared to the wild-type plants, the transgenic plants had higher levels of enzymatic activities for both holoenzyme and α subunit. The transgenic plants had 9 to 68 times the amount of free Trp as the wild-type plants, which was more pronounced than plants overexpressing ASA2 alone. This study demonstrates the potential of co-expressing AS α and β subunits as a robust plant transformation system as well as overcoming feedback inhibition to obtain high levels of Trp biosynthesis.

Conserved sequence of replicase gene mediated resistance in Nicotiana tabacum L. cv Abirami through RNA silencing

Tobacco streak virus (TSV) is economically the most important virus infecting several crop plants in India. Symptomatic samples collected from sunflower and okra fields were subjected to RT-PCR with TSV replicase gene specific primers. The replicase (Rep) genes of these isolates were sequenced in order to develop transgenic resistance to Tobacco streak virus (TSV) in tobacco (Nicotiana tabacum L.) cv Abirami by expressing hairpin RNA transcript (hpRNA). The sequence analysis of replicase gene of these isolates showed a sequence identity of around 99% at nucleotide level with Tamil Nadu okra isolate. The hairpin construct was generated using pHANNIBAL vector with the conserved nucleotide sequence corresponding to position 3065 to 3405 of the TSV replicase gene. The Rep hairpin construct was cloned into pART27 and mobilized into Agrobacterium tumefaciens LBA4404 and introduced into tobacco by Agrobacterium mediated transformation. The T0 plants obtained were screened by PCR and Southern blot analysis using the genomic DNA from transformed tobacco plants. The transformants produced ~299 bp and 340 bp amplicons corresponding to nptII gene and Rep gene respectively. The Southern blot analysis showed single and multiple-copy integration of the transgenes. The transgenic T0 tobacco plants showed resistance against TSV upon mechanical inoculation of TSV without producing any visible symptoms; resistance was also confirmed by DAC-ELISA.

Tobacco plants over-expressing the sweet orange tau glutathione transferases (CsGSTUs) acquire tolerance to the diphenyl ether herbicide fluorodifen and to salt and drought stresses

The glutathione transferases (GSTs) are members of a superfamily of enzymes with pivotal role in the detoxification of both xenobiotic and endogenous compounds. In this work, the generation and characterization of transgenic tobacco plants over-expressing tau glutathione transferases from Citrus sinensis (CsGSTU1 and CsGSTU2) and several cross-mutate forms of these genes are reported. Putative transformed plants were verified for the presence of the transgenes and the relative quantification of transgene copy number was evaluated by Taqman real time PCR. The analysis of gene expression revealed that transformed plants exhibit high levels of CsGSTU transcription suggesting that the insertion of the transgenes occurred in transcriptional active regions of the tobacco genome. In planta studies demonstrate that transformed tobacco plants gain tolerance against fluorodifen. Simultaneously, the wild type CsGSTU genes were in vitro expressed and their kinetic properties were determined using fluorodifen as substrate. The results show that CsGSTU2 follows a Michaelis–Menten hyperbolic kinetic, whereas CsGSTU1 generates a sigmoid plot typical of the regulatory enzymes, thus suggesting that when working at sub-lethal fluorodifen concentrations CsGSTU2 can counteract the herbicide injury more efficiently than the CsGSTU1. Moreover, the transgenic tobacco plant over-expressing CsGSTs exhibited both drought and salinity stress tolerance. However, as we show that CsGSTUs do not function as glutathione peroxidase in vitro, the protective effect against salt and drought stress is not due to a direct scavenging activity of the oxidative stress byproducts. The transgenic tobacco plants, which are described in the present study, can be helpful for phytoremediation of residual xenobiotics in the environment and overall the over-expression of CsGSTUs can be helpful to develop genetically modified crops with high resistance to abiotic stresses.