No abstract

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.

Metabolism of pyroxasulfone in a tolerant crop, wheat and a susceptible plant, rigid ryegrass, was studied using 14 C-pyroxasulfone. The main metabolites were a cysteine conjugate of the isoxazoline ring (M-26), deaminated M-26 (M-29) and a glucose conjugate of M-29, suggesting that the main metabolic route in both plants was the cleavage of the methylenesulfonyl linkage caused by glutathione conjugation. The difference in the metabolic activity was assumed to be one of the factors in determining the selectivity of pyroxasulfone between wheat and rigid ryegrass.

A two-point mutated gene of acetolactate synthase (ALS) was isolated from rice cells, which was cultured together with an ALS-inhibiting herbicide, bispyribac-sodium (BS). The mutations involved residues of tryptophan at position 548 to leucine (W548L) and serine at position 627 to isoleucine (S627I). The ALS expressed in Escherichia coli from this gene showed resistance to multiple herbicides including pyrimidinylcarboxylate (PC), sulfonylurea and imidazolinone herbicides, and showed stronger resistance to PC herbicides than to other herbicides. BS, a PC herbicide, had almost no effect on the enzyme even at 100 mM, which is an approximately 10,000-fold higher concentration than the concentration required for 50% inhibition of the wild-type. The resistance level of W548L/S627I mutating ALS to BS was stronger than the additive effect predicted from the degree of resistance of each single amino acid mutated ALS. Transformed rice cells carrying this gene and a regenerated rice plant expressed resistance to BS, suggesting that this gene is useful as a selectable marker for introducing foreign traits into rice when used with PC herbicides.

We have established a simple diagnosis method for rice blast fungus resistant to MBI-D. This involves the preparation of PCR templates directly from the lesions in combination with primer-introduced restriction enzyme analysis PCR (PIRA-PCR).