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Isoflavones belong to the class of flavonoid compounds, which are important secondary metabolites that play a crucial role in plant development and defense. Acetyl-CoA carboxylase (ACCase) is a biotin-dependent enzyme that catalyzes the conversion of Acetyl-CoA into Malonyl-CoA in plants. It is a key enzyme in fatty acid synthesis and also catalyzes the production of various secondary metabolites. However, information on the ACC gene family in the soybean (Glycine max L. Merr.) genome and the specific members involved in isoflavone biosynthesis is still lacking. In this study, we identified 20 ACC family genes (GmACCs) from the soybean genome and further characterized their evolutionary relationships and expression patterns. Phylogenetic analysis showed that the GmACCs could be divided into five groups, and the gene structures within the same groups were highly conserved, indicating that they had similar functions. The GmACCs were randomly distributed across 12 chromosomes, and collinearity analysis suggested that many GmACCs originated from tandem and segmental duplications, with these genes being under purifying selection. In addition, gene expression pattern analysis indicated that there was functional divergence among GmACCs in different tissues. The GmACCs reached their peak expression levels during the early or middle stages of seed development. Based on the transcriptome and isoflavone content data, a weighted gene co-expression network was constructed, and three candidate genes (Glyma.06G105900, Glyma.13G363500, and Glyma.13G057400) that may positively regulate isoflavone content were identified. These results provide valuable information for the further functional characterization and application of GmACCs in isoflavone biosynthesis in soybean. [ABSTRACT FROM AUTHOR]

Isoflavones belong to the class of flavonoid compounds, which are important secondary metabolites that play a crucial role in plant development and defense. Acetyl-CoA carboxylase (ACCase) is a biotin-dependent enzyme that catalyzes the conversion of Acetyl-CoA into Malonyl-CoA in plants. It is a key enzyme in fatty acid synthesis and also catalyzes the production of various secondary metabolites. However, information on the ACC gene family in the soybean (Glycine max L. Merr.) genome and the specific members involved in isoflavone biosynthesis is still lacking. In this study, we identified 20 ACC family genes (GmACCs) from the soybean genome and further characterized their evolutionary relationships and expression patterns. Phylogenetic analysis showed that the GmACCs could be divided into five groups, and the gene structures within the same groups were highly conserved, indicating that they had similar functions. The GmACCs were randomly distributed across 12 chromosomes, and collinearity analysis suggested that many GmACCs originated from tandem and segmental duplications, with these genes being under purifying selection. In addition, gene expression pattern analysis indicated that there was functional divergence among GmACCs in different tissues. The GmACCs reached their peak expression levels during the early or middle stages of seed development. Based on the transcriptome and isoflavone content data, a weighted gene co-expression network was constructed, and three candidate genes (Glyma.06G105900, Glyma.13G363500, and Glyma.13G057400) that may positively regulate isoflavone content were identified. These results provide valuable information for the further functional characterization and application of GmACCs in isoflavone biosynthesis in soybean.

Acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) is one of the essential biotins containing enzymes required by plants for fatty acid synthesis and elongation. The unique enzyme is present in its homomeric form in all the Gramineae family, making it a suitable target for developing herbicides selectively against weeds of the Gramineae family. One such example is infestation of Phalaris minor in winter wheat crop fields, where aryloxyphenoxypropionates (FOP); cyclohexanediones (DIM) and phenyl pyrazoline (DEN) group of ACCase inhibiting herbicides are used. However, the increasing number of ACCase herbicide resistant weed populations has compelled agro-scientists to seek varied possibilities for weed control, through Integrated Weed Management (IWM) strategies. Developing new potential herbicides to regain sensitivity in weeds could be an approach to weed control. The current advancement in computational techniques could be of aid in developing new herbicide-like molecules by exploring the genomics, proteomics and structural details of catalytic sites of herbicide action in crops as well as weeds.

Asia minor bluegrass (Polypogon fugax) is a common and problematic weed throughout China. P. fugax that is often controlled by acetyl-CoA carboxylase (ACCase) inhibitors in canola fields. Herein, we confirmed a P. fugax population (R) showing resistance to all ACCase inhibitors tested with resistance indexes ranging from 5.4–18.4. We further investigated the resistance mechanisms of this R population. Molecular analyses revealed that an amino acid mutation (Asp-2078-Gly) was present in the R population by comparing ACCase gene sequences of the sensitive population (S). In addition, differences in susceptibility between the R and S population were unlikely to be related to herbicide metabolism. Furthermore, a new derived cleaved amplified polymorphic sequence (dCAPS) method was developed for detecting the Asp-2078-Gly mutation in P. fugax efficiently. We found that 93.75% of plants in the R population carried the Asp-2078-Gly mutation, and all the herbicide-resistant phenotype of this R population is inseparable from this mutation. This is the first report of cross resistance to ACCase inhibitors conferred by the Asp-2078-Gly target-site mutation in P. fugax. The research suggested the urgent need to improve the diversity of weed management practices to prevent the widespread evolution of herbicide resistance in P. fugax in China. [ABSTRACT FROM AUTHOR]

Significance Statement: The limiting subunit of plant acetyl‐CoA carboxylase was identified as α‐carboxyltransferase via absolute protein quantification; overexpression of this subunit in Arabidopsis and Camelina led to an increase of both fatty acid synthesis rate and seed oil content. [ABSTRACT FROM AUTHOR]

BACKGROUND Metabolic resistance is a worldwide concern for weed control but has not yet been well‐characterized at the genetic level. Previously, we have identified an Asia minor bluegrass (Polypogon fugax Nees ex Steud.) population AHHY exhibiting cytochrome P450 (P450)‐involved metabolic resistance to fenoxaprop‐P‐ethyl. In this study, we aimed to confirm the metabolic fenoxaprop‐P‐ethyl resistance in AHHY and uncover the potential herbicide metabolism‐related genes in this economically damaging weed species. RESULTS: Liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays indicated the metabolic rates of fenoxaprop‐P‐ethyl were significantly faster in resistant (R, AHHY) than in susceptible (S, SDTS) plants. The amount of phytotoxic fenoxaprop‐P peaked at 12 h after herbicide treatment (HAT) and started to decrease at 24 HAT in both biotypes. R and S plants at 24 HAT were sampled to conduct isoform‐sequencing (Iso‐Seq) and RNA‐sequencing (RNA‐Seq). A reference transcriptome containing 24 972 full‐length isoforms was obtained, of which 24 329 unigenes were successfully annotated. Transcriptomic profiling identified 28 detoxifying enzyme genes constitutively and/or herbicide‐induced up‐regulated in R than in S plants. Real‐time quantitative polymerase chain reaction (RT‐qPCR) confirmed 17 genes were consistently up‐regulated in R and its F1 generation plants. They were selected as potential fenoxaprop‐P‐ethyl metabolism‐related genes, including ten P450s, one glutathione‐S‐transferase, one UDP‐glucosyltransferase, and five adenosine triphosphate (ATP)‐binding cassette transporters. CONCLUSION: This study revealed that the enhanced rates of fenoxaprop‐P‐ethyl metabolism in P. fugax were very likely driven by the herbicide metabolism‐related genes. The transcriptome data generated by Iso‐Seq combined with RNA‐Seq will provide abundant gene resources for understanding the molecular mechanisms of resistance in P. fugax. [ABSTRACT FROM AUTHOR]

Asia minor bluegrass (Polypogon fugax) is a common and problematic weed throughout China. P. fugax that is often controlled by acetyl-CoA carboxylase (ACCase) inhibitors in canola fields. Herein, we confirmed a P. fugax population (R) showing resistance to all ACCase inhibitors tested with resistance indexes ranging from 5.4–18.4. We further investigated the resistance mechanisms of this R population. Molecular analyses revealed that an amino acid mutation (Asp-2078-Gly) was present in the R population by comparing ACCase gene sequences of the sensitive population (S). In addition, differences in susceptibility between the R and S population were unlikely to be related to herbicide metabolism. Furthermore, a new derived cleaved amplified polymorphic sequence (dCAPS) method was developed for detecting the Asp-2078-Gly mutation in P. fugax efficiently. We found that 93.75% of plants in the R population carried the Asp-2078-Gly mutation, and all the herbicide-resistant phenotype of this R population is inseparable from this mutation. This is the first report of cross resistance to ACCase inhibitors conferred by the Asp-2078-Gly target-site mutation in P. fugax. The research suggested the urgent need to improve the diversity of weed management practices to prevent the widespread evolution of herbicide resistance in P. fugax in China.

To explore resistant mechanism of wild oat to fenoxaprop-p-ethyl, the susceptibility of Acetyl-CoA Carboxylase (ACCase) from 24 wild oat populations to fenoxaprop-p-ethyl, the level of gene expression and mutation site of ACCase were conducted. In vitro ACCase activities were solated and measured by enzyme-linked immunosorbent assay kit (ELISA) assays, the results indicated that the IC50 value of the ACCase of the most unsusceptible to fenoxaprop-p-ethyl in the wild oat population from Yexian2017 (\¥24) was 7206.557-fold compared to that of the ACCase of the most susceptible to fenoxaprop-p-ethyl in the wild oat population from Queshan (\Vl 1 ). The level of gene expression of ACCase was significantly higher in the resistant populations than in the relative susceptible populations, and was significantly more suppressed in the relative susceptible populations than in the resistant populations by quantitative Real-time PCR (qPCR). The mutations point of ACCase, Ile-1781-Leu, Trp-1999- Cys, Trp-2027-Cys, Ile-2041-Asn, Asp-2078-Gly, Cys-2088-Arg to have been published were not found in the populations tested by multiple sequence alignment with a model complete ACCase sequence of Alopecurus myosuroides. These findings suggest that ACCase plays a critical role in the development of wild oat resistant mechanism to fenoxaprop-pethyl. [ABSTRACT FROM AUTHOR]

BACKGROUND Asia minor bluegrass (Polypogon fugax Nees ex Steud.) is an invasive grass species severely infesting wheat and canola fields in China. In May 2017, a suspected resistant P. fugax population AHHY that survived fenoxaprop‐P‐ethyl applied at its field‐recommended rate was collected from a wheat field in Huaiyuan County, Anhui Province, China. This study aimed to determine the resistance profile of AHHY to acetyl‐CoA carboxylase (ACCase) inhibitors and to investigate its mechanisms of resistance to fenoxaprop. RESULTS: Single‐dose testing indicated that the AHHY population had evolved resistance to fenoxaprop. The partial carboxyltransferase domain of ACCase in P. fugax was amplified and compared. Four loci encoding plastidic ACCase were isolated from both the resistant and sensitive individuals. Combining gene sequencing with the derived cleaved amplified polymorphic sequence assay, we found that 100% of the plants of AHHY carried Trp‐1999‐Ser mutation in their ACCase1,1–2 allele. Whole‐plant dose–response bioassay indicated that AHHY was highly resistant to fenoxaprop and pinoxaden (resistance index (RI) ≥ 10) with low resistance to clodinafop‐propargyl, sethoxydim, and clethodim (2 ≤ RI < 5). Pre‐treatment with piperonyl butoxide largely reduced (55%) the weed's resistance to fenoxaprop. Both basal and fenoxaprop‐induced glutathione S‐transferases activities toward 1‐chloro‐2, 4‐dinitrobenzene were significantly higher in resistant plants than in susceptible plants. CONCLUSION: This study revealed that P. fugax had multiple alleles encoding plastidic ACCase, and reported for the first time the occurrence of Trp‐1999‐Ser mutation and non‐target‐site resistance in this species. Fenoxaprop resistance in AHHY plants was conferred by target‐site mutation and P450s‐involved enhanced metabolism. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Wild oat (Avena fatua L.) is a common and problematic weed in wheat fields in China. In recent years, farmers found it increasingly difficult to control A. fatua using acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. The purpose of this study was to identify the molecular basis of clodinafop-propargyl resistance in A. fatua. In comparison to the S1496 population, whole dose response studies revealed that the R1623 and R1625 populations were 71.71- and 67.76-fold resistant to clodinafop-propargyl, respectively. The two resistant A. fatua populations displayed high resistance to fenoxaprop-p-ethyl (APP) and low resistance to clethodim (CHD) and pinoxaden (PPZ), but they were still sensitive to the ALS inhibitors mesosulfuron-methyl and pyroxsulam. An Ile-2041-Asn mutation was identified in both resistant individual plants. The copy number and relative expression of the ACCase gene in the resistant population were not significantly different from those in the S1496 population. Under the application of 2160 g ai ha −1 of clodinafop-propargyl, the fresh weight of the R1623 population was reduced to 74.9%; however, pretreatment with the application of the cytochrome P450 inhibitor malathion and the GST inhibitor NBD-Cl reduced the fresh weight to 50.91% and 47.16%, respectively, which proved the presence of metabolic resistance. This is the first report of an Ile-2041-Asn mutation and probable metabolic resistance in A. fatua , resulting in resistance to clodinafop-propargyl. [Display omitted] • Avena fatua L. has evolved high resistance to clodinafop-propargyl in Xinjiang, China. • An Ile-2041-Asn mutation was first identified in resistant A. fatua populations in China. • TSR and NTSR mechanisms were coexisted in resistant A. fatua. [ABSTRACT FROM AUTHOR]

Acetyl-CoA carboxylase (ACCase) is the key regulator of fatty acid biosynthesis. In most plants, ACCase exists in two locations (cytosol and plastids) and in two forms (homomeric and heteromeric). Heteromeric ACCase comprises four subunits, three of them (ACCA–C) are nuclear encoded (nr) and the fourth (ACCD) is usually plastid encoded. Homomeric ACCase is encoded by a single nr-gene (ACC). We investigated the ACCase gene evolution in gymnosperms by examining the transcriptomes of newly sequenced Gnetum ula , combined with 75 transcriptomes and 110 plastomes of other gymnosperms. AccD- coding sequences are elongated through the insertion of repetitive DNA in four out of five cupressophyte families (except Sciadopityaceae) and were functionally transferred to the nucleus of gnetophytes and Sciadopitys. We discovered that, among the three genera of gnetophytes, only Gnetum has two copies of nr- accD. Furthermore, using protoplast transient expression assays, we experimentally verified that the nr- accD precursor proteins in Gnetum and Sciadopitys can be delivered to the plastids. Of the two nr- accD copies of Gnetum , one dually targets plastids and mitochondria, whereas the other potentially targets plastoglobuli. The distinct transit peptides, gene architectures, and flanking sequences between the two Gnetum accD s suggest that they have independent origins. Our findings are the first account of two distinctly targeted nr- accD s of any green plants and the most comprehensive analyses of ACCase evolution in gymnosperms to date. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Alopecurus japonicus is a serious grass weed species in wheat fields in eastern Asia, and has evolved strong resistance to acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicides. Although target‐site resistance (TSR) to ACCase inhibitors in A. japonicus has been reported, non‐target site resistance (NTSR) has not. This study investigated both TSR and NTSR in a fenoxaprop‐P‐ethyl‐resistant A. japonicus population (AHFD‐3), which was collected in Feidong County, Anhui Province, China. RESULTS: We found that AHFD‐3 exhibited high resistance to fenoxaprop‐P‐ethyl and low resistance to flucarbazone‐sodium. The sensitivity of AHFD‐3 to fenoxaprop‐P‐ethyl increased significantly after treatment with cytochrome P450 (P450) inhibitors; however, such synergies between P450 inhibitors and fenoxaprop‐P‐ethyl were not found in two control populations. Sequences of the entire carboxyltransferase domain of A. japonicus ACCase were obtained, and AHFD‐3 plants showed an Asp‐2078‐Gly substitution in the ACCase. With the derived cleaved amplified polymorphic sequence (dCAPS) method, we found that 85.4% of the plants of AHFD‐3 carried this mutation. The P450 content in AHFD‐3 plants was significantly higher than those of the two control populations after treatment with fenoxaprop‐P‐ethyl. Ten partial sequences of P450 genes in A. japonicus were cloned. Three P450 genes were up‐regulated 12 h after fenoxaprop‐P‐ethyl treatment, which were all from the P450 subfamily CYP72A. Moreover, a P450 gene from the P450 family CYP81 was up‐regulated after fenoxaprop‐P‐ethyl treatment in all populations studied. CONCLUSION: Fenoxaprop‐P‐ethyl resistance in AHFD‐3 plants was conferred by up‐regulation of cytochrome P450s in the CYP72A subfamily and target site mutation of the ACCase gene. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Most methods developed for detecting known single nucleotide polymorphisms (SNP) and deletion−insertion polymorphisms (DIP) are dependent on sequence conservation around the SNP/DIP and are therefore not suitable for application to heterogeneous organisms. Here we describe a novel, versatile and simple PCR-RFLP procedure baptised ‘derived Polymorphic Amplified Cleaved Sequence’ (dPACS) for genotyping individual samples. The notable advantage of the method is that it employs a pair of primers that cover the entire fragment to be amplified except for one or few diagnostic bases around the SNP/DIP being investigated. As such, it provides greater opportunities to introduce mismatches in one or both of the 35−55 bp primers for creating a restriction site that unambiguously differentiates wild from mutant sequences following PCR-RFLP and horizontal MetaPhorTM gel electrophoresis. Selection of effective restriction enzymes and primers is aided by the newly developed dPACS 1.0 software. The highly transferable dPACS procedure is exemplified here with the positive detection (in up to 24 grass and broadleaf species tested) of wild type proline106 of 5-enolpyruvylshikimate-3-phosphate synthase and its serine, threonine and alanine variants that confer resistance to glyphosate, and serine264 and isoleucine2041 which are key target-site determinants for weed sensitivities to some photosystem II and acetyl-CoA carboxylase inhibiting herbicides, respectively.

American sloughgrass ( Beckmannia syzigachne Steud.) is a weed widely distributed in wheat fields of China. In recent years, the evolution of herbicide (fenoxaprop- P -ethyl)-resistant populations has decreased the susceptibility of B. syzigachne . This study compared 4 B. syzigachne populations (3 resistant and 1 susceptible) using iTRAQ to characterize fenoxaprop- P -ethyl resistance in B. syzigachne at the proteomic level. Through searching the UniProt database, 3104 protein species were identified from 13,335 unique peptides. Approximately 2834 protein species were assigned to 23 functional classifications provided by the COG database. Among these, 2299 protein species were assigned to 125 predicted pathways. The resistant biotype contained 8 protein species that changed in abundance relative to the susceptible biotype; they were involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis pathways. In contrast to previous studies comparing only 1 resistant and 1 susceptible population, our use of 3 fenoxaprop-resistant B. syzigachne populations with different genetic backgrounds minimized irrelevant differential expression and eliminated false positives. Therefore, we could more confidently link the differentially expressed proteins to herbicide resistance. Proteomic analysis demonstrated that fenoxaprop- P -ethyl resistance is associated with photosynthetic capacity, a connection that might be related to the target-site mutations in resistant B. syzigachne . This is the first large-scale proteomics study examining herbicide stress responses in different B. syzigachne biotypes. Biological significance This study has biological relevance because it is the first to employ proteomic analysis for understanding the mechanisms underlying Beckmannia syzigachne herbicide resistance. The plant is a major weed in China and negatively affects crop yield, but has developed considerable resistance to the most common herbicide, fenoxaprop- P -ethyl. Through comparisons of resistant and sensitive biotypes, our study identified multiple proteins (involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis) that are putatively linked to B. syzigachne herbicide response. This large-scale proteomics study, sorely lacking in weed science, contributes valuable data that can be applied to more fine-tuned analyses on the functions of specific proteins in herbicide resistance. [ABSTRACT FROM AUTHOR]

Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose–response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes. [ABSTRACT FROM AUTHOR]

Asia minor bluegrass ( Polypogon fugax ) is a common and problematic weed throughout China. P. fugax that is often controlled by acetyl-CoA carboxylase (ACCase) inhibitors in canola fields. Herein, we confirmed a P. fugax population (R) showing resistance to all ACCase inhibitors tested with resistance indexes ranging from 5.4-18.4. We further investigated the resistance mechanisms of this R population. Molecular analyses revealed that an amino acid mutation (Asp-2078-Gly) was present in the R population by comparing ACCase gene sequences of the sensitive population (S). In addition, differences in susceptibility between the R and S population were unlikely to be related to herbicide metabolism. Furthermore, a new derived cleaved amplified polymorphic sequence (dCAPS) method was developed for detecting the Asp-2078-Gly mutation in P. fugax efficiently. We found that 93.75% of plants in the R population carried the Asp-2078-Gly mutation, and all the herbicide-resistant phenotype of this R population is inseparable from this mutation. This is the first report of cross resistance to ACCase inhibitors conferred by the Asp-2078-Gly target-site mutation in P. fug ax. The research suggested the urgent need to improve the diversity of weed management practices to prevent the widespread evolution of herbicide resistance in P. fug ax in China.

Most methods developed for detecting known single nucleotide polymorphisms (SNP) and deletion–insertion polymorphisms (DIP) are dependent on sequence conservation around the SNP/DIP and are therefore not suitable for application to heterogeneous organisms. Here we describe a novel, versatile and simple PCR-RFLP procedure baptised 'derived Polymorphic Amplified Cleaved Sequence' (dPACS) for genotyping individual samples. The notable advantage of the method is that it employs a pair of primers that cover the entire fragment to be amplified except for one or few diagnostic bases around the SNP/DIP being investigated. As such, it provides greater opportunities to introduce mismatches in one or both of the 35–55 bp primers for creating a restriction site that unambiguously differentiates wild from mutant sequences following PCR-RFLP and horizontal MetaPhorTM gel electrophoresis. Selection of effective restriction enzymes and primers is aided by the newly developed dPACS 1.0 software. The highly transferable dPACS procedure is exemplified here with the positive detection (in up to 24 grass and broadleaf species tested) of wild type proline106 of 5-enolpyruvylshikimate-3-phosphate synthase and its serine, threonine and alanine variants that confer resistance to glyphosate, and serine264 and isoleucine2041 which are key target-site determinants for weed sensitivities to some photosystem II and acetyl-CoA carboxylase inhibiting herbicides, respectively. [ABSTRACT FROM AUTHOR]