Acetolactate Synthase Research Articles

Investigating non-target site resistance to pyroxsulam in a glyphosate-resistant Lolium rigidum population.

Resistance to multiple herbicides is common in Lolium rigidum. Here, resistance to acetolactate synthase (ALS)- and susceptibilityto acetyl-CoA carboxylase (ACCase)-inhibiting herbicides was confirmed in a glyphosate-resistant L. rigidum population (NLR70) from Australia and the mechanisms of pyroxsulam resistance were examined. No ALS target-site mutations nor gene overexpression were detected. Cytochrome P450 monooxygenase (P450) and glutathione S-transferase (GST) inhibitors (indicators of some certain P450s or GSTs) did not significantly affect the resistance to pyroxsulam. Nevertheless, HPLC analysis showed that plants of the NLR70 population metabolized pyroxsulam faster than plants of the herbicide-susceptible population (SVLR1). RNA sequencing analysis and RT-qPCR validation confirmed that four P450s (CYP709B2, CYP72A14, CYP89A2, CYP94B3), one GT (UGT79), and one ABC transporter (ABCG41) genes were constitutively upregulated in NLR70 plants. This study demonstrates that the glyphosate-resistant L. rigidum population (NLR70) also exhibits resistance to pyroxsulam and identifies six candidate genes associated with non-target site resistance to pyroxsulam. © 2025 Society of Chemical Industry.

In search of herbistasis: COT-metsulfuron methyl displays rare herbistatic properties.

Weed management is an essential intervention for maintaining food security and protecting biodiversity but is heavily reliant on chemical control measures (i.e., herbicides). Concerningly, only one herbicide has been developed with a new mode of action (MOA) since the 1980s. Therefore, alternative strategies for preventing weed growth need to be explored. The lesser-known concept of halting weed growth through herbistasis could be one strategy to alleviate the lack of success in obtaining new MOA leads, but this type of activity has rarely been investigated. Herein reported is a bioisosteric cyclooctatetraene (COT) for phenyl ring replacement tactic, using the commercial acetolactate synthase (ALS) inhibitor metsulfuron methyl, that has unearthed a rare agent displaying herbistatic properties against the weed, Cryptostegia grandiflora (rubber vine).

Pyroxsulam Resistance in Apera spica-venti: An Emerging Challenge in Crop Protection.

Apera spica-venti, a prevalent weed in Czech winter wheat fields, has developed resistance to ALS-inhibiting herbicides due to their frequent use. This study reports a biotype of A. spica-venti resistant to pyroxsulam, with cross and multiple resistance to iodosulfuron, propoxycarbazone, pinoxaden, and chlortoluron. Dose-response experiments revealed high resistance of both R1 and R2 biotypes to pyroxsulam, with resistance factors (RF) of 6.69 and 141.65, respectively. Pre-treatment with malathion reduced RF by 2.40× and 1.25× in R1 and R2, indicating the potential involvement of cytochrome P450 (CytP450). NBD-Cl pre-treatment decreased RF only in R2, suggesting possible GST involvement. Gene analysis revealed no mutations (at previously reported sites) or overexpression in the acetolactate synthase (ALS) gene. However, a significant difference in ALS enzyme activity between resistant and susceptible biotypes points to target-site resistance mechanisms. Studies with 14C-labeled pyroxsulam showed that reduced absorption and translocation were not likely resistance mechanisms. In summary, herbicide resistance in A. spica-venti appears to result from multiple mechanisms. Possible causes include target-site resistance from an unidentified ALS mutation (within coding or regulatory regions). Enhanced herbicide metabolism via CytP450s and GSTs is also a contributing factor. Further experimental validation is needed to confirm these mechanisms and fully understand the resistance. This evolution underscores the adaptive capacity of weed populations under herbicide pressure, emphasizing the need for alternative control strategies.

Effect of the growth-stimulating bacterium <i>Pseudomonas protegens</i> DA1.2 and its metabolites on damage to rapeseed by soil residues of metsulfuron-methyl

The biochemical processes mediating the positive effect of bacteria on plants experiencing herbicidal stress were investigated. For this purpose the effect of the Pseudomonas protegens DA1.2 bacterial strain, low molecular weight (5 kDa) and high molecular weight (5 kDa) fractions of its culture fluid (CF) on the activity of acetolactate synthase (ALS) and the antioxidant status of rapeseed (Brassica napus L.) of the Kupol variety grown under artificial lighting in methsulfuron-methyl contaminated soil was evaluated. Strain P. protegens DA1.2 and its metabolites contributed to an increase in the mass of rapeseed shoots by 21–68%, reduced the inhibition of the ALS enzyme by 11–24% and mitigated the manifestations of oxidative stress. The protective effect of the treatments decreased in a row: CF with living bacterial cells-low molecular weight fraction of CF-high molecular weight fraction of CF. An increase in the activity of superoxide dismutase by 51–94% and glutathione reductase by 17–20% in plants treated with bacteria or their metabolites indicated the possible participation of these antioxidant enzymes in reducing the phytotoxicity of metsulfuron-methyl soil residues for rapeseed plants.

Inhibitors of acetohydroxyacid synthase as promising agents against non-tuberculous mycobacterial diseases.

Acetohydroxyacid synthase (AHAS), exclusively present in microorganisms and plants, is a promising target for several herbicides due to its catalytic role in the branched-chain amino acid biosynthetic pathway. Previous studies have shown that K13787, a pyrazolopyrimidine sulfonamide AHAS inhibitor, was moderately effective against pulmonary infection caused by M. tuberculosis and nontuberculous mycobacteria (NTM). In this study, we synthesized various structural derivatives of K13787 based on the molecular docking studies and assessed their MICs against mycobacteria species. Among the synthetic compounds screened, K13787, along with KNT2077 and KNT2099, exhibited inhibitory efficacy against M. avium and M. abscessus, including CLR-resistant NTM species. Notably, these compounds displayed a synergistic effect (FIC ≤ 0.5) when combined with CLR against M. avium and M. abscessus. Our findings suggest that these newly identified AHAS-targeted compounds hold promise as lead candidates for novel antimycobacterial agents against NTM infections. Considering the structure-activity relationship, K13787, KNT2077, and KTN2099 emerge as potential treatments for NTM species.

Pro-197-Ser mutation combinations in acetolactate synthase (ALS) homoeologous genes affect ALS inhibitor herbicide resistance levels in Monochoria korsakowii.

Monochoria korsakowii is a common broadleaf weed found in rice (Oryza sativa) fields. Acetolactate synthase (ALS) inhibitor herbicides are commonly used to control broadleaf weeds in rice fields. However, prolonged herbicide use has exacerbated resistance issues. In this study, we evaluated the resistance to ALS inhibitors in populations where the same mutation occurred separately and simultaneously in the two ALS homoeologous genes (ALS1 and ALS2) and investigated the resistance mechanisms in M. korsakowii. Monochoria korsakowii exhibited high resistance to bensulfuron-methyl, low resistance to penoxsulam, and sensitivity to imazethapyr. Three resistant populations were identified: M-1 and M-2, which independently evolved the Pro-197-Ser mutation in ALS1 and ALS2, respectively, and M-3, which harbored this mutation in both ALS1 and ALS2. The sensitivity of ALS isolated from these populations to herbicide inhibition corresponded to the whole-plant resistance levels. Subsequently, we cloned and transformed Pro-197-Ser-mutated ALS1 and ALS2 into Arabidopsis thaliana. The resistance of homozygous A. thaliana to bensulfuron-methyl and penoxsulam aligned with bioassay trends. Furthermore, we measured the ploidy, relative expression, and copy number of ALS1 and ALS2, and found no significant differences, suggesting that the evolution of resistance was primarily attributed to the Pro-197-Ser mutation. Finally, we developed a derived cleaved amplified polymorphic sequence marker for detecting Pro-197-Ser mutation in ALS. The same mutation occurring separately in homoeologous genes resulted in similar resistance levels, whereas simultaneous mutations in homoeologous genes led to increased resistance levels. © 2024 Society of Chemical Industry.

The Influence of Plant Growth-Stimulating Bacteria on the Glutathione-S-Transferase Activity and the Toxic Effect of the Herbicide Metsulfuron-Methyl in Wheat and Canola Plants.

The ability of some rhizosphere bacteria to mitigate herbicidal stress in cultivated plants may be useful in agriculture and bioremediation. There is poor understanding of how bacteria directly or through herbicide degradation affect the biochemical processes in plants exposed to sulfonylurea herbicides. In this study, treatment with a combination of herbicide metsulfuron-methyl (MSM) and bacteria (Pseudomonas protegens DA1.2 or P. chlororaphis 4CH) of wheat (Triticum aestivum L.) and canola (Brassica napus L.) plants was carried out. Activity of glutathione-S-transferase (GST), an important enzyme for the herbicide detoxification, and acetolactate synthase (ALS), a target for MSM in plants, was measured by spectrophotometric assays. MSM residues were analyzed using the HPLC-MS. Then, 24 h after bacterial treatment, GST activity increased by 75-91% in wheat and by 38-94% in canola. On the 30th day, a decrease in MSM in the soil associated with bacterial treatment was 54.6-79.7%. An increase in GST activity and acceleration of MSM degradation were accompanied by a decrease in inhibition of the ALS enzyme in plants, which indicated a mitigation of the toxic effect. The results obtained are evidence that rhizospheric bacteria can have beneficial effects on plants exposed to MSM due to the combination of abilities to directly affect detoxification enzymes in plants and degrade MSM in the soil.

Mutations on the A and C genomes of Brassica napus confer herbicide resistance to acetohydroxyacid synthase inhibitors

ABSTRACT The point mutations conferring resistance to the sulfonylurea herbicide, chlorsulfuron, are reported for two Brassica napus lines (19c and 30a) developed by seed mutagenesis using ethyl methanesulfonate. Crosses with the herbicide-susceptible wild-type line generated F2 and backcrossed progeny and confirmed that chlorsulfuron resistance was inherited as a dominant mutation at a single locus in both lines. F2 progeny from crossing 19c and 30a segregated in a 15 resistant: 1 susceptible ratio, establishing that the two herbicide-resistant loci are independently inherited and that the mutations for resistance occur at different loci. PCR amplification and DNA sequencing of regions from the acetohydroxyacid synthase gene revealed two separate point mutations at different nucleotides in the same codon. This results in the substitution of proline-197 for leucine in 19c and proline-197 for serine in 30a. The different amino acid substitutions in the acetohydroxyacid synthase enzyme explain the differing responses of 19c and 30a to sulfonylurea and imidazolinone herbicides. Based on attempts at introgression of chlorsulfuron resistance to a wide range of forage brassicas, we conclude that the 19c mutation resides in the Brassica C genome, whereas the 30a mutation is in the Brassica A genome.

Effects of Pro197 resistance mutations occurring in different acetolactate synthase (ALS) isozymes on Descurainia sophia L. resistance to tribenuron-methyl

Effects of Pro197 resistance mutations occurring in different acetolactate synthase (ALS) isozymes on Descurainia sophia L. resistance to tribenuron-methyl

Design, Synthesis and Biological Evaluation of 4,6-Substituted 2-Styrylquinoline Derivatives: Potential Antimicrobial and Anticancer Agents

A series of novel 4,6-substituted 2-styrylquinoline derivatives (3a-h) was designed and synthesized based on the substitution in quinoline scaffolds (with Br, H, NO2 at C6 position) as well as the substitution in the styryl moeity (at C4' position). The target compounds were investigated for their antimicrobial activity against Gram-positive Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212) and Gram-negative bacterium Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC 10231) and anticancer activity against human colon adenocarcinoma HT29 cells. Compared to standard drug ciprofloxacin, compounds 3e, 3b and 3c showed significant antibacterial activity against Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Additionally, synthesized compound 3e had significant activity against Candida albicans with MIC value of 8 µg/mL. In anticancer study, the maximum cell death rate was found in compounds 3e, 3b and 3c at the dose of 100 µM i.e., 72.9%., 44.01% and 35.46%, respectively and the measured IC50 were 58.84, 114.53 and 130 µM. The cytotoxic potential of compound 3e was confirmed by AO-EtBr dual staining assay in which red fluorescence of necrotic cells lost their membrane integrity due to toxicity induced by compound 3e. This growth inhibition and apoptotic activity of compound 3e was dose-dependent when the colour changed from green to red. Furthermore, in silico studies were conducted on the synthesized compounds to elucidate the potential mechanisms of action against dihydrofolate reductase (DHFR) from E. coli, DNA gyrase from P. aeruginosa, DHFR from S. aureus, acetolactate synthase from K. pneumoniae and DHFR from C. albicans. The derivatives also exhibited the strongest ligand-receptor binding affinity. Additionally, ADME analysis was performed to predict the pharmacokinetic profiles of the compounds, indicating good oral drug-like properties.

Characterization of acetolactate synthase genes and resistance mechanisms of multiple herbicide resistant Lolium multiflorum

Combining imidazolinone-tolerant wheat with imazamox presents an effective solution to combat weed resistance. However, Lolium multiflorum, a troublesome resistant weed infesting wheat fields, may have developed resistance to imazamox, and the potential resistance mechanisms are intriguing. In this study, we explored the susceptibility of L. multiflorum to imazamox and investigated the resistance mechanisms, including the contribution of the target enzyme acetolactate synthase (ALS) to resistance and the presence of non-target-site resistance (NTSR). Eight L. multiflorum populations suspected of being resistant to imazamox were collected, and six populations exhibited resistance, ranging from 2.45-fold to 16.32-fold. The LmALS1 gene from susceptible population D3 plants and multiple copies of the LmALS gene (LmALS1, LmALS2, LmALS2α, LmALS3, LmALS3α, LmALS3β) from resistant populations D5 and D8 plants were separately amplified. Two mutations (Pro/Gln197 to Thr, Trp574 to Leu) were found in LmALS1 in the resistant populations. Compared to D3, LmALS1 was overexpressed in D5 but not in D8. The presence of LmALS1 mutants (LmALS1-Thr197 and LmALS1- Leu574), along with LmALS2, LmALS3, and their subunits, contribute to the resistance phenotype by increasing bonding energies, weakening hydrogen bonds, or decreasing protein binding pocket volumes and surface area. Additionally, D5 and D8 populations exhibited multiple resistance (>40-fold) to three other ALS inhibitors: pyroxsulam, flucarbazone-sodium, and mesosulfuron-methyl. Pre-treatment with malathion and 4-chloro-7-nitrobenzoxadiazole (cytochrome P450 monooxygenase and glutathione S-transferase inhibitors respectively) reversed the resistance of the D8 population and partially reversed the resistance of the D5 population to imazamox. This study characterizes ALS genes and extends our knowledge into the ALS resistance mechanisms involved in L. multiflorum. It also deepens our understanding of the complex diversification resistance mechanisms, thereby facilitating advances in weed resistance management strategies in wheat fields.

Herbicide resistance is complex: a global review of cross-resistance in weeds within herbicide groups

AbstractHerbicides have been placed in global Herbicide Resistance Action Committee (HRAC) herbicide groups based on their sites of action (e.g., acetolactate synthase–inhibiting herbicides are grouped in HRAC Group 2). A major driving force for this classification system is that growers have been encouraged to rotate or mix herbicides from different HRAC groups to delay the evolution of herbicide-resistant weeds, because in theory, all active ingredients within a herbicide group physiologically affect weeds similarly. Although herbicide resistance in weeds has been studied for decades, recent research on the biochemical and molecular basis for resistance has demonstrated that patterns of cross-resistance are usually quite complicated and much more complex than merely stating, for example, a certain weed population is Group 2-resistant. The objective of this review article is to highlight and describe the intricacies associated with the magnitude of herbicide resistance and cross-resistance patterns that have resulted from myriad target-site and non–target site resistance mechanisms in weeds, as well as environmental and application timing influences. Our hope is this review will provide opportunities for students, growers, agronomists, ag retailers, regulatory personnel, and research scientists to better understand and realize that herbicide resistance in weeds is far more complicated than previously considered when based solely on HRAC groups. Furthermore, a comprehensive understanding of cross-resistance patterns among weed species and populations may assist in managing herbicide-resistant biotypes in the short term by providing growers with previously unconsidered effective control options. This knowledge may also inform agrochemical company efforts aimed at developing new resistance-breaking chemistries and herbicide mixtures. However, in the long term, nonchemical management strategies, including cultural, mechanical, and biological weed management tactics, must also be implemented to prevent or delay increasingly problematic issues with weed resistance to current and future herbicides.

Trp-574-Leu and the novel Pro-197-His/Leu mutations contribute to penoxsulam resistance in Echinochloa crus-galli (L.) P. Beauv.

Recently, due to the widespread use of the acetolactate synthase (ALS)-inhibiting herbicide penoxsulam in paddy fields in China, Echinochloa crus-galli (L.) P. Beauv. has become a problematic grass weed that is frequently not controlled, posing a threat to weed management and rice yield. There are many reports on target-site mutations of ALS inhibiting herbicides; however, the detailed penoxsulam resistance mechanism in E. crus-galli remains to be determined. Greenhouse and laboratory studies were conducted to characterize target-site resistance mechanisms in JL-R, AH-R, and HLJ-R suspected resistant populations of E. crus-galli survived the field-recommended dose of penoxsulam. The whole-plant dose-response testing of E. crus-galli to penoxsulam confirmed the evolution of moderate-level resistance in two populations, JL-R (9.88-fold) and HLJ-R (8.66-fold), and a high-level resistance in AH-R (59.71-fold) population. ALS gene sequencing identified specific mutations in resistant populations, including Pro-197-His in ALS1 for JL-R, Trp-574-Leu in ALS1 for AH-R, and Pro-197-Leu in ALS2 for HLJ-R. In vitro ALS activity assays demonstrated a significantly higher activity in AH-R compared to the susceptible population (YN-S). Molecular docking studies revealed that Trp-574-Leu mutation primarily reduced the enzyme's ability to bind to the triazole-pyrimidine ring of penoxsulam due to decreased π-π stacking interactions, while Pro-197-His/Leu mutations impaired binding to the benzene ring by altering hydrogen bonds and hydrophobic interactions. Additionally, the Pro-197-His/Leu amino acid residue changes resulted in alterations in the shape of the active channel, impeding the efficient entry of penoxsulam into the binding site in the ALS protein. The three mutant ALS proteins expressed via the Bac-to-Bac baculovirus system exhibited notably lower activity inhibition rates than the non-mutant ALS proteins to penoxsulam, indicating all three ALS mutations reduce sensitivity to penoxsulam. This study elucidated the distinct impacts of the Pro-197-His/Leu and Trp-574-Leu mutations in E. crus-galli to penoxsulam resistance. Notably, the Trp-574-Leu mutation conferred stronger resistance to penoxsulam compared to the Pro-197-His/Leu mutations in E. crus-galli. The Pro-197-His/Leu mutations were first detected in E. crus-galli conferring penoxsulam resistance. These findings provide deeper insights into the molecular mechanisms underlying target-site resistance to penoxsulam in E. crus-galli.

Sensitivity of Southcentral U.S. Johnsongrass Accessions to Selected Herbicides

Abstract New technologies in grain sorghum allow for the use of multiple acetyl CoA carboxylase- (ACCase) or acetolactate synthase- (ALS) inhibiting herbicides for johnsongrass control. With the growing issue of herbicide resistance, producers need to understand which herbicides will successfully control johnsongrass accessions. To determine the efficacy of herbicides recently registered or ones with potential to become available for use in grain sorghum, johnsongrass seeds were collected from 2017 to 2021 in Arkansas, Kansas, Texas, and Oklahoma and were screened for sensitivity to fluazifop, quizalofop, nicosulfuron, and imazamox. Additionally, glyphosate sensitivity was evaluated because of its use before planting or postharvest. Quizalofop resulted in 100% mortality of all johnsongrass accessions. Of the johnsongrass accessions evaluated, 89% were completely controlled with glyphosate. The ALS inhibitors nicosulfuron and imazamox resulted in 100% mortality of all Oklahoma accessions, but failures occurred on samples from other states. One accession from Kansas, 12 from Texas, and eight from Arkansas were found to have reduced sensitivity to nicosulfuron and imazamox. If producers plan to plant grain sorghum in areas with johnsongrass populations, an ACCase-inhibitor herbicide will most likely provide effective control. Imazamox and nicosulfuron, in conjunction with the appropriate trait, can be utilized in areas with sensitive johnsongrass populations or where other sensitive grass species are present.

Emergence of multiple resistance to EPSPS and ALS herbicides in smooth pigweed (Amaranthus hybridus): a growing concern in Brazil

Abstract Recently, farmers in Brazil have observed a decline in efficacy of glyphosate, chlorimuron, and imazethapyr control of smooth pigweed (Amaranthus hybridus L.). The objectives of this study were to quantify the resistance of Amaranthus in Brazil to glyphosate and acetolactate synthase (ALS)-inhibiting herbicides, elucidate the mechanism of resistance, and assess the frequency of sensitivity shifts to glyphosate and chlorimuron in Brazil. Dose–response assays were conducted in a greenhouse with glyphosate, chlorimuron, and imazethapyr. This was followed by sequencing of the EPSPS and ALS genes. Additionally , 740 Amaranthus populations across several Brazilian states were monitored over 4 yr, subjected to a single discriminatory dose of glyphosate and chlorimuron. The populations BR18Asp051 and BR21Asp205 were resistant to glyphosate, chlorimuron, and imazethapyr. The elevated resistance level to glyphosate in these populations is attributed to multiple amino acid substitutions (TAP-IVS) in the EPSPS gene; and cross-resistance to sulfonylureas and imidazolinones is conferred by the Trp-574-Leu substitution in the ALS gene in both populations. Overall, resistance distribution indicated that 88% of the sampled populations were considered sensitive to glyphosate, while 66% were sensitive to chlorimuron. Furthermore, 10% of the samples demonstrated multiple resistance to both active ingredients. A shift in glyphosate sensitivity was observed in four states in Brazil; however, sensitivity shifts to chlorimuron were more widely dispersed in Brazilian agricultural regions.

Transcriptomics-guided rational engineering in Bacillus licheniformis for enhancing poly-γ-glutamic acid biosynthesis using untreated molasses

The utilization of non-food raw materials for microbial synthesis of poly-γ-glutamic acid (γ-PGA) presents a promising alternative to conventional food-based biosynthesis. However, the complex carbon source and composition of molasses, a prevalent non-food raw material, may impose constraints on its conversion and utilization by microorganisms. This study aimed to enhance the capacity of Bacillus licheniformis to convert untreated molasses into γ-PGA through transcriptomic analysis to guide metabolic modifications. Initial results from the transcriptomic analysis indicated that the strain utilizing molasses exhibited decreased expression of genes associated with substrate utilization (Module 1) and by-product synthesis (Module 2), while upregulating genes related to precursor synthesis (Module 3). Furthermore, we performed a knockout of the acetolactate synthase (AlsS) to reduce the synthesis of metabolic by-products and a knockout of the global regulator (CcpA) to alleviate carbon catabolite repression (CCR) and then promote substrate utilization. Ultimately, following the tandem overexpression of precursor supplying key genes, the titer of γ-PGA reached 48.26 g/L with a productivity of 1.15 g/L/h, using untreated molasses as the sole carbon source, which was 3.12-fold of the starting strain. These findings offer significant insights into the cost-effective synthesis of γ-PGA by bioconversion of untreated molasses during fermentation.

Effects of a Novel Tripyrasulfone Herbicide on Key Soil Enzyme Activities in Paddy Rice Soil.

Weeds significantly impact paddy yields, and herbicides offer a cost-effective, rapid, and efficient solution compared to manual weeding, ensuring agricultural productivity. Tripyrasulfone, a novel 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor developed by Qingdao Kingagroot Chemicals Co., Ltd., has demonstrated high efficacy when applied post-emergence, causing characteristic foliar bleaching in susceptible weed species, distinct from conventional acetolactate synthase, acetyl-CoA carboxylase, and synthetic auxin herbicides. This study investigates the impact of tripyrasulfone on the activity of key soil enzymes (urease (UE), acid phosphatase (ACP), sucrase (SC), catalase (CAT), and dehydrogenase (DHA)) in paddy soils from Jilin Province and Shandong Province. Different doses of tripyrasulfone (0.1, 1.0, and 2.5 mg kg-1) were applied, and the enzymatic activities were measured. Results indicated that tripyrasulfone initially inhibited UE and ACP activities before activating them. On the 20th day after treatment, UE activity had returned to control levels, whereas ACP activity remained significantly higher, showing long-lasting activation. SC and CAT activities were inhibited but gradually recovered to control levels. Furthermore, DHA activity was activated with a sustained effect, remaining significantly higher than the control group even 20 days after treatment. Overall, the impact of tripyrasulfone on soil enzyme activities diminished over time, suggesting that tripyrasulfone posed minimal long-term ecological risk to soil health.

Catalytic Insights of Acetolactate Synthases from Different Bacteria

Acetolactate synthase (ALS) is an essential enzyme involved in the biosynthesis of platform chemicals acetoin and 2,3-butanediol in several microorganisms. In this study, we investigated the catalytic differences among three bacterial ALSs involved in the ligation of two molecules of pyruvate or 2-ketobutyrate. Based on the findings, we predicted three amino acid residues in each enzyme that caused a discrepancy in accordance with the multi-sequence alignment and molecular docking experiments: I398, A402, and T480 in Bacillus subtilis ALS; V400, Y404, and S482 in Listeria seleigeri serovar 1/2b ALS; and M394, H398, and G476 in Klebsiella pneumoniae ALS. Subsequently, we mutually mutated the residues in the three ALSs. The data obtained confirmed our inference that these three residues in each enzyme are truly correlated with substrate recognition, particularly in recognizing compounds that are larger than pyruvate, such as 2-ketobutyrate, benzaldehyde, and nitrosobenzene. This study further clarifies the biochemical traits of ALSs derived from various bacteria and expands the scope of ALS research.

Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in rice

In molecular design breeding, the simultaneous introduction of desired functional genes through specific nucleotide modifications and the elimination of genes regulating undesired phenotypic traits or agronomic components require advanced gene editing tools. Due to limited editing efficiency, even with the use of highly precise editing tools, such as prime editing (PE), simultaneous editing of multiple mutation types poses a challenge. Here, we replaced Cas9 nickase (nCas9) with Cas9 to construct a Cas9-mediated PE (Cas9-PE) system in rice. This system not only enables precise editing, but also allows for site-specific random mutation. Moreover, leveraging the precision of Cas9-PE, we established a transgene-free multiplex gene editing system using a co-editing strategy. This strategy involved the Agrobacterium-mediated transient expression of the precise editing rice endogenous acetolactate synthase gene ALSS627I to confer herbicide bispyribac-sodium (BS) resistance as a selection marker. This study provides a versatile and efficient multiplex gene editing tool for molecular design breeding.

Field-evolved cross-resistance to ALS-inhibiting herbicides in redroot pigweed (Amaranthus retroflexus) populations and alternative chemical options for effective control

Abstract Twelve putative-resistant (R) redroot pigweed populations were collected in sunflower and soybean fields located in northeastern Greece, after repeated exposure to the acetolactate synthase (ALS)–inhibiting herbicides imazamox and tribenuron-methyl. Studies were conducted to determine the resistance status to these two ALS-inhibiting herbicides and evaluate alternative postemergence and preemergence herbicides for effective control. Two susceptible (S) populations were also included for comparison. Among the 12 putative-R populations studied in the whole-plant dose–response pot experiments, 11 were characterized as cross-resistant (R) to the imidazolinone imazamox and the sulfonylurea tribenuron-methyl. In contrast, the putative R5 and the two reference populations (S1, S2) populations were found to be susceptible. Sequencing of the ALS gene revealed that a point mutation (TGG to TTG at position 574) was selected in domain B, where in combination with domain A the majority of point mutations conferring resistance have been detected, resulting in an amino acid substitution from tryptophan (Trp) to leucine (Leu) in the 11 R populations. By contrast, all sequenced plants of the three susceptible populations were found with the wild-type allele encoding Trp574. The labeled rate of the postemergence herbicides tembotrione and dicamba provided fair to excellent control of the populations with ALS cross-resistance. In contrast, at this rate the preemergence herbicides S-metolachlor + terbuthylazine, isoxaflutole, aclonifen, metribuzin, and pendimethalin provided excellent control. These findings strongly suggest that 11 redroot pigweed populations have evolved cross-resistance to ALS-inhibiting herbicides, but viable options for chemical control of this weed still exist.