Mechanisms of evolved herbicide resistance.

The widely successful use of synthetic herbicides over the past 70 years has imposed strong and widespread selection pressure, leading to the evolution of herbicide resistance in hundreds of weed species. Both target-site resistance (TSR) and nontarget-site resistance (NTSR) mechanisms have evolved to most herbicide classes. TSR often involves mutations in genes encoding the protein targets of herbicides, affecting the binding of the herbicide either at or near catalytic domains or in regions affecting access to them. Most of these mutations are nonsynonymous SNPs, but polymorphisms in more than one codon or entire codon deletions have also evolved. Some herbicides bind multiple proteins, making the evolution of TSR mechanisms more difficult. Increased amounts of protein target, by increased gene expression or by gene duplication, are an important, albeit less common, TSR mechanism. NTSR mechanisms include reduced absorption or translocation and increased sequestration or metabolic degradation. The mechanisms that can contribute to NTSR are complex and often involve genes that are members of large gene families. For example, enzymes involved in herbicide metabolism-based resistances include cytochromes P450, GSH S-transferases, glucosyl and other transferases, aryl acylamidase, and others. Both TSR and NTSR mechanisms can combine at the individual level to produce higher resistance levels. The vast array of herbicide-resistance mechanisms for generalist (NTSR) and specialist (TSR and some NTSR) adaptations that have evolved over a few decades illustrate the evolutionary resilience of weed populations to extreme selection pressures. These evolutionary processes drive herbicide and herbicide-resistant crop development and resistance management strategies.

Identification and characterization of wheat drought-responsive MYB transcription factors involved in the regulation of cuticle biosynthesis.

A plant cuticle forms a hydrophobic layer covering plant organs, and plays an important role in plant development and protection from environmental stresses. We examined epicuticular structure, composition, and a MYB-based regulatory network in two Australian wheat cultivars, RAC875 and Kukri, with contrasting cuticle appearance (glaucousness) and drought tolerance. Metabolomics and microscopic analyses of epicuticular waxes revealed that the content of ß-diketones was the major compositional and structural difference between RAC875 and Kukri. The content of ß-diketones remained the same while those of alkanes and primary alcohols were increased by drought in both cultivars, suggesting that the interplay of all components rather than a single one defines the difference in drought tolerance between cultivars. Six wheat genes encoding MYB transcription factors (TFs) were cloned; four of them were regulated in flag leaves of both cultivars by rapid dehydration and/or slowly developing cyclic drought. The involvement of selected MYB TFs in the regulation of cuticle biosynthesis was confirmed by a transient expression assay in wheat cell culture, using the promoters of wheat genes encoding cuticle biosynthesis-related enzymes and the SHINE1 (SHN1) TF. Two functional MYB-responsive elements, specifically recognized by TaMYB74 but not by other MYB TFs, were localized in the TdSHN1 promoter. Protein structural determinants underlying the binding specificity of TaMYB74 for functional DNA cis-elements were defined, using 3D protein molecular modelling. A scheme, linking drought-induced expression of the investigated TFs with downstream genes that participate in the synthesis of cuticle components, is proposed.

Current status of community resources and priorities for weed genomics research.

Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.

Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters.

Constitutive over-expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3- to 6-week delays in flowering compared to control plants. In this work, two cold-inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low-to-moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up-regulation of cold-responsive target genes. The OsWRKY71 promoter-driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild-type plants. The WRKY71-TaDREB3 promoter-transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.

Nontarget-site resistance due to rapid physiological response in 2,4-D resistant Conyza sumatrensis: reduced 2,4-D translocation and auxin-induced gene expression.

BACKGROUND: Resistance to 2,4-Dichlorophenoxyacetic acid (2,4-D) has been reported in several weed species since the 1950s; however, a biotype of Conyza sumatrensis showing a novel physiology of the rapid response minutes after herbicide application was reported in 2017. The objective of this research was to investigate the mechanisms of resistance and identify transcripts associated with the rapid physiological response of C. sumatrensis to 2,4-D herbicide. RESULTS: Differences were found in 2,4-D absorption between the resistant and susceptible biotypes. Herbicide translocation was reduced in the resistant biotype compared to the susceptible. In resistant plants 98.8% of [14 C] 2,4-D was found in the treated leaf, whereas ≈13% translocated to other plant parts in the susceptible biotype at 96 h after treatment. Resistant plants did not metabolize [14 C] 2,4-D and had only intact [14 C] 2,4-D at 96 h after application, whereas susceptible plants metabolized [14 C] 2,4-D into four detected metabolites, consistent with reversible conjugation metabolites found in other 2,4-D sensitive plant species. Pre-treatment with the cytochrome P450 inhibitor malathion did not enhance 2,4-D sensitivity in either biotype. Following treatment with 2,4-D, resistant plants showed increased expression of transcripts within plant defense response and hypersensitivity pathways, whereas both sensitive and resistant plants showed increased expression of auxin-response transcripts. CONCLUSION: Our results demonstrate that reduced 2,4-D translocation contributes to resistance in the C. sumatrensis biotype. The reduction in 2,4-D transport is likely to be a consequence of the rapid physiological response to 2,4-D in resistant C. sumatrensis. Resistant plants had increased expression of auxin-responsive transcripts, indicating that a target-site mechanism is unlikely. © 2023 Society of Chemical Industry.

Survey of ACCase and ALS resistance in winter annual grasses identifies target-site and nontarget-site imazamox resistance in Secale cereale.

BACKGROUND: Early detection of herbicide resistance in weeds is crucial for successful implementation of integrated weed management. We conducted a herbicide resistance survey of the winter annual grasses feral rye (Secale cereale), downy brome (Bromus tectorum), and jointed goatgrass (Aegilops cylindrica) from Colorado winter wheat production areas for resistance to imazamox and quizalofop. RESULTS: All samples were susceptible to quizalofop. All downy brome and jointed goatgrass samples were susceptible to imazamox. Out of 314 field collected samples, we identified three feral rye populations (named A, B, and C) that were imazamox resistant. Populations B and C had a target-site mechanism with mutations in the Ser653 residue of the acetolactate synthase (ALS) gene to Asn in B and to Thr in C. Both populations B and C had greatly reduced ALS in vitro enzyme inhibition by imazamox. ALS feral rye protein modeling showed that steric interactions induced by the amino acid substitutions at Ser653 impaired imazamox binding. Individuals from population A had no mutations in the ALS gene. The ALS enzyme from population A was equally sensitive to imazamox as to known susceptible feral rye populations. Imazamox was degraded two times faster in population A compared with a susceptible control. An oxidized imazamox metabolite formed faster in population A and this detoxification reaction was inhibited by malathion. CONCLUSION: Population A has a nontarget-site mechanism of enhanced imazamox metabolism that may be conferred by cytochrome P450 enzymes. This is the first report of both target-site and metabolism-based imazamox resistance in feral rye. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors.

Transcription factors have been shown to control the activity of multiple stress response genes in a coordinated manner and therefore represent attractive targets for application in molecular plant breeding. We investigated the possibility of modulating the transcriptional regulation of drought and cold responses in the agriculturally important species, wheat and barley, with a view to increase drought and frost tolerance. Transgenic wheat and barley plants were generated showing constitutive (double 35S) and drought-inducible (maize Rab17) expression of the TaDREB2 and TaDREB3 transcription factors isolated from wheat grain. Transgenic populations with constitutive over-expression showed slower growth, delayed flowering and lower grain yields relative to the nontransgenic controls. However, both the TaDREB2 and TaDREB3 transgenic plants showed improved survival under severe drought conditions relative to nontransgenic controls. There were two components to the drought tolerance: real (activation of drought-stress-inducible genes) and 'seeming' (consumption of less water as a result of smaller size and/or slower growth of transgenics compared to controls). The undesired changes in plant development associated with the 'seeming' component of tolerance could be alleviated by using a drought-inducible promoter. In addition to drought tolerance, both TaDREB2 and TaDREB3 transgenic plants with constitutive over-expression of the transgene showed a significant improvement in frost tolerance. The increased expression of TaDREB2 and TaDREB3 lead to elevated expression in the transgenics of 10 other CBF/DREB genes and a large number of stress responsive LEA/COR/DHN genes known to be responsible for the protection of cell from damage and desiccation under stress.

Synthetic auxin herbicides: finding the lock and key to weed resistance.

Synthetic auxin herbicides are designed to mimic indole-3-acetic acid (IAA), an integral plant hormone affecting cell growth, development, and tropism. In this review, we explore target site genes in the auxin signaling pathway including SCFTIR1/AFB, Aux/IAA, and ARFs that are confirmed or proposed mechanisms for weed resistance to synthetic auxin herbicides. Resistance to auxin herbicides by metabolism, either by enhanced cytochrome P450 detoxification or by loss of pro-herbicide activation, is a major non-target-site resistance pathway. We speculate about potential fitness costs of resistance due to effects of resistance-conferring mutations, provide insight into the role of polyploidy in synthetic auxin resistance evolution, and address the genetic resources available for weeds. This knowledge will be the key to unlock the long-standing questions as to which components of the auxin signaling pathway are most likely to have a role in resistance evolution. We propose that an ambitious research effort into synthetic auxin herbicide/target site interactions is needed to 1) explain why some synthetic auxin chemical families have activity on certain dicot plant families but not others and 2) fully elucidate target-site cross-resistance patterns among synthetic auxin chemical families to guide best practices for resistance management.

Evolution of EPSPS double mutation imparting glyphosate resistance in wild poinsettia (Euphorbia heterophylla L.).

The evolution of glyphosate resistance (GR) in weeds is an increasing problem. Glyphosate has been used intensively on wild poinsettia (Euphorbia heterophylla L.) populations for at least 20 years in GR crops within South America. We investigated the GR mechanisms in a wild poinsettia population from a soybean field in southern Brazil. The GR population required higher glyphosate doses to achieve 50% control (LD50) and 50% dry mass reduction (MR50) compared to a glyphosate susceptible (GS) population. The ratio between the LD50 and MR50 of GR and GS resulted in resistance factors (RF) of 6.9-fold and 6.1-fold, respectively. Shikimate accumulated 6.7 times more in GS than in GR when leaf-discs were incubated with increasing glyphosate concentrations. No differences were found between GR and GS regarding non-target-site mechanisms. Neither population metabolized glyphosate to significant levels following treatment with 850 g ha-1 glyphosate. Similar levels of 14C-glyphosate uptake and translocation were observed between the two populations. No differences in EPSPS expression were found between GS and GR. Two target site mutations were found in all EPSPS alleles of homozygous resistant plants: Thr102Ile + Pro106Thr (TIPT-mutation). Heterozygous individuals harbored both alleles, wild-type and TIPT. Half of GR individuals were heterozygous, suggesting that resistance is still evolving in the population. A genotyping assay was developed based on the Pro106Thr mutation, demonstrating high efficiency to identify homozygous, heterozygous or wild-type EPSPS sequences across different plants. This is the first report of glyphosate-resistant wild-poinsettia harboring an EPSPS double mutation (TIPT) in the same plant.

EPSPS duplication and mutation involved in glyphosate resistance in the allotetraploid weed species Poa annua L.

BACKGROUND: Poa annua is a widespread winter annual weed species in California. Recently, poor control of this species with glyphosate was reported by growers in an almond orchard in California with a history of repetitive glyphosate use. The objectives of this research were to evaluate the level of glyphosate resistance in a developed S4 P. annua line (R) and identify the mechanisms of resistance involved. RESULTS: Whole-plant dose-response experiments confirmed glyphosate resistance in R, which required 18-fold more glyphosate to achieve a 50% growth reduction compared with a susceptible line (S), results that were supported by the lower shikimate accumulation observed in R compared with S. No differences in glyphosate absorption, translocation, or metabolism were observed, suggesting that non-target-site mechanisms of resistance are not involved in the resistance phenotype. A missense single nucleotide polymorphism was observed in EPSPS coding position 106 in R, resulting in a leucine to proline substitution. This polymorphism was observed exclusively in P. supina EPSPS homeologs. A seven-fold increase in the number of copies of EPSPS alleles was observed in R compared with S. CONCLUSIONS: We report the first case of glyphosate resistance associated with both EPSPS duplication and target-site mutation at position 106, leading to high levels of glyphosate resistance in the allotetraploid weed species Poa annua L. Data obtained in this research will be useful for the development of diagnostic tools for rapid glyphosate resistance identification, monitoring and containment. © 2018 Society of Chemical Industry.

Multiple target site resistance to glyphosate in junglerice (Echinochloa colona) lines from California orchards.

BACKGROUND: In California specialty cropping systems such as vineyards and orchards, Echinochloa colona is present as a summer annual weed. It is able to germinate throughout the growing season whenever favorable conditions are present, and management relies heavily on glyphosate applications. Glyphosate-resistant (GR) E. colona biotypes are present in the state, but the levels of resistance observed suggest that there may be differences in mechanisms of resistance among populations. RESULTS: Echinochloa colona lines collected from different regions of California's Central Valley presented resistance levels ranging from 1.4 to 4.3-fold compared to susceptible lines. No differences in the absorption and translocation of [14 C]-glyphosate were observed among lines. Resistant lines accumulated eight-fold less shikimic acid after treatment with 435 and 870 g a.e. ha-1 glyphosate compared to the most susceptible line. Sequencing of a region of the EPSPS gene revealed three single nucleotide changes leading to amino acid substitutions at Proline 106, including Pro106Leu, Pro106Thr and Pro106Ser. CONCLUSION: These results indicate that an altered target site in EPSPS is contributing to resistance in these lines and resistance has evolved independently, multiple times in the Central Valley of California. Additional research is needed to further understand the genomic contributions of resistance loci in this polyploid weed species. © 2018 Society of Chemical Industry.

Transcription of putative tonoplast transporters in response to glyphosate and paraquat stress in Conyza bonariensis and Conyza canadensis and selection of reference genes for qRT-PCR.

Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines. Transcription of ABC transporters also increased after paraquat treatment in all three lines of C. bonariensis. However, in C. canadensis, paraquat treatment increased transcription of only one ABC transporter gene in the susceptible line. The increase in transcription of ABC transporters after herbicide treatment is likely a stress response based on similar response observed across all Conyza lines regardless of resistance or sensitivity to glyphosate or paraquat, thus these genes do not appear to be directly involved in the mechanism of resistance in Conyza spp.

EPSPS gene amplification in glyphosate-resistant Bromus diandrus.

BACKGROUND: Glyphosate is the most widely used herbicide in the world and has been intensively used to control B. diandrus, a problematic weed of crops and pastures in southern Australia. RESULTS: Resistance to glyphosate was identified in two populations of B. diandrus that were nearly fivefold more resistant to glyphosate than wild-type plants. Both populations contained EPSPS gene amplification, with resistant plants having an average of around 20-fold the number of copies of EPSPS compared with susceptible plants. EPSPS expression was also increased in resistant plants of both populations; however, expression levels were not correlated with the number of EPSPS copies. Amplification of only one of the four EPSPS genes present in B. diandus was detected. Investigation into the inheritance of glyphosate resistance found no segregation in the F2 generation. Every individual in the F2 populations contained between three and 30 copies of EPSPS; however, on average they contained fewer copies compared with the parent resistant population. CONCLUSIONS: Glyphosate resistance in B. diandrus is due to EPSPS gene amplification. Resistance is heritable but complex.

Isolation of plant transcription factors using a modified yeast one-hybrid system.

BACKGROUND: The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontechtrade mark product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination.Unfortunately, such cDNA libraries prepared directly in yeast can not be used for the efficient recovery of purified plasmids and thus are incompatible with existing yeast one-hybrid systems, which use yeast transformation for the library screen. RESULTS: Here we propose an adaptation of the yeast one-hybrid system for identification and cloning of transcription factors using a MATCHMAKER cDNA library. The procedure is demonstrated using a cDNA library prepared from the liquid part of the multinucleate coenocyte of wheat endosperm. The method is a modification of a standard one-hybrid screening protocol, utilising a mating step to introduce the library construct and reporter construct into the same cell. Several novel full length transcription factors from the homeodomain, AP2 domain and E2F families of transcription factors were identified and isolated. CONCLUSION: In this paper we propose a method to extend the compatibility of MATCHMAKER cDNA libraries from yeast two-hybrid screens to one-hybrid screens. The utility of the new yeast one-hybrid technology is demonstrated by the successful cloning from wheat of full-length cDNAs encoding several transcription factors from three different families.