Differential Resistance to Acetyl-CoA Carboxylase Inhibitors in Rice: Insights from Two Distinct Target-Site Mutations.

Weeds present a significant challenge to agricultural productivity, and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides have proven to be effective in managing weed populations in rice fields. To develop ACCase-inhibiting herbicide-resistant rice, we generated mutants of rice ACCase (OsACC) featuring Ile-1792-Leu or Gly-2107-Ser substitutions through ethyl methyl sulfonate (EMS) mutagenesis. The Ile-1792-Leu mutant displayed cross-resistance to aryloxyphenoxypropionate (APP) and phenylpyrazoline (DEN) herbicides, whereas the Gly-2107-Ser mutants primarily exhibited cross-resistance to APP herbicides with diminished resistance to the DEN herbicide. In vitro assays of the OsACC activity revealed an increase in resistance to haloxyfop and quizalofop, ranging from 4.84- to 29-fold in the mutants compared to that in wild-type. Structural modeling revealed that both mutations likely reduce the binding affinity between OsACC and ACCase inhibitors, thereby imparting resistance. This study offers insights into two target-site mutations, contributing to the breeding of herbicide-resistant rice and presenting alternative weed management strategies in rice cultivation.

Discovery of Novel Pyridazine Herbicides Targeting Phytoene Desaturase with Scaffold Hopping.

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 µg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

Design, Synthesis, and Biological Evaluation of Pyridazinone-Containing Derivatives As Novel Protoporphyrinogen IX Oxidase Inhibitor.

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.

Design, Synthesis, and Herbicidal Evaluation of Pyrrolidinone-Containing 2-Phenylpyridine Derivatives as Novel Protoporphyrinogen Oxidase Inhibitors.

In this work, a series of pyrrolidinone-containing 2-phenylpyridine derivatives were synthesized and evaluated as novel protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors for herbicide development. At 150 g ai/ha, compounds 4d, 4f, and 4l can inhibit the grassy weeds of Echinochloa crus-galli (EC), Digitaria sanguinalis (DS), and Lolium perenne (LP) with a range of 60 to 90%. Remarkably, at 9.375 g ai/ha, these compounds showed 100% inhibition effects against broadleaf weeds of Amaranthus retroflexus (AR) and Abutilon theophrasti (AT), which were comparable to the performance of the commercial herbicides flumioxazin (FLU) and saflufenacil (SAF) and better than that of acifluorfen (ACI). Molecular docking analyses revealed significant hydrogen bonding and π-π stacking interactions between compounds 4d and 4l with Arg98, Asn67, and Phe392, respectively. Additionally, representative compounds were chosen for in vivo assessment of PPO inhibitory activity, with compounds 4d, 4f, and 4l demonstrating excellent inhibitory effects. Notably, compounds 4d and 4l induced the accumulation of reactive oxygen species (ROS) and a reduction in the chlorophyll (Chl) content. Consequently, compounds 4d, 4f, and 4l are promising lead candidates for the development of novel PPO herbicides.

Cyhalofop-butyl resistance conferred by a novel Trp-2027-Leu mutation of acetyl-CoA carboxylase and enhanced metabolism in Leptochloa chinensis.

BACKGROUND: Chinese sprangletop (Leptochloa chinensis (L.) Nees) is an invasive grass weed severely infesting rice fields across China. In October 2020, a suspected resistant Leptochloa chinensis population HFFD3 that survived the acetyl-CoA carboxylase (ACCase)-inhibiting herbicide cyhalofop-butyl applied at its field-recommended rate was collected from a rice field in Feidong County, Anhui Province, China. This study aimed to determine the resistance profile of HFFD3 to ACCase inhibitors and to investigate its mechanisms of resistance to cyhalofop-butyl. RESULTS: Single-dose testing confirmed that HFFD3 had evolved resistance to cyhalofop-butyl. Two loci encoding plastidic ACCase were each amplified from the susceptible (S) and resistant (R, HFFD3) individual plants. Target gene sequencing and derived cleaved amplified polymorphic sequence assay revealed all the R plants carried a Trp-2027-Leu substitution in their ACCase1,2 copies. Dose-response bioassays revealed that HFFD3 was highly resistant to cyhalofop-butyl and exhibited cross-resistance to metamifop, fenoxaprop-P-ethyl, quizalofop-P-ethyl, and clethodim. Pre-treatment with piperonyl butoxide and 4-chloro-7-nitrobenzoxadiazole considerably reversed the resistance of the R plants to cyhalofop-butyl, by 23% and 43%, respectively. Liquid chromatography-tandem mass spectrometry analysis suggested the metabolic rates of cyhalofop-butyl were significantly faster in the R than in the S plants. CONCLUSION: This study confirmed the first case of an arable weed species featuring cross-resistance to ACCase-inhibiting herbicides due to a novel Trp-2027-Leu mutation of ACCase. Target gene mutation and cytochrome P450s- and glutathione-S-transferases-involved enhanced metabolism may have simultaneously participated in the resistance of HFFD3 population to cyhalofop-butyl.

Resistance to post-emergent herbicides is becoming common for grass weeds on New Zealand wheat and barley farms.

To estimate the prevalence of herbicide-resistant weeds, 87 wheat and barley farms were randomly surveyed in the Canterbury region of New Zealand. Over 600 weed seed samples from up to 10 mother plants per taxon depending on abundance, were collected immediately prior to harvest (two fields per farm). Some samples provided by agronomists were tested on an ad-hoc basis. Over 40,000 seedlings were grown to the 2-4 leaf stage in glasshouse conditions and sprayed with high priority herbicides for grasses from the three modes-of-action acetyl-CoA carboxylase (ACCase)-inhibitors haloxyfop, fenoxaprop, clodinafop, pinoxaden, clethodim, acetolactate synthase (ALS)-inhibitors iodosulfuron, pyroxsulam, nicosulfuron, and the 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS)-inhibitor glyphosate. The highest manufacturer recommended label rates were applied for the products registered for use in New Zealand, often higher than the discriminatory rates used in studies elsewhere. Published studies of resistance were rare in New Zealand but we found weeds survived herbicide applications on 42 of the 87 (48%) randomly surveyed farms, while susceptible reference populations died. Resistance was found for ALS-inhibitors on 35 farms (40%) and to ACCase-inhibitors on 20 (23%) farms. The number of farms with resistant weeds (denominator is 87 farms) are reported for ACCase-inhibitors, ALS-inhibitors, and glyphosate respectively as: Avena fatua (9%, 1%, 0% of farms), Bromus catharticus (0%, 2%, 0%), Lolium spp. (17%, 28%, 0%), Phalaris minor (1%, 6%, 0%), and Vulpia bromoides (0%, not tested, 0%). Not all farms had the weeds present, five had no obvious weeds prior to harvest. This survey revealed New Zealand's first documented cases of resistance in P. minor (fenoxaprop, clodinafop, iodosulfuron) and B. catharticus (pyroxsulam). Twelve of the 87 randomly sampled farms (14%) had ALS-inhibitor chlorsulfuron-resistant sow thistles, mostly Sonchus asper but also S. oleraceus. Resistance was confirmed in industry-supplied samples of the grasses Digitaria sanguinalis (nicosulfuron, two maize farms), P. minor (iodosulfuron, one farm), and Lolium spp. (cases included glyphosate, haloxyfop, pinoxaden, iodosulfuron, and pyroxsulam, 9 farms). Industry also supplied Stellaria media samples that were resistant to chlorsulfuron and flumetsulam (ALS-inhibitors) sourced from clover and ryegrass fields from the North and South Island.

Phi class glutathione transferases as molecular targets towards multiple-herbicide resistance: Inhibition analysis and pharmacophore design.

Multiple-herbicide resistance (MHR) is a global threat to weed control in cereal crops. MHR weeds express a specific phi class glutathione transferase (MHR-GSTF) that confers resistance against multiple herbicides and therefore represents a promising target against MHR weeds. Kinetics inhibition analysis of MHR-GSTFs from grass weeds Lolium rigidum (LrGSTF) Alopecurus myosuroides (AmGSTF) and crops Hordeum vulgare (HvGSTF) and Triticum aestivum (TaGSTF) allowed the identification of the acetanilide herbicide butachlor as a potent and selective inhibitor towards MHR-GSTFs. Also, butachlor is a stronger inhibitor for LrGSTF and AmGSTF compared to HvGSTF and TaGSTF from crops. The crystal structure of LrGSTF was determined at 1.90 Å resolution in complex with the inhibitor S-(4-nitrobenzyl)glutathione. A specific 3D pharmacophore targeting the MHR-GSTFs was designed and used to identify structural elements important for potent and selective inhibition. Structural analysis of GSTFs revealed a decisive role of conserved Tyr118 in ligand binding and pharmacophore design. Its positioning is dependent on an outer patch of adjacent residues that span from position 132 to 134 which are similar for both LrGSTF and AmGSTF but different in HvGSTF and TaGSTF. The results presented here provide new knowledge that may be adopted to cope with MHR weeds.

Discovery of Novel Pyrazole-Quinazoline-2,4-dione Hybrids as 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) has been identified as one of the most significant targets in herbicide discovery for resistant weed control. In a continuing effort to discover potent novel HPPD inhibitors, we adopted a ring-expansion strategy to design a series of novel pyrazole-quinazoline-2,4-dione hybrids based on the previously discovered pyrazole-isoindoline-1,3-dione scaffold. One compound, 3-(2-chlorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-1,5-dimethylquinazoline-2,4(1H,3H)-dione (9bj), displayed excellent potency against AtHPPD, with an IC50 value of 84 nM, which is approximately 16-fold more potent than pyrasulfotole (IC50 = 1359 nM) and 2.7-fold more potent than mesotrione (IC50 = 226 nM). Furthermore, the co-crystal structure of the AtHPPD-9bj complex (PDB ID 6LGT) was determined at a resolution of 1.75 Å. Similar to the existing HPPD inhibitors, compound 9bj formed a bidentate chelating interaction with the metal ion and a π-π stacking interaction with Phe381 and Phe424. In contrast, o-chlorophenyl at the N3 position of quinazoline-2,4-dione with a double conformation was surrounded by hydrophobic residues (Met335, Leu368, Leu427, Phe424, Phe392, and Phe381). Remarkably, the greenhouse assay indicated that most compounds displayed excellent herbicidal activity (complete inhibition) against at least one of the tested weeds at the application rate of 150 g of active ingredient (ai)/ha. Most promisingly, compounds 9aj and 9bi not only exhibited prominent weed control effects with a broad spectrum but also showed very good crop safety to cotton, peanuts, and corn at the dose of 150 g of ai/ha.

Design, Synthesis, and Herbicidal Activity of Novel Diphenyl Ether Derivatives Containing Fast Degrading Tetrahydrophthalimide.

To seek new protoporphyrinogen oxidase (PPO) inhibitors with better biological activity, a series of novel diphenyl ether derivatives containing tetrahydrophthalimide were designed based on the principle of substructure splicing and bioisomerization. PPO inhibition experiments exhibited that 6c is the most potential compound, with the half-maximal inhibitory concentration (IC50) value of 0.00667 mg/L, showing 7 times higher activity than Oxyfluorfen (IC50 = 0.0426 mg/L) against maize PPO and similar herbicidal activities to Oxyfluorfen in weeding experiments in greenhouses and field weeding experiments. In view of the inspected bioactivities, the structure-activity relationship (SAR) of this series of compounds was also discussed. Crop selection experiments demonstrate that compound 6c is safe for soybeans, maize, rice, peanuts, and cotton at a dose of 300 g ai/ha. Accumulation analysis experiments showed that the accumulation of 6c in some crops (soybeans, peanuts, and cotton) was significantly lower than Oxyfluorfen. Current work suggests that compound 6c may be developed as a new herbicide candidate in fields.

Design and Synthesis of N-phenyl Phthalimides as Potent Protoporphyrinogen Oxidase Inhibitors.

Protoporphyrinogen oxidase (PPO) has been identified as one of the most promising targets for herbicide discovery. A series of novel phthalimide derivatives were designed by molecular docking studies targeting the crystal structure of mitochondrial PPO from tobacco (mtPPO, PDB: 1SEZ) by using Flumioxazin as a lead, after which the derivatives were synthesized and characterized, and their herbicidal activities were subsequently evaluated. The herbicidal bioassay results showed that compounds such as 3a (2-(4-bromo-2,6-difluorophenyl) isoindoline-1,3-dione), 3d (methyl 2-(4-chloro-1,3-dioxoisoindolin-2-yl)-5-fluorobenzoate), 3g (4-chloro-2-(5-methylisoxazol-3-yl) isoindoline-1,3-dione), 3j (4-chloro-2-(thiophen-2-ylmethyl) isoindoline-1,3-dione) and 3r (2-(4-bromo-2,6-difluorophenyl)-4-fluoroisoindoline-1,3-dione) had good herbicidal activities; among them, 3a showed excellent herbicidal efficacy against A. retroflexus and B. campestris via the small cup method and via pre-emergence and post-emergence spray treatments. The efficacy was comparable to that of the commercial herbicides Flumioxazin, Atrazine, and Chlortoluron. Further, the enzyme activity assay results suggest that the mode of action of compound 3a involves the inhibition of the PPO enzyme, and 3a showed better inhibitory activity against PPO than did Flumioxazin. These results indicate that our molecular design strategy contributes to the development of novel promising PPO inhibitors.

Discovery of Novel N-Isoxazolinylphenyltriazinones as Promising Protoporphyrinogen IX Oxidase Inhibitors.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is a promising target for herbicide discovery. Search for new compounds with novel chemotypes is a key objective for agrochemists. Here, we describe the discovery and systematic SAR-based structure optimization of novel N-isoxazolinylphenyltriazinones 5-9 as PPO inhibitors. The in vivo herbicidal activity and in vitro Nicotiana tabacum PPO (NtPPO) inhibitory activity were explored in detail. A number of the new synthetic compounds displayed strong PPO inhibitory activity with Ki values in the nanomolar range. Some compounds exhibited excellent and broad-spectrum weed control at the rate of 9.375-37.5 g ai/ha by postemergence application and showed improved monocotyledonous weed control compared to saflufenacil. Most promisingly, ethyl 3-(2-chloro-5-(3,5-dimethyl-2,6-dioxo-4-thioxo-1,3,5-triazinan-1-yl)-4-fluorophenyl)-5-methyl-4,5-dihydroisoxazole-5-carboxylate, 5a, with a Ki value of 4.9 nM, displayed over 2- and 6-fold higher potency than saflufenacil (Ki = 10 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 5a showed excellent and broad-spectrum weed control against 32 kinds of weeds at 37.5-75 g ai/ha. Rice exhibited relative tolerance to 5a at 150 g ai/ha by postemergence application, indicating that 5a could be a potential herbicide candidate for weed control in paddy fields.

Discovery of N-Aroyl Diketone/Triketone Derivatives as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibiting-Based Herbicides.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an important target site for discovering new bleaching herbicides. To explore novel HPPD inhibitors with excellent herbicidal activity, a series of novel N-aroyl diketone/triketone derivatives were rationally designed by splicing active groups and bioisosterism. Bioassays revealed that most of these derivatives displayed preferable herbicidal activity against Echinochloa crus-galli (EC) at 0.045 mmol/m2 and Abutilon juncea (AJ) at 0.090 mmol/m2. In particular, compound I-f was more potent compared to the commercialized compound mesotrione. Molecular docking indicated that the corresponding active molecules of target compounds and mesotrione shared similar interplay with surrounding residues, which led to a perfect interaction with the active site of Arabidopsis thaliana HPPD.

Design, Herbicidal Activity, and QSAR Analysis of Cycloalka[d]quinazoline-2,4-dione-Benzoxazinones as Protoporphyrinogen IX Oxidase Inhibitors.

In continuation of our search for potent protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors, we designed and synthesized a series of novel herbicidal cycloalka[d]quinazoline-2,4-dione-benzoxazinones. The bioassay results of these synthesized compounds indicated that most of the compounds exhibited very strong Nicotiana tabacum PPO (NtPPO) inhibition activity. More than half of the 37 synthesized compounds displayed over 80% control of all three tested broadleaf weeds at 37.5-150 g ai/ha by postemergent application, and a majority of them showed no phytotoxicity toward at least one kind of crop at 150 g ai/ha. Promisingly, 17i (Ki = 6.7 nM) was 6 and 4 times more potent than flumioxazin (Ki = 46 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 17i displayed excellent, broad-spectrum herbicidal activity, even at levels as low as 37.5 g ai/ha, and it was determined to be safe for wheat at 150 g ai/ha in postemergent application, indicating the great potential for 17i development as a herbicide for weed control in wheat fields.

Effects of drought-stress on seed germination and growth physiology of quinclorac-resistant Echinochloa crusgalli.

Echinochloa crusgalli (L.) Beauv. (barnyard grass) is considered a noxious weed worldwide, and is the most pernicious weed decreasing rice yields in China. Recently, E. crusgalli has evolved quinclorac resistance, making it among the most serious herbicide resistant weeds in China. The present study explored differences in germination and growth between quinclorac-resistant and -susceptible E. crusgalli collected in Hunan Province. The order of the seven E. crusgalli biotypes assessed, from high to low quinclorac-resistance, was: quinclorac-resistant, Chunhua, Hanshou, Shimen, Hekou, Dingcheng, and quinclorac-susceptible. With an increased in the level of quinclorac-resistance, the germination rate, length of young shoots and roots, and fresh weight of E. crusgalli were all decreased compared with that in more susceptible biotypes. However, there were no significant differences between quinclorac-resistant and susceptible E. crusgalli biotypes without polyethylene glycol 6000 treatment. Drought had a more obvious effect on glutathione S-transferases (GST) activity, determined by spectrophotometric method, in quinclorac-resistant E. crusgalli. Higher resistance level biotypes showed greater activity, and when treated with polyethylene glycol 6000 for 3 days, all E. crusgalli biotypes showed the highest GST activity. This study demonstrated that as the level of quinclorac-resistance increased, the rate of seed germination decreased, while the growth of young buds, young roots, and fresh weight decreased. Increased quinclorac-resistance may be related to the increased metabolic activity of GST in E. crusgalli.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

A novel genomic approach to herbicide and herbicide mode of action discovery.

A herbicide with a new mode of action has not been commercialized for more than 30 years. A recent paper describes a novel genomic approach to herbicide and herbicide mode of action discovery. Analysis of a microbial gene cluster revealed that it encodes genes for both the biosynthetic pathway for production of the sesquiterpene aspterric acid and an aspterric acid-resistant form of dihydroxy acid dehydratase (DHAD), its target enzyme. Aspterric acid is weak compared with commercial synthetic herbicides, and whether DHAD is a good herbicide target is unclear from this study. Nevertheless, this genomic approach provides a novel strategy for the discovery of herbicides with new modes action. © 2018 Society of Chemical Industry.

Acetolactate Synthase Overexpression in Mesosulfuron-Methyl-Resistant Shortawn Foxtail ( Alopecurus aequalis Sobol.): Reference Gene Selection and Herbicide Target Gene Expression Analysis.

Severe infestations of shortawn foxtail ( Alopecurus aequalis Sobol.), a noxious weed in wheat and canola cropping systems in China, remain standing even after the application of the herbicides, fenoxaprop- P-ethyl and mesosulfuron-methyl. Analysis of gene expression in weed plants subjected to herbicide treatment is a key step toward more mechanistic studies. Since such an analysis often involves quantitative real-time PCR (qRT-PCR), endogenous reference genes with stable expression are required. Herein, we identified specific gene sets, suitable as references for qRT-PCR data normalization in A. aequalis plants under different experimental conditions, using geNorm, NormFinder, BestKeeper, and RefFinder software. Additionally, the reliability of reference genes was verified by analyzing the expression of genes encoding two major herbicide target enzymes: acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS). Furthermore, functional ALS gene amplification was likely present in resistant plants, although it may make no contribution to the resistant phenotypes.

Structural basis for specific inhibition of the highly sensitive ShHTL7 receptor.

Striga hermonthica is a root parasitic plant that infests cereals, decimating yields, particularly in sub-Saharan Africa. For germination, Striga seeds require host-released strigolactones that are perceived by the family of HYPOSENSITIVE to LIGHT (ShHTL) receptors. Inhibiting seed germination would thus be a promising approach for combating Striga However, there are currently no strigolactone antagonists that specifically block ShHTLs and do not bind to DWARF14, the homologous strigolactone receptor of the host. Here, we show that the octyl phenol ethoxylate Triton X-100 inhibits S. hermonthica seed germination without affecting host plants. High-resolution X-ray structures reveal that Triton X-100 specifically plugs the catalytic pocket of ShHTL7. ShHTL7-specific inhibition by Triton X-100 demonstrates the dominant role of this particular ShHTL receptor for Striga germination. Our structural analysis provides a rationale for the broad specificity and high sensitivity of ShHTL7, and reveals that strigolactones trigger structural changes in ShHTL7 that are required for downstream signaling. Our findings identify Triton and the related 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]acetic acid as promising lead compounds for the rational design of efficient Striga-specific herbicides.

Design, Synthesis, and Herbicidal Activity of Pyrimidine-Biphenyl Hybrids as Novel Acetohydroxyacid Synthase Inhibitors.

The issue of weed resistance to acetohydroxyacid synthase (EC 2.2.1.6, AHAS) inhibitors has become one of the largest obstacles for the application of this class of herbicides. In a continuing effort to discover novel AHAS inhibitors to overcome weed resistance, a series of pyrimidine-biphenyl hybrids (4aa-bb and 5aa-ah) were designed and synthesized via a scaffold hopping strategy. Among these derivatives, compounds 4aa ( Ki = 0.09 µM) and 4bb ( Ki = 0.02 µM) displayed higher inhibitory activities against Arabidopsis thaliana AHAS than those of the controls bispyribac ( Ki = 0.54 µM) and flumetsulam ( Ki = 0.38 µM). Remarkably, compounds 4aa, 4bb, 5ah, and 5ag exhibited excellent postemergence herbicidal activity and a broad spectrum of weed control at application rates of 37.5-150 g of active ingredient (ai)/ha. Furthermore, 4aa and 4bb showed higher herbicidal activity against AHAS inhibitor-resistant Descurainia sophia, Ammannia arenaria, and the corresponding sensitive weeds than that of bispyribac at 0.94-0.235 g ai/ha. Therefore, the pyrimidine-biphenyl motif and lead compounds 4aa and 4bb have great potential for the discovery of novel AHAS inhibitors to combat AHAS-inhibiting herbicide-resistant weeds.

Metabolic Resistance to Acetolactate Synthase Inhibiting Herbicide Tribenuron-Methyl in Descurainia sophia L. Mediated by Cytochrome P450 Enzymes.

Descurainia sophia is one of the most notorious broadleaf weeds in China and has evolved extremely high resistance to acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl. The target-site resistance due to ALS gene mutations was well-known, while the non-target-site resistance is not yet well-characterized. Metabolic resistance, which is conferred by enhanced rates of herbicide metabolism, is the most important NTSR. To explore the mechanism of metabolic resistance underlying resistant (R) D. sophia plants, tribenuron-methyl uptake and metabolism levels, qPCR reference gene stability, and candidate P450 genes expression patterns were investigated. The results of liquid chromatography-mass spectrometry (LC-MS) analysis indicated that the metabolic rates of tribenuron-methyl in R plants was significantly faster than in susceptible (S) plants, and this metabolism differences can be eliminated by P450 inhibitor malathion. The genes for 18S rRNA and TIP41-like were identified as the most suitable reference genes using programs of BestKeeper, NormFinder, and geNorm. The P450 gene CYP96A146 constitutively overexpressed in R plants compared to S plants; this overexpression in R plants can be suppressed by malathion. Taken together, a higher expression level of P450 genes, leading to higher tribenuron-methyl metabolism, appears to be responsible for metabolic resistance to tribenuron-methyl in R D. sophia plants.