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RNA-Seq analsis of rye-grass transcriptomic response to an herbicide inhibiting acetolactate-synthase identifies transcripts linked to non-target-site-based resistance
Abstract
Non-target-site resistance (NTSR) to herbicides that disrupts agricultural weed control is a worldwide concern for food security. NTSR is considered a polygenic adaptive trait driven by differential gene regulation in resistant plants. Little is known about its genetic determinism, which precludes NTSR diagnosis and evolutionary studies. We used Illumina RNA-sequencing to investigate transcriptomic differences between plants from the global major weed rye-grass sensitive or resistant to the acetolactate-synthase (ALS) inhibiting herbicide pyroxsulam. Plants were collected before and along a time-course after herbicide application. De novo transcriptome assembly yielded a resource (LOLbase) including 92,381 contigs representing potentially active transcripts that were assigned putative annotations. Early effects of ALS inhibition consistent with the literature were observed in resistant and sensitive plants, proving LOLbase data were relevant to study herbicide response. Comparison of resistant and sensitive plants identified 30 candidate NTSR contigs. Further validation using 212 plants resistant or sensitive to pyroxsulam and/or to the ALS inhibitors iodosulfuron + mesosulfuron confirmed four contigs (two cytochromes P450, one glycosyl-transferase and one glutathione-S-transferase) were NTSR markers which combined expression levels could reliably identify resistant plants. This work confirmed that NTSR is driven by differential gene expression and involves different mechanisms. It provided tools and foundation for subsequent NTSR investigations.
... Some studies on herbicide stress to plants were also carried out using RNA-seq. For example, after treatment with TBM, single nucleotide polymorphisms (SNPs) [12] and non-targetsite resistance (NTSR)-related genes such as for glutathiones, peroxidases, oxidases, hydrolases, and transporter proteins were identified in Myosoton aquaticum L. (water chickweed) [13], short-awn foxtail [14], grain sorghum [12] and rye grass [15]. Application of TBM affected root and above-ground growth of cornflower [16], and reduced the biomass of foxtail millet [17]. ...
... The response to herbicide stress involves various antioxidant defense mechanisms [15], including enzymatic and non-enzymatic antioxidant detoxification pathways. The most common non-enzymatic antioxidant pathways include the defense response activated by plant hormones and signal transduction, as well as osmotic regulation [12]. ...
... This was consistent with our research, in which the expression of ABA 8′hydroxylase was inhibited in the S line, and germination ability and root length were reduced. GST catalyzes the reaction between herbicide and glutathione, or acts as a peroxidase to reduce oxidative damage [15]. The active site, Cys-19 in GSTZ1, has an especially strong catalytic effect [32]. ...
Background
Tribenuron methyl (TBM) is an herbicide that inhibits sulfonylurea acetolactate synthase (ALS) and is one of the most widely used broad-leaved herbicides for crop production. However, soil residues or drifting of the herbicide spray might affect the germination and growth of rapeseed, Brassica napus, so it is imperative to understand the response mechanism of rape to TBM during germination. The aim of this study was to use transcriptome analysis to reveal the gene responses in herbicide-tolerant rapeseed to TBM stress during seed germination.
Results
2414, 2286, and 1068 differentially expressed genes (DEGs) were identified in TBM-treated resistant vs sensitive lines, treated vs. control sensitive lines, treated vs. control resistant lines, respectively. GO analysis showed that most DEGs were annotated to the oxidation-reduction pathways and catalytic activity. KEGG enrichment was mainly involved in plant-pathogen interactions, α-linolenic acid metabolism, glucosinolate biosynthesis, and phenylpropanoid biosynthesis. Based on GO and KEGG enrichment, a total of 137 target genes were identified, including genes involved in biotransferase activity, response to antioxidant stress and lipid metabolism. Biotransferase genes, CYP450, ABC and GST, detoxify herbicide molecules through physical or biochemical processes. Antioxidant genes, RBOH, WRKY, CDPK, MAPK, CAT, and POD regulate plant tolerance by transmitting ROS signals and triggering antioxidant enzyme expression. Lipid-related genes and hormone-related genes were also found, such as LOX3, ADH1, JAZ6, BIN2 and ERF, and they also played an important role in herbicide resistance.
Conclusions
This study provides insights for selecting TBM-tolerant rapeseed germplasm and exploring the molecular mechanism of TBM tolerance during germination.
... NTSR mechanisms are increasingly considered an important method for weed resistance to ALS inhibitor herbicides (Deĺye, 2013). NTSR mainly refers to the physiological metabolic detoxification process of herbicides through non-target pathways and the process of limiting the ability of herbicides to reach their target sites so that they lose activity, which can help to achieve detoxification (Duhoux et al., 2015). After the herbicide enters the plant, there are three main stages to the metabolic process. ...
... Unmetabolized herbicides will destroy membrane lipid peroxidation, promote the production of superoxide anion, hydrogen peroxide, and malondialdehyde, and damage the balance of production and scavenging of reactive oxygen species (ROS) (Liu et al., 2018). The main metabolic enzymes involved include CYP450, superoxide dismutase, and glutelin reductase (Duhoux et al., 2015). CYP450-mediated enhanced herbicide metabolism is one of the most important NTSR mechanisms. ...
Introduction
Nicosulfuron is the leading acetolactate synthase inhibitor herbicide product, and widely used to control gramineous weeds. Here, we investigated the metabolic process of nicosulfuron into foxtail millet and maize, in order to clarify the mechanism of the difference in sensitivity of foxtail millet and maize to nicosulfuron from the perspective of physiological metabolism and provide a theoretical basis for the breeding of nicosulfuron-resistant foxtail millet varieties.
Methods
We treated foxtail millet (Zhangzagu 10, Jingu 21) and maize (Nongda 108, Ditian 8) with various doses of nicosulfuron in both pot and field experiments. The malonaldehyde (MDA) content, target enzymes, detoxification enzymes, and antioxidant enzymes, as well as related gene expression levels in the leaf tissues of foxtail millet and maize were measured, and the yield was determined after maturity.
Results
The results showed that the recommended dose of nicosulfuron caused Zhangzagu 10 and Jingu 21 to fail to harvest; the yield of the sensitive maize variety (Ditian 8) decreased by 37.09%, whereas that of the resistant maize variety (Nongda 108) did not decrease. Nicosulfuron stress increased the CYP450 enzyme activity, MDA content, and antioxidant enzyme activity of foxtail millet and maize, reduced the acetolactate synthase (ALS) activity and ALS gene expression of foxtail millet and Ditian 8, and reduced the glutathione S-transferase (GST) activity and GST gene expression of foxtail millet. In conclusion, target enzymes, detoxification enzymes, and antioxidant enzymes were involved in the detoxification metabolism of nicosulfuron in plants. ALS and GST are the main factors responsible for the metabolic differences among foxtail millet, sensitive maize varieties, and resistant maize varieties.
Discussion
These findings offer valuable insights for exploring the target resistance (TSR) and non-target resistance (NTSR) mechanisms in foxtail millet under herbicide stress and provides theoretical basis for future research of develop foxtail millet germplasm with diverse herbicide resistance traits.
... The present study also focused on the differential gene expression of these enzymes. The CYP 450 family protein was identified as differentially expressed in various herbicideresistant weeds [36][37][38][39][40]. This protein manages phase I to activate the herbicide for further detoxification in phase II mediated by GST and GT [36]. ...
... The CYP 450 family protein was identified as differentially expressed in various herbicideresistant weeds [36][37][38][39][40]. This protein manages phase I to activate the herbicide for further detoxification in phase II mediated by GST and GT [36]. In glyphosate-resistant morning glory, Ipomoea purpurea, the mechanism of glyphosate resistance could be explained by CYP 450 expression, while the expression of the EPSPS gene was unchanged [41]. ...
The evolution of herbicide-resistant weed species is a serious threat for weed control. Therefore, we need an improved understanding of how gene regulation confers herbicide resistance in order to slow the evolution of resistance. The present study analyzed differentially expressed genes after glyphosate treatment on a glyphosate-resistant Tennessee ecotype (TNR) of horseweed (Conyza canadensis), compared to a susceptible biotype (TNS). A read size of 100.2 M was sequenced on the Illumina platform and subjected to de novo assembly, resulting in 77,072 gene-level contigs, of which 32,493 were uniquely annotated by a BlastX alignment of protein sequence similarity. The most differentially expressed genes were enriched in the gene ontology (GO) term of the transmembrane transport protein. In addition, fifteen upregulated genes were identified in TNR after glyphosate treatment but were not detected in TNS. Ten of these upregulated genes were transmembrane transporter or kinase receptor proteins. Therefore, a combination of changes in gene expression among transmembrane receptor and kinase receptor proteins may be important for endowing non-target-site glyphosate-resistant C. canadensis.
... Metabolism-based resistance to herbicides that inhibit ACCases has been reported extensively, with instances increasing more in recent years [14]. RNA-seq has been successfully used to identify genes involved in metabolic resistance to acetolactate synthase (ALS) herbicides in two grass weed species, including Wimmera ryegrass (Lolium rigidum Gaud.) [15] and slender foxtail (Alopecurus myosuroides) [16]. However, no study has yet been conducted at the whole transcriptome level to understand potential NTSR mechanisms involved in L. chinensis resistance. ...
... However, there are few studies on herbicide metabolism and resistance genes at the whole transcriptome level, which has slowed the development of new control mechanisms. RNA-seq has been successfully used to identify genes involved in metabolic resistance to ALS herbicides in two grass weed species: L. rigidum [15] and A. myosuroides [16]. However, there has been no study focused on the NTSR mechanisms of L. chinensis at the whole transcriptome level. ...
Background
Chinese sprangletop [Leptochloa chinensis (L.) Nees] is an annual invasive weed, which can often be found in paddy fields. Cyhalofop-butyl is a specialized herbicide which is utilized to control L. chinensis. However, in many areas, L. chinensis has become resistant to this key herbicide due to its continuous long-term use.
Results
In this study, we utilized a resistant (LC18002) and a sensitive (LC17041) L. chinensis populations previously identified in our laboratory, which were divided into nine different treatment groups. We then employed whole transcriptome analysis to identify candidate genes which may be involved in cyhalofop-butyl tolerance. This analysis resulted in the identification of eight possible candidate genes, including six cytochrome P450 monooxygenase genes and two ATP-binding cassette transporter genes. We then carried out a phylogenetic analysis to identify homologs of the differentially expressed P450 genes. This phylogenetic analysis indicated that every genes have close homologs in pattern species, some of which have been implicated in non-target site resistance (NTSR).
Conclusions
This study is the first to use whole transcriptome analysis to identify herbicide non-target resistance genes in L. chinensis. The differentially expressed genes represent promising targets for better understanding herbicide tolerance in L. chinensis. The eight genes belonging to classes already associated in herbicide tolerance may play important roles in the metabolic resistance of L. chinensis to cyhalofop-butyl, although the exact mechanisms require further study.
... Herbicide detoxification is a complex metabolic process involving herbicide transformation, conjugation and exportation for final processing, coordinated by enzymes and regulatory genes [55,68]. Herbicide metabolism studies indicate that tolerance or resistance is mostly due to increased rates of herbicide detoxification, in both crops and weeds, and generally involves the activity of cytochrome P450 monooxygenases [12,14,[69][70][71][72][73]. In this study, the expression of CYP72A14 and CYP72A15 increased when exposed to florpyrauxifen-benzyl-benzyl, while CYP709B1, CYP709B2, CYP72A14 and CYP72A15 were induced by glufosinate-ammonium treatment, especially when subjected to high temperature stress. ...
... Increased UGT expression was also observed after glufosinate-ammonium and imazethapyr application. Enzymes in the glycosyltransferase family have been reported to play a role in phase II of xenobiotic detoxification, specifically herbicide metabolism, and are associated with abiotic stress tolerance [55,69,102,103]. ...
Global climate change, specifically rising temperature, can alter the molecular physiology of weedy plants. These changes affect herbicide efficacy and weed management. This research aimed to investigate the combined effect of heat stress (HS) and sublethal doses of herbicides (four active ingredients) on adaptive gene expression and efficacy of herbicide on Echinochloa colona (L.) Link (junglerice). Three factors were evaluated; factor A was E. colona generation (G0-original population from susceptible standard; G1 and G2 were progenies of recurrent selection), factor B was herbicide treatment (florpyrauxifen-benzyl, glufosinate-ammonium, imazethapyr, quinclorac and nontreated check) and factor C was HS (30 and 45 °C). The herbicides were applied at 0.125× the recommended dose. Recurrent exposure to HS, combined with sublethal doses of herbicides, favors the selection of plants less susceptible to the herbicide. Upregulation of defense (antioxidant) genes (APX: Ascorbate peroxidase), herbicide detoxification genes (CYP450 family: Cytochrome P450), stress acclimation genes (HSP: Heat shock protein, TPP: Trehalose phosphate phosphatase and TPS: Trehalose phosphate synthase) and genes related to herbicide conjugation (UGT: UDP Glucosyltransferase) was significant. The positive regulation of these genes may promote increased tolerance of E. colona to these herbicides.
... The significant CM-induced UGTs identified by RNA-Seq also warrant examination in the future, especially since reactions catalyzed by CYPs may result in phytotoxic metabolites and still require further detoxification by UGTs [6,13]. In general, herbicide-detoxifying UGTs are not as well characterized compared to CYPs, especially in the Poaceae, but their expression is often induced by herbicides and safeners [38,44,83,84]. Some examples in wheat include detection of glucosylated metabolites of isoproturon [85] and florasulam [86]. ...
Identification and characterization of genes encoding herbicide-detoxifying enzymes is lacking in allohexaploid wheat (Triticum aestivum L.). Gene expression is frequently induced by herbicide safeners and implies the encoded enzymes serve a role in herbicide metabolism and detoxification. Cloquintocet-mexyl (CM) is a safener commonly utilized with halauxifen-methyl (HM), a synthetic auxin herbicide whose phytotoxic form is halauxifen acid (HA). Our first objective was to identify candidate HA-detoxifying genes via RNA-Seq by comparing untreated and CM-treated leaf tissue. On average, 81% of RNA-Seq library reads mapped uniquely to the reference genome and 76.4% of reads were mapped to a gene. Among the 103 significant differentially expressed genes (DEGs), functional annotations indicate the majority of DEGs encode proteins associated with herbicide or xenobiotic metabolism. This finding was further corroborated by gene ontology (GO) enrichment analysis, where several genes were assigned GO terms indicating oxidoreductase activity (34 genes) and transferase activity (45 genes). One of the significant DEGs is a member of the CYP81A subfamily of cytochrome P450s (CYPs; denoted as CYP81A-5A), which are of interest due to their ability to catalyze synthetic auxin detoxification. To investigate CYP expression induced by HM and/or CM, our second objective was to measure gene-specific expression of CYP81A-5A and its homoeologs (CYP81A-5B and CYP81A-5D) in untreated leaf tissue and leaf tissue treated with CM and HM over time using RT-qPCR. Relative to the reference gene (β-tubulin), basal CYP expression is high, expression among these CYPs varies over time, and expression for all CYPs is CM-inducible but not HM-inducible. Further analysis of CYP81A-5A, such as gene knock-out, overexpression experiments, or in vitro activity assays with purified enzyme are necessary to test the hypotheses that the encoded CYP detoxifies HA and that CM upregulates this reaction.
... With the assistance of RNA-Seq, the NTSR mechanism in various weed species has been well investigated, and many functional resistance genes have been identified. RNA-Seq has been recently used in transcriptome analysis of plant responses to herbicide stresses in the grass weeds Eleusine indica and Echinochloa cruss-galli (Yang et al. 2013), and in identifying genes involved in NTSR in Lolium rigidum (Duhoux et al. 2015;Gaines et al. 2014) and Alopecurus myosuroides (Gardin et al. 2015). ...
Background
Bromus japonicus Thunb. is a hazardous weed in wheat fields in China. One B. japonicus population, HB-1, which was collected in Hebei Province, survived field rate flucarbazone-sodium exposure.
Method
To investigate the mechanism of flucarbazone-sodium resistance in the HB-1 population, the herbicide target als gene was isolated, and the in vitro ALS activity was tested. HPLC‒MS/MS was applied to detect flucarbazone-sodium absorption and residue level. Furthermore, RNA-seq and qRT‒PCR were applied to detect genes related to flucarbazone-sodium metabolism or flucarbazone-sodium and its metabolite transport.
Result
The results indicated that in resistant population there were no mutations in the als gene sequences or differences in the IC50 of ALS compared with the sensitive population. In addition, als gene relative copy number and expression also showed no difference. However, significantly increased herbicide metabolism was found in the HB-1 population. In the end, four cytochrome P450 genes (C71D55b, C71A1, C71D55, T5H), three glutathione S-transferase genes (GSTZ, GSTU6b, GSTFB), and three ABC transporter genes (ABCC3, ABCF1, ABCG11) were selected and identified as potentially playing a role in flucarbazone-sodium resistance.
Graphical Abstract
... Transcriptome sequencing (RNA-Seq) technology offers a robust approach for exploring the genetic mechanisms underlying herbicide stress responses in weeds [15,16], especially for identifying the genetic differences between herbicide-resistant and susceptible plants [17][18][19]. Recently, RNA-Seq has been used to identify several genes related to NTSR in weeds, such as Lolium rigidum Gaudin [17,20,21], Alopecurus myosuroides Huds. [22], Beckmannia syzigachne Steud. ...
The evolved resistance of Bromus japonicus Houtt. to ALS-inhibiting herbicides is well established. Previous studies have primarily focused on target-site resistance; however, non-target-site resistance has not been well characterized. This investigation demonstrated that ALS gene sequencing did not detect any previously known resistance mutations in a mesosulfuron-methyl-resistant (MR) population, and notably, treatment with the P450 monooxygenase (P450) inhibitor malathion markedly heightened susceptibility to mesosulfuron-methyl. Utilizing UPLC-MS/MS analysis confirmed elevated mesosulfuron-methyl metabolism in MR plants. The integration of Isoform Sequencing (Iso-Seq) and RNA Sequencing (RNA-Seq) facilitated the identification of candidate genes associated with non-target sites in a subpopulation with two generations of herbicide selection. Through qRT-PCR analysis, 21 differentially expressed genes were characterized, and among these, 10 genes (comprising three P450s, two glutathione S-transferases, one glycosyltransferase, two ATP-binding cassette transporters, one oxidase, and one hydrolase) exhibited constitutive upregulation in resistant plants. Our findings substantiated that increased herbicide metabolism is a driving force behind mesosulfuron-methyl resistance in this B. japonicus population.
... Herbicide-tolerant transgenic plants have been successfully created and used in agriculture for several years. Cytochrome P450 and glutathione Stransferase are essential enzymes that are vital in accelerating the breakdown of herbicides among several groups of enzymes (Duhoux et al., 2015). ...
... Regulatory elements and factors, such as promoters, untranslated regions (UTRs), and transcription factors (TFs), of candidate genes may also contribute to the prominent role of genes on wheat chromosome 5A in controlling HA detoxification compared with its homoeologous chromosomes 5B and 5D. The role of TFs in herbicide detoxification is often inferred, as RNA-seq has identified TFs regulated by herbicides and safeners (Baek et al. 2019;Brazier-Hicks et al. 2020;Chen et al. 2023;Duhoux et al. 2015;Gaines et al. 2014;Zhang et al. 2022). Homoeologue expression bias is commonly observed in allopolyploid plants (Grover et al. 2012), partly attributed to variations in cis-and transregulatory elements between homoeologues (He et al. 2022;Kremling et al. 2018). ...
Allohexaploid wheat ( Triticum aestivum L.) is tolerant to halauxifen-methyl (HM) via rapid detoxification of the phytotoxic form of HM, halauxifen acid (HA), to non-phytotoxic metabolites. Previous research utilizing ‘Chinese Spring’ (CS) wheat, alien substitution (i.e., endogenous chromosome pair substituted with a homoeologous pair from diploid Sear’s goatgrass ( Aegilops searsii M. Feldman & M. Kislev (AS)), or nullisomic-tetrasomic (NT) lines indicated plants lacking chromosome 5A are more sensitive to HM than CS. We hypothesized the increased HM sensitivity of these plants results from losing gene(s) on chromosome 5A associated with HA metabolism, which leads to a reduced HA detoxification rate relative to CS. To compare HA abundance among AS, CS, alien substitution and NT lines during a time course, two excised leaf studies using unlabeled HM and liquid chromatography-mass spectrometry analyses were performed. Aegilops searsii accumulated more HA than CS and each substitution line at 8, 12 and 24 h after treatment (HAT). Furthermore, only the wheat substitution line lacking chromosome 5A displayed greater abundance of HA relative to CS (2.4- to 3.8-fold, depending on the time point). In contrast, HA abundances in lines possessing chromosome 5A were comparable to CS at all points. When comparing NT lines to CS, the nullisomic 5D-tetrasomic 5A (N5D-T5A) line displayed similar HA abundance, whereas the nullisomic 5A-tetrasomic 5D (N5A-T5D) accumulated approximately three-fold more HA at 12 and 24 HAT. These results biochemically support the hypothesis that genes encoding HA-detoxifying enzyme(s) are located on wheat chromosome 5A and corroborate findings from previous greenhouse phenotypic experiments. Future experimentation is needed to identify and characterize genes and enzymes on wheat chromosome 5A involved with HA detoxification, which may include cytochrome P450 monooxygenases, unknown oxidases, UDP-dependent glucosyltransferases, or potentially transcription factors that regulate expression of these genes associated with HA detoxification.
... One of these SNPs (S03_5213977) showed hits with galactinol--sucrose galactosyltransferase (GT) 2 of multiple grass species including H. vulgare, A. tauschii, T. urartu and L. arundinaceum. GTs are a wellresearched group of NTSR enzymes known for herbicide detoxification in several weed species such as L. rigidum (Gaines et al., 2014;Duhoux et al., 2015), A. myosuroides (Gardin et al., 2015) and Eleusine indica (Chen et al., 2015). The second SNP (S02_ 45439268) for reduced sensitivity to haloxyfop was located within 0.1 Mb upstream of a gene Dexi2A01G0009650 in D. exilis (reference genome of a cultivated species of Digitaria) encoding a putative NTSR enzyme UDP-glycosyltransferase 91C1-like protein (UDPGT91C1), the role of which in herbicide detoxification mechanisms has been shown only recently in Arabidopsis Manhattan plots showing marker-trait associations identified for resistance to Clethodim. ...
An in-depth genotypic characterisation of a diverse collection of Digitaria insularis was undertaken to explore the neutral genetic variation across the natural expansion range of this weed species in Brazil. With the exception of Minas Gerais, populations from all other states showed high estimates of expected heterozygosity (HE > 0.60) and genetic diversity. There was a lack of population structure based on geographic origin and a low population differentiation between populations across the landscape as evidenced by average Fst value of 0.02. On combining haloxyfop [acetyl CoA carboxylase (ACCase)-inhibiting herbicide] efficacy data with neutral genetic variation, we found evidence of presence of two scenarios of resistance evolution in this weed species. Whilst populations originating from north-eastern region demonstrated an active role of gene flow, populations from the mid-western region displayed multiple, independent resistance evolution as the major evolutionary mechanism. A target-site mutation (Trp2027Cys) in the ACCase gene, observed in less than 1% of resistant populations, could not explain the reduced sensitivity of 15% of the populations to haloxyfop. The genetic architecture of resistance to ACCase-inhibiting herbicides was dissected using a genome wide association study (GWAS) approach. GWAS revealed association of three SNPs with reduced sensitivity to haloxyfop and clethodim. In silico analysis of these SNPs revealed important non-target site genes belonging to families involved in herbicide detoxification, including UDPGT91C1 and GT2, and genes involved in vacuolar sequestration-based degradation pathway. Exploration of five genomic prediction models revealed that the highest prediction power (≥0.80) was achieved with the models Bayes A and RKHS, incorporating SNPs with additive effects and epistatic interactions, respectively.
... [11][12][13] By contrast, NTSR can be considered as a generalist adaptive response that generally includes altered absorption, translocation, excretion, or sequestration, and increased metabolic degradation. 14 Of these, increased metabolic degradation, also named metabolic resistance, has been identified as a major NTSR mechanism in various herbicide-resistant weed species, such as rigid brome (Bromus rigidus Roth.), 15 ryegrass species (Lolium sp.), [16][17][18] and Asia minor bluegrass (Polypogon fugax Nees ex Steud.). 19,20 An increasing number of studies have indicated that metabolic resistance often involves multiple detoxification enzyme families, including cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), UDP-glucosyltransferases (UDP-GTs), ATP-binding cassette (ABC) transporters, and aldo-keto reductases. ...
BACKGROUND
Echinochloa glabrescens Munro ex Hook. f. is one of the main Echinochloa spp. seriously invading Chinese rice fields and has evolved resistance to commonly used herbicides. Previously, an E. glabrescens population (LJ‐02) with suspected resistance to the acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicide metamifop was collected. This study aimed to determine its resistance status to metamifop and investigate the internal molecular mechanisms of resistance.
RESULTS
Single‐dose testing confirmed that the LJ‐02 population had evolved resistance to metamifop. Gene sequencing and a relative expression assay of ACCase ruled out target‐site based resistance to metamifop in LJ‐02. Whole‐plant bioassays revealed that, compared with the susceptible population XZ‐01, LJ‐02 was highly resistant to metamifop and exhibited cross‐resistance to fenoxaprop‐P‐ethyl. Pretreatment with the known glutathione S‐transferase (GST) inhibitor, 4‐chloro‐7‐nitrobenzoxadiazole (NBD‐Cl), largely reversed the resistance to metamifop by approximately 81%. Liquid chromatography–tandem mass spectrometry analysis indicated that the metabolic rates of one of the major metabolites of metamifop, N‐(2‐fluorophenyl)‐2‐hydroxy‐N‐methylpropionamide (HPFMA), were up to 383‐fold faster in LJ‐02 plants than in XZ‐01 plants. There were higher basal and metamifop‐inducible GST activities toward 1‐chloro‐2,4‐dinitrobenzene (CDNB) in LJ‐02 than in XZ‐01. Six GST genes were metamifop‐induced and overexpressed in the resistant LJ‐02 population.
CONCLUSION
This study reports, for the first time, the occurrence of metabolic metamifop resistance in E. glabrescens worldwide. The high‐level metamifop resistance in the LJ‐02 population may mainly involve specific isoforms of GSTs that endow high catalytic activity and strong substrate specificity. © 2023 Society of Chemical Industry.
... Moreover, designing the experiment according to the Metabolomics Standard Initiative (MSI) is also crucial, which endorses defined measures for the right biological materials preparation, procedures of metabolite extraction, and analytical protocols [30]. Following the regulations that have been stipulated, a sufficient number of sample replications and the conditions under which plant development should occur to be investigated and defined [31]. Similarly, the control of MS parameters in mass spectra registration is necessary. ...
... Metabolic resistance to ACCase-inhibitor herbicides has been offered for a long time in both monocot and dicots weed species increasing (Beckie and Tardif 2012;Yu and Powles 2014b). Most previous research has focused on mechanisms of metabolic herbicide resistance to herbicides, so there is no study on the mechanisms of gene regulation (Duhoux et al. 2015;Pan et al. 2016). The NTSR mechanism is commonly known as a polygenic inheritance because of interference in several genes, including GSTs, GTs, P450s and/or esterases and ABC transporters (Yu and Powles 2014a). ...
Target-site resistance (TSR) and non-target-site resistance (NTSR) to herbicides in arable weeds are increasing rapidly all over the world and threatening universal food safety. Resistance to herbicides that inhibit ACCase activity has been identified in wild oat. In this study, expression of ACC1, ACC2, CYP71R4 and CYP81B1 genes under herbicide stress conditions were studied in two TSR (resistant in the residue Ile1781-Leu and Ile2041-Asn of ACCase) biotypes, two NTSR biotypes and one susceptible biotype of A. ludoviciana for the first time. Treated and untreated biotypes with ACCase-inhibitor clodinafop propargyl herbicide were sampled from the stem and leaf tissues at 24 h after treatment. Our results showed an increase in gene expression levels in different tissues of both types of resistance biotypes that occurred under herbicide treatment compared with non-herbicide treatment. In all samples, the expression levels of leaf tissue in all studied genes were higher than in stem tissue. The results of ACC gene expression showed that the expression level of ACC1 was significantly higher than that of ACC2. Also, expression levels of TSR biotypes were higher than NTSR biotypes for the ACC1 gene. For both CYP71R4 and CYP81B1 genes, the expression ratio increased significantly in TSR and NTSR biotypes in different tissues after herbicide treatment. In contrast, the expression levels of CYP genes in NTSR biotypes were higher than in TSR biotypes. Our results support the hypothesis that the reaction of plants to herbicide is carried out through a different regulation of genes, which can be the result of the interaction of resistance type in the target or non-target-site.
... Moreover, designing the experiment according to the Metabolomics Standard Initiative (MSI) is also crucial, which endorses defined measures for the right biological materials preparation, procedures of metabolite extraction, and analytical protocols [30]. Following the regulations that have been stipulated, a sufficient number of sample replications and the conditions under which plant development should occur to be investigated and defined [31]. Similarly, the control of MS parameters in mass spectra registration is necessary. ...
Several environmental stresses, including biotic and abiotic factors, adversely affect the growth and development of crops, thereby lowering their yield. However, abiotic factors, e.g., drought, salinity, cold, heat, ultraviolet radiations (UVr), reactive oxygen species (ROS), trace metals (TM), and soil pH, are extremely destructive and decrease crop yield worldwide. It is expected that more than 50% of crop production losses are due to abiotic stresses. Moreover, these factors are responsible for physiological and biochemical changes in plants. The response of different plant species to such stresses is a complex phenomenon with individual features for several species. In addition, it has been shown that abiotic factors stimulate multi-gene responses by making modifications in the accumulation of the primary and secondary metabolites. Metabolomics is a promising way to interpret biotic and abiotic stress tolerance in plants. The study of metabolic profiling revealed different types of metabolites, e.g., amino acids, carbohydrates, phenols, polyamines, terpenes, etc, which are accumulated in plants. Among all, primary metabolites, such as amino acids, carbohydrates, lipids polyamines, and glycine betaine, are considered the major contributing factors that work as osmolytes and osmoprotectants for plants from various environmental stress factors. In contrast, plant-derived secondary metabolites, e.g., phenolics, terpenoids, and nitrogen-containing compounds (alkaloids), have no direct role in the growth and development of plants. Nevertheless, such metabolites could play a significant role as a defense by protecting plants from biotic factors such as herbivores, insects, and pathogens. In addition, they can enhance the resistance against abiotic factors. Therefore, metabolomics practices are becoming essential and influential in plants by identifying different phytochemicals that are part of the acclimation responses to various stimuli. Hence, an accurate metabolome analysis is important to understand the basics of stress physiology and biochemistry. This review provides insight into the current information related to the impact of biotic and abiotic factors on variations of various sets of metabolite levels and explores how primary and secondary metabolites help plants in response to these stresses.
... However, this result only indirectly demonstrated the participation of one or more P450s in this resistance, but failed to offer any information regarding the specific P450s involved. Most evidence of P450 involvement in herbicide resistance was obtained by RNA-seq, which is currently the most effective method to identify genes differentially regulated among experimental modalities [34][35][36]. Further research will focus on identifying candidate genes involved in non-target-site-based tribenuron-methyl resistance in S. conoidea. ...
Silene conoidea L. is an annual troublesome broadleaf weed in winter wheat fields in China. In recent years, field applications of tribenuron-methyl have been ineffective in controlling S. conoidea in Hebei Province, China. The aim of this study was to determine the molecular basis of tribenuron-methyl resistance in S. conoidea. Whole-plant response assays revealed that the resistant population (R) exhibited a higher level of resistance (382.3-fold) to tribenuron-methyl. The R population also showed high cross-resistance to other acetolactate synthase (ALS) inhibitors, including imazethapyr, bispyribac-sodium and florasulam. However, the R population could be controlled by the field-recommended rates of bentazone, MCPA, fluroxypyr, carfentrazone-ethyl and bromoxynil. In vitro ALS activity assays indicated that the tribenuron-methyl I50 value for the R population was 18.5 times higher than those for the susceptible population (S). ALS gene sequencing revealed an amino acid mutation, Trp-574-Leu, in the R population. Pretreatment with the P450 inhibitor malathion indicated that the R population might have cytochrome P450-mediated metabolic resistance. These results suggest that the Trp-574-Leu mutation and P450-mediated enhanced metabolism coexist in S. conoidea to generate tribenuron-methyl resistance. This is the first time that target-site and non-target-site resistance to tribenuron-methyl has been reported in S. conoidea.
... However, for many years, agriculture used highly developed herbicide-tolerant transgenic plants. Glutathione S-transferase and cytochrome P450 are two major enzymes that are key factors for an herbicidal breakdown among various major enzymatic groups ( Duhoux et al., 2015). Due to an adapted metabolism, transgenic rice plants with transgenes overexpression such as CYP2C19, CYP1A1, and CYP2B6 were more herbicide-tolerant than non-transgenic rice plants ( Kawahigashi et al., 2006). ...
... However, for many years, agriculture used highly developed herbicide-tolerant transgenic plants. Glutathione S-transferase and cytochrome P450 are two major enzymes that are key factors for an herbicidal breakdown among various major enzymatic groups ( Duhoux et al., 2015). Due to an adapted metabolism, transgenic rice plants with transgenes overexpression such as CYP2C19, CYP1A1, and CYP2B6 were more herbicide-tolerant than non-transgenic rice plants ( Kawahigashi et al., 2006). ...
Global industrialization and intensive agricultural practices after the green revolution have increased the rate of pollutants discharge above nature’'s tolerance limit. Various persistent inorganic (heavy metals) and organic pollutants (e.g., polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and pesticides) are resistant to biodegradation and pose a toxicological risk to both wildlife and humans. Therefore, it is urgently needed to replace the conventional methods of bioremediation with genetically engineered microorganisms (GEMs), which are more effective and environment -friendly. GEMs are created using genetic engineering by modified microorganisms with a more relevant protein with desired function to overexpress the desired character. The “suicidal-GEMs” (S-GEMs), a recent invention on recombinant DNA technology, can help to attain eco-friendly and potential bioremediation of polluted areas. In this chapter, we have critically discussed various GEMs with the ability to remediate organic and inorganic pollutants from the environment in order to achieve United Nations’s Sustainable Development Goals “‘Clean Water and Sanitation’”, “‘Life on Land’”, and “‘Life Below Water’” within 2030.
... However, for many years, agriculture used highly developed herbicide-tolerant transgenic plants. Glutathione S-transferase and cytochrome P450 are two major enzymes that are key factors for an herbicidal breakdown among various major enzymatic groups ( Duhoux et al., 2015). Due to an adapted metabolism, transgenic rice plants with transgenes overexpression such as CYP2C19, CYP1A1, and CYP2B6 were more herbicide-tolerant than non-transgenic rice plants ( Kawahigashi et al., 2006). ...
... De nombreuses études visant à identifier les déterminismes génétiques de la RNLC chez différentes espèces adventices pour différents herbicides ont été conduites (Tableau 4). Études transcriptomiques conduites à ce jour visant à identifier les déterminismes génétiques de la RNLC chez différentes espèces adventices pour différents modes d'action herbicide En ce qui concerne les mécanismes de résistance observés chez différentes espèces aux inhibiteurs de l'ALS (résumé parGaines et al. 2020), la majorité des mécanismes de RNLC identifiés jusqu'ici pour ce mode d'action est due à une métabolisation exacerbée de l'herbicide, impliquant souvent des enzymes de plusieurs familles de cytochromes P450 (e.g.Duhoux et al. 2015 ; Ce type de plan expérimental peut conduire à l'identification de gènes différemment exprimés liés à l'adaptation locale à l'environnement de chaque population et non à la RNLC(Giacomini et al. 2018). La validation des niveaux d'expression est donc une étape cruciale(Giacomini et al. 2018). ...
Common ragweed (Ambrosia artemisiifolia L.), a particularly troublesome and allergenic weed, is mainly controlled in agricultural fields using ALS inhibitor herbicides. Recent cases of herbicide resistance have been reported in France and are jeopardising the efficacy of this mode of action. Both target site resistance (TSR, structural mutation in ALS gene) and non target site resistance (NTSR, regulatory and/or structural mutations in secondary metabolism) are involved. The fundamental aim of this work was to identify the genetic determinisms of resistance to ALS inhibitors that have evolved in common ragweed populations in France. As an applied objective, this work also aimed to prepare the development of a high- throughput molecular diagnostic tool that would ensure a rapid detection of resistance. We first assessed the situation of common ragweed resistance in France and identified the mechanisms involved and their modalities of evolution. Using herbicide sensitivity bioassays coupled with ALS gene sequencing, we showed that ragweed resistance to two active substances, imazamox and tribenuron, is emerging in France and is mainly due to NTSR mechanisms. The observed resistance patterns suggest that a diversity of NTSR mechanisms are evolving in France. Furthermore, we demonstrated that TSR evolved locally, through multiple and independent appearance of mutations in the ALS gene. Thanks to the innovative application of high- throughput sequencing for the diagnosis of TSR on a national scale, we identified several foci of TSR emergence, as well as an unsuspected diversity of mutations in the ALS gene. We then studied the genetic determinisms of NTSR. A transcriptomic approach (RNASeq) associated with an analysis of nucleotide polymorphisms was conducted, based on the hypothesis that genes and/or markers of NTSR differed by their expression level and/or by sequence polymorphisms between plants resistant or sensitive to ALS inhibitors. For the first time, this approach was conducted directly on field plant material, i.e. six populations with distinct geographical origins and/or resistance profiles. Constitutive expression differences between resistant and sensitive plants were identified, especially in genes from families known to be involved in herbicide metabolism (cytochromes P450, transferase enzymes, transporters, etc.), but also in genes that may be involved in regulatory cascades activated by the herbicide. Validation of their relative expression levels and their ability to predict NTSR was performed on a massive sampling of plants. Taken together, the results indicate that a very high diversity of mechanisms is involved in RNLC within and between populations, highlighting the highly polygenic nature of RNLC in ragweed. In addition, assessment of the early response of plants to herbicide application showed that genes involved in secondary plant metabolism are specifically induced by treatment in resistant plants from different populations. Finally, sequence variants potentially correlated with NTSR were identified. Their validity as resistance markers remains to be confirmed. The diversity of resistance mechanisms identified within each population renders the development of a molecular diagnosis tool complex. On the other hand, it opens exciting perspectives for the study of the evolutionary dynamics of the adaptation of an invasive species subjected to a particularly intense anthropogenic selection pressure.
... Despite the fact that the enhanced herbicide detoxification in weedy species has drawn great interest in recent years [35,37], the underlying biochemical mechanisms, enzymes, and specific genes are still not well characterized and remain markedly under-explored. Potential candidates are intensively identified based on large-scale omics [38][39][40][41][42][43], whereas seldom of them have been demonstrated to be associated with specific herbicide metabolism [18,44,45]. In the weedy species A. aequalis, while metabolic resistance has been identified at the whole plant level for several years [46], little progress has been made in resistance gene discovery due to great genetic heterogeneity and lack of genome sequences and genetic linkage maps. ...
Multiple herbicide resistance in diverse weed species endowed by enhanced herbicide detoxification or degradation is rapidly growing into a great threat to herbicide sustainability and global food safety. Although metabolic resistance is frequently documented in the economically damaging arable weed species shortawn foxtail (Alopecurus aequalis Sobol.), relevant molecular knowledge has been lacking. Previously, we identified a field population of A. aequalis (R) that had evolved metabolic resistance to the commonly used acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron-methyl. RNA sequencing was used to discover potential herbicide metabolism-related genes, and four cytochrome P450s (CYP709C56, CYP71R18, CYP94C117, and CYP94E14) were identified with higher expressions in the R vs. susceptible (S) plants. Here the full-length P450 complementary DNA transcripts were each cloned with identical sequences between the S and R plants. Transgenic Arabidopsis overexpressing CYP709C56 became resistant to the sulfonylurea herbicide mesosulfuron-methyl and the triazolo-pyrimidine herbicide pyroxsulam. This resistance profile generally but does not completely in accordance with what is evident in the R A. aequalis. Transgenic lines exhibited enhanced capacity for detoxifying mesosulfuron-methyl into O-demethylated metabolite, which is in line with the detection of O-demethylated herbicide metabolite in vitro in transformed yeast. Structural modeling predicted that mesosulfuron-methyl binds to CYP709C56 involving amino acid residues Thr-328, Thr-500, Asn-129, Gln-392, Phe-238, and Phe-242 for achieving O-demethylation. Constitutive expression of CYP709C56 was highly correlated with the metabolic mesosulfuron-methyl resistance in A. aequalis. These results indicate that CYP709C56 degrades mesosulfuron-methyl and its up-regulated expression in A. aequalis confers resistance to mesosulfuron-methyl.
... 70 To date, more than ten UDP-GT genes are reported to be up-regulated expression by various herbicides and have been identified as herbicide-metabolism related genes. 24,25,36,71,72 In Arabidopsis, UGT91C1 has a strong glycosylating activity toward the triketone herbicide sulcotrione. 72 In this current study, we also found a UDP-GT gene UGT73C5 constitutively up-regulated in R versus S plants. ...
BACKGROUND
Metabolic resistance is a worldwide concern for weed control but has not yet been well‐characterized at the genetic level. Previously, we have identified an Asia minor bluegrass (Polypogon fugax Nees ex Steud.) population AHHY exhibiting cytochrome P450 (P450)‐involved metabolic resistance to fenoxaprop‐P‐ethyl. In this study, we aimed to confirm the metabolic fenoxaprop‐P‐ethyl resistance in AHHY and uncover the potential herbicide metabolism‐related genes in this economically damaging weed species.
RESULTS
Liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays indicated the metabolic rates of fenoxaprop‐P‐ethyl were significantly faster in resistant (R, AHHY) than in susceptible (S, SDTS) plants. The amount of phytotoxic fenoxaprop‐P peaked at 12 h after herbicide treatment (HAT) and started to decrease at 24 HAT in both biotypes. R and S plants at 24 HAT were sampled to conduct isoform‐sequencing (Iso‐Seq) and RNA‐sequencing (RNA‐Seq). A reference transcriptome containing 24 972 full‐length isoforms was obtained, of which 24 329 unigenes were successfully annotated. Transcriptomic profiling identified 28 detoxifying enzyme genes constitutively and/or herbicide‐induced up‐regulated in R than in S plants. Real‐time quantitative polymerase chain reaction (RT‐qPCR) confirmed 17 genes were consistently up‐regulated in R and its F1 generation plants. They were selected as potential fenoxaprop‐P‐ethyl metabolism‐related genes, including ten P450s, one glutathione‐S‐transferase, one UDP‐glucosyltransferase, and five adenosine triphosphate (ATP)‐binding cassette transporters.
CONCLUSION
This study revealed that the enhanced rates of fenoxaprop‐P‐ethyl metabolism in P. fugax were very likely driven by the herbicide metabolism‐related genes. The transcriptome data generated by Iso‐Seq combined with RNA‐Seq will provide abundant gene resources for understanding the molecular mechanisms of resistance in P. fugax.
... CD-HIT-EST was once again used to remove the shorter redundant transcripts (same parameters as previously). To remove additional residual redundancy, we used a custom tool which merges two sequences using successive iterations of CAP3 [136] and NRCL tools with a progressive reduction in the stringency parameters [137]. The final threshold included shared ≥ 94% identity with ≥ 100 bp overlap and overhangs < 40 bp. ...
Background
Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera , being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood.
Results
Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae . Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein.
Conclusions
Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.
... Furthermore, it has been shown that CYP81As can confer resistance to acetyl CoA carboxylase (ACCase)-inhibiting herbicides [48]. In Lolium spp., participation of P450s, GSTs and other genes on herbicide detoxification have been reported [49][50][51]. In A. palmeri accessions resistant to the herbicide S-metolachlor, resistance was attributed to GSTs [10,52]. ...
Amaranthus palmeri S. Watson (Palmer amaranth) is considered a problematic and troublesome weed species in many crops in the USA, partly because of its ability to evolve resistance to herbicides. In this study, we explored the mechanism of resistance in a trifluralin-resistant A. palmeri accession collected from Arkansas, USA. Dose-response assays using agar plates demonstrated an EC50 (effective concentration that reduces root length by 50%) of 1.02 µM trifluralin compared to 0.39 µM obtained in the susceptible accession. Thus, under these conditions, the resistant accession required 2.6 times more trifluralin to inhibit root length by 50%. Seeds in the presence or absence of the cytochrome P450-inhibitior malathion displayed a differential response with no significant influence on root length, suggesting that resistance is not P450-mediated. In addition, application of 4-chloro-7-nitrobenzofurazan (NBD-Cl), a glutathione S-transferase (GST) inhibitor, showed significant differences in root length, indicating that GSTs are most likely involved in the resistance mechanism. Sequencing of α- and β-tubulin genes revealed no single nucleotide polymorphisms (SNPs) previously described between accessions. In addition, relative gene copy number of α- and β-tubulin genes were estimated; however, both resistant and susceptible accessions displayed similar gene copy numbers. Overall, our results revealed that GST-mediated metabolism contributes to trifluralin resistance in this A. palmeri accession from Arkansas.
... To study the molecular mechanism behind this phenomenon and provide a basis for understanding the biological function of genes, it is necessary to analyze the gene expression patterns in plant stress response pathways. Gene expression patterns reflect the tendency of gene activity and are of great significance to the understanding of gene function and gene regulatory network (Gaines et al. 2014;Duhoux et al. 2015). Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is one of the most widely used techniques for gene expression analysis. ...
Trichoderma polysporum was a pathogenic fungi which showed strong pathogenicity to Avena fatua L. in recent study. The stress response of A. fatua to T. polysporum is mediated by the regulation of gene expression. Quantification of the expression of genes requires normalizing RT-qPCR data using reference genes with stable expression in the system studied as internal standards. To construct a RT-qPCR system suitable for response of A. fatua to T. polysporum, and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on eight candidate internal reference genes (18S, 28S, TUA, UBC, ACT, GAPDH, TBP and EF-1α) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP, 18S and UBC were the most stable internal reference genes, TBP and TUA, TBP and GAPDH, 18S and TBP, UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua. This is the first study describing a set of reference genes with a stable expression under fungi stress in A. fatua. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in A. fatua.
... However, there are few studies on herbicide metabolism and resistance genes at the whole transcriptome level, which has slowed the development of new control mechanisms. RNA-seq has been successfully used to identify genes involved in metabolic resistance to ALS herbicides in two grass weed species: L. rigidum [33] and A. myosuroides [34]. However, there has been no study focused on the NTSR mechanisms of L. chinensis at the whole transcriptome level. ...
Background
Chinese sprangletop [ Leptochloa chinensis (L.) Nees] is an annual malignant weed, which can often be found in paddy fields. Cyhalofop-butyl is a specialized herbicide which is utilized to control L. chinensis . However, in many areas, L. chinensis has become tolerant to this key herbicide due to its continuous long-term use.
Results
In this study, we utilized a tolerant (LC18002) and a sensitive (LC17041) L. chinensis populations previously identified in our laboratory, which were divided into four different groups. We then employed whole transcriptome analysis to identify candidate genes which may be involved in cyhalofop-butyl tolerance. This analysis resulted in the identification of six possible candidate genes, including three cytochrome P450 genes and three ATP-binding cassette transporter genes. We then carried out a phylogenetic analysis to identify homologs of the differentially expressed cytochrome P450 genes. This phylogenetic analysis indicated that all genes have close homologs in other species, some of which have been implicated in non-target site resistance (NTSR).
Conclusions
This study is the first to use whole transcriptome analysis to identify herbicide non-target resistance genes in L. chinensis . The differentially expressed genes represent promising targets for better understanding herbicide tolerance in L. chinensis . The six genes belonging to classes already associated in herbicide tolerance may play important roles in the metabolic resistance of L. chinensis to cyhalofop-butyl, although the exact mechanisms require further study.
... Separate trays containing 98 cells (26.2 cm 3 each) were filled with previously described growing media. Individual POAAN-R3 or S plants with a minimum of two tillers were transplanted into each cell and allowed to acclimate to the growth chamber environment before beginning the RNA-Seq experiment, which was designed similar to Duhoux et al. 19 Our aim in this study was to better understand transcriptomic responses of POAAN-R3 and S P. annua in response to an application of the ALS-inhibiting herbicide trifloxysulfuron at label rate (27.8 g ha −1 ). There were six time points in our time-course experiment: 0, 2, 6, 12, 24, and 48 h after treatment (HAT) with five biological replicates of each P. annua biotype at each time point (30 samples total). ...
BACKGROUND
Poa annua is a pervasive grassy, self‐pollinating, weed that has evolved resistance to 10 different herbicide modes‐of‐action, third most of all weed species. We investigated constitutive overexpression of genes associated with non‐target site resistance (NTSR) in POAAN‐R3 and the response of those genes when treated with trifloxysulfuron despite the biotype having a known target site mutation in acetolactate synthase (ALS).
RESULTS
Despite having an ALS target site mutation, POAAN‐R3 still had a transcriptomic response to herbicide application that differed from a susceptible biotype. We observed differential expression of genes associated with transmembrane transport and oxidation–reduction activities, with differences being most pronounced prior to herbicide treatment.
CONCLUSIONS
In the P. annua biotype we studied with confirmed target site resistance to ALS inhibitors, we also observed constitutive expression of genes regulating transmembrane transport, as well as differential expression of genes associated with oxidative stress after treatment with trifloxysulfuron. This accumulation of mechanisms, in addition to the manifestation of target site resistance, could potentially increase the chance of survival when plants are challenged by different modes of action.
... This is the first paper to directly investigate the adaptive potential of plastic expression changes in response to herbicide. Previous work has found evidence of gene expression changes in response to herbicide, often in genes not previously known to be important for herbicide response (Duhoux et al. 2015;Serra et al. 2015;Wright et al. 2018;Zhao et al. 2017;An et al. 2014;Leslie and Baucom 2014), but these studies did not directly investigate whether plastic responses appeared to be adaptive. One hint at the importance of maladaptive plasticity comes from the observation that a susceptible population of Amaranthus tuberculatus increased rapid expression responses to herbicide compared to a naturally resistant population (Kohlhase et al. 2019), suggesting that plastic expression changes in response to herbicide could be maladaptive in this species. ...
Plastic phenotypic responses to environmental change are common, yet we lack a clear understanding of the fitness consequences of these plastic responses. Here, we use the evolution of herbicide resistance in the common morning glory (Ipomoea purpurea) as a model for understanding the relative importance of adaptive and maladaptive gene expression responses to herbicide. Specifically, we compare leaf gene expression changes caused by herbicide to the expression changes that evolve in response to artificial selection for herbicide resistance. We identify a number of genes that show plastic and evolved responses to herbicide and find that for the majority of genes with both plastic and evolved responses, plastic responses appear to be adaptive. We also find that selection for herbicide response increases gene expression plasticity. Overall, these results show the importance of adaptive plasticity for herbicide resistance in a common weed and that expression changes in response to strong environmental change can be adaptive.
Impact Statement
Predicting whether and how organisms will adapt to environmental change is a crucial goal. However, this goal can be complicated because environmental change can alter traits, in a process called plasticity. The extent and fitness consequences of plasticity will have important effects on the adaptive process. In this study, we use adaptation to herbicide in the agricultural weed, the common morning glory, as a model for understanding the extent and fitness consequences of plasticity in gene expression. We find evidence that gene expression plasticity is adaptive in the presence of herbicide, suggesting that understanding plasticity is crucial for understanding how organisms adapt to new environments.
... Indian mustard and poplar tree expressing Glutathione S transferases (GSTs) shows increased tolerance to chloroacetanilide, acetochlor, atrazine, 1-chloro-2,4-dinitrobenzene (CDNB), metolachlor, and phenanthrene [136,137]. Similar report has been found by Duhoux et al. [138] which documented the phytoremediation of herbicide with glutathione S-transferase mediated transgenic plants. ...
Pesticides play a key role in controlling pest attacks and weeds. But, extensive applications of these pesticides and use of inadequate methods results in contamination of our food commodities which now have become a major concern globally. Improper utilization of these low biodegradable chemicals enhances their availability and accumulation in the soil as well as in water. Bio-accumulation of these persistent, toxic compounds imbalances the ecosystem functioning and act as one of the key factor in loss of biodiversity of flora and fauna. Accumulation of these toxic compounds has harmful effect on biology of soil by changing soil flora quantitatively as well as qualitatively, alteration in nutrient balance of the soil, food quality, and human health. Consumption of food containing pesticidal residues causes serious health issues such as impaired developmental processes, effect nervous system functioning, skin irritation. Different approaches are being optimized to reduce pesticide load from the soils. Adsorption, contaminant immobilization, oxidation-reduction, biosulfactants, etc. are among the various approaches which has been categorized under physico-chemical and biological remediation technologies. Present study focused on the various advanced strategies to remove pesticidal contaminants from the soil.
... To study the molecular mechanism behind this phenomenon and provide a basis for understanding the biological function of genes, it is necessary to analyze the gene expression patterns in plant stress response pathways. Gene expression patterns re ect the tendency of gene activity and are of great signi cance to the understanding of gene function and gene regulatory network (Gaines et al. 2014;Duhoux et al. 2015). Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is one of the most widely used techniques for gene expression analysis. ...
In order to construct a RT-qPCR system suitable for response of Avena fatua L. to Trichoderma polysporum , and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on 8 candidate internal reference genes ( 18S , 28S , TUA , UBC , ACT , GAPDH , TBP and EF-1 ) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP , 18S and UBC were the most stable internal reference genes, TBP and TUA , TBP and GAPDH , 18S and TBP , UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as the internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua .
... In order to understand how this resilience is manifested within temperate pasture grasses, a number of studies have looked at the contribution of differential gene expression, usually in leaves and sometimes in roots, in response to various aspects of stress within Lolium and Festuca spp. These studies have involved stresses such as cold-acclimation [3], xenobiotics [4,5], disease resistance [6], submergence [7], salinity [8,9], heavy metals [10,11], as well as water-stress [12][13][14][15][16][17][18][19][20]. Because of the resources required to conduct and analyse transcriptomics experiments, a common approach is to compare gene expression in pairs of tolerant and susceptible genotypes and then to frame observed differences in terms of how expression profiles may be contributing to the differing responses to stress-and this can provide valuable insights. ...
Ryegrasses (Lolium spp.) and fescues (Festuca spp.) are closely related and widely cultivated perennial forage grasses. As such, resilience in the face of abiotic stresses is an important component of their traits. We have compared patterns of differentially expressed genes (DEGs) in roots and leaves of two perennial ryegrass genotypes and a single genotype of each of a festulolium (predominantly Italian ryegrass) and meadow fescue with the onset of water stress, focussing on overall patterns of DEGs and gene ontology terms (GOs) shared by all four genotypes. Plants were established in a growing medium of vermiculite watered with nutrient solution. Leaf and root material were sampled at 35% (saturation) and, as the medium dried, at 15%, 5% and 1% estimated water contents (EWCs) and RNA extracted. Differential gene expression was evaluated comparing the EWC sampling points from RNAseq data using a combination of analysis methods. For all genotypes, the greatest numbers of DEGs were identified in the 35/1 and 5/1 comparisons in both leaves and roots. In total, 566 leaf and 643 root DEGs were common to all 4 genotypes, though a third of these leaf DEGs were not regulated in the same up/down direction in all 4 genotypes. For roots, the equivalent figure was 1% of the DEGs. GO terms shared by all four genotypes were often enriched by both up- and down-regulated DEGs in the leaf, whereas generally, only by either up- or down-regulated DEGs in the root. Overall, up-regulated leaf DEGs tended to be more genotype-specific than down-regulated leaf DEGs or root DEGs and were also associated with fewer GOs. On average, only 5–15% of the DEGs enriching common GO terms were shared by all 4 genotypes, suggesting considerable variation in DEGs between related genotypes in enacting similar biological processes.
... Already improved metabolic diagnostics tools have been used successfully to differentiate resistant Lolium populations from their susceptible counterparts in France. 61,62 One could easily envisage a future in which these and GPS data are collated to enable autonomous field robots to perform precise and targeted herbicide applications. 5 In terms of developing 'narrow-spectrum' herbicides, Mitsui Chemical Agro Inc. is developing a herbicide cyclopyrimorate, specific to weeds invading rice fields, whose structure was optimized for specific target inhibition using the weeds Scirpus juncoides and Sagittaria trifolia, and is expected to be commercialized in the near future. ...
The challenges of resistance to antibiotics and resistance to herbicides have much in common. Antibiotic resistance became a risk in the 1950s, but a concerted global effort to manage it did not begin until after 2000. Widespread herbicide use began during the 1950s and was soon followed by an unabated rise in resistance. Here, we examine what lessons for combatting herbicide resistance could be learnt from the global, coordinated efforts of all stakeholders to avert the antibiotic resistance crisis. © 2021 Society of Chemical Industry.
... By filtering contigs unlikely to be involved in pyroxsulam resistance in Lolium spp. (Duhoux et al., 2015), a list of differentially expressed genes was reduced from > 10,000 to four candidate genes. Similarly, Zhao et al. (2017) focused on the validation of 31 candidate genes from a pool of > 11,000 differentially expressed contigs in Alopecurus aequalis. ...
The Lolium genus encompasses many species that colonize a variety of disturbed and non-disturbed environments. Lolium perenne L. spp. perenne, L. perenne L. spp. multiflorum, and L. rigidum are of particular interest to weed scientists because of their ability to thrive in agricultural and non-agricultural areas. Herbicides are the main tool to control these weeds; however, Lolium spp. populations have evolved multiple- and cross-resistance to at least 14 herbicide mechanisms of action in more than 21 countries, with reports of multiple herbicide resistance to at least seven mechanisms of action in a single population. In this review, we summarize what is currently known about non-target-site resistance in Lolium spp. to acetyl CoA carboxylase, acetohydroxyacid synthase, microtubule assembly, photosystem II, 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthetase, very-long chain fatty acids, and photosystem I inhibitors. We suggest research topics that need to be addressed, as well as strategies to further our knowledge and uncover the mechanisms of non-target-site resistance in Lolium spp.
... Three new pairs of primers were designed for different isoforms according to published L. rigidum transcriptome sequences 26,27 and ⊍-tubulin sequences from Eleusine indica (AJ005599.1), Setaria viridis (AJ586805.1) ...
BACKGROUND
Tubulin, the target site of dinitroaniline herbicides, is encoded by small gene families in plants. To better characterize the mechanisms of target‐site resistance to dinitroaniline herbicides in the globally important weedy species Lolium rigidum, attempts were made to amplify and sequence α‐tubulin transcripts.
RESULTS
Four α‐tubulin isoforms (TUA1, TUA2, TUA3 and TUA4) were identified in L. rigidum. Variations in the number and sequence of transcripts encoding these α‐tubulin proteins were found in individuals from the two L. rigidum populations examined. Within and among populations, differences in the 5′‐ and 3′‐untranslated regions of cDNA in TUA3 and TUA4 were identified. Furthermore, a novel double mutation, Arg‐390‐Cys+Asp‐442‐Glu, in the TUA3 transcript was identified and has the potential to confer dinitroaniline resistance.
CONCLUSION
This research reveals the complexity of the α‐tubulin gene family in individuals/populations of the cross‐pollinated weedy species L. rigidum, and highlights the need for better understanding of the molecular architecture of tubulin gene families for detecting resistance point mutations. Although TUA4 is a commonly expressed α‐tubulin isoform containing most frequently reported resistance mutations, other mutant tubulin isoforms may also have a role in conferring dinitroaniline resistance.
Inhibitors of acetohydroxyacid synthase (also known as acetolactate synthase), the common enzyme of branched-chain amino acid biosynthesis, were used as tools to discern the contribution of newly synthesized precursors (i.e., branched-chain amino acids and α-ketoacids) to branched-chain ester formation in ripening apple (Malus ×domestica Borkh.), banana (Musa spp.), and flowering quince (Chaenomeles ×superba) fruits. After treatment, anteiso- and iso-branched-chain esters (i.e., those related to isoleucine, and valine and leucine, respectively) universally decreased in content by at least 90%. Among free amino acids, only the branched-chain amino acids, with correspondingly reduced branched-chain esters, had a lesser concentration following treatment with the inhibitor. Branched-chain ester production recovered after subsequent feeding with precursor compounds. Our results ultimately reject the hypothesis that anteiso- and iso-branched-chain esters of ripening fruits are primarily derived from preexisting sources and instead support the hypothesis that these esters are largely the product of de novo precursor biosynthesis.
Weeds pose a significant threat to crop production, resulting in substantial yield reduction. In addition, they possess robust weedy traits that enable them to survive in extreme environments and evade human control. In recent years, the application of multi-omics biotechnologies has helped to reveal the molecular mechanisms underlying these weedy traits. In this review, we systematically describe diverse applications of multi-omics platforms for characterizing key aspects of weed biology, including the origins of weed species, weed classification, and the underlying genetic and molecular bases of important weedy traits such as crop–weed interactions, adaptability to different environments, photoperiodic flowering responses, and herbicide resistance. In addition, we discuss limitations to the application of multi-omics techniques in weed science, particularly compared with their extensive use in model plants and crops. In this regard, we provide a forward-looking perspective on the future application of multi-omics technologies to weed science research. These powerful tools hold great promise for comprehensively and efficiently unraveling the intricate molecular genetic mechanisms that underlie weedy traits. The resulting advances will facilitate the development of sustainable and highly effective weed management strategies, promoting greener practices in agriculture.
A sensing mechanism in mammals perceives xenobiotics and induces the transcription of genes encoding proteins that detoxify these molecules. However, it is unclear if plants sense xenobiotics, and activate an analogous signalling system leading to their detoxification. Using the liverwort Marchantia polymorpha, we tested the hypothesis that there is a sensing system in plants that perceives herbicides resulting in the increased transcription of genes encoding proteins that detoxify these herbicides. Consistent with the hypothesis, we show that chlorsulfuron-treatment induces changes in the M. polymorpha transcriptome. However, these transcriptome changes do not occur in chlorsulfuron (CS)-treated target site resistant mutants, where the gene encoding the target carries a mutation that confers resistance to chlorsulfuron. Instead, we show that inactivation of the chlorsulfuron target, acetolactate synthase (ALS) (also known as acetohydroxyacid synthase (AHAS)), is required for the transcriptome response. These data demonstrate that the transcriptome changes in chlorsulfuron-treated plants are caused by disrupted amino acid synthesis and metabolism resulting from acetolactate synthase inhibition, and indicate that the transcriptome changes are not caused by a herbicide sensing mechanism.
Commelina communis L. is a troublesome weed in agronomic fields and increasingly threatens the yield security of corn in north-eastern China. Previously, we found that a C. communis population (JL-1) has evolved resistance to atrazine. Although the potential genetic and enzymic differences contributing to atrazine resistance in this population have been investigated, the specific molecular mechanisms underlying C. communis resistance are still poorly understood. Here, the expression level of the target gene PsbA and the non-target-site resistance (NTSR) mechanism for this population were studied. The results showed that the decline in chlorophyll content in JL-1 leaves was less than in the susceptible JS-10 population following atrazine treatment. JL-1 exhibited an enhanced expression of the PsbA gene compared with JS-10 of 7.28- and 14.28-fold higher at 0 and 24 h after treatment with atrazine, respectively. The cytochrome P450 monooxygenase (P450) inhibitor piperonyl butoxide (PBO) increased the phytotoxicity of atrazine in both populations of C. communis. Seven candidate genes associated with NTSR of Jl-1 were identified through RNA-seq and validated by quantitative real-time PCR, including 5 upregulated genes involved in herbicide metabolism. In addition, the activities of glutathione S-transferases and P450s in JL-1 were increased compared with JS-10. Collectively, PsbA gene overexpression and enhanced metabolism are likely to be responsible for JL-1 resistance to atrazine.
A sensing mechanism in mammals perceives xenobiotics and induces the transcription of genes encoding proteins that detoxify these molecules. However, it is unclear if plants sense xenobiotics, and activate an analogous signalling system leading to their detoxification. Using the liverwort Marchantia polymorpha , we tested the hypothesis that there is a sensing system in plants that perceives herbicides resulting in the increased transcription of genes encoding proteins that detoxify these herbicides. Consistent with the hypothesis, we show that chlorsulfuron-treatment induces changes in the M. polymorpha transcriptome. However, these transcriptome changes do not occur in chlorsulfuron (CS)-treated target site resistant mutants, where the gene encoding the target carries a mutation that confers resistance to chlorsulfuron. Instead, we show that inactivation of the chlorsulfuron target, acetolactate synthase (ALS) (also known as acetohydroxyacid synthase (AHAS)), is required for the transcriptome response. These data are consistent with the changes in the transcriptome of chlorsulfuron-treated plants being caused by disrupted amino acid synthesis and metabolism resulting from acetolactate synthase inhibition. These conclusions suggest that chlorsulfuron is not sensed in M. polymorpha leading to induce a detoxification system.
Author Summary
Herbicide use is increasing throughout the world, however we know little about how plants respond to herbicide treatment and regulate their metabolism. Some plants have evolved resistance to herbicides such as chlorsulfuron by increasing the detoxification of the herbicide compared to sensitive plants. It has been suggested that plants can directly sense the herbicide chemical which activates a detoxification response, in a similar way to the detoxification of foreign chemicals in mammalian cells. The liverwort Marchantia polymorpha is an excellent system to study plant herbicide responses due to its short generation time, ease of propagation and low genetic redundancy. We show that chlorsulfuron treatment alters the expression of many genes in M. polymorpha , however plants with a resistance-conferring mutation in the molecular target of chlorsulfuron do not show any changes in gene expression in response to chlorsulfuron treatment. This result indicates that transcriptome changes caused by chlorsulfuron depend on the inhibition of the target by chlorsulfuron. This suggests that plants do not sense chlorsulfuron and activate a detoxification system. This finding has implications for herbicide use and discovery.
Aegilops tauschii Coss. is a malignant weed in wheat fields in China, its herbicide resistance has been threatening crop production. This study identified one mesosulfuron-methyl-resistant(R) population, JJMHN2018-05 (R), without target resistance mutations. To fully understand the resistance mechanism, non-target site resistance was investigated by using transcriptome sequencing combined with a reference genome. Results showed that the cytochrome P450 monooxygenase (P450) inhibitor malathion significantly increased the mesosulfuron-methyl sensitivity in R plants, and greater herbicide-induced glutathione S-transferase (GST) activity was also confirmed. Liquid chromatography with tandem mass spectrometry analysis further supported the enhanced mesosulfuron-methyl metabolism in R plants. Gene expression data analysis and qRT-PCR validation indicated that eight P450s, six GSTs, two glycosyltransferases (GTs), four peroxidases, and one aldo-keto reductase (AKRs) stably upregulated in R plants. This research demonstrates that the P450s and GSTs involved in enhanced mesosulfuron-methyl metabolism contribute to mesosulfuron-methyl resistance in A. tauschii and identifies potential contributors from metabolic enzyme families.
Herbicide resistance is an evolutionary process that affects entire agricultural regions’ yield and productivity. The high number of farms and the diversity of weed management can generate hot selection spots throughout the regions. Resistant biotypes can present a diversity of mechanisms of resistance and resistance factors depending on selective conditions inside the farm; this situation is similar to predictions by the geographic mosaic theory of coevolution. In Mexico, the agricultural region of the Bajio has been affected by herbicide resistance for 25 years. To date, Avena fatua L. is one of the most abundant and problematic weed species. The objective of this study was to determine the mechanism of resistance of biotypes with failures in weed control in 70 wheat and barley crop fields in the Bajio, Mexico. The results showed that 70% of farms have biotypes with target site resistance (TSR). The most common mutations were Trp–1999–Cys, Asp–2078–Gly, Ile–2041–Asn, and some of such mutations confer cross-resistance to ACCase-inhibiting herbicides. Metabolomic fingerprinting showed four different metabolic expression patterns. The results confirmed that in the Bajio, there exist multiple selection sites for both resistance mechanisms, which proves that this area can be considered as a geographic mosaic of resistance.
Herbicide resistance to chemical herbicide is a global issue that presents an ongoing threat to grain production. Though it has been frequently implicated that the production of detoxification enzymes increased in resistance development, the mechanisms for overexpression of these genes employed by herbicide-resistant weeds remain complicated. In this study, a mesosulfuron-methyl resistant Beckmannia syzigachne population (R) was found to be cross-resistant to another herbicide pyriminobac-methyl. No known target-site mutations were detected in the R population. In contrast, the decreased uptake and enhanced metabolic rates of mesosulfuron-methyl were detected in the R than the susceptible (S) population. Two candidate ATP-binding cassette (ABC) transporter genes (ABCB25 and ABCC14) that were constitutively up-regulated in the R population were identified by RNA-sequencing and validated by RT-qPCR. Alteration of antioxidant enzyme activities and gene expressions implied that mesosulfuron-methyl-induced antioxidant defenses provoked reactive oxygen species (ROS) burst. ROS scavenger assay showed that ROS induces ABCB25 and ABCC14 expression. This study reported for the first time that ABC transporters mediated non-target-site resistance contributes to mesosulfuron-methyl resistance in a B. syzigachne population, and implicated that ROS burst might be involved in the overexpression of ABC transporter genes in weeds.
Global developmental strategies and population expansion are continuously showing their odd impacts on the living world, thereby causing stresses of multiple natures. To combat these stresses, hydrogen sulfide (H2S) is well-examined signaling molecule that acts as a priming agent and helps in regulating the response of plants to various stressful conditions. Hydrogen sulfide is formed in the plant cells as an intermediate of an assimilatory sulfate reduction. Despite the endogenous release of hydrogen sulfide, its exogenous application has been found to be beneficial in the amelioration of multiple abiotic stresses. These responses are also mediated by the expression of genes and proteins that participate in signaling and metabolic pathways induced through several small signaling molecules known as plant hormones or phytohormones. Phytohormones are also found to be involved in regulation of the protective responses under various abiotic and biotic stress conditions. H2S in crosstalk with these phytohormones significantly ameliorates the abiotic stress in plants. In this chapter we have discussed in detail how H2S in crosstalk with phytohormones helps in the enhancement of defense against abiotic stress in plants.
Soil contamination by heavy metals due to rapid industrialization and anthropogenic activities is a serious global environmental issue. Heavy metals adversely affect the soil and plant health. Exposure of plants to heavy metals cause oxidative stress due to excessive production of reactive oxygen species (ROS) which result in decreased crop productivity. Hyperaccumulators have well developed antioxidative defense mechanism which protects the plants against the deleterious effects of the oxidative stress generated by heavy metals. Thiols participate in various chemical reactions that are involved in cellular redox homeostasis and detoxification of ROS. GSH is an important antioxidant that plays vital role in protecting the cell from the damage caused due to ROS generation during abiotic stress. Pytochelatins are also involved in metal stress tolerance due to their high antioxidant ability. This chapter sheds light on the role of thiols in metal tolerance in plants.
Organic xenobiotics are adversely affecting the environment due to their toxicity and longer persistence in the environment. These compounds are hydrophobic in nature and can deteriorate the quality of soil and groundwater. The presence of these organic compounds in the soil retard the growth of plants and inhibit the metabolic activities of microbes. The interactive behavior of plants and microbes is a promising remedial strategy for the removal of these organic pollutants from the environment. In this synergistic association between plants and microbes, plants provide nutrients to the microbes and in return microbes promote the growth of plants, boost the degradation of the organic pollutants and reduce phytotoxicity. Plant root exudates enhance the degradation of organic xenobiotics and increase their bioavailability to microbes by changing soil pH and endow the microbes with substrate for metabolism. This chapter is focused on the degradation mechanism of xenobiotics and plant-bacterial associations for the significant remediation of organic pollutants.
BACKGROUND
Alopecurus myosuroides (blackgrass) is a major weed in Europe with known resistance to multiple herbicide modes of action. In the UK, there is evidence that blackgrass has undergone a range expansion. In this paper, genotyping‐by‐sequencing and population‐level herbicide resistance phenotypes are used to explore spatial patterns of selectively neutral genetic variation and resistance. We also perform a preliminary genome‐wide association study (GWAS) and genomic prediction analysis to evaluate the potential of these approaches for investigating nontarget site herbicide resistance.
RESULTS
Blackgrass was collected from 47 fields across the British Isles and up to eight plants per field population (n = 369) were genotyped by Restriction site‐associated DNA (RAD)‐sequencing. A total of 20 426 polymorphic loci were identified and used for population genetic analyses. Phenotypic assays revealed significant variation in herbicide resistance between populations. Population structure was weak (FST = 0.024–0.048), but spatial patterns were consistent with an ongoing westward and northward range expansion. We detected strong and consistent Wahlund effects (FIS = 0.30). There were no spatial patterns of herbicide resistance or evidence for confounding with population structure. Using a combination of population‐level GWAS and genomic prediction we found that the top 20, 200, and 2000 GWAS loci had higher predictive abilities for fenoxaprop resistance compared to all markers.
CONCLUSION
There is likely extensive human‐mediated gene flow between field populations of the weed blackgrass at a national scale. The lack of confounding of adaptive and neutral genetic variation can enable future, more extensive GWAS analyses to identify the genetic architecture of evolved herbicide resistance. © 2020 Society of Chemical Industry
American sloughgrass (Beckmannia syzigachne Steud.) has become a dominant weed in fields with rice-wheat rotation. Moreover, herbicide resistance has rendered weed control difficult. We identified a biotype showing resistance to ALS inhibitor mesosulfuron-methyl with a resistant index 3.3, but without any ALS mutation. This study aims to identify and confirm the factors associated with non-target site resistance of this biotype to mesosulfuron-methyl using RNA-Seq. 118,111 unigenes were assembled, and 50.9% of these were annotated across seven databases. Eleven contigs related to metabolic resistance were identified based on differential expression via RNA-Seq which include a novel resistance-related transcription factor (MYC3) and two disease resistance proteins were also identified (At1g58602 and At1g15890). Fold changes in expression of these genes in comparison M-R vs. M-S ranged from 3.9 to 11.6, as confirmed by qPCR. The expression of a contig annotated as cytochrome P450 (CYP86B1) in resistant individuals was over 3 times higher than that in sensitive individuals at 0–72 h after mesosulfuron-methyl treatment. A similar trend was noted for three other genes annotated as glutathione S-transferase (GST), namely GST-T3, GST-U6, and GST-U14; the expression of GST-U6 in resistant individuals was up to 142.3 times higher than that in sensitive individuals at 24 h after mesosulfuron-methyl treatment. In addition, GST activity in resistant individuals was 2.1 to 5.3 times higher than that in sensitive individuals. The GR50 of resistant biotype decreased from 24.4 to 11.3 g a.i. ha⁻¹ after P450 inhibitor malathion treatment. This study identified a cytochrome P450 gene CYP86B1 and three GST genes GST-T3, GST-U6, and GST-U14 that have higher expression in mesosulfuron-methyl resistant B. syzigachne, suggesting that both P450- and GST-based activities could be involved in resistance.
*Motivation:* High-throughput nucleotide sequencing provides quantitative readouts in assays for RNA expression (RNA-Seq), protein-DNA binding (ChIP-Seq) or cell counting (barcode sequencing). Statistical inference of differential signal in such data requires estimation of their variability throughout the dynamic range. When the number of replicates is small, error modelling is needed to achieve statistical power.
Results:
We propose an error model that uses the negative binomial distribution, with variance and mean linked by local regression, to model the null distribution of the count data. The method controls type-I error and provides good detection power. *Availability:* A free open-source R software package, DESeq , is available from the Bioconductor project and from "http://www-huber.embl.de/users/anders/DESeq":http://www-huber.embl.de/users/anders/DESeq.
Background
Perennial ryegrass is a highly heterozygous outbreeding grass species used for turf and forage production. Heterozygosity can affect de-Bruijn graph assembly making de novo transcriptome assembly of species such as perennial ryegrass challenging. Creating a reference transcriptome from a homozygous perennial ryegrass genotype can circumvent the challenge of heterozygosity. The goals of this study were to perform RNA-sequencing on multiple tissues from a highly inbred genotype to develop a reference transcriptome. This was complemented with RNA-sequencing of a highly heterozygous genotype for SNP calling.
Result
De novo transcriptome assembly of the inbred genotype created 185,833 transcripts with an average length of 830 base pairs. Within the inbred reference transcriptome 78,560 predicted open reading frames were found of which 24,434 were predicted as complete. Functional annotation found 50,890 transcripts with a BLASTp hit from the Swiss-Prot non-redundant database, 58,941 transcripts with a Pfam protein domain and 1,151 transcripts encoding putative secreted peptides. To evaluate the reference transcriptome we targeted the high-affinity K+ transporter gene family and found multiple orthologs. Using the longest unique open reading frames as the reference sequence, 64,242 single nucleotide polymorphisms were found. One thousand sixty one open reading frames from the inbred genotype contained heterozygous sites, confirming the high degree of homozygosity.
Conclusion
Our study has developed an annotated, comprehensive transcriptome reference for perennial ryegrass that can aid in determining genetic variation, expression analysis, genome annotation, and gene mapping.
The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling.
Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment.
These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined.
Barnyardgrass (Echinochloa crus-galli) is an important weed that is a menace to rice cultivation and production. Rapid evolution of herbicide resistance in this weed makes it one of the most difficult to manage using herbicides. Since genome-wide sequence data for barnyardgrass is limited, we sequenced the transcriptomes of susceptible and resistant barnyardgrass biotypes using the 454 GS-FLX platform.
454 pyrosequencing generated 371,281 raw reads with an average length of 341.8 bp, which made a total length of 126.89 Mb (SRX160526). De novo assembly produced 10,142 contigs (∼5.92 Mb) with an average length of 583 bp and 68,940 singletons (∼22.13 Mb) with an average length of 321 bp. About 244,653 GO term assignments to the biological process, cellular component and molecular function categories were obtained. A total of 6,092 contigs and singletons with 2,515 enzyme commission numbers were assigned to 151 predicted KEGG metabolic pathways. Digital abundance analysis using Illumina sequencing identified 78,124 transcripts among susceptible, resistant, herbicide-treated susceptible and herbicide-treated resistant barnyardgrass biotypes. From these analyses, eight herbicide target-site gene groups and four non-target-site gene groups were identified in the resistant biotype. These could be potential candidate genes involved in the herbicide resistance of barnyardgrass and could be used for further functional genomics research. C4 photosynthesis genes including RbcS, RbcL, NADP-me and MDH with complete CDS were identified using PCR and RACE technology.
This is the first large-scale transcriptome sequencing of E. crus-galli performed using the 454 GS-FLX platform. Potential candidate genes involved in the evolution of herbicide resistance were identified from the assembled sequences. This transcriptome data may serve as a reference for further gene expression and functional genomics studies, and will facilitate the study of herbicide resistance at the molecular level in this species as well as other weeds.
The interaction between environment and genetic traits under selection is the basis of evolution. In this study, we have investigated the genetic basis of herbicide resistance in a highly characterized initially herbicide-susceptible Lolium rigidum population recurrently selected with low (below recommended label) doses of the herbicide diclofop-methyl. We report the variability in herbicide resistance levels observed in F1 families and the segregation of resistance observed in F2 and back-cross (BC) families. The selected herbicide resistance phenotypic trait(s) appear to be under complex polygenic control. The estimation of the effective minimum number of genes (N E), depending on the herbicide dose used, reveals at least three resistance genes had been enriched. A joint scaling test indicates that an additive-dominance model best explains gene interactions in parental, F1, F2 and BC families. The Mendelian study of six F2 and two BC segregating families confirmed involvement of more than one resistance gene. Cross-pollinated L. rigidum under selection at low herbicide dose can rapidly evolve polygenic broad-spectrum herbicide resistance by quantitative accumulation of additive genes of small effect. This can be minimized by using herbicides at the recommended dose which causes high mortality acting outside the normal range of phenotypic variation for herbicide susceptibility.
Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants.
Background
Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm grown in tropical regions. Despite its agronomic importance, previous germplasm assessment studies have relied solely on morphological and agronomical traits. Molecular biology techniques have been scarcely used in assessment of genetic resources and for improvement of important agronomic and quality traits in Cocos nucifera, mostly due to the absence of available sequence information.
Methodology/Principal Findings
To provide basic information for molecular breeding and further molecular biological analysis in Cocos nucifera, we applied RNA-seq technology and de novo assembly to gain a global overview of the Cocos nucifera transcriptome from mixed tissue samples. Using Illumina sequencing, we obtained 54.9 million short reads and conducted de novo assembly to obtain 57,304 unigenes with an average length of 752 base pairs. Sequence comparison between assembled unigenes and released cDNA sequences of Cocos nucifera and Elaeis guineensis indicated that the assembled sequences were of high quality. Approximately 99.9% of unigenes were novel compared to the released coconut EST sequences. Using BLASTX, 68.2% of unigenes were successfully annotated based on the Genbank non-redundant (Nr) protein database. The annotated unigenes were then further classified using the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.
Conclusions/Significance
Our study provides a large quantity of novel genetic information for Cocos nucifera. This information will act as a valuable resource for further molecular genetic studies and breeding in coconut, as well as for isolation and characterization of functional genes involved in different biochemical pathways in this important tropical crop species.
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.
Paraquat is one of the most widely used herbicides worldwide. In green plants, paraquat targets the chloroplast by transferring electrons from photosystem I to molecular oxygen to generate toxic reactive oxygen species, which efficiently induce membrane damage and cell death. A number of paraquat-resistant biotypes of weeds and Arabidopsis mutants have been identified. The herbicide resistance in Arabidopsis is partly attributed to reduced uptake of paraquat through plasma membrane-localized transporters. However, the biochemical mechanism of paraquat resistance remains poorly understood. Here, we report the identification and characterization of an Arabidopsis paraquat resistant 1 (par1) mutant that shows strong resistance to the herbicide without detectable developmental abnormalities. PAR1 encodes a putative transporter protein localized to the Golgi apparatus. Compared with the wild-type plants, the par1 mutant plants show similar efficiency of paraquat uptake, suggesting that PAR1 is not directly responsible for the intercellular uptake of paraquat. However, the par1 mutation caused a reduction in the accumulation of paraquat in the chloroplast, suggesting that PAR1 is involved in the intracellular transport of paraquat into the chloroplast. We identified a PAR1-like gene, OsPAR1, in rice (Oryza sativa). Whereas the overexpression of OsPAR1 resulted in hypersensitivity to paraquat, the knockdown of its expression using RNA interference conferred paraquat resistance on the transgenic rice plants. These findings reveal a unique mechanism by which paraquat is actively transported into the chloroplast, and also provide a practical approach for genetic manipulations of paraquat resistance in crops.
Weeds are among the greatest pests of agriculture, causing billions of dollars in crop losses each year. As crop field management practices have changed over the past 12 000 years, weeds have adapted in turn to evade human removal. This evolutionary change can be startlingly rapid, making weeds an appealing system to study evolutionary processes that occur over short periods of time. An understanding of how weeds originate and adapt is needed for successful management; however, relatively little emphasis has been placed on genetically characterizing these systems. Here, we review the current literature on agricultural weed origins and their mechanisms of adaptation. Where possible, we have included examples that have been genetically well characterized. Evidence for three possible, non-mutually exclusive weed origins (from wild species, crop-wild hybrids or directly from crops) is discussed with respect to what is known about the microevolutionary signatures that result from these processes. We also discuss what is known about the genetic basis of adaptive traits in weeds and the range of genetic mechanisms that are responsible. With a better understanding of genetic mechanisms underlying adaptation in weedy species, we can address the more general process of adaptive evolution and what can be expected as we continue to apply selective pressures in agroecosystems around the world.Heredity advance online publication, 28 November 2012; doi:10.1038/hdy.2012.104.
High-throughput DNA sequencing is a powerful and versatile new technology for ob-taining comprehensive and quantitative data about RNA expression (RNA-Seq), protein-DNA binding (ChIP-Seq), and genetic variations between individuals. It addresses es-sentially all of the use cases that microarrays were applied to in the past, but produces more detailed and more comprehensive results. One of the basic statistical tasks is inference (testing, regression) on discrete count values (e.g., representing the number of times a certain type of mRNA was sampled by the sequencing machine). Challenges are posed by a large dynamic range, heteroskedas-ticity and small numbers of replicates. Hence, model-based approaches are needed to achieve statistical power. I will present an error model that uses the negative binomial distribution, with vari-ance and mean linked by local regression, to model the null distribution of the count data. The method controls type-I error and provides good detection power. I will also discuss how to use the GLM framework to detect alternative transcript isoform usage. A free open-source R software package, DESeq, is available from the Bioconductor project.
The last several years have seen revolutionary advances in DNA sequencing technologies with the advent of next-generation sequencing (NGS) techniques. NGS methods now allow millions of bases to be sequenced in one round, at a fraction of the cost relative to traditional Sanger sequencing. As costs and capabilities of these technologies continue to improve, we are only beginning to see the possibilities of NGS platforms, which are developing in parallel with online availability of a wide range of biological data sets and scientific publications and allowing us to address a variety of questions not possible before. As techniques and data sets continue to improve and grow, we are rapidly moving to the point where every organism, not just select "model organisms", is open to the power of NGS. This volume presents a brief synopsis of NGS technologies and the development of exemplary applications of such methods in the fields of molecular marker development, hybridization and introgression, transcriptome investigations, phylogenetic and ecological studies, polyploid genetics, and applications for large genebank collections.
Even with recent reductions in sequencing costs, most plants lack the genomic resources required for successful short-read transcriptome analyses as performed routinely in model species. Several approaches for the analysis of short-read transcriptome data are reviewed for nonmodel species for which the genome of a close relative is used as the reference genome. Two approaches using a data set from Phytophthora-challenged Rubus idaeus (red raspberry) are compared. Over 70000000 86-nt Illumina reads derived from R. idaeus roots were aligned to the Fragaria vesca genome using publicly available informatics tools (Bowtie/TopHat and Cufflinks). Alignment identified 16956 putatively expressed genes. De novo assembly was performed with the same data set and a publicly available transcriptome assembler (Trinity). A BLAST search with a maximum e-value threshold of 1.0 × 10(-3) revealed that over 36000 transcripts had matches to plants and over 500 to Phytophthora. Gene expression estimates from alignment to F. vesca and de novo assembly were compared for raspberry (Pearson's correlation = 0.730). Together, alignment to the genome of a close relative and de novo assembly constitute a powerful method of transcriptome analysis in nonmodel organisms. Alignment to the genome of a close relative provides a framework for differential expression testing if alignments are made to the predefined gene-space of a close relative and de novo assembly provides a more robust method of identifying unique sequences and sequences from other organisms in a system. These methods are considered experimental in nonmodel systems, but can be used to generate resources and specific testable hypotheses.
Viruses in the genus Bracovirus (BV) (Polydnaviridae) are symbionts of parasitoid wasps that specifically replicate in the ovaries of females. Recent analysis of expressed sequence
tags from two wasp species, Cotesia congregata and Chelonus inanitus, identified transcripts related to 24 different nudivirus genes. These results together with other data strongly indicate
that BVs evolved from a nudivirus ancestor. However, it remains unclear whether BV-carrying wasps contain other nudivirus-like
genes and what types of wasp genes may also be required for BV replication. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV). Here we characterized MdBV replication and performed massively parallel sequencing of M. demolitor ovary transcripts. Our results indicated that MdBV replication begins in stage 2 pupae and continues in adults. Analysis of prereplication- and active-replication-stage ovary
RNAs yielded 22 Gb of sequence that assembled into 66,425 transcripts. This breadth of sampling indicated that a large percentage
of genes in the M. demolitor genome were sequenced. A total of 41 nudivirus-like transcripts were identified, of which a majority were highly expressed
during MdBV replication. Our results also identified a suite of wasp genes that were highly expressed during MdBV replication. Among these products were several transcripts with conserved roles in regulating locus-specific DNA amplification
by eukaryotes. Overall, our data set together with prior results likely identify the majority of nudivirus-related genes that
are transcriptionally functional during BV replication. Our results also suggest that amplification of proviral DNAs for packaging
into BV virions may depend upon the replication machinery of wasps.
Non-target-site based resistance to herbicides is a major threat to the chemical control of agronomically noxious weeds. This adaptive trait is endowed by differences in the expression of a number of genes in plants that are resistant or sensitive to herbicides. Quantification of the expression of such genes requires normalising qPCR data using reference genes with stable expression in the system studied as internal standards. The aim of this study was to validate reference genes in Alopecurus myosuroides, a grass (Poaceae) weed of economic and agronomic importance with no genomic resources.
The stability of 11 candidate reference genes was assessed in plants resistant or sensitive to herbicides subjected or not to herbicide stress using the complementary statistical methods implemented by NormFinder, BestKeeper and geNorm. Ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase were identified as the best reference genes. The reference gene set accuracy was confirmed by analysing the expression of the gene encoding acetyl-coenzyme A carboxylase, a major herbicide target enzyme, and of an herbicide-induced gene encoding a glutathione-S-transferase.
This is the first study describing a set of reference genes (ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase) with a stable expression under herbicide stress in grasses. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in grasses.
HMMER is a software suite for protein sequence similarity searches using probabilistic methods. Previously, HMMER has mainly
been available only as a computationally intensive UNIX command-line tool, restricting its use. Recent advances in the software,
HMMER3, have resulted in a 100-fold speed gain relative to previous versions. It is now feasible to make efficient profile
hidden Markov model (profile HMM) searches via the web. A HMMER web server (http://hmmer.janelia.org) has been designed and implemented such that most protein database searches return within a few seconds. Methods are available
for searching either a single protein sequence, multiple protein sequence alignment or profile HMM against a target sequence
database, and for searching a protein sequence against Pfam. The web server is designed to cater to a range of different user
expertise and accepts batch uploading of multiple queries at once. All search methods are also available as RESTful web services,
thereby allowing them to be readily integrated as remotely executed tasks in locally scripted workflows. We have focused on
minimizing search times and the ability to rapidly display tabular results, regardless of the number of matches found, developing
graphical summaries of the search results to provide quick, intuitive appraisement of them.
High-throughput sequencing assays such as RNA-Seq, ChIP-Seq or barcode counting provide quantitative readouts in the form of count data. To infer differential signal in such data correctly and with good statistical power, estimation of data variability throughout the dynamic range and a suitable error model are required. We propose a method based on the negative binomial distribution, with variance and mean linked by local regression and present an implementation, DESeq, as an R/Bioconductor package.
The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a well-characterized Lolium rigidum biotype. The phenotypic resistance segregation in herbicide resistant and susceptible parents, F1, F2 and backcross (BC) families was analyzed as plant survival following treatment with the chemically unrelated herbicides diclofop-methyl or chlorsulfuron. Dominance and nuclear gene inheritance was observed in F1 families when treated at the recommended field doses of both herbicides. The segregation values of P450 herbicide resistance phenotypic traits observed in F2 and BC families was consistent with resistance endowed by two additive genes in most cases. In obligate out-crossing species such as L. rigidum, herbicide selection can easily result in accumulation of resistance genes within individuals.
In this study, genome-wide expression profiling based on Affymetrix ATH1 arrays was used to identify discriminating responses of Arabidopsis thaliana to five herbicides, which contain active ingredients targeting two different branches of amino acid biosynthesis. One herbicide contained glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), while the other four herbicides contain different acetolactate synthase (ALS) inhibiting compounds. In contrast to the herbicide containing glyphosate, which affected only a few transcripts, many effects of the ALS inhibiting herbicides were revealed based on transcriptional changes related to ribosome biogenesis and translation, secondary metabolism, cell wall modification and growth. The expression pattern of a set of 101 genes provided a specific, composite signature that was distinct from other major stress responses and differentiated among herbicides targeting the same enzyme (ALS) or containing the same chemical class of active ingredient (sulfonylurea). A set of homologous genes could be identified in Brassica napus that exhibited a similar expression pattern and correctly distinguished exposure to the five herbicides. Our results show the ability of a limited number of genes to classify and differentiate responses to closely related herbicides in A. thaliana and B. napus and the transferability of a complex transcriptional signature across species.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-009-9590-y) contains supplementary material, which is available to authorized users.
We conducted a comprehensive metabolic phenotyping of primary metabolism of photosynthetic tissue of Arabidopsis thaliana following spray treatment with a number of commercially used herbicides using a well established gas-chromatography mass-spectrometry profiling method. Applying this technique we were able to identify and quantify in excess of 80 polar metabolites and based on a combination of co-elution with standards and prediction from the mass spectra a similar number of lipophillic components within two chromatographic runs. The herbicides selected were glufosinate, sulcotrione, AE944 [N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine], foramsulfuron, benfuresate and glyphosate. We determined causal changes in the metabolite profiles by following their time-dependent changes using a serial sampling strategy. The resultant profiles were compared both by looking at the largest changes in a metabolite by metabolite manner and by performance of statistical analyses. These data revealed that analysis of the polar metabolites allows clear separation of the compounds under test. This finding is discussed in the context of current strategies for agrochemical discovery.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-008-0149-8) contains supplementary material, which is available to authorized users.
Transcriptome sequencing represents a fundamental source of information for genome-wide studies and transcriptome analysis and will become increasingly important for expression analysis as new sequencing technologies takes over array technology. The identification of the protein-coding region in transcript sequences is a prerequisite for systematic amino acid-level analysis and more specifically for domain identification. In this article, we present FrameDP, a self-training integrative pipeline for predicting CDS in transcripts which can adapt itself to different levels of sequence qualities.
Availability: FrameDP for Linux (web-server and underlying pipeline) is available at {{http://iant.toulouse.inra.fr/FrameDP}} for direct use or a standalone installation.
Contact: thomas.schiex@toulouse.inra.fr
We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample ( RNA - Seq ). This provides a digital measure of the presence and prevalence of transcripts from known ...
Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.
Weed control failures due to herbicide resistance are an increasing and worldwide problem significantly impacting crop yields. Metabolism-based herbicide resistance (referred to as metabolic resistance) in weeds is not well characterized at the genetic level. An RNA-Seq transcriptome analysis was used to find candidate genes conferring metabolic resistance to the herbicide diclofop in a diclofop-resistant population (R) of the major global weed Lolium rigidum. A reference cDNA transcriptome (19,623 contigs) was assembled and assigned putative annotations. Global gene expression was measured using Illumina reads from untreated control, adjuvant-only control, and diclofop treatment of R and susceptible (S). Contigs showing constitutive expression differences between untreated R and untreated S were selected for further validation analysis, including 11 contigs putatively annotated as cytochrome P450 (CytP450), glutathione transferase (GST), or glucosyltransferase (GT), and 17 additional contigs with annotations related to metabolism or signal transduction. In a forward genetics validation experiment, nine contigs had constitutive up-regulation in R individuals from a segregating F2 population, including 3 CytP450, one nitronate monooxygenase (NMO), 3 GST, and 1 GT. Principal component analysis using these nine contigs differentiated F2-R from F2-S individuals. In a physiological validation experiment where 2,4-D pre-treatment induced diclofop protection in S individuals due to increased metabolism, seven of the nine genetically-validated contigs were significantly induced. Four contigs (2 CytP450, NMO, and GT) were consistently highly expressed in nine field-evolved metabolic resistant L. rigidum populations. These four contigs were strongly associated with the resistance phenotype and are major candidates for contributing to metabolic diclofop resistance.This article is protected by copyright. All rights reserved.
Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a non-lethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multi-herbicides. Bispyribac sodium (BS) is a herbicide that inhibits the activity of acetolactate synthase (ALS). Rice of the indica variety show BS tolerance while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 mRNA levels in transgenic plants of rice and Arabidopsis. Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of ALS-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared to CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the field of weed control, herbicide development, and molecular breeding in broad range of crop species.
With no major new site-of-action herbicide introduced into the marketplace in the last 20 years, the stagnation or decline in available herbicides in the past decade in a number of jurisdictions, and ever-increasing incidence of herbicide-resistant (HR) weeds, more efficient use of our existing herbicide tools will be required to proactively or reactively manage HR weed populations. Herbicide-resistant weed management can be aided by crop cultivars with alternative single or stacked herbicide-resistance traits, such as synthetic auxins, which will become increasingly available to growers in the future. An examination of cross-resistance patterns in HR weed populations may inform proactive or reactive HR weed management through better insights into the potential for HR trait-stacked crops to manage HR weed biotypes as well as identify possible effective alternative herbicide options for growers. Clethodim is the lowest resistance risk acetyl-CoA carboxylase (ACC) inhibiting herbicide, with only two of eleven target-site mutations (amino acid substitutions) in weed populations that confer resistance. However, there are no reduced-risk acetolactate synthase/acetohydroxyacid synthase (ALS/AHAS) herbicides or herbicide classes. Growers will be increasingly reliant on reduced-risk herbicide sites of action (groups), such as microtubule assembly inhibitors (e.g., trifluralin, pendimethalin), synthetic auxins (e.g., 2,4-D, dicamba), some photosystem-II inhibitors (nitriles such as bromoxynil), protoporphyrinogen oxidase (PPO) or hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, glyphosate, or glutamine synthetase inhibitor (glufosinate), used in sequences, mixtures, or rotations, to manage HR weed populations.
Resistance to herbicides in arable weeds is increasing rapidly worldwide and threatening global food security. Resistance has now been reported to all major herbicide modes of action despite the development of resistance management strategies in the 1990s. We review here recent advances in understanding the genetic bases and evolutionary drivers of herbicide resistance that highlight the complex nature of selection for this adaptive trait. Whereas early studied cases of resistance were highly herbicide-specific and largely under monogenic control, cases of greatest concern today generally involve resistance to multiple modes of action, are under polygenic control, and are derived from pre-existing stress response pathways. Although 'omics' approaches should enable unraveling the genetic bases of complex resistances, the appearance, selection, and spread of herbicide resistance in weed populations can only be fully elucidated by focusing on evolutionary dynamics and implementing integrative modeling efforts.
Salinity stress is one of the most serious factors that impede the growth and development of various crops. Wild Gossypium species, which are remarkably tolerant to salt water immersion, are valuable resources for understanding salt tolerance mechanisms of Gossypium and improving salinity resistance in upland cotton. To generate a broad survey of genes with altered expression during various stages of salt stress, a mixed RNA sample was prepared from the roots and leaves of G. aridum plants subjected to salt stress. The transcripts were sequenced using the Illumina sequencing platform. After cleaning and quality checks, approximately 41.5 million clean reads were obtained. Finally, these reads were eventually assembled into 98,989 unigenes with a mean size of 452 bp. All unigenes were compared to known cluster of orthologous groups (COG) sequences to predict and classify the possible functions of these genes, which were classified into at least 25 molecular families. Variations in gene expression were then examined after exposing the plants to 200 mM NaCl for 3, 12, 72 or 144 h. Sequencing depths of approximately six million raw tags were achieved for each of the five stages of salt stress. There were 2,634 (1,513 up-regulated/1,121 down-regulated), 2,449 (1,586 up-regulated/863 down- regulated), 2,271 (946 up-regulated/1,325 down-regulated) and 3,352 (933 up-regulated/2,419 down-regulated) genes that were differentially expressed after exposure to NaCl for 3, 12, 72 and 144 h, respectively. Digital gene expression analysis indicated that pathways involved in "transport", "response to hormone stimulus" and "signaling" play important roles during salt stress, while genes involved in "protein kinase activity" and "transporter activity" undergo major changes in expression during early and later stages of salt stress, respectively.
BACKGROUND
Incremental herbicide metabolism by cytochrome P450 monooxygenases (P450s) has been proposed as the basis for resistance to bispyribac-sodium (bispyribac) in a multiple-herbicide-resistant biotype of Echinochloa phyllopogon. Upon exposure to bispyribac, strong induction of bispyribac-metabolising P450 activity has been reported in the resistant line, indicating that P450s induced by bispyribac are involved in the bispyribac resistance. RESULTSA polymerase chain reaction (PCR)-based cloning strategy was used to isolate 39 putative P450 genes from the bispyribac-resistant line of E. phyllopogon. Expression analysis by real-time PCR revealed that seven of the isolated genes were upregulated in response to bispyribac treatment of seedlings at the three-leaf stage. The transcript levels and protein sequences of the seven genes were compared between the bispyribac-resistant line and a susceptible line. CYP71AK2 and CYP72A254 were transcribed prominently in the bispyribac-resistant line. Amino acid polymorphisms were found in three genes, including CYP72A254. CONCLUSION
Upregulated expression of these genes is consistent with the inducible herbicide-metabolising P450 activity under bispyribac stress that was reported in a previous study. This is the first study to compare P450 genes in arable weed species in order to elucidate the mechanism for P450-mediated herbicide resistance. (c) 2013 Society of Chemical Industry
Productivity of crops grown for human consumption is at risk due to the incidence of pests, especially weeds, pathogens and animal pests. Crop losses due to these harmful organisms can be substantial and may be prevented, or reduced, by crop protection measures. An overview is given on different types of crop losses as well as on various methods of pest control developed during the last century.
Estimates on potential and actual losses despite the current crop protection practices are given for wheat, rice, maize, potatoes, soybeans, and cotton for the period 2001–03 on a regional basis (19 regions) as well as for the global total. Among crops, the total global potential loss due to pests varied from about 50% in wheat to more than 80% in cotton production. The responses are estimated as losses of 26–29% for soybean, wheat and cotton, and 31, 37 and 40% for maize, rice and potatoes, respectively. Overall, weeds produced the highest potential loss (34%), with animal pests and pathogens being less important (losses of 18 and 16%). The efficacy of crop protection was higher in cash crops than in food crops. Weed control can be managed mechanically or chemically, therefore worldwide efficacy was considerably higher than for the control of animal pests or diseases, which rely heavily on synthetic chemicals. Regional differences in efficacy are outlined. Despite a clear increase in pesticide use, crop losses have not significantly decreased during the last 40 years. However, pesticide use has enabled farmers to modify production systems and to increase crop productivity without sustaining the higher losses likely to occur from an increased susceptibility to the damaging effect of pests.
The concept of integrated pest/crop management includes a threshold concept for the application of pest control measures and reduction in the amount/frequency of pesticides applied to an economically and ecologically acceptable level. Often minor crop losses are economically acceptable; however, an increase in crop productivity without adequate crop protection does not make sense, because an increase in attainable yields is often associated with an increased vulnerability to damage inflicted by pests.
There are potential advantages and disadvantages associated with grazing spring perennial ryegrass swards designated for first-cut silage. These may differ for intermediate-heading (0·50 ear emergence in the second half of May) and late-heading (0·50 ear emergence in the first half of June) cultivars. The interactions between cultivar type, spring-grazing frequency, silage-harvest date and year were examined in an experiment with a randomized complete block (n = 4) design with a factorial arrangement of treatments, conducted in Ireland. The factors were (i) two perennial ryegrass mixtures: intermediate- vs. late-heading cultivars, (ii) three spring-grazing regimes: no grazing, grazing in mid-March or grazing in both mid-March and mid-April, (iii) four first-cut silage-harvest dates that were at c. 10-d intervals from 19 May and (iv) 2 years (1998 and 1999). The effects of cultivar mixture on herbage mass of the swards in spring were small and not statistically significant. The late-heading cultivars provided lower amounts of herbage dry matter for harvesting for first-cut silage but herbage with higher in vitro organic digestibility values compared with intermediate-heading cultivars. To achieve the same amount of herbage for silage, the late-heading cultivars needed to be harvested 8 d later than the intermediate-heading cultivars. Even with this delay in harvest date, the late-heading cultivars had higher in vitro organic digestibility values than the intermediate-heading cultivars. The late-heading cultivars could be harvested up to 30 d later and produce a higher amount of herbage for first-cut silage with similar digestibility values compared with the intermediate-heading cultivars.
Lolium species (ryegrasses) are genetically highly variable plants that are both forage crops and major weeds across the globe. As weeds, they rapidly evolve resistance under the selective pressure of acetolactate-synthase (ALS) inhibitors, the most resistance-prone herbicide group. Quick and accurate diagnosis is therefore of importance to prevent resistance spread in ryegrass. To develop proactive molecular tools for the detection of mutant, resistant ALS alleles, we assessed variation in the ryegrass ALS gene. Sequencing the full 1929-bp ALS coding sequence in 59 plants from six distant locations revealed a total of 208 polymorphic nucleotide positions (one every 9.3 nucleotides). The heterogeneous distribution of synonymous and non-synonymous substitutions along the ALS coding sequence suggested that nucleotide variation of ALS is shaped by purifying and background selection. Using regions of the ALS coding sequence with a low number of polymorphic nucleotide sites, five derived cleaved amplified polymorphic sequence (dCAPS) assays were developed targeting codons crucial for herbicide sensitivity. These enabled the first detection in ryegrass of a Pro-197-Thr substitution that confers herbicide resistance. Most assays could also be used to genotype Festuca and Vulpia plants. These dCAPS assays should prove powerful tools for both resistance diagnosis and population genetics studies.
Phylogenetic trees of the 8 species of the genus Lolium and of Festuca pratensis have been derived from frequency data at 13 isozyme loci using different distance algorithms. The best tree was obtained by the distance-Wagner method with the Cavalli-Sforza and Edwards chord distance. This tree appears robust when tested by bootstrap resampling of loci. This confirmed previous knowledge on species relationships in the genus Lolium, with supplementary data on the taxonomic position of the less studied L. persicum and L. canariense. Attempts were made to hybridize 4 Lolium species and F. pratensis. True interspecific hybrids, as confirmed by isozyme markers, were obtained for L. perenne x L. temulentum, L. temulentum x L. rigidum, L. temulentum x L. canariense and L. canariense x F. pratensis. This is the first report of this new form of Festulolium, which was obtained at a fairly high frequency, giving argument to a possible genetic proximity between L. canariense, an endemic species restricted to Atlantic islands, and F. pratensis, which has a more widespread, northern distribution. However, chromosome pairing analysis at meiosis did not support this conclusion, as the F1 L. canariense x F. pratensis definitely showed a higher level of asynapsis compared to that reported for L. perenne x F. pratensis. The apparently close relation between L. canariense and F. pratensis seen on the tree is therefore postulated to be an artefact, due to the low number of loci studied.
Non-target-site-based resistance (NTSR) can confer unpredictable cross-resistance to herbicides. However, the genetic determinants of NTSR remain poorly known. The current, urgent challenge for weed scientists is thus to elucidate the bases of NTSR so that detection tools are developed, the evolution of NTSR is understood, the efficacy of the shrinking herbicide portfolio is maintained and integrated weed management strategies, including fully effective herbicide applications, are designed and implemented. In this paper, the importance of NTSR in resistance to herbicides is underlined. The most likely way in which NTSR evolves-by accumulation of different mechanisms within individual plants-is described. The NTSR mechanisms, which can interfere with herbicide penetration, translocation and accumulation at the target site, and/or protect the plant against the consequences of herbicide action, are then reviewed. NTSR is a part of the plant stress response. As such, NTSR is a dynamic process unrolling over time that involves 'protectors' directly interfering with herbicide action, and also regulators controlling 'protector' expression. NTSR is thus a quantitative trait. On this basis, a three-step procedure is proposed, based on the use of the 'omics' (genomics, transcriptomics, proteomics or metabolomics), to unravel the genetic bases of NTSR. Copyright © 2012 Society of Chemical Industry.
Herbicides that target the acetolactate synthase (ALS) are among the most widely used weed control chemicals since their introduction into the marketplace in the early 1980s, including five classes (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthio (or oxy)-benzoates and sulfonylamino-carbonyltriazolinones). The mechanism researches have progressed unprecedentedly in the last two decades. Primary mode of action of the ALS-inhibiting herbicides that interfere with the activity of ALS enzyme seems no longer in doubt. Three lines of investigation from physiology, genetics, molecular and chemical structure aspects came together to prove that ALS is the site of action. Researches on the effects of branched chain amino acids (BCAAs) synthesis or protein metabolism caused by ALS-inhibiting herbicide elicit lots of disputations. Besides these two main works, other secondary effects of ALS inhibition, such as buildup of 2-ketobutyrate (α-ketobutyrate or 2-KB) or 2-aminobutyrate (2-AB, the transamination product of 2-KB), depletion of intermediates of the pathway for some critical processes, disruption of photosynthesis transport and respiration system etc., have also been implicated in the mechanism of plant death. However, there are still some disputations and doubts on the precise mechanisms that need further probing into. Further more, as many ALS-inhibiting herbicides and their derivatives are chiral with one or even more enantiomers, which may behave quite differently in biochemical processes, the effects and the environmental fate of chiral herbicides need to be investigated stereospecifically. By this, we can have a better understanding about the herbicides and avoid unnecessary pollution load.
In recent years, genome sequencing has revealed that cytochromes P450 (P450s) constitute the largest family of enzymatic proteins in higher plants. P450s are mono-oxygenases that insert one atom of oxygen into inert hydrophobic molecules to make them more reactive and hydrosoluble. Besides their physiological functions in the biosynthesis of hormones, lipids and secondary metabolites, P450s help plants to cope with harmful exogenous chemicals including pesticides and industrial pollutants, making them less phytotoxic. The recovery of an increasing number of plant P450 genes in recombinant form has enabled their use in experimentation, which has revealed their extraordinary potential for engineering herbicide tolerance, biosafening, bioremediation and green chemistry.
Transcriptomics studies often rely on partial reference transcriptomes that fail to capture the full catalogue of transcripts and their variations. Recent advances in sequencing technologies and assembly algorithms have facilitated the reconstruction of the entire transcriptome by deep RNA sequencing (RNA-seq), even without a reference genome. However, transcriptome assembly from billions of RNA-seq reads, which are often very short, poses a significant informatics challenge. This Review summarizes the recent developments in transcriptome assembly approaches - reference-based, de novo and combined strategies - along with some perspectives on transcriptome assembly in the near future.
Chlorsulfuron and imazethapyr (herbicides that inhibit acetolactate synthase; ALS, EC 4.1.3.18) produced a strong accumulation of hydroxycinnamic acids that was related to the induction of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 2.5.2.54). The exogenous application of two hydroxycinnamic acids, ferulic and p-coumaric acids, to pea plants resulted in their internal accumulation, arrested growth, carbohydrate and quinate accumulation in the leaves, and the induction of ethanolic fermentation. These effects resemble some of the physiological effects detected after acetolactate synthase inhibition and suggest important roles for ferulic and p-coumaric acids in the mode of action of herbicides inhibiting the biosynthesis of branched chain amino acids.
In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.
Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here we develop a RIP-seq method to capture the PRC2 transcriptome and identify a genome-wide pool of >9000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number of transcripts occur within imprinted regions, oncogene and tumor suppressor loci, and stem cell-related bivalent domains. We provide evidence for direct RNA-protein interactions, most likely via the Ezh2 subunit. We also identify Gtl2 RNA as a PRC2 cofactor that directs PRC2 to the reciprocally imprinted Dlk1 coding gene. Thus, Polycomb proteins interact with a genome-wide family of RNAs, some of which may be used as biomarkers and therapeutic targets for human disease.
The herbicide glyphosate inhibits the biosynthesis of aromatic amino acids by blocking the shikimate pathway. Imazethapyr and chlorsulfuron are two herbicides that act by inhibiting branched-chain amino acid biosynthesis. These herbicides stimulate secondary metabolism derived from the aromatic amino acids. The aim of this study was to test if they cause any cross-effect in the amino acid content and if they have similar effects on the shikimate pathway.
The herbicides inhibiting two different amino acid biosynthesis pathways showed a common pattern in general content of free amino acids. There was a general increase in total free amino acid content, with a transient decrease in the proportion of amino acids whose pathways were specifically inhibited. Afterwards, an increase in these inhibited amino acids was detected; this was probably related to proteolysis. All herbicides caused quinate accumulation. Exogenous application of quinate arrested growth, decreased net photosynthesis and stomatal conductance and was ultimately lethal, similarly to glyphosate and imazethapyr.
Quinate accumulation was a common effect of the two different classes of herbicide. Moreover, exogenous quinate application had phytotoxic effects, showing that this plant metabolite can trigger the toxic effects of the herbicides. This ability to mimic the herbicide effects suggests a possible link between the mode of action of these herbicides and the potential role of quinate as a natural herbicide.
Safeners enhance the selectivity of graminicidal herbicides such as fenoxaprop ethyl in cereals, by increasing their rates of detoxification in the crop. While studying the selectivity of fenoxaprop ethyl in wheat, we determined that the safeners mefenpyr diethyl and fenchlorazole ethyl also enhanced herbicide tolerance in the competing weed black-grass (Alopecurus myosuroides). Fenoxaprop ethyl was detoxified by conjugation with glutathione in both wheat and black-grass, with the resulting metabolites processed to the respective cysteine derivatives, which were then N-glycosylated. In black-grass, these detoxification pathways were only slightly enhanced by safeners, suggesting that metabolism alone was unlikely to account for increased herbicide tolerance. Instead, it was determined that safening was associated with an accumulation of glutathione and hydroxymethylglutathione and enzymes with antioxidant functions including phi and lambda glutathione transferases, active as glutathione peroxidases and thiol transferases respectively. These safener-induced changes closely mirrored those determined in two independent black-grass populations that had acquired multiple herbicide resistance (MHR) in the field. In addition to enhanced glutathione metabolism, both safener treatment and MHR resulted in elevated levels of flavonoids in the foliage of black-grass plants, notably flavone-C-glycosides and anthocyanins. Our results demonstrate that safening in a grass weed is associated with an inducible activation in antioxidant and secondary metabolism which mirrors the biochemical phenotype exhibited in plants that are resistant to multiple classes of herbicides.
Black-grass (Alopecurus myosuroides) is a major weed of wheat in Europe, with several populations having acquired resistance to multiple herbicides of differing modes of action. As compared with herbicide-susceptible black-grass, populations showing herbicide cross-resistance contained greatly elevated levels of a specific type I glutathione transferase (GST), termed AmGST2, but similar levels of a type III GST termed AmGST1. Following cloning and expression of the respective cDNAs, AmGST2 differed from AmGST1 in showing limited activity in detoxifying herbicides but high activities as a glutathione peroxidase (GPOX) capable of reducing organic hydroperoxides. In contrast to AmGST2, other GPOXs were not enhanced in the herbicide-resistant populations. Treatment with a range of herbicides used to control grass weeds in wheat resulted in increased levels of hydroperoxides in herbicide-susceptible populations but not in herbicide-resistant plants, consistent with AmGST2 functioning to prevent oxidative injury caused as a primary or secondary effect of herbicide action. Increased AmGST2 expression in black-grass was associated with partial tolerance to the peroxidizing herbicide paraquat. The selective enhancement of AmGST2 expression resulted from a constitutively high expression of the respective gene, which was activated in herbicide-susceptible black-grass in response to herbicide safeners, dehydration and chemical treatments imposing oxidative stress. Our results provide strong evidence that GSTs can contribute to resistance to multiple herbicides by playing a role in oxidative stress tolerance in addition to detoxifying herbicides by catalysing their conjugation with glutathione.