Hiroaki Saika¹, Fumio Taguchi-Shiobara², Nishizawa-Yokoi Ayako¹, Kiyosumi Hori², Tsuyoshi Tanaka³, Takeshi Itoh³, Seiichi Toki¹

¹Plant Genome Engineering Research Unit, ²Rice Applied Genomics Research Unit, ³Bioinformatics Research Unit

［Abstract］

Map-based cloning revealed that a novel cytochrome P450 gene CYP72A31 is involved in herbicide tolerance. Overexpression of the CYP72A31 gene confers herbicide tolerance in both rice and Arabidopsis.

［Keywords］

rice, acetolactate synthase, herbicide tolerance, detoxification, cytochrome P450

［Background］

Herbicide tolerance mechanisms in plants can be classified roughly into two groups, namely, target-site herbicide tolerance caused by mutations in the target enzyme to which herbicides bind, and decrease of herbicide effective concentration caused by herbicide detoxification. Since herbicide detoxification is involved in tolerance to multiple herbicides, it is an important trait from the standpoint of generation of hard-to-control weeds and the breeding of multi-herbicide tolerant crops. However, there are few reports on the molecular mechanisms of herbicide detoxification. Bispyribac sodium (BS) is one of the herbicides used to inhibit acetolacate synthase (ALS). Recently, it has been suggested that herbicide detoxification is possibly involved in BS tolerance in some rice varieties. In this study, we isolated the gene involved in BS tolerance in rice and attempted the application of molecular breeding of herbicide-tolerant crops.

［Results and Discussion］

1. We performed map-based cloning of a BS-tolerant gene using the progenies derived from a cross between a BS-tolerant indica variety Kasalath and BS-sensitive japonica variety Koshihikari. We succeeded in the identification of CYP72A31 (Os01g0602200) located at 23.6 Mb of chromosome 1 as a BS-tolerant gene (Fig. 1).
2. Overexpression of the CYP72A31 gene confers BS tolerance in the callus and seedlings of both Kasalath and the japonica variety Nipponbare, which shows BS sensitivity similar to Koshihikari (Fig. 2). In both varieties, transgenic callus lines in which CYP72A31 mRNA accumulated at a higher level showed higher tolerance to BS.
3. Bensulfuron-methyl (BSM) is another ALS inhibiting herbicide with a different chemical structure from BS. Arabidopsis seedlings overexpressing CYP72A31 gene showed tolerance to both BS and BSM (Fig. 3).
4. It has been reported that rice CYP81A6, which belongs to a different family as CYP72A31, is involved in BSM tolerance. CYP81A6 knockdown rice plants showed similar BS sensitivity as non-transformants. These results suggest that both CYP72A31 and CYP81A6 are involved in BSM tolerance. Although CYP72A31 is involved in BS tolerance, CYP81A6 has been found not to be involved or is just barely involved, if at all, in BS tolerance.

1. Herbicide-tolerant rice varieties can be produced by introducing the CYP72A31 gene followed by marker-associated selection. It is expected that herbicide-tolerant crops can be produced by transformation with the CYP72A31 gene.
2. Information gained from the study of the biochemical and enzymatic properties of CYP72A31 can be applied in efficient development of novel herbicides and herbicide-tolerant crops.
3. The CYP72A31 gene can be used in a broad array of technologies for selection of transgenic cells and plants, and selection of crossed seeds and plants, e.g. using the F1 hybrid system.

Fig.1. Structure of CYP72A31 gene. The CYP72A31 gene of Kasalath is thought to be functional. In Koshihikari however, sequence analysis of the putative coding region revealed a –1 frame-shift mutation in the 4th exon creating a stop codon. A conserved motif of cytochrome P450 (pink box) is located downstream of this deletion site in the 4th exon. The CYP72A31 gene of Koshihikari is therefore a non-functional gene.

Fig.2. BS sensitivity test of CYP72A31 overexpressing Kasalath. Seeds of CYP72A31 overexpressing Kasalath after 7 days of germination on medium containing BS.

Fig.3. BS sensitivity test of Arabidopsis overexpressing CYP72A31. Seeds of Arabidopsis overexpressing CYP72A31 were germinated and grown for 10 days on medium containing BS. The CYP72A31 mRNA levels in line 1 are higher than those in line 2.

 

[Collaborator]
Junko Horita, Koichiro Kaku and Tsutomu Shimizu (Kumiai Chemical Industry Co., Ltd.), Satoko Nonaka (University of Tsukuba), Satoshi Iwakami (Kyoto University)

[References]

1. International publication #WO2013/054890
2. Saika H, Horita J, Taguchi-Shiobara F, Nonaka S, Nishizawa-Yokoi A, Iwakami S, Hori K, Matsumoto T, Tanaka T, Itoh T, Yano M, Kaku K, Shimizu T, Toki S (2014) A novel rice cytochrome P450 gene, CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis. Plant Physiology DOI:10.1104/pp.113.231266