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ORIGINAL ARTICLE
On the Origin and Evolution of Plant Brassinosteroid ReceptorKinases
Hao Wang • Hongliang Mao
Received: 6 September 2013 / Accepted: 18 December 2013 / Published online: 27 December 2013
� Springer Science+Business Media New York 2013
Abstract Brassinosteroid (BR) signaling pathway is so
far the best-understood receptor-kinase signaling pathway
in plants. In Arabidopsis, the activation of this pathway
requires binding of BRs to the receptor kinase BRASSI-
NOSTEROID-INSENSITIVE I (AtBRI1). Although the
function of AtBRI1 has been extensively studied, it is not
known when the binding function emerged and how this
important component of BR signaling pathway and related
genes (the BRI1–BRL gene family) have evolved in plants.
We define BRI1–BRL genes in sequenced plant genomes,
construct profiles for critical protein domains, scan them
against all accessible plant gene/EST resources, and reveal
the evolution of domain configuration of this family. We
also investigate its evolutionary pattern through phyloge-
netic analysis. The complete BR receptor domain config-
uration originates through two domain gain events in the
ancestral receptor-like kinase: first juxtamembrane domain
gained during the early diversification of land plants, and
then island domain (ID) acquired in the common ancestor
of angiosperms and gymnosperms after its divergence from
spike moss. The 70 amino acid ID has characteristic
sequences of BRI1–BRL family and this family keeps
relative stable copy numbers during the history of angio-
sperms and the majority of duplications and losses have
occurred in terminal taxa in current taxon sampling. This
study reveals important events shaping structural and
functional characteristics of plant BR receptors. It answers
the question of how and when BR receptors originates,
which provide insights into the origin and evolution of the
BR signaling pathway.
Keywords Brassinosteroid � BRI1 � Protein domain �Gene family � Evolution
Introduction
Brassinosteroids (BRs) are a group of plant steroid hor-
mones that play critical roles in a wide range of develop-
mental and physiological processes such as cell elongation,
vascular differentiation, root growth, light response, stres-
ses resistance, and senescence (Clouse and Sasse 1998;
Kim and Wang 2010; Vert et al. 2005). The last two dec-
ades have observed great advances in assembling the BR
signal transduction pathway (or network Wang et al. 2012)
in Arabidopsis. A number of component genes involved in
the BR transduction pathway have been defined and
important signal transduction steps, from BR perception by
the receptor kinase to the activation of the most upstream
transcription factors of the BR-dependent transcriptional
network, have been revealed (e.g. see reviews in Clouse
2011; Kim and Wang 2010).
In early events that activate the Arabidopsis BR sig-
naling network, the binding of BR requires the function of
BR receptor kinase BRASSINOSTEROID-INSENSITIVE
I (BRI1 or AtBRI1). Genetic and biochemical studies have
established AtBRI1 as the major receptor of BRs (Ki-
noshita et al. 2005; Li and Chory 1997) in Arabidopsis, and
great efforts have been made to elucidate the functional
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-013-9609-5) contains supplementarymaterial, which is available to authorized users.
H. Wang � H. Mao
T-Life Research Center, Department of Physics, Fudan
University, Shanghai 200433, People’s Republic of China
H. Wang (&)
Department of Genetics, University of Georgia, 120 Green
Street, Athens, GA 30602, USA
e-mail: wanghao@uga.edu
123
J Mol Evol (2014) 78:118–129
DOI 10.1007/s00239-013-9609-5
regions or protein domains in this gene (see review in Kim
and Wang 2010). The currently accepted domain configu-
ration of AtBRI1 is LRR{20}–ID–LRR21–LRR{3}–TM–
JM–KD–CT (Kim and Wang 2010; Vert et al. 2005), where
LRR, ID, LRR21, TM, JM, KD, and CT denote leucine-
rich repeats, island domain, the 21st LRR domain, trans-
membrane region, juxtamembrane region, kinase domain,
and C-terminal region, respectively. LRR21 and its
upstream adjacent ID play important roles together so this
LRR is marked specially. The numbers in braces are copy
numbers of tandem domain units. For instance, LRR{20}
means 20 tandem LRR domains. AtBRI1 in fact has 25
LRRs (Kinoshita et al. 2005), but the one located at
N-terminal is irregular (She et al. 2011). If taking the
irregular LRR as the first LRR, ID is located between
LRR-21 and -22. In this study, we count the first regular
LRR as the first LRR, following the numbering system
used by several previous works (Kim and Wang 2010; Li
and Chory 1997; Vert et al. 2005).
Several homologs of AtBRI1 have been identified: (1)
AtBRI1 orthologs have been defined in a number of crops
such as tomato (Curl3/tBRI1/SR160; Koka et al. 2000;
Montoya et al. 2002), rice (OsBRI1; Yamamuro et al.
2000), barley (HvBRI1; Chono et al. 2003), cotton
(GhBRI1; Sun et al. 2004), grape (VvBRI1; Symons et al.
2006), and pea (LKA/PsBRI1; Nomura et al. 2003), and
their function in BR perception and plant growth have been
confirmed by mutational researches (see Clouse 2011;
Morillo and Tax 2006 for brief reviews). (2) AtBRI1 is the
major, but not the only BR receptor in Arabidopsis. Three
paralogs of AtBRI1 have been cloned and two of them,
AtBRL1 and AtBRL3, can also bind to BR and mediate
cell-type-specific BR response and rescue the bri1 mutant
when expressed under the control of the BRI1 promoter
(Cano-Delgado et al. 2004). The investigation of the
counterparts of AtBRI1 and AtBRL genes in rice seems to
support the model that BRI1 performing as major BR
receptor and some of its homologs as partial functional
backup also work in rice (Nakamura et al. 2006).
The structure and function of AtBRI1 have been
extensively studied. In contrast, the evolution of AtBRI1
and related genes (called the BRI1–BRL gene family
hereafter) is largely unrevealed. AtBRI1 belongs to LRR
receptor-like kinases (LRR RLKs) which are characterized
by extracellular LRR arrays, a single-pass TM and a
cytoplasmic KD (Shiu and Bleecker 2001a). Though the
relationship and diversity of LRR and KD have attracted
many attentions and these studies have established that
BRI1–BRL genes form a clade in the KD phylogenetic tree
(Dolan et al. 2007; Kobe and Kajava 2001; Lehti-Shiu et al.
2009; Matsushima et al. 2009, 2010; Shiu and Bleecker
2001a, b; Shiu et al. 2004), none of these researches have
focused on the BRI1–BRL family per se. To date, the
relationship between BRI1 and BRL genes only has been
discussed in quite limited organisms (Cano-Delgado et al.
2004; Gish and Clark 2011; Nakamura et al. 2006). The
pioneering work based on the phylogeny of the 10 avail-
able BRI1–BRL genes (Cano-Delgado et al. 2004) dis-
covered that the early duplications splitting BRI2 from
BRI1 and BRL1–3 occurred prior to the divergence of
monocots and dicots. However, due to the lack of data, this
work failed to correctly resolve the time of the split of
BRL1 and BRL3 and could not discuss many other ques-
tions such as the birth and death patterns and lineage dis-
tribution of family members. In short, systematic
investigations of this family with an extensive taxon sam-
pling have not been reported yet and how and when this
family originated still remains an open question.
Recent genomics revolution has uncovered genomes
from many major lineages of plant tree of life and depos-
ited a large number of EST/transcriptome data for lineages
without fully sequenced genomes. With the aim of illu-
minating the origin and evolution of structure and function
of plant BRI1–BRL genes, we have performed comparative
analyses of the BRI1–BRL gene family in major clades of
the plant kingdom. Focusing on the 44 sequenced plant
genomes [32 land plants and 12 green algae (Supporting
Information Table S1)], we have identified the BRI1–BRL
gene repertoire in 29 genomes using bioinformatic method
and tried to answer the following questions: (1) how and
when the family originated, (2) relationship between family
members, and (3) pattern of gene duplication and loss. We
also have discussed a possible picture of the origin of the
single-exon structure of this family.
Materials and Methods
Sequence Data
Fully sequenced plant genomes and gene annotations were
downloaded from phytozome version 8 (http://phytozome.
net/). Data of chlorophytes that were deposited in JGI but
not included in phytozome were downloaded from JGI
genome portal (http://genome.jgi.doe.gov/). The genome
and annotations of Amborella trichopoda were downloaded
from Amborella Genome Database (http://www.amborella.
org/). ESTs, EST assemblies, transcriptome assemblies,
and cDNAs (mRNAs) of conifers as well as other nonan-
giosperm plants were collected from TreeGene (http://den
drome.ucdavis.edu/treegenes/) and plantGDB (http://www.
plantgdb.org/). A translated EST dataset was constructed
by predicting coding regions of the ESTs by ORFPredictor
(http://proteomics.ysu.edu/tools/OrfPredictor.html). Pro-
teins of plants that deposited in Uniprot (http://www.uni
prot.org/) were also downloaded. The combined protein set
J Mol Evol (2014) 78:118–129 119
123
was made of nonredundant sequences from genome
annotation, translated EST dataset, and Uniprot.
BRI1–BRL Gene Identification
The combined protein set was scanned against PFAM
v26.0 (Punta et al. 2012) to obtain all genes containing KD
using the Pfam_scan.pl script (ftp://ftp.sanger.ac.uk/pub/
databases/Pfam/Tools). These genes were then assigned to
subfamilies according to the similarity of their KDs with
known KDs of plant RLK/Pelle genes (Lehti-Shiu et al.
2009). The previous study (Shiu and Bleecker 2003) sug-
gested that AtBRI1 and AtBRL genes belonged to the
LRR-Xb-1 subfamily (called RLK/Pelle-LRR-Xb-1 genes
hereafter). All genes belonged to the RLK/Pelle-LRR-Xb-1
subfamily were extracted and the amino acids neighbor-
joining (NJ) tree was built using their KD sequences (see
below). The genes fell within the same well-supported
clade with known BRI1–BRL genes were extracted as
BRI1–BRL candidates (Supporting Information Fig. S1).
We manually cured the BRI1–BRL candidates from
fully sequenced organisms to improve gene models. Every
gene locus was re-annotated through AUGUSTUS (Stanke
et al. 2008) with EST evidence. Each predicted gene model
was compared to that released by genome sequencing
centers and the better model was chosen based on EST
support and sequence alignment quality. This manual
inspection excluded nine models for further study because
they were far shorter than other BRI1–BRL genes or lacked
other essential domains or had no EST support.
Protein Domain Profile Construction
Multiple sequence alignment (MSA) of protein sequences
of BRI1–BRL genes was constructed using MUSCLE
(Edgar 2004) and the alignment was manually inspected. In
the alignment, blocks representing ID, LRR21, JM, and KD
domains were extracted according to their locations in
AtBRI1 (Vert et al. 2005). Profile hidden Markov models
(HMMs) of domains were then constructed by using
HMMER v3.0 package (Finn et al. 2011).
Domain Configuration Identification
The identification of ID, JM, and KD was performed by
hmmsearch (E value = 1e-5) using profile constructed
above. Besides hmmsearch, PFAM profiles (v26.0) were
also scanned against genome sequences of the 12 chloro-
phytes to detect the LRR–KD configuration in these spe-
cies. The presence of TM in genes was predicted using
TMHMM webserver (http://www.cbs.dtu.dk/services/
TMHMM/).
Phylogenetic Analysis
KD regions were aligned using MUSCLE, and the NJ tree
(Saitou and Nei 1987) of all RLK/Pelle-LRR-Xb-1 genes
was built using MEGA 5 (Tamura et al. 2011) with the
following parameters: Poisson model; uniform rate; and
pairwise deletion of gaps/missing data. Here, we excluded
sequences shorter than 140 aa (50 % of average size of
experimentally verified BRI1–BRL genes) and so the size
of KD sequences used in this NJ tree construction was from
140 to 355 aa.
Amino acid maximum likelihood (ML) phylogenetic
tree of BRI1–BRL genes from sequenced angiosperms was
constructed by RAxML (Stamatakis 2006) using conserved
region stretching from ID to KD (ID–LRR{4}–TM–JM–
KD; sequence lengths 536–648 aa). Sequence alignments
were generated by MUSCLE and low quality regions were
excluded from alignment using trimAL (Capella-Gutierrez
et al. 2009) with the option ‘‘automated1’’. Protein model
selection was performed by Prottest 3.0 (Abascal et al.
2005). Parameters of ML tree construction are as follows:
JTT ? I ? G model; duplications of rapid bootstrap = 100.
In 227 Arabidopsis genes of which KDs belong to the
LRR RLK domain group, 21 highly diverged ones (could
not calculate valid evolutionary distance with others) were
excluded. The DNA MSA of the coding sequences of KD
domain (sequence lengths 417–900 nt) was constructed
with MUSCLE and trimmed with trimAL. ML tree was
built by RAxML with the following parameter settings:
model = GTR ? I ? G; duplications of rapid boot-
strap = 100. Model selection was performed by jModeltest
(Posada 2008).
Gene Duplication–Loss History
The species tree used in this study was constructed by
modifying Phytozome 8 plant tree of life: A. trichopoda
was added as sister taxon of monocots and dicots and
Panicum virgatum as sister species of Setaria italica. Gene
tree and species tree reconciliation was performed by No-
tung 2.6 (Chen et al. 2000). When reconstructing the gene
duplication–loss history, weakly supported edges (boot-
strap value \90 %) of the gene tree were allowed to be
rearranged to minimize duplication and loss events.
Selection Analysis
Coding DNA sequence alignments of paralogous pairs
were obtained under the guidance of protein alignments
using PAL2NAL (Suyama et al. 2006). Dn, Ds, and Dn/Ds
values were calculated by the yn00 program of the PAML
120 J Mol Evol (2014) 78:118–129
123
package (Yang 2007) according to the Nei–Gojobori
method (Nei and Gojobori 1986).
Results
BRI1–BRL Genes in Sequenced Genomes
Phylogenetically, the BRI1–BRL gene family, in this
study, was defined as the sub-clade of RLK/Pelle-LRR-Xb-
1 genes (Lehti-Shiu et al. 2009) that included all of the
previously cloned BRI1–BRL genes. We built a NJ tree for
all detected KD in the combined plant protein set made of
whole gene repertoire of the 44 sequenced genomes, all
plant proteins in the Uniprot database and all translated
ESTs of nonangiosperm plants (see ‘‘Materials and Methods’’
section for details). In the tree (Fig. S1), the BRI1–BRL
clade (bootstrap value = 95 %) included a total of 220
sequences, with 136 being full-length BRI1–BRL gene
models (Table S2). Here, full-length means the open
reading frame containing both a start and stop codon. 117
out of the 136 full-length genes were from 29 sequenced
species (Table S3). Our manual inspection predicted 4
novel models in the apple genome (Malus domestica) and
suggested that 35 gene models should be modified based on
expression and/or conservation evidence. The correspon-
dence between corrected models and automatic gene
annotation was shown in Table S3. The other 19 full-length
BRI1–BRL genes were from Uniprot. They belonged to
organisms without whole-genome sequences when this
study was done (Table S2). It is worth noting that all of the
BRI1–BRL genes are from angiosperms and gymnosperms.
Domain Configuration of BRI1–BRL Family
The MSA of the BRI1–BRL family showed that most
domains were quite conservative (Fig. S2). Low conser-
vation was found at the first several LRRs, CT, and internal
region of TM. Overall, LRR10–24 (covering ID) and KD
were the two most conserved parts in the entire alignment.
We compared the domain configuration of AtBRI1 with
other previously identified BRI1–BRL genes and found all
of them had the identical configuration with AtBRI1.
Therefore, we constructed profiles of the LRR, ID, JM, and
KD domains (see ‘‘Materials and Methods’’ section) and
used them to scan the above 220 BRI1–BRL genes. 152
sequences exhibited a domain configuration like ‘‘LRR
array’’–ID–‘‘LRR array’’–TM–JM–KD (Table S2),
including 136 full-length genes (117 in sequenced spe-
cies ? 19 in other species from Uniprot), 11 partial pro-
teins from conifer EST/transcriptome assemblies, and 5
partial proteins from Uniprot. The other 68 sequences had
KD but not ID: 23 from Uniprot, 6 from plantGDB, and 39
from TreeGene EST/transcriptome assemblies. Unlike
protein sequences, several EST/transcriptome assemblies
might come from the same gene and thus the number of
EST/transcriptome assemblies might not correctly reflect
number of genes. However, the occurrence of a certain
domain in these assemblies was sufficient to confirm their
occurrence in corresponding species.
In this scan, we identified TM domains through the
TMHMM webserver, but not through domain profile ana-
lysis because of low conservation at sequence level.
Sequences of the CT region were also highly divergent
(Fig. S2) and the instability of CT was reported previously
(Xu et al. 2009). Therefore, although it might plays an
inhibitory role in BRI1 function (Wang et al. 2005), CT
was not suitable for profile analysis and excluded from this
study. In short, all full-length genes had a domain config-
uration like ‘‘LRR array’’–ID–‘‘LRR array’’–TM–JM–KD,
so this was the valid definition of BRI1–BRL gene family
in term of domain configuration.
Presence and Absence of Domains in Major Lineages
of the Plant Kingdom
We refined the profile for the four domains (LRR, ID, JM,
and KD) based on the MSA of the 136 full-length BRI1–
BRL genes and scanned them against the combined plant
protein set. The results showed that (1) the LRR–KD
combination was detected in 22 genes from 6 chlorophytes
(Table S4). In contrast, we failed to detect the LRR–KD
configuration in the sequenced red algae (Cyanidioschyzon
merolae). In 13 cases, LRR occurred as long tandem arrays
(C5 LRR units). (2) TM domain was located in between
some (nine cases) but not all of the LRR–KD structure. (3)
JM domain was not detected in any chlorophyte, but was
found in all available major groups of land plants including
liverworts, mosses, lycophytes, and seed plants such as
cycads, gnetophytes, conifers (Table S5), and angiosperms.
We found that whenever full-length proteins are available,
their JMs were found co-occurring with LRR array, TM,
and KD, but not always with ID. (4) IDs were only
detected in gymnosperms (conifers and gnetophytes) and
angiosperms. We found that a total of 29 gymnosperm
EST/transcriptome assemblies contained IDs, with 11 of
them showing complete BRI1–BRL gene domain config-
uration (Table S6). In contrast, this domain configuration
analysis failed to detect BRI1–BRL members in plant
genomes that diverged before seed plants and this result
was consistent with the KD phylogenetic analysis descri-
bed above. In summary, according to current available
data, the first occurrence of LRR–TM–KD, LRR–TM–JM–
KD, and LRR–ID–LRR–TM–JM–KD were observed in
chloroplasts, liverworts, and the common ancestor of seed
plants, respectively (Fig. 1).
J Mol Evol (2014) 78:118–129 121
123
ID Was the Diagnostic Domain of BRI1–BRL Genes
In all full-length sequences within the combined plant
protein set, we found that whenever ID was present, the
entire BRI1–BRL domain configuration, i.e. ‘‘LRR array’’
–ID–‘‘LRR array’’–TM–JM–KD, occurred automatically.
Furthermore, we found that if a sequence contains both ID
and KD, the KD fell in the BRI1–BRL clade. These
observations indicate that domain configuration and phy-
logenetic relationship of KD give equivalent definitions of
BRI1–BRL family and ID is the domain only present in the
BRI1–BRL family. This family now can be described as
plant RLKs containing ID.
Characteristics of ID Domain
The investigation of the alignment profile of ID domain of
BRI1–BRL genes (Figs. 2a; S3) identified six residues
conserved in all genes: C23–G27–L29–E31–C49–Y57,
where letters were codes of residues and numbers were the
locations of residues in the alignment. Besides the CGL-
ECY hexad, another six highly conserved residues or
motifs (i.e., occurring in [95 % of the sequences) were
also revealed within ID domain (Fig. 2a). Moreover, the ID
domain sequences could be further categorized as three
groups according to their conservation pattern (Fig. 2b).
The three ID groups were congruent to the three major
clades of the BRI1–BRL family (Fig. 2b and see below).
Phylogenetics of Plant BRI1–BRL Genes
The ML phylogeny of BRI1–BRL genes from the 29
sequenced angiosperms (Fig. 3) supported with 100 %
bootstrap value that the BRI1–BRL family was composed
of three major clades: the basal split was between BRL2
and all others, and the second split was between BRI1 and
BRL1–3 genes. Here, based on the names of the Arabi-
dopsis and rice members, we called the three clades as
BRI1, BRL1–3 and BRL2, respectively. Reconciliation the
BRI1–BRL gene tree with sequenced angiosperm species
tree (Fig. S4) suggested that the three major clades were
derived from two gene duplication events before the
divergence of angiosperms (Fig. S5). These results were
consistent with the previous results from far less sequences
(Cano-Delgado et al. 2004; Gish and Clark 2011).
We investigated gene duplication and loss pattern of the
family. (1) the majority of duplications (85 %, 28 out the
33) and losses (77 %, 10 out of 13) happened after the
divergence of leaf nodes and their closest relative species
in current taxon sampling (Fig. S5). This indicated that no
drastic expansion or contraction of gene number occurred
in the ancestral nodes. (2) Copy number variation of BRI1–
BRL genes was found in all the three clades (Table 1) with
the highest number of genes in M. domestica (nine; three in
each clade) and lowest in Medicago truncatula (one from
the BRI1 clade). However, 62 % (18 out of 29) species had
three or four genes and, except for M. domestica, all
variations of gene copy number within species were within
mean ± 2 9 SD. In this sense, gene numbers showed no
strong organism bias. (3) The three major clades had
similar numbers of genes (41 in BRI1, 41 in BRL1–3, and
35 in BRL2). These observations support that gene num-
bers were quite stable during the evolution of this gene
family.
According to the phylogeny of BRL1–3 (Fig. 3b), the
Arabidopsis and rice BRL1 and BRL3 genes had inde-
pendent origins. AtBRL1 and AtBRL3 were derived from a
duplication in the common ancestor of Brassicaceae, which
left two descendants in each of the five sequenced
Brassicaceae organisms. Rice BRL1 and BRL3 originated
through a duplication in the common ancestor of grass after
divergence from dicots and both descendants were also
preserved in the most investigated grass species, except for
Brachypodium. In switch grass (P. virgatum), a second
round of duplications occurred independently in each of the
two copies and resulted four BRL1–3 genes.
Selection on Paralogs
The 28 terminal species duplications generated 22 groups
of in-paralogs (Koonin 2005): 17 pairs related by 1
duplication, 4 triplets by 2, and 1 quartets by 3 duplications
(Fig. S5). Investigating selection pressure using Nei–
Gojobori method exhibited Dn/Ds values \1 in all within-
group gene pairs (Table S7) and detected no positive
selection.
Fig. 1 Domain configuration evolution of BRI1–BRL family. The
relationship within seed plants remains unresolved because of
controversial results from different datasets and methods (see e.g.
Mathews 2009). The three stages of BRI1–BRL domain configuration
evolution are mapped to the tree. Arrows point to the internal nodes
which are lower bounds of the first presence of configurations. Right
table shows the presence/absence of domains. ‘‘?’’: presence and
‘‘-’’: absence
122 J Mol Evol (2014) 78:118–129
123
Discussion
Origin of the BRI1–BRL Family: A Three-Stage
Process of Domain Gain
The evolution of the domain configuration provides a
scenario of gene family evolution at the level of organi-
zation of structural and/or functional units. Our results
exhibit a perfect consistency between phylogeny and
domain configuration when defining the BRI1–BRL gene
family. If the origin of the family is marked by formation
of the ‘‘LRR array’’–ID–‘‘LRR array’’–TM–JM–KD
domain configuration, we have revealed a stepwise domain
acquirement process in its ‘‘pre-histoy’’.
LRRs present in numerous proteins from all major
branches of the tree of life and with diverse functions
(Kobe and Kajava 2001). To date, at least seven groups of
LRRs have been identified and at least most LRRs have
been found following the so-called the mutual exclusive
rule, i.e., LRRs from different groups are found to never
occur simultaneously in the same protein. Although phy-
logenetic relationships of the seven groups have not been
well resolved yet (Andrade et al. 2000; Kajava 1998), it is
clear that LRRs presented in BRI1–BRL genes belong to
the plant-specific group that are found in plants and protists
(Kobe and Kajava 2001).
The ancient origin of KDs of BRI1–BRL genes has been
uncovered for more than 10 years: this domain belongs to
plant RLKs domain family, which together with animal
Pelle and related cytoplasmic KDs form the RLK/Pelle
domain family. The RLK/Pelle domain family, animal
receptor serine/threonine kinases, animal receptor tyrosine
kinases, and Raf kinases form a clade within the serine/
threonine/tyrosine kinases (Shiu and Bleecker 2001b). The
domain configuration of RLKs is defined as ‘‘extracelluar
domain’’–KD, where extracellular domain represents a
wide range of domains including LRR (Shiu and Bleecker
2001a). It has been suggested that such domain combina-
tion is the structural basis of new signaling pathway evo-
lution (Lehti-Shiu et al. 2009).
Lehti-Shiu et al. (2009) suggested that the LRR–KD
configuration first occurred in streptophytes after its split
from chlorophytes and before the divergence of land plants
(embryophytes). However, their result of absence of LRR–
KD configuration in chlorophytes was based on limited
data: only two species: Chlamydomonas reinhardtii and
Ostreococcus tauri at that time. In this study, we have
revisited this question by identifying domain configuration
of the 12 sequenced chlorophytes genomes deposited in
DOE-JGI genome portal with two methods (see ‘‘Materials
and Methods’’ section and Table S4). We have added the
red algae C. merolae data (Matsuzaki et al. 2004) in this
investigation since red algae have been placed as the sister
group of green plants in the tree of life by many molecular
phylogenetic studies (Adl et al. 2005). Our results support
that the plant LRR–KD configuration originate in green
plants after the split between them and red algae but before
the split of streptophytes from chlorophytes.
JM domains are found in all land plant major lineages
but not in any of the 12 chlorophytes. In contrast, ID only
occurs in gymnosperms and angiosperms. This indicates
that in the evolutionary trajectory of BRI1–BRL genes, the
acquirement of JM is prior to the acquirement of ID. If the
absence of JM domain in the 12 chlorophytes correctly
reflects the absence of this domain in chlorophytes, the
origin of JM probably is estimated to happen after the
streptophytes diverged from chlorophytes but before the
divergence of liverworts from other land plants. It has been
suggested that Charales and Coleochaetales have a closer
relationship to land plants than other green algae (Karol
et al. 2001; Qiu et al. 2006). However, whole-genome data
on these lineages are not available, so we cannot decide if
the origin of JM is before or after the plant colonization of
land.
Both KD similarity and domain configuration analysis
suggest that the upper and lower bound of the origin of ID
are in euphyllophytes after its split from lycophytes but
before the divergence of angiosperms and extant gymno-
sperms. Ferns and horsetails form a major lineage diverged
after lycophytes but before seed plants. Unfortunately,
current accessible data is not enough to resolve the
occurrence of ID in this lineage.
The diversification of this three clades, and maybe the
differentiation of functions (see below), can also be dated
as before the differentiation of angiosperms and gymno-
sperms: in the RLK/Pelle-LRR-Xb-1 NJ tree (Fig. S1), all
of the three major clades of BRI1–BRL family have
members from conifers (bootstrap support at some nodes
are low). If gymnosperms form the sister clade of angio-
sperms, as argued by the recent molecular studies (see
review in Mathews 2009), the origin of the three clades
probably can be traced to the ancestor of seed plants. If not,
Fig. 2 ID domain of BRI1–BRL family. a Alignment after redun-
dancy elimination at the cutoff of 90 % of sequence identity. The
complete alignment is shown in Fig. S3. In each subgroup, conserved
residues of ID are highlighted with dots. Blue stripes mark the column
of conserved residues. Numbers on the top of alignments shows the
locations of residues in the alignment. b Schematic representation of
the ID domain for each group. ID domains are represented by
horizontal blue stripes. Vertical bars with amino acid symbols above
domains exhibit locations of conserved residues. Vertical bars at two
terminals of domains exhibit locations of the first and last residues.
The relationship between the three groups is shown on the left. This
topology is based on the NJ tree of ID sequences but only the basal
branching pattern of the three groups is shown. Bootstrap values
(1,000 replicates) of the three groups are shown in branches (Color
figure online)
c
J Mol Evol (2014) 78:118–129 123
123
the lower bound can be placed at the common ancestor of
angiosperms and conifers.
In short, the domain configuration evolution of BRI1–
BRL family can be resolved as a three stage process
(Fig. 1): first, the LRR–TM–KD configuration was ancient
to all green plants; then in streptophytes, either before or
after the divergence of Coleochaetales and Charales, JM
domain added in; and at last, ID appeared in the common
ancestor of angiosperms and gymnosperms after differen-
tiated from lycophytes.
124 J Mol Evol (2014) 78:118–129
123
Origin of BR Recognition via BRI1
Although island regions which interrupt LRR in proteins
containing LRR arrays have been widely observed, their
origin, evolution, and function are little known (Matsu-
shima et al. 2009). To date, only in very few genes, e.g.
AtBRI1 in Arabidopsis, DcPSKR in Zinnia elegans and
Toll in Drosophila, island regions have been proposed to
be functional (Gibbard et al. 2006; Kinoshita et al. 2005;
Shinohara et al. 2007). In all the three cases, the functional
island regions (or ID) have been inferred playing a role in
ligand interaction.
In AtBRI1, the essentiality of ID (along with its
downstream LRR) in BR binding has been confirmed
(Kinoshita et al. 2005). Recently resolved three-dimen-
sional structure of AtBRI1 has revealed the details of how
ID functions in BR perception (Hothorn et al. 2011; She
et al. 2011): ID folds back into the interior of the LRR
superhelix to form a surface pocket which the brassinolide,
the first isolated BR, can bind to. Since ID acquirement is
the last step during the domain configuration evolution of
the BRI1–BRL family, this domain gain event provides a
LRR RLK gene the ability to recognize BRs and the
possibility that this new gene is recruited as the receptor to
initiate the BR signaling cascade.
Subsequently, an interesting question is when and how
many times this occurred. Although exceptions have been
reported (Lynch and Wagner 2008; Nehrt et al. 2011), the
orthology conjecture, i.e. orthologues carry out equivalent
functions, whereas paralogues undergo functional diversi-
fication, seems applicable in general (Gabaldon and Koo-
nin 2013). In our case, the AtBRI1 orthologs in tomato
(Holton et al. 2007) have also been established as active
BR receptors. In rice, evidence also suggests that OsBRI1
is a BR receptor (Yamamuro et al. 2000; Zhao et al. 2002).
If the orthology conjecture is valid in our case and the
orthology of BRI1 genes in Arabidopsis, tomato and rice
reflects conservation of ancient function, our results sug-
gest that recruiting of BRI1 gene as a component of BR
signaling pathway has a single origin in plant evolution.
We failed to detect BRI1–BRL homologs with ID in
nonseed plants in this study. However, BRs have been
detected throughout the plant kingdom in every species that
has been examined, including nonseed plants such as fern
(Equisetum arvense), liverwort (Marchantia polymorpha),
and green algae (Chlorella vulgaris, Hydrodictyon
Fig. 3 Amino acids ML phylogeny of BRI1–BRL genes in
sequenced plant genomes. a Relationship of deep nodes and the
BRI1 clade. KD of AT5G07280 is used as outgroup. b, c Phylogeny
of the BRL1–3 and BRL2 clades, respectively. Bootstrap values of
100 replicates are shown in branches. Only values of 50 % or more
are shown. The format of names of leaf nodes is ‘‘three-letter species
code’’ ‘‘Gene ID’’/‘‘location of the region used in tree construction’’.
The correspondence between species code and the standard species
names can be found in Table S1. The detailed information about Gene
ID can be found in Table S3
J Mol Evol (2014) 78:118–129 125
123
reticulatum) (Bajguz and Tretyn 2003; Clouse 2011).
Therefore, if BR signaling exists in these ‘‘lower’’ plant
lineages, they should use a different BR receptor. Collec-
tively, we estimate that the origin of the Arabidopsis BR
signaling paradigm is no earlier than the divergence of
lycophytes.
The Consistency Between Phylogenetic and Functional
Differentiation
Our results show strong correlation between evolutionary
pattern and gene function: it is known (Cano-Delgado et al.
2004; Clay and Nelson 2002; Li and Chory 1997; Na-
kamura et al. 2006) that Arabidopsis BRI1–BRL genes can
be functionally categorized as two groups: (1) AtBRI1,
AtBRL1 and AtBRL3 perform as receptors of BR. (2)
AtBRL2 does not induce BR response but plays a role in
transduction of other extracellular spatial and temporal
signals into downstream cell differentiation responses in
provascular/procambial cells (Ceserani et al. 2009; Clay
and Nelson 2002). In the BR receptor group, AtBRI1 is the
major BR receptor which ubiquitously expresses in grow-
ing cells, while AtBRL1 and AtBRL3 perform as func-
tional redundancy partners of AtBRI1 and induce cell-type-
specific BR response in vascular tissues (Cano-Delgado
et al. 2004). Consistent with this functional diversity, the
ML phylogeny (Fig. 3) exhibits that the basal split of
BRI1–BRL genes is between BRL2 and all others, and the
second split is between BRI1 and BRL1–3 genes and the
two splits are caused by duplications that induce neo- and
sub-functionalization.
At nine sites in the ID, amino acids are conserved in BR
binding genes AtBRI1, AtBRL1–3, and OsBRI1, but not in
the confirmed non-BR-binding gene AtBRL2. Moreover,
seven out of the nine sites (i.e. 9K–11Y/F–61T–64T–68N–
69G–71S) are highly conserved (identity [90 %) in all
BRI1 and BRL1–3 gene models (Fig. 2). Therefore, it is
possible that one or several of these substitutions may have
played a role in the functional diversification of AtBRL2. It
is worth noting that substitution in ID domain may not be
the (or the only) source of the functional alternation of
AtBRL2—changes in other domain may also contribute to
or be fully responsible for that. Further analyses are needed
to reveal why AtBRL2 does not function as a BR receptor.
A Possible Picture of the Evolution of BRI1–BRL
Exon–Intron Structure
95 % (111 out of the 117) BRI1–BRL genes were single-
exon genes. Mapping exon–intron structures into the phy-
logeny of BRI1–BRL family and related genes could
resolve the evolution of the exon–intron structure. How-
ever, if constructing the phylogeny using all BRI1–BRL
related genes (i.e. all LRR RLK genes) from the 29
sequenced species, the relatively short size of KD domain
(\300 aa) and the huge number of genes (Lehti-Shiu et al.
2009) would make the tree highly unreliable. Here, we
used a sampling strategy by investigating LRR RLK genes
in the Arabidopsis genome. The reasons for choosing
Arabidopsis thaliana were as follows: (1) its LRR genes
were distributed in most of the subclades of the LRR RLK
domain family (Shiu et al. 2004) and (2) this genome had
so far the best gene annotation quality in plants. In this
study, we only investigated exon–intron structure within
the protein-coding gene ORFs but not untranslated regions
because the prediction of untranslated regions is less
reliable.
The relationship between DNA sequences of KDs of
Arabidopsis LRR RLK genes is shown in Fig. S6. Con-
sistent with the previous results (Shiu and Bleecker 2003;
Table 1 BRI1–BRL genes in sequenced angiosperm genomes
Organism BRI1 BRL1–3 BRL2 Sum
Aquilegia coerulea 1 0 1 2
Arabidopsis lyrata 1 2 1 4
Arabidopsis thaliana 1 2 1 4
Amborella trichopoda 1 0 2 3
Brachypodium distachyon 1 1 1 3
Brassica rapa 3 2 1 6
Citrus clementina 1 1 1 3
Carica papaya 1 1 1 3
Capsella rubella 1 2 1 4
Cucumis sativus 1 0 1 2
Citrus sinensis 1 1 1 3
Eucalyptus grandis 1 2 2 5
Glycine max 2 2 2 6
Linum usitatissimum 4 0 2 6
Malus domestica 3 3 3 9
Manihot esculenta 2 2 2 6
Mimulus guttatus 2 1 1 4
Medicago truncatula 1 0 0 1
Oryza sativa 1 2 1 4
Panicum virgatum 2 4 0 6
Prunus persica 1 1 1 3
Populus trichocarpa 2 2 2 6
Phaseolus vulgaris 1 1 1 3
Ricinus communis 1 1 1 3
Sorghum bicolor 1 1 1 3
Setaria italica 1 2 1 4
Thellungiella halophila 1 2 1 4
Vitis vinifera 1 1 1 3
Zea mays 1 2 1 4
Total 41 41 35 117
126 J Mol Evol (2014) 78:118–129
123
Shiu et al. 2004), this phylogeny suggested that BRI1–BRL
family and its sister gene EMS1 (EXCESS MICROSP-
OROCYTES1, Ath:AT5G07280, a putative LRR RLK
gene that controls somatic and reproductive cell fates in
Arabidopsis Zhao et al. 2002) formed a monophyletic
group (bootstrap value = 55 %) and the most intron-con-
taining genes were diverged earlier. If the grouping of
BRI1–BRL and EMS1 as a clade was valid and old LRR
RLK genes were intron containing, the origin of single-
exon structure of the BRI1–BRL family could be parsi-
moniously explained as intron loss event(s) in their com-
mon ancestor before the divergence of EMS1. However,
we note that both observations have only rather poor sta-
tistical support and further studies are needed to fully
resolve this question.
Mapping the domain configuration of Arabidopsis LRR
RLK genes to the phylogeny (Fig. S6) identified only five
genes containing JM domain: the four BRI1–BRL genes
and EMS1. With low statistical support, the pattern that all
of the JM containing genes formed a clade indicated that
the acquirement of JM might be posterior to the occurrence
of the single-exon structure of BRI1–BRL family.
Intron-Gain Events
The other six BRI1–BRL genes with two introns were
probably derived from intron gain. Flanking coding
sequences of these introns were highly conservative (two
examples are shown in Fig. S7) and all of these introns
were located at different sites in the genes, and therefore
the six introns were likely to be gained through indepen-
dent events.
Nature of Ancient Intron Loss
Ancient intron loss event(s) that shaping current exon–
intron structure of BRI1–BRL genes may have removed
single or multiple introns at a time. Both reverse trans-
criptase-mediated (RT-mediated) intron loss (Derr 1998;
Roy and Gilbert 2006) and retroposition (Brosius 1991;
McCarrey and Thomas 1987) can lead to simultaneous loss
of multiple introns. Retroposition usually generates an in-
tronless copy that is located at a different locus from the
original copy through the activity of retroelements like
LINEs or LTR retrotransposons. RT-mediated intron loss,
or gene conversion by a cDNA, however, removes introns
in the original gene, but does not change its physical
position in the genome. The two mechanisms can thus be
tested when compared species are evolutionarily close
enough so that synteny is detectable at target loci. In the
BRI1–BRL case, however, it is difficult to distinguish the
two mechanisms because of the rapid erosion of gene
synteny in plant genome evolution.
Acknowledgments This work was supported by the National Basic
Research Program of China (973 Project No. 2007CB814800 and
2013CB834100) and the Shanghai Leading Academic Discipline
Project (No. B111). This study was also supported in part by
resources and technical expertise from the Georgia Advanced Com-
puting Resource Center, a partnership between the University of
Georgia’s Office of the Vice President for Research and Office of the
Vice President for Information Technology.
Conflict of interest The authors declare that they have no conflict
of interest.
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