The Use of Highly Specific GSTs towards the Development of Stress Tolerant Transgenic Plants (pp. 263-274)

Glutathione transferases (GSTs) belong to a super-family of multifunctional proteins. GSTs play a key role in cellular detoxification from xenobiotic substances like herbicides, secondary metabolites and toxic degradation products resulting from oxidative stress and cellular metabolism. Furthermore, environmental conditions generate oxidative stress, the products of which have to be detoxified by plants. It is anticipated that environmental stresses will worsen over the following years due to climate change. Hence, plants must adapt rapidly to the new environmental conditions in order to both survive and satisfy the constantly increasing human demand for agricultural products. Genetic engineering has been successfully used to develop plants resistant to stresses and, having taken all the necessary precautions, could offer a solution as it can help to develop plants with desirable traits in a short period of time. We present here the use of GST isoenzymes in the development of transgenic plants. Although transgenic plants over-expressing various GSTs have been used for “in planta” evaluation of the enzymes used in response to different stresses, the results show that GSTs could be of great value for generating stress tolerant plants. However, the literature is limited and more studies should be performed in order to exploit their full potential

Structure and catalytic properties of human glutathione transferase p1-1

Glutathione transferases (EC 2.5.1.18, GSTs) catalyze the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of electrophilic compounds helping to detoxify a diverse array of toxic xenobiotics including carcinogenic, and pharmacologically active compounds. In this review, detailed descriptions are given on the structure and catalytic properties of human glutathione transferase P1-1 (hGSTP1-1) an enzyme that ubiquitously expressed in human tissues and exhibits many biological functions and multiple roles. The detoxification properties of hGSTP1-1 have been a primary research focus for the last years. However, now it has become apparent that the noncatalytic functions of GSTP1-1 have expanded the biological roles of this enzyme in cell survival, cell death and stress signalling mechanism

Structure and Antioxidant Catalytic Function of Plant Glutathione Transferases

Plant cytosolic glutathione transferases (GSTs) are an ancient enzyme superfamily with multiple and diverse functions which are important in counteracting biotic and abiotic stress. GSTs play an important role in catalyzing the conjugation of xenobiotics and endogenous electrophilic compounds with glutathione (GSH), such as pesticides, chemical carcinogens, environmental pollutants, which leads to their detoxification. GSTs not only catalyze detoxification reactions but they are also involved in GSH-dependent isomerization reactions, in GSH-dependent reduction of organic hydroperoxides formed during oxidative stress, biosynthesis of sulfur-containing secondary metabolites, and exhibit thioltransferase and dehydroascorbate reductase activity. This review focuses on plant GSTs, and attempts to give an overview of the new insights into the catalytic function and structural biology of these enzymes

Morphological, Physiological and Metabolomic Response of transgenic tobacco plants (N. tabacum L.) overexpressing GmGSTU4 under Drought Stress

GSTs appear to have a significant role in plants’ adaptation under abiotic stress as many isoenzymes are found to be differentially expressed under these conditions yet, little is known about the regulatory functions of GSTs. Wild type and transgenic tobacco plants over-expressing the soybean GmGSTU4 of cultivars Basmas, Burley and Virginia were grown in vitro under 100 and 200mM mannitol or in soil (plant pots) by withholding watering for 15 days. However, GmGSTU4 plants did not exhibit significant differences in drought tolerance compared to wild-type plants. Morphological (shoot length, total and root fresh weight) and physiological (chlorophyll content, relative water content and photosynthetic capacity) parameters of transgenic plants did not differ from the wild-type in the presence of 100 or 200mM mannitol or in the soil when watering was halted. Metabolite profiling was used to understand the dynamics between the wild-type and transgenic tobacco response to drought stress. Different metabolic pathways are involved in production of osmoprotectants. These molecules accumulate in plants under stress conditions as adaptive mechanism, which can provide stress tolerance. GmGSTU4 plants did not exhibit difference in drought tolerance compared to wild-type plants, however metabolomics analysis indicated alterations in metabolite profile and increased concentration of sorbitol, glycerol and pyruvic acid. In conclusion, overexpression of GmGSTU4 in transgenic plants did not affect their drought stress tolerance although it has altered their metabolite profile possible because of diverse effects on plant stress tolerance mechanism

Maintenance of metabolic homeostasis and induction of cytoprotectants and secondary metabolites in alachlor-treated GmGSTU4-overexpressing tobacco plants, as resolved by metabolomics

Herbicides are an invaluable tool for agricultural production scaling up. However, their continuous and intensive use has led to an increased incidence of herbicide resistant weeds and environmental pollution. Plant glutathione transferases (GSTs) are tightly connected with crop and weed herbicide tolerance capacitating their efficient metabolic detoxification, thus GSTs can be biotechnologically exploited towards addressing those issues. However, information on their effects at a “systems” level in response to herbicides is lacking. Here, we aimed to study the effects of the chloroacetanilide herbicide alachlor on the metabolome of wild-type and tobacco plants overexpressing the soybean tau class glutathione transferase GmGSTU4. Alachlor-treated wild-type plants This system, naturally serving the detoxification of endogenous exhibited an abiotic stress-like response with increased abundance of compatible solutes, decrease in TCA cycle intermediates and decreased sugar and amino acid content. Transgenic plants responded distinctly, exhibiting an increased induction of abiotic stress responsive metabolites, accumulation of secondary metabolites and its precursors, and metabolic detoxification by-products compared to wild-type plants. These results suggest that the increased metabolic capacity of GmGSTU4 overexpressing plants is accompanied by pleiotropic metabolic alterations, which could be the target for further manipulation in order to develop herbicide resistant crops, plants with increased phytoremediation potential, as well as efficient management of non-target site, GST induced, herbicide resistance in weeds

Over-expression of a specific soybean GmGSTU4 isoenzyme improves chloroacetanilide herbicide tolerance of transgenic tobacco plants

Plant glutathione transferases (GSTs) have a major role in herbicide detoxification. Soybean (Glycine max L.) GmGSTs have been well studied for their correlation in herbicides selectivity towards diphenyl ether, chloroacetanilide and sulfonylurea herbicides. Chloroacetanilide herbicide tolerance was assayed in vitro by measuring the growth inhibition of wild type (wt) and transgenic tobacco seedlings from cultivars (Basmas, Virginia, Burley) in the presence of 7.5 and 15 mg/L of alachlor and metolachlor. Alachlor caused strong inhibition of shoot and root growth of wt tobacco plants. All the transgenic Basmas lines showed significantly higher shoot and root elongation at 7.5 mg/L alachlor, with line BAGST-3 exhibiting the greatest tolerance. However, at 15 mg/L alachlor, growth was highly reduced in transgenic and wt plants. In Burley, only line BUGST-2 has statistically significant greater mean of root and shoot length compared to wt under the two doses. On the contrary, Virginia has reduced growth which was similar to the wt. Metolachlor toxicity was less severe compared to alachlor. Growth of the transgenic lines of the three cultivars was not significantly greater in either metolachlor concentrations tested, compared to wt plants, except line BAGST-3 which exhibited significantly greater mean of shoot and root elongation at 7.5 mg/L. Transgene expression was determined quantitatively using Real Time qPCR, lines BAGST-3 and BUGST-2 showed greater expression of Gmgstu4 in shoot compared to root. These results confirm that overexpression of GmGSTU4 in tobacco provides higher catalytic activities towards xenobiotics, resulting for future use in environmental cleanup of alachlor

Isolation of GST isoenzymes from Phaseolus vulgaris L. and characterization of detoxifying mechanism under biotic and abiotic stress

Three Glutathione transferase (GSTs) isoenzymes have been isolated from P. vulgaris leaves after in vivo treatment with 1/250 fluazifop-p-butyl herbicide. The inducible GST isoenzymes were identified and separated by affinity chromatography. They found to belong to phi and tau classes. Moreover, the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs, termed PvGSTU2-2, PvGSTF1-1 and PvGSTU3-3) were found to catalyze a broad range of reactions and exhibit quite varied substrate specificity. Another GST isoenzyme was isolated from P. vulgaris (PvGST, termed PvGSTU3-3), after its induction with biotic stress treatment (Uromyces appendiculatus infection). PvGSTU3-3 shares high homology the tau class plant and catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance are the high antioxidant catalytic function and hydroperoxidase, thioltransferase, and dehydroascorbate reductase action of Pv-GSTU3-3. Transgenic tobacco plants over-expressing PvGSTU2-2 isoenzyme have been developed via Agrobacteriun tumefaciens in order to study their in planta potential to confer biotic and abiotic resistance as a means of plant breeding. Our results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris. Moreover, highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in P. vulgaris

Expanding the Plant GSTome Through Directed Evolution: DNA Shuffling for the Generation of New Synthetic Enzymes With Engineered Catalytic and Binding Properties

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible multifunctional enzymes that are essential in the detoxification and degradation of toxic compounds. GSTs have considerable biotechnological potential. In the present work, a new method for the generation of synthetic GSTs was developed. Abiotic stress treatment of Phaseolus vulgaris and Glycine max plants led to the induction of total GST activity and allowed the creation of a GST-enriched cDNA library using degenerated GST-specific primers and reverse transcription-PCR. This library was further diversified by employing directed evolution through DNA shuffling. Activity screening of the evolved library led to the identification of a novel tau class GST enzyme (PvGmGSTUG). The enzyme was purified by affinity chromatography, characterized by kinetic analysis, and its structure was determined by X-ray crystallography. Interestingly, PvGmGSTUG displayed enhanced glutathione hydroperoxidase activity, which was significantly greater than that reported so far for natural tau class GSTs. In addition, the enzyme displayed unusual cooperative kinetics toward 1-chloro-2,4-dinitrochlorobenzene (CDNB) but not toward glutathione. The present work provides an easy approach for the simultaneous shuffling of GST genes from different plants, thus allowing the directed evolution of plants GSTome. This may permit the generation of new synthetic enzymes with interesting properties that are valuable in biotechnology