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Brassinosteroid Signal
Transduction from
Receptor Kinases to
Transcription Factors
Tae-Wuk Kim and Zhi-Yong Wang
Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305;
email: [email protected]
Annu. Rev. Plant Biol. 2010. 61:681–704
Key Words
First published online as a Review in Advance on
February 5, 2010
Arabidopsis, GSK3 kinase, plant hormone, receptor kinase, signal
transduction, steroid hormone
The Annual Review of Plant Biology is online at
plant.annualreviews.org
This article’s doi:
10.1146/annurev.arplant.043008.092057
c 2010 by Annual Reviews.
Copyright All rights reserved
1543-5008/10/0602-0681$20.00
Abstract
Brassinosteroids (BRs) are growth-promoting steroid hormones in
plants. Genetic studies in Arabidopsis illustrated the essential roles of BRs
in a wide range of developmental processes and helped identify many
genes involved in BR biosynthesis and signal transduction. Recently,
proteomic studies identified missing links. Together, these approaches
established the BR signal transduction cascade, which includes BR perception by the BRI1 receptor kinase at the cell surface, activation of
BRI1/BAK1 kinase complex by transphosphorylation, subsequent phosphorylation of the BSK kinases, activation of the BSU1 phosphatase,
dephosphorylation and inactivation of the BIN2 kinase, and accumulation of unphosphorylated BZR transcription factors in the nucleus.
Mass spectrometric analyses are providing detailed information on the
phosphorylation events involved in each step of signal relay. Thus, the
BR signaling pathway provides a paradigm for understanding receptor kinase–mediated signal transduction as well as tools for the genetic
improvement of the productivity of crop plants.
681
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Contents
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INTRODUCTION . . . . . . . . . . . . . . . . . .
RECEPTOR ACTIVATION . . . . . . . . .
BRI1 Perceives BRs at the Cell
Surface . . . . . . . . . . . . . . . . . . . . . . . . .
BR-Induced Receptor Kinase
Dimerization and
Oligomerization . . . . . . . . . . . . . . . .
Team Play and Multitasking
of the Receptor Kinases . . . . . . . . .
Regulation by Auto- and
Transphosphorylation . . . . . . . . . . .
Endocytosis of BRI1 and BAK1 . . . . .
Regulation by BKI1 . . . . . . . . . . . . . . . .
SIGNALING OUTPUT FROM
THE RECEPTOR KINASES . . . . .
BZR1 AND BZR2/BES1:
KEY TRANSCRIPTION
FACTORS FOR
BR-REGULATED GENE
EXPRESION . . . . . . . . . . . . . . . . . . . . .
REGULATION OF BZR1 AND
BZR2/BES1 BY BIN2-MEDIATED
PHOSPHORYLATION . . . . . . . . . . .
REGULATION OF BIN2 BY
BSU1-MEDIATED TYROSINE
DEPHOSPHORYLATION AND
PROTEASOME-MEDIATED
DEGRADATION . . . . . . . . . . . . . . . . .
CONCLUDING REMARKS . . . . . . . . .
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INTRODUCTION
Steroids have long been known as hormones
in metazoans (85). Their hormonal activity in
plants was only discovered about 30 years ago,
when a growth-promoting activity in pollen
extract of oilseed rape (Brassica napus) was attributed to a steroid compound named brassinolide (BL) (24). Since then, steroids with
similar structure and activities as BL have
been identified in a variety of plants and are
collectively called brassinosteroids (BRs) (13).
While hormone treatments demonstrated
many physiological and developmental responses induced by exogenous BR, the essential
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function of BRs in plant development was not
widely appreciated until BR-deficient and BRinsensitive mutants were identified in a relative of B. napus, the model organism Arabidopsis thaliana (12, 49, 81). These mutants display
many growth defects, including dwarfism, dark
green leaves, delayed flowering, male sterility,
and photomorphogenesis in complete darkness.
Now BRs are considered essential hormones
that regulate a wide range of developmental and
physiological processes, such as cell elongation,
vascular differentiation, root growth, responses
to light, resistance to stresses, and senescence.
Arabidopsis mutants identified not only BR
biosynthetic enzymes but also signaling proteins that perceive and transduce the BR signal.
Forward genetic screens in Arabidopsis identified multiple alleles of two loci that cause BR
insensitivity, bri1 and bin2. BR INSENSITIVE
1 (BRI1) encodes one of over 200 members
of the leucine-rich repeat-receptor-like kinase
(LRR-RLK) family in Arabidopsis (79), and BR
INSENSITIVE 2 (BIN2) encodes one of the 10
glycogen synthase kinase 3 (GSK3)-like kinases
of Arabidopsis (50). Sensitized genetic screens
using the BR biosynthetic inhibitor brassinazole (BRZ) or weak alleles of bri1 as genetic
background identified additional components
of the pathway, including the BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMSSUPPRESSOR 1 (BES1) transcription factors
(93, 97), BRI1-ASSOCIATED RECEPTOR
KINASE 1 (BAK1) (52, 60), and bri1SUPPRESSOR 1 (BSU1) phosphatase (57)
(Figure 1). BES1 is 89% identical to BZR1 and
was also named BZR2 (93). BAK1 was also identified by yeast two-hybrid screening as a BRI1interacting protein (60).
Additional components of the BR signal transduction pathway have been identified as proteins that interact with these
genetically defined components. The BRI1interacting proteins include BAK1 (60), BRI1
KINASE INHIBITOR 1 (BKI1) (89), and
TRANSTHYRETIN-LIKE (TTL) (61). The
14-3-3 proteins were identified as BZR1- and
BZR2/BES1-interacting proteins (21, 70). In
addition, there is evidence that BES1/BZR2
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BRI1
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LRR1
BAK1
LRR20
LZ
ID
LRR1
LRR21
P -Tyr-831
P -Thr-842
P -Thr-872
P -Thr-880
P -Ser-887
P -Tyr-956
P -Thr-982
P -Ser-1044
P -Thr-1045
P -Thr-1049
P -Tyr-1052
P -Tyr-1057
P -Tyr-1072
LRR24
Transphosphorylation
TM
P -Ser-838
JM
P -Thr-846
P -Ser-290
P -Ser-858
P -Thr-312
KD
LRR5
Pro-rich
TM
P -Thr-446
P -Thr-449
P -Ser-286
KD
AL
P -Thr-450
AL
P -Thr-455
P -Ser-1166
CT
P -Thr-1180
P -Ser-604
P -Ser-612
P -Ser-1162
P -Ser-1168
Figure 1
The structures of BRI1 and BAK1. BRI1 is composed of 24 leucine-rich repeats (LRR) and a 70-amino-acid
island domain (ID), single-pass transmembrane region (TM), juxtamembrane region ( JM), kinase domain
(KD), and C-terminal (CT) region. BAK1 contains 4 leucine zippers (LZ), 5 LRRs, and a proline-rich region
(pro-rich), single-pass TM, KD, and CT. The putative signal peptide region is shown as a black box and
unassigned regions are shown as gray boxes. The activation loop of kinases is designated AL. The confirmed
phosphorylation sites are marked with circles, and putative phosphorylation sites are marked with squares
containing the letter P. The activation phosphorylation sites are shown in red, inhibitory sites in blue, and
residues without significant effect on the kinase activity or not examined experimentally in yellow.
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interacts with other transcription factors, including BIM1, ELF6, and Myb30 (53, 96, 98).
Proteomic studies identified additional components that filled the last gap in the pathway (84),
and biochemical studies assembled these components into a phosphorylation cascasde that
connects BR perception at the cell surface to activation of transcription factors in the nucleus
(42). With the process in hand, detailed biochemical studies are revealing molecular mechanisms of receptor kinase activation (91, 92),
signal relay, and regulation of the BZR transcription factors by phosphorylation (21, 70,
86). BR regulation of gene expression mediated by the BZR transcription factors has been
covered in recent reviews (87). This review focuses on how the BR signal is transduced from
the cell surface receptor kinases to the nuclear
transcription factors.
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RECEPTOR ACTIVATION
BRI1 Perceives BRs at the Cell Surface
The brassinosteroid insensitive 1 (bri1) mutant was
identified as a dwarf insensitive to BR treatment (12, 40). Additional bri1 alleles helped to
clone the BRI1 gene, which encodes an LRRRLK (47). The extracellular domain of BRI1
contains 24 leucine-rich repeats (LRRs) and
an island domain (ID) between the twentieth
and twenty-first LRRs; BRI1’s cytoplasmic domain contains a serine/threonine (Ser/Thr) kinase domain, a juxtamembrane region ( JM),
and the C-terminal extension (CT) (47, 87)
(Figure 1).
The function of BRI1 as the BR receptor
has been demonstrated by several lines of experimental evidence. These include a chimeric
receptor of BRI1-XA21 conferring BR-induced
defense responses (29), binding of BRI1 immunoprecipitated from Arabidopsis plants (94)
or expressed and purified in Escherichia coli
(43) to tritium-labeled BL (3 H-BL), and BRinduced BRI1 auto-phosphorylation (90, 91,
94). It was further shown that the 94-aminoacid ID-LRR21 domain containing ID and its
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flanking LRR21 is sufficient for BR binding
(43). These studies demonstrated that BRI1
is the receptor of BRs and defined a new
steroid-binding motif. BR binding to the
extracellular domain activates the cytoplasmic
kinase, which transduces the signal to other
intracellular proteins.
Most of the mutations identified in the
28 bri1 alleles are clustered in either the IDLRR21 or cytoplasmic kinase domain, consistent with an essential role of the BR binding
and kinase activity for BRI1 function. Interestingly, some of the mutations in the extracellular
domain, such as bri1-5 and bri1-9 alleles, cause
retention of the mutant protein in the endoplasmic reticulum (ER) due to protein misfolding. Genetic screens for suppressors of these
alleles have identified components of the ER
quality-control system that mediate retention
and processing of the misfolded bri1 proteins
(34, 37).
A domain-structure analysis also uncovered
important roles for the JM and CT domains.
Deletion of the JM domain abolished the signaling function of BRI1, suggesting a positive role for the JM in BRI1 function. In contrast, a truncated BRI1, with the C-terminal 49
amino acid residues deleted, displayed higher
kinase activity in vitro and more complete rescue of the bri1 mutant phenotype in transgenic
plants, suggesting an autoinhibitory role of the
CT (92). The CT region contains multiple
phosphorylation sites, and mutation of these
Ser/Thr residues to phosphomimetic acidic
amino acids had effects similar to the deletion of
this region, suggesting that phosphorylation of
the CT region helps to activate the kinase. This
may occur through a conformational change
that releases the autoinhibition of the kinase by
the C-terminal tail (92).
BR-Induced Receptor Kinase
Dimerization and Oligomerization
Studies on animals have shown that receptor kinase activation usually involves ligandinduced homo- or hetero-dimerization or
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hetero-oligomerization (67, 74). For example,
perception of TGFβ involves a homo-dimeric
type I TGFβ receptor and a homo-dimeric
type II TGFβ receptor. TGFβ binds to
the type II receptor to promote its association with and phosphorylation of the type
I receptor, which transduces the signal to
downstream components. Similarly, BR signaling also involves ligand-induced BRI1 homodimerization or homo-oligomerization and
hetero-oligomerization with a coreceptor kinase named BRI1-associated kinase 1 (BAK1).
Homo-dimerization of BRI1 was first
detected using fluorescence resonance energy transfer (FRET) analysis in the plasma
membrane (PM) of cowpea protoplasts (69).
Furthermore, using single molecule detection
methods, it has been demonstrated that about
20% of BRI1 molecules in the PM are present
in homo-dimerized form without higher-order
oligomerization (31). BRI1 homo-dimerization
has also been detected by coimmunoprecipitation of BRI1-GFP and BRI1-FLAG fusion
proteins from transgenic Arabidopsis plants
(92). Although BR had no effect on BRI1
homo-dimerization in protoplasts (31), BR
treatment of Arabidopsis plants increased the
coimmunoprecipitation of BRI1-GFP and
BRI1-FLAG, indicating that BR promotes
or stabilizes BRI1 homo-dimerization (92).
It is yet unclear whether BRs function as
a molecular glue, in a manner similar to
auxin-induced TIR1-IAA protein dimerization
(82), to help stabilize the BRI1 homo-dimer, or
whether BR binding causes a conformational
change that favors homo-dimerization. The
latter possibility is supported by the estimated
one BL molecule that is bound per BRI1-GFP
molecule (43). It is also unclear whether the
kinase activity is required for BR-induced
homo-dimerization of BRI1. Nevertheless,
BR-induced homo-dimerization of BRI1 seems
not sufficient to fully activate the BRI1 kinase,
and further association with the coreceptor
kinase BAK1 is required.
BAK1 was identified independently by two
research groups using a yeast two-hybrid screen
for BRI1 interacting proteins and an acti-
vation tagging screen for bri1-5 suppressors
(52, 60). BAK1 is also named SERK3 and is
one of the five SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERK1
to SERK5) (75), which are LRR-RLKs that
contain a small extracellular domain with five
LRRs (Figure 1). Overexpression of BAK1
suppresses the phenotype of a weak bri1 allele (52, 60). The bak1 loss-of-function mutant
resembles weak bri1 mutants (52), and overexpression of a kinase-inactive bak1 mutant protein causes severe dwarf phenotypes similar to
strong bri1 mutants, presumably due to a dominant negative effect (52). Recent genetic studies
of loss-of-function mutations of BAK1/SERK3
and its homologs (SERK1 and SERK4) confirmed their essential roles in BR signaling, as
well as in additional pathways (3, 28).
The kinase domains of BRI1 and BAK1 interact with and trans-phosphorylate each other
in vitro and in yeast (52, 60), and their in vivo
interaction has been detected by coimmunoprecipitation using transgenic Arabidopsis and
FRET analysis in protoplasts (52, 60, 69). Both
BRI1 and BAK1 show basal levels of kinase activities in vitro, which are increased by the corresponding partner (52). In yeast, kinase activity
is detected only when wild type BRI1 and BAK1
are coexpressed but is not detected when either
BRI1 or BAK1 is mutated or absent (60). These
results suggest that full activation of either receptor kinase requires transphosphorylation by
its partner.
In Arabidopsis, BR treatment promotes the
interaction between BRI1 and BAK1 and increases their phosphorylation (90, 91). The
BR-induced BRI1–BAK1 association seems to
be mediated by their kinase domains rather
than cooperative binding of the extracellular
domains. First, BR binding to BRI1 appears
to be independent of BAK1 and BAK1 does
not participate in BR binding, because the BL
binding activity of BRI1 was not altered in a
bak1 null mutant or by overexpression of BAK1
(43, 92). Second, the interaction between BRI1
and BAK1 requires their kinase activities. In
vitro interaction is reduced by mutations of the
BAK1 kinase domain and almost completely
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abolished by kinase-inactive mutations of the
BRI1 kinase domain (52). A kinase-dead mutant
BRI1 did not show BR-induced association with
BAK1, indicating that intact extracellular domains are not sufficient and BRI1 kinase activity is required for BR-induced association with
BAK1 (91). However, the L46E mutation in the
leucine zipper of BAK1 extracellular domain
appeared to abolish BAK1 binding to BRI1 in
yeast (99). While this result supported a possible role of the extracellular domain in BRI1–
BAK1 association, the lack of BRI1 interaction
could also be due to a possible mislocalization
(such as retention in the ER) or instability of the
mutant BAK1 in the cell. On the other hand, a
kinase-dead mutant BAK1 still associates with
wild type BRI1 upon BR treatment, suggesting that BR first activates BRI1 kinase, and the
association with BAK1 is a result of initial activation of the BRI1 kinase (91). A requirement
of BRI1 kinase activity for BAK1 interaction
and activation is consistent with the observation that BAK1 overexpression suppresses only
weak alleles of bri1 but not the bri1-5 det2 double mutant or bril null mutants (52, 60). These
results suggest that BAK1 is involved in BRI1
activation rather than transducing the BR signal
to downstream substrates.
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Team Play and Multitasking
of the Receptor Kinases
BRI1 is the major BR receptor in higher plants.
Mutations of the BRI1 orthologs in tomato,
rice, and pea cause BR-insensitive phenotypes
(55, 59, 62). However, BRI1 is not the only
BR receptor. There are three close homologs
of BRI1 in Arabidopsis and at least two of them,
BRL1 and BRL3, bind to BR with high affinity
and can rescue the bri1 mutant when expressed
from a BRI1 promoter (8, 43, 101). These BRI1
homologs apparently mediate cell-type-specific
BR response in vascular tissues (8). Such functional specification of BRI1 homologs obviously occurred during evolution before the separation of monocots from dicots, since OsBRL1
and OsBRL3 also seem to mediate tissue- or
cell-type-specific BR responses in rice (59).
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BAK1 also belongs to a small subfamily of
RLKs called SOMATIC EMBRYOGENESIS
RECEPTOR KINASES (SERK1 to SERK5).
In addition to BAK1 (SERK3), two additional
members of the SERK family, SERK1 and
BKK1 (BAK1-like 1, SERK4), appear to play
redundant roles with BAK1 in BR signaling. As with BAK1, overexpression of SERK1
or BKK1 partially suppresses the bri1-5 mutant, and they interact with BRI1 in vivo (3,
28, 38, 69). Whereas the bak1 single lossof-function mutant shows a very weak dwarf
phenotype, the bak1 serk1-1 double mutant
shows a significantly enhanced BR-insensitive
dwarf phenotype, and the bak1-4 bkk1-1 double mutant shows an only slightly enhanced
de-etiolation phenotype compared to bak1 itself. It was recently proposed that SERK1 and
BAK1/SERK3, but not BKK1/SERK4, mediate BR response (3). However, the phenotype
of the bak1 serk1 double mutant is still much
weaker than the strong bri1 alelles or than transgenic plants expressing a dominant negative
form of bak1 (52). The analysis might have been
complicated by the additional roles of BAK1
and BKK1 in pathways that suppress cell death.
Genetic studies have revealed additional
roles of BAK1 and other members of the SERK
family in multiple signaling pathways. First,
BAK1 and BKK1/SERK4 are involved in controlling a light-dependent cell death process
(27). The bak1 bkk1 double mutant displays a
seedling-lethal phenotype in the light but not
in the dark. Many aspects of cell death phenotypes, including the upregulation of defenserelated genes and the accumulation of callose
and H2 O2 , were observed in the bak1 bkk1 double mutant but not in each single mutant or in
the bri1 null mutant, indicating that BAK1 and
BKK1 play a redundant role in a pathway that
suppresses cell death independently of BR signaling (28). Second, BAK1 plays a key role in
pathogenesis. The bak1 mutant displayed enhanced susceptibility to necrotrophic pathogen
infection (41). The application of BR suppresses
only growth defects but not the disease phenotype of the bak1 mutant, supporting the
hypothesis that BAK1 has a BR-independent
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role in defense (41). In addition, the bak1 mutant shows reduced sensitivity to pathogenassociated molecular patterns (PAMPs) such
as flagellin, EF-Tu, INF1, and CSP22. It was
further revealed that BAK1 interacts with the
flagellin receptor FLS2, another LRR-RLK
protein, upon flagellin treatment (10). These
results indicate that BAK1 also functions as a
coreceptor of FLS2 for flagellin signaling. Shan
and coworkers (78) reported that virulence factors of bacterial pathogen target BAK1 to overcome the host defense system and to inhibit BR
signaling as well. The transgenic plants overexpressing the bacterial effector AvrPto show
dwarf phenotypes that mimic weak bri1 mutants, and AvrPto and AvrPtoB interact with
BAK1, leading to disruption of FLS2-BAK1 association (78). Third, SERK1 and SERK2 play
a redundant role in male sporogenesis. The
serk1 serk2 double mutant is male sterile due to
a defect in tapetum development in the anther
(2, 14).
Taken together, it has been shown that the
four members of the SERK family play overlapping roles in at least four distinct pathways:
BAK1, SERK1, and BKK1/SERK4 in BR signaling; BAK1 and BKK1/SERK4 in cell death
control; BAK1 in flagellin signaling and innate
immunity; and SERK1 and SERK2 in male
sporogenesis (3). Members of a gene family
were likely generated by gene duplication,
which initially created copies of genes that were
fully redundant. How different SERK members
gain or retain specific functions is an interesting
evolutionary question. Functional specificity
can be generated by changes in the promoter or
coding sequences, resulting in diverse tissuespecific gene expression or protein activity.
Expression of SERK3 from the SERK1
or SERK2 promoter failed to rescue the
male-sterility phenotype of serk1 serk2 double
mutant, suggesting that the protein specifies
the function in this case (3). In contrast, the
BRI1 homologs appear to have gained functional specificity by changes in the promoters
and gene expression patterns, as expression of
BRL1 from the BRI1 promoter rescued the
bri1 mutant (8).
It should be noted that dual function was also
reported for tomato BRI1, which was shown to
bind systemin, a peptide hormone for wounding response (72, 73); however, this finding was
later disputed (32, 33, 55). The more recent
studies concluded that tomato BRI1 is required
for BR signaling but not for systemin signaling (33, 55). Similarly, progesterone was shown
to bind to the same G-protein-coupled receptor as the peptide oxytocin in certain animal
cells (23), but this finding was also later disputed
(4, 7).
Regulation by Auto- and
Transphosphorylation
To understand the signaling mechanisms of
BRI1 and BAK1, several recent studies have
identified phosphorylation sites in their kinase
domains, mostly by using mass spectrometry
(MS) (39, 64, 90, 91, 99) (Figure 1). The in vitro
phosphorylation sites were identified by analyzing the recombinant intracellular domains
expressed and purified from the bacterium Escherichia coli, and in vivo phosphorylation sites
were identified by analyzing epitope-tagged
BRI1 or BAK1 proteins immunoprecipitated
from transgenic Arabidopsis plants. These studies identified eleven unambiguous phosphorylation sites in BRI1 (six identified in vivo and
five identified only in vitro). MS data also supported the presence of additional phosphorylation sites that could not be assigned to specific amino acid residues, including at least three
sites in the activation loop between domains VII
and VIII. In addition, site-directed mutagenesis
followed by phosphopeptide mapping provided
the evidence for phosphorylation on additional
residues (S1162) (92). Although initial MS analyses identified only phosphorylation of Ser/Thr
residues, a recent study using phosphopeptidespecific antibodies and MS analysis provided
convincing evidence that BRI1 is also phosphorylated at tyrosine residues in vitro and in
vivo, indicating that BRI1 is a dual-specificity
kinase (64). Interestingly, many other LRRRLKs, e.g., BAK1 and BKK1, also contain
phosphorylation at tyrosine residues (64).
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The functions of many identified or putative
phosphorylation sites in BRI1 have been studied using site-directed mutagenesis followed by
in vitro kinase assays and in planta rescue of the
bri1 mutants (64, 90, 91). These studies confirmed an important role of phosphorylation
in the activation loop for the kinase activity
of BRI1. Mutations of S1044/T1045, T1049,
Y1052, or Y1057 nearly abolished BRI1’s kinase activity in vitro and its ability to rescue
the bri1-5 mutant. Mutations of two additional
Tyr residues (Y956 or Y1072) outside the activation loop also abolished the kinase activity,
though phosphorylation of Y1072 has not been
detected in vivo.
Important function was also demonstrated
for phosphorylation in the JM and CT domains
of BRI1. Deletion of the JM domain from BRI1
abolished its signaling function and ability to
rescue the bri1-5 mutant phenotype, without
affecting its localization at the plasma membrane (92). Seven phosphorylation sites in the
JM domain were identified as autophosphorylation sites in vitro and in vivo by MS analysis
(63, 90). Of these, the phosphomimetic substitutions of four phosphorylation sites (S838D,
T842D, T846D, and S858D) enhanced the
phosphorylation of a BRI1 synthetic peptide
substrate in the absence of BAK1, indicating
that phosphorylation of the JM domain activates BRI1 (91). However, the action mechanism by which the JM domain modulates BRI1
activity remains unclear.
By contrast, deletion of the CT domain increases the kinase activity of BRI1 in vitro and in
vivo and promotes hypocotyl growth of bri1-5
and det2 mutant more effectively than wild type
BRI1 (92). Multiple Ser/Thr residues of the
CT domain are phosphorylated. At least four
Ser/Thr residues (S-1162, S-1166, S-1168, and
T-1180) among eight possible sites in the CT
domain were experimentally confirmed (90–
92). Multiple substitutions of Ser/Thr residues
to Asp in the CT domain increase BRI1 kinase activity independent of BAK1 activity (91).
Moreover, mutant BRI1 containing phosphomimetic mutations in the CT domain more
effectively suppressed the dwarf phenotype of
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det2 (91, 92). These results demonstrated that
the CT domain of BRI1 is autoinhibitory of
BRI1 kinase and this inhibitory effect is alleviated by phosphorylation (92). The functions of
the JM and CT domains of BRI1 demonstrate
that noncatalytic regions of the cytoplasmic
domain play important roles in specifically
modulating kinase activity. It is well known that
nonkinase intracellular domains of mammalian
receptor kinases play important roles not only
in autoregulation but also in creating binding sites for substrates in a phosphorylationdependent manner (35, 36). Thus, it is possible
that JM and CT of BRI1 are involved in regulating kinase activity as well as creating binding
sites for BRI1 downstream substrates.
A total of nine phosphorylation sites have
been identified in the intracellular region of
BAK1; five of them were identified in vivo
(S290, T312, T446, T449, and T455) and four
in vitro (S286, T450, S604, and S612) (39, 91,
92, 99). Unlike BRI1, BAK1 is mainly phosphorylated in the activation loop (T446, T449,
T450, and T455) (91), although two phosphorylation sites (S-604 and S-612) in the CT domain were recently detected in vitro (39). The
phosphorylation of the T455 residue, corresponding to T1049 of BRI1 in the activation
loop of BAK1, is essential for BAK1 function,
since mutation of T455A abolished the kinase
activity (91). Although single mutation of the
other phosphorylation sites in the activation
loop had little effect on BAK1 activity, changing
all three Thr to Ala (T446A/T449A/T450A)
abolished the kinase activity (91, 99), indicating that phosphorylation in the activation loop
is critical for BAK1 kinase activity.
While analyses of the in vitro autophosphorylation and in vivo phosphorylation sites
elucidated important residues at which phosphorylation affects kinase activity and signaling
function, the mechanisms of BR regulation of
these phosphorylation events remain unclear.
Recently, it has been proposed that BR-induced
association between BRI1 and BAK1 is a result
of initial activation of the BRI1 kinase (91), and
sequential transphosphorylation between BRI1
and BAK1 activates both receptor kinases.
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BR-induced association between BRI1 and
BAK1 requires kinase activity of BRI1 but not
that of BAK1, since BR increases the interaction
between wild type BRI1 and kinase-dead mutant BAK1 but not between mutant BRI1 and
wild type BAK1. Although the mutant BRI1 still
shows basal level of interaction with BAK1, this
interaction is not increased by BR treatment
(91). In addition, BR-induced BAK1 phosphorylation is enhanced by BRI1 overexpression
but abolished in the bri1 null mutant. By contrast, BRI1 phosphorylation is still induced by
BR treatment even in the bak1 bkk1 (serk3
serk4) double mutant background, though at a
reduced degree compared with the wild type
or BAK1-overexpressing plants. Prephosphorylation of BRI1 by BAK1 in vitro increases
BRI1 phosphorylation of a BRI1-specific substrate, indicating that BRI1 has basal activity
that is further activated by BAK1 transphosphorylation (91). Furthermore, overexpression
of BRI1 rescues hypocotyl elongation of the
bak1 bkk1 double mutant, whereas overexpression of BAK1 fails to suppress null bri1 mutants (91). However, SERK1 might still provide a function in the bak1 bkk1 double mutant,
because SERK1 also associates with BRI1 and
contributes to BR signaling (3, 38). As such,
analysis of a bak1 bkk1 serk1 triple null mutant
is still required to determine if BAK1 and related proteins play essential or supporting roles
in BR activation of BRI1.
Transactivation between BRI1 and BAK1
is mediated by transphosphorylation. The
transphosphorylation sites have been identified
by using in vitro kinase assays performed with
kinase-inactive mutant protein as the substrate
for its wild type partner. MS analyses showed
that BRI1 mainly phosphorylates the kinase domain of BAK1. Of six BAK1 residues (S290,
T312, T446, T449, T450, and T455) transphosphorylated by BRI1, four Thr residues (T446,
T449, T450, and T455) are located in the
activation loop and required for the kinase activity and signaling function of BAK1 (91).
Mutation to Ala either of T455 alone or of
T446, T449, and T450 together abolished the
kinase activity of BAK1 (91, 99). Therefore,
phosphorylation of these residues by BRI1 is
likely to activate BAK1.
In contrast to BRI1 phosphorylation of the
BAK1 activation loop, BAK1 mainly transphosphorylates the JM and CT domains of mutant BRI1 (91). Three phospho-residues in the
JM domain (S838, T846, and S858) and two
in the CT domain (S1166 and T1180) of BRI1
were identified as BAK1 transphosphorylation
sites. In addition, phosphomimetic mutants of
JM and CT domains showed increased BRI1
kinase activity (91), and bri1 mutants carrying
the Ser/Thr substitution to Ala in the JM domain were less effective than wild-type BRI1 in
suppressing the short hypocotyl phenotype of
the bri1-5 mutant. These results support the hypothesis that BAK1 activates BRI1 by phosphorylating the JM and CT domains (91). Together,
the results support a sequential phosphorylation model for BR-activation of the BRI1–
BAK1 receptor kinases: BR binding induces
a basal activation of BRI1 kinase, which then
binds to BAK1 and phosphorylates BAK1 in
the activation loop to activate BAK1; activated
BAK1 then phosphorylates BRI1 in the JM and
CT region to fully activate BRI1’s kinase activity and signaling function through enhanced
phosphorylation of downstream substrates.
While phosphorylation at most sites positively activates the kinases, mutagenesis studies
also revealed phosphorylation sites that possibly inhibit kinase activity. The T872A mutation of BRI1 increased its kinase activity by over
tenfold, due to an increased Vmax and reduced
Km . The results suggest that phosphorylation at
T872 has a negative regulatory effect on the kinase activity, although it is also possible that the
substitution of Ala at this specific Thr residue
changes the protein conformation to a more active state (90). In addition, the S286D mutation
abolished BAK1 activity in vitro and in vivo, although S286A mutation had little effect (91).
Phosphorylation of S286 was detected in vitro
as a BAK1 autophosphorylation site. It remains
unclear whether S286 is phosphorylated in vivo
by BAK1 autophosphorylation as a desensitizing mechanism or by other kinases that modulate the BAK1 activity (91).
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In addition to affecting the kinase activity,
some phosphorylation sites appear to mediate
specific signaling output. Mutation of Y831F in
the JM domain of BRI1 had no obvious effect on
the kinase activity. Expression of mutant BRI1
Y831F rescued the growth phenotype of bri15 but caused larger leaves and early flowering,
suggesting that phosphorylation of Y831 might
provide signal output to leaf development and
flowering regulation. In addition, BAK1 containing the T450A mutation in the activation
loop could rescue the cell-death and dwarf phenotypes of the bak1 bkk1 double mutant but
failed to rescue the flagellin-insensitive phenotype of the mutant (91). These results suggest
that individual phosphorylation sites might affect specific signaling output.
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Endocytosis of BRI1 and BAK1
In mammals and yeast, endocytosis is a common mechanism for delivering activated receptors from the plasma membrane (PM) to its
downstream cellular targets or for clearing the
receptors from the cell surface to terminate the
signaling and desensitize the system (30, 56).
Endocytosis of BRI1 and BAK1 was first observed in cowpea protoplasts (69), where endocytosis was accelerated by coexpression of BRI1
and BAK1. Recently, BRI1-GFP was observed
in the endosomes in Arabidopsis root cells independently of BR treatment (22). Brefeldin
A (BFA), an inhibitor blocking transition from
early to late endosomal compartments, induced
the accumulation of BRI1-GFP in endosomal
compartments. Interestingly, BFA-treatment
activated BR signaling and caused dephosphorylation of BZR2/BES1 and inhibition of
DWF4 expression (22). In contrast, Endosidin1,
a chemical affecting PM/endosome trafficking,
resulted in the accumulation of BRI1-GFP in
large intracellular bodies (68) but caused a dwarf
phenotype similar to the bri1 mutant. BRI1 endocytosis appears not to be regulated by BR but
is increased by overexpression of BAK1 in the
protoplasts.
SERK1 is also internalized, and its internalization involves the KINASE-ASSOCIATED
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PROTEIN PHOSPHATASE (KAPP) (77).
KAPP contains a phosphopeptide-binding domain (FHA) and interacts with SERK1 in a
kinase-activity-dependent manner. Coexpression of KAPP increased SERK1 internalization. Although both SERK1 and KAPP colocalize at the plasma membrane, a FRET signal
that indicates their physical interaction was detected only in the intracellular vesicles. Thus, it
has been proposed that KAPP controls SERK1
internalization (77). KAPP also interacts with
both BRI1 and BAK1 in a phosphorylationdependent manner (17), since it did not interact
with kinase-inactive mutant BRI1 (K911E) and
BAK1 (T546A). The kapp bri1-5 double mutant
showed increased BL sensitivity over the bri15 single mutant, suggesting that KAPP might
be a negative regulator of BR signaling (17).
Whether KAPP inhibits BR signaling by dephosphorylating the receptor kinases or promoting their endocytosis remains to be studied.
While these results support the importance
of correct localization of BRI1 for its signaling
function, the function of BRI1 endocytosis remains unclear. Based on positive signaling from
BFA-sensitive endosomal BRI1, it has been proposed that endocytosis provides additional surface space or proximity for interaction with
cytoplasmic proteins, or traps ligand-receptor
complexes in small vesicle enclosures to maintain BR signaling (22). The latter scenario is
likely to be important in the roots, where water can dilute extracellular signal molecules. In
Arabidopsis, BRI1 is localized in the PM in most
cells and endosomal BRI1 is observed primarily
in root cells (20, 22). On the other hand, we still
do not know how BRI1 is removed from the cell
surface, e.g., in mature tissues that stop growing (20), and whether internalization could be
involved in receptor degradation as shown for
some receptors in animal systems (30).
Regulation by BKI1
The activity of the BRI1/BAK1 receptor
kinases is also regulated by additional interacting proteins. In addition to BAK1,
several novel proteins have been identified
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as BRI1-interacting proteins by yeast twohybrid screens (61, 89). One of these, the
BRI1 KINASE INHIBITOR 1 (BKI1), was
shown to negatively regulate BRI1 function.
RNAi knockdown of BKI1 caused enhanced
hypocotyl elongation, whereas overexpression
of BKI1 resulted in a weak dwarf phenotype.
In addition, BKI1 overexpression reduces the
BZR2/BES1 dephosphorylation induced by BR
treatment and alters BR-regulated gene expression, indicating that BKI1 is a negative regulator of BR signaling. BKI1 is localized at the
plasma membrane in the absence of BR, but
is rapidly relocalized into the cytosol upon BR
treatment (89). These observations suggest that
BKI1 interacts with BRI1 at the plasma membrane to keep BRI1 in an inactive form. BKI1
inhibits BRI1 at least partly by blocking its interaction with BAK1, because incubation with
BKI1 reduced BRI1 interaction with BAK1 in
vitro. Upon initial BR activation of BRI1 kinase,
BKI1 is phosphorylated by BRI1 and rapidly
dissociated from BRI1, allowing BAK1 association and full activation of BRI1 (89). In support of this hypothesis, forced constitutive localization of BKI1 to the plasma membrane by
an artificial N-terminal myristoylation site enhanced the dwarf phenotype caused by overexpression of BKI1. These results demonstrated
that BKI1 provides another level of control that
keeps BRI1 at a basal level when BR levels are
low.
SIGNALING OUTPUT FROM
THE RECEPTOR KINASES
Signaling output from the BRI1–BAK1 receptor complex should be mediated by intracellular
substrates of these kinases. In addition to BAK1
and BKI1, additional BRI1-interacting proteins
have been identified and shown to be phosphorylated by BRI1, at least in vitro. These include
the PM-localized TRANSTHYRETIN-LIKE
(TTL) protein, the homolog of TGFβRECEPTOR-INTERACTING PROTEIN
1 (TRIP-1), and the BR SIGNALING
KINASES (BSKs). While the mechanism of
function remains to be established for TTL
and TRIP-1, the BSKs have been shown to
mediate signaling from BRI1 to downstream
gene expression responses.
TTL interacts with BRI1 in yeast and is
phosphorylated by BRI1 in vitro (61). Overexpression of TTL caused dwarf phenotypes
similar to weak bri1 mutants, and a ttl knockout mutant displayed enhanced BR sensitivity
and plant growth, suggesting that TTL negatively regulates BR signaling via interaction
with BRI1 kinase. TTL binds more strongly to
active BRI1 kinase than to a mutant BRI1 lacking kinase activity (61). This is directly opposite
to BKI1, which binds more strongly to inactive
forms of BRI1 kinase (89). Based on the localization of TTL at the plasma membrane, it was
proposed that TTL might mediate direct BR
regulation of membrane activities that control
cell elongation (61). Whether TTL interaction
with BRI1 affects downstream BR-responsive
gene expression remains unknown.
TRIP-1 shares sequence similarity with the
mammalian TGFβ receptor–interacting protein, which plays a role in TBFβ signaling but is
also an essential subunit of the eIF3 eukaryotic
translation initiation factor. TRIP-1 interacts
with BRI1 in vitro and in vivo and is phosphorylated by BRI1 on at least three residues (T14,
T89, and either T197 or S198) in vitro, suggesting that TRIP-1 is a cytoplasmic substrate
of the BRI1 kinase. It was proposed that TRIP1 might play a role in BR-mediated translational regulation (18); however, direct evidence
for BR-regulation of protein translation is still
lacking. Recently, quantitative proteomics analyses identified a large number of BR-regulated
proteins, but a change at the protein level without a change in the level of the coding RNA has
not been found (16), except for the BR signaling
components, which are regulated by phosphorylation at the posttranslational level.
Using two-dimensional difference gel electrophoresis, proteomic analyses of total protein samples identified only late response
proteins, but analyses of phosphoprotein or
plasma membrane fractions identified early BRresponse proteins that mediate BR signal transduction (83). These include BAK1 and the
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PM-localized BR signaling kinases (BSK1 and
BSK2), which are phosphorylated upon BR
treatment (84). BSKs belong to the cytoplasmic receptor–like kinase (RLCK) subfamily XII
(80), which includes 12 members, all containing
an N-terminal kinase domain and a C-terminal
tertratricopeptide motif. In Arabidopsis, RLKs
are a large protein family with more than 610
members, and 25% of them contain no extracellular domain or transmembrane domain and are
named RLCK (80). BSK1 was phosphorylated
by BRI1 but not by BAK1 in vitro and BRI1
phosphorylated a specific Ser residue (S-230)
of BSK1. In vivo interaction between BRI1 and
BSKs was detected by coimmunoprecipitation,
and the amount of coimmunoprecipitation was
reduced by BR treatment, suggesting that BSKs
disassociate from BRI1 upon phosphorylation
(84). Although most of the knockout mutants of
BSKs did not show an obvious phenotype, the
bsk3 knockout plant showed reduced BR sensitivity and hypersensitivity to the BR biosynthetic inhibitor, BRZ. Overexpression of BSKs
strongly suppressed the dwarf phenotype and
restored BR-regulated gene expression in the
bri1-5 mutant. Furthermore, overexpression of
BSKs partially suppressed a bri1 null mutant but
did not suppress homozygous bin2-1, indicating
that BSKs are positive regulators acting downstream of BRI1 and upstream of BIN2 (84).
AR
1
11
22
NLS
P
DB
40
101
Genetic studies identified two homologous
transcription factors as key components of the
BR signaling pathway and demonstrated that
BR regulates plant development by modulating the expression of nuclear genes. The brassinazole resistant 1 (bzr1-1D) mutant looked like
wild type grown without BRZ when grown on
the BR biosynthetic inhibitor, BRZ, in contrast to the de-eitolation phenotype of wild type
seedlings (93). The bzr1-1D mutant fully suppresses the de-etiolation phenotype and partly
suppresses the dwarf phenotype of the bri1 mutant. BZR1 is a member of a plant-specific gene
family with five additional members in Arabidopsis (Figure 2). The Pro234 to Leu mutation in bzr1-1D stabilizes the protein, which
causes constitutive BR responsive growth in the
dark (93). The exact amino acid change P to
L mutation in BZR1’s closest homolog BZR2
(88% identical amino acid sequence) was found
in a bri1 suppressor mutant named bes1-D (bri1EMS-suppressor 1), and thus BZR2 was also
named BES1 (97). Overexpression of BZR1 increased hypocotyl length and a T-DNA knockout mutant bzr2-1 shows a slight reduction in
hypocotyl length (25, 93). Furthermore, RNAi
P
P
P
P
P
P
P
20 224
85
71 173
77 81
-r 2
-r
-r 1
-r
-r 1 r-1
Se
Se
Se
Se Se
Se Se
r-1
14-3-3
02
r-1
e
S
BZR1 AND BZR2/BES1:
KEY TRANSCRIPTION
FACTORS FOR BR-REGULATED
GENE EXPRESION
169 176
PEST
229
248
335
bzr1-1D
(P234L)
Figure 2
The structure of the transcription factor BZR1. BZR1 contains an alanine-rich (AR) domain, nuclear localization signal (NLS), DNA
binding (DB) domain, BIN2 phosphorylation domain, 14-3-3 binding motif, and PEST (proline, glutamic acid, serine, and threoninerich) domain. Putative BIN2 phosphorylation sites are indicated by asterisks. Yellow circles containing the letter P indicate sites
phosphorylated by BIN2 in vitro, and red circles indicate in vivo phosphorylation sites. (Adapted from References 16, 21; T-W. Kim
and Z. Wang, unpublished data).
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suppression of BZR2/BES1 and its close homologs caused a dwarf phenotype (96), indicating that BZR1 and BZR2/BES1 are positive
regulators of the BR pathway with redundant
roles.
The BZR1 and BZR2/BES1 proteins were
recently shown to be transcription factors
that directly regulate BR-responsive gene expression (25, 96). In one set of experiments, BZR1 fused to the DNA-binding
domain of GAL4 was able to repress the
transcription from a promoter–reporter gene
containing GAL4 binding sites, demonstrating BZR1’s transcription-repressor capacity.
Chromatin-immunoprecipitation experiments
demonstrated that BZR1 binds to the promoters of the BR-biosynthetic genes CPD and
DWF4, which are rapidly suppressed by BR
treatment as a mechanism for feedback regulation of BR biosynthesis. A series of in vitro
DNA-binding experiments demonstrated that
BZR1, through its N-terminal domain, directly bound optimally to the CGTGT/CG
sequence, which is required for the CPD promoter to respond to BR and is thus named
BR-response element (BRRE). Consistent with
BZR1 repressing gene expression through
BRRE, BRRE was found to be enriched in a
large number of BR-repressed genes, including additional BR biosynthetic genes. Interestingly, BRRE is also slightly enriched in promoters of BR-induced genes. Because BZR1 plays
dual roles in feedback inhibition of BR biosynthesis and BR promotion of plant growth, it
is likely that BZR1 also activates certain promoters, possibly by interacting with different
partners (25).
In contrast to the transcription-repressing
activity of BZR1, BZR2/BES1 was shown to
activate a BR-induced gene SAUR-AC1 (96).
BZR2/BES1 interacts with a bHLH transcription factor named BIM1, and together they
bind to the E-box (CANNTG) of the SAURAC1 promoter. Transient co-overexpression
of BZR2/BES1 and BIM1 activates a SAURAC1 promoter–GUS reporter gene, indicating
that BZR2/BES1 and BIM1 positively regulate SAUR-AC1 expression. While both BIM1
overexpression and knockdown experiments
support a role in plant growth regulation, the
phenotypes of these plants are subtle (96). It
is possible that additional bHLH proteins play
redundant roles or that BIMs control only a
subset of BR-regulated genes and developmental processes. In fact, a recent study identified two Jumonji domain–containing proteins,
ELF6 and REF6, as additional BZR2/BES1interacting proteins. Both proteins were
previously found to regulate flowering (98).
Furthermore, BIM1 was shown to interact with
two AP2/ERF transcription factors, DORNROSCHEN and DORNROSCHEN-LIKE,
involved in embryo patterning, and the bim1
mutant also shows embryo-patterning defects
at low penetrance (9). These results suggest
that BZR2/BES1 recruits other transcriptional
regulators to modulate the expression of subsets of target genes and specific developmental
processes.
The opposite transcription activities of
BZR1 and BZR2/BES1 provide a possible explanation for the differences in the light-grown
phenotypes of the bzr1-1D and bes1-D mutants. While both mutants were insensitive to
BRZ and suppress bri1 mutants in the dark,
they showed opposite phenotypes when grown
in the light, with bzr1-1D being a semi-dwarf
and bes1-D having long petioles and pale leaves,
reminiscent of BR-treated plants (93, 97). The
dwarf phenotype of bzr1-1D can be rescued
by BR treatment or overexpression of a BRbiosynthetic gene and is thus at least partly
caused by excessive feedback inhibition of BR
synthesis (25). Opposite transcription activities
would explain such opposite effects on cell elongation of the light-grown mutants. However,
this would be inconsistent with the similar overall constitutive BR-response phenotype in the
dark and under BR-deficient conditions. In fact,
it has been reported that BZR2/BES1 also mediates feedback inhibition of CPD and DWF4
expression (57, 87) and that BZR1 also mediates activation of BR-induced genes (25). Given
the almost identical amino acid sequences of
BZR1 and BZR2/BES1, especially in the DNAbinding domain, it is possible that BZR1 and
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BZR2/BES1 have similar DNA-binding and
transcription activities but are expressed in different tissues and thus have different effects on
BR production in BR source tissues and on cell
elongation in BR target tissues. A detailed analysis of tissue-specific expression of BZR family members will shed light on their functional
specification. Moreover, identification of all the
direct target genes of BZR1 and BZR2/BES1
will be important for understanding the function of the BR signaling pathway.
REGULATION OF BZR1 AND
BZR2/BES1 BY BIN2-MEDIATED
PHOSPHORYLATION
As key transcription factors of the BR signaling pathway, BZR1 and BZR2/BES1 are tightly
controlled by upstream BR signaling. BR treatment induces rapid dephosphorylation of BZR1
and BZR2/BES1, which can be detected as band
shift in immunoblotting (26, 93, 97). As such,
BZR1 and BZR2/BES1 should be phosphorylated by a kinase that negatively regulates BR
response. Such a kinase has been identified by
the second BR-insensitive mutant bin2.
In addition to bri1, bin2 (also named dwf12
or ucu1) is another locus identified based on BRinsensitive dwarf phenotypes. Unlike bri1 mutants, bin2 mutants are due to semi-dominant
gain-of-function mutations (11, 51, 66). BIN2
encodes a protein kinase that shares 70% sequence similarity to the mammalian Glycogen
Synthase Kinase-3 (GSK3). GSK3s are conserved in all eukaryotes from yeast to humans
and play important roles in a wide range of signaling pathways, usually as a negative regulator.
Overexpression of BIN2 caused a dwarf phenotype similar to that of bin2-1, and loss-offunction of BIN2 and its homologs increases
cell elongation and partially suppresses the bri15 mutant, indicating an inhibitory role for BIN2
in BR signaling (50, 95).
Several lines of evidence demonstrate
that BIN2 negatively regulates BR signaling
by phosphorylating BZR1 and BZR2/BES1.
First, the bzr1-1D and bes1-D mutations
fully suppress the bin2-1 mutant phenotype,
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supporting the idea that BZR1 and BZR2/BES1
are downstream of BIN2 in the BR signaling pathway (93, 97). Second, BIN2 interacts
directly with BZR1 and BZR2/BES1 in yeast
and in vitro (93, 97, 100). Third, BIN2 effectively phosphorylates BZR1 and BZR2/BES1
in vitro, causing a mobility shift that is opposite to that caused by BR treatment of plants.
Fourth, BZR1 and BZR2/BES1 are more phosphorylated and accumulate at a reduced level in
the bin2-1 mutant (93, 97). Furthermore, inhibition of BIN2 by chemical inhibitors, such
as LiCl and bikinin, causes dephosphorylation
of BZR1 and BZR2/BES1 (15, 95). There are
about 25 putative GSK3 phosphorylation sites
conserved in BZR1, BZR2/BES1, and the rice
homolog OsBZR1, and these phosphorylation
sites appear to provide multiple mechanisms for
regulating the transcription factors.
BIN2 phosphorylation has multiple inhibitory effects on the function of BZR1 and
BZR2/BES1. First, phosphorylated BZR1 undergoes 26S proteasome–mediated degradation, since treatment with the proteasome inhibitor MG132 causes accumulation of the
phosphorylated BZR1 but not the dephosphorylated form. While there have been conflicting observations of whether BR treatment affects the accumulation of BZR2/BES1 (86,
97), BZR2/BES1 level is much reduced in the
bri1 and bin2-1 mutants (97). Second, phosphorylation of BZR1 and BZR2/BES1 inhibits their DNA-binding activity. The BIN2phosphorylated BZR1 and BZR2/BES1 fail to
bind to DNA fragments derived from CPD or
SAUR-AC1 promoter in vitro, and the transcriptional activity of BZR2/BES1 in yeast was
suppressed in the presence of BIN2 (21, 86).
Third, phosphorylation of S173 causes binding
to 14-3-3 proteins, which reduce nuclear localization of BZR1 and BZR2/BES1 (21).
There have been controversial observations
of whether BR induces nuclear localization of
BZR1 and BZR2/BES1 (21, 86, 93, 97, 100).
The discrepancy is likely to reflect nonuniform
responses of different cells or cells under
different growth conditions (21). For example,
BR rapidly increases the ratio of nuclear to
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cytoplasmic BZR1 in hypocotyls of young
seedlings, but the effect is less pronounced
in mature seedlings. Subcellular fractionation
experiments revealed association of phosphorylated BZR1 with plasma membranes,
suggesting retention of phospho-BZR1 in the
cytoplasm (21). Furthermore, studies in both
Arabidopsis and rice confirmed the important
role of the 14-3-3 proteins in cytoplasmic
localization of phosphorylated BZR factors (5,
21, 70).
Yeast two-hybrid screens identified five
of the fourteen Arabidopsis 14-3-3 proteins
as BZR1-interacting proteins and all eight
rice 14-3-3 proteins as OsBZR1-interacting
proteins (5, 21). 14-3-3s are phosphopeptidebinding proteins that are highly conserved in all
eukaryotes (6). They are involved in numerous
signaling pathways and regulate many cellular
processes in yeast and mammals (6). In plants,
14-3-3 proteins are known to regulate
metabolic enzymes, ion channels, and response to hormones such as abscisic acid
and gibberellins (76). 14-3-3s interact with
target proteins in a sequence-specific and
phosphorylation-dependent manner. They
bind to two types of specific sequences: mode
I, RXXpSXP, or mode II, RXXXpSXP (X =
any amino acid, R = arginine, pS = phosphoserine, and P = proline), in which the S
residue needs to be phosphorylated (1, 58).
BZR1, BZR2/BES1, and OsBZR1 all contain
a conserved 14-3-3 binding motif (amino acids
169-175 of BZR1: RISNSCP), in which the
required S173 of BZR1 is phosphorylated by
BIN2 in vitro (5, 21). Mutations of S173A
of BZR1 and S156A of OsBZR1 abolish
14-3-3 binding in vitro and in vivo. Expression
of BZR1-S173A and OsBZR1-S156A in
transgenic Arabidopsis caused constitutive BR
response phenotypes similar to the bzr1-1D
mutant, indicating that binding by the 14-3-3
proteins inhibits BZR1 function (5, 21, 70).
These mutant proteins seem to have normal
DNA-binding activity and stability in plant
cells but show increased accumulation in the
nucleus and reduced levels in the cytoplasm
(5, 21, 70). Furthermore, treatment with the
14-3-3 inhibitor AICAR (5-aminoimidazole4-carboxamide-1-b-d-ribofuranoside) also increases the nuclear accumulation of BZR1-YFP
protein and reduces the expression level of
BZR1-repressed target genes CPD and DWF4
(21). Another study using the protoplast
system also demonstrated that BIN2-mediated
phosphorylation regulates nucleocytoplasmic
shuttling of BZR1 and the 14-3-3 binding
site and another phosphorylation domain are
involved in the regulation (70). Taken together,
these studies provide convincing evidence
for an essential role of the phosphorylationdependent and 14-3-3 protein–mediated
cytoplasmic localization of the BZR factors in
BR signal transduction. The 14-3-3 proteins
are likely to increase cytoplasmic localization
of phospho-BZR1 by promoting both nuclear
exporting and cytoplasmic retention (21, 70).
The Arabidopsis genome contains 10 GSK3like kinases, also named AtSKs (Arabidopsis
thaliana Shaggy-like Kinases), which can be
divided into four subgroups. Recent studies show that three AtSKs (AtSK21, AtSK22,
and AtSK23/BIN2) belonging to subgroup II
function redundantly in BR signaling. Their
triple loss-of-function mutant shows elongated
petiole and BRZ-resistant phenotype. However, considerable amounts of phosphorylated
BZR2/BES1 are still present in the triple mutant, indicating that other AtSKs might be involved in BR signaling (86, 95). Indeed, yeast
two-hybrid assay using nine AtSKs covering all
four subgroups shows that all six AtSKs belonging to subgroup I and II bind to BZR1 (42). Further study of AtSK12, a member of subgroup I,
demonstrated that AtSK12 interacts with BZR1
in vivo and phosphorylates BZR1 in vitro and
is inactivated by BR treatment (42). Thus, it is
believed that at least six AtSKs can play negative roles in BR signaling by phosphorylating
BZR1.
In addition to BZR1 and BZR2/BES1, BIN2
also phosphorylates the ARF2 transcription factor (88). ARF2 is a transcriptional repressor that
negatively regulates BR and auxin responses,
possibly by competing with activator ARFs
that promote auxin-induced gene expression.
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Loss-of-function arf2 mutant shows reduced
sensitivity to BR inhibitor, BRZ, suggesting a
negative role of ARF2 in BR-mediated growth.
BIN2 alleviated ARF2 transcriptional repression activity and inhibited its DNA-binding activity in vitro (88). It was proposed that BIN2
inactivates ARF2 to promote BR and auxin (88).
However, as a negative regulator of BR signaling, BIN2 is inactivated by BR (42, 65),
which would lead to BR activation of ARF2.
This seems contradictory to the negative role
of ARF2 in hypocotyl elongation but would be
consistent with ARF2’s positive role in promoting stamen elongation and leaf senescence (19).
The mechanism of ARF2 regulation by BR signaling and the function of ARF2 in BR response
require further study.
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REGULATION OF BIN2 BY
BSU1-MEDIATED TYROSINE
DEPHOSPHORYLATION AND
PROTEASOME-MEDIATED
DEGRADATION
It is believed that BR induces dephosphorylation of BZR factors by inhibiting BIN2, but
how BIN2 is inactivated by upstream BR signaling has remained an outstanding question.
In animal systems, GSK3β plays a role in Wnt
signaling similar to BIN2’s role in BR signaling.
GSK3β phosphorylates β-catenin to promote
its degradation and cytoplasmic retention,
similar to BIN2 regulation of BZR1/2. In
mammals, GSK3β action is modulated by
phosphorylation in its N-terminal domain,
protein complex formation, and by priming
phosphorylation of its substrate (54). However,
these mechanisms seem not to be used in BIN2
regulation of the BR signaling pathway (48). A
12-amino-acid BIN2–binding motif in BZR1/2
functions as a docking site for BIN2, though it
remains unclear whether BR regulates BIN2BZR1/2 binding (48). Meanwhile, Peng and
coworkers (65) have reported that BR induces
rapid degradation of BIN2, which is blocked
by MG132, the inhibitor of the 26S proteasome. Interestingly, the bin2-1 mutant protein
696
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·
Wang
accumulates in plants and is not degraded upon
BR treatment, providing genetic evidence for
an important role for BIN2 degradation by
the proteasome in BR signaling. However,
the primary mechanism that triggers BIN2
degradation was not identified (65).
Recently, Kim and coworkers (42) detected
BR-induced dephosphorylation of BIN2 using
immuno-blotting following two-dimensional
gel electrophoresis. This result indicated that
a protein phosphatase might be involved in the
regulation of BIN2. This phosphatase turned
out to be BSU1 (bri1 SUPPRESSOR 1), which
was initially thought to mediate dephosphorylation of BZR2/BES1.
BSU1 was identified by activation tagging
screening for suppressors of a weak bri1-5 allele (57). BSU1 contains an N-terminal kelchrepeat domain and a C-terminal phosphatase
domain homologous to protein phosphatase 1
(PP1). Overexpression of BSU1 suppresses a
bri1 mutant and causes accumulation of dephosphorylated BZR2/BES1. RNAi suppression of
BSU1 homologs caused a weak dwarf phenotype (57). Quadruple loss-of-function of BSU1
family members, by T-DNA knockout of bsu1
bsl1, and RNAi suppression of BSL2 and BSL3
caused an extreme dwarf phenotype similar to
the bri1 null mutant, confirming a positive role
of BSU1 in BR signaling (42).
When BZR1 or BZR2/BES1 and BIN2
were mixed with BSU1 in vitro, BZR1 or
BZR2/BES1 phosphorylation was reduced by
BSU1. However, the effect of BSU1 in the
in vitro assay was mild, and it was proposed that BSU1 might dephosphorylate BZR1
and BZR2/BES1 to activate BR responses in
vivo but need to be activated by posttranslational modification or by a partner protein
(46, 57). However, Kim and coworkers recently found that recombinant BSU1 or the
immunopurified plant BSU1 does not dephosphorylate BZR1 that was prephosphorylated by BIN2, if BIN2 was removed before
adding BSU1 (42). Reduced phosphorylation
of BZR1 and BZR2/BES1 was observed only
when BIN2 was present with BSU1, as reported
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originally (42, 57). Using BZR1 incompletely
phosphorylated by 32 P-ATP to distinguish
dephosphorylation from further phosphorylation by cold ATP, it was shown that BSU1
inhibits BIN2 phosphorylation but does not
dephosphorylate BZR1 directly. Furthermore,
BSU1 inhibition of wild type BIN2 is increased
by BR treatment of the plants from which BSU1
was immunopurified, suggesting that BR signaling activates BSU1. BSU1 fails to inhibit
phosphorylation of BZR1 by the mutant bin21, which causes BR-insensitive phenotypes in
planta. Similar to BR treatment, overexpression of BSU1 reduces the level of BIN2 protein
but not of bin2-1 (42). BSU1 overexpression
suppresses bri1 null allele but not homozygous
bin2-1, although it partly suppresses the dwarf
phenotype of heterozygous bin2-1. These results demonstrate that BSU1 functions downstream of BRI1 but upstream of BIN2 (42). It
was further shown that BSU1 interacts with
BIN2 in vitro and in vivo (42).
Mass spectrometry analysis of recombinant
BIN2 identified Tyr200 as an autophosphorylation site (42). Tyr200 is absolutely conserved
in all GSK3s and phosphorylation at this site is
required for kinase activity of BIN2 as it is for
mammalian GSK3s. The Tyr200-Phe substitution abolished the ability of BIN2 and bin2-1
to cause a dwarf phenotype when overexpressed
in transgenic Arabidopsis, suggesting that phosphorylation of Tyr200 is essential for BIN2
function in vivo. The level of Tyr200 phosphorylation of wild type BIN2 protein was reduced
by incubation with BSU1 in vitro and by BR
treatment of plants in vivo (42). Interestingly,
Tyr200 phosphorylation of mutant bin2-1 protein was not affected by BSU1 in vitro or by
BR treatment in vivo, indicating that the primary effect of the bin2-1 mutation is to block
BSU1 dephosphorylation of Tyr200. These results further demonstrate that BSU1-mediated
dephosphorylation of Tyr200 is the primary
mechanism by which BR signaling inactivates
BIN2 (42). It was also shown that Tyr233 of
AtSK12 (corresponding to Tyr200 of BIN2)
is also dephosphorylated upon BR treatment.
Thus, BSU1-mediated Tyr200 dephosphorylation appears to be a general mechanism for
inactivating GSK3s in BR signaling (42).
Placing BSU1 upstream of BIN2 raised a
possibility that BSU1 might directly interact
with BSKs at the plasma membrane, which
would close the last gap in the BR signaling
pathway. Although BSU1 is primarily localized
in the nucleus, small amounts of BSU1 can be
observed in the cytoplasm (42, 57). Microscopic
observation and proteomic studies revealed that
the BSU1 homologs (BSL1, BSL2, and BSL3)
are localized at the plasma membrane and in the
cytoplasm, raising the possibility of direct interaction with components localized at the plasma
membrane (42). Indeed, in vitro interaction
assays showed that BSU1 specifically interacts
with BSK1 but not with BRI1 and BAK1 (42).
This interaction was increased by BRI1 phosphorylation of BSK1 and abolished by mutation
of the BRI1 phosphorylation site in BSK1
(S230A). In vivo interaction between BSU1 and
BSK1 was also confirmed by coimmunoprecipitation and BiFC assays (42). These results show
that BRI1 phosphorylation of S230 of BSK1
promotes interaction with BSU1 (42). As such,
all major BR signaling components from cell
surface receptor BRI1 kinase to nuclear transcription factors are now physically connected.
The completed BR signaling pathway illustrates the molecular details of sequential signaling events: BR binding to BRI1 at the cell surface, BRI1 kinase activation by ligand-induced
association and transphosphorylation with the
BAK1 coreceptor and dissociation of the BKI1
inhibitor, BRI1 phosphorylation of BSKs, activation of BSU1, inactivation of BIN2 by BSU1catalyzed tyrosine dephosphorylation, and nuclear accumulation of dephosphorylated BZR
factors and BR-responsive gene expression
(42).
As the first complete signal transduction
pathway mediated by a receptor kinase in plants
(Figure 3), the BR signaling pathway provides a paradigm for understanding plant signal transduction. Many signaling mechanisms
revealed in the BR pathway are likely used
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a
BRI1
BAK1
Plasma membrane
BSKs
BKI1
BSU1
14-3-3
P
P
P
BZR1/2
P
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BIN2
P
Proteasomal
degradation
Nucleus
P
BIN2
P
BZR1/2
P
P
BZR1/2
P
P
BZR1/2
P
BSU1
P
BZR1/2-target genes
Cytoplasm
OH
OH
b
BRI1
HO
P
P
HO
BL
HO
O
BL
Plasma membrane
BSU1
P
P
P
HO
BSKs
P
O
BSKs
BKI1
P
HO
OH
OH
HO
BAK1
P
BIN2
BIN2
14-3-3
BZR1/2
Proteasomal
degradation
Nucleus
BSU1
BZR1/2
P
BIN2
BZR1/2
Unknown
factor
BZR1/2-target genes
Cytoplasm
698
Kim
·
Wang
BIN2
Gene expression
and cell growth
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broadly in other pathways. For example, the
mechanism of BRI1-BAK1 kinase activation by
ligand induced oligomerization and transphosphorylation is likely shared by other RLKs. The
regulation of GSK3-like kinases through
tyrosine dephosphorylation by BSU1 (a PP1related phosphatase) is likely a conserved mechanism used in mammals, because phosphorylation of the same tyrosine residue also affects
the activity of mammalian GSK3s and PP1 also
plays a role in the Wnt signaling pathway upstream of GSK3β. The regulation of BIN2 and
BRI1 by tyrosine phosphorylation clearly establishes the role of tyrosine phosphorylation as a
common mechanism of signal transduction in
plants. Although homologs of animal tyrosine
kinases have not been found in plants, many
plant kinases seem to have dual specificity for
Ser/Thr and Tyr.
CONCLUDING REMARKS
The combination of genetics, biochemistry, and
proteomics approaches has contributed to rapid
progress toward a deep understanding of the
BR signaling mechanism. The completed BR
signaling pathway illustrates the molecular details of sequential signaling events: BR binding to BRI1 at the cell surface, BRI1 kinase
activation by ligand-induced association and
transphosphorylation with the BAK1 coreceptor and dissociation of the BKI1 inhibitor, BRI1
phosphorylation of BSKs, activation of BSU1,
inactivation of BIN2 by BSU1-catalyzed tyrosine dephosphorylation, and nuclear accumulation of dephosphorylated BZR factors and BRresponsive gene expression. However, many
more questions are yet to be answered. For
example, how is BSU1 activity regulated by
BSKs? What kind of phosphatase dephosphorylates BZR1/BES1 and how is it regulated by
BR signaling? Do any BR signaling kinases and
phosphatases directly regulate any cellular processes independent of the transcriptional pathway? Yet the more exciting and challenging
question is how such a linear signaling pathway regulates diverse developmental and physiological responses. Identification of direct target genes of BZR1 and BZR2/BES1 is key
for identifying downstream branch pathways
leading to specific responses and for dissecting the transcriptional network that integrates
with other signaling pathways. Ultimately, we
will need to understand the operation of the
BR signaling pathway in the context of tissue,
organ, and whole plant development (44, 45,
71). Most important though, the knowledge of
BR function needs to be applied to genetic engineering that improves food and bio-energy
production.
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 3
The brassinosteroid signal transduction pathway. The signaling components in active and inactive status are
shown in pink and blue, respectively. Phosphorylation confers positive effects (yellow circles) and negative
effects (red circles). (a) In the absence of BR, BKI1 interacts with inactive forms of BRI1, leading to
interruption of BRI1 binding to BAK1. BRI1-bound BSKs are kept in inactive status. Consequently, BSU1 is
inactive. Active BIN2 (harboring phospho-tyrosine at the 200th amino acid residue) constitutively
phosphorylates BZR1 and BZR2/BES1, leading to nuclear exporting and cytoplasmic retention by the
14-3-3 proteins, loss of DNA-binding activity, and proteasomal degradation of BZR1 and BZR2/BES1.
(b) In the presence of BR, BR binds to BRI1, which induces association with BAK1 and disassociation of
BKI1. By sequential transphosphorylation between BRI1 and BAK1, BRI1 is fully activated and
phosphorylates BSKs. Then the phosphorylated BSKs are released from the receptor complex and bind to
BSU1, presumably to enhance BSU1 activity. Activated BSU1 inhibits BIN2 through dephosphorylation of
the phospho-tyrosine residue of BIN2, which allows accumulation of unphosphorylated BZR1 and
BZR2/BES1 in the nucleus. Active BZR1 and BZR2/BES1 bind to genomic DNA to regulate BR-target
gene expression, thereby modulating growth and development of plants.
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DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
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Contents
Volume 61, 2010
A Wandering Pathway in Plant Biology: From Wildflowers to
Phototropins to Bacterial Virulence
Winslow R. Briggs p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Structure and Function of Plant Photoreceptors
Andreas Möglich, Xiaojing Yang, Rebecca A. Ayers, and Keith Moffat p p p p p p p p p p p p p p p p p p p p p21
Auxin Biosynthesis and Its Role in Plant Development
Yunde Zhao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p49
Computational Morphodynamics: A Modeling Framework to
Understand Plant Growth
Vijay Chickarmane, Adrienne H.K. Roeder, Paul T. Tarr, Alexandre Cunha,
Cory Tobin, and Elliot M. Meyerowitz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p65
Female Gametophyte Development in Flowering Plants
Wei-Cai Yang, Dong-Qiao Shi, and Yan-Hong Chen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p89
Doomed Lovers: Mechanisms of Isolation and Incompatibility in Plants
Kirsten Bomblies p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 109
Chloroplast RNA Metabolism
David B. Stern, Michel Goldschmidt-Clermont, and Maureen R. Hanson p p p p p p p p p p p p p p 125
Protein Transport into Chloroplasts
Hsou-min Li and Chi-Chou Chiu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 157
The Regulation of Gene Expression Required for C4 Photosynthesis
Julian M. Hibberd and Sarah Covshoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 181
Starch: Its Metabolism, Evolution, and Biotechnological Modification
in Plants
Samuel C. Zeeman, Jens Kossmann, and Alison M. Smith p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 209
Improving Photosynthetic Efficiency for Greater Yield
Xin-Guang Zhu, Stephen P. Long, and Donald R. Ort p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 235
Hemicelluloses
Henrik Vibe Scheller and Peter Ulvskov p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 263
Diversification of P450 Genes During Land Plant Evolution
Masaharu Mizutani and Daisaku Ohta p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 291
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Evolution in Action: Plants Resistant to Herbicides
Stephen B. Powles and Qin Yu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 317
Insights from the Comparison of Plant Genome Sequences
Andrew H. Paterson, Michael Freeling, Haibao Tang, and Xiyin Wang p p p p p p p p p p p p p p p p 349
High-Throughput Characterization of Plant Gene Functions by Using
Gain-of-Function Technology
Youichi Kondou, Mieko Higuchi, and Minami Matsui p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 373
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Histone Methylation in Higher Plants
Chunyan Liu, Falong Lu, Xia Cui, and Xiaofeng Cao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 395
Genetic and Molecular Basis of Rice Yield
Yongzhong Xing and Qifa Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 421
Genetic Engineering for Modern Agriculture: Challenges and
Perspectives
Ron Mittler and Eduardo Blumwald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 443
Metabolomics for Functional Genomics, Systems Biology, and
Biotechnology
Kazuki Saito and Fumio Matsuda p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 463
Quantitation in Mass-Spectrometry-Based Proteomics
Waltraud X. Schulze and Björn Usadel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 491
Metal Hyperaccumulation in Plants
Ute Krämer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 517
Arsenic as a Food Chain Contaminant: Mechanisms of Plant Uptake
and Metabolism and Mitigation Strategies
Fang-Jie Zhao, Steve P. McGrath, and Andrew A. Meharg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 535
Guard Cell Signal Transduction Network: Advances in Understanding
Abscisic Acid, CO2 , and Ca2+ Signaling
Tae-Houn Kim, Maik Böhmer, Honghong Hu, Noriyuki Nishimura,
and Julian I. Schroeder p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 561
The Language of Calcium Signaling
Antony N. Dodd, Jörg Kudla, and Dale Sanders p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 593
Mitogen-Activated Protein Kinase Signaling in Plants
Maria Cristina Suarez Rodriguez, Morten Petersen, and John Mundy p p p p p p p p p p p p p p p p p 621
Abscisic Acid: Emergence of a Core Signaling Network
Sean R. Cutler, Pedro L. Rodriguez, Ruth R. Finkelstein, and Suzanne R. Abrams p p p p 651
Brassinosteroid Signal Transduction from Receptor Kinases to
Transcription Factors
Tae-Wuk Kim and Zhi-Yong Wang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 681
vi
Contents
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Directional Gravity Sensing in Gravitropism
Miyo Terao Morita p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 705
Indexes
Cumulative Index of Contributing Authors, Volumes 51–61 p p p p p p p p p p p p p p p p p p p p p p p p p p p 721
Cumulative Index of Chapter Titles, Volumes 51–61 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 726
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Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://plant.annualreviews.org
Contents
vii