Cloning and Characterization of a Biotic-Stress-Inducible Glutathione Transferase from Phaseolus vulgaris

Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its Km for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris

Multiple evidence for the role of an Ovate-like gene in determining fruit shape in pepper

<p>Abstract</p> <p>Background</p> <p>Grafting is a widely used technique contributing to sustainable and ecological production of many vegetables, but important fruit quality characters such as taste, aroma, texture and shape are known for years to be affected by grafting in important vegetables species including pepper. From all the characters affected, fruit shape is the most easily observed and measured. From research in tomato, fruit shape is known to be controlled by many QTLs but only few of them have larger effect on fruit shape variance. In this study we used pepper cultivars with different fruit shape to study the role of a pepper <it>Ovate</it>-like gene, <it>CaOvate</it>, which encodes a negative regulator protein that brings significant changes in tomato fruit shape.</p> <p>Results</p> <p>We successfully cloned and characterized <it>Ovate</it>-like genes (designated as <it>CaOvate</it>) from two pepper cultivars of different fruit shape, cv. "Mytilini Round" and cv. "Piperaki Long", hereafter referred to as cv. "Round" and cv. "Long" after the shape of their mature fruits. The <it>CaOvate </it>consensus contains a 1008-bp ORF, encodes a 335 amino-acid polypeptide, shares 63% identity with the tomato OVATE protein and exhibits high similarity with OVATE sequences from other Solanaceae species, all placed in the same protein subfamily as outlined by expert sequence analysis. No significant structural differences were detected between the <it>CaOvate </it>genes obtained from the two cultivars. However, relative quantitative expression analysis showed that the expression of <it>CaOvate </it>followed a different developmental profile between the two cultivars, being higher in cv. "Round". Furthermore, down-regulation of <it>CaOvate </it>through VIGS in cv. "Round" changes its fruit to a more oblong form indicating that <it>CaOvate </it>is indeed involved in determining fruit shape in pepper, perhaps by negatively affecting the expression of its target gene, <it>CaGA20ox1</it>, also studied in this work.</p> <p>Conclusions</p> <p>Herein, we clone, characterize and study <it>CaOvate </it>and <it>CaGA20ox1 </it>genes, very likely involved in shaping pepper fruit. The oblong phenotype of the fruits in a plant of cv. "Round", where we observed a significant reduction in the expression levels of <it>CaOvate</it>, resembled the change in shape that takes place by grafting the round-fruited cultivar cv. "Round" onto the long-fruited pepper cultivar cv. "Long". Understanding the role of <it>CaOvate </it>and <it>CaGA20ox1</it>, as well as of other genes like <it>Sun </it>also involved in controlling fruit shape in Solanaceae plants like tomato, pave the way to better understand the molecular mechanisms involved in controlling fruit shape in Solanaceae plants in general, and pepper in particular, as well as the changes in fruit quality induced after grafting and perhaps the ways to mitigate them.</p

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

Highly conserved motifs in non-coding regions of Sirevirus retrotransposons: the key for their pattern of distribution within and across plants?

<p>Abstract</p> <p>Background</p> <p>Retrotransposons are key players in the evolution of eukaryotic genomes. Moreover, it is now known that some retrotransposon classes, like the abundant and plant-specific Sireviruses, have intriguingly distinctive host preferences. Yet, it is largely unknown if this bias is supported by different genome structures.</p> <p>Results</p> <p>We performed sensitive comparative analysis of the genomes of a large set of Ty1/<it>copia </it>retrotransposons. We discovered that Sireviruses are unique among <it>Pseudoviridae </it>in that they constitute an ancient genus characterized by vastly divergent members, which however contain highly conserved motifs in key non-coding regions: multiple polypurine tract (PPT) copies cluster upstream of the 3' long terminal repeat (3'LTR), of which the terminal PPT tethers to a distinctive attachment site and is flanked by a precisely positioned inverted repeat. Their LTRs possess a novel type of repeated motif (RM) defined by its exceptionally high copy number, symmetry and core CGG-CCG signature. These RM boxes form CpG islands and lie a short distance upstream of a conserved promoter region thus hinting towards regulatory functions. Intriguingly, in the envelope-containing Sireviruses additional boxes cluster at the 5' vicinity of the envelope. The 5'LTR/internal domain junction and a polyC-rich integrase signal are also highly conserved domains of the Sirevirus genome.</p> <p>Conclusions</p> <p>Our comparative analysis of retrotransposon genomes using advanced <it>in silico </it>methods highlighted the unique genome organization of Sireviruses. Their structure may dictate a life cycle with different regulation and transmission strategy compared to other <it>Pseudoviridae</it>, which may contribute towards their pattern of distribution within and across plants.</p

Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris

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

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

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