Isopimaric acid, an ion channel regulator, regulates calcium and oxidative phosphorylation pathways to inhibit breast cancer proliferation and metastasis.

Breast cancer is the globally most common malignant tumor and the biggest threat to women. Even though the diagnosis and treatment of breast cancer are progressing continually, a large number of breast cancer patients eventually develop a metastatic tumor, especially triple-negative breast cancer (TNBC). Recently, metal ion homeostasis and ion signaling pathway have become important targets for cancer therapy. In this study, We analyzed the effects and mechanisms of isopimaric acid (IPA), an ion channel regulator, on the proliferation and metastasis of breast cancer cells (4 T1, MDA-MB-231and MCF-7) by cell functional assay, flow cytometry, western blot, proteomics and other techniques in vitro and in vivo. Results found that IPA significantly inhibited the proliferation and metastasis of breast cancer cells (especially 4 T1). Further studies on the anti-tumor mechanism of IPA suggested that IPA might affect EMT and Wnt signaling pathways by targeting mitochondria oxidative phosphorylation and Ca2+ signaling pathways, and then inducing breast cancer cell cycle arrest and apoptosis. Our research reveals the therapeutic value of IPA in breast cancer and provides a theoretical basis for the new treatment of breast cancer.

LncRNA CYP4A22-AS1 promotes the progression of lung adenocarcinoma through the miR-205-5p/EREG and miR-34c-5p/BCL-2 axes.

OBJECTIVES: Lung adenocarcinoma (LUAD) exhibits a higher fatality rate among all cancer types worldwide, yet the precise mechanisms underlying its initiation and progression remain unknown. Mounting evidence suggests that long non-coding RNAs (lncRNAs) exert significant regulatory roles in cancer development and progression. Nevertheless, the precise involvement of lncRNA CYP4A22-AS1 in LUAD remains incompletely comprehended. METHODS: Bioinformatics analyses evaluated the expression level of CYP4A22-AS1 in lung adenocarcinoma and paracancer. The LUAD cell line with a high expression of CYP4A22-AS1 was constructed to evaluate the role of CYP4A22-AS1 in the proliferation and metastasis of LUAD by CCK8, scratch healing, transwell assays, and animal experiments. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. Luciferase reporter gene analyses, west-blotting, and qRT-PCR were carried out to reveal the interaction between CYP4A22-AS1, miR-205-5p/EREG, and miR-34c-5p/BCL-2 axes. RESULTS: CYP4A22-AS1 expression was significantly higher in LUAD tissues than in the adjacent tissues. Furthermore, we constructed a LUAD cell line with a high expression of CYP4A22-AS1 and noted that the high expression of CYP4A22-AS1 significantly enhanced the proliferation and metastasis of LUAD. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. CYP4A22-AS1 increased the expression of EREG and BCL-2 by reducing the expression of miR-205-5p and miR-34-5p and activating the downstream signaling pathway of EGFR and the anti-apoptotic signaling pathway of BCL-2, thereby triggering the proliferation and metastasis of LUAD. The transfection of miR-205-5p and miR-34-5p mimics inhibited the role of CYP4A22-AS1 in enhancing tumor progression. CONCLUSION: This study elucidates the molecular mechanism whereby CYP4A22-AS1 overexpression promotes LUAD progression through the miR-205-5p/EREG and miR-34c-5p/BCL-2 axes.

Up-regulation of bZIP88 transcription factor is involved in resistance to three different herbicides in both Echinochloa crus-galli and E. glabrescens.

The resistance of weeds to herbicides poses a major threat to agricultural production, and non-target-site resistance (NTSR) is often a serious problem as its mechanisms can in some cases confer resistance to herbicides with different modes of action. In this study, we hypothesized that bZIP transcription factors (TFs), which regulate abiotic stress responses in many plants, play a regulatory role in NTSR. Whole-plant assays indicated that the wild grasses Echinochloa crus-galli and E. glabrescens are resistant to the herbicides penoxsulam, cyhalofop-butyl, and quintrione. Transcriptome sequencing then identified 101 and 49 bZIP TFs with differential expression following penoxsulam treatment in E. crus-galli and E. glabrescens, respectively. Twelve of these genes had >60% homology with rice genes. The expression of bZIP88 was considerably up-regulated 6 h after treatment with the three different herbicides, and it was similar between resistant and susceptible populations; however, the relative expression levels before herbicide treatment and 24 h after were the same. We used rice (Oryza sativa ssp. japonica cv Nipponbare) as a model system for functional validation and found that CRISPR-Cas9-knockout of the rice bZIP88 ortholog increased the sensitivity to herbicide, whereas overexpression reduced it. The OsbZIP88 protein was localized to the nucleus. Using ChIP coupled with high-throughput sequencing, OsbZIP88 was found to form a network regulatory center with other TFs such as bZIP20/52/59 to regulate OsKS1, OsCOE1, and OsIM1, which are related to auxin, abscisic acid, brassinosteroids, and gibberellic acid. Based on these results, we have established a database of bZIP TFs corresponding to herbicide stress, and resolved the mechanisms of the positive regulation of herbicide resistance by bZIP88, thereby providing new insights for NTSR.

Mechanistic insights on cytotoxicity of KOLR, Cinnamomum pauciflorum Nees leaf derived active ingredient, by targeting signaling complexes of phosphodiesterase 3B and rap guanine nucleotide exchange factor 3.

Protein signaling complexes play important roles in prevention of several cancer types and can be used for development of targeted therapy. The roles of signaling complexes of phosphodiesterase 3B (PDE3B) and Rap guanine nucleotide exchange factor 3 (RAPGEF3), which are two important enzymes of cyclic adenosine monophosphate (cAMP) metabolism, in cancer have not been fully explored. In the current study, a natural product Kaempferol-3-O-(3'',4''-di-E-p-coumaroyl)-α-L-rhamnopyranoside designated as KOLR was extracted from Cinnamomum pauciflorum Nees leaves. KOLR exhibited higher cytotoxic effects against BxCP-3 pancreatic cancer cell line. In BxPC-3 cells, the KOLR could enhance the formation of RAPGEF 3/ PDE3B protein complex to inhibit the activation of Rap-1 and PI3K-AKT pathway, thereby promoting cell apoptosis and inhibiting cell metastasis. Mutation of RAPGEF3 G557A or low expression of PDE3B inactivated the binding action of KOLR resulting in KOLR resistance. The findings of this study show that PDE3B/RAPGEF3 complex is a potential therapeutic cancer target.

Mefenacet resistance in multiple herbicide-resistant Echinochloa crus-galli L. populations.

Echinochloa crus-galli L., a notorious weed in rice paddy fields, is usually kept under control by mefenacet application at the pre-emergence or early post-emergence stage. Due to continuous and repeated usage, E. crus-galli is developing resistance to mefenacet in China. Two putative resistant and one susceptible E. crus-galli populations were collected from paddy fields in Jiangsu Province to characterize their herbicide resistance. Compared with the susceptible population, the two mefenacet-resistant populations had 2.8- and 4.1-times greater pre-emergence resistance, and 10- and 6.8-times greater early post-emergence resistance to mefenacet. These mefenacet-resistant E. crus-galli populations also exhibited cross- or multiple-resistance to acetochlor, pyraclonil, imazamox, and quinclorac. However, when the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) was applied prior to post-emergence treatment, mefenacet resistance levels were reduced in both populations. Additionally, GST activity in vivo in one resistant population was much higher than the susceptible population after mefenacet application. The very long chain fatty acid elongases (VLCFAEs) from both mefenacet-resistant populations required much higher mefenacet concentration to inhibit their activity. The reduced sensitivity of VLCFAEs to mefenacet indicates the presence of a target-site resistance mechanism and induction of high GST activity may provide additional contribution to E. crus-galli mefenacet resistance through a non-target-site mechanism.

Comparison of quintrione and quinclorac on mechanism of action.

Quintrione is a new post-emergence herbicide developed for use in rice; however, the mechanism of action remains unclear. We determined the phytotoxicity of quintrione, and the contributions of hormone levels and lipid peroxidation to phytotoxicity, by comparing them to those induced by quinclorac. We also investigated 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity and carotenoid content following treatment with quintrione by comparing them to those induced by quinclorac and mesotrione. We found that quintrione and quinclorac both inhibited the growth of Echinochloa crusgalli var. zelayensis, but that quinclorac was a little more effective. At 24 h, quintrione and quinclorac significantly increased ethylene production and the contents of abscisic acid (ABA) and indole acetic acid (IAA) compared with the control. No significant differences were observed between quintrione and quinclorac on the three plant hormones. Quintrione and quinclorac also induced the formation of malondialdehyde (MDA), which is associated with lipid peroxidation, with no significant difference between them. Carotenoid content was reduced in E. crusgalli var. zelayensis following treatments with quintrione, quinclorac, and mesotrione. At 120 h, carotenoid contents were significantly higher following the quintrione and quinclorac treatments, in comparison with mesotrione treatment. There were no significant differences between quintrione and quinclorac in the inhibition of HPPD activity, and the effects of both were significantly less than the effect of mesotrione. In summary, E. crusgalli var. zelayensis was susceptible to both quintrione and quinclorac. The mechanism of action of quintrione, like that of quinclorac, was related to levels of plant hormones and lipid peroxidation; however, quintrione was a poor inhibitor of HPPD activity compared to mesotrione.

Two mechanisms provide tolerance to cyhalofop-butyl in pond lovegrass [Eragrostis japonica (Thunb.) Trin.].

Pond lovegrass [Eragrostis japonica (Thunb.) Trin.] is an annual grass weed of rice fields worldwide. Cyhalofop-butyl has been widely used for controlling annual grass weeds in rice fields. However, E. japonica is tolerant to cyhalofop-butyl. The effective dose values of cyhalofop-butyl for 29 E. japonica populations causing 50% inhibition of fresh weight (GR50: 130.15 to 187.61 g a.i. ha-1) were much higher than the recommended dose of cyhalofop-butyl (75 g a.i. ha-1) in the field. The mechanisms of tolerance to cyhalofop-butyl in E. japonica were identified. In vitro activity assays revealed that the cyhalofop-butyl concentration required to inhibit 50% of the acetyl-coenzyme A carboxylase (ACCase) activity (IC50) was 6.22-fold higher in E. japonica than that in the cyhalofop-butyl-susceptible Chinese sprangletop [Leptochloa chinensis (L.) Nees]. However, mutations in the ACCase gene, previously found to endow target-site resistance in weeds, were not detected in the sequences obtained. Additionally, the expression level of genes encoding ACCase in E. japonica was found to be as similar to L. chinensis. Tolerance was reduced by two cytochrome P450 monooxygenases (Cyt P450s) inhibitors (1-aminobenzotriazole and piperonyl butoxide) and the activity of NADPH-dependent cytochrome P450 reductase in E. japonica was approximately 4.46-fold higher than that of L. chinensis after cyhalofop-butyl treatment. Taken together, it is concluded that two co-existing mechanisms, an insensitive target ACCase and an enhanced metabolism mediated by Cyt P450s, endow tolerance to cyhalofop-butyl in E. japonica.

The phytotoxicity mechanism of florpyrauxifen-benzyl to Echinochloa crus-galli (L.) P. Beauv and weed control effect.

Weeds infest rice causing high yield losses, leading to the increasing use of herbicides for weed control. However, many weeds have evolved resistance to common commercial herbicides, including penoxsulam, metamifop and quinclorac. This study investigated the weed control effect and the phytotoxicity mechanism of florpyrauxifen-benzyl, a novel synthetic auxin herbicide registered for weed management in rice fields in China. The greenhouse study showed that florpyrauxifen-benzyl was highly efficient (GR50 < 6 and GR90 < 15 g a.i ha-1) at controlling 10 weed species commonly found in rice fields, including penoxsulam- and quinclorac- resistant(R) biotypes of Echinochloa Beauv. and bensulfuron-methyl-R biotype of Ammannia arenaria. The typical plant hormone content showed that following florpyrauxifen-benzyl treatment, indole-3-acetic acid (IAA) production changed only slightly at 12 h, while abscisic acid (ABA) production increased with time in the treated group, whose content was significantly higher than that of the control. Besides, ethylene biosynthesis was stimulated by florpyrauxifen-benzyl, ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) content, and 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO) activities, which evidently increased in the treated group, and ethylene peaked at 36 h. For the antioxidant enzyme activities and malondialdehyde (MDA) content in the treated group, results showed that MDA content continuously increased with time and was greater than that in the untreated group at 48 h and 72 h, superoxide dismutase (SOD) activity changed with exposure time and was significantly higher in the treatment group than the control at 48 h. A similar phenomenon was observed in peroxidase (POD) activity, which reached a peak at 48 h, and no distinct difference in catalase (CAT) activity was observed among groups except for the higher activity in the treated groups than control at 36 h and 48 h. Our results showed that that the stimulation ethylene biosynthesis and accumulation of ABA and reactive oxygen species (ROS) play important roles in the phytotoxicity mechanism of florpyrauxifen-benzyl in plants. Our findings demonstrate the potential of florpyrauxifen-benzyl to provide an alternative weed management strategy for rice fields.

A novel mutation Asp-2078-Glu in ACCase confers resistance to ACCase herbicides in barnyardgrass (Echinochloa crus-galli).

Multiple-herbicide resistance (MHR) in barnyardgrass (Echinochloa crus-galli) is a threat to rice production. The Ala-205-Val mutation in acetolactate synthase (ALS) conferred resistance to several ALS inhibitors in the E. crus-galli population AXXZ-2; consequently, ALS-inhibitors were unable to control this noxious weed species. In the present study, the sensitivity to acetyl-coenzyme A carboxylase (ACCase) herbicides and other herbicides having different modes of action was evaluated to determine an effective strategy for chemical weed control. Compared with that of the reportedly sensitive population JLGY-3, the AXXZ-2 population showed differential resistance to three ACCase-inhibitors (cyhalofop-butyl, fenoxaprop-P-ethyl, and pinoxaden), in addition to quinclorac and pretilachlor. A novel substitution (Asp-2078-Glu) in ACCase was detected as the main target-site resistance mechanisms in the AXXZ-2 population. Structural modeling of the mutant ACCase protein predicted that Asp-2078-Glu confers resistance to three ACCase inhibitors by reducing the binding affinity between them and the ACCase protein. To the best of our knowledge, this is the first study to report that the novel Asp-2078-Glu mutation confers resistance to several ACCase inhibitors. Target-site mutations in ALS and ACCase were detected in this MHR population. Except for quinclorac, pretilachlor, ALS inhibitors, and the three ACCase inhibitors, a number of herbicides remain effective in controlling this MHR E. crus-galli population.

Transcriptome profiling to identify cytochrome P450 genes involved in penoxsulam resistance in Echinochloa glabrescens.

Cytochrome P450s (P450s) confer resistance against herbicides, and this is increasingly becoming a concern for weed control. As a widespread Gramineae weed in paddy fields, Echinocloa glabrescens has become resistant to the acetolactate synthase (ALS)-inhibiting triazolopyrimidine herbicide penoxsulam. In this study, we found that the GR50 of the resistant population (SHQP-R) decreased substantially from 25.6 to 5.0 and 6.2 g a.i. ha-1 after treatment with the P450 inhibitors piperonyl butoxide (PBO) and malathion, respectively. However, P450 inhibitors almost had no effects on the susceptibility of the sensitive population (JYJD-S) to penoxsulam. To investigate the mechanisms of metabolic resistance, transcriptome sequencing analysis was performed to find candidate genes that may confer resistance to penoxsulam in E. glabrescens. A total of 233 P450 differentially expressed genes (DEGs) were identified by transcriptome sequencing. We found that the metabolic process and metabolic pathways were the most highly enriched in DEGs. Further, twenty-seven candidate P450 DEGs were selected for qPCR validation analyses. After penoxsulam treatment, the relative expression levels were significantly higher in SHQP-R than in JYJD-S. Among these, the relative expression of twenty-three P450 DEGs (eighteen from the CYP72A-71C-74A-96A-734A subfamily; five from CYP81E1-94C1-94B3-714C1-714C2) were upregulated and four P450 DEGs (from CYP724B1-711A1-707A7-97B2) were downregulated. Changes in the expression of these candidate P450 genes in E. glabrescens were in response to penoxsulam, which provides preliminary evidence for the role of P450s in herbicide metabolism in E. glabrescens. However, further functional studies on metabolic resistance to penoxsulam in a resistant E. glabrescens population are required.

Na+/K+ Balance and Transport Regulatory Mechanisms in Weedy and Cultivated Rice (Oryza sativa L.) Under Salt Stress.

BACKGROUND: Salinization is a primary abiotic stress constraining global plant growth and production. Weedy rice, though highly homologous to cultivated rice, is more salt tolerant during seed germination and seedling growth; we hypothesize that this is owing to ionic homeostasis and changes in the expression of genes encoding ion transport regulators. RESULTS: The four different genotypes of weedy (JYGY-1 and JYFN-4) and cultivated (Nipponbare and 9311) rice have different salt-tolerance during seed germination and seedling vegetative growth under salt stress. In this study, Na+ and Ca2+content increased in weedy and cultivated rice genotypes under salt stress while K+ and Mg2+decreased; however, JYGY-1 had the lowest Na+/K+ ratio of assessed genotypes. Genes in the high-affinity K+ transporter (HKT) and tonoplast sodium-hydrogen exchanger (NHX) families, and salt overly sensitive 1 (OsSOS1) have more than 98% homology in amino acid sequences between weedy and cultivated rice genotypes. Under salt stress, the HKT family members were differentially expressed in the roots and shoots of four different genotypes. However, the NHX family transcripts were markedly up-regulated in all genotypes, but there are significant differences between different genotypes. OsSOS1 was significantly up-regulated in roots, especially in JYGY-1genotype. CONCLUSIONS: The results showed that different genotypes had different germination and nutrient survival under salt stress, which was related to the difference of ion content and the difference of a series of ion transport gene expression. At the same time this study will provide new insight into the similarities and differences in ion homeostasis and gene regulatory mechanisms between weedy and cultivated rice under salt stress, which can aid in novel rice breeding and growth strategies.

Quinclorac resistance induced by the suppression of the expression of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase genes in Echinochloa crus-galli var. zelayensis.

We previously reported that the mechanism of quinclorac resistance in Echinochloa crus-galli var. zelayensis may be closely related to ethylene biosynthesis and the detoxification of cyanide. Differences in EcCAS gene sequences and expression levels may result in higher capacity to detoxify cyanide in resistant biotypes, which may avoid cyanide accumulation and avoid more ethylene and cyanide production and then avoid damage. In the present study, we focused on the mechanism of resistance related to ethylene biosynthesis in E. crus-galli var. zelayensis. The fresh weight of susceptible and moderately resistant biotypes were significantly reduced after treatment with quinclorac. However, AOA, an ethylene biosynthesis inhibitor, reduced the impact of quinclorac. On pretreatment with AOA, ethylene production was significantly reduced in the three biotypes. The highly resistant biotype produced less ethylene compared to the other two biotypes. Three ACS and seven ACO genes, which are the key genes in ethylene biosynthesis, were obtained. The expression levels of EcACS-like, EcACS7, and EcACO1 varied in the three biotypes upon treatment with quinclorac, which could be manipulated by AOA. In summary, it is inferred that the expression of EcACS-like, EcACS7, and EcACO1 can be stimulated to varying extent after quinclorac treatment in three E. crus-galli var. zelayensis biotypes, which consequently results in varying levels of ethylene production. Lower expression of these three genes results in more resistance to quinclorac, which may also be related to quinclorac resistance in E. crus-galli var. zelayensis.

Mechanism of resistance to cyhalofop-butyl in Chinese sprangletop (Leptochloa chinensis (L.) Nees).

Chinese sprangletop (Leptochloa chinensis (L.) Nees) is a serious grass weed in rice paddies. In some areas, L. chinensis has become resistant to the herbicide cyhalofop-butyl because of its frequent and extensive use over the past five years. In this study, whole-plant dose-response assays were conducted, and a L. chinensis population (ZHYH) had a 75.8-fold resistance index to cyhalofop-butyl. Molecular analyses revealed that this resistance was attributed to a tryptophan (Trp)-2027-to-cysteine (Cys) substitution in the CT domain of the ACCase gene. To our knowledge, this is the first report revealing the mechanism underlying cyhalofop-butyl resistance in L. chinensis. Furthermore, a derived cleaved amplified polymorphic (dCAPS) assay was developed to rapidly detect the Trp-2027-Cys mutation. Of the 100 ZHYH plants analyzed, 52 were heterozygous mutants and 48 were susceptible homozygous plants. In addition, the cyhalofop-butyl-resistant L. chinensis was cross-resistant to aryloxyphenoxypropionate and phenylpyrazoline herbicides, but not to cyclohexanedione, acetolactate synthase-inhibiting, protoporphyrinogen oxidase, and urea herbicides, and had only slight resistance to the hormonal herbicide quinclorac.

Resistance to quinclorac caused by the enhanced ability to detoxify cyanide and its molecular mechanism in Echinochloa crus-galli var. zelayensis.

Quinclorac, an auxin-type herbicide, is widely used to control barnyardgrass and some dicotyledon weeds. Echinochloa crus-galli var. zelayensis, a variety of E. crus-galli (L.) Beauv., is widespread in China and some populations have resistance to quinclorac. E. crus-galli var. zelayensis seeds with varying sensitivity to quinclorac were used in the present study. The expression of the ADP/ATP carrier protein (ANT) gene, which plays an important role in the maintenance of cellular energy balance, dramatically rose in the S biotype after exposure to quinclorac, while no change was found in two R biotypes. The activity of ß-cyanoalanine synthase (ß-CAS), which is the key enzyme for cyanide degradation, was higher in two R biotypes than in the S biotype before and after treatment with quinclorac. One single-nucleotide difference was detected in the EcCAS gene of two R biotypes compared with the S biotype. The nucleotide change, which caused one amino acid substitution, replacing Methionine (Met)-295 with Lysine (Lys)-295 in the two R biotypes, which are same as the rice ß-CAS gene at this position. In addition, EcCAS gene expression was higher in the two R biotypes than in the S biotype. In conclusion, ß-CAS may play a crucial role in the resistance of E. crus-galli var. zelayensis to quinclorac. EcCAS gene mutation and higher gene expression may enhance the activity of ß-CAS to avoid the accumulation of toxic cyanide in resistant populations, thus contributing to the resistance mechanism of E. crus-galli var. zelayensis. to quinclorac.

Establishing a herbicide-metabolizing enzyme library in Beckmannia syzigachne to identify genes associated with metabolic resistance.

Non-target site resistance (NTSR) to herbicides is an increasing concern for weed control. Metabolic herbicide resistance is an important mechanism for NTSR. However, little is known about metabolic resistance at the genetic level. In this study, we have identified three fenoxaprop-P-ethyl-resistant American sloughgrass (Beckmannia syzigachne Steud.) populations, in which the molecular basis for NTSR remains unclear. To reveal the mechanisms of metabolic resistance, the genes likely to be involved in herbicide metabolism (e.g. for cytochrome P450s, esterases, hydrolases, oxidases, peroxidases, glutathione S-transferases, glycosyltransferases, and transporter proteins) were isolated using transcriptome sequencing, in combination with RT-PCR (reverse transcription-PCR) and RACE (rapid amplification of cDNA ends). Consequently, we established a herbicide-metabolizing enzyme library containing at least 332 genes, and each of these genes was cloned and the sequence and the expression level compared between the fenoxaprop-P-ethyl-resistant and susceptible populations. Fifteen metabolic enzyme genes were found to be possibly involved in fenoxaprop-P-ethyl resistance. In addition, we found five metabolizing enzyme genes that have a different gene sequence in plants of susceptible versus resistant B. syzigachne populations. These genes may be major candidates for herbicide metabolic resistance. This established metabolic enzyme library represents an important step forward towards a better understanding of herbicide metabolism and metabolic resistance in this and possibly other closely related weed species. This new information may help to understand weed metabolic resistance and to develop novel strategies of weed management.

An effective method, composed of LAMP and dCAPS, to detect different mutations in fenoxaprop-P-ethyl-resistant American sloughgrass (Beckmannia syzigachne Steud.) populations.

The decreased susceptibility of Beckmannia syzigachne to fenoxaprop-P-ethyl is due to the reliance on it to control grass weeds since the 1990s. Loop-mediated isothermal amplification (LAMP), which is a proven simple, rapid, specific, sensitive and inexpensive assay method, has been used to detect the I1781L mutation in B. syzigachne. In the present study, four sets of primers detected four mutations in B. syzigachne, W2027C, I2041A, D2078G and G2096A, using the LAMP method. Additionally, five newly derived cleaved amplified polymorphic sequence (dCAPS) markers were developed to detect five different mutations. With a method composed of LAMP and dCAPS, 19 fenoxaprop-P-ethyl-resistant B. syzigachne populations collected in 2013 were studied. An effective method, composed of LAMP and dCAPS, to detect five mutations, I1781L, W2027C, I2041A, D2078G and G2096A, in B. syzigachne populations was developed. With this method, a B. syzigachne population resistant to fenoxaprop-P-ethyl can be studied to confirm its constitution. And we determined that the resistance level might be relevant to the mutation type and mutation frequency. The type of mutation and its frequency in fenoxaprop-P-ethyl-resistant B. syzigachne populations can be confirmed to provide appropriate herbicide management.

Molecular basis of ALS- and/or ACCase-inhibitor resistance in shortawn foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis, a predominant weed species in wheat and oilseed rape fields, can no longer be controlled by mesosulfuron-methyl application after continuous use over several years. Based on dose-response studies, the putative resistant populations, JTJY-1 and JHHZ-1, were found to be resistant to mesosulfuron-methyl, with resistance index values of 5.5 and 14, respectively. Sensitivity assays of the mesosulfuron-methyl-resistant populations to other herbicides revealed that the JTJY-1 population had moderate or high cross resistance to sulfonylureas (SUs) and triazolopyrimidines (TPs), but displayed a low level resistance to imidazolinones (IMIs). JTJY-1 also had high multi-resistance to ACCase inhibitors, but remained susceptible to photosystem II inhibitors. The JHHZ-1 population was resistant to all ALS inhibitors tested, but was sensitive to ACCase inhibitors and photosystem II inhibitors. To clarify the molecular basis of resistance in JTJY-1 and JHHZ-1 population, the ALS and ACCase gene were sequenced. Two ALS mutations (Pro-197-Thr or Trp-574-Leu) were detected in the mesosulfuron-methyl-resistant plants. The ACCase gene analysis revealed that the resistant JTJY-1 population had an Ile-1781-Leu mutation. Furthermore, the presence of two different target site resistance (TSR) mechanisms (ALS and ACCase mutations) existing simultaneously in individual A. aequalis was firstly documented in the presented study.

Epigenetic Regulation of CYP72A385-Mediated Metabolic Resistance to Novel Auxin Herbicide Florpyrauxifen-benzyl in Echinochloa crus-galli (L.) P. Beauv.

Weed's metabolic resistance to herbicides has undermined the sustainability of herbicides and global food security. Notably, we identified an Echinochloa crus-galli (L.) P. Beauv population (R) that evolved resistance to the never-used florpyrauxifen-benzyl, in which florpyrauxifen-benzyl was metabolized faster than the susceptible E. crus-galli population (S). RNA-seq identified potential metabolism-related genes, EcCYP72A385 and EcCYP85A1, whose expression in yeast exhibited the capacity to degrade florpyrauxifen-benzyl. Region-2 in the EcCYP72A385 promoter showed significant demethylation after florpyrauxifen-benzyl treatment in the R population. DNA methyltransferase inhibitors induce EcCYP72A385 overexpression in the S population and endow it with tolerance to florpyrauxifen-benzyl. Moreover, methyltransferase-like 7A (EcMETTL7A) was overexpressed in the S population and specifically bound to the EcCYP72A385 promoter. Transgenic EcCYP72A385 in Arabidopsis and Oryza sativa L. exhibited resistance to florpyrauxifen-benzyl, whereas EcMETTL7A transgenic plants were sensitive. Overall, EcCYP72A385 is the principal functional gene for conferring resistance to florpyrauxifen-benzyl and is regulated by EcMETTL7A in E. crus-galli.

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.).

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b5 in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.

Mechanism of resistance to fenoxaprop in Japanese foxtail (Alopecurus japonicus) from China.

Japanese foxtail is one of the most common and troublesome weeds infesting cereal and oilseed rape fields in China. Repeated use during the last three decades of the ACCase-inhibiting herbicide fenoxaprop-P-ethyl to control this weed has resulted in the occurrence of resistance. Dose-response tests established that a population (AHFD-1) from eastern China had evolved high-level resistance to fenoxaprop-P-ethyl. Based on the resistance index, this resistant population of A. japonicus is 60.31-fold resistant to fenoxaprop-P-ethyl. Subsequently, only a tryptophan to cysteine substitution was identified to confer resistance to fenoxaprop-P-ethyl in this resistant population. ACCase activity tests further confirmed this substitution was linked to resistance. This is the first report of the occurrence of Trp-2027-Cys substitution of ACCase in A. japonicus. From whole-plant pot dose-response tests, we confirmed that this population conferred resistance to other APP herbicides, including clodinafop-propargyl, fluazifop-P-butyl, quizalofop-P-ethyl, haloxyfop-R-methyl, cyhalofop-butyl, metamifop, DEN herbicide pinoxaden, but not to CHD herbicides clethodim, sethoxydim. There was also no resistance observed to ALS-inhibiting herbicides sulfosulfuron, mesosulfuron-methyl, flucarbazone-sodium, pyroxsulam, Triazine herbicide prometryne and glyphosate. However, this resistant population was likely to confer slightly (or no) resistant to Urea herbicides chlortoluron and isoproturon.