Herbicide sensitivities of mutated enzymes expressed from artificially generated genes of acetolactate synthase

Abstract

Mutated acetolactate synthase (ALS) genes of rice and Arabidopsis, which confer resistance to ALS-inhibiting herbicides, were generated using PCR and overlap extension. Recombinant ALSs from these mutated genes were prepared as glutathione S-transferase-fused proteins, and sensitivities of the proteins to ALS-inhibiting herbicides were examined. Nine kinds of rice ALSs that have mutations at the P171 position (each amino acid is represented by one letter) showed high resistance to a herbicide, bensulfuron-methyl, but the resistance level to a herbicide, bispyribac-sodium (BS), changed among ALSs. S627I ALS of rice expressed high resistance to herbicides: pyriminobac and pyrithiobac-sodium. P171H/R172S ALS of rice showed greater resistance to a herbicide, chlorsulfuron than the additive effect predicted from the resistance of each single mutated ALS. P171H/W548L ALS and P171H/S627I ALS of rice showed similar synergistic resistance to BS. On the other hand, P197S, W574L, S653I, P197H/R198S and W574L/S653I-mutated ALSs of Arabidopsis expressed similar sensitivities to herbicides as those of rice ALSs with the corresponding mutations. These results proposed that P171-mutated ALSs of rice can be used as model enzymes for resistant weed management to ALS-inhibiting herbicides, and rice-mutated ALS genes encoding mutated ALSs such as S627I, P171H/R172S, P171H/W548L, P171H/S627I, and Arabidopsis-mutated ALS genes encoding mutated ALSs, such as S653I, P197H/R198S and W574L/S653I, are useful as new selectable marker genes for genetic transformation of plants when used together with ALS-inhibiting herbicides to which mutated ALSs express high resistance.

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

“…The ALS activity of W574L/ S653I plants was strongly resistant to PS, BM and IP (Figure 8). The selectable tolerance of plants transplastomic with G121A, A122V and W574L/ S653I ( Figure 9) were similar to those obtained when using the same recombinant mALSs that only expressed the catalytic subunit in E. coli, to which endogenous E. coli regulatory subunits, it was concluded, were not associated (Tables 1 and 2) (Kawai et al, 2008;Okuzaki et al, 2007). Therefore, the regulatory subunits do not affect the sensitivity of these mALSs to herbicides in transplastomic plants.…”

“…Recombinant Arabidopsis mALSs, AtALS-W574L/ S653I and -S653I, were expressed in Escherichia coli cells. 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).…”

“…Herbicide-tolerant plants have been reported for rice, tobacco and Arabidopsis (Chang et al, 1998;Tan et al, 2005;Shimizu et al, 2002;Kawai et al, 2007b;Okuzaki et al, 2007). Several mutated species of Arabidopsis ALS have been expressed in Escherichia coli, and their sensitivity to inhibitors was examined (Tables 1 and 2) (Kawai et al, 2008). These results showed that P197S, W574L, S653I, P197H/ R198S and W574L/ S653I were resistant to SUs, IMs, SUs, IMs, and all three types of herbicides, respectively.…”

“…Recombinant ALS proteins from these genes were prepared as glutathione S-transferasefused proteins, and the sensitivity of the proteins to ALS-inhibiting herbicides were examined. It was found that the level of resistance of these recombinant ALS proteins to ALS-inhibiting herbicides varied for the compounds tested (Table 2), while mALS-P197S, W574L and P197H/ R198S proteins showed similar sensitivity to herbicides to that of rice ALS proteins with the corresponding mutations (Kawai et al, 2008 …”

“…It has recently been reported that mutation of W548L/ S627I and G95A in rice ALS imparts tolerance to all three types of herbicides and pyrimidinylcarboxylate herbicides, respectively (Kawai et al, 2007b;Okuzaki et al, 2007). We examined whether introduction of mALSs into the chloroplast genome can be applied to a strategy involving the rotation supply of different herbicides by characterizing the transplastomic lines with respect to: (i) the influence of hyper-expression of mALSs on plant growth, (ii) feedback regulation by the regulatory subunit in vivo, (iii) the dependency of herbicide resistance on each mutation similarly observed in vitro (Tables 1 and 2) (Kawai et al, 2008;Okuzaki et al, 2007), and (iv) the availability of multiple combinations of different mutations and herbicides. We have reported on the introduction of some mALS genes into the chloroplast genome and examined the sensitivity of transformants to ALS-inhibiting herbicides.…”