1. Brassinosteroids
Brassinolide (BL)
Brassinosteroids (BRs) are a family of about 70 structurally related compounds that contribute to:
Growth
Cell division, elongation, and differentiation
Stress tolerance
Reproductive development
Castasterone (CS)
Brassinolide is the most active compound by weight, but probably not synthesized in at least some monocots. Castasterone is abundant in some tissues and plants. Other less-active BRs are also produced.
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:

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

3. Characterization of det2 and cpd showed that BRs are essentialWT
det2
det2 + BL
cpd
In 1996, two Arabidopsis mutants, de-etiolated2 (det2) and constitutive photomorphogenic dwarf (cpd) were shown to be BR-deficient.
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 Arabidopsis dwarf1 mutant is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis. Plant Physiol. 119: ; 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: ; Nomura, T., et al., (1997). Blockage of brassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea. Plant Physiol. 113:

5. Multiple pathways for BR synthesis
DWF4
CPD
DET2
DWF1 / LKB / BRD2
Campesterol
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:

6. DWF4 encodes a CYP C22-oxidase
dwf4 wild-type
DWF4 encodes cytochrome P450 CYP90B1, a C22-oxidase. Loss-of-function mutants are dwarfed
DWF4
Four dwarf4 mutants
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: ; 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: ; Nomura, T., et al., (1997). Blockage of brassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea. Plant Physiol. 113:

7. BRI1 encodes the main BR receptor; bri1 mutants are BR-insensitive
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:
Wild-type
bri1
The Arabidopsis bri1 mutant is very small and insensitive to exogenous BR
1 cm
BRI1
bri1
Wild-type
In wild-type plants, root growth is inhibited by elevated BR levels
Scale bars in a and b are 1 cm.

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)
bri1
The BRI1 gene was identified through map-based cloning
Extracellular leucine-rich repeat (LRR)
Yellow indicates BR-binding domain
Cytosolic Kinase domain
Reprinted from Li, J., and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90: , with permission from Elsevier; Bishop, G.J., and Koncz, C. (2002). Brassinosteroids and plant steroid hormone signaling. Plant Cell 14: S

9. BRs bind to BRI1’s extracellular leucine-rich repeat domain
Top view
BRs bind through an induced fit mechanism
The LRR domain forms a helical solenoid
70 Å
Binding site
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: 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:

10. BRI1 signaling is negatively regulated by BKI1
Receptor
(BRI1)
Wild-type Arabidopsis
BKI1 overexpression
Receptor
(BRI1)
Inhibitor
(BKI1)
Co-receptor
(BAK1)
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: 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
A genetic screen was used to look for genes that partially reverted the bri1 mutant phenotype. In this case, an activation screen was used, so the identified protein is overexpressed. Overexpression of BAK1 partially reverts the bri1 mutant phenotype
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: 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:

13. Summary – BR receptor action
(BRI1)
Co-receptor
(BAK1)
Phosphorylated, dissociated inhibitor
Inhibitor
(BKI1)
Phosphorylated, activated kinase domain
Next step:
Signal transduction
Wang, X., and Chory, J. (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313: 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
BIN2
BRs
Inactive
Active
BIN2 is a protein kinase that is inactivated by BR signaling
When BIN2 is inactive, BZR1/BES1 are 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: 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: by permission of Oxford University Press

18. Brassinosteroids promote cell elongation
Wild-type
Receptor mutant
Cell elongation and microtubule organization are abnormal in a rice BR receptor mutant
BR application promotes hypocotyl elongation
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: 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:

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:

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: e 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 developmentWT
det2
det2 + BL
The original BR synthesis mutants were identified by their de-etiolated growth pattern in the dark
LIGHT
BRs
Light responsive genes and growth patterns
BRs interfere with light responses, and light interferes with BR synthesis and promotes its inactivation

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

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