Biology subjects

Iwakami, S.

Publications and source records attributed to Iwakami, S..

7 recordsLinked to original sources

bioRxiv2025

Insights into the molecular mechanism and management of resistant Echinochloa crus-galli biotypes from the Philippines to acetolactate synthase inhibitor bispyribac-sodium

Rice production in the Philippines is heavily reliant on herbicide application for weed control. Acetolactate synthase (ALS) inhibitors are the mostly used herbicides for combating major weeds associated to rice, such as Echinochloa crus-galli. In separate locations from the Philippines, rice farmers expressed concerns that the application of ALS inhibitor bispyribac-sodium has been showing poor control of E. crus-galli. Here, we confirmed and quantified the level of bispyribac-sodium resistance in the putative resistant biotypes. Resistance was confirmed in R4 and R12 biotypes collected from the northern and central parts of the Philippines, respectively. The estimated GR50 (dose resulting in 50% growth reduction) revealed a 36.7-and 60.3-fold resistance in R4 and R12 biotypes (106.8 g a.i. ha-1 and 175.4 g a.i. ha-1) compared to the S biotype (2.91 g a.i. ha-1). The R4 biotype carries a single nucleotide mutation in the ALS2 gene translating to Trp-574-Leu amino acid substitution, while there was no mutation detected in any of the ALS conserved regions of R12. Pre-treatment of malathion increased the sensitivity of R12 to bispyribac-sodium, suggesting the involvement of cytochrome P450s. Sensitivity assays to alternative post-emergence herbicides showed that both R4 and R12 can be effectively controlled by metamifop [acetyl-CoA carboxylase (ACCase) inhibitor]. However, R12 survived the application of fenoxaprop-P-ethyl + ethoxysulfuron (ACCase inhibitor + ALS inhibitor). This study unravels the molecular mechanism of the first resistance case to ALS inhibitor in the Philippines and provide practical implications for efficient herbicide resistance management in the affected rice areas.

plant biology↗

bioRxiv2025

Promiscuous cytochrome P450s confer metabolic resistance to synthetic auxin herbicides in the weedy grass Echinochloa phyllopogon

Auxinic herbicides are critically important tools for weed control in modern agriculture. Although resistant weed populations have been increasing in recent years, the underlying mechanisms remain largely unknown. In a multiple-herbicide resistant line of the paddy weed Echinochloa phyllopogon, overexpression of catalytically promiscuous CYP81A P450 enzymes has been shown to confer resistance to diverse herbicides. Here, we investigated the role of these P450s in quinclorac resistance in E. phyllopogon. Arabidopsis thaliana plants expressing CYP81A12 and CYP81A21 genes, previously demonstrated to metabolize other herbicides, showed low but detectable levels of quinclorac resistance. A similar activity was also observed for CYP81A24, another promiscuous enzyme present in E. phyllopogon. Moreover, Escherichia coli strains expressing these genes produced putative hydroxylated metabolites of quinclorac. In silico analyses revealed that quinclorac binds stably near the catalytic heme center of CYP81A12, CYP81A21, and CYP81A24, but not CYP81A18, the importance of spatial alignment between the substrate recognition site and the heme center in determining metabolic activity and resistance potential. Consistent with the phenotype of resistant E. phyllopogon, A. thaliana expressing CYP81A12 and CYP81A21 produced significantly less ethylene following quinclorac treatment, linking P450-mediated metabolism to the suppression of quinclorac-induced phytotoxic responses. Finally, we demonstrated that CYP81A enzymes also confer resistance to other auxinic herbicides, florpyrauxifen-benzyl and 2,4-D. These findings provide new insights into the molecular basis of auxinic herbicide resistance and underscore the role of CYP81A P450s in metabolic detoxification and the prevention of herbicide-induced phytotoxic responses.

plant biology↗

bioRxiv2023

Unraveling the Role of P450 Reductase in Herbicide Metabolic Resistance Mechanism

Plants require cytochrome P450 reductase (CPR) to supply two electrons for cytochrome P450 monooxygenase enzymes (P450) to react with an organic substrate. The transfer of electrons to the P450 active site in the P450 catalytic site relies on a robust and intricate CPR:P450 complex in the endoplasmic reticulum membrane. Transgenic Arabidopsis plants carrying CYP81A12 from Echinochloa phyllopogon, which metabolizes a broad spectrum of herbicides, were crossed with CPR knockout atr1 or atr2 mutant lines. Homozygous gene knockout was confirmed using PCR, and gene copy number of CYP81A12 was determined using ddPCR. Arabidopsis lines expressing CYP81A12 in combination with atr1 or atr2 knockout were used for herbicide dose-response and metabolism studies. Knocking out ATR1 in transgenic Arabidopsis CYP81A12 significantly reduced herbicide resistance. Transgenic mutant plants (CYP81A12 atr1-b) had a 3.6-, 5.6-, 6.8- and at least 26-fold reduction in resistance to mesotrione, 2,4-D, penoxsulam and chlorsulfuron, respectively, in the dose-response assay. Knockouts of the ATR2 also decreased herbicide resistance, but to a lower magnitude than ATR1. These results corroborate [1/2] MS medium assay, and herbicide resistance reduction was observed for additional tested herbicides, bensulfuron, propoxycarbazone and bentazon. Our findings highlight the importance of CPRs in metabolic herbicide resistance in plants, by identifying that a single CPR knockout can reverse herbicide sensitivity . The different CPRs found in weeds have potential as target genes to manage metabolic herbicide resistance evolution. We further provide an in-depth exploration of the evolutionary implications in weed management arising from the results. HIGHLIGHTSKnocking out cytochrome P450 reductase 1 in herbicide tolerant Arabidopsis reduces herbicide resistance, highlighting CPRs as targets for managing herbicide resistance evolution in weeds.

plant biology↗

bioRxiv2023

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 exhibits 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 genomes 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 would contribute to elucidating the population structure of this species and could be a milestone in understanding the molecular mechanisms of the pernicious traits and to developing effective weed control strategies to avoid yield loss in rice production.

genomics↗

bioRxiv2022

Simultaneous overexpression of multiple herbicide-metabolizing genes for broad-spectrum resistance in an agricultural weed Echinochloa phyllopogon

O_LIPrevious research unveiled that the overexpression of catalytically promiscuous CYP81A cytochrome P450s underlies the multiple-herbicide resistance (MHR) in a Californian population of Echinochloa phyllopogon. However, it does not fully accommodate the resistance to diverse herbicides in MHR E. phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. C_LIO_LIWe investigated the high-level resistance to diclofop-methyl in MHR E. phyllopogon. Detailed diclofop-methyl metabolism was analyzed, followed by gene expression study and functional characterization of P450 genes. The generality of the MHR mechanism was investigated using another MHR line. C_LIO_LIThe MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81A12/21. Gene expression study identified the genetically linked overexpression of a novel gene CYP709C69 with CYP81A12/21 in the MHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated-diclofop-acid in yeast. The activity was observed in some CYP709C in plants. Unlike the broad substrate-specificity in CYP81As, CYP709C69 showed narrow substrate-specificity. The overexpression of the CYP81A and CYP709C69 was also observed in another MHR line. C_LIO_LIThe present findings establish a novel concept that genetically-linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds. C_LI

plant biology↗

bioRxiv2021

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 characterisation 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.

plant biology↗

bioRxiv2020

Drastic shift in flowering phenology, an instant reproductive isolation mechanism, explains the population structure of Imperata cylindrica in Japan

Reproductive isolation plays an important role in population differentiation and speciation, thus enhancing biodiversity in wild plants. Hybridisation sometimes involves rapid reproductive isolation between parents and their hybrids through the novel traits of hybrids derived from a new combination of genomes. Here, we report how a hybrids new phenotype contributes to rapid reproductive isolation between two ecotypes of Imperata cylindrica. The two ecotypes differ in their flowering phenology and habitats. An analysis with genetic markers revealed that hybrid populations consisted of only F1 individuals. Both parental ecotypes flowered in spring, but F1s flowered in fall. This drastic shift in flowering phenology prevented backcrossing parental ecotypes to F1. F1s flowered in fall and dispersed seeds in winter. The germination percentage of seeds set on F1 was extremely low in their habitats, and seedlings did not survive due to the low temperatures in winter, resulting in the absence of a F2 generation. In conclusion, flowering phenology mismatch promotes reproductive isolation between parents and F1, resulting in a hybrid population consisting of only F1s.

evolutionary biology↗