Transcriptome Profiling and Genome-Wide Association Studies Reveal GSTs and Other Defense Genes Involved in Multiple Signaling Pathways Induced by Herbicide Safener in Grain Sorghum.
ABSTRACT: Herbicide safeners protect cereal crops from herbicide injury by inducing genes and proteins involved in detoxification reactions, such as glutathione S-transferases (GSTs) and cytochrome P450s (P450s). Only a few studies have characterized gene or protein expression profiles for investigating plant responses to safener treatment in cereal crops, and most transcriptome analyses in response to safener treatments have been conducted in dicot model species that are not protected by safener from herbicide injury. In this study, three different approaches were utilized in grain sorghum (Sorghum bicolor (L.) Moench) to investigate mechanisms involved in safener-regulated signaling pathways. An initial transcriptome analysis was performed to examine global gene expression in etiolated shoot tissues of hybrid grain sorghum following treatment with the sorghum safener, fluxofenim. Most upregulated transcripts encoded detoxification enzymes, including P450s, GSTs, and UDP-dependent glucosyltransferases (UGTs). Interestingly, several of these upregulated transcripts are similar to genes involved with the biosynthesis and recycling/catabolism of dhurrin, an important chemical defense compound, in these seedling tissues. Secondly, 761 diverse sorghum inbred lines were evaluated in a genome-wide association study (GWAS) to determine key molecular-genetic factors governing safener-mediated signaling mechanisms and/or herbicide detoxification. GWAS revealed a significant single nucleotide polymorphism (SNP) associated with safener-induced response on chromosome 9, located within a phi-class SbGST gene and about 15-kb from a different phi-class SbGST. Lastly, the expression of these two candidate SbGSTs was quantified in etiolated shoot tissues of sorghum inbred BTx623 in response to fluxofenim treatment. SbGSTF1 and SbGSTF2 transcripts increased within 12-hr after fluxofenim treatment but the level of safener-induced expression differed between the two genes. In addition to identifying specific GSTs potentially involved in the safener-mediated detoxification pathway, this research elucidates a new direction for studying both constitutive and inducible mechanisms for chemical defense in cereal crop seedlings.
Project description:Plants respond to synthetic chemicals by eliciting a xenobiotic response (XR) that enhances the expression of detoxifying enzymes such as glutathione transferases (GSTs). In agrochemistry, the ability of safeners to induce an XR is used to increase herbicide detoxification in cereal crops. Based on the responsiveness of the model plant Arabidopsis thaliana to the rice safener fenclorim (4,6-dichloro-2-phenylpyrimidine), a series of related derivatives was prepared and tested for the ability to induce GSTs in cell suspension cultures. The XR in Arabidopsis could be divided into rapid and slow types depending on subtle variations in the reactivity (electrophilicity) and chemical structure of the derivatives. In a comparative microarray study, Arabidopsis cultures were treated with closely related compounds that elicited rapid (fenclorim) and slow (4-chloro-6-methyl-2-phenylpyrimidine) XRs. Both chemicals induced major changes in gene expression, including a coordinated suppression in cell wall biosynthesis and an up-regulation in detoxification pathways, whereas only fenclorim selectively induced sulfur and phenolic metabolism. These transcriptome studies suggested several linkages between the XR and oxidative and oxylipin signaling. Confirming links with abiotic stress signaling, suppression of glutathione content enhanced GST induction by fenclorim, whereas fatty acid desaturase mutants, which were unable to synthesize oxylipins, showed an attenuated XR. Examining the significance of these studies to agrochemistry, only those fenclorim derivatives that elicited a rapid XR proved effective in increasing herbicide tolerance (safening) in rice.
Project description:Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S-transferase (GST) activity. In previous research, two atrazine-resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine-glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F2 population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity-purified fractions compared to an atrazine-sensitive population (WCS). One-dimensional electrophoresis of these fractions displayed an approximate 26-kDa band, typical of GST subunits. Several phi- and tau-class GSTs were identified by LC-MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi-class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F2 population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.
Project description:The safener fenclorim (4,6-dichloro-2-phenylpyrimidine) increases tolerance to chloroacetanilide herbicides in rice by enhancing the expression of detoxifying glutathione S-transferases (GSTs). Fenclorim also enhances GSTs in Arabidopsis thaliana, and while investigating the functional significance of this induction in suspension cultures, we determined that these enzymes glutathionylated the safener. The resulting S-(fenclorim)-glutathione conjugate was sequentially processed to S-(fenclorim)-gamma-glutamyl-cysteine and S-(fenclorim)-cysteine (FC), the latter accumulating in both the cells and the medium. FC was then either catabolized to 4-chloro-6-(methylthio)-phenylpyrimidine (CMTP) or N-acylated with malonic acid. These cysteine derivatives had distinct fates, with the enzymes responsible for their formation being induced by fenclorim and FC. Fenclorim-N-malonylcysteine was formed from FC by the action of a malonyl-CoA-dependent N-malonyltransferase. A small proportion of the fenclorim-N-malonylcysteine then underwent decarboxylation to yield a putative S-fenclorim-N-acetylcysteine intermediate, which underwent a second round of GST-mediated S-glutathionylation and subsequent proteolytic processing. The formation of CMTP was catalyzed by the concerted action of a cysteine conjugate beta-lyase and an S-methyltransferase, with the two activities being coordinately regulated. Although the fenclorim conjugates tested showed little GST-inducing activity in Arabidopsis, the formation of CMTP resulted in metabolic reactivation, with the product showing good enhancing activity. In addition, CMTP induced GSTs and herbicide-safening activity in rice. The bioactivated CMTP was in turn glutathione-conjugated and processed to a malonyl cysteine derivative. These results reveal the surprisingly complex set of competing catabolic reactions acting on xenobiotics entering the S-glutathionylation pathway in plants, which can result in both detoxification and bioactivation.
Project description:Non-target site resistance (NTSR) to herbicides in black-grass (Alopecurus myosuroides) results in enhanced tolerance to multiple chemistries and is widespread in Northern Europe. To help define the underpinning mechanisms of resistance, global transcriptome and biochemical analysis have been used to phenotype three NTSR black-grass populations. These comprised NTSR1 black-grass from the classic Peldon field population, which shows broad-ranging resistance to post-emergence herbicides; NTSR2 derived from herbicide-sensitive (HS) plants repeatedly selected for tolerance to pendimethalin; and NTSR3 selected from HS plants for resistance to fenoxaprop-P-ethyl. NTSR in weeds is commonly associated with enhanced herbicide metabolism catalyzed by glutathione transferases (GSTs) and cytochromes P450 (CYPs). As such, the NTSR populations were assessed for their ability to detoxify chlorotoluron, which is detoxified by CYPs and fenoxaprop-P-ethyl, which is acted on by GSTs. As compared with HS plants, enhanced metabolism toward both herbicides was determined in the NTSR1 and NTSR2 populations. In contrast, the NTSR3 plants showed no increased detoxification capacity, demonstrating that resistance in this population was not due to enhanced metabolism. All resistant populations showed increased levels of AmGSTF1, a protein functionally linked to NTSR and enhanced herbicide metabolism. Enhanced AmGSTF1 was associated with increased levels of the associated transcripts in the NTSR1 and NTSR2 plants, but not in NTSR3, suggestive of both pre- and post-transcriptional regulation. The related HS, NTSR2, and NTSR3 plants were subject to global transcriptome sequencing and weighted gene co-expression network analysis to identify modules of genes with coupled regulatory functions. In the NTSR2 plants, modules linked to detoxification were identified, with many similarities to the transcriptome of NTSR1 black-grass. Critical detoxification genes included members of the CYP81A family and tau and phi class GSTs. The NTSR2 transcriptome also showed network similarities to other (a)biotic stresses of plants and multidrug resistance in humans. In contrast, completely different gene networks were activated in the NTSR3 plants, showing similarity to the responses to cold, osmotic shock and fungal infection determined in cereals. Our results demonstrate that NTSR in black-grass can arise from at least two distinct mechanisms, each involving complex changes in gene regulatory networks.
Project description:The closely related sulphonamide safeners, metcamifen and cyprosulfamide, were tested for their ability to protect rice from clodinafop-propargyl, a herbicide normally used in wheat. While demonstrating that both compounds were equally bioavailable in planta, only metcamifen prevented clodinafop from damaging seedlings, and this was associated with the enhanced detoxification of the herbicide. Transcriptome studies in rice cultures demonstrated that whereas cyprosulfamide had a negligible effect on gene expression over a 4 h exposure, metcamifen perturbed the abundance of 590 transcripts. Changes in gene expression with metcamifen could be divided into three phases, corresponding to inductions occurring over 30 min, 1.5 h and 4 h. The first phase of gene induction was dominated by transcription factors and proteins of unknown function, the second by genes involved in herbicide detoxification, while the third was linked to cellular homeostasis. Analysis of the inducible genes suggested that safening elicited similar gene families to those associated with specific biotic and abiotic stresses, notably those elicited by abscisic acid, salicylic acid, and methyl jasmonate. Subsequent experiments with safener biomarker genes induced in phase 1 and 2 in rice cell cultures provided further evidence of similarities in signalling processes elicited by metcamifen and salicylic acid.
Project description:Multiple-herbicide resistance (MHR) in black-grass (Alopecurus myosuroides) and annual rye-grass (Lolium rigidum) is a global problem leading to a loss of chemical weed control in cereal crops. Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to detoxify xenobiotics. In humans, MDR is linked to the overexpression of a pi class glutathione transferase (GSTP1), which has both detoxification and signaling functions in promoting drug resistance. In both annual rye-grass and black-grass, MHR was also associated with the increased expression of an evolutionarily distinct plant phi (F) GSTF1 that had a restricted ability to detoxify herbicides. When the black-grass A. myosuroides (Am) AmGSTF1 was expressed in Arabidopsis thaliana, the transgenic plants acquired resistance to multiple herbicides and showed similar changes in their secondary, xenobiotic, and antioxidant metabolism to those determined in MHR weeds. Transcriptome array experiments showed that these changes in biochemistry were not due to changes in gene expression. Rather, AmGSTF1 exerted a direct regulatory control on metabolism that led to an accumulation of protective flavonoids. Further evidence for a key role for this protein in MHR was obtained by showing that the GSTP1- and MDR-inhibiting pharmacophore 4-chloro-7-nitro-benzoxadiazole was also active toward AmGSTF1 and helped restore herbicide control in MHR black-grass. These studies demonstrate a central role for specific GSTFs in MHR in weeds that has parallels with similar roles for unrelated GSTs in MDR in humans and shows their potential as targets for chemical intervention in resistant weed management.
Project description:Plants contain large numbers of family 1 UDP-glucose-dependent glycosyltransferases (UGTs), including members that conjugate xenobiotics. Arabidopsis contains 107 UGT genes with 99 family members successfully expressed as glutathione transferase (GST)-fusion proteins in E. coli. A high-throughput catalytic screen was developed based on quantification of the fusion by measuring GST activity. UGT activity using UDP-glucose as donor was then determined using 11 synthetic acceptors bearing hydroxyl, amino and thiol groups that had been shown to undergo conjugation in plant extracts. In total, 44 UGTs, largely members of the D and E groups, were active towards xenobiotics, glucosylating phenol and thiol acceptors. In contrast, N-glucosyltransferase (NGT) activity was almost exclusively restricted to a single enzyme, UGT72B1. Using DNA microarrays, the induction of UGT transcripts following treatment with the herbicide safener fenclorim was compared in Arabidopsis and rice. D and L group members were the most safener-inducible UGTs in both species. The respective Arabidopsis enzymes showed low conjugating activity towards xenobiotics. Using Genevestigator, a small group of safened D and L UGTs were consistently induced in response to biotic and abiotic stress suggestive of protective activities beyond xenobiotic detoxification in both species. The induction of other detoxifying gene families following treatment with fenclorim, namely cytochromes P450 and glutathione transferases, further confirmed the selective enhancement of related subfamily members in the two species giving new insight into the safening response in cereals, where herbicide tolerance is enhanced compared with dicots, which are unresponsive to these treatments.
Project description:Natural tolerance in hexaploid bread wheat (Triticum aestivum L.) to synthetic auxin herbicides is primarily due to rapid metabolic detoxification, but genes encoding these herbicide-detoxifying enzymes have yet to be identified. Herbicide safeners are commonly applied in wheat to achieve herbicide tolerance by inducing the expression and activity of herbicide-detoxifying enzymes. While safeners have been utilized for decades, knowledge of mechanisms that induce gene expression is limited. Our objective was to identify wheat chromosomes possessing genes that endow natural or safener-induced tolerance to halauxifen-methyl (HM), a postemergence (POST) wheat-selective synthetic auxin herbicide, using alien substitution (the S genome of Aegilops searsii) and aneuploid lines. Two POST rates of HM were applied to seedlings with 1-2 leaves (Zadoks stages 11-12), and the highest HM rate was also applied with the safener cloquintocet-mexyl (CM). Wheat chromosomes possessing genes associated only with natural HM tolerance were identified because Ae. searsii is HM-sensitive but CM-responsive. Lines with substitutions for 5A and 5B displayed sensitivity to HM, and experiments with nullisomic-tetrasomic (NT) lines further indicated major genes associated with HM tolerance are present on 5A and 5B chromosomes. However, the genes on 5A appear to play a larger role because lines lacking 5A chromosomes displayed more sensitivity than lines lacking 5B. Overall, these results can be utilized to guide future transcriptome analyses to identify candidate genes that confer HM tolerance in wheat.
Project description:The pond wolf spider Pardosa pseudoannulata, an important natural predatory enemy of rice planthoppers, is found widely distributed in paddy fields. However, data on the genes involved in insecticide action, detoxification, and response are very limited for P. pseudoannulata, which inhibits the development and appropriate use of selective insecticides to control insect pests on rice. We used transcriptome construction from adult spider cephalothoraxes to analyze and manually identify genes enconding metabolic enzymes and target receptors related to insecticide action and detoxification, including 90 cytochrome P450s, 14 glutathione S-transferases (GSTs), 17 acetylcholinesterases (AChEs), 17 nicotinic acetylcholine receptors (nAChRs), and 17 gamma-aminobutyric acid (GABA) receptors, as well as 12 glutamate-gated chloride channel (GluCl) unigenes. Sequence alignment and phylogenetic analysis revealed the different subclassifications of P450s and GSTs, some important sequence diversities in nAChRs and GABA receptors, polymorphism in AChEs, and high similarities in GluCls. For P450s in P. pseudoannulata, the number of unigenes belonging to the CYP2 clade was much higher than that in CYP3 and CYP4 clades. The results differed from insects in which most P450 genes were in CYP3 and CYP4 clades. For GSTs, most unigenes belonged to the delta and sigma classes, and no epsilon GST class gene was found, which differed from the findings for insects and acarina. Our results will be useful for studies on insecticide action, selectivity, and detoxification in the spider and other related animals, and the sequence differences in target genes between the spider and insects will provide important information for the design of selective insecticides.