Genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with herbicide resistance evolved before the origin of land plants.

Cytochrome P450 (CYP) monooxygenases and glutathione S-transferases (GST) are enzymes that catalyse chemical modifications of a range of organic compounds. Herbicide resistance has been associated with higher levels of CYP and GST gene expression in some herbicide-resistant weed populations compared to sensitive populations of the same species. By comparing the protein sequences of 9 representative species of the Archaeplastida-the lineage which includes red algae, glaucophyte algae, chlorophyte algae, and streptophytes-and generating phylogenetic trees, we identified the CYP and GST proteins that existed in the common ancestor of the Archaeplastida. All CYP clans and all but one land plant GST classes present in land plants evolved before the divergence of streptophyte algae and land plants from their last common ancestor. We also demonstrate that there are more genes encoding CYP and GST proteins in land plants than in algae. The larger numbers of genes among land plants largely results from gene duplications in CYP clans 71, 72, and 85 and in the GST phi and tau classes [1,2]. Enzymes that either metabolise herbicides or confer herbicide resistance belong to CYP clans 71 and 72 and the GST phi and tau classes. Most CYP proteins that have been shown to confer herbicide resistance are members of the CYP81 family from clan 71. These results demonstrate that the clan and class diversity in extant plant CYP and GST proteins had evolved before the divergence of land plants and streptophyte algae from a last common ancestor estimated to be between 515 and 474 million years ago. Then, early in embryophyte evolution during the Palaeozoic, gene duplication in four of the twelve CYP clans, and in two of the fourteen GST classes, led to the large numbers of CYP and GST proteins found in extant land plants. It is among the genes of CYP clans 71 and 72 and GST classes phi and tau that alleles conferring herbicide resistance evolved in the last fifty years.

Comparison of Glyphosate-Degradation Ability of Aldo-Keto Reductase (AKR4) Proteins in Maize, Soybean and Rice.

Genes that participate in the degradation or isolation of glyphosate in plants are promising, for they endow crops with herbicide tolerance with a low glyphosate residue. Recently, the aldo-keto reductase (AKR4) gene in Echinochloa colona (EcAKR4) was identified as a naturally evolved glyphosate-metabolism enzyme. Here, we compared the glyphosate-degradation ability of theAKR4 proteins from maize, soybean and rice, which belong to a clade containing EcAKR4 in the phylogenetic tree, by incubation of glyphosate with AKR proteins both in vivo and in vitro. The results indicated that, except for OsALR1, the other proteins were characterized as glyphosate-metabolism enzymes, with ZmAKR4 ranked the highest activity, and OsAKR4-1 and OsAKR4-2 exhibiting the highest activity among the AKR4 family in rice. Moreover, OsAKR4-1 was confirmed to endow glyphosate-tolerance at the plant level. Our study provides information on the mechanism underlying the glyphosate-degradation ability of AKR proteins in crops, which enables the development of glyphosate-resistant crops with a low glyphosate residue, mediated by AKRs.

A LuALS Mutation with High Sulfonylurea Herbicide Resistance in Linum usitatissimum L.

The cultivation of herbicide-resistant crops is an effective tool for weed management in agriculture. Weed control in flax (Linum usitatissimum L.) remains challenging due to the lack of available herbicide-resistant cultivars. In this study, a mutant resistant to acetolactate synthase (ALS)-inhibiting herbicides was obtained by ethyl methanesulphonate (EMS) mutagenesis using an elite cultivar, Longya10. Whole-plant dose-response assays revealed that, compared to Longya10, the mutant was 11.57-fold more resistant to tribenuron-methyl (TBM) and slightly resistant to imazethapyr (resistance index (mutant/Longya10) < 3). In vitro acetolactate synthase assays showed that the relative resistance of the mutant was 12.63 times more than that of Longya10. A biochemical analysis indicated that there was a Pro197Ser (relative to the Arabidopsis thaliana ALS sequence) substitution within the LuALS1, conferring high resistance to sulfonylurea herbicides in the mutant. Additionally, two cleaved amplified polymorphic sequence (CAPS) markers, BsaI-LuALS1 and EcoO109I-LuALS1, were developed based on the mutation site for marker assistant selection in breeding. Moreover, the mutant did not cause losses in natural field conditions. We find a mutant with ALS-inhibiting herbicide resistance chemically induced by EMS mutagenesis, providing a valuable germplasm for breeding herbicide-resistant flax varieties.

Improved Method to Characterize Leaf Surfaces, Guide Adjuvant Selection, and Improve Glyphosate Efficacy.

Glyphosate, one of the most widely used herbicides, plays an important role in controlling weeds and ensuring crop production. While using glyphosate, adjuvants are commonly added to improve its deposition on weeds and control efficacy. However, changes in weed leaf surface characteristics may reduce glyphosate penetration and contribute to evolved glyphosate resistance. Therefore, it is significant to introduce an improved method for regularizing leaf surface characterization and guide adjuvant selection to improve glyphosate efficacy. In this work, surface characteristics of typical weed leaves have been systematically investigated by 3D surface analysis and scanning electron microscopy, finally quantified by apparent surface free energy (ASFE) due to its comprehensive and quantitative evaluation of leaf surfaces. Moreover, the relationship between the weed leaf surface characteristics and the retention of glyphosate on weeds was established, further related to the control efficacy against weeds. To maximize the utilization rate of glyphosate, the types and concentrations of adjuvants should be regulated according to the ASFE of weeds. Our findings not only regularize the surface properties of weed leaves but also reveal their influencing mechanism on the deposition and biological activity of glyphosate, which provide effective guidance for the use of glyphosate.

Complementary peptides represent a credible alternative to agrochemicals by activating translation of targeted proteins.

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

Functional characterization of glycine oxidase from Bacillus licheniformis in Escherichia coli and transgenic plants.

OBJECTIVES: To find glycine oxidase genes that can be applied to the breeding of glyphosate resistant crops. RESULTS: The glycine oxidase (GO, EC 1.4.3.19) gene (GenBank No: KC831746) from Bacillus licheniformis (B. licheniformis) was chemically synthesized and transformed into glyphosate-sensitive Escherichia coli (E. coli). The GO gene was transformed into Arabidopsis and rice through Agrobacterium-mediated transformation. The test results confirmed that transgenic plants containing GO genes are more resistant to glyphosate than wild-type plants. On solid Murashige and Skoog (MS) (Murashige and Skoog1962 ) medium containing 200 µM glyphosate, transgenic Arabidopsis thaliana grew normally, while wild-type plants were stunted and root growth was restricted. In a solution containing 500 µM glyphosate, wild-type rice showed severe yellowing, while transgenic rice grew normally. In addition, when sprayed with 10 mM glyphosate solution, wild-type rice withered and died, while transgenic rice grew well. The function of GO gene in glyphosate resistance and the application value of GO gene in the cultivation of glyphosate-resistant crops is proved. CONCLUSIONS: The glycine oxidase gene from B. licheniformis enhances the resistance of E. coli, Arabidopsis and rice to glyphosate.

Aldo-keto reductase may contribute to glyphosate resistance in Lolium rigidum.

BACKGROUND: We have previously demonstrated that an aldo-keto reductase (AKR) from Echinochloa colona (EcAKR4-1) can metabolize glyphosate and confers glyphosate resistance. This study aims to investigate if the EcAKR4-1 orthologs from Lolium rigidum also play a role in glyphosate resistance in non-target-site based, glyphosate-resistant (R) L. rigidum populations from Western Australia. RESULTS: The full-length L. rigidum AKR gene (LrAKR4C10) orthologous to EcAKR4-1, together with a distinct LrAKR1, were cloned from plants of a glyphosate-susceptible (S) (VLR1) and three glyphosate R L. rigidum populations (WALR50, WALR60 and WALR70). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) results showed that basal expression levels of the two LrAKR genes did not differ between the R and S populations, but their expression was significantly induced by glyphosate (up to 4.3-fold) or 2,4-D treatment (up to 3.4-fold) in R populations. Escherichia coli cells transformed respectively with LrAKR4C10 and LrAKR1 were more tolerant to glyphosate. Rice (Oryza sativa) seedlings overexpressing each of the two LrAKR gene survived glyphosate rates that were lethal to the green fluorescence protein (GFP) control plants. Structural modeling predicts a similar way of glyphosate binding and detoxification by LrAKR4C10 and EcAKR4-1, but an alternative way of glyphosate binding by LrAKR1. Relatively lower capacity of the two LrAKRs in conferring glyphosate resistance than the known EcAKR4-1 was discussed in relation to structural interaction. CONCLUSION: Glyphosate-induced higher expression of the two LrAKR genes in L. rigidum populations contributes to a moderate level of glyphosate resistance likely through enhanced glyphosate metabolism. The herbicide 2,4-D can also induce the LrAKR expression, indicating the potential antagonistic effect of 2,4-D to glyphosate. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tribenuron-methyl metabolism and the rare Pro197Phe double mutation together with 2,4-D metabolism and reduced absorption can evolve in Papaver rhoeas with multiple and cross herbicide resistance to ALS inhibitors and auxin mimics.

Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.

Inheritance of 2,4-dichlorophenoxyacetic acid (2,4-D) resistance in Amaranthus palmeri.

In this study, the inheritance of 2,4-D resistance in a multiple herbicide-resistant Palmer amaranth (KCTR) was investigated. Direct and reciprocal crosses were performed using 2,4-D-resistant KCTR and susceptible KSS plants to generate F1 progenies. 2,4-D dose-response assays were conducted to evaluate the response of progenies from each F1 family along with KCTR and KSS plants in controlled environmental growth chambers. Additionally, 2,4-D-resistant male and female plants from each of the F1 families were used in pairwise crosses to generate pseudo-F2 families. Segregation (resistance or susceptibility) of progenies from the F2 families in response to a discriminatory rate of 2,4-D (i.e., 560 g ae ha-1) was evaluated. Dose-response analysis of F1 progenies derived from direct and reciprocal crosses suggested that the 2,4-D resistance in KCTR is a nuclear trait. Chi-square analyses of F2 segregation data implied that 2,4-D resistance in KCTR is controlled by multiple gene(s). Overall, our data suggest that the 2,4-D resistance in KCTR Palmer amaranth is a nuclear inherited trait controlled by multiple genes. Such resistance can spread both via pollen or seed-mediated gene flow. In future, efforts will be directed towards identifying genes mediating 2,4-D resistance in KCTR population.

Molecular Mechanisms of Herbicide Resistance in Weeds.

Herbicides have become one of the most widespread weed-control tools in the world since their advent in the mid-20th century [...].

First report of glyphosate-resistant downy brome (Bromus tectorum L.) in Canada.

Glyphosate is the most used herbicide worldwide, and is an important source of economical weed control in glyphosate-resistant crops, and conservation tillage systems, among other uses. Downy brome (Bromus tectorum L.), otherwise known as cheatgrass, is a highly invasive winter-annual grass weed in cropping systems, pastureland, and naturalized or ruderal areas in western North America. In 2021, a downy brome population remained uncontrolled following four applications of glyphosate in a glyphosate-resistant canola (Brassica napus L.) field located in Taber County, Alberta, Canada. All individuals from the subsequent generation of the population survived glyphosate treatment at the typical field rate (900 g ae ha-1). In dose-response bioassays, the putative glyphosate-resistant population exhibited 10.6- to 11.9-fold, 7.7- to 8.7-fold, 7.8- to 8.8-fold, and 8.3- to 9.5-fold resistance to glyphosate based on plant survival, visible control, and biomass fresh weight and dry weight, respectively, compared with two susceptible populations 21 days after treatment. Estimated glyphosate rates for 80% control of this population ranged from 2795 to 4511 g ae ha-1; well above common usage rates. This downy brome population represents the first glyphosate-resistant grass weed confirmed in Canada, and the second weed species exhibiting glyphosate resistance in the Canadian prairie region.

Inheritance of dicamba-resistance in allotetraploid Chenopodium album.

BACKGROUND: Chenopodium album L. is a troublesome weed in spring-planted crops, and different levels of ploidy have been documented for this weed species. A population of C. album has evolved resistance to dicamba. The level of ploidy and inheritance of dicamba resistance was studied in this population. RESULTS: The resistant and susceptible individuals of C. album were confirmed as tetraploid by flow cytometry. Pair-crosses were made between ten resistant and susceptible individuals. Eight F1 individuals from five crosses were confirmed resistant after treating with dicamba at 400 g a.e. ha-1 . These individuals were selfed, and the response of their progenies to dicamba was assessed in dose-response experiments, and the results confirmed the resistance trait was dominant. Furthermore, an analysis of the segregation patterns revealed that the segregation response of all F2 progenies fitted a 3:1 (resistant/susceptible) ratio when treated with dicamba at 200, 400 and 800 g a.e. ha-1 , suggesting a single gene was responsible for dicamba resistance. CONCLUSIONS: Dicamba resistance in the studied tetraploid population of C. album is governed by a single dominant gene. This type of inheritance suggests that selection for dicamba resistance can occur readily. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetic Analysis and Fine Mapping of ZmGHT1 Conferring Glufosinate Herbicide Tolerance in Maize (Zea mays L.).

Weed interference in the crop field is one of the major biotic stresses causing dramatic crop yield losses, and the development of herbicide-resistant crops is critical for weed control in the application of herbicide technologies. To identify herbicide-resistant germplasms, we screened 854 maize inbreed lines and 25,620 seedlings by spraying them with 1 g/L glufosinate. One plant (L336R), possibly derived from a natural variation of line L336, was identified to have the potential for glufosinate tolerance. Genetic analysis validated that the glufosinate tolerance of L336R is conferred by a single locus, which was tentatively designated as ZmGHT1. By constructing a bi-parental population derived from L336R, and a glufosinate sensitive line L312, ZmGHT1 was mapped between molecular markers M9 and M10. Interestingly, genomic comparation between the two sequenced reference genomes showed that large scale structural variations (SVs) occurred within the mapped region, resulting in 2.16 Mb in the inbreed line B73, and 11.5 kb in CML277, respectively. During the fine mapping process, we did not detect any additional recombinant, even by using more than 9500 F2 and F3 plants, suspecting that SVs should also have occurred between L336R and L312 in this region, which inhibited recombination. By evaluating the expression of the genes within the mapped interval and using functional annotation, we predict that the gene Zm00001eb361930, encoding an aminotransferase, is the most likely causative gene. After glufosinate treatment, lower levels of ammonia content and a higher activity of glutamine synthetase (GS) in L336R were detected compared with those of L336 and L312, suggesting that the target gene may participate in ammonia elimination involving GS activity. Collectively, our study can provide a material resource for maize herbicide resistant breeding, with the potential to reveal a new mechanism for herbicide resistance.

Cytochrome P450 BsCYP99A44 and BsCYP704A177 Confer Metabolic Resistance to ALS Herbicides in Beckmannia syzigachne.

Beckmannia syzigachne is a noxious grassy weed that infests wheat fields in China. Previously, we identified that mesosulfuron-methyl resistance in a B. syzigachne population (R, SD04) was conferred by non-target resistance, such as cytochrome P450 mixed-function oxidases (P450s)-based metabolism. RNA sequencing and real-time PCR (qRT-PCR) were used to discover potential P450s-resistant-related genes. Five cytochrome P450s (CYP704A177, CYP96B84, CYP71D7, CYP93A1, and CYP99A44) were found to be highly expressed in R plants. In this study, CYP99A44 and CYP704A177 were cloned from B. syzigachne and transferred into Arabidopsis thaliana to test the sensitivity of Arabidopsis with and without P450s genes to mesosulfuron-methyl and other acetolactate synthase (ALS)-inhibiting herbicides. Transgenic Arabidopsis overexpressing CYP99A44 became resistant to the sulfonylurea herbicide mesosulfuron-methyl, but showed no resistance to pyroxsulam, imazethapyr, flucarbazone, and bispyribac-sodium. Notably, those overexpressing CYP704A177 showed resistance to pyroxsulam and bispyribac-sodium, but not to mesosulfuron-methyl, imazethapyr, and flucarbazone. These results indicated that B. syzigachne and transgenic Arabidopsis displayed different cross-resistance patterns to ALS-inhibiting herbicides. Subcellular localization revealed that CYP99A44 and CYP704A177 protein were located in the endoplasmic reticulum. Furthermore, these results clearly indicated that CYP99A44-mediated mesosulfuron-methyl resistance in B. syzigachne and CYP704A177 may be involved in B. syzigachne cross-resistance to pyroxsulam and bispyribac-sodium.

Molecular diagnostics for real-time determination of herbicide resistance in wild grasses.

The growth of resistance to multiple herbicides in grass weeds is a major threat to global cereal production and in the UK, is epitomized by the loss of control of blackgrass (Alopecurus myosuroides), causing losses in winter wheat production equating to 5% of national consumption. With an urgent need to develop new black-grass management tools, we have developed a lateral flow assay (LFA) that can predict resistance to multiple herbicides within 10 min.

Increase in glutathione S-transferase activity and antioxidant damage ability drive resistance to bensulfuron-methyl in Sagittaria trifolia.

Weeds tend to develop resistance to herbicides with time. Understanding the resistance mechanisms evolved by weeds would help manage weed infestation. Sagittaria trifolia, a paddy weed found in the rice fields of Liaoning, China, has developed resistance to bensulfuron-methyl, causing severe yield losses in rice. This study deciphers the underlying mechanisms in terms of non-target-site resistance toward bensulfuron-methyl. We compared the ability of glutathione S-transferase (GST) mediated detoxification metabolism and reactive oxygen species (ROS) scavenging between sensitive (NHS) and resistant (NHR) populations of S. trifolia. The resistance ratio of NHR was 210; but the ratio was significantly decreased after GST-inhibitor treatment (44.9). This indicated that a GST-mediated enhancement of detoxification metabolism stimulated the development of resistance. Similarly, higher GST activity was observed in NHR; but the activity equaled that of NHS after GST-inhibitor treatment. However, treatment with the GST-inhibitor did not completely reverse bensulfuron-methyl resistance in NHR, indicating that additional factors contributed to herbicide resistance in these plants. We observed a rapid increase in H2O2 and malondialdehyde accumulation in the case of NHS after bensulfuron-methyl application, whereas those of NHR remained relatively stable, implying that NHR exhibited higher ROS-scavenging capacity under herbicide stress. Further, NHR showed higher glutathione and ascorbic acid contents and higher activities of glutathione reductase and dehydrogenase reductase, all of which contribute towards herbicide resistance in these plants. Our results indicate that GST-mediated detoxification metabolism of bensulfuron-methyl and ROS scavenging capacity contributed to the development of resistance in S. trifolia.

Stacking herbicide detoxification and resistant genes improves glyphosate tolerance and reduces phytotoxicity in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.).

Glyphosate residues retained in the growing meristematic tissues or in grains of glyphosate-resistant crops affect the plants physiological functions and crop yield. Removing glyphosate residues in the plants is desirable with no penalty on crop yield and quality. We report a new combination of scientific strategy to detoxify glyphosate that reduces the residual levels and improve crop resistance. The glyphosate detoxifying enzymes Aldo-keto reductase (AKR1) and mutated glycine oxidase (mGO) with different modes of action were co-expressed with modified EPSPS, which is insensitive to glyphosate in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.). The transgenic tobacco plants expressing individual PsAKR1, mGO, CP4-EPSPS, combinations of PsAKR1:CP4EPSPS, PsAKR1:mGO, and multigene with PsAKR1: mGO: CP4EPSPS genes were developed. The bio-efficacy studies of in-vitro leaf regeneration on different concentrations of glyphosate, seedling bioassay, and spray on transgenic tobacco plants demonstrate that glyphosate detoxification with enhanced resistance. Comparative analysis of the transgenic tobacco plants reveals that double and multigene expressing transgenics had reduced accumulation of shikimic acid, glyphosate, and its primary residue AMPA, and increased levels of sarcosine were observed in all PsAKR1 expressing transgenics. The multigene expressing rice transgenics showed improved glyphosate resistance with yield maintenance. In summary, results suggest that stacking genes with two different detoxification mechanisms and insensitive EPSPS is a potential approach for developing glyphosate-resistant plants with less residual content.

Multiple resistance mechanisms to penoxsulam in Echinochloa crus-galli from China.

Penoxsulam is an important herbicide for the control of Echinochloa crus-galli (L.) P. Beauv. Two resistant populations 17GA (R1) and 16NXB (R2) showed 17- and 3-fold resistance to penoxsulam, respectively. A known resistance mutation of Trp-574-Leu in ALS gene and enhanced rates of penoxsulam metabolism likely involving GST contribute to penoxsulam resistance in R1 population. This population had resistance to the ALS-inhibitors pyribenzoxim and bispyribac­sodium and the auxin herbicide quinclorac, but was susceptible to ACCase-inhibitors quizalofop-p-ethyl and cyhalofop-butyl. No known mutations in the ALS gene conferring target site resistance to ALS-inhibiting herbicides were presented in R2 population. However, penoxsulam metabolism in R2 plants was about 4-fold greater than in susceptible population 14YC (S0) plants. The enzyme inhibitors piperonyl butoxide, malathion and 4-chloro-7-nitrobenzoxadiazole reversed penoxsulam resistance in this population. GST and P450 enzyme activities and the genes of GST1-1, GST1-2, GST1-3, CYP81A18, CYP81A12, CYP81A21 were increased significantly in R2 population. These results indicate that multiple resistance mechanisms had occurred in E. crus-galli populations in central China and resistance needs to be managed effectively by diverse chemical and non-chemical methods.