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.
© 2014 American Society of Plant Biologists
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.
© 2014 American Society of Plant Biologists
Characterization of det2 and cpd
showed that BRs are essential
WT
det2
+ BL
det2
In 1996, two Arabidopsis
mutants, de-etiolated2 (det2) and
constitutive photomorphogenic
dwarf (cpd) were shown to be
BR-deficient.
WT
WT
det2 + BL
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.
© 2014 American Society of Plant Biologists
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: 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.
© 2014 American Society of Plant Biologists
Multiple pathways for BR synthesis
DWF1 / LKB /
BRD2
DET2
Campesterol
DWF4
DWF4
CPD
DET2
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.
© 2014 American Society of Plant Biologists
DWF4 encodes a CYP C22-oxidase
DWF4 encodes cytochrome
P450 CYP90B1, a C22oxidase. Loss-of-function
mutants are dwarfed
dwf4 wild-type
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: 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.
© 2014 American Society of Plant Biologists
BRI1 encodes the main BR receptor;
bri1 mutants are BR-insensitive
The Arabidopsis bri1 mutant
is very small and insensitive
to exogenous BR
1 cm
BRI1
Wild-type
bri1
Wild-type
1 cm
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.
In wild-type plants, root
growth is inhibited by
elevated BR levels
© 2014 American Society of Plant Biologists
The BRI1 receptor is a plasmamembrane localized receptor kinase
BRI1 is a Leucine-rich repeat –
receptor kinase (LRR-RK) (also
known as a LRR Receptor-Like
Kinase (LRR-RLK)
bri1
Extracellular leucine-rich repeat (LRR)
Yellow indicates BR-binding domain
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.
© 2014 American Society of Plant Biologists
BRs bind to BRI1’s extracellular
leucine-rich repeat domain
Top view
The LRR domain
forms a helical
solenoid
70 Å
Binding
site
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.
© 2014 American Society of Plant Biologists
BRI1 signaling is negatively
regulated by BKI1
Receptor
(BRI1)
Co-receptor
(BAK1)
Receptor
(BRI1)
Inhibitor
(BKI1)
Wild-type
BKI1
Arabidopsis 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
© 2014 American Society of Plant Biologists
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.
© 2014 American Society of Plant Biologists
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.
© 2014 American Society of Plant Biologists
Summary – BR receptor action
Receptor
(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 313:1118-1122. Reprinted with permission from AAAS
© 2014 American Society of Plant Biologists
Downstream of the receptor, BRs
activate transcription factors
Activated BRI1
kinase triggers a
kinase cascade
that activates
key 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
KEY OUTCOME OF BR SIGNALING
BIN2
BIN2 is a protein
kinase that is
inactivated by
BR signaling
When BIN2 is
inactive,
BZR1/BES1 are
active
BRs
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.
© 2014 American Society of Plant Biologists
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..
© 2014 American Society of Plant Biologists
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 BRregulated genes
© 2014 American Society of Plant Biologists
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
© 2014 American Society of Plant Biologists
Brassinosteroids promote cell
elongation
Wild-type
Receptor
mutant
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 LeiChoi). 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.
© 2014 American Society of Plant Biologists
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.
© 2014 American Society of Plant Biologists
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.
© 2014 American Society of Plant Biologists
BRs contribute to light-regulated
development
WT
det2
+ BL
det2
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
© 2014 American Society of Plant Biologists
BRs contribute to stress tolerance
STRESS
Tolerance
BRs
Antioxidants
BRs
Detox
Heavy
metals
(OUT)
Uptake
Heavy
metals
(IN)
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.
© 2014 American Society of Plant Biologists
Brassinosteroids – Summary
BRs are perceived
extracellularly by plasmamembrane localized coreceptors BRI1/BAK1
BRI1 activity is controlled
positively and negatively by
binding partners
BRs are
synthesized in
young, growing
tissues
Downstream signaling is
mediated by protein
kinases and phosphatases
Phosphorylated,
dissociated inhibitor
Many transcriptional targets
have been identified
TRANSCRIPTIONAL
AND OTHER
RESPONSES
© 2014 American Society of Plant Biologists
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 determines
when and where
BRs are
synthesized ?
What is the potential for
crop enhancement?
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
© 2014 American Society of Plant Biologists