Satoshi Iwakami

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.

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

Resistance to two herbicides in Echinochloa phyllopogon is associated with overexpression of two cytochrome P450s that are simultaneously controlled by a putative single genetic element. 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

A novel cytochrome P450 monooxygenase is involved in multiple-herbicide detoxification and could be useful in herbicide development and molecular breeding in crops. 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.

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.