Generation of CsGSTUs over-expressing tobacco plants and their role in abiotic and biotic stress tolerance

Xenobiotics are toxic chemicals that are normally not the natural substrates for enzymes or transporters involved in plant resistance. Plants have developed a three phases detoxification system from toxic compounds. Xenobiotic are firstly activated so that certain functional groups can be exposed to the successive action of several modifying enzymes. Among them, the glutathione transferases (GSTs) catalyze the nucleophilic addition of glutathione (GSH) to the electrophilic groups of a large variety of hydrophobic toxic molecules. Previously, two gstu genes have been isolated from sweet orange leaves [(Citrus sinensis) L. Osbeck)] namely GSTU1 and GSTU2. The encoded proteins differ in three amino acids, all of them included in the C-terminal domain of the enzymes (R89P, E117K, I172V). In order to evaluate the contribution of the mismatched amino acids on the catalytic activity of enzymes, several cross-mutant genes were produced by site-directed mutagenesis followed by the biochemical characterization of the in vitro expressed enzymes. In this work, transgenic tobacco plants via Agrobacterium tumefaciens mediated transformation over-expressing both the wild type and mutant CsGSTU genes were generated. Along with the molecular characterization of transformed plants, an in planta study to assess their ability in detoxifying herbicides was also performed. Therefore, transgenic plants were subjected to the action of fluorodifen, a diphenyl ether herbicide that cause photooxidative stress by inhibition of the plastid protoporphyrinogen oxidase and alachlor a chloroacetanilide herbicide which is used to control the growth of broad-leafed weeds and grasses in many crops. The electrolytic leakage assay was carried out to test the damage caused by fluorodifen treatment upon transformed and untransformed tobacco plants. The data revealed that the transgenic lines show a sharp reduction of membrane damage compared with the wild type tobacco plants. To study the tolerance towards alachlor in planta, we assayed the growth inhibition of untrasformed wild type and transgenic tobacco seedlings in the presence of 7.5 mg/l of alachlor. Alachlor negatively influences the growth of roots and stems of untransformed an transformed tobacco seedlings with the exception of the transgenic plants over-expressing CsGSTU2 which are clearly unaffected by herbicide treatment considering either stem or root lenght. Consequently, the herbicide-tolerant transgenic tobacco plants, which are described in the present study, can be utilized for phytoremediation of residual xenobiotics in the environment. Drought and salinity stress tolerance was also assessed. When exposed to 200 mM NaCl both the wild type and transgenic seedlings exhibit a reduction of root lenght, with the exception of the CsGSTU2 over-expressing tobacco line whose root length is as long as untreated control roots indicating a high level of tolerance to NaCl. The effect of drought stress upon root elongation was measured by growing seedlings in the presence of 8% mannitol. In this case all treated tobacco seedlings disclose a sharp decrease of root length, although transgenic lines appear to better tolerate drought stress conditions as the mean root length is significantly higher than that of treated tobacco wild type seedlings. In order to understand the response of tobacco plants over-expressing the CsGSTU genes to biotic stress, untransformed and transformed tobacco leaves were infiltrated with a bacterial suspension of the P. syringae pv. tabaci Tox+ DAPPG-PG 676 strain. The differences observed in symptomatology indicate that the over-expression of CsGSTU1 and CsGSTU2 in tobacco plant bestow the capability to avoid active toxin diffusion in plant tissues blocking chlorotic halos formation probably because tabtoxin is head towards a modification pathway in which CsGSTs could be involved in. This result was confirmed when tobacco leaves was treated with culture filtrates.