Tau Class Glutathione S-transferase Research Articles

CIPK11 phosphorylates GSTU23 to promote cold tolerance in Camellia sinensis.

Cold stress negatively impacts the growth, development, and quality of Camellia sinensis (Cs, tea) plants. CBL-interacting protein kinases (CIPK) comprise a pivotal protein family involved in plant development and response to multiple environmental stimuli. However, their roles and regulatory mechanisms in tea plants (Camellia sinensis (L.) O. Kuntze) remain unknown. Here we show that CsCBL-interacting protein kinase 11 (CsCIPK11), whose transcript abundance was significantly induced at low temperatures, interacts and phosphorylates tau class glutathione S-transferase 23 (CsGSTU23). CsGSTU23 was also a cold-inducible gene and has significantly higher transcript abundance in cold-resistant accessions than in cold-susceptible accessions. CsCIPK11 phosphorylated CsGSTU23 at Ser37, enhancing its stability and enzymatic activity. Overexpression of CsCIPK11 in Arabidopsis thaliana resulted in enhanced cold tolerance under freezing conditions, while transient knockdown of CsCIPK11 expression in tea plants had the opposite effect, resulting in decreased cold tolerance and suppression of the C-repeat-binding transcription factor (CBF) transcriptional pathway under freezing stress. Furthermore, the transient overexpression of CsGSTU23 in tea plants increased cold tolerance. These findings demonstrate that CsCIPK11 plays a central role in the signaling pathway to cold signals and modulates antioxidant capacity by phosphorylating CsGSTU23, leading to improved cold tolerance in tea plants.

Cr(VI) behaves differently than Cr(III) in the uptake, translocation and detoxification in rice roots

Excessive accumulation of chromium (Cr) causes severe damage to both physiological and biochemical processes and consequently growth repression in plants. Hexavalent chromium [Cr(VI)]-elicited alterations in plants have been widely elucidated at either physiological or molecular level, whereas little is known about trivalent chromium [Cr(III)]. Here, we found that both Cr(III) and Cr(VI) significantly inhibited root growth in rice plants. However, rice plants under Cr(VI) showed significantly less inhibition in root growth than those under Cr(III) at low levels, which might be attributed to the different hormetic effects of Cr(III) and Cr(VI) on rice plants. It was unexpected that Cr(III) could be actively taken up by rice roots similarly to Cr(VI); whereas they exhibited different kinetic uptake patterns. Furthermore, root-to-shoot Cr translocation under Cr(VI) was much lower than that under Cr(III). These results indicate that the uptake, translocation, and toxicity of Cr(III) differed greatly from those of Cr(VI). Transcriptome profiling of rice roots revealed that a series of gene families involved in detoxification, including ATP-binding cassette (ABC) transporters, multidrug and toxic compound extrusion proteins (MATEs), and Tau class glutathione S-transferases (GSTUs), were significantly associated with Cr accumulation and detoxification in rice roots. In addition, much more members of these gene families were upregulated by Cr(VI) compared to Cr(III), suggesting their vital roles in Cr uptake, translocation, and detoxification, especially under Cr(VI) stress. Further comparison of gstu9 and gstu10/50 mutants with their wild type confirmed that GSTUs play complex roles in the intracellular Cr transport and redox homeostasis during Cr(III) or Cr(VI) stress. Taken together, our findings provides new insights into the differential behaviors of Cr(III) and Cr(VI) in rice roots, as well as new candidate genes such as OsABCs and OsGSTUs, to further elucidate the mechanisms of the uptake, translocation, and detoxification of Cr(III) and Cr(VI).

Metabolism-Based Nontarget-Site Mechanism Is the Main Cause of a Four-Way Resistance in Shortawn Foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis Sobol. is a predominant grass weed in Chinese winter wheat fields, posing a substantial threat to crop production owing to its escalating herbicide resistance. This study documented the initial instance of an A. aequalis population (AHFT-3) manifesting resistance to multiple herbicides targeting four distinct sites: acetyl-CoA carboxylase (ACCase), acetolactate synthase, photosystem II, and 1-deoxy-d-xylulose-5-phosphate synthase. AHFT-3 carried an Asp-to-Gly mutation at codon 2078 of ACCase, with no mutations in the remaining three herbicide target genes, and exhibited no overexpression of any target gene. Compared with the susceptible population AHFY-3, AHFT-3 metabolized mesosulfuron-methyl, isoproturon, and bixlozone faster. The inhibition and comparison of herbicide-detoxifying enzyme activities indicated the participation of cytochrome P450s in the resistance to all four herbicides, with glutathione S-transferases specifically linked to mesosulfuron-methyl. Three CYP72As and a Tau class glutathione S-transferase, markedly upregulated in resistant plants, potentially played pivotal roles in the multiple-herbicide-resistance phenotype.

A tau class glutathione S-transferase in tea plant, CsGSTU45, facilitates tea plant susceptibility to Colletotrichum camelliae infection mediated by jasmonate signaling pathway.

Tea plant [Camellia sinensis (L.) O. Kuntze], as one of the most important commercial crops, frequently suffers from anthracnose caused by Colletotrichum camelliae. The plant-specific tau (U) class of glutathione S-transferases (GSTU) participates in ROS homeostasis. Here, we identified a plant-specific GST tau class gene from tea plant, CsGSTU45, which is induced by various stresses, including C. camelliae infection, by analyzing multiple transcriptomes. CsGSTU45 plays a negative role in disease resistance against C. camelliae by accumulating H2 O2 . JA negatively regulates the resistance of tea plants against C. camelliae, which depends on CsGSTU45. CsMYC2.2, which is the key regulator in the JA signaling pathway, directly binds to and activates the promoter of CsGSTU45. Furthermore, silencing CsMYC2.2 increased disease resistance associated with reduced transcript and protein levels of CsGSTU45, and decreased contents of H2 O2 . Therefore, CsMYC2.2 suppresses disease resistance against C. camelliae by binding to the promoter of the CsGSTU45 gene and activating CsGSTU45. CsJAZ1 interacts with CsMYC2.2. Silencing CsJAZ1 attenuates disease resistance, upregulates the expression of CsMYC2.2 elevates the level of the CsGSTU45 protein, and promotes the accumulation of H2 O2 . As a result, CsJAZ1 interacts with CsMYC2.2 and acts as its repressor to suppress the level of CsGSTU45 protein, eventually enhancing disease resistance in tea plants. Taken together, the results show that the JA signaling pathway mediated by CsJAZ1-CsMYC2.2 modulates tea plant susceptibility to C. camelliae by regulating CsGSTU45 to accumulate H2 O2 .

OsGSTU17, a Tau Class Glutathione S-Transferase Gene, Positively Regulates Drought Stress Tolerance in Oryza sativa.

As a great threat to the normal growth of rice, drought not only restricts the growth of rice, but also affects its yield. Glutathione S-transferases (GSTs) have antioxidant and detoxification functions. In rice, GSTs can not only effectively cope with biological stress, but also play a defense role against abiotic stress. In this study, we selected OsGSTU17, a member gene that was induced by drought, to explore the role of GSTs and analyze their physiological mechanisms that are involved in rice drought tolerance. With the CRISPR/Cas9 knockout system techniques, we obtained two independent mutant lines of osgstu17. After 14 days of drought stress treatment, and then re-supply of the water for 10 days, the survival rate of the osgstu17 mutant lines was significantly reduced compared to the wild-type (WT). Similarly, with the 10% (w/v) PEG6000 hydroponics experiment at the seedling stage, we also found that compared with the WT, the shoot and root biomass of osgstu17 mutant lines decreased significantly. In addition, both the content of the MDA and H2O2, which are toxic to plants, increased in the osgtu17 mutant lines. On the other hand, chlorophyll and proline decreased by about 20%. The activity of catalase and superoxide dismutase, which react with peroxides, also decreased by about 20%. Under drought conditions, compared with the WT, the expressions of the drought stress-related genes OsNAC10, OsDREB2A, OsAP37, OsP5CS1, OsRAB16C, OsPOX1, OsCATA, and OsCATB in the osgtu17 mutant lines were significantly decreased. Finally, we concluded that knocking out OsGSTU17 significantly reduced the drought tolerance of rice; OsGSTU17 could be used as a candidate gene for rice drought-tolerant cultivation. However, the molecular mechanism of OsGSTU17 involved in rice drought resistance needs to be further studied.

Genome-wide identification and characterization of glutathione S-transferase gene family in quinoa (Chenopodium quinoa Willd.).

The present investigation was envisaged for large scale in-silico genome wide identification and characterization of glutathione S-transferases (GSTs) in Chenopodium quinoa. In this study, a total of 120 GST genes (CqGSTs) were identified and divided into 11 classes of which tau and phi were highest in numbers. The average protein length of protein was found to be 279.06 with their corresponding average molecular weight of 31,819.4kDa. The subcellular localization analysis results showed that proteins were centrally localized in the cytoplasm followed by chloroplast, mitochondria and plastids. Structural analysis revealed the presence of 2 -14 exons in CqGST genes. Most of the proteins possessed two exon one intron organization. MEME analysis identified 15 significantly conserved motifs with a width of 6-50 amino acids. Motifs 1, 3, 2, 5, 6, 8, 9 and 13 were found specifically in tau class family; motifs 3, 4, 5, 6, 7 and 9 were found in phi class gene family, while motifs 3, 4, 13 and 14 were found in metaxin class. Multiple sequence alignment revealed highly conserved N-terminus with active site serine (Ser; S) or cysteine (Cys; C) residue for the activation of GSH binding and GST catalytic activity. The gene loci were found to be unevenly distributed across 18 different chromosomes with a maximum of 17 genes located on chromosome number 7. Dominance of alpha helix was followed by coil, extended strand and beta turns. Gene duplication analysis revealed that segmental duplication and purifying type selection were highest in number and found to be main source of expansion of GST gene family. Cis acting regulatory elements analysis showed the presence of 21 different elements involved in stress, hormone and light response and cellular development. The evolutionary relationship of CqGST proteins carried out using maximum likelihood method revealed that all the tau and phi class GSTs were closely associated with those of G. max, O. sativa and A. thaliana. Molecular docking of GST molecules with the fungicide metalaxyl showed that the CqGSTF1 had the lowest binding energy. The comprehensive study of CqGST gene family in quinoa provides groundwork for further functional analysis of CqGST genes in the species at molecular level and has potential applications in plant breeding.

PeGSTU58, a Glutathione S-Transferase from Populus euphratica, Enhances Salt and Drought Stress Tolerance in Transgenic Arabidopsis.

Glutathione S-transferases (GSTs) play a crucial role in responding to abiotic stress and are an important target for research on plant stress tolerance mechanisms. Populus euphratica is a promising candidate species for investigating the abiotic tolerance mechanisms in woody plants. In our previous study, PeGSTU58 was identified as being associated with seed salinity tolerance. In the present study, PeGSTU58 was cloned from P. euphratica and functionally characterized. PeGSTU58 encodes a Tau class GST and is located in both the cytoplasm and nucleus. Transgenic Arabidopsis overexpressing PeGSTU58 displayed enhanced tolerance to salt and drought stress. Under salt and drought stress, the transgenic plants exhibited significantly higher activities of antioxidant enzymes, including SOD, POD, CAT, and GST, compared to the wild-type (WT) plants. Additionally, the expression levels of several stress-responsive genes, including DREB2A, COR47, RD22, CYP8D11, and SOD1 were upregulated in PeGSTU58 overexpression lines compared to those in WT Arabidopsis under salt and drought stress conditions. Furthermore, yeast one-hybrid assays and luciferase analysis showed that PebHLH35 can directly bind to the promoter region of PeGSTU58 and activate its expression. These results indicated that PeGSTU58 was involved in salt and drought stress tolerances by maintaining ROS homeostasis, and its expression was positively regulated by PebHLH35.

Walnut JrGSTU23 and JrVHAc4 involve in drought tolerance via JrWRKY2-mediated upstream regulatory pathway

Drought stress negatively affects the walnut productivity. To ensure the comprehensive benefits of walnut, this study aims to explore the molecular mechanism of drought stress response. Therefore, the downstream target genes of JrWRKY2, a drought tolerance regulator of Juglans regia, are identified by analyses of promoter expression, yeast one-hybrid and co-expression. The results showed that JrWRKY2 could bind to the W-BOX like motifs from the promoters of JrGSTU23 (a tau class glutathione S-transferase) and JrVHAc4 (a c subunit of vacuole H+-ATPase). JrGSTU23 and JrVHAc4 could be up-regulated by drought stress and abscisic acid (ABA). The drought tolerance of transgenic Arabidopsis overexpression of JrGSTU23 or JrVHAc4 were promoted significantly compared to wild type (WT) plants; More importantly, the degree of improvement in drought resistance is enhanced by application of ABA. The transcription levels of the genes from signaling pathways of ABA and mitogen-activated protein kinases (MAPKs) in JrGSTU23 and JrVHAc4 overexpression plants were higher than those in WT with different profiles. These results propose a trifurcate feed-forward pathway involving in JrWRKY2, JrGSTU23, and JrVHAc4 functions on drought stress response that associated with ABA and MAPK signaling pathways, which will offer new insights to reveal plant adaptation mechanism to adverse stimulus.

Effects of conserved Arg20, Glu74 and Asp77 on the structure and function of a tau class glutathione S-transferase in rice.

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.

OsGSTU6 Contributes to Cadmium Stress Tolerance in Rice by Involving in Intracellular ROS Homeostasis

Glutathione S-transferases (GSTs) are multifunctional proteins that play important roles in cellular detoxification as well as in plant growth and development. Here, we cloned a new rice tau class GST gene, OsGSTU6, and evaluated its roles in regulating cadmium (Cd) stress tolerance of rice. OsGSTU6 is located both in the cytosol and nucleus of cells. The transcripts of OsGSTU6 were detected in the whole life cycle of rice with higher levels in the leaf blade and sheath at heading stage. The transcripts of OsGSTU6 were significantly stimulated by a number of environmental factors including several phytohormones, abiotic stresses, and heavy metals, suggesting its stress-responsive characteristics. Moreover, the overexpression (OE) of OsGSTU6 in rice reduced the accumulation of Cd in leaf and enhanced the tolerance of the plant to Cd stress, while knockdown (RNAi) of OsGSTU6 triggered Cd accumulation in rice leaves and decreased stress tolerance. Meanwhile, the expression levels of several candidate genes responsible for scavenging reactive oxygen species (ROS) were downregulated in OE of OsGSTU6 lines. In addition, the content of superoxide anions (O2−) and glutathione showed reduction in OE plants, but was augmented in RNAi plants under both normal growth and Cd stress, compared with wild type. Furthermore, both the yeast two-hybrid assay and firefly luciferase complementation imaging (LCI) assay demonstrated that OsGSTU6 might function with dimers in plant cells. All these results suggest that OsGSTU6 may play an important role in Cd stress tolerance of rice by involving in the intracellular ROS homeostasis.

The AtGSTU7 gene influences glutathione-dependent seed germination under ABA and osmotic stress in Arabidopsis

Glutathione S-transferases (GSTs) play important roles in metabolism and detoxification of plant cells. However, their functions during development are less well understood. Arabidopsis AtGSTU7 (AT2G29420) encodes a Tau class GST. Here we provide the AtGSTU7 was abundantly expressed in seeds and in roots at an early stage of germination. AtGSTU7 expression was repressed by exogenous ABA and promoted by osmotic stress. A null mutant of AtGSTU7 (atgstu7) accumulated higher contents of reduced GSH and decreased amounts of endogenous H2O2 in seedlings. The atgstu7 plants showed decreased osmotic tolerance during seed germination, which was influenced by GSH and ABI3 gene expression. The results suggested that AtGSTU7 involvement in seed germination is mediated by GSH–ROS homeostasis and ABA signaling.

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 Kₘ 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 °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.

Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa.

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

Phytoremediation of Ni-contaminated soil by Salicornia iranica

ABSTRACTAlthough nickel (Ni) is useful and is used in various industries, it is one of the most usual and important sources of heavy metals pollutants in the world. In this study, Salicornia iranica was used in order to phytoremediate Ni-contaminated soil. Possible mechanisms of plant tolerance to Ni pollution and its detoxification were studied through using expression analysis of glutathione-S-transferase (GST) and measurement of involved key physiological components. The concentration of the chlorophylls a, b, total chlorophyll, and carotenoids were significantly decreased in 500 mg/kg Ni at 3, 24, 48 h, and 90 days after the treatment. Free proline significantly increased in the tissues. The absorption and concentration of Ni increased in tissues, so that Ni concentration at 50, 250, and 500 mg Ni/kg soil significantly increased to 2.5, 3.5, and 4.5 fold compared with the lowest Ni level respectively. In addition, the GST expression was significantly increased both in the 50 and 500 mg/kg Ni treatment. The highest concentration of Ni affected plant growth parameters such as the root and shoot lengths. Therefore, S. iranica is able to accumulate Ni and it can be used as an environmental biotechnological study for phytoremediation of Ni-polluted soils.Abbreviations: ABA: abscisic acid; ABRE: ABA-responsive element; As+3: arsenic; Cd2+: cadmium; ef1: elongation factor; FW: fresh weight; GSH: glutathione; GST: glutathione-S-transferase; GSTU: tau class GST; Hcl: hydrochloric acid; Hg2+: mercury; HgCl2: mercury(II) chloride; MYB: myeloblastosis viral oncogene homolog; Ni+2: nickel; Pb: lead; SiGSTU: Salicornia iranica GSTU; ZnSO4: zinc sulfate

A Tau Class Glutathione-S-Transferase is Involved in Trans-Resveratrol Transport Out of Grapevine Cells.

Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or β-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium.

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM min−1 and 68.49 s−1 respectively and 0.693 mM, 105.32 mM min−1 and 89.57 s−1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.

Functional Characterization of the Tau Class Glutathione-S-Transferases Gene (SbGSTU) Promoter of Salicornia brachiata under Salinity and Osmotic Stress.

Reactive oxygen or nitrogen species are generated in the plant cell during the extreme stress condition, which produces toxic compounds after reacting with the organic molecules. The glutathione-S-transferase (GST) enzymes play a significant role to detoxify these toxins and help in excretion or sequestration of them. In the present study, we have cloned 1023 bp long promoter region of tau class GST from an extreme halophyte Salicornia brachiata and functionally characterized using the transgenic approach in tobacco. Computational analysis revealed the presence of abiotic stress responsive cis-elements like ABRE, MYB, MYC, GATA, GT1 etc., phytohormones, pathogen and wound responsive motifs. Three 5’-deletion constructs of 730 (GP2), 509 (GP3) and 348 bp (GP4) were made from 1023 (GP1) promoter fragment and used for tobacco transformation. The single event transgenic plants showed notable GUS reporter protein expression in the leaf tissues of control as well as treated plants. The expression level of the GUS gradually decreases from GP1 to GP4 in leaf tissues, whereas the highest level of expression was detected with the GP2 construct in root and stem under control condition. The GUS expression was found higher in leaves and stems of salinity or osmotic stress treated transgenic plants than that of the control plants, but, lower in roots. An efficient expression level of GUS in transgenic plants suggests that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). And this property, make it as a potential candidate to be used as an alternative promoter for crop genetic engineering.

Genome-wide association analysis and differential expression analysis of resistance to Sclerotinia stem rot in Brassica napus.

Brassica napus is one of the most important oil crops in the world, and stem rot caused by the fungus Sclerotinia sclerotiorum results in major losses in yield and quality. To elucidate resistance genes andpathogenesis-relatedgenes, genome-wide association analysis of 347 accessions was performed using the Illumina 60K Brassica SNP (single nucleotide polymorphism) array. In addition, the detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B.napus lines, 48h postinoculation with S.sclerotiorum for transcriptome sequencing. We identified 17 significant associations for stem resistance on chromosomes A8 and C6, five of which were on A8 and 12 on C6. The SNPs identified on A8 were located in a 409-kb haplotype block, and those on C6 were consistent with previous QTL mapping efforts. Transcriptome analysis suggested that S.sclerotiorum infection activates the immune system, sulphur metabolism, especially glutathione (GSH) and glucosinolates in both R and S genotypes. Genes found to be specific to the R genotype related to the jasmonic acid pathway, lignin biosynthesis, defence response, signal transduction and encoding transcription factors. Twenty-four genes were identified in both the SNP-trait association and transcriptome sequencing analyses, including a tau class glutathione S-transferase (GSTU) gene cluster. This study provides useful insight into the molecular mechanisms underlying the plant's response to S.sclerotiorum.

Isolation and characterization of three cadmium-inducible promoters from Oryza sativa

Cadmium (Cd) is an important soil pollutant. Developing genetically engineered crops might be a feasible strategy for Cd decontamination and damage prevention. Both genes and promoters are critical for the effective construction of genetically modified plants. Although many functional genes for Cd tolerance and accumulation have been identified, few reports have focused on plant Cd-inducible promoters. Here, we identified three Cd-inducible genes in the rice genome: two tau class glutathione S-transferase (GSTU) genes, OsGSTU5 and OsGSTU37, and an HSP20/alpha crystallin family protein gene, OsHSP18.6. The promoter sequences were isolated and tested in transgenic rice lines using a GUSplus reporter gene. All of the promoters exhibited low background expression under normal conditions and could be strongly induced by Cd stress. Although their strength was comparable to that of the constitutive OsACTIN promoter under Cd stress, their time-dependent expression patterns under both short- and long-term Cd exposure were markedly different. The responses of the three promoters to other heavy metals were also examined. Furthermore, heavy metal-responsive cis elements in the promoters were computationally analyzed, and regions determining the Cd stress response were analyzed using a series of truncations. Our results indicate that the three Cd-inducible rice promoters described herein could potentially be used in applications aimed at improving heavy metal tolerance in crops or for the bio-monitoring of environmental contamination.

Overexpression of GsGSTU13 and SCMRP in Medicago sativa confers increased salt-alkaline tolerance and methionine content.

Tau-class glutathione S-transferases (GSTUs) are ubiquitous proteins encoded by a large gene family in plants, which play important roles in combating different environmental stresses. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three GSTUs (GsGSTU13, GsGSTU14 and GsGSTU19) as potential salt-alkaline stress-responsive genes. Two of them, GsGSTU14 and GsGSTU19, have been shown to positively regulate plant salt-alkaline tolerance. In this study, we further demonstrated the positive function of GsGSTU13 in plant salt-alkaline stress responses by overexpressing it in Medicago sativa. Stress tolerance tests suggested that GsGSTU13 transgenic lines showed better growth and physiological indicators than wild alfalfa (cv. Zhaodong) under alkaline stress. Considering the shortage of methionine in alfalfa, we then co-transformed GsGSTU13 into two main alfalfa cultivars in Heilongjiang Province (cv. Zhaodong and cv. Nongjing No. 1) together with SCMRP, a synthesized gene that could improve the methionine content. We found that GsGSTU13/SCMRP transgenic alfalfa displayed not only higher methionine content but also higher tolerance to alkaline and salt stresses, respectively. Taken together, our results demonstrate that GsGSTU13 acts as a positive regulator in plant responses to salt and alkaline stresses, and can be used as a good candidate for generation of salt-alkaline tolerant crops.