CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASES FROM NEEDLES OF Pinus brutia Ten. TREES

Glutathione S-transferases (GST, E.C. 2.5.1.18) are generally dimeric and multifunctional enzyme family which catalyse the nucleophilic attack of the glutathione on lipophilic compounds with electrophilic centres. Since the 70's GSTs in plant species have been intensively studied, as their role discovered in herbicide detoxification. However, there is only a limited number of studies considering the GST enzyme composition from forest trees, especially not in Pinus brutia, Ten. The trees that exhibited healthy appearance were selected and all belong to the same altitude profile which is located in METU / Yalincak area (Ankara, Turkey). GST activities in the supernatant fractions prepared from needles of P.brutia were determined spectrophotometrically by using 1-chloro-2,4-dinitrobenzene, 2,3-dichloro-4-(2-methylene butyryl)-phenoxy acetic acid (ethacrynic acid), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(p-nitrophenoxy) propane and p-nitrobenzyl chloride as substrates. Only 1-chloro-2,4-dinitrobenzene (160 +/- 10 nmoles min(-1) mg(-1)) and 1,2-dichloro-4-nitrobenzene (2.30 +/- 0.38 nmoles min(-1) mg(-1)) activities were detected and the rest were found as negligible. Accordingly, during purification of GSTs from needles of P.brutia, 1-chloro-2,4-dinitrobenzene was used as the substrate. Purification of GSTs was performed by sequential application of supernatant to gel filtration column chromatography on Sephadex G-25, anion exchange diethylaminoethyl cellulose column chromatography and S-hexylglutathione agarose affinity chromatography. After the final step of purification procedure, 1-chloro-2,4-dinitrobenzene conjugating activity of P. brutia cytosolic GSTs was purified about 15.45 fold with 1.95% yield. Sodium dodecyl sulfate polyacrylamide gel electophoresis results showed that the purified GST isozyme had an Mr of 24 kDa. With this study, we report for the first time the GST isozymes in a gymnosperm, P. brutia

New food for an old mouth: New enzyme for an ancient archaea

As a multifunctional group of enzymes, glutathione S-transferases (GSTs) are capable of inactivation, degradation or excretion of wide range of compounds catalytically or non-catalytically. However, to date, no study has been addresses the presence of GSTs in archaea based on their enzymatic functions. In this study, beside glutathione (GSH) amount measurement, the determination of GST activity in halophilic archaeon called Haloarcula hispanica ATCC 33960 were aimed. According to the results, specific activity was determined as 19.68 nmol min(-1) mg(-1) protein and GSH content were found to be as 194 mu g g(-1) K-m and V-max values for CDNB and GSH calculated from Lineweaver-Burk plot were 0.46 mM and 27.93 nmol min(-1) mg(-1),0.13 mM and 22.03 nmol min(-1) mg(-1), respectively. Hanes-Woolf and Eadie-Hofstee plots for CDNB and GSH were also found to be in co-relation with the results obtained from Lineweaver-Burk plot. To the best of our knowledge, GST enzymes have not been identified in archaea yet, at least based on their catalytic activities. Therefore, it is the first report on this area. (C) 2013 Elsevier Inc. All rights reserved

Molecular characterization of zeta class glutathione S-transferases from Pinus brutia Ten.

Glutathione transferases (GSTs; EC 2.5.1.18) play important roles in stress tolerance and metabolic detoxification in plants. In higher plants, studies on GSTs have focussed largely on agricultural plants. There is restricted information about molecular characterization of GSTs in gymnosperms. To date, only tau class GST enzymes have been characterized from some pinus species. For the first time, the present study reports cloning and molecular characterization of two zeta class GST genes, namely PbGSTZ1 and PbGSTZ2 from Pinus brutia Ten., which is an economically important pine native to the eastern Mediterranean region and have to cope with several environmental stress conditions. The PbGSTZ1 gene was isolated from cDNA, whereas PbGSTZ2 was isolated from genomic DNA. Sequence analysis of PbGSTZ1 and PbGSTZ2 revealed the presence of an open reading frame of 226 amino acids with typical consensus sequences of the zeta class plant GSTs. Protein and secondary structure prediction analysis of two zeta class PbGSTZs have shared common features of other plant zeta class GSTs. Genomic clone, PbGSTZ2 gene, is unexpectedly intronless. Extensive sequence analysis of PbGSTZ2, with cDNA clone, PbGSTZ1, revealed 87% identity at nucleotide and 81% identity at amino acid levels with 41 amino acids differences suggesting that genomic PbGSTZ2 gene might be an allelic or a paralogue version of PbGSTZ1

Molecular cloning and biochemical characterization of a Tau class glutathione S-transferase from Pinus brutia Ten

Key message A new Tau class GST gene was cloned from Pinus brutia Ten. cDNA sequence was analysed for conserved sequences. Substrate specificity, optimum pH, and temperature values of the recombinant PbGST Tau enzyme were determined. Tau class glutathione S-transferases (GSTs) are essential enzymes for detoxification in plants. To date, a lot of the members of this family have been characterized from different plants but the studies on the conifers are very scarce. This study investigates for the first time molecular cloning and biochemical characterization of a Tau class GST gene (PbGST Tau) from Pinus brutia Ten. The full length PbGST Tau ORF was 687 bp having a molecular mass of 27.37 kDa. Catalytic and ligand binding sites of PbGST Tau are well conserved and shared maximum identity with Pinus tabulaeformis GST Tau. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ECA) as substrates exhibited a K-m of 3.66 mM and 0.3 mM, respectively. PbGST Tau enzyme had an optimum activity at pH 6.0 and 8.0 when CDNB and ECA were used as substrate, respectively. The highest activity was measured at 25 degrees C. Through enzyme assays, phylogenetic analysis and structural modelling, we provide a detailed characterization of the PbGST Tau gene and the enzyme. This study is going to provide new insights into the phylogenetic and biochemical analysis of GST family in conifers

Transcriptomics of induced defense responses to greenbug aphid feeding in near isogenic wheat lines.

The greenbug aphid, Schizaphis graminum (Rondani) is an important cereal pest, periodically threatening wheat yields in the United States and around the world. The single dominant gene, Gb3-based resistance is highly durable against prevailing greenbug biotypes under field conditions; however, the molecular mechanisms of Gb3-mediated defense responses remain unknown. We used Affymetrix GeneChip Wheat Genome Arrays to investigate the transcriptomics of host defense responses upon greenbug feeding on resistant and susceptible bulks (RB and SB, respectively) derived from two near-isogenic lines. The study identified 692 differentially expressed transcripts and further functional classification recognized 122 transcripts that are putatively associated to mediate biotic stress responses. In RB, Gb3-mediated resistance resulted in activation of transmembrane receptor kinases and signaling-related transcripts involved in early signal transduction cascades. While in SB, transcripts mediating final steps in jasmonic acid biosynthesis, redox homeostasis, peroxidases, glutathione S-transferases, and notable defense-related secondary metabolites were induced. Also transcripts involved in callose and cell wall decomposition were elevated SB, plausibly to facilitate uninterrupted feeding operations. These results suggest that Gb3-mediated resistance is less vulnerable to cell wall modification and the data provides ample tools for further investigations concerning R gene based model of resistance