Brassinosteroids

1. Brassinosteroids • Brassinosteroids (BRs) are a family of about 70 structurally related compounds that contribute to: • Growth • Cell division, elongation, and differentiation • Stress tolerance • Reproductive development Brassinolide (BL) Castasterone (CS) BR structures are drawn in Vriet, C., Russinova, E. and Reuzeau, C. (2013). From squalene to brassinolide: The steroid metabolic and signaling pathways across the plant kingdom. Mol. Plant. 6: 1738-1757.

2. Brassinolide and castasterone are steroids like some animal hormones Plant Mammal Insect Bishop, G.J., and Koncz, C. (2002). Brassinosteroids and plant steroid hormone signaling. Plant Cell 14:S97-110.

3. Characterization of det2 and cpd showed that BRs are essential In 1996, two Arabidopsis mutants, de-etiolated2 (det2) and constitutive photomorphogenic dwarf (cpd) were shown to be BR-deficient. WT det2 + BL WT WT det2 + BL det2 det2 cpd From Li, J. et al. (1996) A role for brassinosteroids in light-dependent development of Arabidopsis. Science 272: 398–401; reprinted with permission from AAAS. Reprinted from Szekeres, M. et al. (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85: 171–182 with permission from Elsevier.

4. Biosynthesis and homeostasis BR-deficient plants are severely dwarfed Choe, S., et al. (1999). The Arabidopsisdwarf1 mutant is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis. Plant Physiol. 119:897-908; Hong, Z., et al. (2005). The rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, is rescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone. Plant Cell 17:2243-2254; Nomura, T., et al., (1997). Blockage of brassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea. Plant Physiol. 113: 31-37.

5. Multiple pathways for BR synthesis DET2 DWF1 / LKB / BRD2 DWF4 CPD Campesterol DET2 DWF4 DET2 CPD Castasterone Brassinolide Reprinted from Ohnishi, T., Godza, B., Watanabe, B., Fujioka, S., Hategan, L., Ide, K., Shibata, K., Yokota, T., Szekeres, M. and Mizutani, M. (2012). CYP90A1/CPD, a Brassinosteroid biosynthetic cytochrome P450 of Arabidopsis, catalyzes C-3 oxidation. J. Biol. Chem. 287: 31551-31560.

6. DWF4 encodes a CYP C22-oxidase DWF4 encodes cytochrome P450 CYP90B1, a C22-oxidase. Loss-of-function mutants are dwarfed DWF4 Four dwarf4 mutants dwf4 wild-type Azpiroz, R., Wu, Y., LoCascio, J.C., and Feldmann, K.A. (1998). An Arabidopsis brassinosteroid-dependent mutant Is blocked in cell elongation. Plant Cell 10:219-230; Choe, S., Dilkes, B.P., Fujioka, S., Takatsuto, S., Sakurai, A., and Feldmann, K.A. (1998). The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10:231-244; Nomura, T., et al., (1997). Blockage of brassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea. Plant Physiol. 113: 31-37.

7. BRI1 encodes the main BR receptor; bri1 mutants are BR-insensitive bri1 1 cm BRI1 Wild-type Wild-type The Arabidopsis bri1 mutant is very small and insensitive to exogenous BR 1 cm In wild-type plants, root growth is inhibited by elevated BR levels bri1 bri1 Clouse, S.D., Langford, M., and McMorris, T.C. (1996). A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol. 111: 671-678.

8. The BRI1 receptor is a plasma-membrane localized receptor kinase BRI1 is a Leucine-rich repeat – receptor kinase (LRR-RK) (also known as a LRR Receptor-Like Kinase (LRR-RLK) Extracellular leucine-rich repeat (LRR) Yellow indicates BR-binding domain bri1 Cytosolic Kinase domain The BRI1 gene was identified through map-based cloning Reprinted from Li, J., and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90:929-938, with permission from Elsevier; Bishop, G.J., and Koncz, C. (2002). Brassinosteroids and plant steroid hormone signaling. Plant Cell 14:S97-110.

9. BRs bind to BRI1’s extracellular leucine-rich repeat domain Top view The LRR domain forms a helical solenoid Binding site 70 Å BRs bind through an induced fit mechanism Side view Reprinted by permission from Macmillan Publishers Ltd: She, J., Han, Z., Kim, T.-W., Wang, J., Cheng, W., Chang, J., Shi, S, Wang, J. ,Yang, M., Wang, Z.-Y., and Chai, J. (2011). Structural insight into brassinosteroid perception by BRI1. Nature 474: 472-477.Hothorn, M., Belkhadir, Y., Dreux, M., Dabi, T., Noel, J.P., Wilson, I.A., and Chory, J. (2011). Structural basis of steroid hormone perception by the receptor kinase BRI1. Nature 474:467-471.

10. BRI1 signaling is negatively regulated by BKI1 Receptor (BRI1) Receptor (BRI1) Inhibitor (BKI1) Co-receptor (BAK1) Wild-type Arabidopsis BKI1 overexpression BKI1 was identified through a search for proteins that physically interact with BRI1 Inhibitor (BKI1) Wang, X., and Chory, J. (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313:1118-1122. Reprinted with permission from AAAS

11. BRI1 signaling requires activation by a coreceptor BAK1 BAK1 was identified by an activation screen – a screen for plants that partially reverse the dwarf bri1-5 phenotype when overexpressed Upon BR binding, BRI1/BAK1 auto- and transphosphorylate (red circles), fully activating BRI1 kinase activity Reprinted from Li, J., Wen, J., Lease, K.A., Doke, J.T., Tax, F.E., and Walker, J.C. (2002). BAK1, an Arabidopsis LRR Receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110:213-222 with permission from Elsevier.

12. BRI1 and BAK1 interact through a “double lock mechanism” Unoccupied BRI1: Extracellular and cytoplasmic domains prevent BAK1 interaction Occupied BRI1: Phosphorylation of BKI1 causes it to dissociate, and interactions in both domains of BRI1 promote association with coreceptor BAK1 Reprinted by permission from Jaillais, Y., Belkhadir, Y., Balsemão-Pires, E., Dangl, J.L. and Chory, J. (2011). Extracellular leucine-rich repeats as a platform for receptor/coreceptor complex formation. Proc. Natl. Acad. Sci. USA 108: 8503-8507.

13. Summary – BR receptor action Receptor (BRI1) Co-receptor (BAK1) Phosphorylated, dissociated inhibitor Inhibitor (BKI1) Next step: Signal transduction Phosphorylated, activated kinase domain Wang, X., and Chory, J. (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313:1118-1122. Reprinted with permission from AAAS

14. Downstream of the receptor, BRs activate transcription factors When transcription factors BZR1 and BES1are phosphorylated (left) they are inactive and degraded or sequestered. When dephosphorylated they are active and move into nucleus Activated BRI1 kinase triggers a kinase cascade that activates key transcription factors KEY OUTCOME OF BR SIGNALING BRs BIN2 BIN2 is a protein kinase that is inactivated by BR signaling When BIN2 is inactive, BZR1/BES1 are active Inactive Active Reprinted from Belkhadir, Y., Yang, L., Hetzel, J., Dangl, J.L. and Chory, J. (2014). The growth–defense pivot: crisis management in plants mediated by LRR-RK surface receptors. Trends Biochem. Sci. 39: 447-456 with permission from Elsevier.

15. BIN2 kinase targets BES1, BZR1 and other transcription factors BIN2 can integrate BR signals with other signals to effect its diverse roles Reprinted from Guo, H., Li, L., Aluru, M., Aluru, S. and Yin, Y. (2013) Mechanisms and networks for brassinosteroid regulated gene expression. Curr. Opin. Plant Biol. 16: 545–553 with permission from Elsevier..

16. BR signaling - summary • BR is perceived by a complex of plasma-membrane localized co-receptors: BRI1/BAK1 • In the absence of BR, BRI1 is inactivated by its interaction with BKI1 • The BRI1/BAK1 co-receptor complex initiates a signaling cascade that leads to BIN2 dephosphorylation and inactivation • BIN2 targets several transcription factors including BES1 and BZR1. When BIN2 is inactive, BES1 and BZR1 move into the nucleus move activate or repress BR-regulated genes

17. BRs in whole-plant processes • BRs regulate diverse processes that include cell elongation, growth, developmental patterning and cell differentiation • Many BR effects are mediated by cross-talk with other hormones, or transcriptional responses Reprinted from Yang, C.-J., Zhang, C., Lu, Y.-N., Jin, J.-Q. and Wang, X.-L. (2011). The mechanisms of brassinosteroids' action: From signal transduction to plant development. Mol. Plant. 4: 588-600 by permission of Oxford University Press

18. Brassinosteroids promote cell elongation Receptor mutant Wild-type BR application promotes hypocotyl elongation Cell elongation and microtubule organization are abnormal in a rice BR receptor mutant Wang, T.W., Cosgrove, D.J., and Arteca, R.N. (1993). Brassinosteroid stimulation of hypocotyl elongation and wall relaxation in pakchoi (Brassica chinensis cv Lei-Choi). Plant Physiol. 101: 965-968. Yamamuro, C., Ihara, Y., Wu, X., Noguchi, T., Fujioka, S., Takatsuto, S., Ashikari, M., Kitano, H., and Matsuoka, M. (2000). Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina Joint. Plant Cell 12: 1591-1606.

19. Brassinosteroids are necessary for stamen and pollen development Filament elongation Pollen development Pollen tube elongation Image credits: Safro; Graham Matthews; Szumlanski, A.L., and Nielsen, E. (2009). The Rab GTPase RabA4d regulates pollen tube tip growth in Arabidopsis thaliana. Plant Cell 21: 526-544.

20. BRs are at the center of multiple interactions with other factors Oh, E., Zhu, J.-Y., Bai, M.-Y., Arenhart, R.A., Sun, Y. and Wang, Z.-Y. (2014). Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl. eLife. 3:e03031. See also Wang, W., Bai, M.-Y. and Wang, Z.-Y. (2013) The brassinosteroid signaling network — a paradigm of signal integration. Curr. Opin. Plant Biol. 21: 147–153.

21. BRs contribute to light-regulated development The original BR synthesis mutants were identified by their de-etiolated growth pattern in the dark WT det2 + BL BRs LIGHT Light responsive genes and growth patterns det2 BRs interfere with light responses, and light interferes with BR synthesis and promotes its inactivation

22. BRs contribute to stress tolerance BRs contribute to the production of antioxidants, which protect cells from damage. STRESS BRs BRs may also interfere with the uptake of heavy metals and promote their detoxification through antioxidant production and other means. Tolerance Antioxidants BRs Detox Heavy metals (OUT) Heavy metals (IN) Uptake CELLULAR DAMAGE

23. Brassinosteroids – Summary BRs are perceived extracellularly by plasma-membrane localized co-receptors BRI1/BAK1 BRI1 activity is controlled positively and negatively by binding partners BRs are synthesized in young, growing tissues Phosphorylated, dissociated inhibitor Many transcriptional targets have been identified TRANSCRIPTIONAL AND OTHER RESPONSES Downstream signaling is mediated by protein kinases and phosphatases

24. Brassinosteroids – Ongoing research What are the roles of the other BRI1-LIKE receptors and SERK co-receptors? What about the other LRR-RLKs that BAK1 interacts with? What determines how the BR signal integrates with other signals (PHYs, DELLAs)? BRs are synthesized in young, growing tissues What is the potential for crop enhancement? What determines when and where BRs are synthesized ? TRANSCRIPTIONAL AND OTHER RESPONSES Reprinted from Wang, W., Bai, M.-Y. and Wang, Z.-Y. (2013) The brassinosteroid signaling network — a paradigm of signal integration. Curr. Opin. Plant Biol. 21: 147–153 with permission from Elsevier