Plant Physiology Preview. Published on May 1, 2017, as DOI:10.1104/pp.17.00118
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Short title: New BRI1 mutations
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Corresponding author: Jia Li
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Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations,
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School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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+86 931 8915662
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Email: [email protected]
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Copyright 2017 by the American Society of Plant Biologists
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Scanning for new BRI1 receptor mutations via TILLING analysis
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Chao Sun1, Kan Yan1, Jian-Ting Han1, Liang Tao1, Ming-Hui Lv1, Tao Shi1,
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Yong-Xing He1, Michael Wierzba2, Frans E. Tax2, Jia Li1,*
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Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations,
School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ
85721, USA
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*Corresponding author: Jia Li, [email protected]
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Present address of Kan Yan: School of Chemical and Biological Engineering,
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Lanzhou Jiaotong University, Lanzhou 730000, China
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One sentence summary: Nine morphologically altered new BRI1 mutants were identified
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from 83 independent TILLING mutations in Arabidopsis thaliana.
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List of author contributions: J. L. and F. E. T. conceived the research plans,
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supervised the experiments and edited the manuscript; C. S. designed the experiments,
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analyzed the data and prepared the draft of the article; C. S. and K. Y. performed most
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of the experiments; L. T., M-H. L. and M. W. performed part of the experiments; J-T.
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H. and Y-X. H. performed the molecular dynamic simulation analysis; T. S. helped
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with the sequence analysis.
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Funding information: This project is supported by the National Natural Science
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Foundation of China (31470380 and 31530005 to Jia Li)
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ABSTRACT
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Identification and characterization of a mutational spectrum for a specific protein can
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help to elucidate its detailed cellular functions. BRI1, a multidomain transmembrane
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receptor-like kinase, is a major receptor of brassinosteroids in Arabidopsis. Within the
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last two decades, over 20 different bri1 mutant alleles have been identified, which
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helped to determine the significance of each domain within BRI1. To further
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understand molecular mechanisms of BRI1, we tried to identify additional alleles via
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TILLING. Here we report our identification of 83 new point mutations in BRI1,
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including 9 mutations that exhibit an allelic series of typical bri1 phenotypes, from
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subtle to severe morphological alterations. We carried out biochemical analyses to
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investigate possible mechanisms of these mutations in affecting BR signaling. A
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number of interesting mutations have been isolated via this study. For example,
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bri1-702, the only weak allele identified so far with a mutation in the activation loop,
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showed reduced autophosphorylation activity. bri1-705, a subtle allele with a
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mutation in the extracellular portion, disrupts the interaction of BRI1 with its ligand
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brassinolide (BL) and co-receptor BAK1. bri1-706, with a mutation in the
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extracellular portion, is a subtle defective mutant. Surprisingly, root inhibition
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analysis indicated that it is largely insensitive to exogenous BL treatment. In this study,
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we found bri1-301 possess kinase activity in vivo, clarified a previous report arguing
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that kinase activity may not be necessary for the function of BRI1. These data provide
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additional insights into our understanding of the early events in the BR signaling
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pathway.
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INTRODUCTION
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Brassinosteroids (BRs) are known to play critical roles in regulating many aspects of
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plant growth and development, including germination, cell elongation, flowering time
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control, reproduction, photomorphogenesis, and skotomorphogenesis (Clouse and
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Sasse, 1998; Gudesblat and Russinova, 2011; Wang et al., 2012; Zhu et al., 2013).
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Recent studies revealed that BRs are also involved in many additional physiological
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processes, such as root meristem maintenance (Gonzalez-Garcia et al., 2011; Hacham
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et al., 2011; Vilarrasa-Blasi et al., 2014; Chaiwanon and Wang, 2015; Wei and Li,
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2016), stomatal development (Gudesblat et al., 2012; Kim et al., 2012), root hair
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differentiation (Cheng et al., 2014), leaf angle regulation in monocots (Zhang et al.,
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2012), shoot branching control (Wang et al., 2013), and organ boundary determination
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(Bell et al., 2012; Gendron et al., 2012).
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In Arabidopsis, BRs are perceived by their major receptor BRASSINOSTEROID
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INSENSITIVE 1 (BRI1) and co-receptor BRI1-ASSOCIATE RECEPTOR KINASE 1
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(BAK1), both of which are single-pass transmembrane leucine-rich repeat
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receptor-like protein kinases (LRR-RLKs) (Li and Chory, 1997; Li et al., 2002; Nam
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and Li, 2002; Gou et al., 2012). There are 25 total extracellular LRRs in BRI1.
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Between 21st and 22nd LRR, there is an amino acid sequence containing 68 residues
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named as an “island”. Genetic and structural analyses indicated that the “island” and
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the 22nd LRR are directly involved in BR binding. In the absence of BRs, the kinase
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activity of BRI1 is inhibited by its carboxyl terminus (Wang et al., 2005) and by its
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associated inhibitory protein named as BRI1 KINASE INHIBITOR 1 (BKI1) (Wang
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and Chory, 2006). When BRs are present, on the other hand, they can directly interact
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with the extracellular pocket of BRI1, causing BKI1 to dissociate from the complex
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(Wang and Chory, 2006; Hothorn et al., 2011; She et al., 2011; Wang et al., 2014). The
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newly formed BRI1-BL surface can directly interact with the extracellular residues of
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BAK1 via several hydrogen bonds (Santiago et al., 2013; Sun et al., 2013). The
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BRI1-BL-BAK1 complex can then initiate early BR signaling events and activate its
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downstream signaling cascade (Wang et al., 2008). Genetic and structural analyses
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demonstrated the essential roles of both BRI1 and BAK1 in controlling BR signal
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transduction (Clouse et al., 1996; Li and Chory, 1997; Hothorn et al., 2011; She et al.,
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2011; Gou et al., 2012; Santiago et al., 2013; Sun et al., 2013).
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During the past two decades, over 20 unique bri1 alleles have been isolated
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(Clouse et al., 1996; Li and Chory, 1997; Noguchi et al., 1999; Friedrichsen et al.,
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2000; Xu et al., 2008; Belkhadir et al., 2010; Shang et al., 2011; Gou et al., 2012).
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Analyses of these mutants have contributed significantly to our better understanding
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of BRI1 in regulating plant growth and development (Vert et al., 2005). These alleles
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share similar phenotypes in various degrees, including dwarfism, dark-green, compact
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rosette leaves with short petioles, delayed flowering time and senescence, reduced
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male fertility, and altered both photomorphogenesis and skotomorphogenesis (Clouse,
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1996; Kwon and Choe, 2005). Most alleles exhibiting the strongest morphological
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defects contain point mutations in the “island” or kinase domain (Friedrichsen et al.,
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2000). BRI1 was first cloned via a map based strategy (Li and Chory, 1997). Many of
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the early isolated bri1 alleles are strong alleles with largely reduced male sterility,
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preventing their use in large scale genetic transformation assays. Fertile weak bri1
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alleles, such as bri1-5 and bri1-9, on the other hand, are excellent genetic tools used
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for characterizing the entire BR signaling pathway via extragenic modifier screens
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(Noguchi et al., 1999). For example, several key components regulating BR signal
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transduction or BR homeostasis were identified via activation tagging genetic screens
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using bri1-5 as background, including BRS1 (Li et al., 2001), BAK1 (Li et al., 2002),
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BSU1 (Mora-Garcia et al., 2004), BRL1 (Zhou et al., 2004), BEN1 (Yuan et al., 2007),
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and TCP1 (Guo et al., 2010). EBS1 to EBS7, a series of functionally related proteins
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mediating endoplasmic reticulum (ER) quality control, were isolated via a suppressor
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screen from ethylmethanesulfonate (EMS) mutagenized bri1-9 (Jin et al., 2007; Jin et
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al., 2009; Su et al., 2011; Hong et al., 2012; Su et al., 2012; Liu et al., 2015). BES1, a
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key downstream transcription factor in the BR signaling pathway, was identified via a
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suppressor screen utilizing an EMS mutagenized bri1-119 library (Yin et al., 2002).
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Identification of these key regulatory components by using weak bri1 mutants has
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contributed significantly to our current knowledge of the BR signaling pathway, from
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BR perception to downstream responsive gene regulation.
In order to deeply understand the molecular mechanisms of BRI1 in regulating
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the BR signaling pathway, we analyzed all BRI1 point mutations generated from a
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Targeted Induced Local Lesions IN Genomes (TILLING) project (McCallum et al.,
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2000). Among 83 total BRI1 point mutations isolated, we identified 9 mutants,
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including 4 subtle, 1 weak, and 4 strong alleles. For example, bri1-702 is a new weak
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allele with a mutation in the activation loop of the BRI1 kinase domain. Several
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previously identified bri1 activation loop point mutants are all strong alleles.
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Therefore, bri1-702 represents the first weak allele bearing a point mutation in the
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activation loop. bri1-705 is a new subtle allele containing a point mutation in the
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extracellular portion. Structural analysis suggests that this mutation likely disrupts the
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formation of hydrogen bonds among BRI1, brassinolide (BL), and BAK1. A third
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interesting mutant is bri1-706, containing a point mutation in the 8th LRR of the
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extracellular portion of BRI1. bri1-706 shows a subtle aerial part defective phenotype
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compared to wild-type (WT) plants, but its root growth is largely insensitive to
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exogenously applied BL. The new mutants identified from these studies can be used
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as genetic tools for further dissecting the functions of BRI1, as well as utilizing as
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references for studying the biological functions of other RLKs in plants.
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RESULTS
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A series of new bri1 alleles were identified via TILLING analyses
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BRI1, the major receptor of brassinosteroids in Arabidopsis, contains multiple
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functional domains (Li and Chory, 1997; Friedrichsen et al., 2000). Mutational
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analyses have helped to characterize the biological significance of each of these
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domains. To further dissect the roles of BRI1 in the BR signaling pathway, we tried to
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identify all possible BRI1 mutations and identify the mutants with altered phenotypes.
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TILLING is a reverse genetic method using a high-throughput screening
technology to detect point mutations from an EMS mutagenized library (McCallum et
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al., 2000). Using this approach, a large number of mutations can be isolated for a
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specific target gene. TILLING has been used to generate mutations for a number of
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important genes including BRI1 in Arabidopsis (Till et al., 2003). All 87 BRI1
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TILLING mutations were obtained from the Arabidopsis Biological Resource Center
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(ABRC) and 83 mutations were confirmed by a dCAPs method (Neff et al., 1998).
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These mutations are distributed throughout the entire BRI1 sequence, especially in
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LRR1-10 of its extracellular portion, “island” and kinase domain (Supplemental
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Figure S1). These mutations were generated in Columbia (Col-0) background
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containing an er mutation. A high dose of EMS could introduce mutations in other
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genes that could result in BRI1-unrelated phenotypes. In order to remove those
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additional mutations, we backcrossed the mutants with Col-0 for at least three
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generations. Offspring plants containing BRI1 mutations were selected by using the
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dCAPs approach (Neff et al., 1998). After backcrossing three times, most mutations
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were found to exhibit no morphological differences from wild-type plants. Only 9
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mutations consistently exhibit typical bri1-like phenotypes. These mutants were
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named in the order of their identification, from bri1-702 to bri1-711 (Figure 1, Table
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1).
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To further confirm their bri1-related phenotypes, we carried out additional two
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backcrosses for the 9 new mutants isolated via TILLING. In the F2 generation of the
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third backcrossing, the phenotypes of bri1-702, bri1-705, bri1-706, bri1-710 and
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bri1-711 were segregated out as single recessive alleles with a ratio close to 3:1.
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However, for strong alleles, the ratio is less than 4:1, which might be caused by lower
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germination rates as observed previously (Steber and McCourt, 2001). Thus, the
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phenotype appears to be caused by a single locus for all 9 different mutants. bri1-702
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shows a weak bri1 phenotype similar to bri1-5, bri1-9 and bri1-301 (Figure 2A)
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(Noguchi et al., 1999; Xu et al., 2008). In comparison, bri1-705, bri1-706, bri1-710
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and bri1-711 show even weaker phenotypes than bri1-702, which were therefore
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named as subtle alleles in this study. Whereas bri1-703, bri1-704, bri1-708, and
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bri1-709 show severe phenotypes similar to a previously reported bri1 null allele,
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bri1-701 (Figure 2A) (Gou et al., 2012). Interestingly, bri1-707 and bri1-301 both
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have mutations in Gly989, but with different amino acid substitutions. As a
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consequence, bri1-301 shows a weak bri1 phenotype (Xu et al., 2008), whereas
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bri1-707 does not show any defective phenotypes. Similarly, bri1-708 and
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bri1-8/108-112 bear different mutations at the same residue. While bri1-8/108-112 are
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intermediate bri1 mutants (Noguchi et al., 1999), bri1-708 shows a null bri1
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phenotype (Figure 2A). Statistically, all these 9 new bri1 mutants show significantly
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reduced growth (Figure 2B).
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To exclude the possibility that the phenotypes are caused by mutations of other
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genes closely linked to BRI1, which are not easily segregated out by backcrossing, we
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crossed these mutants with a number of known bri1 weak alleles. If the phenotype is
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caused by a mutation in a different gene, the F1 seedlings should show a wild-type
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phenotype due to functional complementation. All the obtained F1 seedlings show
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bri1 like phenotypes (Figure 3A). In addition, we transformed the wild-type BRI1
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sequence driven by its native promoter into these 9 different bri1 mutant alleles. The
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transgenic plants showed BRI1-overexpressing phenotypes in WT background (Figure
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3B) (Wang et al., 2001; Nam and Li, 2002). We next transformed the genomic DNA
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sequences (containing BRI1 promoter and its coding sequences) of bri1-702 and
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bri1-705 to the null mutant, bri1-701. The resulting transgenic plants resembled the
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phenotypes of bri1-702 and bri1-705, respectively (Figure 3C). These genetic and
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transgenic results demonstrated that all 9 new mutants are truly allelic to bri1.
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Characterization of newly identified bri1 mutants
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Root inhibition analysis indicated that bri1-702 and bri1-705 are less sensitive to
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exogenously applied BL (Figure 4A). It is interesting that the rosette of bri1-706 is
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significantly bigger than bri1-702 and bri1-705 (Figure 2A), but its root growth is
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almost completely insensitive to BL treatment, similar to that of null bri1 mutants
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(Figure 4A and 4D). bri1-707, bri1-710 and bri1-711 show slightly reduced
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sensitivity to BL compared to Col-0 (Figure 4B). All new bri1 strong mutants,
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including bri1-703, bri1-704, bri1-708 and bri1-709, are insensitive to BL similar to
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bri1-701 (Figure 4C) (Gou et al., 2012). Previous studies indicated that bri1 weak
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mutant show reduced sensitivity to BL but increased sensitivity to brassinazole (BRZ),
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a specific BR biosynthetic inhibitor (Asami et al., 2000; Yin et al., 2002). We
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therefore tested the sensitivity of these new bri1 alleles to BRZ under darkness. As
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expected, the hypocotyl growth of weak and subtle alleles exhibited hypersensitivity
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to BRZ, whereas all strong alleles are insensitive to the BRZ treatment (Figure 4E).
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These data indicated the new bri1 alleles exhibit typical bri1 phenotypes and reduced
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sensitivity to exogenously applied BL.
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Previous studies also indicated that, upon application of exogenous BL, the
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expression of the BR biosynthesis gene CPD is down-regulated and the expression of
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a BR response gene, SAUR-AC1, is up-regulated (Li et al., 2001; Yin et al., 2005). In
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addition, the phosphorylation status of BES1 also can be used as an indicator of
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downstream signaling response to the BL treatment (Yin et al., 2002; Sun et al., 2010;
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Yu et al., 2011). Quantitative real-time PCR was performed to examine the expression
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levels of CPD and SAUR-AC1 in WT and newly identified bri1 mutants treated with
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or without 1 μM BL (Figure 5A and 5B). Similar to previous reports, the expression
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of SAUR-AC1 was increased dramatically in Col-0 but had no significant change in
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bri1-701 after the BL treatment. The expression of SAUR-AC1 in the newly identified
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weak and subtle bri1 alleles exhibited reduced sensitivity to the BL treatment,
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whereas in newly identified strong bri1 alleles, the expression of SAUR-AC1 is
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insensitive to the treatment of BL and remained at a lower level of expression with or
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without the treatment (Figure 5A). Similar to the SAUR-AC1 response, the CPD
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responses of bri1-702 and bri1-705 showed reduced sensitivity to the BL treatment
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(Figure 5B). Although the expression levels of CPD in BR-treated bri1-706, bri1-707,
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bri1-710, and bri1-711 decreased relatively to the same degree compared to Col-0,
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basal expression levels of CPD without BL treatment were higher than WT,
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suggesting that the BR signaling is indeed partially altered in these mutants (Figure
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5B).
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We next investigated the phosphorylation status of BES1 in WT and in the newly
identified bri1 mutants with or without 1 μM BL application (Figure 5C and 5D). In
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Col-0, two almost equal intensity bands, representing phosphorylated and
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unphosphorylated forms of BES1 were detected without the treatment of BL. After
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the BL treatment, phosphorylated BES1 was greatly reduced and unphosphorylated
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BES1 was significantly accumulated in Col-0, suggesting that the BR signaling
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pathway is functional. In bri1-702 and bri1-705, only a small amount of
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unphosphorylated BES1 was detected after the BL treatment. In the strong bri1 alleles
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bri1-701, bri1-703, bri1-704, bri1-708, and bri1-709, the accumulation of
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unphosphorylated BES1 could not be easily observed after the treatment of BL. In
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subtle alleles, bri1-711, bri1-710, and bri1-706, the accumulation of
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unphosphorylated BES1 is similar to that in Col-0 upon the treatment of 1μM BL. It is
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intriguing that bri1-301 and bri1-707 contain different missense mutations at the same
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residue, but the BES1 response to BL is totally different. bri1-301 remains partial
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sensitivity while bri1-707 is completely sensitive to the treatment of BL (Figure 5C
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and 5D). These molecular and biochemical results confirmed that bri1-706, bri1-710,
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and bri1-711 are subtle alleles of bri1.
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bri1-702, with a point mutation in the activation loop of BRI1, shows reduced
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autophosphorylation activity
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Autophosphorylation, especially the ‘pocket opening’ of the activation loop, is a
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common activation mechanism for protein kinases (Oh et al., 2000). In order to
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understand whether the mutations in the newly identified bri1 mutants affect the
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kinase activity of BRI1, we used the phospho-amino acid stain Pro-Q Diamond to
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detect the autophosphorylation activity of E. coli expressed mutant BRI1 cytoplasmic
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domain (CD) fused with a maltose binding protein (MBP-BRI1-CD) (Figure 6A and
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6B). After sequential staining with the Pro-Q Diamond and Coomassie blue silver, the
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E. coli cell lysate which contains the expressed bri1-301-CD, exhibited an
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undetectable kinase activity in vitro, similar to the results from a previous report (Xu
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et al., 2008). However, bri1-707-CD, which bears a different substitution at the same
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residue with bri1-301-CD, showed reduced but detectable autophosphorylation
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activity (Figure 6A and 6B). Interestingly, compared to the undetectable kinase
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activities of other intracellular bri1 mutants, bri1-702, a weak allele with a mutation
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in the activation loop, still shows autophosphorylation activity, although it is partially
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reduced compared to WT (Figure 6A and 6B). This result indicated that mutation of
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bri1-702 leads to reduced kinase activity of BRI1 and therefore attenuated BR signal
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transduction.
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To confirm these results in vivo, we generated transgenic plants overexpressing
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BAK1-GFP in Col-0 and various genotypic backgrounds. A phosphothreonine
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antibody was used to detect the phosphorylation on threonine residues of
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immunoprecipitated BAK1-GFP from different backgrounds applied with or without
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BL (Figure 6C). Similar to a previous report (Wang et al., 2008), the phosphorylation
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level of BAK1 was strongly enhanced by the BL treatment or by overexpressing BRI1
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in Col-0. The phosphorylation of BAK1 was reduced to an almost undetectable level
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and exhibited no response to BL treatment in bri1 null alleles. In bri1-702 and
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bri1-301, the phosphorylation level of BAK1 was slightly induced by exogenously
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applied BL. Interestingly, without BL, the phosphorylation level of BAK1 was much
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higher in these two weak alleles than even in WT (Figure 6C). These results suggested
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that bri1-702, the only weak allele in activation loop identified so far, exhibited not
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only a reduced autophosphorylation activity of BRI1 in vitro but also a reduced
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sensitivity of BAK1 phosphorylation response to exogenously applied BL in vivo.
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Even though in vitro bri1-301 autophosphorylation could not be detected as
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previously reported (Xu et al., 2008), BAK1 phosphorylation in bri1-301 background
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is much higher than that in bri1-701 and is induced by the addition of BL. This result
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suggests that bri1-301 is indeed partially functional towards its substrate in vivo,
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clarifying a previous argument regarding whether the kinase activity of BRI1 is even
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critical to the BR signaling transduction (Xu et al., 2008).
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Overexpression of mBAK1 failed to generate a dominant negative phenotype in
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bri1-705
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Structural analysis suggested the extracellular portions of BRI1 and BAK1 can form a
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BL-induced complex (Santiago et al., 2013; Sun et al., 2013). Several crucial surfaces
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for the interaction of BRI1 and BAK1 were also identified, among which were two
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residues of BRI1 (Thr726 and Met727) directly associated with BAK1 via
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hydrophobic interactions (Santiago et al., 2013; Sun et al., 2013). Coincidentally,
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bri1-705, a new subtle allele with a typical BR-deficient phenotype, contained a
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nearby mutation at Pro719 to Leu which may affect interaction between BRI1 and
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BAK1. In order to test this hypothesis, a series of transgenic analyses were carried out.
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Based on previous studies, overexpression of BAK1 can suppress the phenotype of
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bri1-5, while overexpression of a kinase-inactive version of BAK1 (mBAK1), in
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which a point mutation is introduced on a conserved lysine residue within the ATP
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binding site, results in a dominant negative phenotype (Li et al., 2002; Gou et al.,
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2012). Both genetic suppression and dominant negative phenotypes rely on
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interactions between the extracellular portion of BRI1 and BAK1. If a mutation in
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BRI1 disrupts the interaction with BAK1 or mBAK1, overexpression of mBAK1 may
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not dramatically alter the phenotype of the bri1 mutant. BAK1-GFP and mBAK1-GFP
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were transformed to a series of weak bri1 alleles. Consistent with previous reports (Li
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et al., 2002), the defective phenotype of bri1-5 was partially rescued by
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overexpressing BAK1 but greatly enhanced by overexpressing mBAK1 (Supplemental
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Figure S2). Overexpression of BAK1 or mBAK1 in bri1-301 and bri1-702 resulted in
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phenotypes similar to those in bri1-5 (Figure 7) (Li et al., 2002). However,
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overexpression of BAK1 or mBAK1 failed to dramatically alter the phenotype of
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bri1-705 (Figure 7), suggesting the mutation in bri1-705 may interfere with the
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interaction between bri1-705 and BAK1 or mBAK1. These genetic data demonstrated
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that the BL-dependent extracellular heterodimerization of BRI1 and BAK1 is an
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important step in the initiation of BR signaling.
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To further confirm this notion, we performed a molecular dynamic simulation
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(MD) analysis of the BRI1-BL-BAK1 structure. The fluctuation of 120 ns MD
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trajectories obtained from two simulation systems (the wild-type complex and
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BRI1-Pro719Leu complex) were monitored by using the root mean square deviation
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(RMSD) of the backbone atoms of BRI1, BAK1 and BL (Supplemental Figure S3). It
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can be clearly seen that the RMSD values of both BRI1-Pro719Leu and wild-type
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BRI1 oscillate at ~ 4 Å after 20 ns whereas the structural fluctuations of BAK1 and
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BL are significantly higher in the BRI1-Pro719Leu complex compared to the
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wild-type complex, implying that the Pro719Leu mutation of BRI1 may destabilize
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the interactions of BRI1-BL and BRI1-BAK1. The simulation analysis also showed
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that the mutation leads to significant conformation alterations of Arg717, which
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precedes Pro719 and has the propensity to change from a random coil to a β-turn in
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the mutant complex. Meanwhile, the β-turn structure formed by Asn705 and Ile706 in
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the wild-type complex seems to be destabilized and prone to show a random coil
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conformation in the mutant complex (Figure 8A). Analysis of the interaction
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interfaces revealed the hydrogen bonding of the residue Asn705, which bridges the
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side-chain of His61 from BAK1 and the hydroxyl group of BL, was continuously
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interrupted in the BRI1-Pro719Leu complex during the last 20 ns simulation (Figure
378
8B). Additionally, the hydrogen bond between the main-chain oxygen atom of His61
379
from BAK1 and the hydroxyl group of BL also breaks up more frequently in the
380
BRI1-Pro719Leu complex compared to the wild-type complex (Figure 8B). Taken
381
together, these results suggest the mutation in bri1-705 results in significant local
382
conformational changes involving the residue Asn705 that interacts with both BAK1
383
and BL, thus destabilizing the pairwise interactions among BRI1, BAK1 and BL
384
(Figure 8C).
385
386
bri1 point mutants exhibiting defective phenotypes all bear point mutations
387
within conserved residues among BRI1s and BRLs
388
389
Eighty-three missense mutations of BRI1 were isolated in this study, among which 9
13
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390
point mutations resulted in typical bri1 phenotypes including one mutation that
391
generated a premature stop codon, and 74 point mutations did not show any
392
significant phenotypes in normal laboratory growing conditions. There were 16
393
different bri1 point mutants identified in previous reports (premature stop mutants are
394
not included). Altogether, 98 point mutations of BRI1 have been found so far. We
395
were curious whether these 24 bri1 point mutants (16 previously identified plus 8
396
newly identified) with mutant phenotypes all contain point mutations in conserved
397
residues. We thus carried out a sequence alignment among 70 BRI1s or BRLs from 18
398
sequenced flowering plant species (Figure 9A). As expected, all of these mutants are
399
mutated at conserved residues, but their phenotypes are not positively related to the
400
degree of conservation. Surprisingly, not all mutations of conserved residues exhibit
401
defective phenotypes (Figure 9A). As BRI1 is a typical LRR-RLK with multiple
402
functional domains, we also performed sequence analysis among all LRR-RLKs from
403
Arabidopsis thaliana (Figure 9B). These results indicated that not all mutation
404
residues of 24 bri1mutants are conserved among all LRR-RLKs. These residues may
405
be particularly important for specific BRI1 functions which have been evolutionarily
406
selected. We also found that not all mutations of conserved residues among all
407
LRR-RLKs exhibit defective phenotypes (Figure 9B).
408
409
DISCUSSION
410
411
The activation loop is a crucial region for protein kinase activity (Oh et al., 2000).
412
Sequence analysis indicated most residues in the activation loop are extremely
413
conserved in 70 BRI1s/BRLs obtained from 18 sequenced flowering plant species
414
(Supplemental Figure S4A). Besides, there are several residues in the activation loop
415
of BRI1 that are also conserved in all Arabidopsis LRR-RLKs (Supplemental Figure
416
S4B). Two new bri1 strong alleles, bri1-703 and bri1-704, contain mutations in the
417
last and first residues of the BRI1 activation loop respectively, and both of which are
418
extremely conserved not only in BRI1s or BRLs but also in all other Arabidopsis
419
LRR-RLKs. Two previously reported alleles, bri1-103/104 and bri1-115, with
14
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420
mutations on extremely conserved residues among BRI1s or BRLs but less conserved
421
among all other LRR-RLKs, also show strong bri1 phenotypes (Supplemental Figure
422
S4) (Li and Chory, 1997; Friedrichsen et al., 2000). We found that bri1-702, the only
423
weak allele in the activation loop of BRI1, contains a mutation in Pro1050 which is
424
extremely conserved in BRI1s or BRLs but not conserved in all other LRR-RLKs
425
(Supplemental Figure S4). However, there is also a mutation found via TILLING in
426
Arg1032Lys which has the same degree of conservation with bri1-103/104 among
427
BRI1s or BRLs but showed no defective phenotypes, possibly because it is mutated to
428
lysine, which shows similar degree of conservation with arginine in other LRR-RLKs
429
(Supplemental Figure S4). Among all Arabidopsis LRR-RLKs, several residues
430
mutated in bri1 alleles are not even conserved. We suggest that these mutation
431
residues may be related to the specificity of the BRI1-mediated signaling pathway
432
(Figure 9B).
433
In previous studies, the importance of the BRI1 kinase activity was questioned
434
because bri1-301-KD showed no kinase activity in vitro, either via an
435
autophosphorylation assay or an analysis for its phosphorylation activity towards
436
BAK1, but exhibits a weak bri1 phenotype (Xu et al., 2008). In our studies,
437
bri1-301-CD fusion protein extracted from E. coli also could not be stained by
438
phosphoprotein diamond Pro-Q (Figure 6A and 6B). However, we noticed that unlike
439
a bri1 null allele, the BAK1 phosphorylation level in bri1-301 is even higher than that
440
of wild-type as detected by immunoblotting using a phosphothreonine antibody
441
(Figure 6C). This is possibly caused by higher endogenous levels of BL in bri1-301 as
442
was found in other bri1 mutants (Noguchi et al., 1999). The phosphorylation level of
443
BAK1 in bri1-301 can be significantly induced by BL in vivo (Figure 6C). This result
444
indicated bri1-301-CD possesses kinase activity for its substrate BAK1 in vivo. The
445
undetectable autophosphorylation of bri1-301-CD in vitro may be caused by protein
446
misfolding when expressed in E. coli.
447
In this study, we also have some interesting findings from analyzing new bri1
448
mutant alleles. For example, bri1-707 (Gly989Glu, Col-0 accession) contains the
449
same mutation as bri1-301 (Gly989Ile, Col-0 accession) but with a different amino
15
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450
acid substitution. As a result, bri1-707 shows a WT-like phenotype (Figure 2).
451
Actually, the change of Gly to Glu is predicted to substitute a larger side chain than
452
Gly to Ile because of an additional carboxyl group in Glu. Similarly, the new strong
453
allele bri1-708 (Arg983Gly, Col-0 accession) also bears a point mutation at the same
454
residue as bri1-8 (Arg983Gln, WS2 accession) and bri1-108-112 (Arg983Gln, Col-0
455
accession). However, the Arg to Gly substitution causes a greater phenotypical
456
alteration than the Arg to Gln substitution. These results indicated that the
457
mechanisms of point mutation are complicated and may involve in structural changes.
458
We identified an extracellular subtle mutant bri1-705, which has an amino acid
459
substitution close to the BRI1-BAK1 interaction surface. Unlike other weak alleles,
460
such as bri1-5, bri1-702 and bri1-301, overexpression of mBAK1 in bri1-705 failed to
461
generate a dominant negative phenotype (Figure 7). This result suggested that the
462
mutation in bri1-705 may affect the interaction between the extracellular portions of
463
BRI1 and BAK1. This is further supported by the MD simulation results, indicating
464
that the mutation in bri1-705 results in significant local conformational changes,
465
causing the disruption of three pairs of hydrogen bonds formed between
466
BRI1-Asn705 and BAK1-His61, BRI1-Asn705 and BL, and BAK1-His61 and BL.
467
Alterations of this hydrogen bonding network could potentially destabilize the
468
pairwise interactions among BRI1, BAK1 and BL and lead to decreased binding
469
affinity of the BRI1-BAK1 extracellular portions. Our data therefore provide
470
additional genetic and structural evidence for the crucial role of BL-dependent
471
BRI1-BAK1 heterodimerization in the early events of BR signaling.
472
During our study, we identified another surprising bri1 mutant, bri1-706.
473
bri1-706 only shows a subtle morphological phenotype compared to other weak
474
alleles isolated so far (Figure 2). However, root inhibition and hypocotyl promotion
475
assays showed that bri1-706 is greatly insensitive to the treatment of BL (Figure 4A,
476
Supplemental Figure S5A). When pBRI1::BRI1 was introduced in bri1-706, both the
477
phenotype and insensitivity to BL were completely restored (Figure 3B, Supplemental
478
Figure S5B). Endo H analyses indicated that bri1-706 proteins are partially retained in
479
ER in 10-day-old seedlings, in which roots and shoots of the seedlings showed no
16
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
480
significant differences (Supplementation Figure S5C-D and S6). But CPD
481
down-regulation and unphosphorylated BES1 accumulation in response to exogenous
482
BL in bri1-706 are similar to those of wild-type seedlings (Supplemental Figure
483
S5E-G). This is the first bri1 mutant showing that phenotypic severity is not
484
positively correlated to the degree of insensitivity to exogenous BL application. These
485
data suggest that the specific mutation found in bri1-706 may have destructed the
486
interaction of bri1-706 with an unknown protein which may regulate a branch of the
487
BR signaling pathway. Detailed molecular mechanisms regarding how bri1-706
488
affects root growth sensitivity to BL need to be further investigated in the near future.
489
We identified a strong allele, bri1-709, in which a point mutation results in a
490
premature stop codon within the juxtamembrane region (Supplemental Figure S7A).
491
Interestingly, a specific band which is smaller than that in Col-0 can be detected by an
492
immunoblotting assay using total proteins from bri1-709 plants with an anti-BRI1
493
antibody (Supplemental Figure S7B). This result suggests that the early stop codon of
494
bri1-709 did not disrupt the protein transport of BRI1, and that a partial BRI1 protein
495
without the kinase domain and the carboxyl terminus is expressed in bri1-709. This
496
mutant can be used for future genetic and biochemical analyses.
497
In previous studies, two extracellular weak alleles, bri1-5 and bri1-9, were found
498
to be retained in the endoplasmic reticulum (ER), because the mutational BRI1
499
proteins interact with ER chaperones and fail to pass the ER quality control system
500
(Jin et al., 2007; Hong et al., 2008; Jin et al., 2009). In order to test whether BRI1 is
501
retained in the ER for our newly identified extracellular mutation alleles, we
502
performed an Endo H analysis (Supplemental Figure S6). The results indicated that
503
BRI1 is not retained on ER in bri1-710 and bri1-711, and partially retained on ER in
504
bri1-705 and bri1-706. In bri1-705 and bri1-706, there is still a considerable amount
505
of BRI1 which is resistant to Endo H cleavage, suggesting partially retained bri1-705
506
or bri1-706 on ER may not be the main reason causing their defective phenotypes.
507
508
509
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510
CONCLUSIONS
511
512
In this study, we analyzed 83 BRI1 point mutations generated by TILLING, among
513
which 9 mutants showed typical bri1 mutant phenotypes. bri1-702 is a weak allele.
514
bri1-705, bri1-706, bri1-710, and bri1-711, are subtle alleles. bri1-703, bri1-704,
515
bri1-708, and bri1-709 are strong alleles, similar to bri1-701 (Figure 1, Table 1).
516
bri1-702, the first weak allele in the activation loop of BRI1 identified so far,
517
exhibited not only reduced autophosphorylation activity of BRI1 in vitro but also
518
reduced sensitivity of BAK1 phosphorylation response to exogenously applied BL in
519
vivo (Figure 6). bri1-705, a new subtle allele in the extracellular portion, contains a
520
BRI1 mutation that may disrupts the formation of hydrogen bonds among BRI1, BL,
521
and BAK1 (Figure 8). Surprisingly, bri1-706, a subtle morphological mutant with a
522
mutation in the extracellular portion of BRI1, showed insensitive root growth
523
inhibition to exogenously applied BL which is similar to the null bri1 mutants (Figure
524
2A and 4A). Identification of unexpected characteristics of bri1-706 suggests there are
525
still many unknown aspects in the early events of the BR signaling pathway waiting to
526
be elucidated in the future.
527
528
MATERIALS AND METHODS
529
530
Plant materials, growth conditions and mutants detection
531
532
Original bri1 TILLING seeds obtained from ABRC are all in Col-0 accession which
533
contains an er mutation. After er and additional mutations were segregated out via
534
backcrossing for at least three generations, each homozygous bri1 was isolated and
535
used for phenotypic determination and detailed analyses. Other known bri1 mutants
536
used in this study include bri1-5 (WS2 background), bri1-9 (Col-0 background)，
537
bri1-301 (Col-0 background) and bri1-701 (SALK_116202). All plants were grown in
538
a greenhouse under a long-day lighting condition (16 h light and 8 h dark) or under
539
darkness at 22˚C. Seedlings were grown in 1/2 MS agar plates or liquid media
18
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
540
541
supplemented with 1% (w/v) sucrose.
dCAPs approach was used for confirming the point mutations (Neff et al., 1998).
542
dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html) was used for designing
543
mismatched PCR primers to generate or disrupt a restriction enzyme recognition site
544
relative to the mutation (Neff et al., 2002). All primers and enzymes used in this study
545
for genotyping bri1 TILLING mutations are listed in Supplemental Table S1.
546
547
Root inhibition and hypocotyl growth assays
548
549
Seeds were surface sterilized with 75% (w/v) ethanol for 30 seconds, followed by 1%
550
NaClO for additional 7 min, and washed with desterilized water for 5 times. The
551
seeds were then placed on 1/2 MS plates with 0.8% (w/v) agar, 1% (w/v) sucrose, and
552
different concentrations of 24-epiBL (Sigma, St. Louis, MO) or brassinazole (Santa
553
Cruz Biotechnology). The seeds were vernalized at 4˚C for 3 days and transferred to
554
different growth conditions as indicated in the figure legends. Before placed in the
555
dark, plates were exposed to white light for 6 hours to induce germination. All
556
measurements were carried out using ImageJ (http://rsb.info.nih.gov/ij/).
557
558
Vector construction and transgenic plants generation
559
560
Gateway technology (Invitrogen, Life Technologies) was used in cloning all genes
561
into expression vectors in this study. The full-length CDS fragments of BRI1,
562
BRI1-CD and BAK1 were amplified and cloned into a pDONR/Zeo entry clone vector.
563
Site-directed mutagenesis was carried out for generating mutated entry clones of BRI1,
564
BRI1-CD and BAK1 based on the method described previously (Gou et al., 2010). All
565
the cloned genes were sequence verified. Genes in the entry clone vectors were then
566
cloned into various destination vectors for different purposes such as
567
pBRI1-GWR-Flag, pBIB-BASTA-35s-GWR-Flag and pBIB-HYG-35s-GWR-GFP. All
568
expression constructs were transferred into appropriate plant materials by a floral dip
569
method (Clough and Bent, 1998).
19
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570
RNA extraction and quantitative Real-time PCR
571
572
Seven-day-old seedlings of Col-0 and bri1 mutants grown on 1/2 MS plates were
573
collected and then grown with or without 1 μM 24-epiBL in liquid media for 4 hours.
574
Total RNAs were extracted using an RNA simple total RNA kit (Tiangen, China) and
575
reverse transcribed to total cDNAs utilizing M-MLV a reverse transcriptase
576
(Invitrogen, Life Technologies). The quantitative Real-time PCR was performed by a
577
StepOnePlus Real-Time PCR System (Applied Biosystems) utilizing SYBR Green
578
reagent (Takara, Japan). Detailed experimental procedures were the same as
579
previously reported (Zhao et al., 2016).
580
581
Membrane protein isolation, immunoprecipitation, Western blotting and
582
phosphoprotein staining
583
584
Ten-day-old seedlings of 35S::BAK1-GFP 35S::BRI1-FLAG, Col-0 35S::BAK1-GFP,
585
bri1-702 35S::BAK1-GFP, bri1-301 35S::BAK1-GFP, or bri1-701 35S::BAK1-GFP
586
were treated with or without 100 nM 24-epiBL for 1.5 hours and then grounded to
587
fine powder in liquid N2. The membrane protein isolation procedure was the same as
588
previously reported (Li et al., 2002). The soluble protein samples were
589
immunoprecipitated utilizing an anti-GFP antibody (Invitrogen, Carlsbad, CA)
590
combined with Protein A/G Plus-Sefinose Resin (BBI, Canada). The
591
immunoprecipitated proteins were separated on 10% SDS-PAGE and tested by
592
Western analyses with anti-GFP and anti-phosphothreonine antibodies.
593
The same plant materials used for quantitative Real-time PCR were also used in
594
detection of BES1 phosphorylation status. Total proteins were separated on 12%
595
SDS-PAGE and detected via a specific anti-BES1 antibody (made by Jia Li’s lab).
596
The transgenetic plants in Figure 7 were identified by detecting their GFP tag utilizing
597
an anti-GFP antibody (Invitrogen, Carlsbad, CA). The Endo H analyses shown in
598
Supplemental Figure S5 and S6 were performed as previously described (Hong et al.,
599
2008). The total proteins of WT and mutants in Supplemental Figure S7 were
20
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
600
separated on 8% SDS-PAGE and detected via a specific anti-BRI1 antibody (Agrisera,
601
A177246, Sweden). Horseradish peroxidase-linked anti-rabbit or anti-mouse antibody
602
was used as a secondary antibody, and the signal was detected by Western Lightning
603
Chemiluminescence Reagent Plus (Perkin-Elmer, Waltham, MA) in all Western blot
604
experiments.
605
For the phosphoprotein staining assay, the cytoplasmic domain sequences of WT
606
BRI1 and mutated BRI1 (residues 815–1196) were cloned into the expression vector
607
pMAL-cRI-MBP-GWR. The recombinant proteins were expressed in E. coli and
608
induced by 0.1mM IPTG for 6 hours at 18˚C. The cell lysates were boiled and
609
centrifuged. The supernatant was separated on 10% SDS-PAGE and sequentially
610
stained with Pro-Q Diamond (Invitrogen, Carlsbad, CA) and Coomassie blue silver as
611
previously described (Taylor et al., 2013).
612
613
Molecular dynamics simulation analysis
614
615
The initial complex coordinates of BRI1-BAK1-BL were retrieved from the PDB
616
database (http://www.rcsb.org) under the accession code 4M7E (Sun et al., 2013). MD
617
simulations were carried out using the AMBER 10 software package (Case et al.,
618
2005) to check the stability of the BRI1 and BAK1 in complex with BL. We used the
619
AMBER ff12SB (Hornak et al., 2006) force field for the protein and water and the
620
general AMBER force field (GAFF) (Wang et al., 2004) for the BL molecule. Atom
621
types and AM1-BCC atomic charges were generated for BL using the Antechamber
622
module. The LEaP module of the AMBER10 software package was used to prepare
623
the protein-ligand complex. The complex was solvated using the explicit TIP3P
624
(Jorgensen et al., 1983) water model, which extended a minimum 10 Å distance from
625
the box surface to any atom of the solute. Two consecutive steps of minimization were
626
carried out. All bond lengths were constrained using the SHAKE algorithm (Ryckaert
627
et al., 1977), and the equations of motion were integrated with 2 fs time step using the
628
Verlet leapfrog algorithm. To eliminate possible bumps between the solute and the
629
solvent, the entire system was minimized in two steps. First, the complex was
21
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630
restrained with a harmonic potential with a force constant k=50 kcal mol-1Å-2. The
631
water molecules and counter ions were optimized using the steepest descent method
632
of 1,000 steps, followed by the conjugate gradient method for 2,000 steps. Second, the
633
entire system was optimized using the first step method without any constraints.
634
Subsequently, the entire system was heated gradually in the NVT ensemble from 0 to
635
300k over 400ps, with a weak restraint (k=5 kcal mol-1Å-2) for the complex (Wu et al.,
636
2016). The final coordinates obtained after the temperature equilibration simulation
637
were used for a 120 ns MD simulation during which the temperature was kept by the
638
Langevin thermostat with a collision frequency of 2 ps-1. A constant pressure periodic
639
boundary was used to maintain the pressure of the system using an isotropic position
640
scaling algorithm with a relaxation time of 2 ps. After MD simulation, distances
641
between two atoms were calculated using the cpptraj program, and the dictionary of
642
secondary structure of protein (DSSP) program developed by Kabsch and Sander was
643
used to assign the secondary structures of the BRI1-BAK1-BL complex (Kabsch and
644
Sander, 1983). All the structure figures were prepared using PyMol (www.pymol.org).
645
646
Sequence analysis
647
648
For sequence analysis, the sequences of 70 BRI1s or BRLs from 18 flowering plants,
649
and 223 LRR-RLK sequences from Arabidopsis were downloaded and analyzed in
650
software Mega (http://www.megasoftware.net/), the final alignment data was
651
generated to a logo (Figure 9 and Supplemental Figure S4) on a website
652
(http://weblogo.berkeley.edu/logo.cgi).
653
654
SUPPLEMENTAL MATERIALS
655
656
Supplemental Table S1. List of primers and enzymes used in this study for
657
genotyping bri1 TILLING mutations.
658
Supplemental Figure S1. Eighty-three total mutations of BRI1 identified in this study
659
and 22 previously reported bri1 mutants.
22
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
660
Supplemental Figure S2. Overexpression of mBAK1 in bri1-5 resulted in a dominant
661
negative phenotype.
662
Supplemental Figure S3. Monitoring of the equilibration of the MD trajectories of
663
the wild-type complex (colored in black) and BRI1-Pro719Leu complex (colored in
664
red).
665
Supplemental Figure S4. Sequence analyses of the BRI1 activation loop.
666
Supplemental Figure S5. Endo H cleavage of BRI1, responses of BES1
667
phosphorylation and CPD expression to BL treatment showed no significant
668
differences between shoots and roots of bri1-706 seedlings.
669
Supplemental Figure S6. Endo H analysis indicated that BRI1 is not retained in the
670
endoplasmic reticulum (ER) in bri1-710 and bri1-711, and partially retained in the ER
671
in bri1-705 and bri1-706.
672
Supplemental Figure S7. In bri1-709, the mutation results in a premature stop
673
codon.
674
675
ACKNOWLEDGMENTS
676
677
We thank Jia Li lab member, Yao Xiao, for generating the BES1 antibody. We also
678
thank Steven Henikoff and his team for their hard work on the
679
Arabidopsis TILLING Project. All the seeds were purchased from ABRC.
680
681
682
683
684
685
686
687
688
23
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Table 1. bri1 alleles (For references, please see Figure 1)
Allele
Base pair
change
Accession
Allelic
strength
Possible mechanism
bri1-1
G2725A
Col-0
Strong
unknown
bri1-3
4-bp deletion
after 2745
WS2
Strong
Premature stop
bri1-4
10-bp deletion
after 459
WS2
Strong
Premature stop
bri1-5
G206A
WS2
Weak
ER retention
bri1-5R1
G260A
bri1-5
Weak
Partially restore ER retention of
bri1-5
bri1-6/119
G1931A
En-2
Weak
unknown
bri1-7
G1838A
WS2
Weak
unknown
bri1-8/108
G2948A
-112
WS2/
Col-0
Intermediate
Autophosphorylation cannot be
detected in vitro
bri1-9
C1985T
WS2
Weak
ER retention
bri1-101
G3232A
Col-0
Strong
Autophosphorylation cannot be
detected in vitro
bri1-102
C2249T
Col-0
Strong
unknown
bri1-103/
104
G3091A
Col-0
Strong
unknown
bri1-105107
C3175T
Col-0
Strong
unknown
bri1-113
G1832A
Col-0
Strong
unknown
bri1-114/
116
C1747T
Col-0
Strong
unknown
bri1-115
G3143A
Col-0
Strong
unknown
bri1-117/
118
G3415A
Col-0
Strong
unknown
bri1-120
T1196C
Ler
Weak
unknown
24
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
bri1-201
G1831A
WS2
Strong
unknown
bri1-202
C2854T
WS2
Strong
unknown
bri1-301
GG2965/6AT
Col-0
Weak
Autophosphorylation of bri1-301
cannot be detected in vitro but
transphosphorylation of BAK1
can be detected in vivo
bri1-701
T-DNA
insertion
Col-0
Strong
Knock-out of BRI1
bri1-702
C3148T
Col-0
Weak
Reduced autophosphorylation
in vitro
bri1-703
G3166A
Col-0
Strong
Autophosphorylation cannot be
detected in vitro
bri1-704
G3079A
Col-0
Strong
Autophosphorylation cannot be
detected in vitro
bri1-705
C2156T
Col-0
Subtle
Disrupt the formation of
hydrogen bonds among BRI1,
BL, and BAK1
bri1-706
C758T
Col-0
Subtle
unknown
bri1-708
C2947G
Col-0
Strong
Autophosphorylation cannot be
detected in vitro
bri1-709
G2543A
Col-0
Strong
Premature stop
bri1-710
G1858A
Col-0
Subtle
unknown
bri1-711
G2236A
Col-0
Subtle
unknown
689
690
691
692
693
694
695
696
697
25
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
698
FIGURE LEGENDS
699
700
Figure 1. Diagram showing all bri1 mutants identified thus far, including 9 new
701
alleles identified by this study. New mutants are labeled in red and known mutants
702
are labeled in black. Known bri1 mutants include bri1-1 (Clouse et al., 1996;
703
Friedrichsen et al., 2000), bri1-3, bri1-4, bri1-5, bri1-6, bri1-7, bri1-8, bri1-9
704
(Noguchi et al., 1999), bri1-5R1 (Belkhadir et al., 2010), bri1-101, bri1-102
705
bri1-103/104, bri1-105-107, bri1-108-112, bri1-113, bri1-114/116, bri1-115,
706
bri1-117/118, bri1-119 (Li and Chory, 1997; Friedrichsen et al., 2000), bri1-120
707
(Shang et al., 2011), bri1-201, bri1-202 (Domagalska et al., 2007), bri1-301 (Xu et al.,
708
2008), and bri1-701 (Gou et al., 2012).
709
710
Figure 2. New bri1 mutants identified in this study and their phenotypes. (A)
711
Phenotypes of mutants and wild-type seedlings. The picture was taken 3 weeks after
712
germination. (B) Rosette width and height of the mutants and wild-type seedlings.
713
Rosette width was measured 3 weeks after germination. Height was measured when
714
growth was completely finished. T-tests were carried out to show the significance
715
between each allele and Col-0. Each data shown is the average and SD (n≥20). ***,
716
P<0.001. ns, not significant.
717
718
Figure 3. Complementation analyses confirm that all mutants are alleles of bri1.
719
(A) The phenotypes of new bri1 weak or subtle alleles cannot be complemented by
720
other bri1 weak alleles. (B) BRI1 driven by its native promoter can completely rescue
721
the phenotypes of new bri1 weak or subtle alleles. (C) The phenotypes of bri1-702
722
and bri1-705 can be recapitulated by expressing their corresponding genomic DNA in
723
a null BRI1 mutant allele, bri1-701. Pictures were taken 4 weeks after germination.
724
725
Figure 4. All mutants show either insensitive or reduced sensitivity to
726
exogenously applied BL. (A to C) Response of root growth to the treatment of BL.
727
Seven-day-old seedlings grown on 1/2 MS media supplemented with different
26
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
728
concentrations of BL. (D) bri1-706 is almost completely insensitive to BL.
729
Seven-day-old seedlings grown on 1/2 MS media supplemented with or without 1μM
730
BL. The seedlings were grown under long-day light conditions in a 22˚C growth
731
chamber for A to D. (E) Response of hypocotyl growth to the treatment of BRZ.
732
Four-day-old seedlings were grown on 1/2 MS media with or without BRZ
733
application under darkness in a 22˚C growth chamber. T-tests were performed to show
734
significance between each allele and Col-0. Each data represents average and SD
735
(n≥20). ***, P<0.001.
736
737
Figure 5. Responses of downstream BR signaling components of the new mutants
738
to BL. (A and B) Transcriptional responses of SAUR-AC1 and CPD to the treatment
739
of BL. ACTIN2 was used as a reference gene. Error bars represent SD (n= 3). (C)
740
Response of BES1 phosphorylation status to the treatment of BL. Total protein was
741
extracted and immuno-blotted with an anti-BES1 antibody. (D) Coomassie blue
742
staining showing equal loading between each pair of samples in C. Seven-day-old
743
long-day grown seedlings treated with or without 1 μM BL for 4 h were used in A-D.
744
-, without BL treatment; +, with BL treatment; *, non-specific band. BES1-P,
745
phosphorylated BES1; BES1, unphosphorylated BES1.
746
747
Figure 6. In vitro and in vivo phosphorylation analyses of the BRI1, bri1, and
748
BAK1. (A) Pro-Q diamond analyses of BRI1 and various bri1 cytoplasmic domains.
749
The recombinant proteins were expressed in E. coli and total protein extracts were
750
separated on 10% SDS-PAGE. The gel was stained by Pro-Q diamond. (B) Coomassie
751
blue silver staining to show equal loading of the samples. mBRI1 contains a mutation
752
within the ATP binding site (Lys911Glu). (C) In vivo phosphorylation analyses of
753
BAK1-GFP from various bri1 mutants in response to the treatment of BL. Membrane
754
proteins were extracted from 10-day-old long-day grown seedlings treated with or
755
without 100 nM BL for 1.5h. BAK1-GFP proteins from different bri1 alleles were
756
immunoprecipitated with an anti-GFP antibody. The phosphorylation levels of BAK1
757
were detected with a phosphothreonine antibody. The immunoprecipitated
27
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
758
BAK1-GFP was immuno-blotted with an anti-GFP antibody to show equal loading. -,
759
without BL treatment; +, with BL treatment.
760
761
Figure 7. Overexpression of mBAK1 in bri1-301 and bri1-702 but not in bri1-705
762
resulted in dominant negative phenotypes. (A) Phenotypes of the transgenic plants
763
and their backgrounds. The pictures were taken 3 weeks after germination. (B)
764
Western blotting analyses showing the expressed BAK1-GFP and mBAK1-GFP
765
fusion proteins in transgenic plants. Total proteins from rosette leaves were extracted
766
and immuno-blotted with an anti-GFP antibody. mBAK1 contains a mutation within
767
the ATP binding site (Lys317Glu).
768
769
Figure 8. Conformational changes monitored during the last 20 ns MD
770
simulation. (A) Time evolution of the secondary structures of residue 703-721 of
771
BRI1 in the wild-type complex (left) and mutant complex (right). Compared with the
772
wild-type complex, the residue Arg717 preceding Pro719 has the propensity to change
773
from a random coil to a β-turn while Asn705 and Ile706 seem to be destabilized and
774
prone to adopt a random coil conformation in the mutant complex. (B) Monitoring
775
time evolution of the distances between the atom OD1 of BRI1-Asn 705 and ND1 of
776
BAK1-His61 (upper left panel), atom OD1 of BRI1-Asn 705 and O02 from C2 of BL
777
(upper right panel) and atom ND1 of BAK1-His61 and O03 from C3 of BL (lower
778
panel) , respectively. The distances in the wild-type complex and mutant complex are
779
colored in black and red, respectively. (C) The interaction modes of BRI1-Asn705
780
(cyan), BAK1-His61 (purple) and BL (yellow). At 110 ns, as shown in upper right
781
inset, Asn705, His61 and BL form pairwise hydrogen bonds, indicated by the close
782
distances of 2.91, 2.81 and 3.03 Å between Asn705, His61 and BL, respectively.
783
However, in the bri1-705 mutant complex shown in the lower right inset, these
784
hydrogen bonds are disrupted as evidenced by the much larger pairwise distances
785
between Asn705, His61 and BL.
786
787
28
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788
Figure 9. Sequence analyses of all 98 BRI1 point mutations. (A) Sequence
789
analyses among 70 BRI1s or BRLs from 18 sequenced flowering plant species.
790
Mutants showing defective phenotypes all result from mutations of conserved
791
residues. But not all mutations in the conserved residues show defective phenotypes.
792
(B) Sequence analyses among all 223 LRR-RLKs of Arabidopsis thaliana. Not all
793
mutation sites from mutants with defective phenotypes are conserved, and vice versa.
794
Only the amino acid sites with mutations are shown. New mutants from this study are
795
labeled in red. Previously known mutants are labeled in black. New mutations without
796
significant phenotypes are labeled in grey. The size of the logo is positively correlated
797
to the degree of conservation.
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
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818
REFERENCES
819
820
Asami T, Min YK, Nagata N, Yamagishi K, Takatsuto S, Fujioka S, Murofushi N,
821
Yamaguchi I, Yoshida S (2000) Characterization of brassinazole, a
822
triazole-type brassinosteroid biosynthesis inhibitor. Plant Physiol 123: 93-99
823
Belkhadir Y, Durbak A, Wierzba M, Schmitz RJ, Aguirre A, Michel R, Rowe S,
824
Fujioka S, Tax FE (2010) Intragenic Suppression of a Trafficking-Defective
825
Brassinosteroid Receptor Mutant in Arabidopsis. Genetics 185: 1283-1296
826
Bell EM, Lin WC, Husbands AY, Yu L, Jaganatha V, Jablonska B, Mangeon A,
827
Neff MM, Girke T, Springer PS (2012) Arabidopsis LATERAL ORGAN
828
BOUNDARIES negatively regulates brassinosteroid accumulation to limit
829
growth in organ boundaries. Proc Natl Acad Sci U S A 109: 21146-21151
830
Case DA, Cheatham TE, Darden TA, Gohlke H, Luo R, Merz KM, Onufriev AV,
831
Simmerling C, Wang B, Woods RJ (2005) The Amber biomolecular
832
simulation programs. J Comput Chem 26: 1668-1688
833
Chaiwanon J, Wang ZY (2015) Spatiotemporal brassinosteroid signaling and
834
antagonism with auxin pattern stem cell dynamics in Arabidopsis roots. Curr
835
Biol 25: 1031-1042
836
Cheng Y, Zhu W, Chen Y, Ito S, Asami T, Wang X (2014) Brassinosteroids control
837
root epidermal cell fate via direct regulation of a MYB-bHLH-WD40 complex
838
by GSK3-like kinases. Elife 02525
839
Clough SJ, Bent AF (1998) Floral dip: a simplified method for
840
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:
841
735-743
842
843
844
Clouse SD (1996) Molecular genetic studies confirm the role of brassinosteroids in
plant growth and development. Plant J 10: 1-8
Clouse SD, Langford M, McMorris TC (1996) A brassinosteroid-insensitive mutant
845
in Arabidopsis thaliana exhibits multiple defects in growth and development.
846
Plant Physiol 111: 671-678
847
Clouse SD, Sasse JM (1998) Brassinosteroids: Essential regulators of plant growth
30
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
848
and development. Annu Rev of Plant Physiol Plant Mol Biol 49: 427-451
849
Domagalska MA, Schomburg FM, Amasino RM, Vierstra RD, Nagy F, Davis SJ
850
(2007) Attenuation of brassinosteroid signaling enhances FLC expression and
851
delays flowering. Development 134: 2841-2850
852
Friedrichsen DM, Joazeiro CA, Li J, Hunter T, Chory J (2000)
853
Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat
854
receptor serine/threonine kinase. Plant Physiol 123: 1247-1256
855
Gendron JM, Liu JS, Fan M, Bai MY, Wenkel S, Springer PS, Barton MK, Wang
856
ZY (2012) Brassinosteroids regulate organ boundary formation in the shoot
857
apical meristem of Arabidopsis. Proc Natl Acad Sci U S A 109: 21152-21157
858
Gonzalez-Garcia M-P, Vilarrasa-Blasi J, Zhiponova M, Divol F, Mora-Garcia S,
859
Russinova E, Cano-Delgado AI (2011) Brassinosteroids control meristem
860
size by promoting cell cycle progression in Arabidopsis roots. Development
861
138: 849-859
862
Gou X, Yin H, He K, Du J, Yi J, Xu S, Lin H, Clouse SD, Li J (2012) Genetic
863
evidence for an indispensable role of somatic embryogenesis receptor kinases
864
in brassinosteroid signaling. PLoS genet 8: e1002452
865
866
867
Gudesblat GE, Russinova E (2011) Plants grow on brassinosteroids. Curr Opin in
Plant Biol 14: 530-537
Gudesblat GE, Schneider-Pizon J, Betti C, Mayerhofer J, Vanhoutte I, van
868
Dongen W, Boeren S, Zhiponova M, de Vries S, Jonak C, Russinova E
869
(2012) SPEECHLESS integrates brassinosteroid and stomata signalling
870
pathways. Nat Cell Biol 14: 548-554
871
Guo Z, Fujioka S, Blancaflor EB, Miao S, Gou X, Li J (2010) TCP1 Modulates
872
Brassinosteroid Biosynthesis by Regulating the Expression of the Key
873
Biosynthetic Gene DWARF4 in Arabidopsis thaliana. Plant Cell 22:
874
1161-1173
875
Hacham Y, Holland N, Butterfield C, Ubeda-Tomas S, Bennett MJ, Chory J,
876
Savaldi-Goldstein S (2011) Brassinosteroid perception in the epidermis
877
controls root meristem size. Development 138: 839-848
31
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
878
Hong Z, Jin H, Tzfira T, Li J (2008) Multiple Mechanism-Mediated Retention of a
879
Defective Brassinosteroid Receptor in the Endoplasmic Reticulum of
880
Arabidopsis. Plant Cell 20: 3418-3429
881
Hong Z, Kajiura H, Su W, Jin H, Kimura A, Fujiyama K, Li J (2012)
882
Evolutionarily conserved glycan signal to degrade aberrant brassinosteroid
883
receptors in Arabidopsis. Proc Natl Acad Sci U S A 109: 11437-11442
884
Hornak V, Abel R, Okur A, Strockbine B, Roitberg AE, Simmerling C (2006)
885
Comparison of multiple Amber force fields and development of improved
886
protein backbone parameters. Proteins 65: 712-725
887
Hothorn M, Belkhadir Y, Dreux M, Dabi T, Noel JP, Wilson IA, Chory J (2011)
888
Structural basis of steroid hormone perception by the receptor kinase BRI1.
889
Nature 474: 467-471
890
Jin H, Hong Z, Su W, Li J (2009) A plant-specific calreticulin is a key retention
891
factor for a defective brassinosteroid receptor in the endoplasmic reticulum.
892
Proc Natl Acad Sci U S A 106: 13612-13617
893
Jin H, Yan Z, Nam KH, Li J (2007) Allele-Specific Suppression of a Defective
894
Brassinosteroid Receptor Reveals a Physiological Role of UGGT in ER
895
Quality Control. Mol Cell 26: 821-830
896
Jorgensen WL, Chandrasekhar J, Madura JD, Impey R, Klein ML (1983)
897
Comparison of simple potential functions for simulating liquid water. J Chem
898
Phys 79: 926-935
899
Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern
900
recognition of hydrogen-bonded and geometrical features. Biopolymers 22:
901
2577-2637
902
Kim TW, Michniewicz M, Bergmann DC, Wang ZY (2012) Brassinosteroid
903
regulates stomatal development by GSK3-mediated inhibition of a MAPK
904
pathway. Nature 482: 419-422
905
906
907
Kwon M, Choe S (2005) Brassinosteroid biosynthesis and dwarf mutants. J Plant
Biol 48: 1-15
Li J, Chory J (1997) A Putative Leucine-Rich Repeat Receptor Kinase Involved in
32
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
908
909
Brassinosteroid Signal Transduction. Cell 90: 929-938
Li J, Lease KA, Tax FE, Walker JC (2001) BRS1, a serine carboxypeptidase,
910
regulates BRI1 signaling in Arabidopsis thaliana. Proc Natl Acad Sci U S A 98:
911
5916-5921
912
Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC (2002) BAK1, an Arabidopsis
913
LRR Receptor-like Protein Kinase, Interacts with BRI1 and Modulates
914
Brassinosteroid Signaling. Cell 110: 213-222
915
Li JM, Nam KH, Vafeados D, Chory J (2001) BIN2, a new
916
917
brassinosteroid-insensitive locus in Arabidopsis. Plant Physiol 127: 14-22
Liu Y, Zhang C, Wang D, Su W, Liu L, Wang M, Li J (2015) EBS7 is a
918
plant-specific component of a highly conserved endoplasmic
919
reticulum-associated degradation system in Arabidopsis. Proc Natl Acad Sci U
920
S A 112: 12205-12210
921
McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeting induced local
922
lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol
923
123: 439-442
924
Mora-Garcia S, Vert G, Yin YH, Cano-Delgado A, Cheong H, Chory J (2004)
925
Nuclear protein phosphatases with Kelch-repeat domains modulate the
926
response to brassinosteroids in Arabidopsis. Genes Dev 18: 448-460
927
928
929
Nam KH, Li J (2002) BRI1/BAK1, a Receptor Kinase Pair Mediating
Brassinosteroid Signaling. Cell 110: 203-212
Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple technique for the
930
genetic analysis of single nucleotide polymorphisms: experimental
931
applications in Arabidopsis thaliana genetics. Plant J 14: 387-392
932
Neff MM, Turk E, Kalishman M (2002) Web-based primer design for single
933
nucleotide polymorphism analysis. Trends Genet 18: 613-615
934
Noguchi T, Fujioka S, Choe S, Takatsuto S, Yoshida S, Yuan H, Feldmann KA,
935
Tax FE (1999) Brassinosteroid-insensitive dwarf mutants of Arabidopsis
936
accumulate brassinosteroids. Plant Physiol 121: 743-752
937
Oh MH, Ray WK, Huber SC, Asara JM, Gage DA, Clouse SD (2000)
33
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
938
Recombinant brassinosteroid insensitive 1 receptor-like kinase
939
autophosphorylates on serine and threonine residues and phosphorylates a
940
conserved peptide motif in vitro. Plant Physiol 124: 751-765
941
Ryckaert J, Ciccotti G, Berendsen HJC (1977) Numerical integration of the
942
cartesian equations of motion of a system with constraints: molecular
943
dynamics of n-alkanes. J Comput Phys 23: 327-341
944
Santiago J, Henzler C, Hothorn M (2013) Molecular mechanism for plant steroid
945
receptor activation by somatic embryogenesis co-receptor kinases. Science
946
341: 889-892
947
Shang Y, Lee MM, Li J, Nam KH (2011) Characterization of cp3 reveals a new bri1
948
allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR
949
signaling. BMC Plant Biol 11: 8
950
She J, Han Z, Kim TW, Wang J, Cheng W, Chang J, Shi S, Wang J, Yang M,
951
Wang ZY, Chai J (2011) Structural insight into brassinosteroid perception by
952
BRI1. Nature 474: 472-476
953
954
955
Steber CM, McCourt P (2001) A role for brassinosteroids in germination in
Arabidopsis. Plant Physiol 125: 763-769
Su W, Liu Y, Xia Y, Hong Z, Li J (2011) Conserved endoplasmic
956
reticulum-associated degradation system to eliminate mutated receptor-like
957
kinases in Arabidopsis. Proc Natl Acad Sci U S A 108: 870-875
958
Su W, Liu Y, Xia Y, Hong Z, Li J (2012) The Arabidopsis homolog of the
959
mammalian OS-9 protein plays a key role in the endoplasmic
960
reticulum-associated degradation of misfolded receptor-like kinases. Mol Plant
961
5: 929-940
962
Sun Y, Fan X-Y, Cao D-M, Tang W, He K, Zhu J-Y, He J-X, Bai M-Y, Zhu S, Oh
963
E, Patil S, Kim T-W, Ji H, Wong WH, Rhee SY, Wang Z-Y (2010)
964
Integration of Brassinosteroid Signal Transduction with the Transcription
965
Network for Plant Growth Regulation in Arabidopsis. Developmental Cell 19:
966
765-777
967
Sun Y, Han Z, Tang J, Hu Z, Chai C, Zhou B, Chai J (2013) Structure reveals that
34
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
968
BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell Res 23:
969
1326-1329
970
Taylor I, Seitz K, Bennewitz S, Walker JC (2013) A simple in vitro method to
971
measure autophosphorylation of protein kinases. Plant Methods 9: 22
972
Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner
973
C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, Henikoff S
974
(2003) Large-scale discovery of induced point mutations with high-throughput
975
TILLING. Genome Res 13: 524-530
976
Vert G, Nemhauser JL, Geldner N, Hong F, Chory J (2005) Molecular
977
mechanisms of steroid hormone signaling in plants. Annu Rev Cell Dev Biol
978
21: 177-201
979
Vilarrasa-Blasi J, Gonzalez-Garcia MP, Frigola D, Fabregas N, Alexiou KG,
980
Lopez-Bigas N, Rivas S, Jauneau A, Lohmann JU, Benfey PN, Ibanes M,
981
Cano-Delgado AI (2014) Regulation of plant stem cell quiescence by a
982
brassinosteroid signaling module. Dev Cell 30: 36-47
983
Wang J, Jiang J, Wang J, Chen L, Fan SL, Wu JW, Wang X, Wang ZX (2014)
984
Structural insights into the negative regulation of BRI1 signaling by
985
BRI1-interacting protein BKI1. Cell Res 24: 1328-1341
986
Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and
987
testing of a general amber force field. J Comput Chem 25: 1157-1174
988
Wang X, Chory J (2006) Brassinosteroids regulate dissociation of BKI1, a negative
989
regulator of BRI1 signaling, from the plasma membrane. Science 313:
990
1118-1122
991
Wang X, Kota U, He K, Blackburn K, Li J, Goshe MB, Huber SC, Clouse SD
992
(2008) Sequential Transphosphorylation of the BRI1/BAK1 Receptor Kinase
993
Complex Impacts Early Events in Brassinosteroid Signaling. Dev Cell 15:
994
220-235
995
Wang X, Li X, Meisenhelder J, Hunter T, Yoshida S, Asami T, Chory J (2005)
996
Autoregulation and homodimerization are involved in the activation of the
997
plant steroid receptor BRI1. Dev Cell 8: 855-865
35
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
998
999
1000
1001
1002
1003
Wang Y, Sun S, Zhu W, Jia K, Yang H, Wang X (2013)
Strigolactone/MAX2-induced degradation of brassinosteroid transcriptional
effector BES1 regulates shoot branching. Dev Cell 27: 681-688
Wang ZY, Bai MY, Oh E, Zhu JY (2012) Brassinosteroid signaling network and
regulation of photomorphogenesis. Annu Rev Genet 46: 701-724
Wang ZY, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 is a critical
1004
component of a plasma-membrane receptor for plant steroids. Nature 410:
1005
380-383
1006
1007
1008
Wei Z, Li J (2016) Brassinosteroids Regulate Root Growth, Development, and
Symbiosis. Mol Plant 9: 86-100
Wu D, Tao X, Chen Z, Han J, Jia W, Zhu N, Li X, Wang Z, He Y (2016) The
1009
environmental endocrine disruptor p -nitrophenol interacts with FKBP51, a
1010
positive regulator of androgen receptor and inhibits androgen receptor
1011
signaling in human cells. J Hazard Mater 307: 193-201
1012
1013
1014
Xu W, Huang J, Li B, Li J, Wang Y (2008) Is kinase activity essential for biological
functions of BRI1? Cell Res 18: 472-478
Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J (2005) A new class of
1015
transcription factors mediates brassinosteroid-regulated gene expression in
1016
arabidopsis. Cell 120: 249-259
1017
Yin Y, Wang Z-Y, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J (2002) BES1
1018
Accumulates in the Nucleus in Response to Brassinosteroids to Regulate Gene
1019
Expression and Promote Stem Elongation. Cell 109: 181-191
1020
Yu X, Li L, Zola J, Aluru M, Ye H, Foudree A, Guo H, Anderson S, Aluru S, Liu
1021
P, Rodermel S, Yin Y (2011) A brassinosteroid transcriptional network
1022
revealed by genome-wide identification of BESI target genes in Arabidopsis
1023
thaliana. Plant J 65: 634-646
1024
Yuan T, Fujioka S, Takatsuto S, Matsumoto S, Gou X, He K, Russell SD, Li J
1025
(2007) BEN1, a gene encoding a dihydroflavonol 4-reductase (DFR)-like
1026
protein, regulates the levels of brassinosteroids in Arabidopsis thaliana. Plant J
1027
51: 220-233
36
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1028
Zhang C, Xu Y, Guo S, Zhu J, Huan Q, Liu H, Wang L, Luo G, Wang X, Chong
1029
K (2012) Dynamics of brassinosteroid response modulated by negative
1030
regulator LIC in rice. PLoS Genet 8: e1002686
1031
Zhao B, Lv M, Feng Z, Campbell T, Liscum E, Li J (2016) TWISTED DWARF 1
1032
Associates with BRASSINOSTEROID-INSENSITIVE 1 to Regulate Early
1033
Events of the Brassinosteroid Signaling Pathway. Mol Plant 9: 582-592
1034
Zhou A, Wang HC, Walker JC, Li J (2004) BRL1, a leucine-rich repeat
1035
receptor-like protein kinase, is functionally redundant with BRI1 in regulating
1036
Arabidopsis brassinosteroid signaling. Plant J 40: 399-409
1037
1038
Zhu JY, Sae-Seaw J, Wang ZY (2013) Brassinosteroid signalling. Development 140:
1615-1620
37
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
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Parsed Citations
Asami T, Min YK, Nagata N, Yamagishi K, Takatsuto S, Fujioka S, Murofushi N, Yamaguchi I, Yoshida S (2000) Characterization of
brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor. Plant Physiol 123: 93-99
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Belkhadir Y, Durbak A, Wierzba M, Schmitz RJ, Aguirre A, Michel R, Rowe S, Fujioka S, Tax FE (2010) Intragenic Suppression of a
Trafficking-Defective Brassinosteroid Receptor Mutant in Arabidopsis. Genetics 185: 1283-1296
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bell EM, Lin WC, Husbands AY, Yu L, Jaganatha V, Jablonska B, Mangeon A, Neff MM, Girke T, Springer PS (2012) Arabidopsis
LATERAL ORGAN BOUNDARIES negatively regulates brassinosteroid accumulation to limit growth in organ boundaries. Proc Natl
Acad Sci U S A 109: 21146-21151
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Case DA, Cheatham TE, Darden TA, Gohlke H, Luo R, Merz KM, Onufriev AV, Simmerling C, Wang B, Woods RJ (2005) The Amber
biomolecular simulation programs. J Comput Chem 26: 1668-1688
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Chaiwanon J, Wang ZY (2015) Spatiotemporal brassinosteroid signaling and antagonism with auxin pattern stem cell dynamics in
Arabidopsis roots. Curr Biol 25: 1031-1042
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cheng Y, Zhu W, Chen Y, Ito S, Asami T, Wang X (2014) Brassinosteroids control root epidermal cell fate via direct regulation of a
MYB-bHLH-WD40 complex by GSK3-like kinases. Elife 02525
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
J 16: 735-743
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clouse SD (1996) Molecular genetic studies confirm the role of brassinosteroids in plant growth and development. Plant J 10: 1-8
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clouse SD, Langford M, McMorris TC (1996) A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects
in growth and development. Plant Physiol 111: 671-678
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clouse SD, Sasse JM (1998) Brassinosteroids: Essential regulators of plant growth and development. Annu Rev of Plant Physiol
Plant Mol Biol 49: 427-451
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Domagalska MA, Schomburg FM, Amasino RM, Vierstra RD, Nagy F, Davis SJ (2007) Attenuation of brassinosteroid signaling
enhances FLC expression and delays flowering. Development 134: 2841-2850
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Friedrichsen DM, Joazeiro CA, Li J, Hunter T, Chory J (2000) Brassinosteroid-insensitive-1 is a ubiquitously expressed leucinerich repeat receptor serine/threonine kinase. Plant Physiol 123: 1247-1256
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gendron JM, Liu JS, Fan M, Bai MY, Wenkel S, Springer PS, Barton MK, Wang ZY (2012) Brassinosteroids regulate organ
boundary formation in the shoot apical meristem of Arabidopsis. Proc Natl Acad Sci U S A 109: 21152-21157
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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Gonzalez-Garcia M-P, Vilarrasa-Blasi J, Zhiponova M, Divol F, Mora-Garcia S, Russinova E, Cano-Delgado AI (2011)
Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots. Development 138: 849-859
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gou X, Yin H, He K, Du J, Yi J, Xu S, Lin H, Clouse SD, Li J (2012) Genetic evidence for an indispensable role of somatic
embryogenesis receptor kinases in brassinosteroid signaling. PLoS genet 8: e1002452
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gudesblat GE, Russinova E (2011) Plants grow on brassinosteroids. Curr Opin in Plant Biol 14: 530-537
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gudesblat GE, Schneider-Pizon J, Betti C, Mayerhofer J, Vanhoutte I, van Dongen W, Boeren S, Zhiponova M, de Vries S, Jonak C,
Russinova E (2012) SPEECHLESS integrates brassinosteroid and stomata signalling pathways. Nat Cell Biol 14: 548-554
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Guo Z, Fujioka S, Blancaflor EB, Miao S, Gou X, Li J (2010) TCP1 Modulates Brassinosteroid Biosynthesis by Regulating the
Expression of the Key Biosynthetic Gene DWARF4 in Arabidopsis thaliana. Plant Cell 22: 1161-1173
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hacham Y, Holland N, Butterfield C, Ubeda-Tomas S, Bennett MJ, Chory J, Savaldi-Goldstein S (2011) Brassinosteroid perception
in the epidermis controls root meristem size. Development 138: 839-848
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hong Z, Jin H, Tzfira T, Li J (2008) Multiple Mechanism-Mediated Retention of a Defective Brassinosteroid Receptor in the
Endoplasmic Reticulum of Arabidopsis. Plant Cell 20: 3418-3429
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hong Z, Kajiura H, Su W, Jin H, Kimura A, Fujiyama K, Li J (2012) Evolutionarily conserved glycan signal to degrade aberrant
brassinosteroid receptors in Arabidopsis. Proc Natl Acad Sci U S A 109: 11437-11442
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hornak V, Abel R, Okur A, Strockbine B, Roitberg AE, Simmerling C (2006) Comparison of multiple Amber force fields and
development of improved protein backbone parameters. Proteins 65: 712-725
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hothorn M, Belkhadir Y, Dreux M, Dabi T, Noel JP, Wilson IA, Chory J (2011) Structural basis of steroid hormone perception by the
receptor kinase BRI1. Nature 474: 467-471
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jin H, Hong Z, Su W, Li J (2009) A plant-specific calreticulin is a key retention factor for a defective brassinosteroid receptor in the
endoplasmic reticulum. Proc Natl Acad Sci U S A 106: 13612-13617
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jin H, Yan Z, Nam KH, Li J (2007) Allele-Specific Suppression of a Defective Brassinosteroid Receptor Reveals a Physiological
Role of UGGT in ER Quality Control. Mol Cell 26: 821-830
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jorgensen WL, Chandrasekhar J, Madura JD, Impey R, Klein ML (1983) Comparison of simple potential functions for simulating
liquid water. J Chem Phys 79: 926-935
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical
features. Biopolymers 22: 2577-2637
Pubmed: Author and Title
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kim TW, Michniewicz M, Bergmann DC, Wang ZY (2012) Brassinosteroid regulates stomatal development by GSK3-mediated
inhibition of a MAPK pathway. Nature 482: 419-422
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kwon M, Choe S (2005) Brassinosteroid biosynthesis and dwarf mutants. J Plant Biol 48: 1-15
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li J, Chory J (1997) A Putative Leucine-Rich Repeat Receptor Kinase Involved in Brassinosteroid Signal Transduction. Cell 90:
929-938
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li J, Lease KA, Tax FE, Walker JC (2001) BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana. Proc
Natl Acad Sci U S A 98: 5916-5921
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC (2002) BAK1, an Arabidopsis LRR Receptor-like Protein Kinase, Interacts with
BRI1 and Modulates Brassinosteroid Signaling. Cell 110: 213-222
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li JM, Nam KH, Vafeados D, Chory J (2001) BIN2, a new brassinosteroid-insensitive locus in Arabidopsis. Plant Physiol 127: 14-22
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Liu Y, Zhang C, Wang D, Su W, Liu L, Wang M, Li J (2015) EBS7 is a plant-specific component of a highly conserved endoplasmic
reticulum-associated degradation system in Arabidopsis. Proc Natl Acad Sci U S A 112: 12205-12210
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeting induced local lesions IN genomes (TILLING) for plant functional
genomics. Plant Physiol 123: 439-442
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mora-Garcia S, Vert G, Yin YH, Cano-Delgado A, Cheong H, Chory J (2004) Nuclear protein phosphatases with Kelch-repeat
domains modulate the response to brassinosteroids in Arabidopsis. Genes Dev 18: 448-460
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Nam KH, Li J (2002) BRI1/BAK1, a Receptor Kinase Pair Mediating Brassinosteroid Signaling. Cell 110: 203-212
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide
polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J 14: 387-392
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Neff MM, Turk E, Kalishman M (2002) Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 18:
613-615
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Noguchi T, Fujioka S, Choe S, Takatsuto S, Yoshida S, Yuan H, Feldmann KA, Tax FE (1999) Brassinosteroid-insensitive dwarf
mutants of Arabidopsis accumulate brassinosteroids. Plant Physiol 121: 743-752
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Oh MH, Ray WK, Huber SC, Asara JM, Gage DA, Clouse SD (2000) Recombinant brassinosteroid insensitive 1 receptor-like kinase
autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro. Plant Physiol 124:
751-765
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ryckaert J, Ciccotti G, Berendsen HJC (1977) Numerical integration of the cartesian equations of motion of a system with
constraints: molecular dynamics of n-alkanes. J Comput Phys 23: 327-341
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Santiago J, Henzler C, Hothorn M (2013) Molecular mechanism for plant steroid receptor activation by somatic embryogenesis coreceptor kinases. Science 341: 889-892
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shang Y, Lee MM, Li J, Nam KH (2011) Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR
domain of BRI1 mediating BR signaling. BMC Plant Biol 11: 8
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
She J, Han Z, Kim TW, Wang J, Cheng W, Chang J, Shi S, Wang J, Yang M, Wang ZY, Chai J (2011) Structural insight into
brassinosteroid perception by BRI1. Nature 474: 472-476
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Steber CM, McCourt P (2001) A role for brassinosteroids in germination in Arabidopsis. Plant Physiol 125: 763-769
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Su W, Liu Y, Xia Y, Hong Z, Li J (2011) Conserved endoplasmic reticulum-associated degradation system to eliminate mutated
receptor-like kinases in Arabidopsis. Proc Natl Acad Sci U S A 108: 870-875
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Su W, Liu Y, Xia Y, Hong Z, Li J (2012) The Arabidopsis homolog of the mammalian OS-9 protein plays a key role in the endoplasmic
reticulum-associated degradation of misfolded receptor-like kinases. Mol Plant 5: 929-940
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sun Y, Fan X-Y, Cao D-M, Tang W, He K, Zhu J-Y, He J-X, Bai M-Y, Zhu S, Oh E, Patil S, Kim T-W, Ji H, Wong WH, Rhee SY, Wang ZY (2010) Integration of Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth Regulation in
Arabidopsis. Developmental Cell 19: 765-777
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sun Y, Han Z, Tang J, Hu Z, Chai C, Zhou B, Chai J (2013) Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound
brassinolide. Cell Res 23: 1326-1329
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Taylor I, Seitz K, Bennewitz S, Walker JC (2013) A simple in vitro method to measure autophosphorylation of protein kinases. Plant
Methods 9: 22
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG,
Comai L, Henikoff S (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13: 524530
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Vert G, Nemhauser JL, Geldner N, Hong F, Chory J (2005) Molecular mechanisms of steroid hormone signaling in plants. Annu Rev
Cell Dev Biol 21: 177-201
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Vilarrasa-Blasi J, Gonzalez-Garcia MP, Frigola D, Fabregas N, Alexiou KG, Lopez-Bigas N, Rivas S, Jauneau A, Lohmann JU,
Benfey PN, Ibanes M, Cano-Delgado AI (2014) Regulation of plant stem cell quiescence by a brassinosteroid signaling module. Dev
Cell 30: 36-47
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang J, Jiang J, Wang J, Chen L, Fan SL, Wu JW, Wang X, Wang ZX (2014) Structural insights into the negative regulation of BRI1
signaling by BRI1-interacting protein BKI1. Cell Res 24: 1328-1341
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput
Chem 25: 1157-1174
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang X, Chory J (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma
membrane. Science 313: 1118-1122
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang X, Kota U, He K, Blackburn K, Li J, Goshe MB, Huber SC, Clouse SD (2008) Sequential Transphosphorylation of the
BRI1/BAK1 Receptor Kinase Complex Impacts Early Events in Brassinosteroid Signaling. Dev Cell 15: 220-235
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang X, Li X, Meisenhelder J, Hunter T, Yoshida S, Asami T, Chory J (2005) Autoregulation and homodimerization are involved in
the activation of the plant steroid receptor BRI1. Dev Cell 8: 855-865
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang Y, Sun S, Zhu W, Jia K, Yang H, Wang X (2013) Strigolactone/MAX2-induced degradation of brassinosteroid transcriptional
effector BES1 regulates shoot branching. Dev Cell 27: 681-688
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang ZY, Bai MY, Oh E, Zhu JY (2012) Brassinosteroid signaling network and regulation of photomorphogenesis. Annu Rev Genet
46: 701-724
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang ZY, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 is a critical component of a plasma-membrane receptor for plant
steroids. Nature 410: 380-383
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wei Z, Li J (2016) Brassinosteroids Regulate Root Growth, Development, and Symbiosis. Mol Plant 9: 86-100
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wu D, Tao X, Chen Z, Han J, Jia W, Zhu N, Li X, Wang Z, He Y (2016) The environmental endocrine disruptor p -nitrophenol
interacts with FKBP51, a positive regulator of androgen receptor and inhibits androgen receptor signaling in human cells. J
Hazard Mater 307: 193-201
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xu W, Huang J, Li B, Li J, Wang Y (2008) Is kinase activity essential for biological functions of BRI1? Cell Res 18: 472-478
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J (2005) A new class of transcription factors mediates brassinosteroidregulated gene expression in arabidopsis. Cell 120: 249-259
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yin Y, Wang Z-Y, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J (2002) BES1 Accumulates in the Nucleus in Response to
Brassinosteroids to Regulate Gene Expression and Promote Stem Elongation. Cell 109: 181-191
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Yu X, Li L, Zola J, Aluru M, Ye H, Foudree A, Guo H, Anderson S, Aluru S, Liu P, Rodermel S, Yin Y (2011) A brassinosteroid
transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J 65: 634-646
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yuan T, Fujioka S, Takatsuto S, Matsumoto S, Gou X, He K, Russell SD, Li J (2007) BEN1, a gene encoding a dihydroflavonol 4reductase (DFR)-like protein, regulates the levels of brassinosteroids in Arabidopsis thaliana. Plant J 51: 220-233
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhang C, Xu Y, Guo S, Zhu J, Huan Q, Liu H, Wang L, Luo G, Wang X, Chong K (2012) Dynamics of brassinosteroid response
modulated by negative regulator LIC in rice. PLoS Genet 8: e1002686
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhao B, Lv M, Feng Z, Campbell T, Liscum E, Li J (2016) TWISTED DWARF 1 Associates with BRASSINOSTEROID-INSENSITIVE 1
to Regulate Early Events of the Brassinosteroid Signaling Pathway. Mol Plant 9: 582-592
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhou A, Wang HC, Walker JC, Li J (2004) BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with
BRI1 in regulating Arabidopsis brassinosteroid signaling. Plant J 40: 399-409
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhu JY, Sae-Seaw J, Wang ZY (2013) Brassinosteroid signalling. Development 140: 1615-1620
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
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