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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Biochemical and Expression Analyses of the Rice Cinnamoyl-CoA Reductase Gene Family</title><author><name sortKey="Park, Hye Lin" sort="Park, Hye Lin" uniqKey="Park H" first="Hye Lin" last="Park">Hye Lin Park</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Bhoo, Seong Hee" sort="Bhoo, Seong Hee" uniqKey="Bhoo S" first="Seong Hee" last="Bhoo">Seong Hee Bhoo</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kwon, Mi" sort="Kwon, Mi" uniqKey="Kwon M" first="Mi" last="Kwon">Mi Kwon</name><affiliation><nlm:aff id="aff2"><institution>Institute of Biological Chemistry, Washington State University</institution>,<addr-line>Pullman, WA</addr-line>,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Lee, Sang Won" sort="Lee, Sang Won" uniqKey="Lee S" first="Sang-Won" last="Lee">Sang-Won Lee</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Cho, Man Ho" sort="Cho, Man Ho" uniqKey="Cho M" first="Man-Ho" last="Cho">Man-Ho Cho</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29312373</idno><idno type="pmc">5732984</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732984</idno><idno type="RBID">PMC:5732984</idno><idno type="doi">10.3389/fpls.2017.02099</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000843</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000843</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Biochemical and Expression Analyses of the Rice Cinnamoyl-CoA Reductase Gene Family</title><author><name sortKey="Park, Hye Lin" sort="Park, Hye Lin" uniqKey="Park H" first="Hye Lin" last="Park">Hye Lin Park</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Bhoo, Seong Hee" sort="Bhoo, Seong Hee" uniqKey="Bhoo S" first="Seong Hee" last="Bhoo">Seong Hee Bhoo</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Kwon, Mi" sort="Kwon, Mi" uniqKey="Kwon M" first="Mi" last="Kwon">Mi Kwon</name><affiliation><nlm:aff id="aff2"><institution>Institute of Biological Chemistry, Washington State University</institution>,<addr-line>Pullman, WA</addr-line>,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Lee, Sang Won" sort="Lee, Sang Won" uniqKey="Lee S" first="Sang-Won" last="Lee">Sang-Won Lee</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Cho, Man Ho" sort="Cho, Man Ho" uniqKey="Cho M" first="Man-Ho" last="Cho">Man-Ho Cho</name><affiliation><nlm:aff id="aff1"><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></nlm:aff></affiliation></author></analytic><series><title level="j">Frontiers in Plant Science</title><idno type="eISSN">1664-462X</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Cinnamoyl-CoA reductase (CCR) is the first committed enzyme in the monolignol pathway for lignin biosynthesis and catalyzes the conversion of hydroxycinnamoyl-CoAs into hydroxycinnamaldehydes. In the rice genome, 33 genes are annotated as <italic>CCR</italic> and <italic>CCR-like</italic> genes, collectively called <italic>OsCCR</italic>s. To elucidate the functions of <italic>OsCCR</italic>s, their phylogenetic relationships, expression patterns at the transcription levels and biochemical characteristics were thoroughly analyzed. Of the 33 <italic>OsCCR</italic>s, 24 of them encoded polypeptides of lengths similar to those of previously identified plant CCRs. The other nine OsCCRs had much shorter peptide lengths. Phylogenetic tree and sequence similarities suggested OsCCR4, 5, 17, 18, 19, 20, and 21 as likely candidates for functional CCRs in rice. To elucidate biochemical functions, OsCCR1, 5, 17, 19, 20, 21, and 26 were heterologously expressed in <italic>Escherichia coli</italic> and the resulting recombinant OsCCRs were purified to apparent homogeneity. Activity assays of the recombinant OsCCRs with hydroxycinnamoyl-CoAs revealed that OsCCR17, 19, 20, and 21 were biochemically active CCRs, in which the NAD(P)-binding and NADP-specificity motifs as well as the CCR signature motif were fully conserved. The kinetic parameters of enzyme reactions revealed that feruloyl-CoA, a precursor for the guaiacyl (G)-unit of lignin, is the most preferred substrate of OsCCR20 and 21. This result is consistent with a high content (about 70%) of G-units in rice lignins. Phylogenetic analysis revealed that OsCCR19 and 20 were grouped with other plant CCRs involved in developmental lignification, whereas OsCCR17 and 21 were closely related to stress-responsible CCRs identified from other plant species. In agreement with the phylogenetic analysis, expression analysis demonstrated that <italic>OsCCR20</italic> was constitutively expressed throughout the developmental stages of rice, showing particularly high expression levels in actively lignifying tissues, such as roots and stems. These results suggest that <italic>OsCCR20</italic> is primarily involved in developmental deposition of lignins in secondary cell walls. As expected, the expressions of <italic>OsCCR17</italic> and <italic>21</italic> were induced in response to biotic and abiotic stresses, such as <italic>Magnaporthe grisea</italic> and <italic>Xanthomonas oryzae</italic> pv. <italic>oryzae</italic> (<italic>Xoo</italic>) infections, UV-irradiation and high salinity, suggesting that these genes play a role in defense-related processes in rice.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Ahuja, I" uniqKey="Ahuja I">I. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Front Plant Sci</journal-id><journal-id journal-id-type="iso-abbrev">Front Plant Sci</journal-id><journal-id journal-id-type="publisher-id">Front. Plant Sci.</journal-id><journal-title-group><journal-title>Frontiers in Plant Science</journal-title></journal-title-group><issn pub-type="epub">1664-462X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29312373</article-id><article-id pub-id-type="pmc">5732984</article-id><article-id pub-id-type="doi">10.3389/fpls.2017.02099</article-id><article-categories><subj-group subj-group-type="heading"><subject>Plant Science</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Biochemical and Expression Analyses of the Rice Cinnamoyl-CoA Reductase Gene Family</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Park</surname><given-names>Hye Lin</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/472567/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Bhoo</surname><given-names>Seong Hee</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kwon</surname><given-names>Mi</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Sang-Won</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Cho</surname><given-names>Man-Ho</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="author-notes" rid="fn002"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/472579/overview"></uri></contrib></contrib-group><aff id="aff1"><sup>1</sup><institution>Graduate School of Biotechnology and College of Life Sciences, Kyung Hee University</institution>,<addr-line>Yongin</addr-line>,<country>South Korea</country></aff><aff id="aff2"><sup>2</sup><institution>Institute of Biological Chemistry, Washington State University</institution>,<addr-line>Pullman, WA</addr-line>,<country>United States</country></aff><author-notes><fn fn-type="edited-by"><p>Edited by: Danièle Werck, Centre National de la Recherche Scientifique (CNRS), France</p></fn><fn fn-type="edited-by"><p>Reviewed by: Soren K. Rasmussen, University of Copenhagen, Denmark; John Sedbrook, Illinois State University, United States</p></fn><corresp id="fn001">*Correspondence: Sang-Won Lee <email xlink:type="simple">swlee6803@khu.ac.kr</email></corresp><corresp id="fn002">Man-Ho Cho <email xlink:type="simple">manhocho@khu.ac.kr</email></corresp><fn fn-type="other" id="fn003"><p>This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science</p></fn></author-notes><pub-date pub-type="epub"><day>12</day><month>12</month><year>2017</year></pub-date><pub-date pub-type="collection"><year>2017</year></pub-date><volume>8</volume><elocation-id>2099</elocation-id><history><date date-type="received"><day>07</day><month>9</month><year>2017</year></date><date date-type="accepted"><day>24</day><month>11</month><year>2017</year></date></history><permissions><copyright-statement>Copyright © 2017 Park, Bhoo, Kwon, Lee and Cho.</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>Park, Bhoo, Kwon, Lee and Cho</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Cinnamoyl-CoA reductase (CCR) is the first committed enzyme in the monolignol pathway for lignin biosynthesis and catalyzes the conversion of hydroxycinnamoyl-CoAs into hydroxycinnamaldehydes. In the rice genome, 33 genes are annotated as <italic>CCR</italic> and <italic>CCR-like</italic> genes, collectively called <italic>OsCCR</italic>s. To elucidate the functions of <italic>OsCCR</italic>s, their phylogenetic relationships, expression patterns at the transcription levels and biochemical characteristics were thoroughly analyzed. Of the 33 <italic>OsCCR</italic>s, 24 of them encoded polypeptides of lengths similar to those of previously identified plant CCRs. The other nine OsCCRs had much shorter peptide lengths. Phylogenetic tree and sequence similarities suggested OsCCR4, 5, 17, 18, 19, 20, and 21 as likely candidates for functional CCRs in rice. To elucidate biochemical functions, OsCCR1, 5, 17, 19, 20, 21, and 26 were heterologously expressed in <italic>Escherichia coli</italic> and the resulting recombinant OsCCRs were purified to apparent homogeneity. Activity assays of the recombinant OsCCRs with hydroxycinnamoyl-CoAs revealed that OsCCR17, 19, 20, and 21 were biochemically active CCRs, in which the NAD(P)-binding and NADP-specificity motifs as well as the CCR signature motif were fully conserved. The kinetic parameters of enzyme reactions revealed that feruloyl-CoA, a precursor for the guaiacyl (G)-unit of lignin, is the most preferred substrate of OsCCR20 and 21. This result is consistent with a high content (about 70%) of G-units in rice lignins. Phylogenetic analysis revealed that OsCCR19 and 20 were grouped with other plant CCRs involved in developmental lignification, whereas OsCCR17 and 21 were closely related to stress-responsible CCRs identified from other plant species. In agreement with the phylogenetic analysis, expression analysis demonstrated that <italic>OsCCR20</italic> was constitutively expressed throughout the developmental stages of rice, showing particularly high expression levels in actively lignifying tissues, such as roots and stems. These results suggest that <italic>OsCCR20</italic> is primarily involved in developmental deposition of lignins in secondary cell walls. As expected, the expressions of <italic>OsCCR17</italic> and <italic>21</italic> were induced in response to biotic and abiotic stresses, such as <italic>Magnaporthe grisea</italic> and <italic>Xanthomonas oryzae</italic> pv. <italic>oryzae</italic> (<italic>Xoo</italic>) infections, UV-irradiation and high salinity, suggesting that these genes play a role in defense-related processes in rice.</p></abstract><kwd-group><kwd>rice</kwd><kwd>cinnamoyl-CoA reductase</kwd><kwd>monolignol pathway</kwd><kwd>lignin</kwd><kwd>biotic/abiotic stress</kwd></kwd-group><counts><fig-count count="6"></fig-count><table-count count="2"></table-count><equation-count count="0"></equation-count><ref-count count="70"></ref-count><page-count count="14"></page-count><word-count count="10603"></word-count></counts></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>Plants are constantly confronted by both biotic and abiotic stresses, leading to significant reductions in their productivity (Strange and Scott, <xref rid="B62" ref-type="bibr">2005</xref>; Vinocur and Altman, <xref rid="B69" ref-type="bibr">2005</xref>; Oerke, <xref rid="B51" ref-type="bibr">2006</xref>; Chakraborty and Newton, <xref rid="B13" ref-type="bibr">2011</xref>). Abiotic stresses, including drought, salinity, and extreme temperature, are the primary factors in crop loss and can reduce the average yields of major crop plants by more than 50% (Boyer, <xref rid="B10" ref-type="bibr">1982</xref>; Oerke, <xref rid="B51" ref-type="bibr">2006</xref>). Biotic stresses, such as infection by pathogens, can cause serious reduction of cereal production (Strange and Scott, <xref rid="B62" ref-type="bibr">2005</xref>; Oerke, <xref rid="B51" ref-type="bibr">2006</xref>; Chakraborty and Newton, <xref rid="B13" ref-type="bibr">2011</xref>). It has been reported that actual losses of worldwide rice production due to biotic stresses, in the period of 2001–2003, comprised an estimated 37.4% of the total attainable production (Oerke, <xref rid="B51" ref-type="bibr">2006</xref>). To cope with biotic and abiotic stresses, plants have developed a wide array of defense mechanisms such as the fortification of cell walls, production of phytoalexins, and accumulation of reactive oxygen species (Moura et al., <xref rid="B49" ref-type="bibr">2010</xref>; Ahuja et al., <xref rid="B1" ref-type="bibr">2012</xref>; Großkinsky et al., <xref rid="B26" ref-type="bibr">2012</xref>; Miedes et al., <xref rid="B48" ref-type="bibr">2014</xref>; Rejeb et al., <xref rid="B57" ref-type="bibr">2014</xref>).</p><p>Lignin is complex aromatic polymer primarily composed of <italic>p</italic>-hydroxyphenyl (H)-, G- and syringyl (S)-units derived from monolignols. Lignin is predominantly deposited in the secondary cell walls of xylem and fiber cells and makes the cell walls rigid and impervious (Campbell and Sederoff, <xref rid="B11" ref-type="bibr">1996</xref>; Donaldson, <xref rid="B20" ref-type="bibr">2001</xref>; Bonawitz and Chapple, <xref rid="B9" ref-type="bibr">2010</xref>; Vanholme et al., <xref rid="B68" ref-type="bibr">2010</xref>). The lignified secondary cell walls are important for the water conduction and mechanical support of vascular plants, and serve as a physical barrier against pathogens and herbivores (Campbell and Sederoff, <xref rid="B11" ref-type="bibr">1996</xref>; Donaldson, <xref rid="B20" ref-type="bibr">2001</xref>; Vanholme et al., <xref rid="B68" ref-type="bibr">2010</xref>; Miedes et al., <xref rid="B48" ref-type="bibr">2014</xref>). In addition to developmental deposition, the synthesis and deposition of lignin-related phenolics are induced in response to biotic and abiotic stresses (Moura et al., <xref rid="B49" ref-type="bibr">2010</xref>; Hamann, <xref rid="B28" ref-type="bibr">2012</xref>; Miedes et al., <xref rid="B48" ref-type="bibr">2014</xref>).</p><p>The biosynthetic pathway of lignin is divided into two branches: the general phenylpropanoid pathway from phenylalanine to hydroxycinnamoyl-CoAs, and the monolignol pathway from hydroxycinnamoyl-CoAs to monolignols. These monolignols include <italic>p</italic>-coumaroyl, coniferyl, and sinapyl alcohols (Davin et al., <xref rid="B18" ref-type="bibr">2008</xref>; Vanholme et al., <xref rid="B68" ref-type="bibr">2010</xref>; Miedes et al., <xref rid="B48" ref-type="bibr">2014</xref>). In addition to lignin biosynthesis, the phenylpropanoid pathway is used in the synthesis of a vast array of phenolic compounds including phytoalexins, phenylpropanoid conjugates and flavonoids (Dixon et al., <xref rid="B19" ref-type="bibr">2002</xref>; Ahuja et al., <xref rid="B1" ref-type="bibr">2012</xref>; Großkinsky et al., <xref rid="B26" ref-type="bibr">2012</xref>; Cho and Lee, <xref rid="B15" ref-type="bibr">2015</xref>). Hydroxycinnamoyl-CoA esters are subsequently channeled into the lignin branch pathway to produce monolignols through hydroxycinnamaldehydes via two reductive steps catalyzed by CCR and cinnamyl alcohol dehydrogenase (Nimz et al., <xref rid="B50" ref-type="bibr">1975</xref>; Gross, <xref rid="B25" ref-type="bibr">1981</xref>; Lüderitz and Grisebach, <xref rid="B45" ref-type="bibr">1981</xref>; Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>). In addition to serving as intermediates in lignin biosynthesis, hydroxycinnamaldehydes, and monolignols can play a role as defensive compounds and act as precursors for lignan biosynthesis (Keen and Littlefield, <xref rid="B36" ref-type="bibr">1979</xref>; Barber et al., <xref rid="B6" ref-type="bibr">2000</xref>; Davin et al., <xref rid="B18" ref-type="bibr">2008</xref>; König et al., <xref rid="B37" ref-type="bibr">2014</xref>; Satake et al., <xref rid="B59" ref-type="bibr">2015</xref>; Teponno et al., <xref rid="B66" ref-type="bibr">2016</xref>). In rice, the expression of phenylpropanoid pathway genes were induced in response to biotic and abiotic stresses, such as <italic>M. grisea</italic> infection and UV-irradiation, and led to the synthesis of phenolic phytoalexins (Ishihara et al., <xref rid="B31" ref-type="bibr">2008</xref>; Park et al., <xref rid="B53" ref-type="bibr">2013</xref>, <xref rid="B54" ref-type="bibr">2014</xref>; Cho and Lee, <xref rid="B15" ref-type="bibr">2015</xref>). It has been also reported that hydroxycinnamic acid amides were synthesized and deposited in the cell walls of <italic>Bipolaris oryzae</italic> infected tissues, suggesting that these amides were involved in physical defense against the pathogen (Ishihara et al., <xref rid="B31" ref-type="bibr">2008</xref>, <xref rid="B32" ref-type="bibr">2011</xref>).</p><p>CCR is the first enzyme of the monolignol pathway, and catalyzes the conversion of <italic>p</italic>-coumaroyl-, feruloyl-, and sinapoyl-CoAs to <italic>p</italic>-coumaraldehyde, coniferaldehyde, and sinapaldehyde, respectively (Gross, <xref rid="B25" ref-type="bibr">1981</xref>; Davin et al., <xref rid="B18" ref-type="bibr">2008</xref>; Vanholme et al., <xref rid="B68" ref-type="bibr">2010</xref>). Homologs of <italic>CCR</italic> gene families have been reported to be diverse in plant species, including 11 genes in <italic>Arabidopsis thaliana</italic>, nine in <italic>Populus trichocarpa</italic>, 26 in rice (<italic>Oryza sativa</italic>) and 10 in <italic>Eucalyptus grandis</italic> (Costa et al., <xref rid="B17" ref-type="bibr">2003</xref>; Kawasaki et al., <xref rid="B35" ref-type="bibr">2006</xref>; Shi et al., <xref rid="B60" ref-type="bibr">2010</xref>; Carocha et al., <xref rid="B12" ref-type="bibr">2015</xref>). Functional studies of CCRs has been performed in some plant species including <italic>A. thaliana, Eucalytus gunnii</italic>, soybean (<italic>Glycine max</italic>), poplar (<italic>P. euramericana</italic>), maize (<italic>Zea mays</italic>), switchgrass (<italic>Panicum virgatum</italic>), and wheat (<italic>Triticum aestivum</italic>) (Lüderitz and Grisebach, <xref rid="B45" ref-type="bibr">1981</xref>; Sarni et al., <xref rid="B58" ref-type="bibr">1984</xref>; Goffner et al., <xref rid="B23" ref-type="bibr">1994</xref>; Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>; Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Goujon et al., <xref rid="B24" ref-type="bibr">2003</xref>; Ma, <xref rid="B46" ref-type="bibr">2007</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>). Multiple homologs of <italic>CCR</italic> genes have been reported to play different roles in the same plant species (Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Ma, <xref rid="B46" ref-type="bibr">2007</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>). In <italic>A. thaliana, AtCCR1</italic> is involved in developmental lignification, while <italic>AtCCR2</italic> participates in stress and elicitor responses (Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>). Down-regulation of <italic>AtCCR1</italic> has been observed to lead the reduction of lignin contents up to 50% in <italic>A. thaliana</italic> (Goujon et al., <xref rid="B24" ref-type="bibr">2003</xref>). Although several studies have reported defense-related functions of <italic>CCR-like</italic> genes in rice (Kawasaki et al., <xref rid="B35" ref-type="bibr">2006</xref>; Bart et al., <xref rid="B7" ref-type="bibr">2010</xref>), biochemical and physiological roles of rice <italic>CCR</italic> and <italic>CCR-like</italic> genes are largely unknown.</p><p>In the MSU Rice Genome Annotation Project (RGAP) database, we found 33 genes annotated as <italic>CCR</italic> and <italic>CCR-like</italic> genes, collectively called <italic>OsCCR</italic>s. The gene expression profiles of different developmental stages, organs and stress conditions, and the activity of enzyme toward hydroxycinnamoyl-CoA substrates were examined for the functional characterization of <italic>OsCCR</italic>s in rice. An activity assay of recombinant OsCCR proteins revealed that OsCCR17, 19, 20, and 21 were biochemically functional CCRs in rice. Expression and phylogenetic analyses were performed to elucidate the physiological role of <italic>OsCCR</italic>s, and suggested that <italic>OsCCR19</italic> and <italic>20</italic> are primarily involved in developmental lignification, while <italic>OsCCR17</italic> and <italic>21</italic> likely play a role in defense responses.</p></sec><sec sec-type="materials and methods" id="s2"><title>Materials and methods</title><sec><title>Plant growth and materials</title><p>Sterilized seeds of wild-type rice plants (<italic>O. sativa</italic> L. spp. <italic>Japonica</italic> cv. <italic>Dongjin</italic>) were germinated on Murashige and Skoog (MS) medium (Duchefa, Harlem, Netherlands) in a growth chamber with a 12 h photoperiod and temperature of 28°C. Ten-day old seedlings were transferred to soil and grown in a greenhouse at 28°C during the day and 20°C at night. Stem and leaf samples were collected from 10-week-old rice plants, and panicle samples were collected from 14-week-old rice plants. Root and shoot samples were collected from 10-day old rice seedlings.</p><p><italic>p</italic>-Coumaric acid, ferulic acid, sinapic acid, coenzyme-A (CoA) and reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) for hydroxycinnamoyl-CoA production were purchased from Sigma-Aldrich (St. Louis, MO, USA). Reagents for buffers, media and other solutions were obtained from Sigma-Aldrich and Duchefa.</p></sec><sec><title>Multiple sequence alignments and phylogenetic analysis of OsCCRs</title><p>Deduced protein sequences of OsCCRs and functional CCRs identified from other plant species were retrieved from the MSU RGAP database (<ext-link ext-link-type="uri" xlink:href="http://rice.plantbiology.msu.edu/">http://rice.plantbiology.msu.edu/</ext-link>, Kawahara et al., <xref rid="B34" ref-type="bibr">2013</xref>) and the National Center for Biotechnological Information (<ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/">https://www.ncbi.nlm.nih.gov/</ext-link>) database, respectively. Multiple amino acid sequence alignment was performed with Clustal-W (Thompson et al., <xref rid="B67" ref-type="bibr">1994</xref>), and a phylogenetic analysis was conducted with MEGA ver. 6 (Tamura et al., <xref rid="B65" ref-type="bibr">2013</xref>) using the neighbor-joining method.</p></sec><sec><title>Cloning of <italic>OsCCR</italic>s</title><p>Total RNA was isolated from 8-week-old rice leaves with RNAiso (Takara, Shiga, Japan). The first cDNA was synthesized using the total RNA and SuPrimeScript RT premix with an oligo dT primer (GeNet Bio, Daejeon, Korea). Cloning primers for <italic>OsCCR</italic> genes were designed according to the sequences in the MSU RGAP database. The amplification primers and polymerase chain reaction (PCR) conditions are provided in Supplementary Table <xref ref-type="supplementary-material" rid="SM1">1</xref>. PCR was performed using Solg™ Pfu DNA Polymerase (SolGent, Daejeon, Korea). The resulting PCR products were subcloned into the pGEM™-T Easy vector (Promega, Madison, WI, USA) or pJET 1.2 blunt cloning vector (Thermo Scientific, Carlsbad, CA, USA). After sequence confirmation, each <italic>OsCCR</italic> gene was cut out with the appropriate restriction enzymes and inserted into the pET28a(+) vector (Novagen, Madison, WI, USA). The resulting <italic>OsCCR</italic>/pET28a(+) constructs were individually transformed into <italic>E. coli</italic> BL21(DE3) cells for heterologous expression of OsCCRs.</p></sec><sec><title>Expression and purification of recombinant OsCCRs</title><p>The <italic>E. coli</italic> transformants harboring the <italic>OsCCR</italic>/pET28a(+) construct were grown at 37°C until an OD<sub>600</sub> of ~0.6 in LB medium containing kanamycin (25 μg/mL) was achieved. At that point, 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added in the culture for induction. After additional incubation at 18 or 25°C for 16 h, the cells were harvested by centrifugation (5,000 g for 15 min). Cell pellets were resuspended in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, 2 mM KH<sub>2</sub>PO<sub>4</sub>) supplemented with lysozyme (1 mg/mL) and phenylmethylsulfonyl fluoride (1 mM). The resuspended cells were sonicated on ice, and the crude protein extracts were obtained by centrifugation (15,900 g for 20 min, 4°C). The crude protein samples were mixed with Ni-NTA Agarose beads (Qiagen, Hilden, Germany) and incubated at 4°C for 2 h with agitation. The mixtures were packed into a chromatography column and washed three times with a five-column volume of 20 mM imidazole in Tris buffer (50 mM Tris, pH 8.0, 300 mM NaCl). The recombinant OsCCRs were eluted with 50–100 mM imidazole in Tris buffer. The eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).</p></sec><sec><title>Enzymatic synthesis of hydroxycinnamoyl-CoAs</title><p>For the OsCCR activity assay, hydroxycinnamoyl-CoAs were synthesized by the method described by Beuerle and Pichersky (<xref rid="B8" ref-type="bibr">2002</xref>). <italic>Arabidopsis</italic> 4-coumarate:CoA ligase 1 (<italic>At4CL1</italic>) was cloned from <italic>A. thaliana</italic> cDNA, and the resulting gene was inserted into pET28a(+) vector (Supplementary Table <xref ref-type="supplementary-material" rid="SM1">1</xref>) (Stuible and Kombrink, <xref rid="B63" ref-type="bibr">2001</xref>). The recombinant At4CL1 was expressed in <italic>E. coli</italic> and purified with Ni-NTA Agarose beads according to the methods described above. To synthesize the hydroxycinnamoyl-CoA esters, 3.3 mg hydroxycinnamic acid (<italic>p</italic>-coumaric, ferulic, or sinapic acids), 2 mg CoA, and 6.9 mg ATP were dissolved into a total volume of 10 mL of 50 mM Tris-HCl pH 7.5 buffer containing 2.5 mM MgCl<sub>2</sub>. The reaction was initiated by the addition of 0.25 mg purified At4CL1. After a 5 h incubation at room temperature with agitation, 6.9 mg ATP, 2 mg CoA, and 0.25 mg purified At4CL1 enzyme were added to the reaction mixture, and the incubation was continued at room temperature for 12 h. Ammonium acetate (0.4 g) was added to the mixture to halt the reaction. Hydroxycinnamoyl-CoA esters were purified using Sep-Pak® Vac tC<sub>18</sub> cartridge (Waters, Milford, MA, USA) preconditioned with consecutive washes of MeOH, H<sub>2</sub>O, and 4% ammonium acetate solution (five column-volumes each). The reaction mixture was loaded on the preconditioned cartridge, and the column was rinsed with 4% ammonium acetate solution. The hydroxycinnamoyl-CoA esters were eluted with H<sub>2</sub>O. Fractions containing the hydroxycinnamoyl-CoA esters were identified by UV/Vis spectra recorded using a V-550 UV/Vis-spectrophotometer (Jasco, Tokyo, Japan), and the purified products were lyophilized for storage.</p></sec><sec><title>CCR activity assay and determination of kinetic parameters</title><p>OsCCR activity was measured according to the methods of Lüderitz and Grisebach (<xref rid="B45" ref-type="bibr">1981</xref>). The reaction mixture consisted of 0.1 mM NADPH, 30 μM hydroxycinnamoyl-CoA, and 5 μg of purified recombinant OsCCR protein in 100 mM sodium/potassium phosphate buffer (pH 6.25) to a total volume of 500 μL. The enzyme reactions were carried out at 30°C. The reaction was initiated by an addition of recombinant OsCCR protein, and decreases in A<sub>366</sub> were monitored for 10 min by a Cary 300 Bio UV/Vis-spectrophotometer (Varian, Mulgrave, Victoria, Australia). For determination of <italic>K</italic><sub>M</sub> and <italic>V</italic><sub>max</sub>, the substrates were used at concentrations of 5–50 μM. <italic>K</italic><sub>M</sub> and <italic>V</italic><sub>max</sub> were determined by extrapolation from Lineweaver-Burk plots. The enzyme assays were carried out in quadruplicate and the result represented the mean ± standard deviation.</p></sec><sec><title>UV and salt treatment</title><p>Wild-type Dongjin rice plants were grown in a greenhouse for 8 weeks after germination. UV-C treatment of rice plants were performed using the methods described by Park et al. (<xref rid="B53" ref-type="bibr">2013</xref>). UV-treated rice leaves were collected 1, 24, and 48 h after UV treatment.</p><p>To treat salt stress, rice seedlings were hydroponically grown on MS medium (Duchefa), and 10 day-old rice seedlings were treated with 250 mM NaCl. After 1, 3, 6, 12, and 24 h salt treatments, rice seedlings were collected for the analysis of <italic>OsCCR</italic> expression.</p></sec><sec><title>Analysis of <italic>OsCCR</italic> gene expression</title><p>The public transcriptomic analysis data of <italic>OsCCR</italic> genes in various rice developmental stages as well as under biotic [<italic>M. grisea, Xoo</italic>, and <italic>X. oryzae</italic> pv. <italic>oryzicolar</italic> (<italic>Xoc</italic>) infections] and abiotic stresses (drought, salt and cold) were downloaded from the Genevestigator plant biology database (<ext-link ext-link-type="uri" xlink:href="https://genevestigator.com/gv/doc/intro_plant.jsp">https://genevestigator.com/gv/doc/intro_plant.jsp</ext-link>) (Hruz et al., <xref rid="B29" ref-type="bibr">2008</xref>). Microarray data of UV-C treated rice were obtained from the transcriptomic analysis conducted by Park et al. (<xref rid="B53" ref-type="bibr">2013</xref>). The genes that changed more than two-fold, with a <italic>p</italic> < 0.05, were identified as being differentially expressed genes. The normalized data was uploaded and heatmap expression patterns were generated using the Multi Experiment Viewer program (<ext-link ext-link-type="uri" xlink:href="http://mev.tm4.org/#/welcome">http://mev.tm4.org/#/welcome</ext-link>).</p></sec><sec><title>RNA isolation and quantitative real-time PCR analysis</title><p>Total RNA extraction from rice samples and cDNA synthesis were accomplished using the methods described above. Quantitative real-time PCR (qRT-PCR) was performed using a Prime Q-Mastermix (GeNet Bio, Daejeon, Korea) on a Rotor-Gene Q instrument system (Qiagen). For normalization of transcript levels, rice ubiquitin5 (<italic>UBQ5</italic>) gene (Os01g22490) was used as a reference gene, which expresses stably in rice (Jain et al., <xref rid="B33" ref-type="bibr">2006</xref>). The <sub>ΔΔ</sub>Ct method was applied to calculate expression levels (Choi et al., <xref rid="B16" ref-type="bibr">2014</xref>). To ensure primer specificity, we used the data when the melting curve showed a single peak. Primers for qRT-PCR are listed in Supplementary Table <xref ref-type="supplementary-material" rid="SM2">2</xref>. The primer sequences for <italic>OsCCR19, 20, 21</italic>, and <italic>UBQ5</italic> were followed by Koshiba et al. (<xref rid="B38" ref-type="bibr">2013</xref>). To assess the expression of <italic>OsCCR17, 19, 20</italic>, and <italic>21</italic> in different tissues and stress conditions, qRT-PCR analysis was performed on triplicated biological samples, and each sample was analyzed twice for technical replicate. The results represent the mean ± standard deviation. One-way ANOVA and Tukey's HSD <italic>post-hoc</italic> test for qRT-PCR data were performed and significant differences (<italic>p</italic> < 0.05) were represented with the letters a and b. All statistical analysis was carried out using SPSS statistics.</p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><sec><title>The CCR gene family in rice</title><p><italic>CCR</italic>s are a large gene family in plants, and belong to the mammalian 3β-hydroxysteroid dehydrogenase (HSD)/plant dihydroflavonol reductase (DFR) superfamily (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>). In the MSU RGAP Database (Kawahara et al., <xref rid="B34" ref-type="bibr">2013</xref>), 33 genes were annotated as CCRs or CCR-like (CCR/DFR/epimerase 3β-HSD) proteins in the rice genome (Table <xref ref-type="table" rid="T1">1</xref>). The open reading frame (ORF) and peptide lengths of functional CCR genes from <italic>A. thaliana</italic>, maize, wheat, switchgrass and <italic>E. gunnii</italic> are 999–1,125 nucleotides and 332–374 amino acids long, respectively (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>; Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Ma, <xref rid="B46" ref-type="bibr">2007</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>). Of the 33 <italic>OsCCR</italic>s, 24 <italic>OsCCR</italic>s had ORFs of a comparable size (960–1,140 nucleotides) to known functional CCR genes, which encode 319–379 amino acids (Table <xref ref-type="table" rid="T1">1</xref>), indicating that there is no C-terminal extension in OsCCRs. These OsCCRs contained highly homologous sequences to the characteristic NAD(P)-binding and catalytic domains of CCR proteins (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>). This finding agreed well with the rice <italic>CCR</italic> gene family previously identified by a homology search (Kawasaki et al., <xref rid="B35" ref-type="bibr">2006</xref>). Therefore, the naming of <italic>OsCCR</italic>s identified in this study (<italic>OsCCR1</italic>–<italic>8, 10</italic>–<italic>24</italic>, and <italic>26</italic>) followed that of Kawasaki et al. (<xref rid="B35" ref-type="bibr">2006</xref>). The other <italic>OsCCR</italic>s had short ORFs encoding <229 amino acids, and lacked one or both conserved regions (Table <xref ref-type="table" rid="T1">1</xref> and Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>). These short <italic>OsCCR</italic>s were designated as <italic>OsCCR27</italic>-<italic>35</italic> (Table <xref ref-type="table" rid="T1">1</xref>).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Rice <italic>CCR</italic> and <italic>CCR-like</italic> gene family<xref ref-type="table-fn" rid="TN1"><sup>a</sup></xref>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Locus ID</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Name</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Gene description</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>ORF<xref ref-type="table-fn" rid="TN2"><sup>b</sup></xref></bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Protein size (aa)<xref ref-type="table-fn" rid="TN3"><sup>c</sup></xref></bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Theoretical MW<xref ref-type="table-fn" rid="TN4"><sup>d</sup></xref> (kDa)</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">Os01g18110</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR4</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase, putative, expressed</td><td valign="top" align="center" rowspan="1" colspan="1">981</td><td valign="top" align="center" rowspan="1" colspan="1">326</td><td valign="top" align="center" rowspan="1" colspan="1">36.2</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os01g18120</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR5</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase, putative, expressed</td><td valign="top" align="center" rowspan="1" colspan="1">987</td><td valign="top" align="center" rowspan="1" colspan="1">328</td><td valign="top" align="center" rowspan="1" colspan="1">36.5</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os01g45200</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR2</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl-CoA reductase-related, putative, expressed/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,092</td><td valign="top" align="center" rowspan="1" colspan="1">363</td><td valign="top" align="center" rowspan="1" colspan="1">39.5</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os01g61230</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR6</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl-CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">981</td><td valign="top" align="center" rowspan="1" colspan="1">326</td><td valign="top" align="center" rowspan="1" colspan="1">35.8</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os01g74660</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR26</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl-CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">984</td><td valign="top" align="center" rowspan="1" colspan="1">327</td><td valign="top" align="center" rowspan="1" colspan="1">35.2</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os02g08420</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR21</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase, putative, expressed/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,035</td><td valign="top" align="center" rowspan="1" colspan="1">344</td><td valign="top" align="center" rowspan="1" colspan="1">37.9</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os02g56460</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR1</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,017</td><td valign="top" align="center" rowspan="1" colspan="1">338</td><td valign="top" align="center" rowspan="1" colspan="1">37.4</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os02g56680</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR12</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,014</td><td valign="top" align="center" rowspan="1" colspan="1">337</td><td valign="top" align="center" rowspan="1" colspan="1">37.3</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os02g56690</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR13</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,065</td><td valign="top" align="center" rowspan="1" colspan="1">354</td><td valign="top" align="center" rowspan="1" colspan="1">38.5</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os02g56700</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR10</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,020</td><td valign="top" align="center" rowspan="1" colspan="1">339</td><td valign="top" align="center" rowspan="1" colspan="1">37.6</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os02g56720</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR11</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,005</td><td valign="top" align="center" rowspan="1" colspan="1">334</td><td valign="top" align="center" rowspan="1" colspan="1">37</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os03g60279</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR27</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl-CoA reductase family, putative, expressed</td><td valign="top" align="center" rowspan="1" colspan="1">411</td><td valign="top" align="center" rowspan="1" colspan="1">136</td><td valign="top" align="center" rowspan="1" colspan="1">14.5</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os03g60380</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR22</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,005</td><td valign="top" align="center" rowspan="1" colspan="1">334</td><td valign="top" align="center" rowspan="1" colspan="1">35.4</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os05g50250</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR23</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl-CoA reductase-related/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,140</td><td valign="top" align="center" rowspan="1" colspan="1">379</td><td valign="top" align="center" rowspan="1" colspan="1">41.3</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os06g41800</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR28</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">435</td><td valign="top" align="center" rowspan="1" colspan="1">144</td><td valign="top" align="center" rowspan="1" colspan="1">15.9</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os06g41810</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR8</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">966</td><td valign="top" align="center" rowspan="1" colspan="1">321</td><td valign="top" align="center" rowspan="1" colspan="1">35.2</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os06g41840</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR7</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">966</td><td valign="top" align="center" rowspan="1" colspan="1">321</td><td valign="top" align="center" rowspan="1" colspan="1">34.7</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os08g08500</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR29</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">690</td><td valign="top" align="center" rowspan="1" colspan="1">229</td><td valign="top" align="center" rowspan="1" colspan="1">24.9</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os08g17500</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR18</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,029</td><td valign="top" align="center" rowspan="1" colspan="1">342</td><td valign="top" align="center" rowspan="1" colspan="1">34.9</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os08g34280</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR20</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,086</td><td valign="top" align="center" rowspan="1" colspan="1">361</td><td valign="top" align="center" rowspan="1" colspan="1">38.7</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g04050</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR17</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,044</td><td valign="top" align="center" rowspan="1" colspan="1">347</td><td valign="top" align="center" rowspan="1" colspan="1">37.9</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g08720</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR24</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">975</td><td valign="top" align="center" rowspan="1" colspan="1">324</td><td valign="top" align="center" rowspan="1" colspan="1">35.9</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g09230</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR30</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase, putative, expressed</td><td valign="top" align="center" rowspan="1" colspan="1">447</td><td valign="top" align="center" rowspan="1" colspan="1">148</td><td valign="top" align="center" rowspan="1" colspan="1">16.5</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g09270</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR31</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">321</td><td valign="top" align="center" rowspan="1" colspan="1">106</td><td valign="top" align="center" rowspan="1" colspan="1">11.5</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g25150</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR19</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,074</td><td valign="top" align="center" rowspan="1" colspan="1">357</td><td valign="top" align="center" rowspan="1" colspan="1">38.6</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g31490</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR15</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,032</td><td valign="top" align="center" rowspan="1" colspan="1">343</td><td valign="top" align="center" rowspan="1" colspan="1">37.9</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g31498</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR32</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">633</td><td valign="top" align="center" rowspan="1" colspan="1">210</td><td valign="top" align="center" rowspan="1" colspan="1">22.7</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g31502</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR16</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">1,047</td><td valign="top" align="center" rowspan="1" colspan="1">348</td><td valign="top" align="center" rowspan="1" colspan="1">38.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g31506</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR33</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase/epimerase 3β _HSD protein</td><td valign="top" align="center" rowspan="1" colspan="1">663</td><td valign="top" align="center" rowspan="1" colspan="1">220</td><td valign="top" align="center" rowspan="1" colspan="1">24.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g31514</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR14</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase family/dihydroflavonol-4-reductase/epimerase 3β _HSD protein</td><td valign="top" align="center" rowspan="1" colspan="1">1,044</td><td valign="top" align="center" rowspan="1" colspan="1">347</td><td valign="top" align="center" rowspan="1" colspan="1">38.7</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g31518</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR34</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl-CoA reductase, putative</td><td valign="top" align="center" rowspan="1" colspan="1">501</td><td valign="top" align="center" rowspan="1" colspan="1">166</td><td valign="top" align="center" rowspan="1" colspan="1">18.3</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os09g31522</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR35</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl CoA reductase/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">384</td><td valign="top" align="center" rowspan="1" colspan="1">127</td><td valign="top" align="center" rowspan="1" colspan="1">13.7</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Os10g42620</td><td valign="top" align="left" rowspan="1" colspan="1">OsCCR3</td><td valign="top" align="left" rowspan="1" colspan="1">Cinnamoyl-CoA reductase-related, putative, expressed/dihydroflavonol-4-reductase</td><td valign="top" align="center" rowspan="1" colspan="1">960</td><td valign="top" align="center" rowspan="1" colspan="1">319</td><td valign="top" align="center" rowspan="1" colspan="1">35.4</td></tr></tbody></table><table-wrap-foot><fn id="TN1"><label>a</label><p><italic>Rice genes annotated as CCRs and CCR-like (CCR/DFR/epimerase 3β-HSD) proteins were retrieved from the MSU RGAP database</italic>.</p></fn><fn id="TN2"><label>b</label><p>ORF, Open reading frame;</p></fn><fn id="TN3"><label>c</label><p>aa, Amino acid;</p></fn><fn id="TN4"><label>d</label><p><italic>MW, Molecular weight</italic>.</p></fn></table-wrap-foot></table-wrap><p><italic>OsCCR</italic>s were distributed across rice chromosomes 1, 2, 3, 5, 6, 8, 9, and 10 (Table <xref ref-type="table" rid="T1">1</xref>). Chromosome 9 included 12 <italic>OsCCR</italic>s, and chromosomes 1 and 2 contained five and six <italic>OsCCR</italic>s, respectively. Chromosomes 3, 5, 6, 8, and 10 contained 1–3 <italic>OsCCR</italic>s. These genes were composed of one to six exons (Figure <xref ref-type="fig" rid="F1">1</xref>). Based on the number of exons and exon-intron structures, Barakat et al. (<xref rid="B5" ref-type="bibr">2011</xref>) grouped <italic>P. trichocarpa CCR</italic> and <italic>CCR-like</italic> genes into three exon-intron patterns (Patterns 1–3). Most previously studied functional <italic>CCR</italic>s, such as <italic>AtCCR1, EuCCR</italic> (<italic>E. gunnii CCR</italic>), <italic>ZmCCR1</italic> (<italic>Z. mays CCR1</italic>), and <italic>SbCCR1</italic> (<italic>Sorghum bicolor CCR1</italic>), are composed of five exons, as in the exon-intron structure Pattern 2 (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>). Of the rice genes studied in this study, <italic>OsCCR1, 4, 5, 12, 19, 20</italic>, and <italic>24</italic> had five exons, with a Pattern 2 exon-intron structure, and <italic>OsCCR6, 7, 8, 14, 15, 16</italic>, and <italic>22</italic> had six exons as in Pattern 3. Although <italic>OsCCR21</italic> had six exons, its exon-intron structure was more similar to that of Pattern 2; we term this Pattern 2-like (Figure <xref ref-type="fig" rid="F1">1</xref>). The exon-intron structures of <italic>OsCCR2</italic> and <italic>23</italic> were similar to Pattern 3. <italic>OsCCR3, 10, 11</italic>, and <italic>13</italic> consisted of four exons, although they had a different exon-intron pattern than Pattern 1. We designated this group as Pattern 4 (Figure <xref ref-type="fig" rid="F1">1</xref>). <italic>OsCCR17</italic> and <italic>18</italic> had exceptionally long exons, with the last exons consisting of 540 and 704 nucleotides, respectively, and were grouped into Pattern 5 (Figure <xref ref-type="fig" rid="F1">1</xref>). <italic>OsCCR26</italic> was composed of one exon encoding a polypeptide of 327 amino acids, which was designated Pattern 6. Although an activity assay was not performed, <italic>IiCCR</italic> was identified from <italic>Isatis indigotica</italic> and its genomic sequence was found to have no intron (Hu et al., <xref rid="B30" ref-type="bibr">2011</xref>). The other <italic>OsCCR</italic>s were composed of two to five exons with much shorter ORF lengths than those of functional <italic>CCR</italic> genes (Figure <xref ref-type="fig" rid="F1">1</xref>).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Exon-intron structures of <italic>OsCCR</italic>s and other plant <italic>CCR</italic> genes. <italic>OsCCR</italic>s and other plant <italic>CCR</italic>s were divided into nine patterns based on their exon-intron structures. Exons and introns are indicated by boxes and lines, respectively. Numbers in the boxes represent the exon sizes. The intron sizes are not to scale. The pattern names of exon-intron structures are indicated in on the right side of figure. <italic>P. trichocarpa CCR</italic> (<italic>PoptrCCR</italic>), <italic>A. thaliana CCR</italic> (<italic>AtCCR</italic>), <italic>Z. mays CCR</italic> (<italic>ZmCCR</italic>), <italic>I. indigotica CCR</italic> (<italic>IiCCR</italic>), <italic>S. bicolor CCR</italic> (<italic>SbCCR</italic>).</p></caption><graphic xlink:href="fpls-08-02099-g0001"></graphic></fig></sec><sec><title>Sequence homology and phylogenetic analysis of OsCCRs</title><p>Multiple aignments of CCR protein sequences revealed that OsCCR1–25 had about 30–90% similarity to functional CCRs from other plant species (Supplementary Table <xref ref-type="supplementary-material" rid="SM3">3</xref>). In particular, OsCCR20, 21, 19, and 17 were highly homologous (62–92% similarity) with known CCRs. The short length OsCCR27–35 had a low sequence homology at <39% similarity to other plant CCRs (Supplementary Table <xref ref-type="supplementary-material" rid="SM3">3</xref>). A phylogenetic analysis showed that OsCCR19, 20, 17, 18, and 21 were grouped with known plant CCRs (Figure <xref ref-type="fig" rid="F2">2</xref> and Supplementary Figure <xref ref-type="supplementary-material" rid="SM7">2</xref>). In particular, OsCCR20 and 19 were closely related to PvCCR1 (<italic>P. virgatum</italic> CCR1), SbCCR1, ZmCCR1, LpCCR (<italic>Lolium perenne</italic> CCR), HvCCR (<italic>Hordeum vulgare</italic> CCR) and TaCCR1 (<italic>T. aestivum</italic> CCR1). These CCRs have been suggested as monocot functional CCRs involved in developmental lignification (Figure <xref ref-type="fig" rid="F2">2</xref> and Supplementary Figure <xref ref-type="supplementary-material" rid="SM7">2</xref>) (Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>; Larsen, <xref rid="B40" ref-type="bibr">2004a</xref>,<xref rid="B41" ref-type="bibr">b</xref>; Ma, <xref rid="B46" ref-type="bibr">2007</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>; Li et al., <xref rid="B43" ref-type="bibr">2016</xref>). OsCCR17, 18, and 21 were grouped with ZmCCR2, SbCCR2, TaCCR2, and PvCCR2, suggesting that they play a role in defense-related processes under biotic and abiotic stresses (Figure <xref ref-type="fig" rid="F2">2</xref> and Supplementary Figure <xref ref-type="supplementary-material" rid="SM7">2</xref>) (Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Li et al., <xref rid="B43" ref-type="bibr">2016</xref>).</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Phylogenetic analysis of OsCCRs belonging to the clade 1 of Class I (Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>) and 17 characterized CCRs from other plant species. The neighbor-joining tree was built using MEGA6. The gray shade indicates dicot CCRs, and other shades indicate monocot CCRs. Blue and orange shades indicate the functional CCRs involved in developmental lignification and defense-related processes, respectively. <italic>A. thaliana</italic> CCRs (AtCCR1, AAG46037; AtCCR2, AAG53687); <italic>H. vulgare</italic> CCR (HvCCR, AAN71760); <italic>L. esculentum</italic> CCRs (LeCCR1, AAY41879.1; LeCCR2, AAT41880.1); <italic>L. perenne</italic> CCR (LpCCR, AAG09817.1); <italic>P. trichocarpa</italic> CCR12 (PoptrCCR12, CAA12276.1); <italic>P. virgatum</italic> CCRs (PvCCR1a, GQ450297; PvCCR1e, GQ450301; PvCCR2a, GQ450302); <italic>S. bicolor</italic> CCRs (SbCCR1, XP002445566.1; SbCCR2-1, EER98579.1; SbCCR2-2, EES04640.1); <italic>T. aestivum</italic> CCRs (TaCCR1, ABE01883; TaCCR2, AY771357); <italic>Z. mays</italic> CCRs (ZmCCR1, CAA74071; ZmCCR2, NP_001005715).</p></caption><graphic xlink:href="fpls-08-02099-g0002"></graphic></fig><p>The most striking homology between the predicted peptide sequences of OsCCRs and functional CCRs was found in regions covered by two highly conserved motifs. These were the NAD(P)-binding motif at the N-terminus, and the catalytic motif for CCR activity (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>) (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>; Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). The former is a well-conserved motif for cofactor binding in the mammalian 3β-HSD/plant DFR protein superfamily (Baker et al., <xref rid="B3" ref-type="bibr">1990</xref>; Baker and Blasco, <xref rid="B2" ref-type="bibr">1992</xref>; Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). The latter is a CCR signature motif (NWYCYGK), in which the NWYCY sequence may be crucial for CCR activity (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>; Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). Barakat et al. (<xref rid="B5" ref-type="bibr">2011</xref>) applied maximum likelihood phylogenetic analysis to CCRs and CCR-like proteins from several land plant species, including <italic>A. thaliana</italic>, rice, <italic>P. trichocarpa</italic>, sorghum, grape (<italic>Vitis vinifera</italic>) and alfalfa (<italic>Medicago truncatula</italic>), and divided these proteins into three classes (Classes I–III). Class I was subdivided into three clades (clades 1–3), with the clade 1 containing functional CCRs. Most rice CCRs belonged to Class I, with OsCCR1, 4, 5, 10, 11, 12, 17, 18, 19, 20, 21, and 24 falling into the clade 1 of Class I (Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>). Among the OsCCRs belonging to the clade 1 of Class I, OsCCR4, 5, 17, 18, 19, 20, and 21 contained both conserved motifs (Figure <xref ref-type="fig" rid="F3">3</xref>). The NWYCYGK motif was fully conserved in OsCCR19 and 20 (Figure <xref ref-type="fig" rid="F3">3</xref>). One amino acid variation was found in OsCCR4, 5, 21, 17, and 18, a substitution of the similar amino acid A for G in the NWYCYGK sequence. This variation also occurred in PvCCR2a, SbCCR2-1, SbCCR2-2, and ZmCCR2 (Figure <xref ref-type="fig" rid="F3">3</xref>). All other OsCCRs besides the Class I-clade 1 OsCCRs had neither one nor both conserved motifs (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>). A phylogenetic analysis also showed that OsCCR19, 20, 17, 18, and 21 were grouped with functional plant CCRs (Figure <xref ref-type="fig" rid="F2">2</xref>). OsCCR1, 4, 5, 10, 11, 12, and 24 were clearly separated from functional CCRs (Figure <xref ref-type="fig" rid="F2">2</xref>). Overall, these results suggest that OsCCR4, 5, 17, 18, 19, 20, and 21 were likely candidates for functional CCRs in rice, with OsCCR17, 18, 19, 20, and 21 being the most plausible candidates.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Multiple alignments of the NAD(P)-binding, NADP specificity and CCR catalytic motifs of OsCCRs belonged to the clade 1 of Class I (Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>) with functional CCRs from other plant species. Amino acid sequences were aligned using Clustal-W. Shaded amino acids denote identical or similar amino acids. NAD(P)-binding and NADP specificity motifs are boxed in blue and green, respectively. The catalytic signature motif of CCRs are boxed in red. Consensus amino acid sequences are displayed below the boxes. <italic>A. thaliana</italic> CCRs (AtCCR1, AAG46037; AtCCR2, AAG53687); <italic>H. vulgare</italic> CCR (HvCCR, AAN71760); <italic>L. perenne</italic> CCR (LpCCR, AAG09817.1); <italic>P. virgatum</italic> CCRs (PvCCR1a, GQ450297; PvCCR2a, GQ450302); <italic>T. aestivum</italic> CCRs (TaCCR1, ABE01883; TaCCR2, AY771357); <italic>Z. mays</italic> CCRs (ZmCCR1, CAA74071; ZmCCR2, NP_001005715).</p></caption><graphic xlink:href="fpls-08-02099-g0003"></graphic></fig></sec><sec><title>Cloning and heterologous expression of <italic>OsCCR</italic>s</title><p>To elucidate biochemical functions of OsCCRs, we attempted to clone the likely functional candidates (<italic>OsCCR</italic>4<italic>, 5, 17, 18, 19, 20</italic>, and <italic>21</italic>) from wild type rice plants. The cDNAs of <italic>OsCCR5, 17, 19, 20</italic>, and <italic>21</italic> were successfully cloned from rice leaves. Despite many attempts, the cDNAs of <italic>OsCCR4</italic> and <italic>18</italic> could not be cloned from rice, which was likely a result of very low expression levels of these genes throughout all developmental stages (Supplementary Figure <xref ref-type="supplementary-material" rid="SM8">3</xref>). <italic>OsCCR1</italic> and <italic>26</italic> were also cloned to examine their CCR activity. Heterologous expressions of the His-tagged OsCCR proteins were attempted under various growth temperatures and IPTG concentrations. OsCCR1, 5, 19, 20, 21, and 26 were successfully expressed as soluble protein in <italic>E. coli</italic> by 0.1 mM IPTG at an induction temperature of 25°C. Only limited amounts of OsCCR17 soluble proteins were expressed at 18 and 25°C, with most expressed proteins being in an insoluble form. Recombinant OsCCRs were purified with Ni<sup>2+</sup> affinity chromatography to apparent homogeneity (Figure <xref ref-type="fig" rid="F4">4</xref>). The purified OsCCR proteins exhibited molecular masses of 40.5–46.4 kDa on SDS-PAGE, which agreed well with their theoretical molecular masses (Figure <xref ref-type="fig" rid="F4">4</xref> and Table <xref ref-type="table" rid="T1">1</xref>).</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Purification of recombinant OsCCRs expressed in <italic>E.coli</italic>. The His-tagged OsCCR1, 5, 19, 20, 21, and 26 were expressed in <italic>E. coli</italic> as a soluble form. The recombinant proteins were purified by Ni<sup>2+</sup>-affinity chromatography. M, Molecular weight marker; 1, OsCCR1; 2, OsCCR5, 3, OsCCR19; 4, OsCCR20; 5, OsCCR21; 6, OsCCR26.</p></caption><graphic xlink:href="fpls-08-02099-g0004"></graphic></fig></sec><sec><title>CCR activity and kinetic parameters of the recombinant OsCCRs</title><p>To investigate the enzymatic properties of OsCCRs, the activities of recombinant OsCCRs were assayed using <italic>p</italic>-coumaroyl-, feruloyl-, and sinapoyl-CoAs, precursors for the H-, G-, and S-units of lignin, respectively. OsCCR17, 19, 20, and 21 showed the reductase activity to the examined substrates (Supplementary Table <xref ref-type="supplementary-material" rid="SM4">4</xref>). In these OsCCRs, the NAD(P)-binding and catalytic motifs were fully conserved (Figure <xref ref-type="fig" rid="F3">3</xref>). OsCCR1, 5, and 26 showed no detectable activity toward the hydroxycinnamoyl-CoA substrate (Supplementary Table <xref ref-type="supplementary-material" rid="SM4">4</xref>). In OsCCR1 and OsCCR26, the signature NWYCY motif essential for CCR activity was replaced by NLYCC and KWYPV, respectively (Figure <xref ref-type="fig" rid="F3">3</xref>). Although OsCCR5 contained a fully conserved catalytic motif, it had no detectable activity toward the hydroxycinnamoyl-CoA substrate. This was likely caused by a polymorphism in the corresponding residue of H208, which is important in substrate binding as identified by a functional analysis of PtoCCRs (<italic>P. tomentosa</italic> CCRs) (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>) (Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). The polymorphism of H208 to A, R, M, V, K, L, M, and P residues are found in OsCCRs. This likely occurred by various duplication and retention events in CCR gene family during the evolution (Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>). In OsCCR5, H208 was replaced by an R residue (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>). Of the likely functional OsCCRs, OsCCR4, and 18 had well-conserved NWYCY motif (Figure <xref ref-type="fig" rid="F3">3</xref>). OsCCR4, however, featured an H208R replacement similar to that observed in OsCCR5 (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>). OsCCR18 included one amino acid insertion (RNPDDAAK) in the NADP specificity motif [R(X)<sub>5</sub>K]. In functional CCRs, this motif includes five amino acids between R and K residues and forms a short loop. The R and K residues form salt bridges with the phosphate in NADPH. This motif is important in distinguishing CCR from NAD(H)-dependent short-chain dehydrogenase/reductases (SDRs) (Figure <xref ref-type="fig" rid="F3">3</xref>) (Pan et al., <xref rid="B52" ref-type="bibr">2014</xref>; Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). Chao et al. (<xref rid="B14" ref-type="bibr">2017</xref>) suggested that mutations of this motif cause the loss of enzyme activity of PtoCCR8. Therefore, we speculate that neither OsCCR4 nor 18 have any CCR activity.</p><p>To elucidate the enzymatic properties of OsCCRs, the kinetic parameters of the recombinant OsCCR19, 20, and 21 catalyzed reactions were determined toward the hydroxycinnamoyl-CoA substrate (Table <xref ref-type="table" rid="T2">2</xref>). Although OsCCR17 displayed enzyme activity, the amount of purified proteins from the <italic>E. coli</italic> culture was too small for kinetic analysis. The <italic>K</italic><sub>M</sub>-values of OsCCR20 for <italic>p</italic>-coumaroyl-, feruloyl-, and sinapoyl-CoA were 24.08, 15.71, and 23.34 μM, respectively (Table <xref ref-type="table" rid="T2">2</xref>). The <italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>M</sub>-values of OsCCR20 for feruloyl-CoA (1.41 μM<sup>−1</sup> min<sup>−1</sup>) was about five-fold higher than those for <italic>p</italic>-coumaroyl- and sinapoyl-CoAs (0.32 and 0.24 μM<sup>−1</sup> min<sup>−1</sup>, respectively), indicating that it has a greater catalytic efficiency toward feruloyl-CoA than toward the other substrates (Table <xref ref-type="table" rid="T2">2</xref>). The <italic>K</italic><sub>M</sub>-values of OsCCR21 for <italic>p</italic>-coumaroyl-, feruloyl- and sinapoyl-CoA were 16.36, 2.70, and 10.20 μM, respectively, indicating that OsCCR21 has a higher substrate affinity toward feruloyl-CoA than the other substrates (Table <xref ref-type="table" rid="T2">2</xref>). The <italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>M</sub>-values also revealed a greater catalytic efficiency of OsCCR21 toward feruloyl-CoA (0.77 μM<sup>−1</sup> min<sup>−1</sup>) than toward <italic>p</italic>-coumaroyl- or sinapoyl-CoAs (0.08 and 0.07 μM<sup>−1</sup> min<sup>−1</sup>, respectively). This result indicates that among three hydroxycinnamoyl-CoA substrates, both OsCCR20 and 21 have substrate preferences for feruloyl-CoA. The substrate preferences of both OsCCR20 and 21, with the strongest preference being toward feruloyl-CoA, is consistent with the lignin composition of rice. Gui et al. (<xref rid="B27" ref-type="bibr">2011</xref>) reported that rice lignin is composed of 70, 20, and 10% of G-, S-, and H-units, respectively. We also analyzed the lignin contents in stems of the Dongjin rice cultivar used in this study and found that the lignin composition was 62, 35, and 3% of G-, S-, and H-units, respectively (Supplementary Table <xref ref-type="supplementary-material" rid="SM10">5</xref>). The <italic>K</italic><sub>M</sub>-values of OsCCR19 for <italic>p</italic>-coumaroyl-, feruloyl-, and sinapoyl-CoA were 36.66, 26.85, and 62.54 μM, respectively (Table <xref ref-type="table" rid="T2">2</xref>). OsCCR19 showed similar catalytic efficiency toward <italic>p</italic>-coumaroyl-, feruloyl-, and sinapoyl-CoAs with the <italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>M</sub>-values of 0.60, 0.43, and 0.55 μM<sup>−1</sup> min<sup>−1</sup>, respectively (Table <xref ref-type="table" rid="T2">2</xref>).</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Kinetic parameters of recombinant OsCCR19, 20 and 21 catalyzed reaction with hydroxycinnamoyl-CoAs<xref ref-type="table-fn" rid="TN5"><sup>a</sup></xref>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>OsCCR</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Substrate</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold><italic>K</italic><sub>M</sub> (μM)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold><italic>V</italic><sub>max</sub> (μmol min<sup>−1</sup>mg<sup>−1</sup>)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold><italic>k</italic><sub>cat</sub> (min<sup>−1</sup>)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold><italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>M</sub> (μM<sup>−1</sup> min<sup>−1</sup>)</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">OsCCR19</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>p</italic>-Coumaroyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">36.66 ± 3.06</td><td valign="top" align="center" rowspan="1" colspan="1">0.51 ± 0.11</td><td valign="top" align="center" rowspan="1" colspan="1">21.17</td><td valign="top" align="center" rowspan="1" colspan="1">0.60</td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Feruloyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">26.85 ± 6.68</td><td valign="top" align="center" rowspan="1" colspan="1">0.28 ± 0.12</td><td valign="top" align="center" rowspan="1" colspan="1">11.48</td><td valign="top" align="center" rowspan="1" colspan="1">0.43</td></tr><tr style="border-bottom: thin solid #000000;"><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Sinapoyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">62.54 ± 14.08</td><td valign="top" align="center" rowspan="1" colspan="1">0.83 ± 0.04</td><td valign="top" align="center" rowspan="1" colspan="1">34.19</td><td valign="top" align="center" rowspan="1" colspan="1">0.55</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">OsCCR20</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>p</italic>-Coumaroyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">24.08 ± 3.05</td><td valign="top" align="center" rowspan="1" colspan="1">0.17 ± 0.02</td><td valign="top" align="center" rowspan="1" colspan="1">7.72</td><td valign="top" align="center" rowspan="1" colspan="1">0.32</td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Feruloyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">15.71 ± 1.55</td><td valign="top" align="center" rowspan="1" colspan="1">0.58 ± 0.12</td><td valign="top" align="center" rowspan="1" colspan="1">22.61</td><td valign="top" align="center" rowspan="1" colspan="1">1.41</td></tr><tr style="border-bottom: thin solid #000000;"><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Sinapoyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">23.34 ± 2.60</td><td valign="top" align="center" rowspan="1" colspan="1">0.13 ± 0.01</td><td valign="top" align="center" rowspan="1" colspan="1">5.64</td><td valign="top" align="center" rowspan="1" colspan="1">0.24</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">OsCCR21</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>p</italic>-Coumaroyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">16.36 ± 4.69</td><td valign="top" align="center" rowspan="1" colspan="1">0.02 ± 0.003</td><td valign="top" align="center" rowspan="1" colspan="1">1.12</td><td valign="top" align="center" rowspan="1" colspan="1">0.08</td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Feruloyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">2.70 ± 1.69</td><td valign="top" align="center" rowspan="1" colspan="1">0.03 ± 0.003</td><td valign="top" align="center" rowspan="1" colspan="1">1.59</td><td valign="top" align="center" rowspan="1" colspan="1">0.77</td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Sinapoyl-CoA</td><td valign="top" align="center" rowspan="1" colspan="1">10.20 ± 1.84</td><td valign="top" align="center" rowspan="1" colspan="1">0.02 ± 0.004</td><td valign="top" align="center" rowspan="1" colspan="1">0.72</td><td valign="top" align="center" rowspan="1" colspan="1">0.07</td></tr></tbody></table><table-wrap-foot><fn id="TN5"><label>a</label><p><italic>The results represent the mean ± standard deviation of four independent experiments</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title><italic>In silico</italic> and qRT-PCR analyses of <italic>OsCCR</italic> gene expression</title><p>Expression of <italic>OsCCR</italic>s were investigated with the microarray data obtained from the Genevestigator database. Some <italic>OsCCR</italic>s (<italic>OsCCR3, 6, 7, 8, 16, 19, 20, 21, 22</italic>, and <italic>26</italic>) displayed a high level of expression throughout all developmental stages including germination, seedling, tillering, stem elongation, booting, heading, flowering, milk, and dough stages (Supplementary Figure <xref ref-type="supplementary-material" rid="SM8">3</xref>). Of these constitutively expressed <italic>OsCCR</italic>s, <italic>OsCCR19, 20</italic>, and <italic>21</italic> were found to encode biochemically active CCRs (Table <xref ref-type="table" rid="T2">2</xref> and Supplementary Table <xref ref-type="supplementary-material" rid="SM4">4</xref>), suggesting that these genes likely play a physiological role in rice. The <italic>OsCCR17</italic> gene encoding enzymatically active CCR was expressed only during the early growth stages (Supplementary Figure <xref ref-type="supplementary-material" rid="SM8">3</xref>). Expression of <italic>OsCCR17, 19, 20</italic>, and <italic>21</italic> in different developmental stages and tissues of rice were also examined by qRT-PCR analysis. Similar with the microarray data, <italic>OsCCR20</italic> and <italic>21</italic> were expressed in all examined stages and tissues, and <italic>OsCCR17</italic> was expressed primarily in rice seedling shoots and roots (Figure <xref ref-type="fig" rid="F5">5</xref>). Although both <italic>OsCCR20</italic> and <italic>21</italic> were constitutively expressed in the examined rice tissues, the <italic>OsCCR20</italic> expressions were much higher in actively lignifying organs, such as roots and stems than those of <italic>OsCCR21</italic>. qRT-PCR analysis showed that expression of <italic>OsCCR19</italic> was very low in most examined rice tissues (Figure <xref ref-type="fig" rid="F5">5</xref>). The phylogenetic analysis revealed that OsCCR19 and 20 was closely related to functional CCRs, including ZmCCR1, PvCCR1, LpCCR, and HvCCR. These CCRs may participate in developmental lignin deposition in secondary cell walls (Larsen, <xref rid="B40" ref-type="bibr">2004a</xref>,<xref rid="B41" ref-type="bibr">b</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>). This evidence suggests that <italic>OsCCR</italic>20 acts as a functional rice CCR and involved in developmental lignification.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p>Quantitative real-time PCR analysis of <italic>OsCCR17, 19, 20</italic>, and <italic>OsCCR21</italic> gene expression in rice seedlings and different organs. Root and shoot samples were collected from 10-day old rice seedlings. Ten-week old rice plants yielded leaf, leaf sheath and stem samples. Panicles were obtained from 14-week old rice plants. An ubiquitin gene (<italic>OsUBQ5</italic>) was amplified using specific primers and used as an internal control. Expression levels of each <italic>OsCCR</italic> gene are presented as the relative expression compared to the <italic>OsUBQ5</italic> mRNA level. qRT-PCR analysis was performed on the triplicated biological samples. The results represent the mean ± standard deviation.</p></caption><graphic xlink:href="fpls-08-02099-g0005"></graphic></fig><p>Expression profiles of <italic>OsCCR</italic>s were also altered by biotic and abiotic stresses. Transcriptomic analysis showed that expression of <italic>OsCCR</italic>s was significantly induced during abiotic stress conditions, such as exposure to cold (<italic>OsCCR1, 2, 6, 21, 23</italic>, and <italic>33</italic>), drought (<italic>OsCCR3, 7</italic>, and <italic>15</italic>), and high salinity (<italic>OsCCR3, 7, 17</italic>, and <italic>18</italic>) (Supplementary Figure <xref ref-type="supplementary-material" rid="SM9">4A</xref>). Under abiotic stress conditions, the biochemically functional genes <italic>OsCCR21</italic> and <italic>17</italic> were induced by cold and salt stresses, respectively. In our previous microarray data of UV-treated rice leaves (Park et al., <xref rid="B53" ref-type="bibr">2013</xref>), <italic>OsCCR1, 3, 17, 20, 21</italic>, and <italic>23</italic> were found to be up-regulated in response to UV-irradiation (Supplementary Figure <xref ref-type="supplementary-material" rid="SM9">4B</xref>). <italic>In silico</italic> analysis of public microarray data also showed that the expression of several <italic>OsCCR</italic>s was up-regulated by biotic stresses, such as <italic>M. grisea, Xoo</italic>, and <italic>Xoc</italic> infections. The expression of <italic>OsCCR1, 2, 3, 5, 17, 18, 20</italic>, and <italic>21</italic> were induced by <italic>M. grisea</italic> infection. The expression of functional <italic>OsCCR17, 20</italic>, and <italic>21</italic> were induced by both UV-irradiation and <italic>M. grisea</italic> infection. Infection with <italic>Xoo</italic> stimulated the expression of <italic>OsCCR20</italic> and <italic>21</italic>, and the <italic>Xoc</italic> infection induced <italic>OsCCR21</italic> expression (Supplementary Figure <xref ref-type="supplementary-material" rid="SM10">5</xref>). Among these stress-inducible <italic>OsCCR</italic>s, expression of <italic>OsCCR17</italic> and <italic>21</italic> was frequently observed to be stimulated by multiple abiotic stresses. To confirm the stress-inducible expression of <italic>OsCCR</italic>s, qRT-PCR analysis was performed with UV-treated rice leaves and salt-treated rice seedlings (Figure <xref ref-type="fig" rid="F6">6</xref>). The expression level of <italic>OsCCR17</italic> and <italic>21</italic> in UV-treated rice leaves increased about 70- and 10-fold compared to those of the non-treated control an hour after UV treatment, respectively (Figure <xref ref-type="fig" rid="F6">6A</xref>). The expressions of <italic>OsCCR17</italic> and <italic>21</italic> were also significantly increased by salt treatment compared to a control, which received a mock treatment (Figure <xref ref-type="fig" rid="F6">6B</xref>). The qRT-PCR analysis showed that the transcript levels of <italic>OsCCR19</italic> and <italic>20</italic> were not significantly changed by both stress conditions (Figure <xref ref-type="fig" rid="F6">6</xref>). These results suggest that <italic>OsCCR17</italic> and <italic>21</italic> are most likely involved in the stress responses of rice.</p><fig id="F6" position="float"><label>Figure 6</label><caption><p>Quantitative real-time PCR analysis of <italic>OsCCR17, 19, 20</italic> and <italic>OsCCR21</italic> gene expression in response to UV-irradiation and salt treatment. Rice plants treated with UV <bold>(A)</bold> and salt <bold>(B)</bold> were collected at designated time points and used to examine the expression of <italic>OsCCR17, 19, 20</italic>, and <italic>OsCCR21</italic>. An ubiquitin gene (<italic>OsUBQ5</italic>) was amplified using specific primers and used as an internal control. Expression level of each <italic>OsCCR</italic> gene was presented as the relative expression compared to the <italic>OsUBQ5</italic> mRNA level. qRT-PCR analysis was performed on the triplicated biological samples. One-way ANOVA and Tukey's HSD <italic>post-hoc</italic> test for qRT-PCR data were performed and significant differences (<italic>p</italic> < 0.05) were represented with the letters a and b. The results represent the mean ± standard deviation.</p></caption><graphic xlink:href="fpls-08-02099-g0006"></graphic></fig></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p>Although plant CCRs comprise a large gene family, only a small number of CCR genes have been reported to encode biochemically active CCRs for the biosynthesis of lignin and defense-related phenolic compounds (Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Costa et al., <xref rid="B17" ref-type="bibr">2003</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>). Xu et al. (<xref rid="B70" ref-type="bibr">2009</xref>) suggested that the expansion of lignin biosynthetic gene families was rapidly occurred after divergence of monocots and dicots at 120 million years ago. The large gene family of <italic>CCR</italic>s in plants was suggested to occur by various duplication and retention events during the evolution and indeed, 67% of rice <italic>CCR</italic> and <italic>CCR-like</italic> genes were located on duplicated chromosome regions (Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>). A large member of CCR gene family in plants has also been supposed to because of their substrate diversity (Xu et al., <xref rid="B70" ref-type="bibr">2009</xref>). All previously characterized CCRs showed similar peptide lengths ranging from 332 to 374 amino acids in <italic>A. thaliana</italic>, wheat, sorghum, switchgrass, and <italic>E. gunnii</italic> (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Ma, <xref rid="B46" ref-type="bibr">2007</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Li et al., <xref rid="B43" ref-type="bibr">2016</xref>). Twenty-four OsCCRs had peptide lengths similar to known CCRs (Table <xref ref-type="table" rid="T1">1</xref>). These OsCCRs showed high homology to well-conserved NAD(P)-binding and catalytic motifs of functional CCRs (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref> and Table <xref ref-type="table" rid="T1">1</xref>). As a member of the mammalian 3β-HSD/plant DFR superfamily, CCRs share the NAD(P)-binding domain with DFRs. CCRs, however, have the distinct catalytic motifs with signature NWYCYGK sequence different from DFRs (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>; Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). Of OsCCRs with appropriate peptide lengths, OsCCR19 and 20 exhibited the fully conserved catalytic motif, and OsCCR4, 5, 17, 18, and 21 had the signature motif with one amino acid variation (G to A) (Figure <xref ref-type="fig" rid="F3">3</xref>). The G to A variation in the CCR catalytic motifs has been frequently found in other active CCRs, such as PvCCR2a and ZmCCR2 (Figure <xref ref-type="fig" rid="F3">3</xref>) (Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Li et al., <xref rid="B43" ref-type="bibr">2016</xref>). Indeed, our biochemical assays confirmed that OsCCR17, 19, 20 and 21 had CCR activity to hydroxycinnamoyl-CoAs (Table <xref ref-type="table" rid="T2">2</xref> and Supplementary Table <xref ref-type="supplementary-material" rid="SM4">4</xref>). OsCCR1 and 26, with two and four mismatches in this motif, respectively, had no CCR activity (Supplementary Table <xref ref-type="supplementary-material" rid="SM4">4</xref>). This evidence suggests that the NWYCY(G/A)K sequence is crucial for CCR activity. An activity assay also revealed that OsCCR5 had no CCR activity, although it contained the signature catalytic motif. A recent study demonstrated that H208 in PtoCCRs is indispensable for substrate binding, and is conserved in the functional CCRs from other plant species (Supplementary Figure <xref ref-type="supplementary-material" rid="SM6">1</xref>) (Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). In OsCCR5, H208 was replaced by R, which likely caused the loss of its CCR activity. Like OsCCR5, OsCCR4 had the H208R replacement. In addition to the NAD(P)-binding motif, the NADP-specific R(X)<sub>5</sub>K motif was identified by structural analysis of <italic>M. truncatula</italic> CCR2 and petunia CCR1. This NADP-specificity motif is a key structure distinguishing CCRs from NAD(H)-dependent SDRs (Pan et al., <xref rid="B52" ref-type="bibr">2014</xref>). This motif was well-conserved in the active OsCCRs (Figure <xref ref-type="fig" rid="F3">3</xref>). OsCCR18 showed one amino acid insertion in the NADP specificity motif (Figure <xref ref-type="fig" rid="F3">3</xref>). Although no activity assay was performed, for these reasons, we speculate that OsCCR4 and 18 had no CCR activity. Altogether, this evidence suggested that of the 33 OsCCRs studied here, OsCCR17, 19, 20, and 21 may encode biochemically functional CCRs in rice. In addition, a previous study reported that the enzyme activity of OsCCR1 is activated by the small GTPase <italic>OsRac1</italic> that controls defense-related lignin synthesis (Kawasaki et al., <xref rid="B35" ref-type="bibr">2006</xref>).</p><p>Plant <italic>CCR</italic> and <italic>CCR-like</italic> genes are composed of different numbers of exons and exon-intron structures. The <italic>A. thaliana CCR</italic> gene family has been suggested to have seven patterns of exon-intron structures (Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>). The <italic>OsCCR</italic>s examined in this study also exhibited eight exon-intron patterns (Figure <xref ref-type="fig" rid="F1">1</xref>). Barakat et al. (<xref rid="B5" ref-type="bibr">2011</xref>) divided the exon-intron structures of <italic>PoptrCCR</italic>s into three patterns (Patterns 1–3) comprised of 4, 5, and 6 exons, respectively. In Pattern 2, the fourth exon is about two times longer than other exons. The length of the fourth exon in Pattern 2 is similar to the combined lengths of the fourth and fifth exons of Pattern 3 (Barakat et al., <xref rid="B5" ref-type="bibr">2011</xref>). Most functional <italic>CCR</italic>s, such as <italic>AtCCR1, EuCCR, ZmCCR1</italic>, and <italic>SbCCR1</italic>, involved in developmental lignification are grouped into Pattern 2 (Figure <xref ref-type="fig" rid="F1">1</xref>) (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>). Consistently, <italic>OsCCR19</italic> and <italic>20</italic> were composed of five exons with the exon-intron structure of Pattern 2. Although <italic>OsCCR21</italic>, which encoded biochemically active CCR, had six exons, the exon-intron structure differed from that of Pattern 3. Rather, the exon-intron structure of <italic>OsCCR21</italic> was more similar to Pattern 2 (Pattern 2-like), with the length of the fourth exon equaling that seen in Pattern 2 (Figure <xref ref-type="fig" rid="F1">1</xref>). <italic>SbCCR2-2</italic> has been reported to also exhibit a Pattern 2-like exon-intron structure (Figure <xref ref-type="fig" rid="F1">1</xref>) (Li et al., <xref rid="B43" ref-type="bibr">2016</xref>). The biochemically active <italic>OsCCR17</italic> was composed of four exons with an exceptionally long fourth exon (Pattern 5) (Figure <xref ref-type="fig" rid="F1">1</xref>). <italic>ZmCCR2</italic> showed the exon-intron structure of Pattern 5 (Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>). <italic>AtCCR2</italic> exhibited the exon-intron structure of Pattern 4, being composed of four exons (Figure <xref ref-type="fig" rid="F1">1</xref>) (Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>). Unlike <italic>CCR</italic> genes involved in developmental lignification, which were mostly grouped into Pattern 2, stress-related <italic>CCR</italic>s such as <italic>AtCCR2, ZmCCR2</italic>, and <italic>SbCCR2-2</italic> exhibited diverse exon-intron patterns.</p><p>Enzymatic properties of CCRs have been elucidated from many plants, and observed to reflect the lignin compositions of the source plant species (Piquemal et al., <xref rid="B56" ref-type="bibr">1998</xref>; Ma and Tian, <xref rid="B47" ref-type="bibr">2005</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>). Lignins of gymnosperm wood are predominantly composed of G-units (Campbell and Sederoff, <xref rid="B11" ref-type="bibr">1996</xref>; Donaldson, <xref rid="B20" ref-type="bibr">2001</xref>; Vanholme et al., <xref rid="B68" ref-type="bibr">2010</xref>). Unlike gymnosperm lignins, most angiosperm lignins are a mixture of G- and S-units (Donaldson, <xref rid="B20" ref-type="bibr">2001</xref>; Vanholme et al., <xref rid="B68" ref-type="bibr">2010</xref>). The proportion of H-units is variable within plant species and even between tissues in the same plant (Campbell and Sederoff, <xref rid="B11" ref-type="bibr">1996</xref>; Vanholme et al., <xref rid="B68" ref-type="bibr">2010</xref>). Wheat CCRs (TaCCR1 and 2) and switchgrass PvCCR1 have substrate preference for feruloyl-CoA, a precursor for the G-unit (Ma and Tian, <xref rid="B47" ref-type="bibr">2005</xref>; Ma, <xref rid="B46" ref-type="bibr">2007</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>). Similarly, OsCCR20 and 21 showed a preference for feruloyl-CoA over other CoA esters (Table <xref ref-type="table" rid="T2">2</xref>). This result agrees well with rice lignin compositions, which has a high G-unit content and a relatively small portion of S- and H-units (Gui et al., <xref rid="B27" ref-type="bibr">2011</xref> and Supplementary Table <xref ref-type="supplementary-material" rid="SM5">5</xref>). Different from OsCCR20, OsCCR19 showed similar catalytic efficiency toward three examined substrates. It has been known that the substrate preferences of CCRs vary between CCRs from different plant species, even in isozymes from the same species (Goffner et al., <xref rid="B23" ref-type="bibr">1994</xref>; Baltas et al., <xref rid="B4" ref-type="bibr">2005</xref>; Li et al., <xref rid="B44" ref-type="bibr">2005</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>).</p><p>During development, lignin is deposited in the thickened secondary cell walls. In addition, its synthesis can be induced by diverse biotic and abiotic stresses (Moura et al., <xref rid="B49" ref-type="bibr">2010</xref>; Miedes et al., <xref rid="B48" ref-type="bibr">2014</xref>). In <italic>A. thaliana</italic>, maize and switchgrass, <italic>CCR1</italic> genes are related to lignin biosynthesis during development and <italic>CCR2</italic> genes are involved in stress-related processes (Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>; Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>). Phylogenetic analysis of functional CCRs has revealed that constitutive CCRs involved in developmental lignification are grouped separately from CCRs implicated in defense-related processes (Figure <xref ref-type="fig" rid="F2">2</xref> and Supplementary Figure <xref ref-type="supplementary-material" rid="SM7">2</xref>) (Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Li et al., <xref rid="B43" ref-type="bibr">2016</xref>; Chao et al., <xref rid="B14" ref-type="bibr">2017</xref>). OsCCR19 and 20 were closely related to constitutive CCRs, such as ZmCCR1, PvCCR1, and LpCCR (Figure <xref ref-type="fig" rid="F2">2</xref>). CCRs in this group have been observed to be highly expressed in actively lignifying tissues, including stems and roots (Pichon et al., <xref rid="B55" ref-type="bibr">1998</xref>; Larsen, <xref rid="B40" ref-type="bibr">2004a</xref>,<xref rid="B41" ref-type="bibr">b</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>). <italic>In silico</italic> transcriptomic analysis showed that <italic>OsCCR19</italic> and <italic>20</italic> are constitutively expressed throughout all developmental stages of rice (Supplementary Figure <xref ref-type="supplementary-material" rid="SM8">3</xref>). Our qRT-PCR analysis also revealed strong expressions of <italic>OsCCR20</italic> in lignifying tissues such as roots and stems (Figure <xref ref-type="fig" rid="F5">5</xref>). Unlike the microarray data, <italic>OsCCR19</italic> was rarely expressed in most examined tissues. The kinetic analysis also showed that <italic>OsCCR20</italic> was more enzymatically efficient toward feruloyl-CoA, a precursor of the lignin G-unit (Table <xref ref-type="table" rid="T2">2</xref>). These results suggest that <italic>OsCCR20</italic> primarily participates in developmental deposition of lignins in secondary cell wall. Lignification occurs prominently in differentiating xylem tissues and interfascicular fibers in stems and roots (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>; Goujon et al., <xref rid="B24" ref-type="bibr">2003</xref>; Tamasloukht et al., <xref rid="B64" ref-type="bibr">2011</xref>). Functional CCRs involved in developmental lignification have been found to localize in these tissues. <italic>In situ</italic> hybridization of the CCR antisense probe shown that the CCR transcripts are localized in the differentiating xylem tissues of poplar stems (Lacombe et al., <xref rid="B39" ref-type="bibr">1997</xref>). In <italic>Leucaena leucocephala</italic> seedlings, the CCR proteins are localized in the developing xylem tissues of stems and roots (Srivastava et al., <xref rid="B61" ref-type="bibr">2015</xref>). Transient expression of SbCCR-GFP in tobacco leaves indicated that CCR proteins are localized in the cytoplasm (Li et al., <xref rid="B43" ref-type="bibr">2016</xref>). Kawasaki et al. (<xref rid="B35" ref-type="bibr">2006</xref>) also reported that OsCCR1 is localized in the cytoplasm. Analysis of N-terminal sequence of OsCCRs using the SignalP tool (<ext-link ext-link-type="uri" xlink:href="http://www.cbs.dtu.dk/services/SignalP/">http://www.cbs.dtu.dk/services/SignalP/</ext-link>) showed that all OsCCRs, except OsCCR27, have no signal sequence. The expression of <italic>CCR</italic>s in other groups are induced under various stress conditions (Lauvergeat et al., <xref rid="B42" ref-type="bibr">2001</xref>; Fan et al., <xref rid="B22" ref-type="bibr">2006</xref>; Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>; Li et al., <xref rid="B43" ref-type="bibr">2016</xref>). For instance, <italic>ZmCCR2</italic> expression was highly induced by water deficit in the root elongation zone of maize (Fan et al., <xref rid="B22" ref-type="bibr">2006</xref>). <italic>PvCCR2</italic> was highly induced after the rust disease infection (Escamilla-Treviño et al., <xref rid="B21" ref-type="bibr">2010</xref>), and <italic>SbCCR2-2</italic> expression was stimulated by sorghum aphid infection (Li et al., <xref rid="B43" ref-type="bibr">2016</xref>). Phylogenetic analysis indicated that OCCR17 and 21 were grouped with the stress-inducible CCRs (Figure <xref ref-type="fig" rid="F2">2</xref>). Although transcriptomic analysis revealed constitutive expression of <italic>OsCCR21</italic>, its expression was strongly stimulated by infections of rice pathogens (<italic>M. grisea, Xoo</italic> and <italic>Xoc</italic>) (Supplementary Figure <xref ref-type="supplementary-material" rid="SM10">5</xref>). Expression of <italic>OsCCR17</italic> was also induced by <italic>M. grisea</italic> and <italic>Xoo</italic> infections. In addition, <italic>OsCCR17</italic> and <italic>21</italic> expressions were strongly induced by abiotic stresses, such as cold, high salinity, and UV-irradiation (Supplementary Figure <xref ref-type="supplementary-material" rid="SM9">4</xref>). Transcriptomic analysis of UV-treated rice has revealed that a set of phenylpropanoid and monolignol pathway genes are co-expressed immediately after UV-treatment, with the response involving biosynthesis of defense-related compounds such as phytoalexins (Park et al., <xref rid="B53" ref-type="bibr">2013</xref>, <xref rid="B54" ref-type="bibr">2014</xref>; Cho and Lee, <xref rid="B15" ref-type="bibr">2015</xref>). Our qRT-PCR analysis also observed strong induction of <italic>OsCCR17</italic> and <italic>21</italic> in response to UV and salt-treatment (Figure <xref ref-type="fig" rid="F5">5</xref>). These results strongly suggest that <italic>OsCCR17</italic> and <italic>21</italic> likely participates in defense-related lignification and synthesis of phenolic compounds.</p></sec><sec sec-type="conclusions" id="s5"><title>Conclusion</title><p>Expression patterns and biochemical properties of the rice <italic>CCR</italic> gene family were thoroughly analyzed in the present study. OsCCR17, 19, 20, and 21 were found to have NAD(P)-binding and NADP-specific motifs as well as the CCR signature motif. The recombinant OsCCR17, 19, 20, and 21 showed enzyme activity toward hydroxycinnamoyl-CoA substrates, indicating that these OsCCRs are biochemically functional CCRs in rice. Phylogenetic analysis revealed that OsCCR19 and 20 were closely related to other plant CCRs involved in developmental lignification. <italic>In silico</italic> transcriptomic analysis and qRT-PCR consistently demonstrated that <italic>OsCCR20</italic> were constitutively expressed throughout all developmental stages of rice, with especially high expression levels in actively lignifying tissues such as roots, stems and panicles. These results suggest that <italic>OsCCR20</italic> are primarily involved in the developmental deposition of lignins in secondary cell walls. Meanwhile, the expressions of <italic>OsCCR17</italic> and <italic>21</italic> were induced in response to biotic and abiotic stresses, such as <italic>M. grisea</italic> and <italic>Xoo</italic> infections, UV-irradiation and high salinity. OsCCR17 and 21 were also grouped with stress-responsible CCRs identified from other plant species. Therefore, we suggest that <italic>OsCCR17</italic> and <italic>21</italic> play a role in defense-related processes of rice under biotic and abiotic stress conditions.</p></sec><sec id="s6"><title>Author contributions</title><p>M-HC and S-WL: conceived and designed the experiments; HLP and M-HC: performed the experiments and conducted bioinformatics analyses; MK: performed the experiments. M-HC, HLP, SHB, and S-WL: analyzed the data, and wrote the manuscript.</p><sec><title>Conflict of interest statement</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></sec></body><back><ack><p>This work was supported by the Next-Generation BioGreen 21 Program (Project No: PJ01107501) funded by the Rural Development Administration, and the Mid-career Researcher Program (NRF-2016R1A2B4014276) through NRF grant funded by the Ministry of Education, Science and Technology, Republic of Korea.</p></ack><sec sec-type="supplementary-material" id="s7"><title>Supplementary material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fpls.2017.02099/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fpls.2017.02099/full#supplementary-material</ext-link></p><supplementary-material content-type="local-data" id="SM1"><media xlink:href="Table1.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM2"><media xlink:href="Table2.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM3"><media xlink:href="Table3.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM4"><media xlink:href="Table4.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM5"><media xlink:href="Table5.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM6"><media xlink:href="Image1.PDF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM7"><media xlink:href="Image2.PDF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM8"><media xlink:href="Image3.PDF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM9"><media xlink:href="Image4.PDF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM10"><media xlink:href="Image5.PDF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id="B1"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ahuja</surname><given-names>I.</given-names></name><name><surname>Kissen</surname><given-names>R.</given-names></name><name><surname>Bones</surname><given-names>A. 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