Koichiro Kaku

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.

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T1 soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

Transgenic Tall Fescue and Maize with Resistance to ALS-Inhibiting Herbicides

Transgenic crops such as maize (Zea mays L.), soybeans (Glycine max L. Merr.), canola (Brassica napus L.) and cotton (Gossypium hirsutum L.) have been widely used in the field. In 2009, transgenic crops were cultivated in approximately 134 million hectares in 25 countries, mainly USA, Brazil, Argentina, India, Canada and China (http://www.isaaa.org). The adoption of transgenic crops has steadily increased since 1996 because of their many benefits for farmers. Herbicide resistance is one of the most important agronomic traits conferred onto transgenic crops. Herbicide-resistant crops comprise 62 percent of all transgenic crops (http://www.isaaa.org), and are produced by the introduction of herbicide-resistant genes using genetic transformation. Herbicide resistance can be used as an efficient tool to allow easier weed management. It facilitates control of weed species and contributes to reducing costs, labor, and the waste of chemical spray. Herbicide resistance can also facilitate the selection of transgenic cells from non-transgenic cells as a selectable marker in genetic transformation. Acetolactate synthase (ALS)-inhibiting herbicides are widely used around the world. ALSinhibiting herbicide-resistant weeds were first found in kochia (Kochia scoparia L. Shrad) (Primiani et al., 1990) and prickly lettuce (Lactuca serriola L.) (Mallory-Smith et al., 1990). Subsequently, plants and cultured cells resistant to ALS-inhibiting herbicides have been generated using both conventional mutation breeding and somatic cell selection. Since then, the ALS genes have been cloned and characterized. In most cases, resistance to ALSinhibiting herbicides has been found to be conferred by single or double amino-acid mutations at a particular position in ALS. Mutated ALS genes can be used not only for the generation of herbicide-resistant crops, but also as selectable markers. We are now producing transgenic tall fescue (Festuca arundinacea Schreb.) and maize that are resistant to ALS-inhibiting herbicides using novel mutated ALS genes. This chapter focuses on mutated ALS genes and their application to the production of herbicide-resistant crops and selection of transgenic cells as selectable markers.