Transgenic tobacco plants overexpressing cotton glutathione S-transferase (GST) show enhanced resistance to methyl viologen

A GST (EC 2.5.1.18) gene (Gst-cr 1) from cotton was introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants overexpressing Gst-cr1 were normal in growth and mature compared with control, but had much higher levels of GST and GPx activities and showed an enhanced resistance to oxidative stress induced by a low concentration of methyl viologen (MV). Six antioxidant enzymes, glutathione S-transferase, glutathione peroxidase (EC 1.11.1.9), superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), and ascorbate peroxidase (EC 1.11.1.11) were monitored in transgenic lines and non-transgenic control during MV treatments. When they were treated with 0.03 mmol/L of MV, both transgenic lines and control showed a rapid increase in the activities of GST, GPx, SOD, POD, APx, while the activity of CAT seemed to be irregular. The percent of the increase in SOD and POD activities was much higher in control than in transgenic plants. When treated with 0.05 mmol/L of MV, both control and transgenic plants were severely damaged, and the activities of the six enzymes decreased sharply.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

Transgenic tobacco plants expressingBoRS1 gene fromBrassica oleracea var.acephala show enhanced tolerance to water stress

Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, theBrassica oleracea var.acephala BoRS1 gene was transferred into tobacco throughAgrobacterium- mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows thatBoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.     

Adaptations to biotic and abiotic stress: Macaranga-ant plants optimize investment in biotic defence

Obligate ant plants (myrmecophytes) in the genus Macaranga produce energy- and nutrient-rich food bodies (FBs) to nourish mutualistic ants which live inside the plants. These defend their host against biotic stress caused by herbivores and pathogens. Facultative, 'myrmecophilic' interactions are based on the provision of FBs and/or extrafloral nectar (EFN) to defending insects that are attracted from the vicinity. FB production by the myrmecophyte, M. triloba, was limited by soil nutrient content under field conditions and was regulated according to the presence or absence of an ant colony. However, increased FB production promoted growth of the ant colonies living in the plants. Ant colony size is an important defensive trait and is negatively correlated to a plant's leaf damage. Similar regulatory patterns occurred in the EFN production of the myrmecophilic M. tanarius. Nectar accumulation resulting from the absence of consumers strongly decreased nectar flow, which increased again when consumers had access to the plant. EFN flow could be induced via the octadecanoid pathway. Leaf damage increased levels of endogenous jasmonic acid (JA), and both leaf damage and exogenous JA application increased EFN flow. Higher numbers of nectary visiting insects and lower numbers of herbivores were present on JA-treated plants. In the long run, this decreased leaf damage significantly. Ant food production is controlled by different regulatory mechanisms which ensure that costs are only incurred when counterbalanced by defensive effects of mutualistic insects.     

Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors

The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H(2)O(2) by PAO, the latter was below the detection limits. To show the mode(s) of H(2)O(2) scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild-type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H(2)O(2) generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H(2)O(2), or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H(2)O(2) generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.     

Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens

 The rice chitinase gene (RCC2), classified as class I chitinase, was introduced into the somatic embryos of grapevine (Vitis vinifera L. cv. Neo Muscut) by Agrobacterium infection. After co-cultivation with Agrobacterium, somatic embryos were transferred onto Murashige and Skoog hormone-free medium supplemented with 50 mg/l kanamycin. Transformed secondary or tertiary embryos were selected, and then more than 20 transgenic plantlets were recovered. Two transformants showed enhanced resistance against powdery mildew caused by Uncinula necator. Few disease symptoms were observed on leaves of these transformants compared with those of the non-transformant, although browning and necrotic symptoms, which seemed to constitute a hypersensitive reaction, were observed. Scanning electron microscopic observation revealed that conidial germination, mycelial growth and conidial formation were suppressed on the leaf surface of the transformant. The transgenic grapevines obtained also exhibited slight resistance against Elisinoe ampelina inducing anthracnose, resulting in a reduction in disease lesions. The relationship between the expression of the foreign chitinase gene and the disease resistance is discussed. Received: 5 April 1999 / Revision received: 13 September 1999 / Accepted: 6 October 1999     

Increased resistance to oxidative stress in transgenic tobacco plants overexpressing bacterial serine acetyltransferase

Plant expression cassettes containing the Escherichia coli cysE gene alleles (encoding SAT) were constructed. After the Agrobacterium-mediated transformation of tobacco, we identified stable transformed plants containing several-fold higher SAT activity in comparison to the control plant. Determination of non-protein thiol contents indicated two- to threefold higher cysteine and glutathione levels in some of these transgenic plants. The maximal elevation of the cysteine level was about fourfold while that of GSH was about twofold higher than in the controls. The most striking physiological consequence of the modification of sulfur metabolite levels in the transgenic plants, however, was their several-fold increased resistance to oxidative stress generated by exogenous hydrogen peroxide.     

RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.     

Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress

Summary. The cysteine biosynthesis pathway differs between plants and the yeast Saccharomyces cerevisiae. The yeast MET25 gene encoded to O-acetylhomoserine sulfhydrylase (AHS) catalyzed the reaction that form homocysteine, which later can be converted into cystiene. In vitro studies show that this enzyme possesses also the activity of O-acetyl(thiol)lyase (OASTL) that catalyzes synthesis of cysteine in plants. In this study, we generated transgenic tobacco plants expressing the yeast MET25 gene under the control of a constitutive promoter and targeted the yeast protein to the cytosol or to the chloroplasts. Both sets of transgenic plants were taller and greener than wild-type plants. Addition of SO₂, the substrate of the yeast enzyme caused a significant elevation of the glutathione content in representative plants from each of the two sets of transgenic plants expressing the yeast gene. Determination of non-protein thiol content indicated up to four-folds higher cysteine and 2.5-fold glutathione levels in these plants. In addition, the leaf discs of the transgenic plants were more tolerant to toxic levels of sulphite, and to paraquat, an herbicide generating active oxygen species.     

Transgenic tobacco plants accumulating osmolytes show reduced oxidative damage under freezing stress.

We studied the reaction to the oxidative component of freezing in several tobacco lines, transformed with genes coding for enzymes involved in the synthesis of osmoprotectants (proline, fructan or glycine betaine) along with their wild type. The levels of some oxidative stress markers (leakage of electrolytes, hydrogen peroxide and malondialdehyde) as well as the activity of antioxidative enzymes catalase (EC 1.11.1.6.) and guaiacol peroxidase (EC 1.11.1.7.) have been followed at acclimation, 12 and 24 h freezing and at recovery. Freezing for 24 h resulted in severe damages for the wild type. A corresponding increase of electrolyte leakage, hydrogen peroxide and malondialdehyde contents, a rise of peroxidase activity and inhibition of catalase activity occurred in the non-transformants. Similar, but significantly lower trend of the same parameters has been found for the transgenic lines. Moreover, the oxidative markers returned to their normal levels when the transformants were able to recover from freezing. It could be speculated that transfer of genes, coding for accumulation of osmoprotectants, is related to reduced intensity of freezing-induced oxidative processes. Our lines and model system could serve as a good prerequisite for additional studies to gain further insights into the complex role of osmoprotectants in freezing tolerance.     

Transgenic Nicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper

The diets of two-thirds of the world’s population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T₀ and T₁) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd²⁺ and Zn²⁺ did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper.     

Thaumatin gene confers resistance to fungal pathogens as well as tolerance to abiotic stresses in transgenic tobacco plants

We report here the development of transgenic tobacco plants with thaumatin gene of Thaumatococcus daniellii under the control of a strong constitutive promoter-CaMV 35S. Both polymerase chain reaction and genomic Southern analysis confirmed the integration of transgene. Transgenic plants exhibited enhanced resistance with delayed disease symptoms against fungal diseases caused by Pythium aphanidermatum and Rhizoctonia solani. The leaf extract from transgenic plants effectively inhibited the mycelial growth of these pathogenic fungi in vitro. The transgenic seeds exhibited higher germination percentage and seedling survival under salinity and PEG-mediated drought stress as compared to the untransformed controls. These observations suggest that thaumatin gene can confer tolerance to both fungal pathogens and abiotic stresses.     

Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress

A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.     

Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis

Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress responses. In this study, the transgenic Arabidopsis thaliana plants with modulated exp...     

Transgenic tobacco plants overexpressing a cold-adaptive nitroreductase gene exhibited enhanced 2,4-dinitrotoluene detoxification rate at low temperature

Abstract

Plants encounter many environmental factors such as low and high temperatures during phytoremediation processes. In this study, our aim was to produce the transgenic tobacco plants by using a newly characterized bacterial nitroreductase, Ntr, which was active at a broad range temperature in order to detoxify 2,4-dinitrotoluene (2,4-DNT) at lower temperature. The presence of Ntr and its heterologous expression was verified in T1 transgenic plants and their growing ability were determined under toxic amount of 2,4-DNT (35?µM). Fresh weight and dry weight of transgenic plants were significantly higher than wild type (WT) under toxic 2,4-DNT at 22?°C, indicating higher growth capacity of the transgenics. Transgenic plants also showed a higher tolerance than WT when exposed to 2,4-DNT at 15?°C. Moreover, transformation rate of 2,4-DNT was gradually decreased through decreasing temperatures in WT media, however, it was increased through decreasing temperatures in transgenic plant TR3-25 media and it had the highest transformation rate (54%) of 2,4-DNT at 4?°C. Correlatively, 2,4-DNT treatment at 4?°C led to a significant decrease in H₂O₂ level in transgenic plants. Thus, transgenic plants overexpressing nitroreductase might have an important advantage for phytoremediation of toxic nitroaromatic compounds in field applications at low temperatures.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.