Non-target Site Resistance Research Articles

Metribuzin Resistance in a Wild Radish ( Raphanus raphanistrum) Population via Both psbA Gene Mutation and Enhanced Metabolism.

There have been many studies on target-site resistance (TSR) to PSII-inhibiting herbicides, but only a few on the non-target-site resistance (NTSR). Here, we reported both TSR and NTSR to metribuzin in a wild radish population. Dose-response studies revealed a higher level of resistance to metribuzin in the resistant (R) compared to the susceptible (S) population. Sequencing of the target psbA gene revealed the known Ser-264-Gly mutation in R plants. In addition, a higher level of [14C]-metribuzin metabolism and, consequently, a lower level of [14C] translocation were also detected in the R plants. These results demonstrated that both psbA gene mutation and enhanced metabolism contribute to metribuzin resistance in this wild radish population. Furthermore, this resistant population showed resistance to ALS-inhibiting herbicides due to multiple ALS gene mutations. This is the first report in wild radish of metabolic herbicide resistance, in addition to the target-site psbA gene mutation.

Reduced Absorption and Impaired Translocation Endows Glyphosate Resistance in Amaranthus palmeri Harvested in Glyphosate-Resistant Soybean from Argentina.

Amaranthus palmeri S. Watson is probably the worst glyphosate-resistant (GR) weed worldwide. The EPSPS (5-enolpyruvylshikimate-3-phosphate-synthase) gene amplification has been reported as the major target-site-resistance (TSR) mechanism conferring resistance to glyphosate in this species. In this study, TSR and non-target-site-resistance (NTSR) mechanisms to glyphosate were characterized in a putative resistant A. palmeri population (GRP), harvested in a GR soybean crop from Argentina. Glyphosate resistance was confirmed for the GRP population by dose-response assays. No evidence of TSR mechanisms, as well as glyphosate metabolism, was found in this population. Moreover, a susceptible population (GSP) that absorbed about 10% more herbicide than the GRP population was evaluated at different periods after treatment. The GSP population translocated about 20% more glyphosate to the remainder of the shoots and roots at 96 h after treatment than the control, while the GRP population retained 62% of herbicide in the treated leaves. This is the first case of glyphosate resistance in A. palmeri involving exclusively NTSR mechanisms.

Target-site mutation accumulation among ALS inhibitor-resistant Palmer amaranth.

Palmer amaranth (Amaranthus palmeri S. Wats) is one of the most common and troublesome weeds in the USA. Palmer amaranth resistance to acetolactate synthase (ALS) inhibitors is widespread in the USA, as in Arkansas. The cross-resistance patterns and mechanism of resistance are not known. Experiments were conducted to determine cross-resistance to ALS inhibitors and identify target-site mutations in 20 Palmer amaranth localities from 13 counties in Arkansas. All Palmer amaranth localities tested had plants cross-resistant to imazethapyr, flumetsulam, primisulfuron, pyrithiobac and trifloxysulfuron. The dose of trifloxysulfuron that caused 50% control was 21-56-fold greater for resistant accessions than for susceptible ones. All but three resistant plants analyzed had one or two relative copies of ALS; one plant had seven relative copies. All resistant plants tested (18 localities) carried the Trp574Leu mutation, which is known to confer broad resistance to ALS inhibitors, supporting the cross-resistance pattern observed. Besides the Trp574Leu mutation, 30% of localities had individuals with one additional resistance-conferring mutation including Ala122Thr, Pro197Ala or Ser653Asn. The Trp574Leu mutation in ALS is the primary mechanism of resistance to ALS inhibitors in Palmer amaranth from Arkansas, USA. In some localities, multiple mutations have accumulated in one plant. All localities tested contained plants with resistance to five families of ALS inhibitors. Localities with extremely high resistance to ALS inhibitors, and those outside the subset we studied, may harbor non-target site resistance mechanisms. ALS inhibitors are generally no longer effective on Palmer amaranth in these localities from the US mid-south. © 2018 Society of Chemical Industry.

Characterization of Non-Target-Site Mechanisms in Imidazolinone-Resistant Sunflower by RNA-seq

AbstractImisun sunflowers (Helianthus annuus L.) are imidazolinone-resistant cultivars in which the two mechanisms of herbicide resistance coexist: (i) mutation in herbicide target-site (target-site resistance) and (ii) non-target-site resistance (NTSR). In Imisun technology, NTSR could be related to herbicide metabolism and might occur as a result of a constitutive up-regulation of resistance genes, or it can appear only after herbicide treatment. The objective of this study was to characterize NTSR in Imisun sunflower in response to imazethapyr using RNA-Seq and to determine whether these mechanisms are constitutive or herbicide-induced. Cypsels were germinated in plastic pots, watered by capillarity and growth in chamber under controlled conditions. Seven-day-old plants were treated with 0 (control) and 1 μM imazethapyr for 12 h. After leaf RNA purification, stranded, paired-end cDNA libraries were constructed. Sequencing was performed with Illumina HiSeq2000. Local mapping, with and without multihits, was carried out over the reference transcriptome HaT13l and differential expression was analysed. Sixty one and 47 contigs (according to mapping strategy) related to xenobiotic metabolism were found: cytochromes P450s, ABC transporters, glycosyltransferases, UDPglucuronosyl/glucosyltransferases and glutathione S-transferases. None of these contigs showed differential expression between control and imazethapyr-treated plants. Seventeen interesting contigs were verified by qRT-PCR. These results suggest that constitutive NTSR mechanisms may account for imidazolinone resistance in Imisun sunflower.

Climate change exacerbates pest damage through reduced pesticide efficacy.

Pesticide efficacy is strongly associated with environmental conditions. Conditional resistance defined as a reduction in pesticide sensitivity under changed environmental conditions has been widely detected under climatic changes such as elevated temperatures and CO2 enrichment. Given the effects of environmental conditions on pesticide sensitivity, many of the putative resistance reports made by farmers may be due to pesticide application followed by non-optimal environmental conditions rather than the evolution of resistance. This type of conditional resistance may be the result of phenotypic plasticity or epigenetic changes in response to environmental changes. Elevated temperatures and CO2 enrichment can directly lead to reduced pesticide efficacy by altering pesticide metabolism and translocation, or indirectly increasing pesticide detoxification in host-plants thus reducing pesticide availability for the target pest. Stress-related signal transduction pathways, as well as physiological changes, can both be associated with accelerated pesticide detoxification under climatic changes. The possibility for parallel mechanisms controlling these responses in different pest species should be considered. It is proposed that the same mechanisms leading to non-target site resistance in pests may also play a role in conditional resistance, suggesting we can predict the pesticides to which pests are likely to be less responsive under changing climatic conditions. Using adjuvants to improve pesticide translocation or reduce pesticide metabolism, alongside with new technologies such as using nanoparticles may result in higher pesticide functionality under the projected climate change. Exploring the physiological, transcriptional and biochemical basis underlying conditional resistance is crucial in maintaining future pest management under changing environmental conditions. © 2018 Society of Chemical Industry.

Enhanced Herbicide Metabolism and Metabolic Resistance Genes Identified in Tribenuron-Methyl Resistant Myosoton aquaticum L.

The evolved resistance of Myosoton aquaticum L. to acetolactate synthase (ALS) inhibitors is well established, but most research has focused on target-site resistance, while nontarget-site resistance remains neglected. Here, we investigated mechanisms of the latter. The pretreatment with the P450 inhibitor malathion significantly increased the sensitivity of resistant plants to tribenuron-methyl. The rapid P450-mediated tribenuron-methyl metabolism in resistant plants was confirmed by LC-MS/MS analysis. Besides, GST activity was higher among resistant than susceptible individuals. The next transcriptome analysis generated 544,102,236 clean reads from RNA sequencing libraries. De novo assembly yielded 102,529 unigenes with an average length of 866 bp, annotated across seven databases. Digital gene expression selected 25 differentially expressed genes, further validated with qRT-PCR. Three P450 genes, two GST genes, two glucosyltransferase genes, four ABC transporter genes, and four additional contigs were constitutively up-regulated in resistant individuals. Overall, our research confirmed that enhanced herbicide metabolism drives tribenuron-methyl resistance in M. aquaticum.

Enhanced Trifluralin Metabolism Can Confer Resistance in Lolium rigidum.

Resistance to the pre-emergence herbicide trifluralin is increasing in Australian annual ryegrass ( Lolium rigidum) populations. Three L. rigidum populations (R1, R2, and R3) collected from Australian grain fields were identified with trifluralin resistance. Both target-site and nontarget-site resistance mechanisms were investigated. No target-site α-tubulin mutations were detected in populations R1 and R3, while an Arg-243-Lys mutation was found in R2. Compared with the three trifluralin-susceptible populations, enhanced [14C]-trifluralin metabolism, quantified by measuring the amount of [14C] label partitioning into the polar phase of a hexane:methanol system, was identified in all the three resistant populations. This is the first report of metabolic resistance to trifluralin. Coevolution of target-site and nontarget-site resistance to trifluralin is occurring, and metabolic resistance is not rare in L. rigidum populations in Australia. A method was established for trifluralin metabolic resistance detection, overcoming the difficulties of quantifying this highly volatile herbicide by chromatographic methods.

Validation of expression stability of reference genes in response to herbicide stress in wild oat (Avena ludoviciana)

Weeds are serious problem in crop production and wild oat is a grass weed of economic and agronomic significance. We need to extend our basic knowledge of weeds especially in molecular genetics and gene expression. For study of gene expression by semi-quantitative and quantitative PCR, it is recommended that normalization of reference genes be carried out in order to select the most stable reference gene for a precise gene expression study. The purpose of this research was evaluation of four reference genes in response to treated and untreated (control) by herbicide in two tissues (stem and leaf) of non-target site resistance wild oat (A. ludoviciana). Four candidate reference genes including Actin, Ef1α (elongation factor 1 alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and TBP (TATA-box-binding protein) were used to determine stable reference gene exposed to the herbicide using the statistical methods of NormFinder, BestKeeper and delta-Ct. NormFinder indicated that TBP and Actin genes are the best combination of two genes for normalizing calculations (with a combined gene stability value of 0.012) for qPCR analysis under herbicide stress in different tissues of non-target site resistance wild oat. Based on the statistical results, the Ef1α gene was identified as the unstable reference gene. Totally, according to results of this study, TBP gene is the most stable reference gene and therefore, this gene can be used as a reference gene for future studies of quantitative PCR analysis of herbicide stress-responsive gene expression in wild oat and potentially in other grass weed species.

Fenoxaprop-P-ethyl resistance conferred by cytochrome P450s and target site mutation in Alopecurus japonicus.

Alopecurus japonicus is a serious grass weed species in wheat fields in eastern Asia, and has evolved strong resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Although target-site resistance (TSR) to ACCase inhibitors in A. japonicus has been reported, non-target site resistance (NTSR) has not. This study investigated both TSR and NTSR in a fenoxaprop-P-ethyl-resistant A. japonicus population (AHFD-3), which was collected in Feidong County, Anhui Province, China. We found that AHFD-3 exhibited high resistance to fenoxaprop-P-ethyl and low resistance to flucarbazone-sodium. The sensitivity of AHFD-3 to fenoxaprop-P-ethyl increased significantly after treatment with cytochrome P450 (P450) inhibitors; however, such synergies between P450 inhibitors and fenoxaprop-P-ethyl were not found in two control populations. Sequences of the entire carboxyltransferase domain of A. japonicus ACCase were obtained, and AHFD-3 plants showed an Asp-2078-Gly substitution in the ACCase. With the derived cleaved amplified polymorphic sequence (dCAPS) method, we found that 85.4% of the plants of AHFD-3 carried this mutation. The P450 content in AHFD-3 plants was significantly higher than those of the two control populations after treatment with fenoxaprop-P-ethyl. Ten partial sequences of P450 genes in A. japonicus were cloned. Three P450 genes were up-regulated 12 h after fenoxaprop-P-ethyl treatment, which were all from the P450 subfamily CYP72A. Moreover, a P450 gene from the P450 family CYP81 was up-regulated after fenoxaprop-P-ethyl treatment in all populations studied. Fenoxaprop-P-ethyl resistance in AHFD-3 plants was conferred by up-regulation of cytochrome P450s in the CYP72A subfamily and target site mutation of the ACCase gene. © 2018 Society of Chemical Industry.

Optimizing RNA-seq studies to investigate herbicide resistance.

Transcriptomic profiling, specifically via RNA sequencing (RNA-seq), is becoming one of the more commonly used methods for investigating non-target site resistance (NTSR) to herbicides due to its high throughput capabilities and utility in organisms with little to no previous sequence information. A review of the weed science RNA-seq literature revealed some basic principles behind generating quality data from these types of studies. First, studies that included more replicates per biotype and took steps to control for genetic background had significantly better control of false positives and, consequently, shorter lists of potential resistance genes to sift through. Pooling of biological replicates prior to sequencing was successful in some cases, but likely contributed to an overall increase in the false discovery rate. Although the inclusion of herbicide-treated samples was common across most of the studies, it ultimately introduced difficulties in interpretation of the final results due to challenges in capturing the right sampling window after treatment and to the induction of stress responses in the injured herbicide-sensitive plants. RNA-seq is an effective tool for NTSR gene discovery, but careful consideration should be given to finding the most powerful and cost-effective balance between replicate number, sequencing depth and treatment number. © 2017 Society of Chemical Industry.

Gene expression in response to glyphosate treatment in fleabane (Conyza bonariensis)-glyphosate death response and candidate resistance genes.

This study takes a whole-transcriptome approach to assess gene expression changes in response to glyphosate treatment in glyphosate-resistant fleabane. We assessed gene expression changes in both susceptible and resistant lines so that the glyphosate death response could be quantified, and constitutively expressed candidate resistance genes identified. There are three copies of the glyphosate target site (5-enolpyruvylshikimate-3-phosphate; EPSPS) gene in Conyza and because Conyza bonariensis is allohexaploid, there is a baseline nine copies of the gene in any individual. Many genes were differentially expressed in response to glyphosate treatment. Known resistance mutations are present in EPSPS2 but they are present in a glyphosate-susceptible line as well as resistant lines and therefore not sufficient to confer resistance. EPSPS1 is expressed four times more than EPSPS2, further reducing the overall contribution of these mutations. We demonstrate that glyphosate resistance in C. bonariensis is not the result of EPSPS mutations or overexpression, but due to a non-target-site mechanism. A large number of genes are affected by glyphosate treatment. We present a list of candidate non-target-site-resistance (NTSR) genes in fleabane for future studies into these mechanisms. © 2017 Society of Chemical Industry.

A quantitative genetic examination of non‐target‐site resistance applied to Avena species

SummaryQuantitative genetics tools can be used to assess whether using herbicides at low doses drive selection on standing genetic variation in populations leading to non‐target‐site resistance (NTSR). These tools are particularly important for estimating the number of genes involved and the potential speed of evolution. A short cut to answering questions about the evolution of NTSR may be to measure heritability. The heritability index (H) provides a measure of the potential to develop NTSR and can be simply calculated from classical dose–response experiments. This measure and the associated experimental designs are discussed with two applied examples on Avena spp. (A. fatua and A. sterilis). In these examples, H values ranged from 0.24 to 0.73, which means that selection for NTSR is highly probable in cases with high H value. We suggest that structuring plants into genetic groups (e.g. families or populations) can contribute to our understanding of the evolutionary potential of populations and plant species to evolve resistance, without increasing experimental cost and time.

Possible involvement of glutathione S-transferases in imazamox detoxification in an imidazolinone-resistant sunflower hybrid

The resistance of crops to herbicides can be due to target site based resistance or non-target site based resistance mechanisms or a combination of both. In non-target site resistance, the detoxification efficiency plays a major role by involvement of enzymes such as P450s, GTs, GSTs and ABC transporters. The resistance of the first commercial Clearfield sunflower hybrids (Imisun trait) to herbicides of imidazolinone group is based on a combination of both types of resistance. The target site resistance consists of a mutation in Ahasl1 gene, encoding the synthesis of the AHAS enzyme. The non-target site resistance is supposed to be due to intensified herbicide disposal and is not fully understood. The objective of this study was to detect the fast response of the glutathione-mediated detoxification system in IMI-R and IMI-S sunflower hybrids to the herbicide imazamox and to study the possible participation of GSTs in the enhancement of the hybrids' tolerance. The obtained results allow to presume that GSTs are involved in imazamox detoxification in the sunflower Imisun trait and thus contributing to its non-target site resistance.

Non‐target site resistance to flucarbazone, imazamethabenz and pinoxaden is controlled by three linked genes in Avena fatua

SummaryExtensive herbicide usage has led to the evolution of resistant weed populations that cause substantial crop yield losses and increase production costs. The multiple herbicide‐resistant (MHR) Avena fatua populations utilised in this study are resistant to members of all selective herbicide families, across five modes of action, available for A. fatua control in US small grain production, and thus pose significant agronomic and economic threats. Resistance to acetolactate synthase and acetyl‐CoA carboxylase inhibitors is not conferred by known target site mutations, indicating that non‐target site resistance (NTSR) mechanisms are involved. Understanding the inheritance of NTS MHR is of upmost importance for continued agricultural productivity in the face of the rapid increase in resistant weed populations worldwide. As few studies have examined the inheritance of NTSR in autogamous weeds, we investigated the inheritance and genetic control of NTSR in the highly autogamous, allohexaploid species A. fatua. We found that NTSR in MHRA. fatua is controlled by three separate, closely‐linked nuclear genes for flucarbazone‐sodium, imazamethabenz‐methyl and pinoxaden. The single‐gene NTSR inheritance patterns reported here contrast with other examples in allogamous species and illustrate the diversity of evolutionary responses to strong selection.

No Vegetative and Fecundity Fitness Cost Associated with Acetyl-Coenzyme A Carboxylase Non-target-site Resistance in a Black-Grass (Alopecurus myosuroides Huds) Population.

Attention should be devoted to weeds evolving herbicide resistance with non-target-site resistance (NTSR) mechanism due to their unpredictable resistance patterns. Quantification of fitness cost can be used in NTSR management strategies to determine the long-term fate of resistant plants in weed populations. To our knowledge, this is the first report evaluating potential fecundity and vegetative fitness of a NTSR black-grass (Alopecurus myosuroides Huds), the most important herbicide resistant weed in Europe, with controlled genetic background. The susceptible (S) and NTSR sub-populations were identified and isolated from a fenoxaprop-P-ethyl resistant population by a plant cloning technique. Using a target-neighborhood design, competitive responses of S and NTSR black-grass sub-populations to increasing density of winter wheat were quantified for 2 years in greenhouse and 1 year in field. Fitness traits including potential seed production, vegetative biomass and tiller number of both sub-populations significantly decreased with increasing density of winter wheat. More importantly, no statistically significant differences were found in fitness traits between S and NTSR sub-populations either grown alone (no competition) or in competition with winter wheat. According to the results, the NTSR black-grass is probably to persist in field even in the cessation of fenoxaprop-P-ethyl. So, effective herbicide resistant management strategies are strongly suggested to prevent and stop the spread of the NTSR black-grass, otherwise NTSR loci conferring resistance to a range of herbicides in black-grass will persist in the gene pool even in the absence of herbicide application. Consequently, herbicide as an effective tool for control of black-grass will gradually be lost in fields infested by NTSR black-grass.

Constitutive redox and phosphoproteome changes in multiple herbicide resistant Avena fatua L. are similar to those of systemic acquired resistance and systemic acquired acclimation

Plants are routinely confronted with numerous biotic and abiotic stressors, and in response have evolved highly effective strategies of systemic acquired resistance (SAR) and systemic acquired acclimation (SAA), respectively. A much more evolutionarily recent abiotic stress is the application of herbicides to control weedy plants, and their intensive use has selected for resistant weed populations that cause substantial crop yield losses and increase production costs. Non-target site resistance (NTSR) to herbicides is rapidly increasing worldwide and is associated with alterations in generalized stress defense networks. This work investigated protein post-translational modifications associated with NTSR in multiple herbicide resistant (MHR) Avena fatua, and their commonalities with those of SAR and SAA. We used proteomic, biochemical, and immunological approaches to compare constitutive protein profiles in MHR and herbicide susceptible (HS) A. fatua populations. Phosphoproteome and redox proteome surveys showed that post-translational modifications of proteins with functions in core cellular processes were reduced in MHR plants, while those involved in xenobiotic and stress response, reactive oxygen species detoxification and redox maintenance, heat shock response, and intracellular signaling were elevated in MHR as compared to HS plants. More specifically, MHR plants contained constitutively elevated levels of three protein kinases including the lectin S-receptor-like serine/threonine-protein kinase LecRK2, a well-characterized component of SAR. Analyses of superoxide dismutase enzyme activity and protein levels did not reveal constitutive differences between MHR and HS plants. The overall results support the idea that herbicide stress is perceived similarly to other abiotic stresses, and that A. fatua NTSR shares analogous features with SAR and SAA. We speculate that MHR A. fatua’s previous exposure to sublethal herbicide doses, as well as earlier evolution under a diversity of abiotic and biotic stressors, has led to a heightened state of stress preparedness that includes NTSR to a number of unrelated herbicides.

High conservation of the transcriptional response to acetolactate‐synthase‐inhibiting herbicides across plant species

SummaryWe investigated whether specific ALS‐inhibitor response marker‐genes (AIRMs) identified in the model dicotyledonous plant Arabidopsis thaliana (Brassicaceae) were conserved across phylogenetically very distant species. The most probable homologs of the 533 A. thalianaAIRMs were identified in the major monocotyledonous weeds Alopecurus myosuroides and Lolium sp. (Poaceae) using recently established transcriptome resources. Expression patterns of above 70% of the grass AIRMs homologs matched those of their A. thaliana counterparts. Pathways of response to acetolactate‐synthase (ALS)‐inhibiting herbicides seem therefore strongly conserved, even across very distant plant species. This opens new perspectives for unravelling the genetic determinants of non‐target‐site resistance (NTSR) to ALS inhibitors, especially those governing NTSR regulation that remains fully unknown to date. Model plants could be used in a first step to discover the genes involved in ALS‐inhibitor response pathways. Subsequent studies comparing sequence and expression of weed homologs of these genes should expedite the identification of candidates for the role of genes governing NTSR sensing and regulatory pathways.

Fitness of ALS-Inhibitors Herbicide Resistant Population of Loose Silky Bentgrass (Apera spica-venti).

Herbicide resistance is an example of plant evolution caused by an increased reliance on herbicides with few sites of action to manage weed populations. This micro-evolutionary process depends on fitness, therefore the assessment of fitness differences between susceptible and resistant populations are pivotal to establish management strategies. Loose silky bentgrass (Apera spica-venti) is a serious weed in Eastern, Northern, and Central Europe with an increasing number of herbicide resistant populations. This study examined the fitness and growth characteristics of an ALS resistant biotype. Fitness and growth characteristics were estimated by comparing seed germination, biomass, seed yield and time to key growth stages at four crop densities of winter wheat (0, 48, 96, and 192 plants m-2) in a target-neighborhood design. The resistant population germinated 9–20 growing degree days (GDD) earlier than the susceptible population at 10, 16, and 22°C. No differences were observed between resistant and susceptible populations in tiller number, biomass, time to stem elongation, time to first visible inflorescence and seed production. The resistant population reached the inflorescence emergence and flowering stages in less time by 383 and 196 GDD, respectively, at a crop density of 96 winter wheat plants m-2 with no differences registered at other densities. This study did not observe a fitness cost to herbicide resistance, as often hypothesized. Inversely, a correlation between non-target site resistance (NTSR), earlier germination and earlier flowering time which could be interpreted as fitness benefits as these plant characteristics could be exploited by modifying the timing and site of action of herbicide application to better control ALS NTSR populations of A. spica-venti.

Enhanced 2,4-D Metabolism in Two Resistant Papaver rhoeas Populations from Spain.

Corn poppy (Papaver rhoeas), the most problematic broadleaf weed in winter cereals in Southern Europe, has developed resistance to the widely-used herbicide, 2,4-D. The first reported resistance mechanism in this species to 2,4-D was reduced translocation from treated leaves to the rest of the plant. However, the presence of other non-target site resistance (NTSR) mechanisms has not been investigated up to date. Therefore, the main objective of this research was to reveal if enhanced 2,4-D metabolism is also present in two Spanish resistant (R) populations to synthetic auxins. With this aim, HPLC experiments at two 2,4-D rates (600 and 2,400 g ai ha−1) were conducted to identify and quantify the metabolites produced and evaluate possible differences in 2,4-D degradation between resistant (R) and susceptible (S) plants. Secondarily, to determine the role of cytochrome P450 in the resistance response, dose-response experiments were performed using malathion as its inhibitor. Three populations were used: S, only 2,4-D R (R-703) and multiple R to 2,4-D and ALS inhibitors (R-213). HPLC studies indicated the presence of two hydroxy metabolites in these R populations in shoots and roots, which were not detected in S plants, at both rates. Therefore, enhanced metabolism becomes a new NTSR mechanism in these two P. rhoeas populations from Spain. Results from the dose-response experiments also showed that pre-treatment of R plants with the cytochrome P450 (P450) inhibitor malathion reversed the phenotype to 2,4-D from resistant to susceptible in both R populations. Therefore, it could be hypothesized that a malathion inhibited P450 is responsible of the formation of the hydroxy metabolites detected in the metabolism studies. This and previous research indicate that two resistant mechanisms to 2,4-D could be present in populations R-703 and R-213: reduced translocation and enhanced metabolism. Future experiments are required to confirm these hypotheses, understand the role of P450, and the relationship between both NTSR mechanisms. On this basis, selection pressure with synthetic auxins bears the risk of promoting the evolution enhanced metabolism in Papaver rhoeas.

Intensive herbicide use has selected for constitutively elevated levels of stress-responsive mRNAs and proteins in multiple herbicide-resistant Avena fatua L.

Intensive use of herbicides has led to the evolution of two multiple herbicide-resistant (MHR) Avena fatua (wild oat) populations in Montana that are resistant to members of all selective herbicide families available for A. fatua control in US small grain crops. We used transcriptome and proteome surveys to compare constitutive changes in MHR and herbicide-susceptible (HS) plants associated with non-target site resistance. Compared to HS plants, MHR plants contained constitutively elevated levels of differentially expressed genes (DEGs) with functions in xenobiotic catabolism, stress response, redox maintenance and transcriptional regulation that are similar to abiotic stress-tolerant phenotypes. Proteome comparisons identified similarly elevated proteins including biosynthetic and multifunctional enzymes in MHR plants. Of 25 DEGs validated by RT-qPCR assay, differential regulation of 21 co-segregated with flucarbazone-sodium herbicide resistance in F3 families, and a subset of 10 of these were induced or repressed in herbicide-treated HS plants. Although the individual and collective contributions of these DEGs and proteins to MHR remain to be determined, our results support the idea that intensive herbicide use has selected for MHR populations with altered, constitutively regulated patterns of gene expression that are similar to those in abiotic stress-tolerant plants. © 2017 Society of Chemical Industry.