Transformation of Arabidopsis by mutated acetolactate synthase genes from rice and Arabidopsis that confer specific resistance to pyrimidinylcarboxylate-type ALS inhibitors

Abstract

Previously, we showed that four mutated acetolactate synthase (ALS) genes derived from rice and Arabidopsis (W548L/S627IOsALS, S627IOsALS, W574L/S653IAtALS and S653IAtALS) confer high levels of resistance to pyrimidinylcarboxylate type ALS inhibitors (Kawai et al. 2008). Mutated ALS genes of rice were obtained from rice cells cultured in the presence of an ALS-inhibitor. The mutated ALS genes of Arabidopsis, which have the same amino acid substitutions as those of rice, have been generated artificially. Here, we demonstrate that these mutated genes function as effective selectable markers for transformation of Arabidopsis. Specifically, we studied expression of the mutated ALSs in Arabidopsis and their effect on the sensitivity of transgenic Arabidopsis plants to the ALS inhibitors. Our results show that the degree of resistance to the ALS inhibitors of transformants expressing Arabidopsis mutated ALSs was greater than those of transformants expressing rice mutated ALSs. The amino acid sequences of ALSs derived from monocotyledonous plants and those derived from dicotyledonous plants were clearly divided into two clusters in a phylogenetic tree. Based on these results, it would be preferable to use rice and Arabidopsis mutated ALS genes for generating monocotyledonous and dicotyledonous transgenic plants, respectively. Moreover, our findings are particularly useful when generating transgenic plants with a known ALS nucleotide sequence. In such cases, their own ALS gene carrying these mutations could be used as a selectable marker because amino acid residues at the point of mutation are conserved among plant species.Key words: Acetolactate synthase, pyrimidinylcarboxylate, selectable marker gene.Plant Biotechnology 27, 75-84 (2010) Original PaperAbbreviations: ALS, acetolactate synthase; BS, bispyribac-sodium; IM, imidazolinone; PC, pyrimidinylsalicylate; PS, pyrithiobac-sodium; SU, sulfonylurea This article can be found at http://www.jspcmb.jp/ inhibitor, bispyribac-sodium (BS), and demonstrated that it could be used as a selectable marker for generating transgenic rice plants (Kawai et al. 2007a;Kawai et al. 2007b). We also found that a single amino acid substitution (S627I) in the ALS gene (S627IOsALS) displays high levels of resistance to other PC-type ALS inhibitors; pyrithiobac-sodium (PS) and pyriminobac (PM). In addition, artificially generated W574L/S653I and S653I Arabidopsis ALS genes (W574L/S653IAtALS and S653IAtALS) corresponding to the rice mutated ALS genes showed similar sensitivity to PC-type ALS inhibitors (Kawai et al. 2008). We postulated that these mutated ALS genes coupled with the PC-type ALS inhibitors might be promising selectable markers for various plant species. Indeed, previous reports have shown that W548L/S627IOsALS works as an effective selectable marker gene for transformation of wheat (Ogawa et al. 2008) and soybean (Tougou M et al. 2009).In this paper, we generated transgenic Arabidopsis via agrobacterium-mediated transformation with rice mutated ALS genes (W548L/S6...

Application of Mutated Acetolactate Synthase Genes for Herbicide Resistance to Crop Improvement

“…These recombinant mALSs exhibited resistance to PCs, and showed similar sensitivity against herbicides to rice recombinant ALSs with the corresponding mutations (Table 1) (Kawai et al, 2008). We have shown that these Arabidopsis ALS genes can also be utilized as selectable markers for the genetic transformation of Arabidopsis (Kawai et al, 2010). It has been revealed that selection by PCs can clearly distinguish resistant seedlings from non-resistant seedlings of Arabidopsis at very low concentrations of herbicide compared with kanamycin selection (Figure 3).…”

A Novel Rice Cytochrome P450 Gene, CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

“…A BS-insensitive mutant ALS gene has been used widely as a positive marker to obtain transformed cells and plants under BS selection. For example, a combination of a mutated rice ALS gene and BS selection has been applied in rice (Osakabe et al, 2005;Okuzaki et al, 2007;Wakasa et al, 2007;Taniguchi et al, 2010), wheat (Ogawa et al, 2008), tall fescue (Festuca arundinacea; Sato et al, 2013), Arabidopsis (Kawai et al, 2010), and soybean (Glycine max; Tougou et al, 2009). Like the mutated ALS gene, the CYP81A6 gene has also been reported as a selection marker with BSM in Arabidopsis, cotton (Gossypium hirsutum), and tobacco plants (Ke et al, 2012;Liu et al, 2012).…”

A technical breakthrough close at hand: feasible approaches toward establishing a gene-targeting genetic transformation system in seaweeds

“…Acetohydroxyacid synthase (AHAS; also known as acetolactate synthase), which catalyzes the first step in biosynthesis of branched-chain amino acids such as valine, leucine, and isoleucine (Duggleby et al, 2008), is a target of at least three classes of herbicides, including pyrimidinyl carboxylate (PC), sulfonylurea (SU), and imidazolinone herbicides (Kawai et al, 2008; Shimizu et al, 2008). Among these, sulfometuron methyl (SMM) and bensulfuron methyl (BM) (both SU), and bispyribac-sodium, pyrithiobac-sodium, and pyriminobac-methyl (all PC) have been used to screen for genetically transformed cells, for which inhibitor-tolerant variants of AHAS were used as selectable markers (Lapidot et al, 2002; Shimizu et al, 2008; Kawai et al, 2010). Thus, if seaweeds are sensitive to AHAS inhibitors, selection of transformed cells could then be possible, as in the report of successful chloroplast transformation in the red unicellular alga Porphyridium sp.…”