Chemical Genetic Dissection of Brassinosteroid Ethylene Interaction

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1 Molecular Plant Volume 1 Number 2 Pages March 2008 RESEARCH ARTICLE Chemical Genetic Dissection of Brassinosteroid Ethylene Interaction Joshua M. Gendron a,b, Asif Haque b, Nathan Gendron b, Timothy Chang b, Tadao Asami c and Zhi-Yong Wang b,1 a Department of Biological Sciences, Stanford University, Stanford, CA b Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA c Department of Applied Biological Chemistry, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo , Japan ABSTRACT We undertook a chemical genetics screen to identify chemical inhibitors of brassinosteroid (BR) action. From a chemical library of 10,000 small molecules, one compound was found to inhibit hypocotyl length and activate the expression of a BR-repressed reporter gene (CPD::GUS) inarabidopsis, and it was named brassinopride (BRP). These effects of BRP could be reversed by co-treatment with brassinolide, suggesting that BRP either directly or indirectly inhibits BR biosynthesis. Interestingly, the compound causes exaggerated apical hooks, similar to that caused by ethylene treatment. The BRP-induced apical hook phenotype can be blocked by a chemical inhibitor of ethylene perception or an ethylene-insensitive mutant, suggesting that, in addition to inhibiting BR, BRP activates ethylene response. Analysis of BRP analogs provided clues about structural features important for its effects on two separate targets in the BR and ethylene pathways. Analyses of the responses of various BR and ethylene mutants to BRP, ethylene, and BR treatments revealed modes of cross-talk between ethylene and BR in dark-grown seedlings. Our results suggest that active downstream BR signaling, but not BR synthesis or a BR gradient, is required for ethylene-induced apical hook formation. The BRP-related compounds can be useful tools for manipulating plant growth and studying hormone interactions. INTRODUCTION Brassinosteroids (BRs) are a family of poly-hydroxylated steroid hormones that are involved in many aspects of plant growth and development. The biosynthesis of biologically active brassinosteroids involves a series of cytochrome P450 and steroid 5a-reductase enzymes. BRs bind to the extracellular domain of the leucine-rich-repeat receptor-like kinase (LRR RLK) BRI1 (Brassinosteroid Insensitive 1) (Li and Chory, 1997; Kinoshita et al., 2005) and activate its kinase function. BRI1 and its coreceptor BAK1 another LRR RLK (Li et al., 2002; Nam and Li, 2002) transduce the BR signal through an unknown mechanism to soluble downstream components. Two of these components BIN2 (BR-Insensitive 2) (Li et al., 2001; Li and Nam, 2002) and BSU1 (bri1 Suppressor 1) (Mora-Garcia et al., 2004), a GSK3 kinase and a Ser Thr phosphatase, respectively, control the phosphorylation states of a family of nuclear transcription factors that include BZR1 (Brassinazole Resistant 1) (Wang et al., 2002; He et al., 2005) and BES1 (bri1 EMS Suppressor 1), also known as BZR2(Yin et al., 2002, 2005). BZR1 and BZR2/BES1 were identified as dominant gain-of-function mutants that are resistant to brassinazole (BRZ) (a BR biosynthesis inhibitor) and suppress the dwarf phenotype of bri1 mutants. Phosphorylation of BZR1 and BZR2/BES1 inhibits their activity through multiple mechanisms, including proteasome degradation, cytoplasmic retention, and abolishment of DNA binding (Gampala et al., 2007; Gendron and Wang, 2007). BR-induced dephosphorylation activates BZR1 and BZR2/BES1 proteins, which directly regulate the transcription of BR-responsive genes. Ethylene is a gaseous hormone that regulates various developmental processes in plants. It is perceived by a small family of membrane-bound histidine kinase receptors that are similar to bacterial two-component histidine kinases. These include ETR1 (Ethylene Response 1), ERS1 (Ethylene Resistant 1), ETR2 (Ethylene Response 2), ERS2 (Ethylene Resistant 2), and EIN4 (Ethylene Insensitive 4) (Chang et al., 1993; Hua et al., 1995; Roman et al., 1995). Downstream of the receptors, a Ser Thr kinase, CTR1 (Constitutive Triple Response 1), negatively regulates ethylene signaling in the absence of ethylene; however, the substrates of CTR1 remain unknown (Kieber et al., 1993). Further downstream, EIN2 (Ethylene Insensitive 2), which contains a domain similar to an NRAMP-type metal ion transporter, is 1 To whom correspondence should be addressed. zywang24@stanford.edu, fax , tel , ext ª The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: /mp/ssn005

2 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction 369 essential for ethylene response, yet its biochemical role in the pathway remains unclear (Alonso et al., 1999). Finally, ethylene signaling controls transcriptional responses through the regulation of a family of transcription factors called EIN3 (Ethylene Insensitive 3) and the five EILs (EIN3 Like) (Chao et al., 1997). Interestingly, small molecule inhibitors of ethylene biosynthesis and perception have been identified. AgNO3 is one compound that can block the perception of ethylene by the receptors and is useful for chemical genetic studies of ethylene deficiency (Beyer, 1976). Chemical inhibitors of hormone synthesis or signaling are powerful tools widely used for studying hormone function. BRZ was used to identify BZR1 (Wang et al., 2002) and has been used to study the effects of BR on growth and development in various plant species (Asami et al., 2000). Other BR biosynthesis inhibitors have been discovered, including triadimefon (Asami et al., 2003), propiconazole (Sekimata et al., 2002), tebconazole (Sekimata et al., 2002), BRZ2001 (Sekimata et al., 2001), and spironolactone (Asami et al., 2004). All of these compounds contain a triazole functional group except for spironolactone, which is a steroid-like compound that presumably mimics BR and competes for association with BR biosynthesis enzymes. Chemical genetics has led to identification of components of several hormonal pathways, such as auxin signaling and transport, and vesicle trafficking. Sirtinol was used in a screen for mutants that alter the expression pattern of the auxin reporter, DR5-GUS, and enhance the phenotype of the yucca mutant (Grozinger et al., 2001; Zhao et al., 2003). Yokonolides A and B were identified as natural products of the soil organism Streptomyces diastatochromogenes that inhibit auxin-responsive gene expression (Hayashi et al., 2001, 2003). A screen for compounds that affect gravitropic responses identified multiple compounds that affect membrane trafficking in Auxindependent and independent manners (Surpin et al., 2005). A screen of a chemical library identified several compounds that block auxin-induced gene expression (Armstrong et al., 2004). These studies demonstrate the importance of small molecule screens in plant biology. We screened 10,000 small molecules to find inhibitors of BR action. Based on hypocotyl growth inhibition in the dark and activation of the BR-responsive reporter gene (CPD::GUS), we identified one compound that we named brassinopride (BRP). Physiological experiments using combinations of BRP, brassinolide, and BR- and ethylene-signaling mutants show that BRP inhibits BR action and promotes ethylene action. These experiments also provide new insights into BR and ethylene cross-talk in seedling development. RESULTS Screening a Chemical Library for Inhibitors of Brassinosteroid Action Inhibition of BR action causes dwarfism and increased expression of BR-repressed genes. To identify small molecule inhibitors of BR action, we screened a diverse set of 10,000 synthesized chemicals using the CPD::GUS transgenic line, which contains a BR-repressed CPD promoter driving the expression of b-glucuronidase (Mathur et al., 1998) (Figure 1A). The seeds were placed in 96-well plates containing halfstrength MS (MS) agar medium and individual chemicals at various concentrations in each well. Plants were grown in the dark for 5 d at room temperature and examined visually for decreased hypocotyl length. Seedlings showing hypocotyls shorter than wild-type grown on MS alone were placed in Figure 1. A Screen for BR Inhibitors. (A) Seeds of Arabidopsis transformed with the CPD::GUS reporter gene were grown for 5 d in the dark in 96-well plates on medium containing half MS and one of 10,000 compounds. Seedlings with short hypocotyls were then stained for GUS activity. Chemicals that cause darker GUS staining were further tested. One compound (BRP) caused morphology typical of a BR biosynthesis inhibitor. (B) BRP is similar in size to BRZ but does not contain the triazole functional group (highlighted in blue) that is necessary for BRZ function.

3 370 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction GUS substrate medium overnight. The histochemical staining pattern and intensity of GUS were examined and those that exhibited darker staining than the control were marked as positive hits for inhibitors of BR. Ninety chemicals caused short hypocotyls similar to BR inhibitors. To confirm the effects of the chemicals and determine whether they inhibit BR signaling or at a step upstream of BR signaling, we grew wild-type and bzr1-1d mutant plants on medium containing the chemicals and BR. If the wild-type plants were rescued by BR, then we classified the chemical as a potential biosynthesis inhibitor. If the wild-type plants were not rescued by application of BR, but were rescued by the bzr1-1d mutant, then the compound was classified as a possible BRsignaling inhibitor. Identification of Brassinopride After retesting, one compound caused effects similar to a BR biosynthesis inhibitor (Compound , Figure 1B). The hypocotyls of wild-type plants grown on the compound at 33.6 lm (the screening concentration) were one-third the height of those grown on MS medium, while the hypocotyls of bzr1-1d plants were two-thirds the height of those grown on MS medium, indicating that bzr1-1d is resistant to the effects of the compound. The GUS staining in the cotyledons of CPD::GUS plants treated with the compound was darker than the MS control (Figure 1A). These observations indicate that the compound inhibits BR action. The IUPAC name for compound is N-benzyl-N- (1-cyclopropylethyl)-4-fluorobenzamide. Due to the BR inhibition caused by the compound and the presence of a cyclopropyl side group and a di-benzyl amide backbone, we named thecompound Brassinopride (Brassinosteroid inhibitor + cyclopropyl + amide = Brassinopride (BRP)). Characterization of BRP To determine at which concentration BRP is most effective, we grew wild-type and bzr1-1d plants on BRP concentrations ranging from 0 to 500 lm in the dark (Figure 2A). The Figure 2A inset shows that the largest ratio of bzr1-1d to wild-type hypocotyl lengths occurs at 100 lm, but this is close to the saturation point for BRP and causes the plants to die. The concentration of BRP that causes 50% inhibition of hypocotyl length (IC50) is around 17 lm. Since the IC50 of BRP is around 17 lm and BRZ is around 1 lm (Asami et al., 2000), it seems that BRP is less active than BRZ. On 40 lm BRP, hypocotyl lengths of bzr1-1d and WT show a ratio of approximately two, which is similar to that on 2 lm BRZ (hypocotyl lengths on BRZ marked by arrows in Figure 2A). Additionally, we tested the effects of BRP on light-grown wild-type and bzr1-1d plants (Figure 2B). The effects of BRP on plant size were similar to those of BRZ. Increasing concentrations of BRP caused smaller rosettes, shorter hypocotyls and petioles, and shorter roots all typical dwarf phenotypes caused by BR deficiency. The bzr1-1d plants are less sensitive on BRP compared with wild-type, as shown by larger rosettes Figure 2. Dose Response Curves for BRP. Wild-type (WT) and bzr1-1d mutant plants were grown on MS medium containing various concentrations of BRP or BRZ. (A) Hypocotyl lengths of seedlings grown in the dark. Insert panel shows the ratio of height between bzr1-1d and Col-0 at the various concentrations of BRP. Arrows show hypocotyl lengths of WT and bzr1-1d on 2 lm BRZ. (B) Seedlings grown in the light for 10 d. and longer roots (Figure 2B). These data suggest that BRP has similar effects in both light- and dark-grown plants. BRP Affects BR without GA-Related Side Effects We tested whether BRP inhibits BR biosynthesis or signaling by co-treating wild-type and bzr1-1d with either 40 lm BRP or 1 lm BRZ and various concentrations of BL (Figure 3A 3D). Increasing concentrations of BL rescued the BRP-treated plants similarly to BRZ-treated plants (Figure 3B and 3C), suggesting that BRP inhibits BR biosynthesis. Additionally, we examined the expression of the BR marker gene, CPD, using the CPD::GUS transgenic line (Figure 3D). BRP increased CPD::GUS expression in the cotyledons, and this was reversed by co-treatment with BL, providing further evidence that BRP inhibits BR biosynthesis. The optimal BL concentration for rescue of both BRP and BRZ is 5 nm. As expected, bzr1-1d was not rescued by BR treatment and was slightly hypersensitive to BL, as shown by its shorter hypocotyls in BL-treated plants (Figure 3A 3C). These

4 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction 371 Figure 3. BRP Inhibits BR and not GA. Col-0 and bzr1-1d seedlings were grown in the dark on medium containing either no chemical (MS) ((A) and (E)), BRP ((B) and (F)), BRZ ((C) and (H)), or PAC (G) together with various concentrations of BL ((A) (C)) orga((e) (H)). Light microscope images (D) of histochemical staining of CPD::GUS transgenic plants grown on MS, BRP, and BRP with BL. results show that BRP has similar effects on hypocotyl elongation and BR-responsive gene expression as BRZ, and thus BRP most likely inhibits BR biosynthesis rather than signaling. To test whether BRP specifically inhibits BR or also affects other growth-promoting hormones such as gibberellic acid (GA), we co-treated wild-type and bzr1-1d with 40 lm BRP, 1 lm Paclobutrazol (PAC, a GA inhibitor) (Wang et al., 1986), or 1 lm BRZ with various concentrations of GA (Figure 3E 3H). Plants grown in the dark on regular medium showed no further hypocotyl elongation in response to treatment with increasing concentrations of GA (Figure 3E). Similarly, plants grown on BRP medium showed little response to GA (Figure 3F). In contrast, GA increased the hypocotyl length of both PAC-treated plants and BRZ-treated plants (Figure 3G and

5 372 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction 3H), indicating that PAC and BRZ inhibit GA synthesis but BRP does not. As a constitutive BR-response mutant, bzr1-1d responded similarly to GA as wild-type plants (compare Figure 3A 3C with 3G 3H). Reversibility and Inducibility of BRP and BRZ To further characterize BRP and the differences between BRP and BRZ, we tested the reversibility and inducibility of BRP and BRZ (Figure 4). To test reversibility, we grew wild-type plants on either 40 lm BRP or 1 lm BRZ in the dark for 3 d and then transferred half of the plants to MS medium and allowed them to grow for an additional 2 d in the dark. After transfer, plants that were initially grown on BRP had longer hypocotyls than those that were initially grown on BRZ, indicating that they recovered better than those grown on BRZ (Figure 4A). The increased reversibility of BRP suggests that it is metabolized faster than BRZ or that it has a lower affinity for its target, as suggested by its higher physiological IC50. To test the inducibility, we grew wild-type plants on MS medium for 2 d in the dark and then transferred 30 seedlings to MS containing either 40 lm BRP or 1 lm BRZ for an additional 3 d. In this case, the plants transferred to either BRP or BRZ had similar hypocotyl lengths. Thus, both compounds can affect brassinosteroid action after germination. BRP Affects Ethylene Production One characteristic of BRZ-treated or BR-deficient plants is the lack of apical hook formation (Chory et al., 1991; Li et al., 1996; Asami et al., 2000). Surprisingly, BRP shows the opposite effect on apical hook formation to BRZ. BRP causes an enhanced apical hook similar to that caused by ethylene or the ethylene precursor ACC (Figure 5A). In Arabidopsis, ethylene is known to cause the triple response phenotype in dark-grown seedlings: short hypocotyl, short roots, and enhanced apical hook (Guo and Ecker, 2004). Therefore, we tested whether part of the BRP phenotype is due to enhanced ethylene action (Figure 5). Wildtype and ein2-1 (an ethylene-insensitive mutant) (Guzman and Ecker, 1990) were grown on BRP, ACC (an ethylene precursor), and BRZ (Figure 5A), and hypocotyl length and apical hook angle were measured. Interestingly, ein2-1 showed no apical hook formation but increased hypocotyl length on BRP medium. Similar to the ein2-1 mutant, AgNO 3 significantly decreased the effects of BRP on the apical hook and partially reversed its inhibition of hypocotyl length (Figure 5B). In contrast, ein2-1 and AgNO 3 have little effect on hypocotyl lengths of plants grown on BRZ (Figure 5A and data not shown). These results suggest that enhanced ethylene action is responsible for the exaggerated apical hook and contributes partly to the short hypocotyl phenotype caused by BRP. To address the role of BR in formation of the apical hook in BRP-treated plants, wild-type plants were grown on MS, BRP, and BRZ with various concentrations of BL. BL was able to partially suppress the enhanced apical hook phenotype of BRPtreated plants, suggesting that BR does have a role in apical hook formation (Figure 5C). This is consistent with previous Figure 4. Phenotypic Effects of BRP are More Reversible than BRZ. (A) Col-0 plants were grown on regular medium (MS), or medium containing DMSO, 1 lm BRZ, or 40 lm BRP for 3 d in the dark and then half of the plants from the DMSO, BRZ, and BRP plates were transferred to regular medium and grown for 2 d in the dark. (B) Col-0 plants were grown on regular medium, DMSO, 1 lm BRZ, or 40 lm BRP for 2 d and then a portion of the plants on the regular medium plate was transferred to DMSO, BRZ, or BRP and grown for 3 more days. reports showing that ACC controls BR activity to induce apical hook formation (De Grauwe et al., 2005). BR Deficiency and Ethylene Agonism Can Lead To the Phenotypes Seen in BRP-Treated Plants To further test whether BRP functions by activating ethylene and inhibiting BR, wild-type, bzr1-1d, bzr1-1d;bes1-d, and ein2-1 plants were co-treated with 1 lm BRZ and various concentrations of ACC (Figure 6) in an attempt to mimic the BRP phenotype. BRZ and ACC have a similar inhibitory effect on hypocotyl elongation and an opposite effect on apical hook formation. Their effects on hypocotyl elongation are additive when both are applied to plants (Figure 6A). In contrast, the inhibitory effect of BRZ on apical hook formation can be cancelled by increasing concentration of ACC. As such, a combination of 1 lm BRZ and 5 lm ACC yielded similar phenotypes as 40 lm BRP, supporting that the phenotypes caused by BRP can

6 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction 373 Figure 5. BRP Promotes Apical Hook Formation by Activating the Ethylene Pathway. (A) Col-0 and ein2-1 plants were grown on ACC, BRZ, and BRP for 5 d in the dark. Hypocotyl length and apical hook angle were measured. (B) Col-0 plants were grown on the indicated compounds (20 lm ACC, 40 lm BRP, or 20 lm AgNO 3 ) for 5 d in the dark. Hypocotyl length and apical hook angle were measured. (C) Col-0 plants were grown on regular medium (MS), BRP (40 mm), and BRZ (1 mm) with various concentrations of BL for 5 d in the dark. Apical hook angles were measured. be due to inhibition of BR action and promotion of ethylene action. The ein2-1 mutant showed no response to ACC, but similar response as wild-type to BRZ, consistent with EIN2 s specific function in ethylene signaling. In contrast, the bzr1-1d;bes1- D double mutant and bes1-d single mutant (Figure 7A) were not only insensitive to BRZ and BRP, but also less sensitive to ACC in hypocotyl inhibition than wild-type and bzr1-1d (Figure 6A), suggesting that BES1 mediates ethylene inhibition of hypocotyl elongation. These results support that ethyleneinduced hypocotyl shortening is at least in part due to a reduction in BR action. This reduction in BR action can be suppressed by activating the BR pathway through stabilization of BES1 by the bes1-d mutation. In contrast, BR action on the hypocotyl is not mediated by ethylene, since ein2-1 shows normal sensitivity to BRZ treatment (Figure 6A). To further address the relationship between ethylene and BR in dark-grown seedlings, WT (Col-0 and WS), bzr1-1d, bes1-d (WS background), bzr1-1d;bes1-d, bzr1-1d;bri1-116 (a null allele of bri1), and ein2 were grown on MS, BRZ, ACC, and BRP (Figure 7). As expected, bzr1-1d, bes1-d, bzr1-1d;bes1-d, and bzr1-1d;bri1-116 were all resistant to BRZ and BRP in hypocotyl length and apical hook formation (Figure 7A and 7B). On ACC, only ein2-1, bes1-d, and bzr1-1d;bes1-d

7 374 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction mutant has no BR receptor for perception of a BR gradient, it still forms an apical hook similar to wild-type plants (Figure 7B), suggesting that perception of a BR gradient is not required for apical hook formation. The bzr1-1d mutation stabilizes BZR1 and activates the downstream BR-signaling pathway, which is sufficient for hook formation in the absence of BR perception. Therefore, the gradient of CPD expression observed previously (De Grauwe et al., 2005) is unlikely to contribute to the apical hook formation. Ethylene may function through modulating downstream BR signaling rather than BR biosynthesis to regulate apical hook formation. Alternatively, an active BR pathway might be required for sufficient ethylene synthesis or signaling, which is supported by the rescue of apical hook formation of BRZ-treated seedlings by increasing concentration of ACC (Figure 6B). Interestingly, the bzr1-1d;bri1-116 double mutant shows severe hypocotyl bending below the apical hook when the plants are grown on ACC (Figure 7C), suggesting that BR perception is required for maintaining a straight hypocotyl when the ethylene pathway is activated. Proper BR synthesis and signaling appear necessary for maintaining a straight hypocotyl in the presence of ethylene. These results support a complex interaction between BR and ethylene. Analogs of BRP with Altered Effectiveness and Specificity Give Hints about Functionality of Active Groups of BRP Figure 6. Simultaneously Increasing Ethylene and Decreasing BR Levels Mimics BRP Effects. Col-0, bzr1-1d, bzr1-1d;bes1-d, and ein2 plants were grown on the indicated chemicals for 5 d in the dark, and hypocotyl length (A) and apical hook angle (B) were measured. had longer hypocotyls than the wild-type controls, suggesting that ethylene may function through BES1, but not BZR1, to control cell expansion in the hypocotyl (Figure 7A). Interestingly, bzr1-1d, bes1-d, bzr1-1d;bes1-d, and bzr1-1d;bri1-116 were all slightly resistant to ACC in apical hook formation compared with wild-type (Col-0 and WS), confirming that proper BR action is necessary for ethylene-induced hook formation (Figure 7B). Together, these data suggest that ethylene functions partly through BR to regulate both hypocotyl length and apical hook formation, and it is likely that ethylene functions through the BES1-dependent branch of the BR-signaling pathway to control hypocotyl elongation. Previous reports of CPD::GUS expression on the outer side of the apical hook suggested that ethylene establishes a BR gradient in the apical hook that contributes to hook formation (De Grauwe et al., 2005). To test whether a gradient of BR is necessary to form an apical hook, the effect of a bri1-null mutant was examined. In the bri1-116;bzr1-1d double mutant, BR perception is defective but downstream BR responses are activated by the increased stability of BZR1. Although the double It is possible that different structural domains of BRP are important for its effect on BR and ethylene pathways. We used the program ChemMine (Girke et al., 2005) to search for analogs of BRP (Figure 8A). We grew wild-type and bzr1-1d plants on 12 analogs of BRP to test whether any analogs are specific to either BR or ethylene and to identify functional groups of interest on the BRP molecule (Figure 8). One of the analogs tested (a3) showed the same level of biological activity as BRP in both hypocotyl inhibition and apical hook formation (Figure 8A and 8B). Interestingly, a3 does not contain the cyclopropyl group present in BRP, suggesting that the cyclopropyl side group on BRP is not necessary for its biological activity. Furthermore, changes in the backbone and phenyl-based side group of the molecule appear to affect the specificity and effectiveness of these compounds. All compounds missing the alkane located next to the amide (a4, a5, a7, a9), an isopropyl or cyclopropyl side chain (a2, a5, a12), or one of the phenyl rings (a10, a11) are less effective. These observations indicate that the backbone, size, and shape of the molecule are important for the effectiveness of BRP. Compounds with nitro groups replacing the halogen on the phenyl ring (a4 and a8) have decreased effectiveness (Figure 8A and 8B), likely due to the decreased lipophilic nature of the nitro compounds, which could reduce membrane permeability. It would be of interest to find BRP-like molecules that specifically affect either BR or ethylene independently. None of the BRP analogs affected BR specifically, but one of them (a6) seems to have reduced effect on BR action. Wild-type plants grown on compound a6 showed reduced hypocotyl

8 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction 375 Figure 7. Interaction between BR and Ethylene in Dark-Grown Seedlings. Col-0, WS, bzr1-1d, bes1-d, bzr1-1d;bes1-d, bzr1-1d;bri1-116, and ein2-1 were grown on the indicated chemicals for five days in the dark, and the hypocotyl length (A) and apical hook angle (B) were measured. (C) Light microscope images of Col-0, bzr1-1d, and bzr1-1d;bri1-116 grown on MS, 1 lm BRZ, and 20 lm ACC. length and enhanced apical hooks (Figure 8A and 8B). However, the bzr1-1d mutation was less effective in suppressing the effect of a6 than that of BRP or a3, suggesting that a6 may be more specific to the ethylene pathway and less effective in BR inhibition than BRP. The a6 compound contains a urea group in place of the amide group in BRP. These results suggest that modifying the side groups of BRP can have specific effects on the activity of BRP in BR and ethylene functions. The results support the hypothesis that BRP has two targets in plants one involved in BR action and the other involved in ethylene action. DISCUSSION Based on inhibition of cell elongation and activation of BRrepressed gene expression, we identified BRP as a chemical that inhibits BR action. Abolishment of BRP s effects on hypocotyl inhibition and CPD::GUS expression by co-treatment with BR and by bzr1-1d mutation suggests that BRP inhibits BR biosynthesis rather than BR signaling. Interestingly, BRP also causes exaggerated apical hooks in dark-grown seedlings an effect similar to that of ethylene. Physiological experiments using ethylene mutants and treatment with ethylene (ACC) and an ethylene-perception inhibitor support that BRP promotes ethylene action at a step of or upstream of ethylene perception, possibly ethylene synthesis. These experiments establish BRP as a chemical with dual activities of inhibiting BR action and promoting ethylene action. Our experiments, using a combination of BRP with BR or ethylene mutants, further elucidate the mode of cross-talk between the BR and ethylene pathways. BRP is a Non-Triazole Type BR Inhibitor and Ethylene Agonist Most of the compounds known to inhibit BR biosynthesis are triazole-type cytochrome P450 inhibitors, or BR structural mimics such as spirinolactone. BRP is likely to represent a unique class of BR biosynthesis inhibitors that does not contain the traditional triazole structural component or structural similarities to BRs. It is interesting to note that compounds containing cyclopropyl groups have been found to interact with or be catalyzed by cytochrome p450 enzymes (Shaffer et al., 2002; Cryle et al., 2005), but our results suggest that the cyclopropyl group is not necessary for BRP s function in BR inhibition or ethylene agonism (Figure 8A, compound a3). Instead, the common factors necessary for BRP to inhibit BR are the amide backbone, the two benzene rings, and the presence of side chains on the amide backbone and the benzene ring. Interestingly, BRP has been patented (Patent # WO ) (Andersen et al., 2004) as an inhibitor of 11b-hydroxysteroid dehydrogenase in animals. It is possible that BRP also directly inhibits an enzyme involved in steroid modification in Arabidopsis.

9 376 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction Figure 8. BRP Analogs Provide Information about the Functional Groups of BRP. (A) Structures (left) of BRP and analogs a1 to a12 and hypocotyl lengths (right) of Col-0 and bzr1-1d seedlings grown on the indicated compounds in the dark for 5 d. (B) Apical hook angles of Col-0 grown on indicated compounds in the dark for 5 d.

10 Gendron et al. d Dissection of Brassinosteroid Ethylene Interaction 377 BRP Affects the BR and Ethylene Pathway Our data show that BRP not only inhibits BR action, but also activates the ethylene pathway. BRP is likely to increase ethylene synthesis because the ethylene-like effects of BRP are abolished by AgNO 3, which blocks ethylene perception. Furthermore, the BRP-like effects can be mimicked when BRZ is combined with ACC treatment. BRP does contain a cyclopropyl side group that is structurally similar to ACC, but analyses of analogs suggest that the cyclopropyl group is not necessary for BRP s function in ethylene agonism. One possibility is that the alkane linked to the amide backbone and the cyclopropyl can be metabolized either to ACC or ethylene. Alternatively, BRP may directly affect an enzyme in ethylene synthesis. Genetic studies of mutants in the ethylene biosynthesis pathway or direct measurements of ethylene and its precursors in BRP-treated plants will elucidate how BRP acts on the ethylene pathway. Further studies of more varied analogs of BRP could provide more information about the functional groups of BRP and lead to identification of compounds that specifically affect BR or ethylene. BR and Ethylene Interact to Regulate Seedling Development in the Dark The interactions between hormone pathways have become a focus for many labs studying hormone signaling, and the identification of small molecules affecting multiple pathways will aid in the dissection of hormone cross-talk. Paclobutrazol is an example of one small molecule that inhibits both GA and ABA synthesis in plants (Wang et al., 1986, 1987) and ABA synthesis in Cercospora rosicola (a fungal plant pathogen, Norman et al., 1986). BRZ inhibits the synthesis of both BR and GA. BRP is a unique compound that acts on both BR and ethylene in plants. The dual role of BRP in ethylene and BR function makes BRP a useful tool for investigating BR ethylene cross-talk. Recent studies have shown that BR and ethylene have overlapping functions in hypocotyl elongation and apical hook formation (De Grauwe et al., 2005). It was suggested that ethylene controls the biosynthesis of BRs and establishes a gradient of BR in the apical hook region that contributes to the hook formation. Our results refine this conclusion by showing that a null bri1 mutant crossed to the bzr1-1d gain-of-function mutant forms an apical hook similar to wild-type. In this case, BR perception is negated by the lack of BRI1, but the downstream pathway is activated by the bzr1-1d mutation, which stabilizes the BZR1 protein. Therefore, perception of a gradient of BR appears to be not necessary to form a proper apical hook; rather, it is likely the activation of the BR-signaling pathway that is required. Ethylene may control the gradient of activity of BR signaling downstream of BRI1, or activation of certain BR responsive genes by BZR1 is required for ethylene-promoted apical hook formation. Our results showing that the bzr1-1d;bes1-d double mutant is partially resistant to ACC in inhibiting hypocotyl elongation suggest that ethylene inhibition of hypocotyls requires normal BR response. Yet, BR does not seem to act through ethylene in the hypocotyl, since ein2-1 is not resistant to BRZ. Therefore, our results support that ethylene reduces BR action to inhibit hypocotyl elongation, which supports that ethylene, at least in part, acts by recruiting the functions of other hormones to control hypocotyl elongation and hook formation (De Grauwe et al., 2005). It is possible, though, that BR also regulates ethylene action, as BR has been shown to regulate the level of an ACC oxidase (Deng et al., 2007). Further chemical genetic studies using BRP as a tool will shed light on the molecular mechanisms of BR ethylene cross-talk in seedling development. METHODS Plant Materials and Growth Conditions Arabidopsis thaliana ecotype Columbia was used for all experiments (except bes1-d, which is in WS background). Seeds were sterilized in 75% ethanol with 0.01% Triton X-100. Seeds were sown on 0.5 Murashige and Skoog (MS) media containing 0.8% Phytoblend (Caisson, Utah, USA). All seeds were coldtreated for 48 h, placed in the light overnight, and then grown on vertical plates (except screen plates and light-grown plants) in the dark at room temperature for 4 d. Light-grown plants were transferred from the cold to the light and grown on horizontal plates for 10 d. Chemical Library Screen The DIVERSet library (ChemBridge, San Diego, CA), containing 10,000 organic molecules in 96-well-plate format, was used for the screen. 0.1 mg of each compound was dissolved in 20 llof DMSO and then diluted 5-fold in water to approximate concentrations of 2 4 mm in 20% DMSO. 1 ll of the diluted compound was then aliquotted to new 96-well plates and diluted in 99 ll of warm 0.5 MS and 0.8% Phytoblend, which was allowed to solidify before seeds were sown. Approximately 10 CPD::GUS transgenic seeds were placed in each well, and part of each plate was used for MS, BL, and BRZ controls. The plants were screened visually for hypocotyl length, apical hook formation, and cotyledon opening. Histochemical staining was used for the secondary screen. Approximately half of the seedlings from the wells that had plants with short hypocotyls were placed directly in GUS substrate media. 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