Selective Mimics of Strigolactone Actions and Their Potential Use for Controlling Damage Caused by Root Parasitic Weeds

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1 Molecular Plant Volume 6 Number 1 Pages January 2013 RESEARCH ARTICLE Selective Mimics of Strigolactone Actions and Their Potential Use for Controlling Damage Caused by Root Parasitic Weeds Kosuke Fukui a, Shinsaku Ito a and Tadao Asami a,b,1 a Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, Japan b JST, CREST, Yayoi, Bunkyo, Tokyo, Japan ABSTRACT Strigolactones (SLs) are a novel class of plant hormones and rhizosphere communication signals, although the molecular mechanisms underlying their activities have not yet been fully determined. Nor is their application in agriculture well developed. The importance of plant hormone agonists has been demonstrated in both basic and applied research, and chemicals that mimic strigolactone functions should greatly facilitate strigolactone research. Here, we report our discovery of a new phenoxyfuranone compound, 4-Br debranone (4BD), that shows similar activity to that of the major strigolactone (SL) analog GR24 in many aspects of a biological assay on plants. 4BD strongly inhibited tiller bud outgrowth in the SL-deficient rice mutant d10 at the same concentration as GR24, with no adverse effects, even during prolonged cultivation. This result was also observed in the Arabidopsis thaliana SL-deficient mutants max1, max3, and max4. However, the application of 4BD to the Arabidopsis SL-insensitive mutant max2 induced no morphological changes in it. The expression of SL biosynthetic genes was also reduced by 4BD treatment, probably via negative feedback regulation. However, in a seed germination assay on Striga hermonthica, a root parasitic plant, 4BD showed far less activity than GR24. These results suggest that 4BD is the first plant-specific strigolactone mimic. Key words: strigolactones; plant growth regulator; Arabidopsis; rice; debranone; branching; tillering. Introduction Plant hormones play a key role in plant life cycles and the mechanisms underlying their functions have attracted the attention of many scientists. Research into their functions has sometimes involved to the use of functional regulators, such as hormone agonists and/or antagonists. Chemicals that regulate auxin functions have made great contributions to research, especially to the study of auxin signaling and transport, and most of the known factors in auxin signaling and transport have been identified in Arabidopsis thaliana by screening for mutants resistant to various regulators of auxin functions (De Rybel et al., 2009). For example, the synthetic auxin 2,4-D has been widely used in research as a useful chemical probe for the activities of auxin because it is both potent and stable, and several genetic loci have been identified by screening for Arabidopsis mutants that are resistant to the action of 2,4-D (Leyser et al., 1993, 1996). The auxin receptor TRANSPORT INHIBITOR RESPONSE 1 (TIR1) was identified by screening with auxin transport inhibitors, naphthylphthalamic acid and 2-carboxyphenyl-3-phenylpropane-1,2-dione (Ruegger et al., 1998). The receptor of abscisic acid, PYRABACTIN RESISTANCE 1 (PYR1), was isolated by screening mutants with pyrabactin, an agonist of abscisic acid (Park et al., 2009). Similarly, the jasmonoyl isoleucine and methyl jasmonate receptor, CORONATINE INSENSITIVE 1 (COI1), was isolated using coronatine as an agonist of jasmonate (Feys et al., 1994; Sheard et al., 2010). A similar methodology is applicable to research into new plant hormones. Strigolactones (SLs) are a novel class of plant hormones that regulate shoot branching outgrowth (Gomez-Roldan et al., 2008; Umehara et al., 2008) and root development (Kapulnik et al., 2011; Koltai, 2011; Ruyter- Spira et al., 2011). SLs also act as rhizosphere communication signals between plants and arbuscular mycorrhizal (AM) fungi (Akiyama et al., 2005), and between plants and root parasitic weeds, such as Striga and Orobanche (Cook et al., 1966; Xie and Yoneyama, 2010). In the past, several mutants defective in SL 1 To whom correspondence should be addressed. asami@pgr1.ch. a.u-tokyo.ac.jp, tel , fax The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: /mp/sss138, Advance Access publication 30 November 2012 Received 31 July 2012; accepted 21 October 2012

2 Fukui et al. Selective Mimics of Strigolactone Actions 89 production or signaling have been identified by morphological studies of rice, Arabidopsis, pea, and petunia. In this way, some genes related to SL biosynthesis or signaling have been identified. CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) (Booker et al., 2004; Johnson et al., 2006; Zou et al., 2006), CCD8 (Sorefan et al., 2003; Snowden et al., 2005; Arite et al., 2007), D27 (encoding an iron-containing protein) (Lin et al., 2009; Alder et al., 2012), and MAX1 (encoding cytochrome P450) (Stirnberg et al., 2002; Booker et al., 2005) are involved in SL biosynthesis. MAX2, D3, and RMS4 encode F-box proteins and D14 encodes an α/β hydrolase, which act in SL signaling (Stirnberg et al., 2002; Ishikawa et al., 2005; Yan et al., 2007; Arite et al., 2009; Gao et al., 2009; Liu et al., 2009). Recently, an AtD14 paralog, KARRIKIN INSENSITIVE 2 (KAI2), was found by mutant screening with karrikins (Waters et al., 2012). One of the key points to the successful identification of KAI2 was the use of karrikins, which, like SLs, promote the germination of dormant Arabidopsis seeds (Roche et al., 1997; Flematti et al., 2004), and have a butenolide substructure similar to the D-ring of general SLs. Thus, karrikins are similar in structure and bioactivity to the SLs, although it has been demonstrated that karrikins only activate the KAI2 signaling pathway, unlike the synthetic SL agonist GR24, which activates both the KAI2 and D14 signaling pathways. In this context, the development of chemicals that affect SL functions as only a plant hormone should accelerate research into SLs like the case of other plant hormones. Until now, several chemicals have been shown to regulate the hormonal functions of the SLs. GR24 and its derivative GR5 were active SL analogs of plant hormonal action in shoot branching inhibition. Recently, a variety of natural and artificial SLs, including GR24 and a debranone derivative, were evaluated regarding their hormonal activity in a pea branching inhibition test (Boyer et al., 2012). This result provides important information regarding structural requirement in branching inhibition activity. Abamine is a dioxygenase inhibitor and TIS13 and TIS108 are triazole-type compounds, anticipated to target cytochrome P450, that are reported to inhibit SL biosynthesis in rice and Arabidopsis, although their target proteins are still unclear (Kitahata et al., 2006; Ito et al., 2010, 2011). Cotylimide also increases SL biosynthesis by an unknown mechanism (Tsuchiya et al., 2010). Many SL analogs have also been developed to stimulate the seed germination of root parasitic weeds, but their effects on branching in plants have not been thoroughly investigated (Zwanenburg et al., 2009; Yoneyama et al., 2010; Mwakaboko and Zwanenburg, 2011). Here, we propose that SL mimics that have a different character from the existing SL function regulators can be used for both scientific and agricultural purposes. In this paper, we present a new SL mimic that exerts a unique activity that is specific for plant branching. RESULTS 4BD Shows Strigolactone-Like Activity in Rice Seedlings Shoot branching is coordinately regulated by multiple plant hormones, including SL (Ferguson and Beveridge, 2009; Domagalska and Leyser, 2011; Müller and Leyser, 2011). The SL-deficient rice mutant d10 has an enhanced branching phenotype, which is restored to wild-type (WT) phenotype by treatment with exogenous SL (Gomez-Roldan et al., 2008; Umehara et al., 2008). Recently, we found that some phenoxy furanone derivatives inhibit tiller bud outgrowth in the rice d10 mutant, as do strigolactones, and collectively designated these chemicals debranones (debranching furanone). With a structure activity relationship (SAR) study of debranone using a rice d10 assay, we identified 4-Br debranone (5-[4-bromophenoxy]-3-methylfuran-2[5H]-one; 4BD) as the most potent SL mimic among the chemicals synthesized in that study (Fukui et al., 2011), but have not yet investigated its activity as a plant hormone in plants other than rice. In this study, we examined its biological activity in detail and determined its potential as a chemical tool with which to clarify the functions of SLs in plants. Debranone can be hydrolyzed to a phenol and a furanone (5-hydroxy-3-methylfuran-2[5H]-one) in the hydroponic culture medium used in our assay system. To eliminate the possibility that these degradation products of 4BD could be the active ingredients in the SL-like activity of this compound, we examined the effects of its hydrolysates (4-bromophenol and 5-hydroxy-3-methylfuran-2[5H]-one) (Figure 1). In our assay system, 4BD inhibited the axillary shoot outgrowth in the rice d10 mutant, like GR24, whereas neither hydrolysate affected the phenotype (Figure 2A). We also checked the stability of 4BD in our medium and found that it was sufficiently stable and did not produce any detectable hydrolysates under our assay conditions. These data clearly suggest that 4BD itself is the active ingredient involved in the inhibition of shoot branching, and that the structure of 4BD meets the requirements for this activity. Similarly, the cleavage of GR24 at the conjugated enol ether moiety connecting the ABC ring part to the D-ring furanone part inactivated GR24 in a seed germination stimulation assay on root parasitic weeds (Johnson et al., 1981; Zwanenburg et al., 2009). The SAR study of the SL D-ring also revealed that the existence of a 3-methyl group on the butenolide was important for its stimulatory activity in a seed germination assay on root parasitic weeds (Johnson et al., 1981; Mangnus et al., 1992b). Figure 1. Chemical Structures of Two GR Series, Karrikin, 4BD, and Its Hydrolisates.

3 90 Fukui et al. Selective Mimics of Strigolactone Actions Figure 2. Effect of Debranones on Tiller Bud Outgrowth in Rice Seedlings. (A) 16-day-old wild-type (WT) and d10 mutant. Scale bar indicates 2 cm. Arrowheads indicate first and second tiller buds. (B, C) 16-day-old WT and d10 mutant. Scale bars indicate second tiller length of seedlings with or without 1 µm chemical treatment. The data are means ± SE of six samples. A preliminary shoot branching assay of the debranones also demonstrated the importance of the 3-methyl group on the D-ring butenolide for their activity (Figure 2B and C). 4-Cl debranone (4CD), which is a first-obtained active debranone derivative, inhibited the shoot branching of the d10 mutant but its demethyl analog (CPF) was inactive in this assay. This result may also suggest that 4BD acts as an agonist of the SLs. Effects of 4BD on Rice Grown under Long-Term Hydroponic Culture Conditions To evaluate 4BD as a mimic of SL, we wanted to amass more experimental evidence of its similarity to GR24 in terms of the pleiotropic biological activities of the SLs. Another important phenotype of the SL-deficient rice mutants, apart from their more extensive tillering, is the dwarfism that emerges in the

4 Fukui et al. Selective Mimics of Strigolactone Actions 91 Figure 3. Effect of 4BD on Grown Rice Tillering and Height. (A) 47-day-old wild-type (WT) and d10 mutant with or without 1 µm chemical treatment. Scale bar indicates 10 cm. (B) Number of tiller buds in WT and d10 mutant rice with or without 1 µm chemical treatment. The data are means ± SE of four samples. (C) Plant height in WT and d10 mutant rice with or without 1 µm chemical treatment. The data are means ± SE of four samples. adult growth phase. This dwarfism is caused by a lack of SL signaling and is restored by treatment with GR24, an exogenous SL (Umehara et al., 2008). Therefore, we examined the effects of 4BD treatment in a rice hydroponic culture assay system for 47 d (Figure 3). We measured the number of tiller buds and the plant height every 10 d from day 17. The number of tillers on the 47-day-old rice d10 mutant was up to but that on the 47-day-old rice WT was about 5. The application of either 4BD or GR24 to the medium suppressed the tiller buds outgrowth on the 27-day-old rice to the level observed in the WT seedlings but the number of tillers in the rice d10 mutant treated with 1 μm GR24 increased in a later stage of growth relative to that in plants treated with 1 μm 4BD. As for the plant height, the rice d10 mutant was about 150 mm shorter than WT. However, the plant height of the d10 mutants was recovered to that of WT by the treatment with 4BD or GR24, respectively.

5 92 Fukui et al. Selective Mimics of Strigolactone Actions Effects of 4BD on Shoot Branching in Arabidopsis In Figures 2 and 3, we show the SL-like activity of 4BD in the rice plant, a model monocot. Next, we investigated whether 4BD inhibits shoot branching by controlling the SL signaling pathway in Arabidopsis, a model dicot. To examine the effects of 4BD on the increased-branching phenotype of the max3 mutant, which is defective in the SL biosynthesis gene CCD8, we used the hydroponic culture system reported by Umehara et al. (2008; Figure 3A and B). Our results indicate that 5 μm 4BD clearly inhibited the development of axillary shoots and the activity of 5 μm 4BD was similar to that of GR24 at the same concentration in this Arabidopsis assay. We then examined the effects of 4BD on other max mutants. MAX1 encodes a cytochrome P450 involved in SL biosynthesis, MAX4 encodes the SL biosynthetic protein CCD7, and MAX2 encodes an F-box protein that acts in the SL signaling pathway. Both the max1 and max4 mutants are sensitive to exogenous SL, but the max2 mutant is insensitive to it. Our data show that 4BD suppressed axillary shoot development in the SL biosynthesis mutants, but had no significant effect on that in the SL-insensitive mutant. GR24 produced similar results to those of 4BD. These data suggest that 4BD inhibits the enhanced branching of max mutants via the SL signaling pathway. Effects of 4BD on Root Formation in Arabidopsis Several studies have demonstrated that the root architecture of Arabidopsis is drastically altered in response to exogenously applied SLs, which inhibit lateral root formation and induce root hair elongation (Kapulnik et al., 2011; Koltai et al., 2011). In their studies, no SL-induced reduction in the lateral roots was apparent in the SL signaling mutant max2. However, SL increased the root hair length in the WT and the max3 and max4 mutants, but not in the max2 mutant. Because this response of the Arabidopsis roots shows high sensitivity to SLs and can only be observed within a short time after chemical treatment, we thought this biological system should be appropriate for the assessment of the activity of SL mimics. Therefore, we examined the effects of 4BD on the Arabidopsis root architecture (Figure 4A C). We measured the root hair length of Arabidopsis and compared the results with those of a 4BD-treated group (control) and a GR24-treated group. In the WT, 4BD treatment promoted the elongation of the root hairs more than no treatment, but the effect of 4BD was less marked than that of GR24. In the max2 mutant, neither 4BD nor GR24 significantly affected the elongation of the root hairs (Figure 4B). The effect of 4DB on the lateral root number was also evaluated. Treatment with 4BD reduced the lateral root number in the WT, as did the SLs reported in the previous paper (Kapulnik et al., 2011), but the effect of 4BD was not as potent as that of GR24. Unexpectedly, treatment with high concentrations of GR24 affected the lateral root growth in max2, although 4BD had no effect. The possible causes of this effect include the existence of another signaling pathway, other than that via MAX2, a leaky mutant allele that can be overridden by high concentrations of GR24, and the inhibition of lateral root formation by an adverse effect of GR24. Gene Expression in Arabidopsis Seedlings Treated with 4BD In terms of its morphological effects, the activity of 4BD was similar to that of GR24 on the aboveground parts of the plants, but it was less active than GR24 on the underground parts. To investigate the SL-like activity of 4BD at the transcript level in roots, we compared the relative expression levels of SL-related genes in Arabidopsis roots after treatment with each chemical, using quantitative polymerase chain reaction with reverse transcription (RT qpcr). Some SL-related genes have been identified and used as indicators of SL signaling (Mashiguchi et al., 2009; Waters et al., 2012). The expression of MAX3 was repressed and that of STH7 was activated by both 4BD treatment and GR24 treatment. The expression of other previously reported SL-related genes (MAX4, KUF1, and BRC1) was unaffected when the roots were treated with these chemicals under our experimental conditions (data not shown). Effects of 4BD on Root Parasitic Seed Germination and Its Agricultural Application As described above, 4BD functions as an SL mimic, affecting various aspects of the SL-related physiology of model plants, in both monocots and dicots. Because SL is a stimulator of seed germination in root parasitic weeds such as Striga and Orobanche, our interest next shifted to the effects of 4BD on root parasitic seed germination. This functional role of the SLs is so important in protecting crops from these parasites that it has been studied for a long time. We evaluated the stimulatory activity of 4BD on Striga hermonthica in a seed germination assay. Like GR24, almost all the SL analogs developed act as germination stimulants on root parasitic weeds, and their minimal effective concentrations for inducing the germination of S. hermonthica are less than nanomolar (Kondo et al., 2007; Mwakaboko and Zwanenburg, 2011). To explore the potential of debranone as a germination stimulant, we compared the germination rates of seeds treated with 4BD with those of seeds treated with GR24 at concentrations in the range of M (Figure 5A). GR24 activity potently induced germination, even at concentrations below 10 9 M. In contrast, 4BD did not stimulate germination, even at 10 6 M, although 4BD is more potent than GR24 in suppressing tiller bud outgrowth in rice. This result implies that 4BD cannot be used as a seed germination stimulant to induce suicidal germination in root parasitic weeds, but, alternatively, 4BD can be used as an SL mimic for specific target organisms. To demonstrate the usefulness of these characteristics of 4BD in agriculture, we propose a new method of parasite control, involving a combination of SL biosynthetic mutants and 4BD. In general, an SL-deficient plant is infected by fewer Striga plants (Dor et al., 2011; Jamil et al., 2012) but usually with a lower crop yield than the WT. However, 4BD treatment

6 Fukui et al. Selective Mimics of Strigolactone Actions 93 Figure 4. Effect of 4BD on Axillary Shoots of Arabidopsis max Mutants. (A) 30-day-old wild-type and max3 mutant with or without 5 μm chemical treatment. Scale bar indicates 5 cm. (B) Scale bar indicates number of axillary shoots in WT and max3 mutant with or without 5 μm chemical treatment. The data are means ± SE of 11 samples. (C) Scale bar indicates number of axillary shoots in WT, max1, max2, and max4 mutant with or without chemical treatment. The data are means ± SE of five or six samples. restores the mutant phenotype to the WT without stimulating Striga germination. On the basis of these findings, we compared the seed-germination-inducing activity of extracts from several groups of plants (Figure 5C): the 4BD-treated d10 mutant and the WT in hydroponic culture medium; the untreated d10 mutant and the WT in hydroponic culture medium; and the GR24-treated d10 mutant and the WT in hydroponic culture medium. The WT phenotype was restored to the 4BD- or GR24-treated d10 mutants, but extracts of the media from the 4BD-treated d10 mutant and the untreated d10 mutant media induced less S. hermonthica germination than did the medium from the untreated WT, whereas an extract of the medium from the GR24-treated d10 mutant strongly stimulated the germination of the parasite.

7 94 Fukui et al. Selective Mimics of Strigolactone Actions Figure 5. Effect of 4BD on Arabidopsis Root Architecture and on SL-Related Gene Expression. (A) Primary root segments of WT and max2 with or without 10 μm chemical treatment. Scale bars indicate 300 μm. (B) Scale bar indicates root hair length of 10-day-old Arabidopsis with or without 10 μm chemical treatment. The data are means ± SE of 100 samples. (C) Scale bar indicates lateral root number of 10-day-old Arabidopsis with or without 10 μm chemical treatment. The data are means ± SE of 10 samples. (D) Relative transcript levels of MAX3 in 2-week-old Arabidopsis roots with or without 5 μm chemical treatment for 24 h. The data are means ± SD of three samples. (E) Relative transcript levels of STH7 in 2-week-old Arabidopsis roots with or without 5 μm chemical treatment for 24 h. The data are means ± SD of three samples Discussion In this study, we have demonstrated the multiple SL-like activities of 4BD, such as its inhibition of shoot branching in both rice and Arabidopsis, its regulation of SL-regulated gene expression in Arabidopsis, its promotion of root hair elongation and inhibition of lateral root formation in Arabidopsis, and its stimulation of seed germination in S. hermonthica. In

8 Fukui et al. Selective Mimics of Strigolactone Actions 95 Figure 6. Application of 4BD to Striga Seed Germination. (A) Germination rate of Striga seed with chemical treatment at described concentration. The data are means ± SE of three samples. (B) 16-day-old wild-type (WT) and d10 mutant. Scale bars indicate second tiller length of seedlings with or without 1 μm chemical treatment. The data are means ± SE of six samples. (C) Estimation of germination stimulant levels in culture medium of 16-day-old WT and d10 rice seedlings with or without 1 μm chemical treatment using Striga seed. Culture mediums were combined in each group and used for extraction. The data are means ± SE of three samples. DW, distilled water. GR24 was used for positive standard at 1 μm. particular, 4BD strongly inhibited tiller bud outgrowth in the rice d10 mutant and this effect appeared to last longer than that of the SL analog GR24 (Figures 1 and 2). 4BD also inhibited axillary shoot branching in the Arabidopsis max mutants that are defective in SL biosynthesis, but not in the max2 mutant (Figure 3), which is insensitive to SLs. These results suggest that 4BD acts as an SL mimic in the signaling system that regulates shoot branching. The mrna transcript levels of the SL-regulated genes MAX3 and STH7 were regulated by both 4BD and GR24 treatments (Figure 4) in Arabidopsis. However, unlike its inhibition of shoot branching, 4BD was less active than GR24 in promoting the seed germination of S. hermonthica and root hair elongation in Arabidopsis (Figures 5 and 6). 4BD was also less active than GR24 in hyphal branching of AM fungi (personal communication from Dr Akiyama). These differences in the activities of 4BD and GR24 may be attributable to the difference in their structures. Historically, the SLs were first isolated as stimulants of seed germination in root parasitic weeds. Therefore, research into the design of chemicals that mimic the SL function(s) began with the synthesis of natural SL analogs (Johnson et al., 1981) and, until recently, the activity of the SL analogs has been evaluated by measuring their germination-stimulating activity in root parasitic weeds. The most commonly used SL analog, GR24, was thus synthesized and selected based on SAR studies of SL analogs in seed germination assays, using root parasitic weeds (Zwanenburg et al., 2009). These SAR studies revealed several structural requirements for a germination stimulant. A methylbutenolide ring as the D-ring and an enol ether moiety connecting the C-ring and D-ring were thought to be essential for the stimulation of seed germination in root parasitic weeds, but it was recently demonstrated that the imino ether moiety could replace the enol ether moiety (Kondo et al., 2007). According to the SAR of SL agonists involved in the hyphal branching of AM fungi, the SL structural requirement for hyphal branching is slightly different from that required for seed germination in root parasitic weeds (Akiyama et al., 2010). This result suggests that the structural requirements

9 96 Fukui et al. Selective Mimics of Strigolactone Actions for the different activities of SLs depend on the species or organ upon which the SLs act. In this context, there is nothing strange about these differences in the structural requirements for the inhibition of plant shoot branching and other physiological functions. 4BD, which was discovered as an inhibitor of shoot branching in an SL-deficient mutant, has a simple structure, constructed by a coupling reaction between phenol and bromofuranone. Therefore, 4BD is structurally different from other SLs in its lack of an enol ether moiety. This fact suggests that the structural requirements for SL activity in the inhibition of shoot branching are less strict than those for the other biological activities of SLs. In a recent paper, the SARs of various SL agonists demonstrated that SL analogs with a dimethylbutenolide D-ring were also active in inhibiting the branching of an SL-deficient pea mutant, but were less active than SL analogs containing methylbutenolide, the original D-ring of the SLs, in stimulating seed germination in root parasitic weeds (Boyer et al., 2012). These results and the data presented in our study suggest that one of the common structural requirements for SL functions is the D-ring, but that there is much room for the chemical modification of the D-ring. The activity of 4BD varied from subtle to potent, according to the assay system used in this report. It is not strange that the SL target and/or mechanism differ between plants and root parasites because the SL functions differ in the different stages of the plant life cycle. However, the activity of 4BD was distinct from that of GR24 in the Arabidopsis root hair elongation assay, despite the minimal difference between their activities in the Arabidopsis shoot branching assay (Figures 4 and 6). These results raise the question of whether the SL signaling pathway involved in root hair formation is distinct from that involved in shoot branching. At present, the components involved in root-specific SL signaling have not been well characterized, but at least MAX2 must act in this signaling pathway because the response of the max2 mutant to 4BD or GR24 is far smaller than that of the WT in the root hair elongation assay (Figure 4). Similarly, MAX2 is recognized as an essential component of the karrikin signaling pathway (Nelson et al., 2011). Thus, MAX2 is considered to be a common signaling component of the karrikin and SL signaling pathways. The karrikins and SLs contain similar molecular features, such as a butenolide substructure, and the karrikin signaling component KAI2 in Arabidopsis is a member of the α/β hydrolase superfamily, as is the SL signaling component D14 in rice (Waters et al., 2012). Because of the morphological differences between the kai2 mutant and the max1, max3, and max4 mutants, the KAI2 signaling pathway can be distinguished from the SL signaling pathway, in which D14 acts upstream from MAX2. Waters et al. demonstrated that the karrikins activate KAI2 signaling but not D14 signaling, whereas GR24 activates not only D14 signaling, but also KAI2 signaling. In this study, we have demonstrated that GR24 activates not only the SL signaling that underlies the inhibition of shoot branching, but also the SL signaling that underlies root hair development, whereas 4BD only slightly activates the SL signaling that regulates root hair development. In this context, we speculate that there might be a root-specific SL signaling pathway that is not strongly activated by 4BD and that SLs are general activators of these signaling pathways, as discussed above, whereas karrikins and 4BD are specific activators of the KAI2 pathway and the SL signaling pathway involved in branching inhibition, respectively. Further investigation, including the identification of the SL receptor(s) and the binding affinity of 4BD and the karrikins for this receptor, should reveal the mechanisms behind the selective activities of 4BD and the karrikins. Parasitic weeds belonging to the genera Striga and Orobanche invade the roots of host plants, and transfer nutrients and water from the hosts through their haustoria to themselves (Joel, 2000). Devastating losses in crop yields have been brought about in sub-saharan Africa, the Middle East, and Asia by parasitic weed infections in agricultural lands (Parker, 2009). Numerous practices have been developed to protect crops from parasite attack (Hearne, 2009). One promising method is the use of germination stimulants that cause the suicidal germination of seeds and reduce the number of root parasitic seeds in the soil (Zwanenburg et al., 2009). Germination inhibition by inhibitors of germination in root parasites and the reduction of SL biosynthesis in crops by inhibitors of SL biosynthesis may also be useful (Jamil et al., 2010; Ito et al., 2011), if the problem of expense can be resolved. Alternatively, the genetic control of root parasitic weeds through the development of resistant crops should be effective (Yoder and Scholes, 2010). Resistance to parasitic weeds can be induced for each stage of the parasite life cycle. For example, SL-deficient mutants are parasitized by fewer parasites than WT plants and, although haustorial resistance mutants are invaded by parasites, the invasion of their vascular systems by the haustoria is prevented (Gurney et al., 2006; Jamil et al., 2011). The use of cultivars resistant to root parasitic weeds should represent a cost-effective method of control, but this strategy is limited by our lack of resistant mutants and our lack of understanding of the molecular genetic basis of host resistance to root parasitic weeds. Based on the result shown in Figure 5, we propose a potential strategy for the control of root parasitic weed infections, involving the combined use of an SL-deficient mutant and 4BD. The development of an SL-deficient mutant is easier than the development of other cultivars resistant to root parasitic weeds because several SL biosynthetic genes have already been identified and the phenotype of such mutants is very distinct from that of the WT. 4BD can also restore the phenotype of an SL-deficient mutant without stimulating the germination of root parasitic weeds (Figure 5). With this combination, the 4BD treatment does not affect the resistance of the SL-deficient mutants to infection by the root parasitic weed but abolishes the phenotype of the SL-deficient mutant that causes a reduction in the crop yield. The foliar application of 4BD requires smaller amounts of chemical than soil applications. This is one of the

10 Fukui et al. Selective Mimics of Strigolactone Actions 97 advantages of this combinatorial method over the use of stimulators of suicidal germination. The combination of WT and 4BD can be an alternative way for the control of Striga germination because 4DB treatment induces feedback regulation of SL biosynthesis genes and would reduce SL production. However, the exogenous SL treatment to WT also inhibits normal branching outgrowth that is important for normal crop production. That is, 4BD treatment to WT will induce less branching phenotype and less crop yield. In this context, the combination of SL-deficient mutants and 4BD could be preferable. 4BD is a function-selective SL agonist. Therefore, as demonstrated above, we could control the infection of crops by root parasitic weeds with a combination of SL-deficient mutants and 4BD. At present, the mechanisms underlying the differences in the biological functions of 4BD and GR24, a general SL agonist, are unclear, but the identification and structural analysis of the SL receptor(s) should reveal the mechanisms of their selective biological functions at the molecular level. METHODS Chemicals GR24 was synthesized as described previously (Mangnus et al., 1992) to give four stereoisomers. We used (±)-(3aR*,8bS*,2 R*)- GR24 with the same relative stereochemistry as (±)-strigol. All debranones were prepared according to a previously described reaction (Fukui et al., 2011). Plant Materials Rice (Oriza sativa L.) tillering dwarf mutants d10-1 and its WT (cv. Shiokari) were used in this study (Ishikawa et al., 2005). Arabidopsis thaliana more axillary shoot mutants max1-1, max2-1, max3-9, max4-1, and its WT Columbia-0 were used in this study (Mashiguchi et al., 2009). Seed of S. hermonthica harvested in Sudan were kindly probided by Prof. A.E. Babiker (Sudan University of Science and Technology, Sudan). Rice Hydroponic Culture Rice seeds were sterilized in 2.5% sodium hypochlorite solution containing 0.01% Tween-20 for 30 min and washed with sterile water five times, then incubated in sterile water at 25 C in the dark for 2 d. Germinated seeds were transferred into hydroponic culture media (Umehara et al., 2008) solidified with 0.6% agar and cultured at 25 C under fluorescent white light with a 16-h light/8-h dark photoperiod for 7 d. Each seedling was transferred to a glass vial containing a 12-ml sterilized hydroponic culture solution with or without chemicals, and grown under the same conditions for 7 d. For prolonged cultivation, 17-day-old rice seedlings were transferred to a glass bottle containing a 500-ml sterilized hydroponic culture solution with or without chemicals, and grown under the same conditions for 30 d. Each chemical was added to the hydroponic culture media at the first time of transfer to the glass bottle. Each bottle was filled up with hydroponic culture media every 4 d. Arabidopsis Hydroponic Culture Arabidopsis seeds were surface-sterilized with 70% ethanol solution for 30 min, sown on half-strength Murashige and Skoog (MS) medium containing 1% sucrose and 0.7% agar, and placed for 2 d at 4 C (Murashige and Skoog, 1962). After that, seeds were grown up at 22 C under fluorescent white light with a 16-h light/8-h dark photoperiod for 15 d. Plants were then transferred to a plastic pot containing 230 ml hydroponic solution with or without chemicals and grown under the same conditions for an additional 15 d (Norén et al., 2004). The solution was renewed every 4 d. Arabidopsis Root Hair Elongation and Lateral Root Formation Assay Arabidopsis seeds were surface-sterilized with 70% ethanol solution for 30 min, sown on half-strength MS medium containing 1% sucrose and 0.7% agar, and placed for 2 d at 4 C (Murashige and Skoog, 1962). After that, seeds were grown up at 22 C under fluorescent white light with continuous light for 6 d. Then, plants were transferred to half-strength MS medium containing 1% sucrose, 0.7% agar, and chemicals, and grown under the same conditions for an additional 4 d. Plates were incubated vertically in the growing condition. Arabidopsis Relative Expression Levels of mrna Arabidopsis seeds were surface-sterilized with 70% ethanol solution for 30 min., sown on half-strength MS medium containing 1% sucrose and 0.7% agar, and placed for 2 d at 4 C (Murashige and Skoog, 1962). After that, seeds were grown up at 22 C under fluorescent white light with a 16-h light/8- h dark photoperiod for 15 d. Plants were then incubated in sterile water with or without chemicals for 1 d under the same conditions. Total RNA was extracted from roots using the Plant RNA Isolation Reagent (Invitrogen). For qrt PCR, total RNA was treated with DNase I to remove genomic DNA and 1 µg of it was reverse-transcribed using the PrimeScript RT reagent kit with gdna Eraser (TaKaRa). First-strand cdnas were diluted twofold with water and 1 µl of diluted cdna was used for PCR amplification of MAX3 (At2g42620), STH7 (At4g39070), and Act2 (At3g18780) fragments using genespecific primers. Actin expression was used as an internal standard. The primer sets used to amplify the transcripts were previously described sets (Mashiguchi et al., 2009). PCRs were performed with SYBR green I using a Thermal Cycler Dice Real Time System TP800 (TaKaRa). Striga Germination Assay The germination assay using S. hermonthica was performed as described previously (Sugimoto and Ueyama, 2008). For bioassay, de-ionized water and GR24 solution were used as negative and positive controls, respectively. Five ml of each

11 98 Fukui et al. Selective Mimics of Strigolactone Actions hydroponic culture solution was extracted with 1 ml of ethyl acetate three times, and organic phases were combined and dried in vacuo. Then, each sample was diluted with acetone and sterile water to achieve the same concentration as the hydroponic culture solution and used for bioassay. FUNDING Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) (grant to T.A.); Japan Society for the Promotion of Science Fellows (grant to K.F.); Core Research for Evolutional Science and Technology (CREST) Program of Japan Science and Technology Agency (JST) (grant to T.A.). AKNOWLEDGMENTS We thank Dr Junko Kyozuka for kindly providing rice SL mutants and Dr Abdelgabbar Eltayeb Babiker for kindly providing S. hermonthica seed. No conflict of interest declared. References Akiyama, K., Matsuzaki, K., and Hayashi, H. (2005). Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. 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