Functional Characterization of Soybean Glyma04g39610 as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors
Identifieur interne : 000346 ( Pmc/Corpus ); précédent : 000345; suivant : 000347Functional Characterization of Soybean Glyma04g39610 as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors
Auteurs : Suna Peng ; Ping Tao ; Feng Xu ; Aiping Wu ; Weige Huo ; Jinxiang WangSource :
- International Journal of Molecular Sciences [ 1422-0067 ] ; 2016.
Abstract
Brassinosteroids (BR) play important roles in plant growth and development. Although BR receptors have been intensively studied in
Url:
DOI: 10.3390/ijms17060897
PubMed: 27338344
PubMed Central: 4926431
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Functional Characterization of Soybean <italic>Glyma04g39610</italic> as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors</title><author><name sortKey="Peng, Suna" sort="Peng, Suna" uniqKey="Peng S" first="Suna" last="Peng">Suna Peng</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Tao, Ping" sort="Tao, Ping" uniqKey="Tao P" first="Ping" last="Tao">Ping Tao</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Xu, Feng" sort="Xu, Feng" uniqKey="Xu F" first="Feng" last="Xu">Feng Xu</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Wu, Aiping" sort="Wu, Aiping" uniqKey="Wu A" first="Aiping" last="Wu">Aiping Wu</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Huo, Weige" sort="Huo, Weige" uniqKey="Huo W" first="Weige" last="Huo">Weige Huo</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Jinxiang" sort="Wang, Jinxiang" uniqKey="Wang J" first="Jinxiang" last="Wang">Jinxiang Wang</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27338344</idno><idno type="pmc">4926431</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926431</idno><idno type="RBID">PMC:4926431</idno><idno type="doi">10.3390/ijms17060897</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000346</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Functional Characterization of Soybean <italic>Glyma04g39610</italic> as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors</title><author><name sortKey="Peng, Suna" sort="Peng, Suna" uniqKey="Peng S" first="Suna" last="Peng">Suna Peng</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Tao, Ping" sort="Tao, Ping" uniqKey="Tao P" first="Ping" last="Tao">Ping Tao</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Xu, Feng" sort="Xu, Feng" uniqKey="Xu F" first="Feng" last="Xu">Feng Xu</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Wu, Aiping" sort="Wu, Aiping" uniqKey="Wu A" first="Aiping" last="Wu">Aiping Wu</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Huo, Weige" sort="Huo, Weige" uniqKey="Huo W" first="Weige" last="Huo">Weige Huo</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Jinxiang" sort="Wang, Jinxiang" uniqKey="Wang J" first="Jinxiang" last="Wang">Jinxiang Wang</name><affiliation><nlm:aff id="af1-ijms-17-00897">The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</nlm:aff></affiliation><affiliation><nlm:aff id="af2-ijms-17-00897">College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</nlm:aff></affiliation></author></analytic><series><title level="j">International Journal of Molecular Sciences</title><idno type="eISSN">1422-0067</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Brassinosteroids (BR) play important roles in plant growth and development. Although BR receptors have been intensively studied in <italic>Arabidopsis</italic>, the BR receptors in soybean remain largely unknown. Here, in addition to the known receptor gene <italic>Glyma06g15270</italic> (<italic>GmBRI1a</italic>), we identified five putative BR receptor genes in the soybean genome: <italic>GmBRI1b</italic>, <italic>GmBRL1a</italic>, <italic>GmBRL1b</italic>, <italic>GmBRL2a</italic>, and <italic>GmBRL2b</italic>. Analysis of their expression patterns by quantitative real-time PCR showed that they are ubiquitously expressed in primary roots, lateral roots, stems, leaves, and hypocotyls. We used rapid amplification of cDNA ends (RACE) to clone <italic>GmBRI1b</italic> (<italic>Glyma04g39160</italic>), and found that the predicted amino acid sequence of GmBRI1b showed high similarity to those of AtBRI1 and pea PsBRI1. Structural modeling of the ectodomain also demonstrated similarities between the BR receptors of soybean and <italic>Arabidopsis</italic>. GFP-fusion experiments verified that GmBRI1b localizes to the cell membrane. We also explored <italic>GmBRI1b</italic> function in <italic>Arabidopsis</italic> through complementation experiments. Ectopic over-expression of <italic>GmBRI1b</italic> in <italic>Arabidopsis</italic> BR receptor loss-of-function mutant (<italic>bri1-5 bak1-1D</italic>) restored hypocotyl growth in etiolated seedlings; increased the growth of stems, leaves, and siliques in light; and rescued the developmental defects in leaves of the <italic>bri1-6</italic> mutant, and complemented the responses of BR biosynthesis-related genes in the <italic>bri1-5 bak1-D</italic> mutant grown in light. Bioinformatics analysis demonstrated that the six BR receptor genes in soybean resulted from three gene duplication events during evolution. Phylogenetic analysis classified the BR receptors in dicots and monocots into three subclades. Estimation of the synonymous (<italic>K</italic><sub>s</sub>) and the nonsynonymous substitution rate (<italic>K</italic><sub>a</sub>) and selection pressure (<italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub>) revealed that the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> of BR receptor genes from dicots and monocots were less than 1.0, indicating that BR receptor genes in plants experienced purifying selection during evolution.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Mussig, C" uniqKey="Mussig C">C. Müssig</name></author><author><name sortKey="Shin, G H" uniqKey="Shin G">G.H. Shin</name></author><author><name sortKey="Altmann, T" uniqKey="Altmann T">T. Altmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hacham, Y" uniqKey="Hacham Y">Y. Hacham</name></author><author><name sortKey="Holland, N" uniqKey="Holland N">N. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Mol Sci</journal-id><journal-id journal-id-type="iso-abbrev">Int J Mol Sci</journal-id><journal-id journal-id-type="publisher-id">ijms</journal-id><journal-title-group><journal-title>International Journal of Molecular Sciences</journal-title></journal-title-group><issn pub-type="epub">1422-0067</issn><publisher><publisher-name>MDPI</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27338344</article-id><article-id pub-id-type="pmc">4926431</article-id><article-id pub-id-type="doi">10.3390/ijms17060897</article-id><article-id pub-id-type="publisher-id">ijms-17-00897</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Functional Characterization of Soybean <italic>Glyma04g39610</italic> as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Suna</given-names></name><xref ref-type="aff" rid="af1-ijms-17-00897">1</xref><xref ref-type="aff" rid="af2-ijms-17-00897">2</xref></contrib><contrib contrib-type="author"><name><surname>Tao</surname><given-names>Ping</given-names></name><xref ref-type="aff" rid="af1-ijms-17-00897">1</xref><xref ref-type="aff" rid="af2-ijms-17-00897">2</xref></contrib><contrib contrib-type="author"><name><surname>Xu</surname><given-names>Feng</given-names></name><xref ref-type="aff" rid="af1-ijms-17-00897">1</xref><xref ref-type="aff" rid="af2-ijms-17-00897">2</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Aiping</given-names></name><xref ref-type="aff" rid="af1-ijms-17-00897">1</xref><xref ref-type="aff" rid="af2-ijms-17-00897">2</xref></contrib><contrib contrib-type="author"><name><surname>Huo</surname><given-names>Weige</given-names></name><xref ref-type="aff" rid="af1-ijms-17-00897">1</xref><xref ref-type="aff" rid="af2-ijms-17-00897">2</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Jinxiang</given-names></name><xref ref-type="aff" rid="af1-ijms-17-00897">1</xref><xref ref-type="aff" rid="af2-ijms-17-00897">2</xref><xref rid="c1-ijms-17-00897" ref-type="corresp">*</xref></contrib></contrib-group><contrib-group><contrib contrib-type="editor"><name><surname>Iriti</surname><given-names>Marcello</given-names></name><role>Academic Editor</role></contrib></contrib-group><aff id="af1-ijms-17-00897"><label>1</label>The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agriculture University, Guangzhou 510642, China;<email>pengsuna2013@163.com</email> (S.P.);<email>hntaoping@163.com</email> (P.T.);<email>fengxu@hybribio.cn</email> (F.X.);<email>wuaiping1@163.com</email> (A.W.);<email>18819266123@163.com</email> (W.H.)</aff><aff id="af2-ijms-17-00897"><label>2</label>College of Agriculture & Root Biology Center, South China Agricultural University, Guangzhou 510642, China</aff><author-notes><corresp id="c1-ijms-17-00897"><label>*</label>Correspondence: <email>jinxwang@scau.edu.cn</email>; Tel.: +86-20-8528-0156</corresp></author-notes><pub-date pub-type="epub"><day>07</day><month>6</month><year>2016</year></pub-date><pub-date pub-type="collection"><month>6</month><year>2016</year></pub-date><volume>17</volume><issue>6</issue><elocation-id>897</elocation-id><history><date date-type="received"><day>09</day><month>4</month><year>2016</year></date><date date-type="accepted"><day>19</day><month>5</month><year>2016</year></date></history><permissions><copyright-statement>© 2016 by the authors; licensee MDPI, Basel, Switzerland.</copyright-statement><copyright-year>2016</copyright-year><license><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>).</license-p></license></permissions><abstract><p>Brassinosteroids (BR) play important roles in plant growth and development. Although BR receptors have been intensively studied in <italic>Arabidopsis</italic>, the BR receptors in soybean remain largely unknown. Here, in addition to the known receptor gene <italic>Glyma06g15270</italic> (<italic>GmBRI1a</italic>), we identified five putative BR receptor genes in the soybean genome: <italic>GmBRI1b</italic>, <italic>GmBRL1a</italic>, <italic>GmBRL1b</italic>, <italic>GmBRL2a</italic>, and <italic>GmBRL2b</italic>. Analysis of their expression patterns by quantitative real-time PCR showed that they are ubiquitously expressed in primary roots, lateral roots, stems, leaves, and hypocotyls. We used rapid amplification of cDNA ends (RACE) to clone <italic>GmBRI1b</italic> (<italic>Glyma04g39160</italic>), and found that the predicted amino acid sequence of GmBRI1b showed high similarity to those of AtBRI1 and pea PsBRI1. Structural modeling of the ectodomain also demonstrated similarities between the BR receptors of soybean and <italic>Arabidopsis</italic>. GFP-fusion experiments verified that GmBRI1b localizes to the cell membrane. We also explored <italic>GmBRI1b</italic> function in <italic>Arabidopsis</italic> through complementation experiments. Ectopic over-expression of <italic>GmBRI1b</italic> in <italic>Arabidopsis</italic> BR receptor loss-of-function mutant (<italic>bri1-5 bak1-1D</italic>) restored hypocotyl growth in etiolated seedlings; increased the growth of stems, leaves, and siliques in light; and rescued the developmental defects in leaves of the <italic>bri1-6</italic> mutant, and complemented the responses of BR biosynthesis-related genes in the <italic>bri1-5 bak1-D</italic> mutant grown in light. Bioinformatics analysis demonstrated that the six BR receptor genes in soybean resulted from three gene duplication events during evolution. Phylogenetic analysis classified the BR receptors in dicots and monocots into three subclades. Estimation of the synonymous (<italic>K</italic><sub>s</sub>) and the nonsynonymous substitution rate (<italic>K</italic><sub>a</sub>) and selection pressure (<italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub>) revealed that the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> of BR receptor genes from dicots and monocots were less than 1.0, indicating that BR receptor genes in plants experienced purifying selection during evolution.</p></abstract><kwd-group><kwd>brassinosteroids</kwd><kwd>BR receptor</kwd><kwd>soybean</kwd><kwd>gene family</kwd><kwd>evolution</kwd></kwd-group></article-meta></front><body><sec id="sec1-ijms-17-00897"><title>1. Introduction</title><p>The brassinosteroid (BR) phytohormones play important roles in many aspects of plant growth and development, including root growth and development [<xref rid="B1-ijms-17-00897" ref-type="bibr">1</xref>,<xref rid="B2-ijms-17-00897" ref-type="bibr">2</xref>], stomatal development [<xref rid="B3-ijms-17-00897" ref-type="bibr">3</xref>], seed germination [<xref rid="B4-ijms-17-00897" ref-type="bibr">4</xref>], skotomorphogenesis [<xref rid="B5-ijms-17-00897" ref-type="bibr">5</xref>,<xref rid="B6-ijms-17-00897" ref-type="bibr">6</xref>], phototropism [<xref rid="B7-ijms-17-00897" ref-type="bibr">7</xref>], nodulation [<xref rid="B8-ijms-17-00897" ref-type="bibr">8</xref>], immunity responses [<xref rid="B9-ijms-17-00897" ref-type="bibr">9</xref>], and abiotic stress responses [<xref rid="B10-ijms-17-00897" ref-type="bibr">10</xref>,<xref rid="B11-ijms-17-00897" ref-type="bibr">11</xref>]. As early as 1990, scientists reported that BR promoted adventitious rooting in soybean hypocotyl cuttings [<xref rid="B12-ijms-17-00897" ref-type="bibr">12</xref>]. Later work showed that BR can promote stem growth in soybean [<xref rid="B13-ijms-17-00897" ref-type="bibr">13</xref>] and the application of BR increased soybean tolerance to drought by increasing the concentrations of soluble sugars and proline [<xref rid="B14-ijms-17-00897" ref-type="bibr">14</xref>]. Additionally, BR increases the expression of soybean <italic>SAUR 6B</italic>, which promotes epicotyl elongation in a time-dependent manner [<xref rid="B13-ijms-17-00897" ref-type="bibr">13</xref>]. These studies indicate that BRs regulate soybean growth, development, and stress responses at a physiological and molecular level.</p><p>Work in <italic>Arabidopsis</italic> and rice has revealed the BR signaling pathway. BR signals are detected by BR receptors such as BRASSINOSTEROID INSENSITIVE 1 (BRI1) in the cell membrane [<xref rid="B15-ijms-17-00897" ref-type="bibr">15</xref>,<xref rid="B16-ijms-17-00897" ref-type="bibr">16</xref>]. In the absence of BRs, BRI1 is bound by the membrane-localized BRI1 KINASE INHIBITOR 1 (BKI1) [<xref rid="B17-ijms-17-00897" ref-type="bibr">17</xref>]. Upon perception of BR, BRI1 disassociates from BKI1 [<xref rid="B17-ijms-17-00897" ref-type="bibr">17</xref>] and interacts with BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), a membrane kinase and co-receptor of BRI1 [<xref rid="B18-ijms-17-00897" ref-type="bibr">18</xref>]. BRI1 and BAK1 <italic>trans</italic>-phosphorylate each other [<xref rid="B17-ijms-17-00897" ref-type="bibr">17</xref>]. BRASSINOSTEROID INSENSITIVE 2 (BIN2), the downstream regulator of BRI1, is a highly conserved GSK kinase and a negative regulator of BR signaling; BIN2 phosphorylates the transcription factors BRASSINAZOLE-RESISTANT 1 (BZR1) and BZR2 thus inactivating them [<xref rid="B19-ijms-17-00897" ref-type="bibr">19</xref>,<xref rid="B20-ijms-17-00897" ref-type="bibr">20</xref>,<xref rid="B21-ijms-17-00897" ref-type="bibr">21</xref>,<xref rid="B22-ijms-17-00897" ref-type="bibr">22</xref>]. In contrast, PROTEIN PHOSPHATASE 2A (PP2A) mediates the dephosphorylation, and thus activation of BZR1 [<xref rid="B23-ijms-17-00897" ref-type="bibr">23</xref>]. High levels of BR in plants leads to the inactivation of BIN2. The dephosphorylated BZR1 and BZR2 shuttle from the cytoplasm to the nucleus and bind to the promoters of numerous downstream genes, thus strengthening BR signaling [<xref rid="B24-ijms-17-00897" ref-type="bibr">24</xref>]. BIN2 and BZR1 are regulated by proteasome-dependent pathways in <italic>Arabidopsis</italic> [<xref rid="B21-ijms-17-00897" ref-type="bibr">21</xref>,<xref rid="B25-ijms-17-00897" ref-type="bibr">25</xref>].</p><p>AtBRI1, the most important BR receptor in <italic>Arabidopsis</italic>, has a membrane-localization signal peptide in the N-terminus, 25 leucine-rich repeat (LRR) domains, and a 70-amino acid island between LRR XXI and LRR XXII [<xref rid="B26-ijms-17-00897" ref-type="bibr">26</xref>], which is indispensable for the perception of BR [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. Although BAK1 does not directly bind to BR and only has five LRR motifs, BAK1 promotes BR signaling by interacting with and phosphorylating BRI1. Two recent structural biology studies have shown that AtBRI1 is a BR receptor [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. These studies revealed that brassinolide (BL) binds to a highly hydrophobic surface groove on BRI1 (LRR) and the ectodomain is crucial for the binding of BR [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. This insight could be extrapolated to investigate BR receptors in agricultural plants.</p><p>Identifying BR receptors in other plants and deciphering their functions provides an important initial step toward deciphering BR signaling networks and understanding their evolution. <italic>Arabidopsis</italic> has three functional BR receptors, BRI1, BRL1, and BRL3. AtBRL2 appears to be non-functional in BR signaling [<xref rid="B29-ijms-17-00897" ref-type="bibr">29</xref>,<xref rid="B30-ijms-17-00897" ref-type="bibr">30</xref>]. The rice genome contains four BR receptor genes, <italic>OsBRI1</italic>, <italic>OsBRL1</italic>, <italic>OsBRL2</italic>, and <italic>OsBRL3</italic> [<xref rid="B31-ijms-17-00897" ref-type="bibr">31</xref>,<xref rid="B32-ijms-17-00897" ref-type="bibr">32</xref>]. BR receptors have also been identified in tomato [<xref rid="B33-ijms-17-00897" ref-type="bibr">33</xref>], pea [<xref rid="B34-ijms-17-00897" ref-type="bibr">34</xref>], barley [<xref rid="B35-ijms-17-00897" ref-type="bibr">35</xref>], cotton [<xref rid="B36-ijms-17-00897" ref-type="bibr">36</xref>], maize [<xref rid="B37-ijms-17-00897" ref-type="bibr">37</xref>], and wheat [<xref rid="B38-ijms-17-00897" ref-type="bibr">38</xref>]. Although the BR signaling pathway has been well studied in <italic>Arabidopsis</italic> and rice, it is not well understood in soybean. Recent work reported that soybean <italic>Glyma06g12570</italic> encodes a functional BR receptor [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>]. Considering the high levels of duplication in the soybean genome [<xref rid="B40-ijms-17-00897" ref-type="bibr">40</xref>], we postulated that soybean may have other functional BR receptors.</p><p>In this study, we conducted an evolutionary and functional examination of soybean BR receptors. Including the known gene <italic>Glyma06g15270</italic> [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>], we identified six BR receptor genes in the soybean genome and analyzed their expression patterns. We also further examined one gene, <italic>Glycine max Glyma04g39610</italic> (<italic>GmBRI1b</italic>), which encodes a homolog of AtBRI1. GmBRI1b localizes to the membrane and can function as a BR receptor in <italic>Arabidopsis</italic>. Analysis of the evolution of BR receptors in plants showed that BR receptors were subjected to purifying or negative selection.</p></sec><sec id="sec2-ijms-17-00897"><title>2. Results</title><sec id="sec2dot1-ijms-17-00897"><title>2.1. Isolation of Glyma04g39610 (GmBRI1b)</title><p>To clone soybean brassinosteroid receptors, we used the AtBRI1 protein sequence to search the soybean EST database [<xref rid="B41-ijms-17-00897" ref-type="bibr">41</xref>], using the BLASTP algorithm. We found that the amino acid sequence encoded by a tentative contig (TA51665) showed a high similarity to a region of AtBRI1. Therefore, we used the contig to design 5′-RACE and 3′-RACE primers to amplify the flanking regions of TA51665. After cloning and sequencing the flanking region, a cDNA fragment approximately 4 kb long containing a poly (A) tail was obtained (data not shown). Using ORF finder [<xref rid="B42-ijms-17-00897" ref-type="bibr">42</xref>], the full cDNA was predicted to contain a long open reading frame (Genbank Accession No. KU360113) that encodes a protein of 1187 amino acids. Alignment with the soybean genome sequence indicated that this protein is encoded by <italic>Glyma04g39610</italic> [<xref rid="B43-ijms-17-00897" ref-type="bibr">43</xref>]. As <italic>Glyma06g15270</italic> (<italic>GmBRI1</italic>) was reported to encode a BR receptor [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>], we named <italic>Glyma04g39610</italic> as <italic>GmBRI1b</italic> and renamed <italic>Glyma06g15270</italic> as <italic>GmBRI1a</italic>. Further bioinformatics analysis showed that GmBRI1b contains a membrane-localized signal peptide in the N-terminus followed by 25 LRRs, a transmembrane domain, and a Ser/Thr kinase domain in the C-terminus (<xref ref-type="table" rid="ijms-17-00897-t001">Table 1</xref> and <xref ref-type="app" rid="app1-ijms-17-00897">Table S1</xref>). Alignment analysis indicated that GmBRI1b has 69% and 81% identity to AtBRI1 and pea BRI1 (PsBRI1), respectively (<xref ref-type="app" rid="app1-ijms-17-00897">Figure S1</xref>).</p><p>As reported, <italic>AtBRI1</italic> and rice <italic>OsBRI1</italic> lack introns [<xref rid="B15-ijms-17-00897" ref-type="bibr">15</xref>,<xref rid="B31-ijms-17-00897" ref-type="bibr">31</xref>]. To determine whether <italic>GmBRI1b</italic> contains introns, we designed primers covering the initiation codon and stop codon and amplified the genomic DNA. After sequencing, we found that <italic>GmBRI1b</italic> also lacks introns. This indicated that the structure of the BR receptor genes has been highly conserved between these two species.</p></sec><sec id="sec2dot2-ijms-17-00897"><title>2.2. Identification of Other BR Receptor Genes in Soybean</title><p>Given that a whole-genome duplication occurred during soybean evolution [<xref rid="B40-ijms-17-00897" ref-type="bibr">40</xref>], we proposed that <italic>Glycine max</italic> has additional BR receptor genes. Thus, using the released soybean genome from 2010 [<xref rid="B40-ijms-17-00897" ref-type="bibr">40</xref>], we performed a BLAST search against the soybean genome [<xref rid="B43-ijms-17-00897" ref-type="bibr">43</xref>] using the BLASTP algorithm using the sequences of the four Arabidopsis BR receptors as queries. Apart from <italic>GmBRI1a</italic> [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>] and <italic>GmBRI1b</italic>, four additional putative BR receptor genes were found, <italic>Glyma04g12860</italic> (<italic>GmBRL1a</italic>), <italic>Glyma06g47870</italic> (<italic>GmBRL1b</italic>), <italic>Glyma05g26771</italic> (<italic>GmBRL2a</italic>), and <italic>Glyma0809750</italic> (<italic>GmBRL2b</italic>) on chromosomes 4, 5, 6, and 8, respectively (<xref ref-type="table" rid="ijms-17-00897-t001">Table 1</xref>). All six soybean BR receptors contain a kinase domain (KD) and five out of the six have a signal peptide (SP) and a transmembrane domain (TM) as predicted by the SMART program [<xref rid="B44-ijms-17-00897" ref-type="bibr">44</xref>] (<xref ref-type="table" rid="ijms-17-00897-t001">Table 1</xref>).</p><p>GmBRI1a, GmBRI1b, GmBRL1b, and GmBRL2b were predicted to be membrane proteins via the PSORT program [<xref rid="B45-ijms-17-00897" ref-type="bibr">45</xref>], and GmBRL1a and GmBRL2a appeared to be localized in the cytoplasm and nucleus, respectively. In addition, five of the BR genes had no introns, but <italic>GmBRL2a</italic> had one intron (<xref ref-type="table" rid="ijms-17-00897-t001">Table 1</xref>).</p><p>Next, we aligned the full amino acid sequences of the BR receptor proteins from <italic>Arabidopsis</italic>, rice, soybean, tobacco, potato, <italic>Medicago</italic>, and barley. As shown in <xref ref-type="app" rid="app1-ijms-17-00897">Figure S1</xref>, the similarities between the BR receptors were as high as 80%, indicating that the BR receptors evolved slowly in higher plants. Recent structural studies indicated that the ectodomain in BR receptors is the BR-binding domain [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. Thus, we compared the amino acid sequences of the ectodomains of the BR receptors from soybean, <italic>Arabidopsis</italic>, rice, barley, pea, and tomato. As expected, the similarities were high over 77% (<xref ref-type="app" rid="app1-ijms-17-00897">Figure S2</xref>). Additionally, the KDs among the BR receptors from the different species were also highly conserved (data not shown), indicating the importance of KDs for BR function.</p><p>In <italic>Arabidopsis</italic>, the island domain (ID) between LLRS XXI and XXII has been reported to be involved in BR binding [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. To determine whether the soybean BR receptors have the ID, we aligned the sequences with their counterparts in <italic>Arabidopsis</italic> and other species. As shown in <xref ref-type="app" rid="app1-ijms-17-00897">Figure S3</xref>, the sequences of the IDs are highly conserved in BR receptors among the different species, though we did observe that the ID sequences of GmBRI2a and GmBRI2b showed more variation than those of GmBRI1a, GmBRI1b, GmBRL1a, and GmBRL1b.</p><p>To evaluate the duplication of the BR receptor genes in soybean, we used the PGDD software [<xref rid="B46-ijms-17-00897" ref-type="bibr">46</xref>]. Three BR receptor gene duplication events were detected in soybean, <italic>GmBRI1a</italic> VS <italic>GmBRI1b</italic>, <italic>GmBRL1a</italic> VS <italic>GmBRL1b</italic>, and <italic>GmBRL2a</italic> VS <italic>GmBRL2b</italic> (<xref ref-type="app" rid="app1-ijms-17-00897">Figure S4</xref>).</p></sec><sec id="sec2dot3-ijms-17-00897"><title>2.3. Transcript Levels of GmBRI1b and Other BR Receptor Genes in Soybean</title><p>We used quantitative real-time PCR (qRT-PCR) to determine the expression patterns and transcript abundance of <italic>GmBRI1b</italic> in soybean. <italic>GmBRI1b</italic> was universally expressed in the primary roots, lateral roots, hypocotyls, epicotyls, cotyledons, apical buds, and leaves of soybean (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>A).</p><p>The transcript levels of other <italic>Glycine max</italic> BR receptors from different organs were also determined through qRT-PCR. As shown in <xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>B, the expression pattern of <italic>GmBRI1a</italic> was similar to that of <italic>GmBRI1b</italic> (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>A) and the abundances of both were higher in lateral roots, as was <italic>GmBRL2b</italic> (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>F). This indicates their important roles in lateral root development. Similarly, the transcript levels of <italic>GmBRL1a</italic> (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>C), <italic>GmBRL1b</italic> (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>D), and <italic>GmBRL2a</italic> (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>E) were relatively higher in leaves.</p><p>Additionally, we investigated the expression levels of the BR receptors in soybean based on previous RNA-Seq studies [<xref rid="B47-ijms-17-00897" ref-type="bibr">47</xref>]. As shown in <xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>G, the transcript levels of <italic>GmBRI1a</italic> and <italic>GmBRI1b</italic> were relatively high in young leaf, flower, pod, seed, root, and nodule tissues, indicating their important roles in soybean growth and development. <italic>GmBRL1a</italic> and <italic>GmBRL1b</italic> were also expressed in all tested organs although their transcript levels were lower than those of <italic>GmBRI1a</italic> and <italic>GmBRI1b</italic>. By contrast, <italic>GmBRI2a</italic> and <italic>GmBRI2b</italic> had relatively low transcript levels in seeds and nodules (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>G). The relatively high expression levels of <italic>GmBRI1a</italic> and <italic>GmBRI1b</italic> in nodules suggest their important roles in nodulation. Based on similarities of their expression patterns, the six soybean BR receptor genes can be classified into three groups, <italic>GmBRI1a</italic> and <italic>GmBRI1b</italic>; <italic>GmBRL1a</italic> and <italic>GmBRL1b</italic>; and <italic>GmBRI2a</italic> and <italic>GmBRI2b</italic>.</p></sec><sec id="sec2dot4-ijms-17-00897"><title>2.4. Subcellular Localization of GmBRI1b</title><p>As mentioned above, a signal peptide in the N-terminus and a transmembrane (TM) domain were predicted in GmBRI1b [<xref rid="B44-ijms-17-00897" ref-type="bibr">44</xref>] (<xref ref-type="table" rid="ijms-17-00897-t001">Table 1</xref>). This indicated that GmBRI1b might be a cell membrane protein. To determine the subcellular localization of GmBRI1b, we constructed a fusion protein of GmBRI1b::GFP using the gateway vector pMDC43. We then co-transformed tobacco leaf epidermal cells with constructs encoding GmBRI1b::GFP and the plasma membrane marker protein AtPIP2A::mCherry [<xref rid="B48-ijms-17-00897" ref-type="bibr">48</xref>]. Using laser confocal microscopy, we detected fluorescence signals only in the plasma membrane and the GFP fluorescence co-localized with mCherry fluorescence (<xref ref-type="fig" rid="ijms-17-00897-f002">Figure 2</xref>). This suggested that GmBRI1b is a cell membrane protein.</p></sec><sec id="sec2dot5-ijms-17-00897"><title>2.5. Functional Analysis of GmBRI1b in Arabidopsis</title><p>BRs increase cell elongation in higher plants and a deficiency of BR results in smaller, curled leaves and shorter petioles [<xref rid="B15-ijms-17-00897" ref-type="bibr">15</xref>]. The BR receptor AtBRI1 plays crucial roles in <italic>Arabidopsis</italic> growth and development, especially in stem and leaf growth. We hypothesized that GmBRI1b can function as a BR receptor to promote stem and leaf growth. To test this hypothesis and investigate the function of <italic>GmBRI1b</italic>, we tested whether <italic>GmBRI1b</italic> could complement the <italic>Arabidopsis BRI1</italic> loss-of-function mutant <italic>bri1-5 bak1-1D</italic> [<xref rid="B49-ijms-17-00897" ref-type="bibr">49</xref>]. The <italic>bri1-5</italic> allele contains a Tyr-69 substitution at the first cysteine pair of AtBRI1 that appears to be important for its dimerization [<xref rid="B49-ijms-17-00897" ref-type="bibr">49</xref>]. The <italic>bak1-1D</italic> line, in which expression of <italic>BAK1</italic> is activated by an insertion of four tandem copies of the cauliflower mosaic virus (CaMV) 35S promoter, has stronger expression of <italic>BAK1</italic>, compared with wild type [<xref rid="B18-ijms-17-00897" ref-type="bibr">18</xref>]. In contrast to the <italic>bri1-5</italic> mutant, the <italic>bri1-5 bak1-1D</italic> mutant has a relatively higher stature and longer petioles, but still exhibits a deficiency in BR signaling, represented by relatively shorter stems and petioles relative to the Ws-2 wild type [<xref rid="B18-ijms-17-00897" ref-type="bibr">18</xref>].</p><p>To test whether <italic>GmBRI1b</italic> can complement the <italic>Arabidopsis</italic> mutants, we created transgenic <italic>GmBRI1b</italic> over-expression lines (<italic>GmBRI1b-OX</italic>) driven by CaMV 35S promoter in the Ws-2 wild-type plants and in the <italic>bri1-5 bak1-1D</italic> mutant (<xref ref-type="app" rid="app1-ijms-17-00897">Figure S5</xref>A,B). After 50 days, we measured the height of the wild-type plants, <italic>bri1-5 bak1-1D</italic> mutant, and the over-expression lines grown under the same lighting and temperature conditions. Over-expression of <italic>GmBRI1b</italic> had little effect on the height of the transgenic Ws-2 wild-type plants (<xref ref-type="fig" rid="ijms-17-00897-f003">Figure 3</xref>A,C). As reported, the <italic>bri1-5 bak1-1D</italic> mutant was shorter than the wild-type Ws-2 plants [<xref rid="B18-ijms-17-00897" ref-type="bibr">18</xref>] (<xref ref-type="fig" rid="ijms-17-00897-f003">Figure 3</xref>B,D), but over-expression of <italic>GmBRI1b</italic> restored the normal plant height in the transgenic <italic>bri1-5 bak1-1D</italic> mutant. For example, <italic>GmBRI1b</italic> over-expression lines of the <italic>bri1-5 bak1-1D</italic> mutant were 2.6× and 2× taller compared with the non-transformed mutant (<xref ref-type="fig" rid="ijms-17-00897-f003">Figure 3</xref>B,D), further supporting the hypothesis that GmBRI1b functions as a BR receptor.</p><p>We also observed leaf and petiole growth and development in the Ws-2 wild type, and the <italic>bri1-5 bak1-1D</italic> mutant, and their corresponding <italic>GmBRI1b</italic> over-expression lines. The <italic>bri1-5 bak1-1D</italic> mutant had smaller leaves and shorter petioles compared to the Ws-2 wild type at 25 days after germination (<xref ref-type="fig" rid="ijms-17-00897-f004">Figure 4</xref>A), but over-expression of <italic>GmBRI1b</italic> in the transgenic <italic>bri1-5 bak1-1D</italic> mutant resulted in narrower leaves and longer petioles than in the non-transformed mutant (<xref ref-type="fig" rid="ijms-17-00897-f004">Figure 4</xref>A–C). Over-expression of <italic>GmBRI1b</italic> significantly increased the length of the 6th, 7th, and 8th leaf petiole in the Ws-2 wild type plants (<italic>p</italic> < 0.01), but no differences were found in the 1st to 5th leaves (<xref ref-type="fig" rid="ijms-17-00897-f004">Figure 4</xref>D). Over-expression of <italic>GmBRI1b</italic> in the transgenic <italic>bri1-5</italic><italic>bak1-1D</italic> mutant significantly increased elongation of the 3rd to the 8th leaves (<italic>p</italic> < 0.05 or <italic>p</italic> < 0.01), but not the 1st and the 2nd leaves (<xref ref-type="fig" rid="ijms-17-00897-f004">Figure 4</xref>E).</p><p>The <italic>bri1-6</italic> mutant has smaller, curled leaves with very short petioles. Ectopic over-expression of <italic>GmBRI1b</italic> in the transgenic <italic>bri1-6</italic> mutant (<xref ref-type="app" rid="app1-ijms-17-00897">Figure S5</xref>C) significantly increased petiole length and restored the normal wild-type leaf phenotypes in 20-day-old (<xref ref-type="fig" rid="ijms-17-00897-f005">Figure 5</xref>A–C) and 40-day-old (<xref ref-type="fig" rid="ijms-17-00897-f005">Figure 5</xref>D–F) plants.</p><p>In <italic>Arabidopsis,</italic> limitations in BR levels or defects in BR signaling lead to shorter siliques [<xref rid="B50-ijms-17-00897" ref-type="bibr">50</xref>]. The length of siliques in the same position and the same developmental stage were measured in the shoots of the Ws-2 wild type, and <italic>bri1-5 bak1-1D</italic> mutant, and their corresponding <italic>GmBRI1b</italic> over-expression lines. For the Ws-2 wild-type lines, the siliques of <italic>GmBRI1bOX-5</italic> were significantly longer than those of the non-transformed Ws-2 wild type, but this was not true for <italic>GmBRI1bOX-1</italic> (<xref ref-type="fig" rid="ijms-17-00897-f006">Figure 6</xref>A,B). For the <italic>bri1-5 bak1-1D</italic> lines, over-expression of <italic>GmBRI1b</italic> significantly increased the length of the siliques (<xref ref-type="fig" rid="ijms-17-00897-f006">Figure 6</xref>C,D).</p><p>Collectively, these results demonstrate that <italic>GmBRI1b</italic> functions as a BR receptor at the physiological and genetic level.</p></sec><sec id="sec2dot6-ijms-17-00897"><title>2.6. Ectopic Over-Expression of GmBRI1b Increased the Hypocotyl Length of the bri1-5 bak1-1D Mutant and Changed the Responses of the Wild Type and bri1-5 bak1-1D Mutant to Brassinazole</title><p>Brassinazole (Brz) effectively inhibits BR biosynthesis [<xref rid="B51-ijms-17-00897" ref-type="bibr">51</xref>] and Brz treatment decreases the growth of etiolated <italic>Arabidopsis</italic> hypocotyls [<xref rid="B51-ijms-17-00897" ref-type="bibr">51</xref>,<xref rid="B52-ijms-17-00897" ref-type="bibr">52</xref>]. To evaluate the effects of ectopic over-expression of <italic>GmBRI1b</italic> on the response to Brz, <italic>Arabidopsis</italic> seeds were germinated in half-strength MS media and then transferred to different concentrations of Brz-containing MS media after three days. After six days, the lengths of the hypocotyls were measured.</p><p>The differences in hypocotyl lengths between the dark-grown Ws-2 wild-type plants and the two corresponding over-expression lines were not significant under the control conditions (no Brz treatment). In plants treated with 1 µM Brz, the hypocotyl lengths of the two over-expression lines were 1.58× and 1.50× longer than those of the non-transgenic Ws-2 wild-type seedlings (<italic>p</italic> < 0.05). In plants treated with 2 µM Brz, the lengths of the hypocotyls in the two over-expression lines were 1.23× and 1.19× longer than those of the non-transgenic Ws-2 wild-type seedlings (<italic>p</italic> < 0.01) (<xref ref-type="fig" rid="ijms-17-00897-f007">Figure 7</xref>A,B).</p><p>Ectopic over-expression of <italic>GmBRI1b</italic> in the transgenic <italic>bri1-5 bak1-1D</italic> mutant promoted hypocotyl growth in dark-grown seedlings in the untreated and Brz-treated conditions (<xref ref-type="fig" rid="ijms-17-00897-f007">Figure 7</xref>A,C). In the untreated plants, the hypocotyl lengths of the two over-expression lines were increased by 1.53× and 1.40× over those of the non-transformed <italic>bri1-5 bak1-1D</italic> mutant. In the plants treated with 1 µM Brz, over-expression of <italic>GmBRI1b</italic> increased the length of the hypocotyls by 1.68× and 1.54× (<italic>p</italic> < 0.01). In the plants treated with 2 µM Brz, the length of the hypocotyls in the over-expression lines increased by 1.61× and 1.53× (<italic>p</italic> < 0.01) compared with those of the <italic>bri1-5 bak1-1D</italic> mutant lacking the transgene (<xref ref-type="fig" rid="ijms-17-00897-f007">Figure 7</xref>C).</p><p>Taken together, these data demonstrated that ectopic over-expression of <italic>GmBRI1b</italic> decreased the sensitivity of the Ws-2 wild type and the <italic>bri1-5 bak1-1D</italic> mutant to exogenous Brz by restoring hypocotyl growth. These results further suggest that GmBRI1b functions as a BR receptor in <italic>Arabidopsis</italic>.</p></sec><sec id="sec2dot7-ijms-17-00897"><title>2.7. Over-Expression of GmBRI1b Altered the Expression Level of BR Biosynthesis-Related Genes in the bri1-5 bak1-1D Mutant</title><p>Previous studies have revealed that the expression levels of BR biosynthesis-related genes, such as <italic>DWF4</italic>, <italic>CPD</italic>, <italic>BR6ox-1</italic>, and <italic>BR6ox-2</italic>, are regulated by negative feedback by BR itself and by BR signaling [<xref rid="B53-ijms-17-00897" ref-type="bibr">53</xref>,<xref rid="B54-ijms-17-00897" ref-type="bibr">54</xref>,<xref rid="B55-ijms-17-00897" ref-type="bibr">55</xref>]. Therefore, we selected these four marker genes to explore the effects of ectopic over-expression of <italic>GmBRI1b</italic> on the crosstalk between BR signaling and BR biosynthesis at the molecular level. Over-expression of <italic>GmBRI1b</italic> had little effect on <italic>DWF4</italic> transcription in wild type Ws-2, but significantly down-regulated transcription of <italic>DWF4</italic> in the transgenic <italic>bri1-5 bak1-1D</italic> mutant (<xref ref-type="fig" rid="ijms-17-00897-f008">Figure 8</xref>A). Dislike Ws-2, ectopic over-expression of <italic>GmBRI1b</italic> significantly repressed the expression of <italic>CPD</italic> in the <italic>bri1-5 bak1-1D</italic> over-expression line (<italic>p</italic> < 0.001, <xref ref-type="fig" rid="ijms-17-00897-f008">Figure 8</xref>B). Over-expression of <italic>GmBRI1b</italic> repressed the expression of <italic>BR6ox-1</italic> in the <italic>bri1-5 bak1-1D</italic> over-expression line by 0.51× compared with the non-transformed <italic>bri1-5 bak1-1D</italic> mutant (<xref ref-type="fig" rid="ijms-17-00897-f008">Figure 8</xref>C). In addition, over-expression of <italic>GmBBRI1b</italic> repressed the expression of <italic>BR6ox-2</italic> by 0.29× in the <italic>bri1-5 bak1-1D</italic> over-expression line compared with their corresponding non-transformed mutant, but this is not true in wild type (<xref ref-type="fig" rid="ijms-17-00897-f008">Figure 8</xref>D). Thus, these results indicate that ectopic over-expression of <italic>GmBRI1b</italic> enhanced BR signaling in the <italic>bri1-5 bak1-1D</italic> mutant, and that GmBRI1b is functional in <italic>Arabidopsis</italic>.</p></sec><sec id="sec2dot8-ijms-17-00897"><title>2.8. Structural Modeling of Soybean BR Receptors</title><p>Two studies reported the X-ray diffraction structure of the AtBRI1 ligand-binding domain (ectodomain) in 2011 [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. In AtBRI1, a 70-amino-acid ID between LRR XXI and XXII, which folds back into the interior of the super helix, generates a pocket for binding brassinolide [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. A 69 amino acid long ID was found between the LRR XXI and XXII in GmBRI1b (<xref ref-type="app" rid="app1-ijms-17-00897">Table S1</xref> and <xref ref-type="app" rid="app1-ijms-17-00897">Figure S3</xref>). Computational homology modeling is a powerful tool to investigate conservation between homologous proteins across plant species [<xref rid="B56-ijms-17-00897" ref-type="bibr">56</xref>]. After searching the PDB database [<xref rid="B57-ijms-17-00897" ref-type="bibr">57</xref>], 3RGZ and 3RGX were selected as the best templates with which to rebuild the structure of soybean BR receptors. In addition, we also reconstructed AtBRL1, AtBRL2, and AtBRL3. As shown in <xref ref-type="app" rid="app1-ijms-17-00897">Table S2</xref>, 3RGZ and 3RGX were chosen as the best templates for GmBRL1a, GmBRL1b, GmBRL2a, GmBRL2b, AtBRL1, and AtBRL3, or GmBRI1a and GmBRI1b, respectively. The related parameters in <xref ref-type="app" rid="app1-ijms-17-00897">Table S2</xref> indicated the reliability of the homology modeling.</p><p>The α-helix and β-sheet were found in the ectodomain of soybean BR receptors (<xref ref-type="fig" rid="ijms-17-00897-f009">Figure 9</xref>). The structural models of the <italic>Glycine max</italic> BR receptors and <italic>Arabidopsis</italic> BRL1 and BRL3 show high similarities in the 3-D structures of the ectodomains of the BR receptors. The tertiary structures of the BR receptors in each class also show high similarities (<xref ref-type="fig" rid="ijms-17-00897-f009">Figure 9</xref>). This suggested that the protein structures of BR receptors are conserved across plants.</p></sec><sec id="sec2dot9-ijms-17-00897"><title>2.9. Evolutionary Analysis of BR Receptors in Plants</title><p>To analyze the evolutionary relationship among BR receptors across plant species, including receptors from moss, ferns, gymnosperms, and angiosperms, we collected BR receptor sequences using BLASTP searches. First, we performed BLAST searches against different plant genomes with the amino acid sequences of AtBRI1, AtBRL1, AtBRL2, and AtBRL3. We then selected the proteins with high scores as candidate BR receptors in the different plant species. Last, we performed domain analysis with the SMART program and predicted the kinase domain and LRR domains. Based on these criteria, the sequences of 76 putative BR proteins from <italic>Physcomitrella patens</italic>, <italic>Selaginella moelledorffii</italic>, four monocots, three legumes (<italic>Glycine max</italic>, <italic>Medicago truncatula</italic>, and <italic>Phaseolus vulgaris</italic>), and 11 dicots were aligned with ClustalW 2.1. Next, we reconstructed the phylogenetic tree of the BR receptors with MrBayes 3.2 software [<xref rid="B61-ijms-17-00897" ref-type="bibr">61</xref>]. Three proteins from <italic>Physcomitrella patens</italic> and six from <italic>Selaginella moelledorffii</italic> were classified into the same subgroup with 100% bootstrap support (<xref ref-type="fig" rid="ijms-17-00897-f010">Figure 10</xref>) and were considered to be an outgroup in the reconstructed phylogenetic tree. The remaining 67 BR receptor proteins from monocots and dicots were grouped into Clades I, II, and III (100% bootstrap support). In each clade, the proteins from the monocots (rice, maize, sorghum, and <italic>Brachypodium distachyon</italic>) or from the dicots formed well-separated subclades with 100% bootstrap support (<xref ref-type="fig" rid="ijms-17-00897-f010">Figure 10</xref>). The branch length indicates the history of evolution.</p><p>The three clades were represented by BRI1, BRL1/BRL3, and BRL2. As mentioned above, BRL2 in rice and <italic>Arabidopsis</italic> appeared to be non-functional. Two <italic>Glycine max</italic> BR receptors (GmBRL2a and GmBRL2b) also seemed to have no function in BR signaling. When compared with the evolutionary distance of the BR receptors from legumes and other dicots, the BR receptors from legumes showed more conservation than other BR receptors.</p><p>In addition, we also reconstructed the phylogenetic tree with the Maximum Likelihood (ML) method to reconstruct the evolutionary relationship among BR receptors with PhyML [<xref rid="B58-ijms-17-00897" ref-type="bibr">58</xref>]. The phylogenetic tree generated with this method was the same as that generated from the Bayesian method (data not shown).</p><p>We investigated the synonymous (<italic>K</italic><sub>s</sub>) and nonsynonymous substitution rate (<italic>K</italic><sub>a</sub>) and selection pressure (<italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub>) of the BR receptor genes in higher plants during evolution. The aligned BR receptor amino acid sequences and their corresponding cDNA sequences that were conserved across soybean, rice, maize, <italic>Arabidopsis</italic>, and common bean were analyzed using the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> calculator [<xref rid="B62-ijms-17-00897" ref-type="bibr">62</xref>]. As shown in <xref ref-type="fig" rid="ijms-17-00897-f011">Figure 11</xref>, the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> values in all nodes and branches were less than 1.0, indicating that BR receptors were subjected to strong selection pressure.</p></sec></sec><sec id="sec3-ijms-17-00897"><title>3. Discussion</title><p>Although BR signaling has been extensively studied in rice and <italic>Arabidopsis</italic>, BR signaling in soybean is still largely unknown. In this study, we cloned the soybean BR receptor gene <italic>Glyma04g39610</italic> (<italic>GmBRI1b</italic>) and demonstrated that it functions as a BR receptor in <italic>Arabidopsis</italic> at the physiological, genetic, and molecular levels. In addition to this BR receptor gene and another known soybean BR receptor gene <italic>Glymg06g15270</italic> [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>], we identified four other soybean BR receptor genes.</p><p>BRs play important roles in plant growth, development, and stress adaptations such as cell elongation and division, seed germination, photomorphogenesis and skotomorphorgenesis, responses to salts and heavy metals, and pathogen resistance. BRs have been found in 61 species of embryophytes, including 53 angiosperms, six gymnosperms, one pteridophyte (<italic>Equisetum arvense</italic>), and one bryophyte (<italic>Marchantia polymorpha</italic>) [<xref rid="B63-ijms-17-00897" ref-type="bibr">63</xref>]. Additionally, two single-celled green freshwater algae (<italic>Chlorophyta</italic>; <italic>Chlorella vulgaris</italic> and <italic>Hydrodictyon reticulatum</italic>) and the marine brown alga <italic>Cystoseira myrica</italic> biosynthesize BRs [<xref rid="B64-ijms-17-00897" ref-type="bibr">64</xref>]. This indicates that BRs appear to be conserved phytohormones in plants. Considering that BRs even exist in single-celled plants, the identification of BR receptor-like proteins in moss and fern may deepen our understanding of the evolution of BR signaling in plants. Our phylogenetic analysis implies that BR receptors exist universally throughout the plant kingdom (<xref ref-type="fig" rid="ijms-17-00897-f010">Figure 10</xref>).</p><p>Similar to previously reported BR receptor genes in Arabidopsis and rice [<xref rid="B15-ijms-17-00897" ref-type="bibr">15</xref>,<xref rid="B31-ijms-17-00897" ref-type="bibr">31</xref>], <italic>GmBRI1b</italic> does not contain introns. We determined that at least six BR receptor genes exist in the soybean genome, in contrast to only four BR receptor genes in both <italic>Arabidopsis</italic> and rice, indicating that BR signaling may be more complex in soybean. In addition, two soybean BR receptor genes, <italic>GmBRL2a</italic> and <italic>GmBRL2b</italic>, showed a close evolutionary relationship with <italic>AtBRL2</italic> and <italic>OsBRL2</italic>, which have been reported to have no function in BR signaling [<xref rid="B29-ijms-17-00897" ref-type="bibr">29</xref>,<xref rid="B32-ijms-17-00897" ref-type="bibr">32</xref>]. The role of soybean <italic>GmBRI2a</italic> and <italic>GmBRI2b</italic> in BR signaling needs further study.</p><p>As previously reported, functional BR receptors in <italic>Arabidopsis</italic> and rice are localized in cell membranes [<xref rid="B31-ijms-17-00897" ref-type="bibr">31</xref>,<xref rid="B65-ijms-17-00897" ref-type="bibr">65</xref>]. A signal peptide was found in the N-terminus of GmBRI1b (<xref ref-type="table" rid="ijms-17-00897-t001">Table 1</xref>). GFP fusion experiments showed that GmBRI1b localizes in the plasma membrane (<xref ref-type="fig" rid="ijms-17-00897-f002">Figure 2</xref>). This supports the idea that GmBRI1b perceives BR at the cell membrane.</p><p><italic>AtBRI1</italic> and <italic>OsBRI1</italic> are ubiquitously expressed in all tissues [<xref rid="B31-ijms-17-00897" ref-type="bibr">31</xref>,<xref rid="B65-ijms-17-00897" ref-type="bibr">65</xref>]. We found that <italic>GmBRI1b</italic> is expressed in apical buds, cotyledons, epicotyls, hypocotyls, leaves, lateral roots, and primary roots (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>A), implying a crucial and universal role in soybean growth and development. The soybean RNA-Seq data [<xref rid="B47-ijms-17-00897" ref-type="bibr">47</xref>] supported our results (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>G). Moreover, the transcript abundance of two soybean BR receptor genes, <italic>GmBRI1a</italic> and <italic>GmBRI1b</italic>, in nodules was relatively high, suggesting that these two genes play important roles in nodulation (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>G). Application of Brz on mature leaves or into the culture media increased the nodule number and inhibited internode growth in the soybean cultivar Enrei and foliar applications of BR inhibited nodulation and root growth in the super-nodulating mutant <italic>En6500</italic>, indicating the existence of BR signaling modules in soybean [<xref rid="B66-ijms-17-00897" ref-type="bibr">66</xref>]. Moreover, these results indicate that endogenous BR homeostasis or BR signaling might control nodulation. Recent studies demonstrated that pea BR biosynthesis mutant (<italic>lk</italic>), and BR receptor mutant (<italic>lkb</italic>) had fewer lateral roots and nodules and the decreased nodule number did not seem to be attributed to changes in endogenous GA or auxin levels [<xref rid="B8-ijms-17-00897" ref-type="bibr">8</xref>]. Therefore, the roles of BR receptors in legume nodulation need further study.</p><p>In our study, we noticed differences in expression among the soybean BR receptors. For instance, the expression levels of <italic>GmBRL2a</italic> and <italic>GmBRL2b</italic> were very low in seeds, but the levels of <italic>GmBRI1a</italic> and <italic>GmBRI1b</italic> were higher in seeds (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>G). We also found higher expression of <italic>GmBRI1a</italic> and <italic>GmBRI1b</italic> in flowers (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>G), indicating that these genes might regulate flower and seed development. The highest expression of <italic>GhBRI1</italic> was found in hypocotyls, while its transcripts were much lower in mature roots [<xref rid="B36-ijms-17-00897" ref-type="bibr">36</xref>]. <italic>GmBRI1a</italic> was found to be highly expressed in soybean hypocotyls and is up-regulated by exogenous BR [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>]. We detected higher expression of <italic>GmBRI1b</italic> in hypocotyls and lateral roots in fourteen-day-old soybean seedlings, in which active cell proliferation and elongation are occurring. A previous study showed that some gene pairs resulting from gene duplication in soybean showed similar expression patterns during nodulation, while others showed different expression patterns [<xref rid="B67-ijms-17-00897" ref-type="bibr">67</xref>]. Interestingly, we found that three BR receptor gene pairs generally showed similar transcription patterns (<xref ref-type="fig" rid="ijms-17-00897-f001">Figure 1</xref>A–F). As three gene duplication events occurred during soybean evolution (<xref ref-type="app" rid="app1-ijms-17-00897">Figure S4</xref>), it is possible that the promoter sequences in the three gene pairs are similar and some common <italic>cis</italic>-elements might be found among them.</p><p>The loss of function mutation in BR signaling or biosynthesis genes in Arabidopsis results in a dwarf phenotype and late flowering [<xref rid="B6-ijms-17-00897" ref-type="bibr">6</xref>,<xref rid="B68-ijms-17-00897" ref-type="bibr">68</xref>,<xref rid="B69-ijms-17-00897" ref-type="bibr">69</xref>,<xref rid="B70-ijms-17-00897" ref-type="bibr">70</xref>]. We found that ectopic over-expression of <italic>GmBRI1b</italic> in transgenic <italic>bri1-6</italic>, and <italic>bri1-5 bak1-1D</italic> mutant restored the normal wild-type phenotype, including the height of the seedlings and the length of petioles and siliques, leaf growth. Thus, we concluded that <italic>GmBRI1b</italic> functions as a BR receptor in <italic>Arabidopsis</italic> at the physiological and genetic level. We also demonstrated that GmBRI1b acts as a functional BR receptor in <italic>Arabidopsis</italic> at the molecular level. Ectopic over-expression of <italic>GmBRI1b</italic> repressed the relatively high expression of <italic>DWF4</italic>, <italic>CPD</italic>, <italic>BR6OX-1</italic>, and <italic>BR6OX-2</italic> in the transgenic <italic>bri1-5 bak1-1D</italic> mutant. GmBRI1a was classified into the same subclade with GmBRI1b in this study. In a previous study, when <italic>GmBRI1a</italic> was expressed in the <italic>Arabidopsis bri1-5</italic> mutant, reversed the developmental defects of the <italic>bri1-5</italic> mutant [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>], although the subcellular localization of GmBRI1a was not determined [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>]. The BR-binding activity of GmBRI1b remains unknown. Results from this study combined with previous studies suggest that GmBRIa and GmBRI1b are BR receptors.</p><p>Domain analysis showed that GmBRI1b contains an N-terminal signal peptide, a transmembrane domain, a kinase domain, and 25 LRR motifs (<xref ref-type="table" rid="ijms-17-00897-t001">Table 1</xref> and <xref ref-type="app" rid="app1-ijms-17-00897">Table S1</xref>). This is in accordance with the structures of AtBRI1 and GmBRI1a [<xref rid="B39-ijms-17-00897" ref-type="bibr">39</xref>]. We noticed that in higher plants, the complete BR receptor domains evolved through two domain-gain events in the ancestral receptor-like kinase, the juxtamembrane domain (JM) and the island domain (ID) [<xref rid="B71-ijms-17-00897" ref-type="bibr">71</xref>]; the JM domain was acquired during the early diversification of plants and the ID domain formed in the ancestors of angiosperms and gymnosperms after their divergence from moss [<xref rid="B71-ijms-17-00897" ref-type="bibr">71</xref>].</p><p>Based on structural biology studies conducted in 2012 [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>], the ectodomain is responsible for the binding of the BR receptor to brassinolide. As expected, the highly conserved ectodomain sequences of the soybean BR receptors with BR receptors of other plant species were observed (<xref ref-type="app" rid="app1-ijms-17-00897">Figure S2</xref>). Moreover, our structural modeling indicates that the ectodomain of all six soybean BR receptors form similar tertiary structures to that of AtBRI1 (<xref ref-type="fig" rid="ijms-17-00897-f009">Figure 9</xref>). This indicates that the ectodomain of BR receptors in <italic>Glycine max</italic> might have an identical function to that of AtBRI1 <italic>in vivo</italic>.</p><p>The amino acid sequences in the IDs between different plant species are highly conserved and IDs participate directly in BR binding [<xref rid="B27-ijms-17-00897" ref-type="bibr">27</xref>,<xref rid="B38-ijms-17-00897" ref-type="bibr">38</xref>]. The results in <xref ref-type="app" rid="app1-ijms-17-00897">Figure S3</xref> show that the ID in soybean BR receptors is highly conserved, but the ID in GmBRL2a andGmBRL2b show more variation than the other four soybean BR receptors. GmBRL2a and GmBRL2b were classified into the same subclade with AtBRL2, which have been documented to have no function in BR signaling [<xref rid="B29-ijms-17-00897" ref-type="bibr">29</xref>]. This raises the question of whetherGmBRL2a and GmBRL2b play a role in BR signaling, and if no, can this be ascribed to the differences in the IDs? Of note, IDs only exist in gymnosperms (conifers and gnetophytes) and angiosperms. In contrast, the more ancient plant LRR–KD domain configuration generated from green algae after the split between the green algae and red algae [<xref rid="B71-ijms-17-00897" ref-type="bibr">71</xref>].</p><p>We investigated the evolution of BR receptors in plants. BLAST searches indicated that a similar kinase existed in lower plants such as <italic>Physcomitrella patens</italic> and <italic>Selaginella moelledorffii</italic>. This is consistent with a previous study that suggested that the kinase domain (KD) is more ancient [<xref rid="B71-ijms-17-00897" ref-type="bibr">71</xref>]. When we reconstructed the phylogenic tree, we found that the proteins from <italic>Physcomitrella patens</italic> and <italic>Selaginella moelledorffii</italic> had a close relationship, although we did not identify conserved BR receptors in <italic>Chlamydomonas reinhardtii</italic>. Considering the existence of BRs in single-celled plants [<xref rid="B64-ijms-17-00897" ref-type="bibr">64</xref>], we can not rule out the existence of BR receptor-like proteins in single-celled plants as some receptor kinase proteins might act as BR receptors. Interestingly, Cheon <italic>et al.</italic> [<xref rid="B72-ijms-17-00897" ref-type="bibr">72</xref>] reported that <italic>Selaginella</italic> lacks a homolog of AtBRI1, but does have downstream proteins such as BIN2, BSU1, and BZR1. This implies that the BR receptor complex evolved in a common ancestor of lycophytes, gymnosperms, and angiosperms. We found that a total of 67 BR receptors from dicots and monocots can be evolutionarily grouped into three clades represented by AtBRI1/OsBRI1, AtBRL1/OsBRL1, and AtBRL2/OsBRL2 with significant bootstrap support, which is in accordance with a previous study [<xref rid="B72-ijms-17-00897" ref-type="bibr">72</xref>]. Each clade can be divided into two subclades, Ia, Ib, IIa, IIb, IIIa, and IIIb (100% bootstrap support), one subclade from monocots, and the other from dicots (<xref ref-type="fig" rid="ijms-17-00897-f010">Figure 10</xref>). These results indicate that three ancestral BR receptor genes might generate a plethora of BR genes in plants. One subgroup, which was represented by AtRBL2, might have lost its BR receptor function during evolution [<xref rid="B29-ijms-17-00897" ref-type="bibr">29</xref>], but the reason for this is currently unknown.</p><p>We analyzed the selection pressure of BR receptors in plants during evolution. Notably, in each clade, the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> was less than 1.0 (<xref ref-type="fig" rid="ijms-17-00897-f011">Figure 11</xref>). This suggests that the BR receptor genes in higher plants were subjected to negative or purifying selection during evolution and indicates that the amino acid residues in BR receptor proteins are important and that a nonsynonymous mutation would be lethal or harmful for species survival. A previous study showed that the <italic>D</italic><sub>n</sub>/<italic>D</italic><sub>s</sub> values were less than 1.0 in all gene pairs and detected no positive selection during BR receptors evolution [<xref rid="B71-ijms-17-00897" ref-type="bibr">71</xref>]. Similarly, the substitution ratio of non-synonymous to synonymous SNPs (<italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub>) of as high as 76% analyzed <italic>JAZ</italic> genes across 13 monocot and dicot species was less than 1.0 [<xref rid="B73-ijms-17-00897" ref-type="bibr">73</xref>]. Additionally, the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> of the squamosa promoter binding protein (SBP)-box genes that encodes crucial transcription factors in plants were less than 0.5 [<xref rid="B74-ijms-17-00897" ref-type="bibr">74</xref>]. Our results are in accordance with previous reports that the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> of some important genes in multiple plant species are less than 1.0 [<xref rid="B75-ijms-17-00897" ref-type="bibr">75</xref>].</p><p>In the case of soybean, BR receptors have undergone duplication due to whole-genome duplication events [<xref rid="B40-ijms-17-00897" ref-type="bibr">40</xref>]. The soybean genome duplication also makes it much more difficult to identify BR-insensitive mutants. The CRISPR-Cas9 tool, together with artificial microRNA to knock out or knock down genes, would be valuable to further study other soybean BR receptor genes and for investigations of BR receptor functions in soybean.</p></sec><sec id="sec4-ijms-17-00897"><title>4. Materials and Methods</title><sec id="sec4dot1-ijms-17-00897"><title>4.1. Plant Materials and Growth</title><p><italic>Glycine max</italic> cultivar BD2 was used as the soybean material. One week after germination, soybean plants were cultured with half-strength Hoagland’s solution in the greenhouse. The wild type <italic>Arabidopsis</italic> Ws-2, and the <italic>bri1-5</italic><italic>bak1-D</italic> and <italic>bri1-6</italic> mutants were used. Seeds were stratified in the dark at 4 °C for 2 days, then surface-sterilized for 30 s in 75% ethanol followed by 8–10 min in 10% NaClO solution and washed five times with sterilized distilled water, then plated on half-strength MS media containing 1% sucrose and 0.8% agar with a pH of 5.8. Plates were kept in a growth chamber with a 16-h light/8-h dark cycle and a temperature cycle of 23 °C light/21 °C dark. After one week, seedlings were transplanted into soil and cultured as indicated above.</p></sec><sec id="sec4dot2-ijms-17-00897"><title>4.2. Extraction of Genomic DNA and RNA and Reverse Transcription of mRNA</title><p>Soybean and <italic>Arabidopsis</italic> genomic DNA and RNA were extracted with the CTAB and TRIzol methods, respectively. The cDNAs were reverse transcribed through MLV-transcriptase according to the vendor’s instructions. Other molecular experimental procedures were based on standard methods.</p></sec><sec id="sec4dot3-ijms-17-00897"><title>4.3. RACE Cloning of GmBRI1b</title><p>Two-week-old soybean BD2 seedlings were used to extract total RNA with the TRIzol method. Reverse transcription was carried out following standard methods to obtain cDNA. We used the SMART RACE kit (Takara Biomedical Technology, Beijing, China) to clone the <italic>GmBRI1b</italic> cDNA 5′ fragments and 3′fragments. Specific primer pairs were designed with PerlPrimer [<xref rid="B76-ijms-17-00897" ref-type="bibr">76</xref>] and are listed in <xref ref-type="app" rid="app1-ijms-17-00897">Table S3</xref>.</p></sec><sec id="sec4dot4-ijms-17-00897"><title>4.4. Analysis of Expression Patterns of Soybean BR Receptor Genes</title><p>We designed specific primer pairs with PerlPrimer [<xref rid="B76-ijms-17-00897" ref-type="bibr">76</xref>] based on the cDNA sequences and genomic sequences [<xref rid="B43-ijms-17-00897" ref-type="bibr">43</xref>] of the six soybean BR receptors to detect the expression levels of the six genes in different organs. Seven-day-old seedlings of the soybean cultivar BD2 germinated on sands were transferred to half-strength Hoagland’s nutrient solution. Seven days later, the roots, stems, and leaves were sampled for extraction of total RNA. Quantitative real-time PCR (qRT-PCR) was used to test the expression levels of the six BR receptor genes. The PCR thermal cycler parameters used were 40 cycles of 95 °C for 15 s, 60 °C for15 s, and 72 °C for 30 s. <italic>GmEF1a</italic> (<italic>Glyma19g07240</italic>) was used to normalize the expression levels. We used Rotorgen software and absolute quantification method [<xref rid="B77-ijms-17-00897" ref-type="bibr">77</xref>] to calculate the PCR results with the amplicon of <italic>GmEF1a</italic> as standard. The data presented were from three independent biological experiments.</p></sec><sec id="sec4dot5-ijms-17-00897"><title>4.5. Over-Expression of GmBRI1b in Arabidopsis</title><p>The plasmid pCHF3 (a gift from Christian Fankhauser) was digested with <italic>EcoRI</italic> and <italic>SalI</italic> to release the CaMV 35S constitutive promoter and then the 35S promoter was ligated with the <italic>EcoRI</italic> and <italic>SalI</italic>-digested plasmid pPZP221 (a gift from Jianming Li). We named this plasmid p35SPZP221. We amplified <italic>GmBRI1b</italic>cDNA, which contains <italic>SalI</italic> and <italic>SmaI</italic> restriction sites, with primer pairs. Next, we ligated <italic>GmBRI1b</italic> with plasmid p35SPZP221 digested with <italic>SalI</italic> and <italic>SmaI</italic>. After sequencing, <italic>Arabidopsis</italic> wild-type Ws-2, the <italic>bri1-5 bak1-1D</italic> and <italic>bri1-6</italic> mutants were transformed with <italic>Agrobacterium</italic> GV3101 via the floral dipping method [<xref rid="B78-ijms-17-00897" ref-type="bibr">78</xref>]. Then, the transgenic seedlings were screened in half-strength MS media that was solidified with 0.8% agar and contained 100 mg/L gentamycin. The homozygous, one-copy insertion transgenic lines were confirmed with PCR and the χ<sup>2</sup>-test and the qualifying over-expression lines were used for further experiments.</p></sec><sec id="sec4dot6-ijms-17-00897"><title>4.6. Subcellular Localization of GmBRI1b</title><p>To determine the subcellular localization of GmBRI1b, we constructed the fusion protein of GmBRI1b with GFP. The ORF of <italic>GmBRI1b</italic>, in which no stop codon exists, was amplified by PCR using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAAAGCTCTGTACAGAAGCT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATGCTTGCTCAATTCAGGG-3′. The amplified cDNA fragment was then recombined into the pMDC43 vector, thus producing the GFP::BRI1b construct under the control of the 35S promoter. AtPIP2A, which was shown to be a plasma membrane aquaporin [<xref rid="B48-ijms-17-00897" ref-type="bibr">48</xref>], fused with mCherry was used as the plasma membrane marker protein. <italic>Agrobacterium tumefaciens</italic> mediated transient expression in <italic>Nicotiana</italic><italic>benthamiana</italic> tobacco leaves was conducted as described [<xref rid="B79-ijms-17-00897" ref-type="bibr">79</xref>] with minor modifications. The <italic>Agrobacterium</italic> GV3101 strain harboring the constructs of GFP::GmBRI1b and AtPIP2A-mCherry [<xref rid="B56-ijms-17-00897" ref-type="bibr">56</xref>] was inoculated in YEP medium with the appropriate antibiotics and incubated for 16 h with shaking at 28 °C. After centrifugation at 5000 rpm for 10 min, the cell pellet was re-suspended to OD<sub>600</sub> = 1.0 in the infiltration medium (10 mM MgCl<sub>2</sub>, 10 mM MES, and 150 µM acetosyringone). The cell suspension was then allowed to standing at 22 to −24 °C for 2 to 3 h before infection into the tobacco leaves. A mix of cells containing the same quantities of <italic>GFP::GmBRI1b</italic> and <italic>AtPIP2A-mCherry</italic>, were then infiltrated into the leaves of three- to four-week-old tobacco plants. After three days, we observed the fluorescence distribution in the tobacco epidermal cells at 488 nm (GFP) and 587 nm (mCherry) wave lengths by confocal laser scanning microscopy (LSM780, Zeiss, Jena, Germany).</p></sec><sec id="sec4dot7-ijms-17-00897"><title>4.7. Phenotypic Analysis of Transgenic Arabidopsis</title><p>For sterilized solid media culture, seeds were sterilized as described above and sown in solid half-strength MS medium containing 0, 1, or 2 µM Brz. Then, the square petri dishes were wrapped with double layers of foil and were placed vertically in full darkness. After 7 days, the seedlings were scanned and analyzed with ImageJ software to quantify the length of the hypocotyls.</p><p>For determination of height, silique length, and petiole length, the seeds of the wild-type Ws-2, the <italic>bri1-6</italic> and <italic>bri1-5 bak1-1D</italic> mutants, and their corresponding over-expression lines were stratified at 4 °C for 2 days to break dormancy and then sown in soil. After the culture period, the height of the plants and the length of the petioles and siliques were measured.</p></sec><sec id="sec4dot8-ijms-17-00897"><title>4.8. Determination of the Expression Levels of BR Biosynthesis-Related Genes in the Wild Type, the Mutant, and Their Corresponding over-Expression Lines</title><p>To determine the expression levels of the BR biosynthesis-related genes <italic>CPD</italic>, <italic>DWF4</italic>, <italic>BR6ox-1</italic>, and <italic>BR6ox-2</italic>, seedlings of one-week-old Ws-2 wild type, and <italic>bri1-5 bak1-1D</italic> mutant, and the over-expression lines were transplanted into soil for one month under standard growth conditions. Then, whole plants were used to extract total RNA with the TRIzol method. cDNAs were obtained by reverse transcriptase reactions. qRT-PCR was used to determine the transcript abundances of <italic>CPD</italic>, <italic>DWF4</italic>, <italic>BR6ox-1</italic>, and <italic>BR6ox-2</italic>. <italic>AtEF-1a</italic> was used as a reference gene to normalize the qRT-PCR results. The qRT-PCR data were determined by absolute quantification method [<xref rid="B77-ijms-17-00897" ref-type="bibr">77</xref>] with the amplicon of AtEF-1a as standard. Specific primer pairs are listed in <xref ref-type="app" rid="app1-ijms-17-00897">Table S3</xref>.</p></sec><sec id="sec4dot9-ijms-17-00897"><title>4.9. Determination of the GmBRI1b Structure</title><p>Based on the full sequence of the <italic>GmBRI1b</italic> cDNA, we designed primers at the regions of the initiation codon and the stop codon to amply the genomic DNA fragment. After sequencing, we compared the genomic and cDNA sequences of <italic>GmBRI1b</italic> using SIM4 software [<xref rid="B80-ijms-17-00897" ref-type="bibr">80</xref>] to determine the protein structure of GmBRI1b.</p></sec><sec id="sec4dot10-ijms-17-00897"><title>4.10. Alignment of BR Receptors</title><p>T-coffee software [<xref rid="B81-ijms-17-00897" ref-type="bibr">81</xref>] was used to align the sequences of the full BR receptor, ectodomain, and island domain of the BRs from the different plant species <italic>Glycine max</italic>, <italic>Arabidopsis thaliana</italic>, <italic>Solanum</italic><italic>lycopersicum</italic>, <italic>Oryza</italic><italic>sativa</italic>, <italic>Pisum</italic><italic>sativum</italic>, <italic>Hordeum</italic><italic>vulgare</italic>, and <italic>Medicago</italic><italic>truncatula</italic>.</p></sec><sec id="sec4dot11-ijms-17-00897"><title>4.11. Structural Modeling of Soybean BR Receptors</title><p>The sequences of the ectodomain of AtBRI1, AtBRL1, AtBRL3, and the six soybean BR receptors were aligned with T-coffee v11.0 [<xref rid="B81-ijms-17-00897" ref-type="bibr">81</xref>]. We then searched for the best-scoring templates in the Protein Data Bank [<xref rid="B57-ijms-17-00897" ref-type="bibr">57</xref>] and selected 3RGXZ and 3RGXA. Next, we rebuilt nine structural models (six soybean BR receptors, AtBRL1, AtBRL3, and AtBRL2) of each ectodomain using Modeller v9.10 [<xref rid="B56-ijms-17-00897" ref-type="bibr">56</xref>,<xref rid="B82-ijms-17-00897" ref-type="bibr">82</xref>] and reported the results using the best model based on the DOPE score [<xref rid="B83-ijms-17-00897" ref-type="bibr">83</xref>]. The PDB files and images were processed with PyMOL v1.5 [<xref rid="B60-ijms-17-00897" ref-type="bibr">60</xref>].</p></sec><sec id="sec4dot12-ijms-17-00897"><title>4.12. Estimation of Selection and Substitution Rates</title><p>The cDNA and amino acid sequences of the BR receptors from the monocots rice and maize and the dicots soybean, common bean, and <italic>Arabidopsis</italic> were used to calculate nonsynonymous (<italic>K</italic><sub>a</sub>) and synonymous (<italic>K</italic><sub>s</sub>) substitution rates and their ratio (<italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub>) for each node/branch via a <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> online calculator tool [<xref rid="B84-ijms-17-00897" ref-type="bibr">84</xref>].</p></sec><sec id="sec4dot13-ijms-17-00897"><title>4.13. Data Analysis</title><p>Data were analyzed with Excel 2003. The Student’s <italic>t</italic>-test was used to compare the differences. R 3.0.1 package [<xref rid="B85-ijms-17-00897" ref-type="bibr">85</xref>], gplots [<xref rid="B86-ijms-17-00897" ref-type="bibr">86</xref>], and ggplot2 [<xref rid="B87-ijms-17-00897" ref-type="bibr">87</xref>] were used to draw the heatmap and other figures.</p></sec></sec><sec id="sec5-ijms-17-00897"><title>5. Conclusions</title><p><italic>Glyma04g39610</italic> encodes <italic>GmBRI1b</italic>, which functions as a BR receptor. GmBRI1b is cell membrane protein. Ectopic over-expression of <italic>GmBRI1b</italic> in <italic>bri1-6</italic> and <italic>bri1-5 bak1-1D</italic> rescues the BR signaling-related growth and development defects of the two mutants. The <italic>Glycine max</italic> genome contains six BR receptor-encoding genes, which are grouped into three clades and are generated from three gene duplication events during evolution. BR receptors in plants were subjected to purifying selection during evolution.</p></sec></body><back><ack><title>Acknowledgments</title><p>This study was supported by NSFC (No. 31071848). We are in debt to Jianming Li for his critical comments and suggestions during the early stage of this study and for providing the <italic>bri1-6</italic> seeds. We thank Arabidopsis Biological Resource Center (ABRC) for providing <italic>bri1-5 bak1-1D</italic> (CS6126) seeds and Jennifer Mach for her comments and help in English writing.</p></ack><app-group><app id="app1-ijms-17-00897"><title>Supplementary Materials</title><p>Supplementary materials can be found at <uri xlink:type="simple" xlink:href="http://www.mdpi.com/1422-0067/17/6/897/s1">http://www.mdpi.com/1422-0067/17/6/897/s1</uri>.</p><supplementary-material content-type="local-data" id="ijms-17-00897-s001"><media xlink:href="ijms-17-00897-s001.zip"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></app></app-group><notes><title>Author Contributions</title><p>Suna Peng participated in the study design, carried out the experiments and data analysis, and drafted the manuscript; Ping Tao, Feng Xu, Aiping Wu, and Weige Huo carried out the experiments and analyzed data; and Jinxiang Wang conceived, designed experiments, analyzed data, and wrote the paper. All authors read and approved the final manuscript.</p></notes><notes><title>Conflicts of Interest</title><p>The authors declare no conflict of interest.</p></notes><glossary><title>Abbreviations</title><p><array><tbody><tr><td align="left" valign="middle" rowspan="1" colspan="1">AA</td><td align="left" valign="middle" rowspan="1" colspan="1">Amino acid</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">BAK1</td><td align="left" valign="middle" rowspan="1" colspan="1">BRI1 ASSOCIATED RECEPTOR KINASE 1</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">BIN2</td><td align="left" valign="middle" rowspan="1" colspan="1">BRASSINOSTEROID INSENSITIVE2</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">BKI1</td><td align="left" valign="middle" rowspan="1" colspan="1">BRI1 KINASE INHIBITOR 1</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">BR</td><td align="left" valign="middle" rowspan="1" colspan="1">Brassinosteroid</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">BRI1</td><td align="left" valign="middle" rowspan="1" colspan="1">BRASSINOSTEROID INSENSITIVE 1</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Brz</td><td align="left" valign="middle" rowspan="1" colspan="1">Brassinazole</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">BZR1</td><td align="left" valign="middle" rowspan="1" colspan="1">BRASSINAZOLE-RESISTANT1</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">ID</td><td align="left" valign="middle" rowspan="1" colspan="1">Island domain</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">ORF</td><td align="left" valign="middle" rowspan="1" colspan="1">Open reading frame</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">qRT-PCR</td><td align="left" valign="middle" rowspan="1" colspan="1">Quantitative real-time 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Results in (<bold>A</bold>–<bold>F</bold>) were means ± SD from three independent experiments, each of which were technically repeated three times. The normalized RNA-Seq expression data of soybean BR receptor genes were downloaded from SoyBase [<xref rid="B47-ijms-17-00897" ref-type="bibr">47</xref>] (<bold>G</bold>).</p></caption><graphic xlink:href="ijms-17-00897-g001a"></graphic><graphic xlink:href="ijms-17-00897-g001b"></graphic></fig><fig id="ijms-17-00897-f002" position="float"><label>Figure 2</label><caption><p>Subcellular localization of GmBRI1b. The subcellular localization was determined with the constructs GFP::GmBRI1b: (<bold>A</bold>) GFP::BRI1b; (<bold>B</bold>) AtPIP1A::mCherry; and (<bold>C</bold>) merged image. The GFP and mCherry signals were detected at 484 and 544 nm, respectively. Scale bar = 50 µm.</p></caption><graphic xlink:href="ijms-17-00897-g002"></graphic></fig><fig id="ijms-17-00897-f003" position="float"><label>Figure 3</label><caption><p>Over-expression of <italic>GmBRI1b</italic> increased plant height in the <italic>bri1-5 bak1-1D</italic> mutant. The height of 50-day-old Ws-2 wild type and two corresponding <italic>GmBRI1b</italic> over-expression lines (<italic>GmBRI1b-OX</italic>) (<bold>A</bold>,<bold>C</bold>); and the <italic>bri1-5 bak1-1D</italic> mutant and corresponding <italic>GmBRI1b-OX</italic> lines (<bold>B</bold>,<bold>D</bold>). Results are means ± SD from five plants. Experiments were repeated two times with similar trend (Student’s <italic>t</italic>-test, ** <italic>p</italic> < 0.01).</p></caption><graphic xlink:href="ijms-17-00897-g003"></graphic></fig><fig id="ijms-17-00897-f004" position="float"><label>Figure 4</label><caption><p>Ectopic over-expression of <italic>GmBRI1b</italic> increased the length of the petioles in the transgenic <italic>bri1-5</italic><italic>bak1-1D</italic> mutant and wild type Ws-2. The 25-day-old plants (<bold>A</bold>). The 1st to the 8th leaves of the Ws-2 wild type and two corresponding <italic>GmBRI1b</italic> over-expression lines (<italic>GmBRI1b-OX</italic>) (<bold>B</bold>); and the <italic>bri1-5 bak1-1D</italic> mutant and corresponding <italic>GmBRI1b-OX</italic> lines (<bold>C</bold>). The length of the petioles from the 1st to the 8th leaves (L1-8) was measured in 25-day-old seedlings of the Ws-2 wild-type lines (<bold>D</bold>); and the <italic>bri1-5 bak1-1D</italic> mutant lines (<bold>E</bold>). Results are means ± SD from five independent experiments (in total, 25 seedlings were measured) (#, control; Student’s <italic>t</italic>-test, * <italic>p</italic> < 0.05; ** <italic>p</italic> < 0.01). Scale bar = 1 cm.</p></caption><graphic xlink:href="ijms-17-00897-g004"></graphic></fig><fig id="ijms-17-00897-f005" position="float"><label>Figure 5</label><caption><p>Ectopic over-expression of <italic>GmBRI1b</italic> restored the wild-type leaf phenotype in the transgenic <italic>bri1-6</italic> mutant. Leaf phenotypes of the 20-day-old <italic>bri1-6</italic> mutant (<bold>A</bold>); and the two corresponding <italic>GmBRI1b</italic> over-expression lines <italic>GmBRI1bOX-1</italic> (<bold>B</bold>) and <italic>GmBRI1bOX-6</italic> (<bold>C</bold>); Leaf phenotypes of the 40-day-old <italic>bri1-6</italic> mutant (<bold>D</bold>); and the two corresponding <italic>GmBRI1b</italic> over-expression lines <italic>GmBRI1bOX-1</italic> (<bold>E</bold>) and <italic>GmBRI1bOX-6</italic> (<bold>F</bold>).</p></caption><graphic xlink:href="ijms-17-00897-g005"></graphic></fig><fig id="ijms-17-00897-f006" position="float"><label>Figure 6</label><caption><p>Over-expression of <italic>GmBRI1b</italic> increased the length of the siliques in the <italic>bri1-5bak1-1D</italic> mutant. The length of siliques in the Ws-2 wild type and the two corresponding <italic>GmBRI1b</italic> over-expression lines (<italic>GmBRI1b-OX</italic>) (<bold>A</bold>,<bold>B</bold>); and the <italic>bri1-5 bak1-1D</italic> mutant and corresponding <italic>GmBRI1b-OX</italic> lines (<bold>C</bold>,<bold>D</bold>). Results are means ± SD from three independent experiments (a total of 15 seedlings were measured) (Student’s <italic>t</italic>-test, ** <italic>p</italic> < 0.01, *** <italic>p</italic> < 0.001). Scale bar = 1 cm.</p></caption><graphic xlink:href="ijms-17-00897-g006"></graphic></fig><fig id="ijms-17-00897-f007" position="float"><label>Figure 7</label><caption><p>Over-expression of <italic>GmBRI1b</italic> increased the tolerance to Brz in the <italic>Arabidopsis</italic> plants. Hypocotyl measurements were taken after exposure to different concentrations of Brz in seedlings of the Ws-2 wild type and the two corresponding <italic>GmBRI1b</italic> over-expression lines (<italic>GmBRI1b-OX</italic>) (<bold>A</bold>,<bold>B</bold>); and the <italic>bri1-5 bak1-1D</italic> mutant and corresponding <italic>GmBRI1b-OX</italic> lines (<bold>A</bold>,<bold>C</bold>). All seedlings were grown under full darkness for seven days. Results are means ± SD from three independent experiments (a total of 30 seedlings were measured) (#, control; Student’s <italic>t</italic>-test, * <italic>p</italic> < 0.05; ** <italic>p</italic> < 0.01; *** <italic>p</italic> < 0.001). Scale bar = 1 cm.</p></caption><graphic xlink:href="ijms-17-00897-g007"></graphic></fig><fig id="ijms-17-00897-f008" position="float"><label>Figure 8</label><caption><p>Effect of ectopic over-expression of <italic>GmBRI1b</italic> on the expression of BR biosynthesis-related genes in the transgenic <italic>bri1-5 bak1-1D</italic> mutant. Seedlings were grown as described in Materials and Methods. qRT-PCR was used to detect the relative expression levels of: <italic>DWF4</italic> (<bold>A</bold>); <italic>CPD</italic> (<bold>B</bold>); <italic>BR6ox-1</italic> (<bold>C</bold>); and <italic>BR6ox-2</italic> (<bold>D</bold>) in Ws-2 and <italic>bri1-5 bak1-1D</italic> mutant and their corresponding over-expression lines. Results are means ± SD from three independent experiments with three technical replicates (Student’s <italic>t</italic>-test, ** <italic>p</italic>< 0.01, *** <italic>p</italic>< 0.001).</p></caption><graphic xlink:href="ijms-17-00897-g008"></graphic></fig><fig id="ijms-17-00897-f009" position="float"><label>Figure 9</label><caption><p>Structural modeling of six soybean BR receptors. The program PhyML3.0 [<xref rid="B58-ijms-17-00897" ref-type="bibr">58</xref>] was used to reconstruct the phylogenetic tree of BR receptors from <italic>Arabidopsis</italic> and soybean. The evolutionary lineages of four BR receptors in <italic>Arabidopsis</italic> and six BR receptors in soybean were compared. The LG model for amino acid substitutions with estimated Gamma distribution was used to reconstruct the tree and the bootstrap value was set as 1000. A total of ten BR receptors were classified into Clades I, II, and III. The numbers above each branch of the tree are the bootstrap values. Scale bar indicates 0.1 amino acid substitution over evolution. In addition, the ectodomains of ten BR receptors were modeled with MODELLER9.11 software [<xref rid="B59-ijms-17-00897" ref-type="bibr">59</xref>] based on the template of AtBRI1, which was determined with X-ray crystallization on a 3-D level in 2011 [<xref rid="B28-ijms-17-00897" ref-type="bibr">28</xref>]. The PDB files were processed with PyMOL v1.5 software [<xref rid="B60-ijms-17-00897" ref-type="bibr">60</xref>].</p></caption><graphic xlink:href="ijms-17-00897-g009"></graphic></fig><fig id="ijms-17-00897-f010" position="float"><label>Figure 10</label><caption><p>Evolutionary analysis of BR receptors in plants. BLASTP was used to search for BR receptor homologs in different plant species. A total of 76 BR receptors as shown in Spreadsheet S1 were analyzed from monocots (<italic>Oryza sativa</italic> (Os), <italic>Zea mays</italic> (Zm), <italic>Sorghum bicolor</italic> (Sb), <italic>Brachypodium</italic><italic>distachyon</italic> (Bradi)), dicots (<italic>Arabidopsis thaliana</italic> (At), <italic>Glycine max</italic> (Gm), <italic>Solanum</italic><italic>lycopersicum</italic> (Soly), <italic>Medicago</italic><italic>truncatula</italic> (Mt), <italic>Phaseolus vulgaris</italic> (Pv), <italic>Populus</italic><italic>trichocarpa</italic> (Pt), <italic>Eucalyptus grandis</italic> (Eucgr), <italic>Citrus</italic><italic>sinensis</italic> (Cis), <italic>Gossypium</italic><italic>raimondii</italic> (Gora), <italic>Cucumis sativa</italic> (Cusa), <italic>Prunus</italic><italic>persica</italic>(Pp), <italic>Manihot</italic><italic>esculenta</italic> (Me), <italic>Ricinus</italic><italic>communis</italic> (Rc), and <italic>Nicotiana</italic><italic>tabacum</italic> (Nt)), moss (<italic>Physcomitrella patens</italic> (Phpat)), and fern (<italic>Selaginella</italic><italic>moelledorffii</italic> (Smo)) and grouped into three clades, CladesI, II, and III. Additionally, nine BR receptor homologs from moss and fern were determined to be members of an outgroup. The values above the branches are the probability of the bootstrap value with 1000 repeats. MrBayes 3.2 software was used to reconstruct the phylogenetic tree as described in the Methods Section. Scale bar indicates 0.1 amino acid substitution over evolution.</p></caption><graphic xlink:href="ijms-17-00897-g010"></graphic></fig><fig id="ijms-17-00897-f011" position="float"><label>Figure 11</label><caption><p>Estimation of <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> in the BR receptor genes from soybean, common bean, <italic>Arabidopsis,</italic> rice, and maize. The cDNA sequences and amino acid sequences of the BR receptors from dicots (soybean, common bean, and <italic>Arabidopsis</italic>) and monocots (rice and maize) were used to estimate the <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub>. Twenty nodes are shown. The <italic>K</italic><sub>a</sub> and <italic>K</italic><sub>s</sub> values in each node and branch are marked in blue and red, respectively. The <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> in each branch is indicated in parentheses and all <italic>K</italic><sub>a</sub>/<italic>K</italic><sub>s</sub> values were less than 1.0.</p></caption><graphic xlink:href="ijms-17-00897-g011"></graphic></fig><table-wrap id="ijms-17-00897-t001" position="float"><object-id pub-id-type="pii">ijms-17-00897-t001_Table 1</object-id><label>Table 1</label><caption><p>General information about the brassinosteroid (BR) receptor genes in soybean based on bioinformatics analysis.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Gene</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Locus</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">EST</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">TM</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">SP</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">KD</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Length (AA)</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Localization</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Int/Ext</th></tr></thead><tbody><tr><td align="center" valign="middle" rowspan="1" colspan="1"><italic>GmBRI1a</italic></td><td align="center" valign="middle" rowspan="1" colspan="1"><italic>Glyma06g15270</italic></td><td align="center" valign="middle" rowspan="1" colspan="1">Yes</td><td align="center" valign="middle" rowspan="1" colspan="1">784..806</td><td align="center" valign="middle" rowspan="1" colspan="1">1..20</td><td align="center" valign="middle" rowspan="1" colspan="1">871..1143</td><td align="center" valign="middle" rowspan="1" colspan="1">1184</td><td align="center" valign="middle" rowspan="1" colspan="1">Plas</td><td align="center" valign="middle" rowspan="1" colspan="1">0/1</td></tr><tr><td align="center" valign="middle" rowspan="1" colspan="1"><italic>GmBRI1b</italic></td><td align="center" valign="middle" rowspan="1" colspan="1"><italic>Glyma04g39610</italic></td><td align="center" valign="middle" rowspan="1" colspan="1">Yes</td><td align="center" valign="middle" rowspan="1" colspan="1">787..809</td><td align="center" valign="middle" rowspan="1" colspan="1">1..22</td><td align="center" valign="middle" rowspan="1" colspan="1">874..1146</td><td align="center" valign="middle" rowspan="1" colspan="1">1187</td><td align="center" valign="middle" rowspan="1" colspan="1">Plas</td><td align="center" valign="middle" rowspan="1" colspan="1">0/1</td></tr><tr><td align="center" valign="middle" rowspan="1" colspan="1"><italic>GmBRL1a</italic></td><td align="center" valign="middle" rowspan="1" colspan="1"><italic>Glyma04g12860</italic></td><td align="center" valign="middle" rowspan="1" colspan="1">Yes</td><td align="center" valign="middle" rowspan="1" colspan="1">827..849</td><td align="center" valign="middle" rowspan="1" colspan="1">1..43</td><td align="center" valign="middle" rowspan="1" colspan="1">928..1202</td><td align="center" valign="middle" rowspan="1" colspan="1">1207</td><td align="center" valign="middle" rowspan="1" colspan="1">Cyto</td><td align="center" valign="middle" rowspan="1" colspan="1">0/1</td></tr><tr><td align="center" valign="middle" rowspan="1" colspan="1"><italic>GmBRL1b</italic></td><td align="center" valign="middle" rowspan="1" colspan="1"><italic>Glyma06g47870</italic></td><td align="center" valign="middle" rowspan="1" colspan="1">Yes</td><td align="center" valign="middle" rowspan="1" colspan="1">843..865</td><td align="center" valign="middle" rowspan="1" colspan="1">No</td><td align="center" valign="middle" rowspan="1" colspan="1">912..1186</td><td align="center" valign="middle" rowspan="1" colspan="1">1211</td><td align="center" valign="middle" rowspan="1" colspan="1">Plas</td><td align="center" valign="middle" rowspan="1" colspan="1">0/1</td></tr><tr><td align="center" valign="middle" rowspan="1" colspan="1"><italic>GmBRL2a</italic></td><td align="center" valign="middle" rowspan="1" colspan="1"><italic>Glyma05g26771</italic></td><td align="center" valign="middle" rowspan="1" colspan="1">Yes</td><td align="center" valign="middle" rowspan="1" colspan="1">752..774</td><td align="center" valign="middle" rowspan="1" colspan="1">1..32</td><td align="center" valign="middle" rowspan="1" colspan="1">756..1038</td><td align="center" valign="middle" rowspan="1" colspan="1">1053</td><td align="center" valign="middle" rowspan="1" colspan="1">Nucl</td><td align="center" valign="middle" rowspan="1" colspan="1">1/2</td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1"><italic>GmBRL2b</italic></td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1"><italic>Glyma08g09750</italic></td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">Yes</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">no</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">1..29</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">837..1121</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">1136</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">Plas</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">0/1</td></tr></tbody></table><table-wrap-foot><fn><p>The transmembrane domains (TM) signal peptides (SP), and kinase domains (KD) were predicted by the SMART [<xref rid="B44-ijms-17-00897" ref-type="bibr">44</xref>] program and the positions (from the amino terminus to the carboxyl terminus) of the TM, SP, and KD are indicated in the table. Putative cell localization of the soybean BR receptors was predicted by PSORT [<xref rid="B45-ijms-17-00897" ref-type="bibr">45</xref>]. Based on the released genome sequences and cDNA sequences of soybean, the numbers of introns and exons were determined through SIM4 as described in Methods. AA, amino acid; Cyto, cytoplasm; EST, expressed sequence tag; Ext, Extron; Int, intron; Nucl, nucleus; Plas, plasmamembrane.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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