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Functional Characterization of Soybean Glyma04g39610 as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors

Identifieur interne : 000175 ( Pmc/Checkpoint ); précédent : 000174; suivant : 000176

Functional Characterization of Soybean Glyma04g39610 as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors

Auteurs : Suna Peng [République populaire de Chine] ; Ping Tao [République populaire de Chine] ; Feng Xu [République populaire de Chine] ; Aiping Wu [République populaire de Chine] ; Weige Huo [République populaire de Chine] ; Jinxiang Wang [République populaire de Chine]

Source :

RBID : PMC:4926431

Abstract

Brassinosteroids (BR) play important roles in plant growth and development. Although BR receptors have been intensively studied in Arabidopsis, the BR receptors in soybean remain largely unknown. Here, in addition to the known receptor gene Glyma06g15270 (GmBRI1a), we identified five putative BR receptor genes in the soybean genome: GmBRI1b, GmBRL1a, GmBRL1b, GmBRL2a, and GmBRL2b. 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 GmBRI1b (Glyma04g39160), 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 Arabidopsis. GFP-fusion experiments verified that GmBRI1b localizes to the cell membrane. We also explored GmBRI1b function in Arabidopsis through complementation experiments. Ectopic over-expression of GmBRI1b in Arabidopsis BR receptor loss-of-function mutant (bri1-5 bak1-1D) 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 bri1-6 mutant, and complemented the responses of BR biosynthesis-related genes in the bri1-5 bak1-D 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 (Ks) and the nonsynonymous substitution rate (Ka) and selection pressure (Ka/Ks) revealed that the Ka/Ks 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.


Url:
DOI: 10.3390/ijms17060897
PubMed: 27338344
PubMed Central: 4926431


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PMC:4926431

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<email>hntaoping@163.com</email>
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<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;
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(S.P.);
<email>hntaoping@163.com</email>
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<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>
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</TEI>
<pmc article-type="research-article">
<pmc-dir>properties open_access</pmc-dir>
<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>
<floats-group>
<fig id="ijms-17-00897-f001" position="float">
<label>Figure 1</label>
<caption>
<p>Expression of soybean BR receptors:
<italic>GmBRI1b</italic>
(
<bold>A</bold>
);
<italic>GmBRI1a</italic>
(
<bold>B</bold>
);
<italic>GmBRL1a</italic>
(
<bold>C</bold>
);
<italic>GmBRIL1b</italic>
(
<bold>D</bold>
);
<italic>GmBRL2a</italic>
(
<bold>E</bold>
); and
<italic>GmBRL2b</italic>
(
<bold>F</bold>
) in apical buds (b), cotyledons (c), epicotyls (e), hypocotyls (h), leaves (l), lateral roots (lr), and primary roots (pr).
<italic>GmEF1a</italic>
was used to normalize the qRT-PCR data. 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>
<affiliations>
<list>
<country>
<li>République populaire de Chine</li>
</country>
<region>
<li>Guangdong</li>
</region>
<settlement>
<li>Jiangmen</li>
</settlement>
</list>
<tree>
<country name="République populaire de Chine">
<region name="Guangdong">
<name sortKey="Peng, Suna" sort="Peng, Suna" uniqKey="Peng S" first="Suna" last="Peng">Suna Peng</name>
</region>
<name sortKey="Huo, Weige" sort="Huo, Weige" uniqKey="Huo W" first="Weige" last="Huo">Weige Huo</name>
<name sortKey="Tao, Ping" sort="Tao, Ping" uniqKey="Tao P" first="Ping" last="Tao">Ping Tao</name>
<name sortKey="Wang, Jinxiang" sort="Wang, Jinxiang" uniqKey="Wang J" first="Jinxiang" last="Wang">Jinxiang Wang</name>
<name sortKey="Wu, Aiping" sort="Wu, Aiping" uniqKey="Wu A" first="Aiping" last="Wu">Aiping Wu</name>
<name sortKey="Xu, Feng" sort="Xu, Feng" uniqKey="Xu F" first="Feng" last="Xu">Feng Xu</name>
</country>
</tree>
</affiliations>
</record>

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