2009). of a series of branching mutants, ((((((Sergeant et al. 2009). In addition, CCD7 also plays a role in the production of some mycorrhiza-induced apocarotenoids in tomato (Vogel et al. 2010), suggesting that CCD7 inhibitors may affect the production of other apocarotenoids. Besides CCDs, another target enzyme class for developing SL biosynthesis inhibitors is cytochrome P450 monooxygenases (P450s); at least one P450 (CYP711A) is involved in SL biosynthesis. Towards this goal, we screened a chemical library consisting of potential inhibitors of P450s for SL biosynthesis inhibitors and discovered a new lead compound that is able to decrease SL levels in rice seedlings. Results Screening for triazole-type chemicals inducing SL-deficient mutant-like morphology in rice and grow out, while those of wild-type plants remain dormant (Umehara et al. 2008). Therefore, our chemical library was screened for chemicals that induce the first and second tiller bud outgrowth as candidates for SL biosynthesis inhibitors. Unfortunately, none of the chemicals tested induced the outgrowth of the first tiller bud. However, some chemicals induced second tiller bud outgrowth, many of which were found also to reduce plant height. Under our growth conditions, SL-deficient mutants do not show a significant difference in plant height from the wild type (data not shown), so this reduction in plant height caused by chemical treatments could be due to inhibition of other pathway(s). A likely explanation was the inhibition of gibberellin biosynthesis, because, with the exception of TIS13 and TIS15, all chemicals that induced second tiller bud outgrowth were gibberellin biosynthesis inhibitors or their analogs: paclobutrazol (TIS9), paclobutrazol analog (TIS18) and uniconazole analogs (TIS24, TIS29, TIS33 and TIS34) (Fig. 1ACE). Among the tested compounds, TIS13 and TIS29 were the most effective in inducing second tiller bud outgrowth (Fig. 1F, G). Open in a separate window Fig. 1 Screening of the chemicals that induce outgrowth of second tiller bud in 2-week-old rice seedlings. (A and B) Length of the second tiller in seedlings treated with 10?M of the chemicals. (C and D) Plant height of seedlings treated with 10?M of the chemicals. The data are means??SD of three samples. (E) Structures of chemicals that induced tiller IGFBP4 bud outgrowth in A and B. (F and G) Two-week-old rice seedling treated with or without chemicals (TIS13 or TIS29). Scale bars in F and G indicate 5 and 1?cm, respectively. White arrowheads indicate second tillers. Analysis of SL levels in chemical-treated rice Although rice seedlings treated with TIS13 or TIS29 showed second tiller bud outgrowth, plant height was remarkably reduced (Fig. 1). Paclobutrazol (TIS9), a gibberellin biosynthesis inhibitor, and its analogs showed reduced plant height and second tiller bud outgrowth (Fig. 1). A rice gibberellin-deficient Dyphylline mutant which overexpresses gibberellin 2-oxidase has reduced plant height and increased tiller bud outgrowth (Lo et al. 2008). In this context we thought that second tiller bud outgrowth on seedlings treated with TIS13 or TIS29 could be induced by inhibiting gibberellin biosynthesis. To determine whether or not these chemicals inhibit SL biosynthesis, we analyzed the level of 2-and (Cook et al. 1966). We employed a highly sensitive germination assay using seeds as a first step to evaluate TIS13 as a chemical that controls parasitic weed germination. In agreement with the result of germination, because the co-application of 1 1?M GR24 with the TIS13-treated culture medium did not inhibit germination (Fig. 4). These results indicate that the SL biosynthesis inhibitor selected as an inducer of tiller bud outgrowth could have the Dyphylline potential to be applied to control parasitic weed germination. Open in a separate window Fig. 4 Estimation of germination stimulant levels in culture media of 2-week-old seedlings using seeds. DW, distilled water; GR24, 1?M GR24; control, non-treated rice culture media; TIS13, culture media of TIS13-treated plants; TIS13?+?GR24, culture media co-incubated with 1?M GR24.In addition, CCD7 also plays a role in the production of some mycorrhiza-induced apocarotenoids in tomato (Vogel et al. analysis of a series of branching mutants, ((((((Sergeant et al. 2009). In addition, CCD7 also plays a role in the production of some mycorrhiza-induced apocarotenoids in tomato (Vogel et Dyphylline al. 2010), suggesting that CCD7 inhibitors may affect the production of other apocarotenoids. Besides CCDs, another target enzyme class for developing SL biosynthesis inhibitors is cytochrome P450 monooxygenases (P450s); at least one P450 (CYP711A) is involved in SL biosynthesis. Towards this goal, we screened a chemical library consisting of potential inhibitors of P450s for SL biosynthesis inhibitors and discovered a new lead compound that is able to decrease SL levels in rice seedlings. Results Screening for triazole-type chemicals inducing SL-deficient mutant-like morphology in rice and grow out, while those of wild-type plants remain dormant (Umehara et al. 2008). Therefore, our chemical library was screened for chemicals that induce the first and second tiller bud outgrowth as candidates for SL biosynthesis inhibitors. Unfortunately, none of the chemicals tested induced the outgrowth of the first tiller bud. However, some chemicals induced second tiller bud outgrowth, many of which were found also to reduce plant height. Under our growth conditions, SL-deficient mutants do not show a significant difference in plant height from the wild type (data not shown), so this reduction in plant height caused by chemical treatments could be due to inhibition of other pathway(s). A likely explanation was the inhibition of gibberellin biosynthesis, because, with the exception of TIS13 and TIS15, all chemicals that induced second tiller bud outgrowth were gibberellin biosynthesis inhibitors or their analogs: paclobutrazol (TIS9), paclobutrazol analog (TIS18) and uniconazole analogs (TIS24, TIS29, TIS33 and TIS34) (Fig. 1ACE). Among the tested compounds, TIS13 and TIS29 were the most effective in inducing second tiller bud outgrowth (Fig. 1F, G). Open in a separate window Fig. 1 Screening of the chemicals that induce outgrowth of second tiller bud in 2-week-old rice seedlings. (A and B) Length of the second tiller in seedlings treated with 10?M of the chemicals. (C and D) Plant height of seedlings treated with 10?M of the chemicals. The data are means??SD of three samples. (E) Structures of chemicals that induced tiller bud outgrowth in A and B. (F and G) Two-week-old rice seedling treated with or without chemicals (TIS13 or TIS29). Scale bars in F and G indicate 5 and 1?cm, respectively. White arrowheads indicate second tillers. Analysis of SL levels in chemical-treated rice Although rice seedlings treated with TIS13 or TIS29 showed second tiller bud outgrowth, plant height was remarkably reduced (Fig. 1). Paclobutrazol (TIS9), a gibberellin biosynthesis inhibitor, and its analogs showed reduced plant height and second tiller bud outgrowth (Fig. 1). A rice gibberellin-deficient mutant which overexpresses gibberellin 2-oxidase has reduced plant height and increased tiller bud outgrowth (Lo et al. 2008). In this context we thought that second tiller bud outgrowth on seedlings treated with TIS13 or TIS29 could be induced by inhibiting gibberellin biosynthesis. To determine whether or not these chemicals inhibit SL biosynthesis, we analyzed the level of 2-and (Cook et al. 1966). We employed a highly sensitive germination assay using seeds as a first step to evaluate TIS13 as a chemical that controls parasitic weed germination. In agreement with the result of germination, because the co-application of 1 1?M GR24 with the TIS13-treated culture medium did not inhibit germination (Fig. 4). These results indicate that the SL biosynthesis inhibitor selected as an inducer of tiller bud outgrowth could have the potential to be applied to control parasitic weed germination. Open in a separate window Fig. 4 Estimation of germination stimulant levels in culture media of 2-week-old seedlings using seeds. DW, distilled water; GR24, 1?M GR24; control, non-treated rice culture media; TIS13, culture media of TIS13-treated plants; TIS13?+?GR24, culture media co-incubated with 1?M GR24 and TIS13..