Chemical Genetic Dissection of Brassinosteroid–Ethylene Interaction Gendron Joshua M. , Haque Asif , Gendron Nathan , Chang Timothy , Asami Tadao , Wang Zhi-Yong   Molecular Plant  Volume 1, Issue 2, Pages 368-379 (March 2008) DOI: 10.1093/mp/ssn005 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 μM BRZ. (B) Seedlings grown in the light for 10 d. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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)) or GA ((E)–(H)). Light microscope images (D) of histochemical staining of CPD::GUS transgenic plants grown on MS, BRP, and BRP with BL. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 μM BRZ, or 40 μM 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 μM BRZ, or 40 μM 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. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 μM ACC, 40 μM BRP, or 20 μM AgNO3) 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. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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 μM BRZ, and 20 μM ACC. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2008 1, 368-379DOI: (10.1093/mp/ssn005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions