Syntenic analysis of ACCase loci and target-site-resistance mutations in cyhalofop-butyl resistant Echinochloa crus-galli var. crus-galli in Japan.

BACKGROUND: Recently, suspected cyhalofop-butyl-resistant populations of allohexaploid weed Echinochloa crus-galli var. crus-galli were discovered in rice fields in Aichi Prefecture, Japan. Analyzing the target-site ACCase genes of cyhalofop-butyl helps understand the resistance mechanism. However, in E. crus-galli, the presence of multiple ACCase genes and the lack of detailed gene investigations have complicated the analysis of target-site genes. Therefore, in this study, we characterized the herbicide response of E. crus-galli lines and thoroughly characterized the ACCase genes, including the evaluation of gene mutations in the ACCase genes of each line. RESULT: Four suspected resistant lines collected from Aichi Prefecture showed varying degrees of resistance to cyhalofop-butyl and other FOP-class ACCase inhibitors but were sensitive to herbicides with other modes of action. Through genomic analysis, six ACCase loci were identified in the E. crus-galli genome. We renamed each gene based on its syntenic relationship with other ACCase genes in the Poaceae species. RNA-sequencing analysis revealed that all ACCase genes, except the pseudogenized copy ACCase2A, were transcribed at a similar level in the shoots of E. crus-galli. Mutations known to confer resistance to FOP-class herbicides, that is W1999C, W2027C/S and I2041N, were found in all resistant lines in either ACCase1A, ACCase1B or ACCase2C. CONCLUSION: In this study, we found that the E. crus-galli lines were resistant exclusively to ACCase-inhibiting herbicides, with a target-site resistance mutation in the ACCase gene. Characterization of ACCase loci in E. crus-galli provides a basis for further research on ACCase herbicide resistance in Echinochloa spp. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Telomere-to-telomere genome assembly of an allotetraploid pernicious weed, Echinochloa phyllopogon.

Echinochloa phyllopogon is an allotetraploid pernicious weed species found in rice fields worldwide that often exhibit resistance to multiple herbicides. An accurate genome sequence is essential to comprehensively understand the genetic basis underlying the traits of this species. Here, the telomere-to-telomere genome sequence of E. phyllopogon was presented. Eighteen chromosome sequences spanning 1.0 Gb were constructed using the PacBio highly fidelity long technology. Of the 18 chromosomes, 12 sequences were entirely assembled into telomere-to-telomere and gap-free contigs, whereas the remaining six sequences were constructed at the chromosomal level with only eight gaps. The sequences were assigned to the A and B genome with total lengths of 453 and 520 Mb, respectively. Repetitive sequences occupied 42.93% of the A genome and 48.47% of the B genome, although 32,337, and 30,889 high-confidence genes were predicted in the A and B genomes, respectively. This suggested that genome extensions and gene disruptions caused by repeated sequence accumulation often occur in the B genome before polyploidization to establish a tetraploid genome. The highly accurate and comprehensive genome sequence could be a milestone in understanding the molecular mechanisms of the pernicious traits and in developing effective weed control strategies to avoid yield loss in rice production.

Transcriptionally linked simultaneous overexpression of P450 genes for broad-spectrum herbicide resistance.

Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (RT-qPCR)-based segregation screening identified the transcriptionally linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated diclofop acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the 3 herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the 3 genes. We propose that transcriptionally linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass.

Comparison of herbicide specificity of CYP81A cytochrome P450s from rice and a multiple-herbicide resistant weed, Echinochloa phyllopogon.

BACKGROUND: CYP81A cytochrome P450s (CYP81As) play a key role in herbicide detoxification in Poaceae plants. Crop CYP81As confer natural tolerance to multiple herbicides, whereas CYP81As in weeds disrupt herbicide action. Identifying differences in CYP81A herbicide specificity between crops and weeds could provide valuable information for controlling weeds. In this study, we quantitatively compared herbicide specificity between CYP81A6 from rice (Oryza sativa) and CYP81A12 and CYP81A21 from a weed, Echinochloa phyllopogon, using transgenic Escherichia coli and Arabidopsis. RESULTS: All three CYP81As metabolized the five tested herbicides and formed similar metabolites, with the highest relative activities of 400 to 580% toward bentazone compared to their activity on bensulfuron-methyl (defined as 100%). However, they showed differing activity toward propyrisulfuron. The relative activities of Echinochloa phyllopogon CYP81A12 (12.2%) and CYP81A21 (34.4%) toward propyrisulfuron were lower than that of rice CYP81A6 (98.5%). Additionally, rice CYP81A6 produced O-demethylated propyrisulfuron and hydroxylated products, whereas Echinochloa phyllopogon CYP81As produced only hydroxylated products. Arabidopsis expressing CYP81A12 and CYP81A21 exhibited lower levels of resistance against propyrisulfuron compared to that in Arabidopsis expressing CYP81A6. Homology modeling and in silico docking revealed that bensulfuron-methyl docked well into the active centers of all three CYP81As, whereas propyrisulfuron docked into rice CYP81A6 but not into Echinochloa phyllopogon CYP81As. CONCLUSION: The differing herbicide specificity displayed by rice CYP81A6 and Echinochloa phyllopogon CYP81A12 and CYP81A21 will help design inhibitors (synergists) of weed CYP81As, as well as develop novel herbicide ingredients that are selectively metabolized by crop CYP81As, to overcome the problem of herbicide resistance. © 2022 Society of Chemical Industry.

Gene expression shapes the patterns of parallel evolution of herbicide resistance in the agricultural weed Monochoria vaginalis.

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterization and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

Investigation of clomazone-tolerance mechanism in a long-grain cultivar of rice.

BACKGROUND: Clomazone is a potent herbicide for controlling weeds that have evolved resistance to other herbicides due to its unique mode of action. Clomazone is used in rice cultivation, but is limited to long-grain cultivars because other cultivars are highly sensitive to it. In this study, we investigated the mechanism of clomazone tolerance in a long-grain cultivar. RESULTS: The long-grain cultivar Kasalath tolerated approximately five-fold higher doses of clomazone compared to two short-grain cultivars, Nipponbare and Koshihikari. While Arabidopsis thaliana transformed with a rice cytochrome P450, CYP81A6, showed resistance to clomazone, the cyp81a6 knockout Kasalath was unchanged in its clomazone sensitivity. The inheritance of clomazone sensitivity in the F1 and F2 of Kasalath and Nipponbare indicated the involvement of multiple loci for clomazone tolerance. Four chromosome segment substitution lines of Nipponbare/Kasalath and Koshihikari/Kasalath exhibited partial tolerance to clomazone. The overlapping DNA region among the four lines is on chromosome 5 within 11.5 Mb. CONCLUSION: Multiple loci are involved in clomazone tolerance in Kasalath, one of which is located on chromosome 5. This information will help develop short-grain cultivars tolerant to clomazone. © 2021 Society of Chemical Industry.

Precision genome editing in plants via gene targeting and subsequent break-induced single-strand annealing.

Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase-type Cas9 fused with deaminase have been developed. However, the direction and position of practical substitution are still limited. In this context, homologous recombination (HR)-mediated gene targeting (GT) has advantages because any mutations existing on the donor DNA are copied and passed onto the endogenous DNA. As HR-mediated GT is extremely rare in higher plants, positive-negative selection has been used to isolate cells in which GT has occurred. After successful selection, positive selection marker is no longer needed and should ideally be eliminated. In a previous study, we reported a seamless piggyBac-transposon-mediated marker elimination system. Precision marker elimination efficiency in this system is very high. The piggyBac transposon integrates into the host genome at TTAA elements and excises without leaving a footprint at the excised site, so a TTAA sequence is necessary at the location of a positive selection marker. To compensate for this limitation, we have developed a novel marker elimination system using an I-SceI break and subsequent single-strand annealing (SSA)-mediated DNA repair system.

Cytochrome P450-mediated herbicide metabolism in plants: current understanding and prospects.

Cytochrome P450s (P450s) have been at the center of herbicide metabolism research as a result of their ability to endow selectivity in crops and resistance in weeds. In the last 20 years, ≈30 P450s from diverse plant species have been revealed to possess herbicide-metabolizing function, some of which were demonstrated to play a key role in plant herbicide sensitivity. Recent research even demonstrated that some P450s from crops and weeds metabolize numerous herbicides from various chemical backbones, which highlights the importance of P450s in the current agricultural systems. However, due to the enormous number of plant P450s and the complexity of their function, expression and regulation, it remains a challenge to fully explore the potential of P450-mediated herbicide metabolism in crop improvement and herbicide resistance mitigation. Differences in the substrate specificity of each herbicide-metabolizing P450 are now evident. Comparisons of the substrate specificity and protein structures of P450s will be beneficial for the discovery of selective herbicides and may lead to the development of crops with higher herbicide tolerance by transgenics or genome-editing technologies. Furthermore, the knowledge will help design sound management strategies for weed resistance including the prediction of cross-resistance patterns. Overcoming the ambiguity of P450 function in plant xenobiotic pathways will unlock the full potential of this enzyme family in advancing global agriculture and food security. © 2020 Society of Chemical Industry.

Characterization of the acetolactate synthase gene family in sensitive and resistant biotypes of two tetraploid Monochoria weeds, M. vaginalis and M. korsakowii.

Monochoria vaginalis and M. korsakowii are allotetraploid noxious weeds in rice cultivation. Occurrences of resistance to acetolactate synthase (ALS)-inhibiting herbicides have been reported in these weeds in Japan since the 1990s. The existence of multiple copies of ALS genes in both species has hindered and complicated the detailed study of molecular mechanisms in them. To determine the copy number and full-length of ALS genes in both species, we first amplified partial sequences of ALS genes and separated them by cloning. Five and three distinct sequences were identified in M. vaginalis and M. korsakowii, respectively. RACE and TAIL PCR successfully isolated full-length ALS genes, revealing that one copy of ALS genes in both species is a pseudogene formed by a frameshift mutation. Interestingly, one of the four putative functional ALS genes in M. vaginalis contains an intron in the 3'-untranslated region. Amplification and sequencing of the full-length ALS genes in sensitive and suspected resistant lines revealed a non-synonymous point mutation at codon Pro197, resulting in amino acid substitutions (Leu, Ser, or Ala) well known to endow ALS inhibitor resistance. Importantly, codon Pro197 of the M. korsakowii pseudogene encodes leucine (Leu) both in resistant and sensitive plants, which is also known to confer ALS inhibitor resistance when ALS genes are functional. Dose responses to imazosulfuron of the lines analyzed for ALS genes were in agreement with the existence of the mutations. These results suggest that some caution is needed when diagnosing molecular resistance in M. korsakowii. The information of copy number and full-length sequences will help diagnose ALS resistance and make a basis for the study of the evolution of ALS resistance in Monochoria spp.

Functional characterization of cytochrome P450 CYP81A subfamily to disclose the pattern of cross-resistance in Echinochloa phyllopogon.

KEY MESSAGE: CYP81A P450s armor Echinochloa phyllopogon against diverse and several herbicide chemistries. CYP81A substrate preferences can be a basis for cross-resistance prediction and management in E. phyllopogon and other related species. Metabolism-based herbicide resistance is a major threat to agriculture, as it is unpredictable and could extend resistance to different chemical groups and modes of action, encompassing existing, novel and to-be-discovered herbicides. Limited information on the enzymes involved in herbicide metabolism has hindered the prediction of cross-resistance in weeds. Members of CYP81A subfamily in multiple herbicide resistant (MHR) Echinochloa phyllopogon were previously identified for conferring cross-resistance to six unrelated herbicide classes. This suggests a critical role of CYP81As in endowing unpredictable cross-resistances in E. phyllopogon, thus the functions of all its nine putative functional CYP81A genes to 33 herbicides from 24 chemical groups were characterized. Ectopic expression in Arabidopsis thaliana identified the CYP81As that can confer resistance to multiple and diverse herbicides. The CYP81As were further characterized for their enzymatic functions in Escherichia coli. CYP81A expression in E. coli was optimized via modification of the N-terminus, co-expression with HemA gene and culture at optimal temperature. CYP81As metabolized its herbicide substrates into hydroxylated, N-/O-demethylated or both products. The cross-resistance pattern conferred by CYP81As is geared towards all chemical groups of acetolactate synthase inhibitors and is expanded to herbicides inhibiting photosystem II, phytoene desaturase, protoporphyrinogen oxidase, 4-hydroxyphenylpyruvate dioxygenase, and 1-deoxy-D-xylulose 5-phosphate synthase. Cross-resistance to herbicides pyrimisulfan, propyrisulfuron, and mesotrione was predicted and confirmed in MHR E. phyllopogon. This study demonstrated that the functional characterization of the key enzymes for herbicide metabolism could disclose the cross-resistance pattern and identify appropriate chemical options to manage the existing and unexpected cross-resistances in E. phyllopogon.

Quinclorac resistance in Echinochloa phyllopogon is associated with reduced ethylene synthesis rather than enhanced cyanide detoxification by ß-cyanoalanine synthase.

BACKGROUND: Multiple herbicide resistant Echinochloa phyllopogon exhibits resistance to the auxin herbicide quinclorac. Previous research observed enhanced activity of the cyanide-detoxifying enzyme ß-cyanoalanine synthase (ß-CAS) and reduced ethylene production in the resistant line, suggesting ß-CAS-mediated cyanide detoxification and insensitivity to quinclorac stimulation as the resistance mechanisms. To investigate the molecular mechanisms of quinclorac resistance, we characterized the ß-CAS genes alongside plant transformation studies. The association of ß-CAS activity and ethylene production to quinclorac resistance was assayed in the F6 progeny of susceptible and resistant lines of E. phyllopogon. RESULTS: A single nucleotide polymorphism in a ß-CAS1 intron deleted aberrantly spliced mRNAs and enhanced ß-CAS activity in the resistant line. The enhanced activity, however, was not associated with quinclorac resistance in F6 lines. The results were supported by lack of quinclorac resistance in Arabidopsis thaliana expressing E. phyllopogon ß-CAS1 and no difference in quinclorac sensitivity between ß-CAS knockout and wild-type rice. Reduced ethylene production co-segregated with quinclorac resistance in F6 lines which were previously characterized to be resistant to other herbicides by an enhanced metabolism. CONCLUSION: ß-CAS does not participate in quinclorac sensitivity in E. phyllopogon. Our results suggest that a mechanism(s) leading to reduced ethylene production is behind the resistance. © 2019 Society of Chemical Industry.

Role of CYP81A cytochrome P450s in clomazone metabolism in Echinochloa phyllopogon.

Clomazone is a herbicide used in the cultivation of numerous crops due to its unique site of action and effectiveness on weeds. The differences in clomazone susceptibility among plants have been attributed to the differences in their complex clomazone metabolic pathways that are not fully understood. We previously identified two CYP81A cytochrome P450 monooxygenases that metabolize five chemically unrelated herbicides in multiple-herbicide resistant Echinochloa phyllopogon. Since the resistant E. phyllopogon have decreased clomazone susceptibility, involvement of these P450s in clomazone resistance was suggested. In this study, we revealed that each P450 gene endowed Arabidopsis thaliana (Arabidopsis) with clomazone resistance. Consistent with this, clomazone resistance co-segregated with resistance to other herbicides in F6 progenies of crosses between susceptible and resistant E. phyllopogon, suggesting that the P450s are involved in differential clomazone susceptibility in E. phyllopogon. Arabidopsis transformations of the other seven CYP81As of E. phyllopogon found that two more genes, CYP81A15 and CYP81A24, decreased Arabidopsis susceptibility to clomazone. Differences in substrate preference between clomazone and a herbicide that inhibits acetolactate synthase were suggested among the four CYP81A P450s. This study provides insights into clomazone metabolism in plants.

CYP81A P450s are involved in concomitant cross-resistance to acetolactate synthase and acetyl-CoA carboxylase herbicides in Echinochloa phyllopogon.

Californian populations of Echinochloa phyllopogon have evolved multiple-herbicide resistance (MHR), posing a threat to rice production in California. Previously, we identified two CYP81A cytochrome P450 genes whose overexpression is associated with resistance to acetolactate synthase (ALS) inhibitors from two chemical groups. Resistance mechanisms to other herbicides remain unknown. We analyzed the sensitivity of an MHR line to acetyl-CoA carboxylase (ACCase) inhibitors from three chemical groups, followed by an analysis of herbicide metabolism and segregation of resistance of the progenies in sensitive (S) and MHR lines. ACCase herbicide metabolizing function was investigated in the two previously identified P450s. MHR plants exhibited resistance to all the ACCase inhibitors by enhanced herbicide metabolism. Resistance to the ACCase inhibitors segregated in a 3 : 1 ratio in the F2 generation and completely co-segregated with ALS inhibitor resistance in F6 lines. Expression of the respective P450 genes conferred resistance to the three herbicides in rice, which is in line with the detection of hydroxylated herbicide metabolites in vivo in transformed yeast. CYP81As are super P450s that metabolize multiple herbicides from five chemical classes, and concurrent overexpression of the P450s induces metabolism-based resistance to the three ACCase inhibitors in MHR E. phyllopogon, as it does to ALS inhibitors.

Copy Number Variation in Acetolactate Synthase Genes of Thifensulfuron-Methyl Resistant Alopecurus aequalis (Shortawn Foxtail) Accessions in Japan.

Severe infestations of Alopecurus aequalis (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of A. aequalis growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in ALS gene copy numbers among accessions were revealed. Two copies, ALS1 and ALS2, were conserved in all accessions, while some carried two additional copies, ALS3 and ALS4. A single-base deletion in ALS3 and ALS4 further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the ALS gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the ALS1 or ALS2 gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each ALS gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of ALS genes in A. aequalis will aid future analysis of the mechanism of resistance.

Multiple-herbicide resistance in Echinochloa crus-galli var. formosensis, an allohexaploid weed species, in dry-seeded rice.

Biotypes of Echinochloa crus-galli var. formosensis with resistance to cyhalofop-butyl, an acetyl-CoA carboxylase (ACCase) inhibitor, have been found in dry-seeded rice fields in Okayama, Japan. We collected two lines with suspected resistance (Ecf27 and Ecf108) from dry-seeded rice fields and investigated their sensitivity to cyhalofop-butyl and other herbicides. Both lines exhibited approximately 7-fold higher resistance to cyhalofop-butyl than a susceptible line. Ecf108 was susceptible to penoxsulam, an acetolactate synthase (ALS) inhibitor. On the other hand, Ecf27 showed resistance to penoxsulam and two other ALS inhibitors: propyrisulfuron and pyriminobac-methyl. The alternative herbicides butachlor, thiobencarb, and bispyribac-sodium effectively controlled both lines. To examine the molecular mechanisms of resistance, we amplified and sequenced the target-site encoding genes in Ecf27, Ecf108, and susceptible lines. Partial sequences of six ACCase genes and full-length sequences of three ALS genes were examined. One of the ACCase gene sequences encodes a truncated aberrant protein due to a frameshift mutation in both lines. Comparisons of the genes among Ecf27, Ecf108, and the susceptible lines revealed that none of the ACCases and ALSs in Ecf27 and Ecf108 have amino acid substitutions that are known to confer herbicide resistance, although a single amino acid substitution was found in each of three ACCases in Ecf108. Our study reveals the existence of a multiple-herbicide resistant biotype of E. crus-galli var. formosensis at Okayama, Japan that shows resistance to cyhalofop-butyl and several ALS inhibitors. We also found a biotype that is resistant only to cyhalofop-butyl among the tested herbicides. The resistance mechanisms are likely to be non-target-site based, at least in the multiple-herbicide resistant biotype.

Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon.

Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon.

A novel rice cytochrome P450 gene, CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis.

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.

Cytochrome P450 genes induced by bispyribac-sodium treatment in a multiple-herbicide-resistant biotype of Echinochloa phyllopogon.

BACKGROUND: Incremental herbicide metabolism by cytochrome P450 monooxygenases (P450s) has been proposed as the basis for resistance to bispyribac-sodium (bispyribac) in a multiple-herbicide-resistant biotype of Echinochloa phyllopogon. Upon exposure to bispyribac, strong induction of bispyribac-metabolising P450 activity has been reported in the resistant line, indicating that P450s induced by bispyribac are involved in the bispyribac resistance. RESULTS: A polymerase chain reaction (PCR)-based cloning strategy was used to isolate 39 putative P450 genes from the bispyribac-resistant line of E. phyllopogon. Expression analysis by real-time PCR revealed that seven of the isolated genes were upregulated in response to bispyribac treatment of seedlings at the three-leaf stage. The transcript levels and protein sequences of the seven genes were compared between the bispyribac-resistant line and a susceptible line. CYP71AK2 and CYP72A254 were transcribed prominently in the bispyribac-resistant line. Amino acid polymorphisms were found in three genes, including CYP72A254. CONCLUSION: Upregulated expression of these genes is consistent with the inducible herbicide-metabolising P450 activity under bispyribac stress that was reported in a previous study. This is the first study to compare P450 genes in arable weed species in order to elucidate the mechanism for P450-mediated herbicide resistance.

Isolation and expression of genes for acetolactate synthase and acetyl-CoA carboxylase in Echinochloa phyllopogon, a polyploid weed species.

BACKGROUND: Target-site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in arable weeds, whereas non-target-site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target-site resistance has not been reported, and non-target-site resistance is assumed to be the basis for resistance. To explore why target-site resistance had not occurred, the target-site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined. RESULTS: Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3. CONCLUSION: E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target-site resistance to ACCase inhibitors in E. phyllopogon.

Concentration-dependent dual effects of hydrogen peroxide on insulin signal transduction in H4IIEC hepatocytes.

BACKGROUND: Oxidative stress induced by the accumulation of reactive oxygen species (ROS) has a causal role in the development of insulin resistance, whereas ROS themselves function as intracellular second messengers that promote insulin signal transduction. ROS can act both positively and negatively on insulin signaling, but the molecular mechanisms controlling these dual actions of ROS are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we directly treated H4IIEC hepatocytes with hydrogen peroxide (H2O2), a representative membrane-permeable oxidant and the most abundant ROS in cells, to identify the key factors determining whether ROS impair or enhance intracellular insulin signaling. Treatment with high concentrations of H2O2 (25-50 µM) for 3 h reduced insulin-stimulated Akt phosphorylation, and increased the phosphorylation of both JNK and its substrate c-Jun. In contrast, lower concentrations of H2O2 (5-10 µM) enhanced insulin-stimulated phosphorylation of Akt. Moreover, lower concentrations suppressed PTP1B activity, suggesting that JNK and phosphatases such as PTP1B may play roles in determining the thresholds for the diametrical effects of H2O2 on cellular insulin signaling. Pretreatment with antioxidant N-acetyl-L-cysteine (10 mM) canceled the signal-promoting action of low H2O2 (5 µM), and it canceled out further impairment of insulin of insulin signaling induced by high H2O2 (25 µM). CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that depending on its concentration, H2O2 can have the positive or negative effect on insulin signal transduction in H4IIEC hepatocytes, suggesting that the concentration of intracellular ROS may be a major factor in determining whether ROS impair or enhance insulin signaling.