Glutathione S-transferases activity in rice
91609858
Glutathione S transferases modulate Cu tolerance in Oryza sativa
Abstract Along with the widespread use of copper (Cu) based fungicides and fertilizers, Cu contaminated soil has been a major issue resulting in the loss of crop yield. To improve Cu tolerance in Oryza sativa, we previously carried out a screen of Cu sensitivity and obtained the Cu tolerant (B1139) and the Cu sensitive (B1195) rice varieties. Here, by analyzing an RNA Seq transcriptome profile of root tips, 20 glutathione S transferase (GST) genes were differently expressed between the Cu tolerant B1139 and the Cu sensitive B1195 when exposed to excess Cu2+ A more rapid decrease of root GST activity occurred in Cu treated B1195 than in Cu treated B1139, suggesting that different GST activity might affect Cu tolerance in rice. This is further supported by the observation of Cu hypersensitivity in rice seedlings deficient of the most abundant GST protein, OsGSTF2, demonstrating that GST proteins modulate Cu tolerance in rice. This, at least partially, leads to different Cu tolerance between B1139 and B1195. Furthermore, homologous modeling showed that a putative Cu2+ binding site, OsGSTF2H41 locates at the active center of GST enzyme. In vitro analysis demonstrated that Cu2+ interferes with the GST activity of OsGSTF2 protein, indicating that GST activity reduction by Cu2+ might be the binding of Cu2+ to the OsGSTF2H41 site. Consistently, amino acid replacement of histidine 41 with glycine (OsGSTF2H41G) resulted in decreased GST activity of OsGSTF2. Additionally, ectopic expression of OsGSTF2 enhances Cu tolerance in Escherichia coli, suggesting that the GST proteins might alleviate Cu toxicity by the binding of free Cu2+ in cells.
Lu Li Meng Hou Lily Cao Yan Xia Zhenguo Shen Zhubing Hu
26506018
Distinct time courses of increase in cytochromes P450 1A2, 2A5 and glutathione S transferases during the progressive hepatitis associated with Helicobacter hepaticus.
Mice naturally infected by Helicobacter hepaticus develop a chronic active hepatitis leading to hepatocellular carcinoma. This mouse model of liver cancer was used to examine the impact of bacterial infection on the hepatic expression and activity of enzymes involved in carcinogen bioactivation (phase I enzymes) and detoxification (phase II enzymes) No major differences in total cytochrome P450 (CYP) content were found between control and infected mice during the course of the study. The most striking modulations of individual isoenzymes were the increases in immunohistochemical staining observed for CYP1A and CYP2A5 in relation to increasing age and liver lesions. The increase in CYP2A5 in mice aged over 12 months was confirmed by the observed increases in coumarin 7 hydroxylation (CYP2A5 substrate) in vitro and CYP2A5 mRNA levels by Northern blot analysis. Immunoblotting confirmed the specific induction of CYP1A2 in infected mice 12 and 18 months of age. Perfusion of liver with nitroblue tetrazolium, an indicator for superoxide formation, demonstrated that in livers of infected mice, hepatocytes often co expressed CYP2A5 and formazan deposition. Concerning phase II enzymes, an enhancement of glutathione S transferase (GST) activities, related to the disease process, was observed in infected mice. An age specific increase of GSTpi and A4.4 (early stage of disease) and GST YaYa >9 months) expression was also demonstrated by immunohistochemical staining. In contrast, catalase and glutathione peroxidase activities, as well as reduced glutathione content were decreased in the early stages of disease (3 9 months) in infected mice compared to age matched control mice. Overall, these results suggest that alterations in CYP and GST expression may contribute to the aetiology of tumour incidence due to H. hepaticus infection via production of reactive oxygen species.
Carcinogenesis
Pascale Chomarat Marek Sipowicz Bhalchandra A Diwan Laura W Fornwald Yogesh C Awasthi Miriam Anver Jerry M Rice Lucy M Anderson Christopher Paul Wild
85388494
Characterization of two cDNAs encoding glutathione S transferases in rice and induction of their transcripts by the herbicide safener fenclorim
The safener fenclorim (4,6 dichloro 2 phenyl pyrimidine) induces glutathione S transferases (GSTs; EC 2.5.18) in rice (Oryza sativa L. cv. Lemont) including those with activity toward pretilachlor [2 chloro 2',6' diethyl N (2 propoxyethyl)acetamide] and this is associated with increased tolerance to this herbicide. In this study, we report on the isolation and characterization of two partial cDNA clones encoding GSTs in rice. The two cDNA clones were isolated by using GST specific degenerate primers and the RT PCR (reverse transcriptase polymerase chain reaction) technique from control (unsafened) and fenclorim treated (safened) rice roots, respectively. Sequence analyses demonstrated that the two cDNAs share 60% amino acid sequence identity and 62% nucleotide sequence identity, appearing to represent two different rice GSTs (RGST I and II) Southern blot analysis with the two clones confirmed the presence of at least three GST genes in the rice genome. Northern blotting with the two clones as probes showed that the GST gene(s) are constitutively expressed in roots of unsafened rice seedlings. Treatment with the safener fenclorim induced the expression of the rice GST gene(s) The fenclorim mediated induction of mRNA expression coincided with a concomitant induction of total GST activity toward pretilachlor in roots of fenclorim safened rice. Phylogenetic analysis based on the amino acid sequence alignment of the two rice cDNAs with sequences of other plant GSTs showed that RGST I seems to be more closely related to wheat GSTs, while RGST II is more closely related to the maize GST I and IV isozymes.
Jingrui Wu Carole L Cramer Kriton K Hatzios
230108023
Effects of conserved Arg20, Glu74 and Asp77 on the structure and function of a tau class glutathione S transferase in rice.
KEY MESSAGE The relative position of domains is critical for enzymatic properties of tau class glutathione S transferases, and altering the position of linker far away from the active center affects catalytic property. Glutathione S transferases (GSTs) are a family of phase II detoxification enzymes whose main function is to improve plant resistance to stresses. To understand the structural effects of tau class GSTs on their function, using OsGSTU17 as an example, we predicted the residues involved in the interactions between its domains and linker region. We further detected the structural changes in mutants and the corresponding changes in terms of substrate activity and kinetic parameters. Four pairs of residues, including Ala14 and Trp165, Arg20 and Tyr154, Glu74 and Arg98, Asp77 and Met87, forming hydrogen bonds and salt bridges were found to play important roles in maintaining the relative position between the domains and linker region inside the protein. The hydrogen bond between Trp165 and Ala14 affected the structural stability has been demonstrated in our previous study. The mutant R20A lost almost all catalytic activity. Interestingly, the mutant E74A exhibited a significant decrease in activity towards 7 chloro 4 nitrobenzo 2 oxa 1, 3 diazole, 1 chloro 2, 4 dinitrobenzene and 4 nitrobenzyl chloride, while its activity towards substrate cumene hydroperoxide remained unchanged. Compared with other mutants, the mutant D77A exhibited decreased affinity to its substrates and increased activity towards 1 chloro 2, 4 dinitrobenzene and cumene hydroperoxide, but its thermodynamic stability did not change significantly. The relative position of individual domain was critical for enzymatic properties, and the linker which is far away from the active site could change the enzymatic properties of GSTs via altering the relative position of the individual domain. Our results provide insights into the relationship between structure and function of tau class GSTs.
Plant molecular biology
Xue Yang Zhi-hai Wu Jie Gao
84094405
Characterization and Safener Induction of Multiple Glutathione S Transferases in Three Genetic Lines of Rice
Abstract Multiple glutathione S transferase (GST; EC 2.5.1.18) activity peaks were characterized by fast protein liquid chromatography (FPLC) in three genetic lines of rice (Oryza sativa L. Root applied pretilachlor (0.15 mM) inhibited root growth of 5 day old etiolated seedlings by 67% in Teqing (indica type) 54% in Koshihikari (japonica type) and 34% in Lemont (indica x japonica type) The safener fenclorim (1 mM) prevented the injury caused by pretilachlor in all genotypes. GST activity from 5 day old untreated or fenclorim treated seedlings of the three genetic lines was assayed using 1 chloro 2,4 dinitrobenzene (CDNB) radiolabeled pretilachlor, and radiolabeled fenclorim as substrates. GST activity with each substrate was greater in roots than in shoots of the three genotypes. Treatment with fenclorim increased GST activity in roots and shoots by 20 to 90% GST activity in all genotypes was greater with CDNB, followed by pretilachlor, and then fenclorim. GST(CDNB) and GST(pretilachlor) activities were greater in Lemont, followed by Koshihikari, and then Teqing. In contrast, GST(fenclorim) activity was greater in Koshihikari and least in Lemont. FPLC elution patterns (MonoQ column) of partially purified GST extracts from untreated and fenclorim treated seedlings of the three rice genotypes revealed the presence of multiple GST activities. Teqing shoots had six GST activity peaks with fenclorim. Four of them were also active with CDNB and pretilachlor. Koshihikari shoots had six GST activity peaks with pretilachlor. Five of them were also active with fenclorim, but only three were active with CDNB. Lemont shoots exhibited three GST activity peaks with fenclorim, five with CDNB, and seven with pretilachlor. Multiple GST activity peaks using CDNB and pretilachlor as substrates were also present in the roots of Teqing, Koshihikari, and Lemont seedlings. Fenclorim increased the activity of GST peaks conjugating pretilachlor and CDNB more than those active with fenclorim. Induction of GST peaks was more evident in Lemont, followed by Koshihikari, and then Teqing. The differential patterns of GST peaks active toward pretilachlor show that rice japonica lines may be more tolerant to pretilachlor than indica lines.
Fan Deng Kriton K Hatzios
85017306
Characterization of glutathione S transferase of the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) Comparison of its properties of glutathione S transferases from other lepidopteran insects
Abstract An enzyme that possesses the glutathione S transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1 chloro 2,4 dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3 10 and at temperatures below 50 degC. The enzyme was also able to conjugate glutathione to 4 hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.
Kohji Yamamoto Satoshi Teshiba Yoichi Aso
54549452
Rice (Oryza sativa L. tau class glutathione S transferase (OsGSTU30) overexpression in Arabidopsis thaliana modulates a regulatory network leading to heavy metal and drought stress tolerance.
Glutathione S transferases (GSTs) are well known enzymes due to their role in detoxification of xenobiotic compounds. However, their biochemical action is still not so clear in imparting tolerance to several abiotic stresses in crop plants. In our previous study, we observed that rice tau class OsGSTU30 plays a significant role in the detoxification of Cr(vi) Interestingly, q RT PCR analysis also revealed higher expression of OsGSTU30 under drought conditions. In this study, we characterize OsGSTU30 in response to drought as well as heavy metal [Cr(vi) stresses through overexpression in Arabidopsis thaliana. Biochemical and physiological analyses revealed that OsGSTU30 overexpression lines have improved tolerance against both stresses as compared to wild type plants. Kinetic analysis and molecular docking confirmed that OsGSTU30 enzyme possesses both GST as well as glutathione peroxidase (GPx) like activity. Differentially expressed stress responsive genes were also identified by transcriptome analysis, involved in different biological pathways during abiotic stresses. These results suggest the signaling functions of OsGSTU30 apart from its catalytic activity during abiotic stress responses and can be further exploited for improving the stress tolerance in crops.
Metallomics integrated biometal science
Dipali Srivastava Giti Verma Abhishek Singh Chauhan Veena Pande Debasis Chakrabarty
73442099
Increasing tolerance to bispyribac sodium is able to allow glutathione homeostasis to recover in indica rice compared with japonica rice.
The high activity and broad weed spectrum of BS has made it widely used in China. However, accidental crop injuries, particularly occurring in Jiangsu, Hunan, Hubei and Heilongjiang provinces in recent years, have resulted in limiting the application of BS in China. In this study, glutathione homeostasis was measured in the contrasting sensitivity of indica and japonica rice cultivar after bispyribac sodium (BS) treatment. The results showed that japonica rice cultivar Nanjing 9108 was more sensitive to BS than indica rice Nanjing 11 and indica hybrid cultivar Guangliangyou 6326. In response to the exposure of BS in all rice cultivars, especially Nanjing 9108, the perturbation of glutathione homeostasis occurred, including the decreased reduced glutathione (GSH) and increased oxidized glutathione (GSSG) These results were supported by increased activities of glutathione S transferases (GSTs) in Nanjing 11 and Guangliangyou 6326. Further tests revealed that when Nanjing 11 was pretreated with the glutathione depleting agents L buthionine sulfoximine (BSO) or diethylmaleate (DEM) the GSH levels, the activity of GSTs, and the gene expression levels of GR and GSTs decreased, finally increasing the phytotoxicity of BS. The aforementioned DEM inhibitory responses were further rescued by exogenously applied GSH. In contrast, the pretreatment of glutathione or N acetyl L cysteine (NAC) not only increased the contents of GSH, the activities of GSTs, and the expression level of GR and GSTs gene, but also alleviated BS phytotoxicity in Nanjing 9108. In both cultivars, DEM increased phytotoxicity and GSH partially reversed this. This study suggests that increasing tolerance to BS was able to allow glutathione homeostasis to recover in indica rice cultivar compared with japonica rice cultivar.
Pesticide biochemistry and physiology
Hongchun Wang Zhijie Guo Wenbiao Shen Yuanlai Lou
24707788
Plasma B esterase and glutathione S transferase activity in the toad Chaunus schneideri (Amphibia, Anura) inhabiting rice agroecosystems of Argentina
B esterase (BChE: butyrylcholinesterase and CbE: carboxylesterase) and glutathione S transferase (GST) activity were measured in the plasma of Chaunus schneideri collected in rice fields and surrounding environments and in a reference pristine forest. The chemical criterion based on in vitro reactivation of BChE activity using pyridine 2 aldoxime methochloride (2 PAM) was also determined. Mean values of plasma BchE, CbE, and GST activity for samples from agricultural areas were different from those for samples from pristine forest. Plasma samples from the two agricultural areas showed positive reactivation of BChE activity after incubation with 2 PAM. Based on our experimental evidence we suggest B esterases and gluthatione S transferases can be used in field monitoring as biomarkers of exposure of wildlife to pesticides, because the analysis in non destructive and is sensitive to anti ChE agrochemicals. Chemical reactivation of BChE is also a complementary method for assessing the effects of pesticides on toads inhabiting rice fields. Further studies are urgently needed to investigate adverse effects of massive exposure to pesticides experienced by native populations of anurans.
Ecotoxicology
Andres M Attademo Paola Mariela Peltzer Rafael Carlos Lajmanovich Mariana C Cabagna Gabriela S Fiorenza
87538589
Purification and Characterization of Glutathione S Transferases of Eoreuma loftini and Diatraea saccharalis (Lepidoptera: Pyralidae)
Clutathione S transferases (CST) from larvae of the sugarcane borer, Diatraea Saccharalis (F. and the Mexican rice borer, Eareuma laftini (Dyar) were characterized. Apparently homogeneous CST isoenzymes from the larvae of both species were purified with CSH affinity chromatography and isoelectric focusing. E. laftini had higher CST activity (31.12 units/g wet weight of tissue) compared with D. saccharalis (24.8 units/g wet weight of tissue) The CST isoenzyme of D. saccharalis with a pi value of 9.3 was a dimer of two identical subunits M 25,000) whereas the CST isoenzyme with a pi value of 8.0 was a heterodimer with subunit M, values of 25,000 and 27,000. The CST isoenzymes of E. laftini with pI values of 9.7 and 7.7 were homo dimers of subunits with M, values of 25,000 and 26,000, respectively. However, the CST isoenzyme with a pI value of 5.3 was a heterodimer of subunits having M, values of 26,000 and 27,000. Peptide fingerprint analysis by SV 8 protease digestion revealed primary structural differences in these isoenzymes. The N termini of all CST isoenzymes from both the species of larvae were blocked. Substrate specificities and kinetic properties of the purified CSTs from both the species have been described. The low Km and high V max values toward l chloro 2,4 dinitrobenzene of CST enzymes isolated from both D. saccharalis and E. laftini indicate that these enzymes have a high affinity for electrophilic substrates compared with most human CSTs.
Narendra Kumar Tiwari Sharad S Singhal Rajendra K Sharma Robert L Meagher Yogesh C Awasthi