Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR signaling
BMC Plant Biology
Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR signaling
Yun Shang 0 3
Myeong Min Lee 2
Jianming Li 1
Kyoung Hee Nam 0 3
0 Division of Biological Science, Sookmyung Women's University , Seoul , Korea
1 Department of Molecular, Cellular, and Developmental Biology, University of Michigan , Ann Arbor, MI , USA
2 College of Life Science and Biotechnology, Yonsei University , Seoul , Korea
3 Division of Biological Science, Sookmyung Women's University , Seoul , Korea
Background: Since the identification of BRI1 (BRASSINOSTEROID-INSENSITIVE1), a brassinosteroids (BRs) receptor, most of the critical roles of BR in plant development have been assessed using various bri1 mutant alleles. The characterization of individual bri1 mutants has shown that both the extracellular and cytoplasmic domains of BRI1 are important to its proper functioning. Particularly, in the extracellular domain, regions near the 70-amino acid island are known to be critical to BR binding. In comparison, the exact function of the leucine rich-repeats (LRR) region located before the 70-amino acid island domain in the extracellular cellular portion of BRI1 has not yet been described, due to a lack of specific mutant alleles. Results: Among the mutants showing altered growth patterns compared to wild type, we further characterized cp3, which displayed defective growth and reduced BR sensitivity. We sequenced the genomic DNA spanning BRI1 in the cp3 and found that cp3 has a point mutation in the region encoding the 13th LRR of BRI1, resulting in a change from serine to phenylalanine (S399F). We renamed it bri1-120. We also showed that overexpression of the wild type BRI1 protein rescued the phenotype of bri1-120. Using a GFP-tagged bri1-120 construct, we detected the bri1-120 protein in the plasma membrane, and showed that the phenotypic defects in the rosette leaves of bri1301, a kinase-inactive weak allele of BRI1, can be restored by the overexpression of the bri1-120 proteins in bri1-301. We also produced bri1-301 mutants that were wild type in appearance by performing a genetic cross between bri1-301 and bri1-120 plants. Conclusions: We identified a new bri1 allele, bri1-120, whose mutation site has not yet been found or characterized. Our results indicated that the extracellular LRR regions before the 70-amino acid island domain of BRI1 are important for the appropriate cellular functioning of BRI1. Also, we confirmed that a successful interallelic complementation occurs between the extracellular domain mutant allele and the cytoplasmic kinase-inactive mutant allele of BRI1 in vivo.
Numerous plant developmental processes, such as
germination, cell elongation, photomorphogenic responses,
and male fertility are regulated by the plant-specific
steroidal hormones, brassinosteroids (BR). BR-biosynthetic
or BR-perceiving mutants have exhibited defective
growth patterns in various tissues that persist
throughout their entire life span, indicating the critical role of
BR in plant development [1,2]. Although studies
researching the BR signaling process began much more
recently than any of the other plant hormones, the
identification of BRASSINOSTEROID-INSENSITIVE1
(BRI1), a receptor of BR , and several other important
components involved in BR signaling have provided
much insight into many important components in plant
development . Plasma membrane-localized BRI1 and
its co-receptor BRI1-ASSOCIATED KINASE1 (BAK1)
are receptor-like serine/threonine kinases containing
leucine-rich repeats (LRR-RLKs) [5,6]. N-terminal LRRs
are found in the extracellular portion of the plasma
membrane. BRI1 constitutively forms a homodimer in
the plasma membrane. In the absence of BR, the activity
of the BRI1 homodimer is inhibited by the BRI1 kinase
inhibitor 1 (BKI1) by binding BKI1 to the C-terminal
portion of BRI1. In the presence of BR, BKI1 is released
by the direct binding of BR to the 70-amino acid island
region in the extracellular domain of BRI1 . Then,
BRI1 recruits BAK1, forming heterodimerized-receptor
complexes in the plasma membrane [8,9], leading to
the activation of the BES1 and BZR1 transcription
factors that regulate the expression of the BR-associated
BRI1 is considered to be a master regulator that plays
a critical role in the direct binding of BR and
subsequent BR signaling processes , while BAK1 has been
found to be a partner not only for BRI1 but also for
other LRR-RLKs, such as FLS2 and EFRs, which are
involved in the plant innate immunity responses [13,14].
To date, genetic screening looking for BR-insensitive
mutants has resulted in the identification of only two
genes, BRI1 and BIN2 [3,15]. Since the first report of
BRI1 in 1997 , more than 30 different mutant alleles
have been identified in several different Arabidopsis
ecotypes, including Col-0, Ws-2, and En-2 during last two
decades. Large numbers of mutant alleles that have
mutations in various positions of a specific gene provide
information regarding how that gene acts, because the
mutation sites themselves are indicators of their
importance to the functioning of the gene. In that sense,
studying multiple mutant alleles of BRI1 will be likely to
reveal important information regarding its function.
Detailed analyses of the characteristics of each mutation
have shown that both the extracellular and cytoplasmic
domains of BRI1 are required for full BRI1 functioning,
because the mutation sites of all of the bri1 mutant
alleles are dispersed in both an extracellular domain and
a cytoplasmic kinase domain [4,16].
The extracellular domain of BRI1 consists of LRRs
and a 70-amino acid island containing unique sequences
that show little homology to any other protein. Since
BRI1 was discovered, it has been considered to have 25
LRRs with a 70-amino acid island flanking the 21st and
22nd LRR. However, Vert et al, (2005)  suggested
that BRI1 contains 24 LRRs, postulating that the 21st
LRR is actually an atypical formation. It appears evident
that the region near the 70-amino acid island allows for
the extracellular binding of BR. It is interesting to note
that most of the mutation sites in the extracellular
domain of BRI1 are clustered in the 70-amino acid
island domain and in the 4 LRRs situated before the
transmembrane domain. There are very few examples of
mutant alleles containing defects in the LRR regions
that occur before the 70-amino acid island. This may be
partially because the mutations in these LRR regions of
BRI1 were neglected due to the lack of any discernible
phenotypic alterations. Or, at the opposite extreme, they
may lethally affect plant development, resulting in no
viable mutants for further analyses. Here, we report a
new mutant allele of BRI1, bri1-120. A point mutation
in the region encoding the 13th LRR of BRI1 in bri1-120
caused the defective growth and reduced BR sensitivity
of the plant. Using this mutant allele, we demonstrated
successful interallelic complementation using a
kinaseinactive mutant allele, bri1-301 and performed a detailed
analysis of BR sensitivity.
Phenotypic analyses of the weak bri1-looking semi-dwarf
To find natural mutants that show altered growth
patterns compared to their corresponding wild type plants,
we searched for and obtained mutant seed stocks from
the Arabidopsis Biological Resource Center (ABRC). We
grew several putative seeds and selected the cp3 mutant
(seed stock No. CS48) for further analysis, because
compared to the corresponding wild type plant Landsberg
(Ler), the phenotypic features of the mutant, including
the downward curling, dark-green compact rosette
leaves, and reduced growth gave the appearance of a
weak bri1 mutant, bri1-301 (Figure 1A and 1B). The cp3
Figure 1 Phenotypic analysis of cp3 mutant compared with
weak bri1 allele, bri1-301. A. 3-week-old soil grown plants of cp3
and bri1-301 were shown with corresponding wild type plants,
Landsberg (Ler) and Columbia (Col), respectively. B. Phenotype of
cp3 and Ler grown for 5 weeks. C. Quantitative determination of
growth in cp3 and Ler. Leaf length (LL), leaf width (LW), and petiole
length (PL) of the 5-week-old plants were measured Silique length
(S), peduncle length (P), and height of individual plants (H) were
measured from the 7-week old plants (n = 60, except height (n =
25). Growth is represented as a relative value compared to that of
Ler. Experiments were repeated twice. Error bars denote standard
mutant exhibited reduced growth in all aspects except
leaf width (Figure 1C), resulting in a semi-dwarf stature
with round and compact rosette leaves. To determine
the BR sensitivity of the cp3 mutant, we applied 1 M
of brassinolide (BL), the most bioactive BR, to the
mutant plants on the region where the plants were
exhibiting growth. Compared with the wild type Ler, which
showed elongated petioles and leaves and faded green
colored leaves upon overnight BR exposure, the leaves
and petioles of the mutant plants were much less
elongated and displayed still green-colored leaves, indicating
reduced sensitivity to BR (Additional file 1). To confirm
this, we analyzed the transcriptional inhibition of the
CPD expression pattern in cp3 mutants with and
without exogenous BL treatment, using the known weak
bri1 mutant, bri1-301, as a control (Figure 2A). As
observed with bri1-301, the cp3 mutant contained
higher levels of CPD transcripts compared to the wild
type Ler in the presence of BL, indicating that the cp3
mutant possesses reduced-BL sensitivity. We also
performed a root growth inhibition assay using the plants
grown on the media containing BL (Figure 2B). Both
the Columbia (Col-0) and Ler wild type plants showed
Figure 2 BR sensitivity of cp3. A. Transcriptional inhibition of CPD
expression in response to BL was determined in cp3, Ler, bri1-301, and
Col. B. Root growth was measured from the indicated plants grown
vertically in 1/2 MS media containing BL. Root length is represented as
the relative growth compared to that of the mock-treated sample. C.
Root growth was measured from Ler and cp3 plants grown vertically
in 1/2 MS media containing the various plant hormones indicated.
Root length of the plants treated with hormones is represented as a
percentage of the root length of the plants grown on the medium
without hormone treatment. Experiments were repeated three times.
Error bars denote standard errors.
more than 50% reductions in root growth in the
presence of 10 nM BL. In contrast, the cp3 mutants showed
30 to 40% reductions in root growth after treatment
with the same concentration of BL. In comparison,
bri1301 displayed almost no sensitivity to BL in terms of
root inhibition. These results indicate that the BL
sensitivity of cp3 is reduced compared to the wild type,
although the degree of reduction is less than that of
bri1-301. We further assessed the response of cp3 to
other plant hormones. Similar degrees of root growth
inhibition were observed in Ler and cp3 when the plants
were treated with a variety of hormones with the
exception of BL (Figure 2C), indicating that the cp3 mutant
specifically has a reduced sensitivity to BL, but not to
any other plant hormones.
Identification of the weak bri1 mutant allele, bri1-120
Based on the morphological phenotypes and reduced BL
sensitivity of the cp3 mutant, we thought that cp3 may
be one of the bri1 mutant alleles. Thus, we sequenced
the genomic DNA spanning the BRI1 region in the cp3
mutant. We also sequenced the same region of Ler as a
control. Since the Arabidopsis whole genome sequences
are derived from the Col-0 ecotype, we found one
mismatched nucleotide in the 3,512th position from the
open reading frame of the BRI1 sequence between Ler
and Col-0. This nucleotide change causes an alteration
from arginine to glycine in the 1171st amino acid of
BRI1. More importantly, we found an additional
mismatched nucleotide at the 1196th position from the
open reading frame with a T to C change, resulting in a
change from serine to phenylalanine at the 399th amino
acid position of BRI1 in the cp3 mutant (Figure 3A).
The wild type Ler has a nucleotide T in the 1196th
position as in Col-0. Therefore, we reasoned that the
nucleotide change at the 3512th position of BRI1 in Ler
is a natural polymorphism due to an ecotype difference
between Ler and Col-0, and that the nucleotide change
at the 1196th position of BRI1 in the cp3 mutant
compared to Ler causes its phenotypic changes.
To verify this notion, we generated a transgenic cp3
plant overexpressing BRI1 by introducing a BRI1
promoter-driven BRI1:BRI1-GFP construct. The growth of
the BRI1-overexpressing cp3 plants was more similar to
that of the wild type as compared to the non-transformed
cp3 plants (Figure 3B). We confirmed that the BRI1-GFP
transgene was highly expressed in the transgenic cp3
plants by RT-PCR analyses using primers that amplified
transgene specifically (Figure 3C). The cp3 plants
overexpressing BRI1-GFP showed nearly normal overall growth
patterns with elongated leaves and petiole length as
well as total height, similar to those observed with Ler
(Figure 3D). In addition, the cp3 transgenic plants
overexpressing BRI1 showed restored BL sensitivity, exhibiting a
Figure 3 Cp3 is allelic to bri1. A. The schematic protein structure
of BRI1 is shown and the mutation site of bri1-120/cp3 is marked in
the 13th LRR of the extracellular domain of BRI1. B. Overexpression
of BRI1 rescued the cp3 mutant phenotypes. The pictures were
taken of 3-week-old plants (left panel) and 5-week-old plants (right
panel). C. RT-PCT analysis shown the expression of endogenous BRI1
and the BRI1 derived from the transgene in the cp3 mutant
overexpressing BRI1 compared with those of Ler and
untransformed cp3 mutant. D. Quantitative growth criteria were
measured from the transgenic cp3 overexpressing BRI1. LL: leaf
length, LW: leaf width, PL: petiole length, and H: total height.
Growth is represented as a relative value compared to that of Ler.
Experiments were repeated twice. Error bars denote standard errors.
E. Pattern of transcriptional inhibition of CPD expression in response
to BL was restored in the transgenic cp3 overexpressing BRI1.
BL-induced transcriptional inhibition of CPD expression
(Figure 3E). These results suggest that the growth
retardation of the cp3 mutant accompanied by the dark green
coloring is caused by a mutation in the extracellular
domain of BRI1. Therefore, we renamed the cp3 mutant
bri1-120, referring to the order of naming for bri1
mutant alleles 
BRI1(S399F) protein is localized in plasma membrane and
the overexpression of BRI1(S399F) in bri1-301 resulted in
the leaf elongation of bri1-301 and co-suppression of the
We introduced nucleotide C instead of T at the 1196th
position of BR1 by site-directed mutagenesis to generate
the bri1-120 mutated BRI1, using the BRI1-GFP
construct as a template. The resulting construct
(BRI1:bri1120-GFP) was transformed into the wild type Col-0,
bri1-301 plants to produce a mutated BRI1(S399F).
After the wild type plant was transformed with BRI1:
bri1-120-GFP, we first observed the intracellular
localization of the BRI1(S399F) protein using a confocal
microscope by detecting the GFP that was fused
with BRI1(S399F) in the plasma membrane of the cells
(Figure 4A), which indicated that bri1-120 possesses the
plasma membrane-localized BL receptor, although BRI1
(S399F) may not be fully functional protein. In
comparison, the mutated BRI1 proteins, BRI1(C69Y) in bri1-5
and BRI1(S662F) in bri1-9, in which both mutations are
in the extracellular domain of BRI1, are known to be
localized to the endoplasmic reticulum (ER) .
Figure 4 Analysis of transgenic plants transformed with BRI1:
bri1-120-GFP. A. Confocal microscopic observations of the GFP
signal fused with BRI1(S399F) or intact BRI1 were performed on the
root tips. Microscopic features of the same root tissues under
bright-field were are shown side-by-side. B. Overexpression of BRI1:
bri1-120-GFP in bri1-301 led to an allelic series of the bri1 phenotype.
Representative fully rescued (Line 1), intermediate (Line 2), and
strong bri1 mutant-looking transgenic bri1-301 plants are shown
with un-transformed bri1-301. C. Analysis of BRI1 expression and
determination of BRI1 protein amount in transgenic bri1-301
overexpressing BRI1:bri1-120-GFP detected by anti-GFP antibodies
and anti-BRI1 antibodies.
We also produced transgenic bri1-301 overexpressing
mutated BRI1(S399F) to examine whether the additional
BRI1 proteins are able to rescue the bri1-301 mutant
phenotypes or not, although the transgenic plants have
two mutated forms of BRI1 derived from the bri1-301
and bri1-120 mutations, respectively. From this analysis,
we found that all of the transgenic bri1-301 displayed
phenotypes that were wild type in appearance, with less
compact rosette leaves due to elongated petioles and
leaves, even in the T1 generation. In subsequent
generations, a phenotypic recovery of bri1-301 achieved by the
overexpression of BRI1:bri1-120-GFP was observed in
most of the plants. However, some plants showed only a
partial recovery of the bri1-301 phenotype, and a few
plants displayed stronger mutant phenotypes compared
to the non-transformed bri1-301 (Figure 4B). And these
phenotypic differences still remained in inflorescent
adult stage (Additional file 2A). We attributed the
phenotypic differences of the transgenic bri1-301
overexpressing BRI1:bri1-120-GFP to the co-suppression of
BRI1 gene. The BRI1 transcripts derived from the
endogenous BRI1 and the transgene were shown to be
inversely correlated with phenotypic severity by RT-PCR
analyses using primers that amplified each gene
specifically (Figure 4C). Co-suppression was first shown in
petunia, in which the transgene, chalcone-synthase A,
caused transcript loss due to the degradation of the
homologous endogenous gene . Since then, it has
been regarded as eukaryotic post-transcriptional gene
silencing. We also performed a western blot analysis of
the total proteins from the plants showing representative
phenotypes using the anti-GFP antibodies and anti-BRI1
antibodies. As shown in Figure 4C in the bottom panel,
all the transgenic plants produced mutated BRI1(S399F)
protein fused to GFP detected by anti-GFP antibodies,
although the protein expression level is higher in the
transgenic bri1-301 plant that was wild type in
appearance. When we used anti-BRI1 antibodies that can
detect both endogenous and transgene-derived BRI1
proteins, the same plant contained more BRI1 proteins.
In contrast, much less BRI1 proteins were detected in
the strong bri1 mutant-looking bri1-301 transgenic
plant compared to the untransformed bri1-301. We also
detected bri1 mutant-looking phenotypic alterations that
were due to co-suppression in the wild type plant
overexpressing BRI1:bri1-120-GFP (Additional file 3).
Bri1-301 and bri1-120 complemented each other to form
a functional BRI1 receptor
Based on the results above, we questioned whether an
increased number of BRI1 proteins, although it is
partially functional, is enough to mediate BR signaling with
heterodimers consisting of mutated proteins, and if the
heterodimerization between the BRI proteins containing
an extracellular LRR domain mutation in bri1-120 and
the BRI1 proteins with a cytoplasmic kinase domain
mutation in bri1-301, reconstituted a fully functional
BRI1 in the cells. To address these questions, we crossed
bri1-120 with bri1-301. We expected that all of the F2
plants from this cross would exhibit semi-dwarf looking
phenotypes, similar to both parental plants. However,
when we analyzed 235 individual plants from the F2
generation, we found that the phenotypic segregation
deviated slightly from the expected one. Thus, we grew
and genotyped all of the plants using CAPS and dCAPS
primers specific for the bri1-301 and bri1-120
mutations, respectively. More attention was directed toward
the plants that were heterozygous both mutations in
each homologous chromosome of the cell: the bri1-301
mutation residing on one homologous chromosome and
the bri1-120 mutation on the other. Among these
plants, approximately half showed compact rosettes and
semi-dwarf statures similar to the parental mutant
phenotypes, and the remaining half displayed rescued
bri1301 phenotypes in terms of overall rosette morphologies
(Figure 5 and Additional file 2B). Because there are no
additional BRI1 proteins added by the transgene in
these crossed plants, it is suggested that the rescued
bri1-301 phenotype resulted from the interallelic
complementation that occurred between the bri1-120 and
bri1-301 mutated alleles.
Different BL sensitivity was observed in the bri1-301
transformed with BRI1:bri1-120-GFP and the bri1-301
crossed with bri1-120
The results above indicate that both the overexpression
of bri1-120 by transformation and the reconstitution of
functional BRI1 by crossing it with bri1-120 restored the
mutant phenotype of bri1-301. We also wanted to know
Figure 5 Interallelic complementation between bri1-120 and
bri1-301. Overall rosette phenotypes of the F2 plants produced by
the genetic crosses of bri1-120 and bri1-301, whose genotypes were
heterozygous for both mutations, are compared with parental
mutant plants and wild type plants. Lower two panels show the
confirmed genotype of the bri1-120 and bri1-301 mutation in each
whether BR sensitivity returns to normal in these plants.
We examined root growth inhibition in the transgenic
bri1-301 transformed with BRI1:bri1-120-GFP in the
presence or absence of BL. Compared with the
untransformed bri1-301 and bri1-120 control plants, the
root length of the transgenic bri1-301 plant in the
absence of BL was shorter than that of non-transformed
bri1-301, similar to that of bri1-120. Moreover, the root
growth inhibition pattern exhibited after the BL
treatment of the transgenic bri1-301 plant was similar to that
of bri1-120 (Figure 6A). The rescue of the transcriptional
inhibition of CPD expression in the transgenic bri1-301
by the overexpression of BRI1:bri1-120-GFP was not as
dramatic as that observed in the wild type, either (Figure
6B). In comparison, the root lengths of the wild
typelooking F2 plants crossed with bri1-301 and bri1-120
were more similar to the root length of the wild type.
Also, the degree of inhibition of root growth showed
similar patterns compared to the wild type (Figure 6A).
The transcript level of CPD was reduced in response to
BL to the same degree as seen in the wild type (Figure
6B). Taken together, these results suggest that the F2
plants crossed with bri1-301 and bri1-120 were similar to
the wild type plant not only morphologically but also in
terms of their cellular responsiveness to BL, leading to
the strong assumption that these F2 plants contain a
functional BRI1 (Figure 6A).These results suggest that
the elongated rosette phenotype that has been frequently
considered to be the BR sensitivity gauge may not
Figure 6 BR sensitivity of the rescued bri1-301 plants. A. Root
growth was measured for the indicated plants grown vertically in
1/2 MS media or 1/2 MS media containing 100 nM of BL.
Experiments were repeated three times. Error bars denote standard
errors. B. Transcriptional inhibition of CPD expression in response to
BL was determined in the rescued bri1-301 plants compared with
that of control plants.
BRI1-120 revealed the importance of the LRR region in
the extracellular domain of BRI1
The degree of phenotypic alteration caused by each bri1
allele depends on the specific affected mutation sites .
Mutants that have amino acid changes in the
cytoplasmic kinase domain usually show very strong mutant
phenotypes, which can be attributed to loss of BRI1
kinase activity. Bri1-301 is an exceptional case. Although
bri1-301 was shown to be a kinase-inactive protein, the
mutant plant exhibits only mild phenotypic changes.
Bri1-301 contains two nucleotide changes (GG to AC)
in the cytoplasmic kinase domain of BRI1, resulting in a
change from Gly989 to Ile . However, Gly989 is not
a conserved amino acid, and its position is slightly out
of the critical region of the kinase domain. So, it is
possible that Gly989 is important for maintaining the
proper conformation of the BRI1 protein to retain its
kinase activity, but, not for controlling the kinase
In comparison, most of the mutations in the
extracellular domain of BRI1 produced relatively mild mutant
phenotypes. A more thorough examination of the
extracellular domain of BRI1 revealed that the 70-amino acid
island domain and the subsequent four LRRs before the
transmembrane domain are frequent mutation sites,
indicating their functional importance to the BRI1
protein. In addition, the first cysteine pair before the
beginning of the LRRs is thought to be critical for BRI1 as
seen in the mutant bri1-5 (C69Y). So far bri1-4 is the
only mutant in which the mutation occurred in the LRR
regions preceding the 70-amino acid island domain .
However, a 10-bp deletion in the 3rd LRR of BRI1 in
bri1-4 introduced a premature stop in translation and
did not provide any clues regarding the functional
importance of the LRR domains of BRI1.
In this study, we analyzed the BR-related phenotypes of
cp3 grown from the CS48 seeds obtained from ABRC
to have more natural mutants with similar morphologies
to known bri1 mutants, although the phenotypic strength
of bri1-120 is relatively weak compared to other bri1
mutants, such as bri1-5 or bri1-9. Cp3 has the
COMPACTA3 (cp3) mutation, and cp3 mutants show altered
phytochrome A signaling . However, the mutated
gene has not been characterized yet. From the direct
sequencing of the genomic DNA region containing
BRI1, we found that this plant contains a mutation in
BRI1 called bri1-120. Bri1-120 contains phenylalanine
instead of serine at the 399th position in the 13th LRR
due to a nucleotide change (T to C) at the 1196th
position (Figure 3A). When we overexpressed wild type
BRI1 in bri1-120, mutant phenotypes of bri1-120 were
rescued not only morphologically but also in terms of
their sensitivities to BR (Figure 3). Overexpression of the
bri1-120 protein in wild type plants produced transgenic
plants with bri1 mutant phenotypes (Figure 4 and
Supplementary Figure 2). We believe that bri1-120 is the first
example of a natural mutant allele with a point mutation
in the LRR region of the extracellular domain of BRI1.
These results suggest that the LRR region before the
70amino acid island domain is also important in
maintaining a fully functional BRI1.
Tandem array of repeating LRR are known to provide
protein-protein interaction motif . The plant-specific
LRR motif out of seven subfamilies contains 23-25
amino acids that form an extended b-strand connected
with an a-helix by a loop . Especially, first 11 amino
acid residues (LxxLxLxxNxL) in LRR are highly
conserved and corresponds the region forming b-strand and
loop [21,23]. Leucine residues can be compatible with
isoleucine (I), valine (V), and phenylalanine (F), which
form the hydrophobic core . Asparagine (N) in the
9th position is important for half-turn in LRR unit, and
serine or threonine are the preferred amino acid in the
8th position, just before the asparagine . We found
that the first part of amino acid sequence in the 13th
LRR of BRI1 (LLTLDLSSNNF from 392nd to 402nd
amino acid in BRI1) is well matched with the known
consensus sequence. Compared with that, the serine
residue at the 399th position of BRI1 in front of the
asparagines is changed to phenylalanine in bri1-120
mutant. Regarding that serine or threonine is able to
form an additional hydrogen bond with other part of
proteins, it is highly possible that hydrophobic
phenylalanine instead of serine residue in bri1-120 causes
conformational change of LRR motif in the BRI1. Among
other genes encoding the LRR-RLKs, CLAVATA1
(CLV1) which involves in meristem differentiation has
been reported to have three missense mutant alleles
within LRRs: cla1-10 in LRR4, clv1-4 in LRR5, and
clv18 in LRR9. These mutations were likely to be harmful
for the dimerization of CLV1 with other receptors .
The HAR receptor that regulates the nodulation in
legumes possesses 21 LRRs. Mutation in the LRR7 in
har1-4, which alters b-strand structure, led to the
reduced ligand binding . Therefore, it is possible
that conformational changes due to a mutation in the
13th LRRs of BRI1 affect receptor dimerization or reduce
ligand binding capacity. Recently, several mutants
generated by the TILLING method were reported to have
amino acid changes in the LRR region of the
extracellular domain of BRI1  (http://tilling.fhcrc.org), and
they are awaiting further analysis to reveal the
functional significance of the LRR domain of BRI1.
Interallelic complemented bri1-301 showed different BL
sensitivity as compared to the bri1-301 overexpressing a
There have been many reports that the compact and
downward-curling rosette leaves that are considered to
be weak bri1 mutant phenotypes can be restored by the
overexpression of the genes encoding the positive
regulators of BR signaling, such as BAK1 [5,6], BSK1 
and BES1 , and BRI1 itself . Bri1-9, bri1-5 and
bri1-301 are frequently used in these types of studies.
Here, we showed that the phenotypic defects in the
rosette leaves of bri1-301 can be restored in two ways.
First, we overexpressed BRI1:bri1-120-GFP, causing the
bri1-120 mutation in bri1-301, and we showed that the
transgenic bri1-301 displayed an elongated leaf and
petiole growth pattern similar to that of the wild type
(Figure 4B and 4C). Secondly, we generated plants by
crossing bri1-120 with bri1-301. Receptors that require
the assembly of homodimers in order to become active
signaling complexes were interallelically complemented
[30,31]. However, to date, it has not been elucidated
that whether the bri1 alleles that have the extracellular
domain mutation are able to complement
kinaseinactive bri1 alleles. By showing that more than half of
the F2 plants had perfectly wild type-looking overall
rosette morphologies, we demonstrated a successful
interallelic complementation with two different bri1
alleles (Figure 5). The possibility that the genetic
recombination between one homologous chromosome with a
bri1-120 mutation and the other homologous
chromosome with a bri1-301 mutation occurs during the self
fertilization of a F1 progeny after the initial cross,
resulting in a homologous chromosome without either
mutation, cannot be completely ruled out. However, that
event seems to occur very rarely, because both
mutations are less than 2 Kb apart.
Interestingly, during our analysis, we found significant
differences in growth patterns and the BR sensitivities
between the bri1-301 plants rescued by the genetic
cross with bri1-120 and the bri1-301 plants rescued by
the transformation of a BRI1:bri1-120-GFP construct.
The overall rosette phenotype of the rescued bri1-301
plants generated by any one of the methods was similar
to that of the wild type plants. However, the bri1-301
plants overexpressing BRI1(S399F) due to the
transformation of BRI1:bri1-120-GFP showed reduced root and
hypocotyl growth in normal growth conditions
compared to the wild type plants. Moreover, the BR
sensitivities of these plants were similar to the BR sensitivity of
bri1-120 based on the inhibition of root growth and
CPD expression in response to BL. On the other hand,
both root and hypocotyl growth and BR sensitivity
almost completely reverted to wild type levels in the
plants heterozygous for each mutated allele due to the
cross of bri1-301 and bri1-120 (Figure 6). It is possible
that although the bri1-301 phenotypes could be rescued
by both a transgenic approach, transformation of BRI1:
bri1-120-GFP gene, and a genetic cross with bri1-120,
different growth pattern in detail and the BR sensitivity
between both lines were resulted from the more
accumulation of the BRI1-120-GFP proteins in transgenic
bri1-301, because expression level of transgene was
diverse in each transgenic plant. We also cannot rule
out the possibility that the increased amount of
BRI1120-GFP proteins in transgenic bri1-301 affected only
rosette development with unknown mechanisms yet.
Taken together, these results suggest that observing the
shape of the rescued rosette, including the elongated
leaves and petioles, is not likely to be a precise way to
determine BR sensitivity. A recent publication supported
this view. Albrecht et al. (2008)  reported that the
overexpression of AtSERK4 in bri1-301 led to the
appearance of the rescued compact rosette leaves but
did not promote hypocotyl growth. Additionally, we
previously showed similar phenomena when BAK1 was
overexpressed in bri1-301 . Conventionally, several
indicators, such as the conversion of the rosette leaf
phenotypes from compact, curled and dark-green
elongated, the inhibition and promotion of the root and
hypocotyl growth, respectively, the transcriptional
inhibition of CPD expression, and the BL-induced
accumulation of dephosphorylated BES1, have been used to
denote normal BR sensitivity. We believe that each
experimental method represents a different degree of BR
sensitivity. In that sense, the rescued rosette phenotype
does not reflect heightened BL sensitivity as compared
to any other method. However, the changes observed in
the outward appearance of the weak bri1 mutant
phenotype can still be regarded as useful indicators the genetic
suppressor screening of bri1 mutants to find additional
regulators involved in BR signaling. A BRI1 co-receptor
BAK1 , BRS1 (a secreted carboxpeptidase) , BRL1
(BRI1-like1) , BSU1 (a serine/threonine protein
phosphatase) , and BEN1 (a dihydroflavonol
4reductase-like protein) , and recently published
TCP1 (a transcriptional modulator of DWARF4, BR
biosynthetic gene)  are examples of bri1 suppressors
identified in the activation-tagged bri1-5. In addition,
the proteins involved in ER quality control were
revealed allele-specifically in the genetic suppressor
screening of EMS-mutagenized bri1-9 [39-41]. Bri1-301
was also used for the suppressor screening in the
activation tagged pools, resulting in the identification of
several ATBS genes, including one encoding a bHLH
transcription factor that regulates BR signaling (ATBS1)
 and YUCCA, which is involved in
tryptophandependent auxin biosynthesis (ATBS3 to ATBS6) .
These results imply that the suppressor screening
of bri1 mutant alleles with rosette leaf phenotypes can
allow for the mining of genes related to diverse cellular
functions in addition to BR signaling. We believe that
bri1-120 is a suitable mutant allele for this purpose. We
are currently performing genetic screening to search for
modulators of bri1-120, to expand the understanding of
the functions of this gene.
In summary we demonstrated that the mutant
previously referred to as cp3 that shows retarded growth
and reduced BR sensitivity is allelic to bri1, and we
renamed it bri1-120. The analysis of a point mutation in
the 13th LRR that resides before the 70-amino acid
island portion of the extracellular domain of BRI1 has
indicated that this specific LRR region is critical for
proper BRI1 functioning. Using bri1-120 and bri1-301,
we revealed that interallelic complementation is able to
occur between the extracellular domain mutant allele
and the cytoplasmic kinase-inactive mutant allele of
BRI1 in vivo.
Plant growth condition
We used Arabidopsis thaliana Landsberg (Ler) as the
wild type for the comparison with phenotypic changes
of bri1-120 (seeds from CS48) and used Arabidopsis
thaliana Columbia (Col-0) as the wild type for the
comparison with phenotypes of the transgenic bri1-301
plants. All transgenic plants used here were made by
floral dipping into suspensions of Agrobacterium
tumerfaciens (GV3101) containing appropriate binary plasmid
constructs. Seed sterilization was performed by washing
the seeds with 75% ethanol containing 0.05% Tween-20
for 15 minutes, and then washing them twice with 95%
ethanol. Sterilized seeds were plated in 1/2 MS
(Duchefa) containing 0.8% phytoagar. After stratification at 4
C for 2 days, plates were transferred to a growth room
set at 22C under long-day conditions (16 hours L/8
hours D). To observe the plant phenotypes, the seeds
were sown directly onto soil (Sunshine #5) top-layered
with fine particles of vermiculite.
Construction of plasmids
The plasmid containing the bri1-120 mutation in BRI1 to
express the mutated BRI1 protein, BRI1(S399F), was made
by in vitro site-directed mutagenesis using a QuickChange
Site-Directed Mutagenesis Kit (Stratagene) with
pPZP212BRI1:BRI1-GFP as a template. The sequences of the
primers used were a 5-cgttagatctcagcttcaacaatttctccgg-3
(forward) and 5-ccggagaaattgttgaagctgagatctaacg-3
(reverse). All of the resulting plasmids were fully
sequenced to confirm the presence of the intended
changes and the absence of other alterations. After
confirmation, the plasmid, BRI1:bri1-120-GFP, was
transformed into wild type and bri1-301 plants by
Agrobacterium tumefaciens-mediated floral dipping.
Confocal microscopic analysis of the subcellular
localization of BRI1(S399F)
The localization pattern of BRI1(S399F) was analyzed by
examining the root tips of 5-day-old BRI1:bri1-120-GFP
transgenic seedlings using a Zeiss LSM510 Meta
confocal microscope with excitation set at 488 nm and a
500530-nm band-path filter was used to detect the GFP.
Root growth inhibition assay
To determine the BR sensitivity of the plants, the
sterilized seeds of interest were placed in a line on 1/2 MS
containing 0.8% phytoagar plates supplemented with or
without brassinolide (BL) at the indicated
concentrations. The seeds of the different plants of interest were
seeded in the same plate to minimize ambient
differences. Three sets of plates were plated vertically and
grown for 10 days at 22C under long-light conditions
(16 hours L/8 hours D) for root elongation. Root lengths
were measured for 20-30 seedlings in each line. To
determine the hormone sensitivity of bri1-120, we added
20 M of IAA, GA, kinetin, and ACC and 50 M of JA
to 1/2 MS MS plates and processed them the same way.
All of the chemicals were purchased from Duchefa
Biochemie except IAA (Sigma Aldrich) and BL (Synthchem.
Inc.) All experiments were repeated twice.
CPD expression analysis
We grew the sterilized seeds of interest on the 1/2 MS
(Duchefa) containing 0.8% phytoagar plates supplemented
with or without brassinolide (BL) for 10 days and
extracted total RNA from each seedling. For the northern
hybridization, the total RNA was run on a
formaldehydecontaining 1% agarose gel, blotted onto a nylon
membrane (GE Healthcare) and hybridized with the
32Plabeled CPD probe (32a-P-dCTP, 10 mCi/mol, IZOTOP)
at 42C in a hybridization solution (1M NaCl, 1% SDS,
1% dextran sulfate (Sigma Aldrich), and 50% formamide).
For the RT-PCR analysis, the RNA was treated with
RNase-free RQ1 DNases (Promega), and the first-strand
cDNA was synthesized using the Superscript-MMLV
reverse transcriptase (Invitrogen) and oligo d(T15) primer.
The same aliquot of first-strand cDNA was used as a
template in the second polymerase chain reaction, in
which the CPD transcript was amplified for 23 cycles
with the primers CPD-RTF: 5-gccttcaccgcttttctcctcctc-3
and CPD-RTR: 5-atttgacggcgagagtcatgatcg-3.
Confirmation of BRI1 expression by RT-PCR analysis
RNAs were purified from the seedlings grown for two
weeks on 1/2 MS plate, and treated with RNase-free
RQ1 DNase (Promega). First-strand cDNA synthesis was
performed using the Superscript-MMLV reverse
transcriptase (Invitrogen) according to manufacturers
protocol. Second step of polymerase chain reactions were
performed with the same aliquot of first-strand cDNA
as a template. Polymerase chain reaction was as
followings: pre-denaturation at 94C for 4 min., denaturation
at 94C for 30sec., primer-annealing at 52C for 30 sec.,
elongation at 72C for 30 sec. for 22 cycles, and
postelongation at 72C for 7 min. The primer sequences for
detection of endogenous BRI1 expression are 15F7:
5-tgcgatggatacgcatttaa-3 (forward) and BRI1 3UTR:
5-tcggactgacccttagatg-3 (reverse). The primer sequences
for detection of transgene-derived BRI1 expression are
GFPSEQF: 5-acaacatcgaagacggcggcgtg-3 (forward) and
KH002: 5-cagtaggattgtggtgtgtgcgc-3 (reverse). The
expression of each gene was normalized to b-Tubulin
with primers of TUBF 5-atgcgtgagattcttcacatcc-3
(forward) and TUBR 5-tgggtactcttcacggatcttag-3 (reverse).
Genotyping of bri1-120 and bri1-301 mutations
For the bri1-301 genotyping, the genomic DNA region
adjescent to the bri1-301 mutation was amplified in a
polymerase chain reaction (PCR) with the primer set
5-ggaaaccattgggaagatca-3 (forward) and
5-gctgtttcacccatccaa-3 (reverse) and then digested with DPN. One
of the restriction sites for DPN in the PCR-amplified
fragment is lost in bri1-301, so DNA fragments with
different sizes can be distinguished in the 1% agarose gels
after electrophoresis. For the bri1-120 genotyping, we
PCR-amplified the genomic DNA with specifically
designed dCAPS primers 5-
ccgcttcgttgctaacgttagatctaagct-3 (forward) and
5-ccagttaagattggtacagttacttaaacc-3 (reverse), to generate a Hind site only in
bri1-120. Hind-digested PCR products were run on a
3% agarose electrophoresis gel. Wild type Col, Ler,
bri1-120, bri1-301, and the F1 plants crossed with
bri1-120 and bri1-301 were always included in the
experiments as controls.
Detection of BRI1 proteins by western blot analysis
Total protein crude extracts were prepared from 3-4
leaves of 3-week-old soil-grown plants with the
extraction buffer (50 mM HEPES (pH 7.4), 10 mM EDTA,
0.1% Triton X-100, and a protease inhibitor cocktail
(1 tablet/50 mL, Roche)). Equal amounts of total protein
were separated by 7.5% SDS-PAGE and blotted onto a
PVDF membrane (Bio-Rad) with the BIO-RAD Mini
PROTEAN and Criterion systems, respectively. A
western blot analysis was carried out with anti-BRI1
antibodies and peroxidase-conjugated secondary antibodies
(Goat anti-rabbit IgG, Pierce). Protein bands were
visualized with an ECL plus western blotting detection
system (GE Healthcare).
Additional file 1: Test for BR sensitivity of cp3. Cp3 and Ler were
grown on 1/2 MS for 9 days, and then 1 M of BL and mock treatment
were applied to the plates. Photos were taken after overnight incubation.
Additional file 2: Plant Phenotypes of inflorescence stage. A. Three
representative transgenic bri1-301 plants overexpressing of
BRI1:bri1-120GFP shown in figure 4B were taken pictures after 7 weeks growth. B.
Adult stage phenotypes of F2 plants produced by the genetic crosses of
bri1-120 and bri1-301 shown in figure 5A are exhibited with a bri1-120
Additional file 3: Overexpression of BRI1:bri1-120-GFP in wild type.
A. Transgenic plants that show no discernible phenotypic changes
(Line1) or display strong bri1 mutant-looking phenotypes (Line 2) are
shown with an un-transformed wild type plant. B. Analysis of BRI1
expression from the phenotypically representative transgenic plants.
This work were supported by the Korean Science and Engineering Foundation
(grant # R01-2007-000-20074-0 to K.H.N.), by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (grant # 2010-0022823 to
K.H.N.) and by the National Institute of Health Grant (GM060519 to J.L).
YS designed and performed all of the experiments. MML participated in
designing the experiment involving the genetic crosses of bri1-120 and
bri1301. JL provided the bri1 mutant seeds and helped with manuscript
preparation. KHN is the primary investigator for this study; she conceived
and coordinated the whole study, and wrote and revised the manuscript. All
authors read and approved the final manuscript.
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