The lycopene β-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm
Identifieur interne : 000627 ( Pmc/Corpus ); précédent : 000626; suivant : 000628The lycopene β-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm
Auteurs : Jian Zeng ; Cheng Wang ; Xi Chen ; Mingli Zang ; Cuihong Yuan ; Xiatian Wang ; Qiong Wang ; Miao Li ; Xiaoyan Li ; Ling Chen ; Kexiu Li ; Junli Chang ; Yuesheng Wang ; Guangxiao Yang ; Guangyuan HeSource :
- BMC Plant Biology [ 1471-2229 ] ; 2015.
Abstract
Lycopene β-cyclase (LCYB) is a key enzyme catalyzing the biosynthesis of β-carotene, the main source of provitamin A. However, there is no documented research about this key cyclase gene’s function and relationship with β-carotene content in wheat. Therefore, the objectives of this study were to clone
In the present study, a lycopene β-cyclase gene, designated
Our results suggest that
The online version of this article (doi:10.1186/s12870-015-0514-5) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s12870-015-0514-5
PubMed: 25943989
PubMed Central: 4433027
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PMC:4433027Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The lycopene β-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm</title><author><name sortKey="Zeng, Jian" sort="Zeng, Jian" uniqKey="Zeng J" first="Jian" last="Zeng">Jian Zeng</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Cheng" sort="Wang, Cheng" uniqKey="Wang C" first="Cheng" last="Wang">Cheng Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Xi" sort="Chen, Xi" uniqKey="Chen X" first="Xi" last="Chen">Xi Chen</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Zang, Mingli" sort="Zang, Mingli" uniqKey="Zang M" first="Mingli" last="Zang">Mingli Zang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Yuan, Cuihong" sort="Yuan, Cuihong" uniqKey="Yuan C" first="Cuihong" last="Yuan">Cuihong Yuan</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Xiatian" sort="Wang, Xiatian" uniqKey="Wang X" first="Xiatian" last="Wang">Xiatian Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Qiong" sort="Wang, Qiong" uniqKey="Wang Q" first="Qiong" last="Wang">Qiong Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Li, Miao" sort="Li, Miao" uniqKey="Li M" first="Miao" last="Li">Miao Li</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Li, Xiaoyan" sort="Li, Xiaoyan" uniqKey="Li X" first="Xiaoyan" last="Li">Xiaoyan Li</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Ling" sort="Chen, Ling" uniqKey="Chen L" first="Ling" last="Chen">Ling Chen</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Li, Kexiu" sort="Li, Kexiu" uniqKey="Li K" first="Kexiu" last="Li">Kexiu Li</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Chang, Junli" sort="Chang, Junli" uniqKey="Chang J" first="Junli" last="Chang">Junli Chang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Yuesheng" sort="Wang, Yuesheng" uniqKey="Wang Y" first="Yuesheng" last="Wang">Yuesheng Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Yang, Guangxiao" sort="Yang, Guangxiao" uniqKey="Yang G" first="Guangxiao" last="Yang">Guangxiao Yang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="He, Guangyuan" sort="He, Guangyuan" uniqKey="He G" first="Guangyuan" last="He">Guangyuan He</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25943989</idno><idno type="pmc">4433027</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433027</idno><idno type="RBID">PMC:4433027</idno><idno type="doi">10.1186/s12870-015-0514-5</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000627</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">The lycopene β-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm</title><author><name sortKey="Zeng, Jian" sort="Zeng, Jian" uniqKey="Zeng J" first="Jian" last="Zeng">Jian Zeng</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Cheng" sort="Wang, Cheng" uniqKey="Wang C" first="Cheng" last="Wang">Cheng Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Xi" sort="Chen, Xi" uniqKey="Chen X" first="Xi" last="Chen">Xi Chen</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Zang, Mingli" sort="Zang, Mingli" uniqKey="Zang M" first="Mingli" last="Zang">Mingli Zang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Yuan, Cuihong" sort="Yuan, Cuihong" uniqKey="Yuan C" first="Cuihong" last="Yuan">Cuihong Yuan</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Xiatian" sort="Wang, Xiatian" uniqKey="Wang X" first="Xiatian" last="Wang">Xiatian Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Qiong" sort="Wang, Qiong" uniqKey="Wang Q" first="Qiong" last="Wang">Qiong Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Li, Miao" sort="Li, Miao" uniqKey="Li M" first="Miao" last="Li">Miao Li</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Li, Xiaoyan" sort="Li, Xiaoyan" uniqKey="Li X" first="Xiaoyan" last="Li">Xiaoyan Li</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Ling" sort="Chen, Ling" uniqKey="Chen L" first="Ling" last="Chen">Ling Chen</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Li, Kexiu" sort="Li, Kexiu" uniqKey="Li K" first="Kexiu" last="Li">Kexiu Li</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Chang, Junli" sort="Chang, Junli" uniqKey="Chang J" first="Junli" last="Chang">Junli Chang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Yuesheng" sort="Wang, Yuesheng" uniqKey="Wang Y" first="Yuesheng" last="Wang">Yuesheng Wang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="Yang, Guangxiao" sort="Yang, Guangxiao" uniqKey="Yang G" first="Guangxiao" last="Yang">Guangxiao Yang</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author><author><name sortKey="He, Guangyuan" sort="He, Guangyuan" uniqKey="He G" first="Guangyuan" last="He">Guangyuan He</name><affiliation><nlm:aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</nlm:aff></affiliation></author></analytic><series><title level="j">BMC Plant Biology</title><idno type="eISSN">1471-2229</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Lycopene β-cyclase (LCYB) is a key enzyme catalyzing the biosynthesis of β-carotene, the main source of provitamin A. However, there is no documented research about this key cyclase gene’s function and relationship with β-carotene content in wheat. Therefore, the objectives of this study were to clone <italic>TaLCYB</italic> and characterize its function and relationship with β-carotene biosynthesis in wheat grains. We also aimed to obtain more information about the endogenous carotenoid biosynthetic pathway and thus provide experimental support for carotenoid metabolic engineering in wheat.</p></sec><sec><title>Results</title><p>In the present study, a lycopene β-cyclase gene, designated <italic>TaLCYB</italic>, was cloned from the hexaploid wheat cultivar Chinese Spring. The cyclization activity of the encoded protein was demonstrated by heterologous complementation analysis. The <italic>TaLCYB</italic> gene was expressed differentially in different tissues of wheat. Although <italic>TaLCYB</italic> had a higher expression level in the later stages of grain development, the β-carotene content still showed a decreasing tendency. The expression of <italic>TaLCYB</italic> in leaves was dramatically induced by strong light and the β-carotene content variation corresponded with changes of <italic>TaLCYB</italic> expression. A post-transcriptional gene silencing strategy was used to down-regulate the expression of <italic>TaLCYB</italic> in transgenic wheat, resulting in a decrease in the content of β-carotene and lutein, accompanied by the accumulation of lycopene to partly compensate for the total carotenoid content. In addition, changes in <italic>TaLCYB</italic> expression also affected the expression of several endogenous carotenogenic genes to varying degrees.</p></sec><sec><title>Conclusion</title><p>Our results suggest that <italic>TaLCYB</italic> is a genuine lycopene cyclase gene and plays a crucial role in β-carotene biosynthesis in wheat. Our attempt to silence it not only contributes to elucidating the mechanism of carotenoid accumulation in wheat but may also help in breeding wheat varieties with high provitamin A content through RNA interference (RNAi) to block specific carotenogenic genes in the wheat endosperm.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1186/s12870-015-0514-5) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bramley, Pm" uniqKey="Bramley P">PM Bramley</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Fraser, Pd" uniqKey="Fraser P">PD Fraser</name></author><author><name sortKey="Bramley, Pm" uniqKey="Bramley P">PM Bramley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Clagett Dame, M" uniqKey="Clagett Dame M">M Clagett-Dame</name></author><author><name sortKey="Knutson, D" uniqKey="Knutson D">D Knutson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Abdel Aal, E Sm" uniqKey="Abdel Aal E">E-SM Abdel-Aal</name></author><author><name sortKey="Akhtar, H" uniqKey="Akhtar H">H Akhtar</name></author><author><name sortKey="Zaheer, K" uniqKey="Zaheer K">K Zaheer</name></author><author><name sortKey="Ali, R" uniqKey="Ali R">R Ali</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hirschberg, J" uniqKey="Hirschberg J">J Hirschberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruiz Sola, Ma" uniqKey="Ruiz Sola M">MA Ruiz-Sola</name></author><author><name sortKey="Rodriguez Concepcion, M" uniqKey="Rodriguez Concepcion M">M Rodriguez-Concepcion</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cunningham, Fx" uniqKey="Cunningham F">FX Cunningham</name></author><author><name sortKey="Sun, Z" uniqKey="Sun Z">Z Sun</name></author><author><name sortKey="Chamovitz, D" uniqKey="Chamovitz D">D Chamovitz</name></author><author><name sortKey="Hirschberg, J" uniqKey="Hirschberg J">J Hirschberg</name></author><author><name sortKey="Gantt, E" uniqKey="Gantt E">E Gantt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cunningham, Fx" uniqKey="Cunningham F">FX Cunningham</name></author><author><name sortKey="Gantt, E" uniqKey="Gantt E">E Gantt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cunningham, Fx" uniqKey="Cunningham F">FX Cunningham</name></author><author><name sortKey="Pogson, B" uniqKey="Pogson B">B Pogson</name></author><author><name sortKey="Sun, Z" uniqKey="Sun Z">Z Sun</name></author><author><name sortKey="Mcdonald, Ka" uniqKey="Mcdonald K">KA McDonald</name></author><author><name sortKey="Dellapenna, D" uniqKey="Dellapenna D">D DellaPenna</name></author><author><name sortKey="Gantt, E" uniqKey="Gantt E">E Gantt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Singh, M" uniqKey="Singh M">M Singh</name></author><author><name sortKey="Lewis, Pe" uniqKey="Lewis P">PE Lewis</name></author><author><name sortKey="Hardeman, K" uniqKey="Hardeman K">K Hardeman</name></author><author><name sortKey="Bai, L" uniqKey="Bai L">L Bai</name></author><author><name sortKey="Rose, Jk" uniqKey="Rose J">JK Rose</name></author><author><name sortKey="Mazourek, M" uniqKey="Mazourek M">M Mazourek</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ampomah Dwamena, C" uniqKey="Ampomah Dwamena C">C Ampomah-Dwamena</name></author><author><name sortKey="Mcghie, T" uniqKey="Mcghie T">T McGhie</name></author><author><name sortKey="Wibisono, R" uniqKey="Wibisono R">R Wibisono</name></author><author><name sortKey="Montefiori, M" uniqKey="Montefiori M">M Montefiori</name></author><author><name sortKey="Hellens, Rp" uniqKey="Hellens R">RP Hellens</name></author><author><name sortKey="Allan, Ac" uniqKey="Allan A">AC Allan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Devitt, Lc" uniqKey="Devitt L">LC Devitt</name></author><author><name sortKey="Fanning, K" uniqKey="Fanning K">K Fanning</name></author><author><name sortKey="Dietzgen, Rg" uniqKey="Dietzgen R">RG Dietzgen</name></author><author><name sortKey="Holton, Ta" uniqKey="Holton T">TA Holton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bouvier, F" uniqKey="Bouvier F">F Bouvier</name></author><author><name sortKey="D Harlingue, A" uniqKey="D Harlingue A">A d'Harlingue</name></author><author><name sortKey="Hugueney, P" uniqKey="Hugueney P">P Hugueney</name></author><author><name sortKey="Marin, E" uniqKey="Marin E">E Marin</name></author><author><name sortKey="Marion Poll, A" uniqKey="Marion Poll A">A Marion-Poll</name></author><author><name sortKey="Camara, B" uniqKey="Camara B">B Camara</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sandmann, G" uniqKey="Sandmann G">G Sandmann</name></author><author><name sortKey="Romer, S" uniqKey="Romer S">S Römer</name></author><author><name sortKey="Fraser, Pd" uniqKey="Fraser P">PD Fraser</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhu, C" uniqKey="Zhu C">C Zhu</name></author><author><name sortKey="Naqvi, S" uniqKey="Naqvi S">S Naqvi</name></author><author><name sortKey="Gomez Galera, S" uniqKey="Gomez Galera S">S Gomez-Galera</name></author><author><name sortKey="Pelacho, Am" uniqKey="Pelacho A">AM Pelacho</name></author><author><name sortKey="Capell, T" uniqKey="Capell T">T Capell</name></author><author><name sortKey="Christou, P" uniqKey="Christou P">P Christou</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fraser, Pd" uniqKey="Fraser P">PD Fraser</name></author><author><name sortKey="Enfissi, E" uniqKey="Enfissi E">E Enfissi</name></author><author><name sortKey="Bramley, Pm" uniqKey="Bramley P">PM Bramley</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Mares, Dj" uniqKey="Mares D">DJ Mares</name></author><author><name sortKey="Campbell, A" uniqKey="Campbell A">A Campbell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qin, X" uniqKey="Qin X">X Qin</name></author><author><name sortKey="Zhang, W" uniqKey="Zhang W">W Zhang</name></author><author><name sortKey="Dubcovsky, J" uniqKey="Dubcovsky J">J Dubcovsky</name></author><author><name sortKey="Tian, L" uniqKey="Tian L">L Tian</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cong, L" uniqKey="Cong L">L Cong</name></author><author><name sortKey="Wang, C" uniqKey="Wang C">C Wang</name></author><author><name sortKey="Li, Zq" uniqKey="Li Z">ZQ Li</name></author><author><name sortKey="Chen, L" uniqKey="Chen L">L Chen</name></author><author><name sortKey="Yang, Gx" uniqKey="Yang G">GX Yang</name></author><author><name sortKey="Wang, Ys" uniqKey="Wang Y">YS Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, J" uniqKey="Wang J">J Wang</name></author><author><name sortKey="He, X" uniqKey="He X">X He</name></author><author><name sortKey="He, Z" uniqKey="He Z">Z He</name></author><author><name sortKey="Wang, H" uniqKey="Wang H">H Wang</name></author><author><name sortKey="Xia, X" uniqKey="Xia X">X Xia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Howitt, Ca" uniqKey="Howitt C">CA Howitt</name></author><author><name sortKey="Cavanagh, Cr" uniqKey="Cavanagh C">CR Cavanagh</name></author><author><name sortKey="Bowerman, Af" uniqKey="Bowerman A">AF Bowerman</name></author><author><name sortKey="Cazzonelli, C" uniqKey="Cazzonelli C">C Cazzonelli</name></author><author><name sortKey="Rampling, L" uniqKey="Rampling L">L Rampling</name></author><author><name sortKey="Mimica, Jl" uniqKey="Mimica J">JL Mimica</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mayer, Kf" uniqKey="Mayer K">KF Mayer</name></author><author><name sortKey="Rogers, J" uniqKey="Rogers J">J Rogers</name></author><author><name sortKey="Dolezel, J" uniqKey="Dolezel J">J Doležel</name></author><author><name sortKey="Pozniak, C" uniqKey="Pozniak C">C Pozniak</name></author><author><name sortKey="Eversole, K" uniqKey="Eversole K">K Eversole</name></author><author><name sortKey="Feuillet, C" uniqKey="Feuillet C">C Feuillet</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, C" uniqKey="Wang C">C Wang</name></author><author><name sortKey="Zeng, J" uniqKey="Zeng J">J Zeng</name></author><author><name sortKey="Li, Y" uniqKey="Li Y">Y Li</name></author><author><name sortKey="Hu, W" uniqKey="Hu W">W Hu</name></author><author><name sortKey="Chen, L" uniqKey="Chen L">L Chen</name></author><author><name sortKey="Miao, Y" uniqKey="Miao Y">Y Miao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hugueney, P" uniqKey="Hugueney P">P Hugueney</name></author><author><name sortKey="Badillo, A" uniqKey="Badillo A">A Badillo</name></author><author><name sortKey="Chen, H C" uniqKey="Chen H">H-C Chen</name></author><author><name sortKey="Klein, A" uniqKey="Klein A">A Klein</name></author><author><name sortKey="Hirschberg, J" uniqKey="Hirschberg J">J Hirschberg</name></author><author><name sortKey="Camara, B" uniqKey="Camara B">B Camara</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alquezar, B" uniqKey="Alquezar B">B Alquezar</name></author><author><name sortKey="Zacarias, L" uniqKey="Zacarias L">L Zacarias</name></author><author><name sortKey="Rodrigo, Mj" uniqKey="Rodrigo M">MJ Rodrigo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Paine, Ja" uniqKey="Paine J">JA Paine</name></author><author><name sortKey="Shipton, Ca" uniqKey="Shipton C">CA Shipton</name></author><author><name sortKey="Chaggar, S" uniqKey="Chaggar S">S Chaggar</name></author><author><name sortKey="Howells, Rm" uniqKey="Howells R">RM Howells</name></author><author><name sortKey="Kennedy, Mj" uniqKey="Kennedy M">MJ Kennedy</name></author><author><name sortKey="Vernon, G" uniqKey="Vernon G">G Vernon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Aluru, M" uniqKey="Aluru M">M Aluru</name></author><author><name sortKey="Xu, Y" uniqKey="Xu Y">Y Xu</name></author><author><name sortKey="Guo, R" uniqKey="Guo R">R Guo</name></author><author><name sortKey="Wang, Z" uniqKey="Wang Z">Z Wang</name></author><author><name sortKey="Li, S" uniqKey="Li S">S Li</name></author><author><name sortKey="White, W" uniqKey="White W">W White</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Diretto, G" uniqKey="Diretto G">G Diretto</name></author><author><name sortKey="Al Babili, S" uniqKey="Al Babili S">S Al-Babili</name></author><author><name sortKey="Tavazza, R" uniqKey="Tavazza R">R Tavazza</name></author><author><name sortKey="Papacchioli, V" uniqKey="Papacchioli V">V Papacchioli</name></author><author><name sortKey="Beyer, P" uniqKey="Beyer P">P Beyer</name></author><author><name sortKey="Giuliano, G" uniqKey="Giuliano G">G Giuliano</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Scolnik, Pa" uniqKey="Scolnik P">PA Scolnik</name></author><author><name sortKey="Bartley, G" uniqKey="Bartley G">G Bartley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guzman, I" uniqKey="Guzman I">I Guzman</name></author><author><name sortKey="Hamby, S" uniqKey="Hamby S">S Hamby</name></author><author><name sortKey="Romero, J" uniqKey="Romero J">J Romero</name></author><author><name sortKey="Bosland, Pw" uniqKey="Bosland P">PW Bosland</name></author><author><name sortKey="O Onnell, Ma" uniqKey="O Onnell M">MA O’Connell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pecker, I" uniqKey="Pecker I">I Pecker</name></author><author><name sortKey="Gabbay, R" uniqKey="Gabbay R">R Gabbay</name></author><author><name sortKey="Cunningham, Fx" uniqKey="Cunningham F">FX Cunningham</name></author><author><name sortKey="Hirschberg, J" uniqKey="Hirschberg J">J Hirschberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Demmig Adams, B" uniqKey="Demmig Adams B">B Demmig-Adams</name></author><author><name sortKey="Gilmore, Am" uniqKey="Gilmore A">AM Gilmore</name></author><author><name sortKey="Adams, Ww" uniqKey="Adams W">WW Adams</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dall Osto, L" uniqKey="Dall Osto L">L Dall'Osto</name></author><author><name sortKey="Fiore, A" uniqKey="Fiore A">A Fiore</name></author><author><name sortKey="Cazzaniga, S" uniqKey="Cazzaniga S">S Cazzaniga</name></author><author><name sortKey="Giuliano, G" uniqKey="Giuliano G">G Giuliano</name></author><author><name sortKey="Bassi, R" uniqKey="Bassi R">R Bassi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nisar, N" uniqKey="Nisar N">N Nisar</name></author><author><name sortKey="Li, L" uniqKey="Li L">L Li</name></author><author><name sortKey="Lu, S" uniqKey="Lu S">S Lu</name></author><author><name sortKey="Khin, Nc" uniqKey="Khin N">NC Khin</name></author><author><name sortKey="Pogson, Bj" uniqKey="Pogson B">BJ Pogson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vallabhaneni, R" uniqKey="Vallabhaneni R">R Vallabhaneni</name></author><author><name sortKey="Bradbury, Lm" uniqKey="Bradbury L">LM Bradbury</name></author><author><name sortKey="Wurtzel, Et" uniqKey="Wurtzel E">ET Wurtzel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Da Silva, Mr" uniqKey="Da Silva M">MR da Silva</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Diretto, G" uniqKey="Diretto G">G Diretto</name></author><author><name sortKey="Tavazza, R" uniqKey="Tavazza R">R Tavazza</name></author><author><name sortKey="Welsch, R" uniqKey="Welsch R">R Welsch</name></author><author><name sortKey="Pizzichini, D" uniqKey="Pizzichini D">D Pizzichini</name></author><author><name sortKey="Mourgues, F" uniqKey="Mourgues F">F Mourgues</name></author><author><name sortKey="Papacchioli, V" uniqKey="Papacchioli V">V Papacchioli</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moreno, Jc" uniqKey="Moreno J">JC Moreno</name></author><author><name sortKey="Pizarro, L" uniqKey="Pizarro L">L Pizarro</name></author><author><name sortKey="Fuentes, P" uniqKey="Fuentes P">P Fuentes</name></author><author><name sortKey="Handford, M" uniqKey="Handford M">M Handford</name></author><author><name sortKey="Cifuentes, V" uniqKey="Cifuentes V">V Cifuentes</name></author><author><name sortKey="Stange, C" uniqKey="Stange C">C Stange</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cunningham, Fx" uniqKey="Cunningham F">FX Cunningham</name></author><author><name sortKey="Gantt, E" uniqKey="Gantt E">E Gantt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rosati, C" uniqKey="Rosati C">C Rosati</name></author><author><name sortKey="Aquilani, R" uniqKey="Aquilani R">R Aquilani</name></author><author><name sortKey="Dharmapuri, S" uniqKey="Dharmapuri S">S Dharmapuri</name></author><author><name sortKey="Pallara, P" uniqKey="Pallara P">P Pallara</name></author><author><name sortKey="Marusic, C" uniqKey="Marusic C">C Marusic</name></author><author><name sortKey="Tavazza, R" uniqKey="Tavazza R">R Tavazza</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fraser, Pd" uniqKey="Fraser P">PD Fraser</name></author><author><name sortKey="Romer, S" uniqKey="Romer S">S Romer</name></author><author><name sortKey="Shipton, Ca" uniqKey="Shipton C">CA Shipton</name></author><author><name sortKey="Mills, Pb" uniqKey="Mills P">PB Mills</name></author><author><name sortKey="Kiano, Jw" uniqKey="Kiano J">JW Kiano</name></author><author><name sortKey="Misawa, N" uniqKey="Misawa N">N Misawa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rodriguez Illal N, A" uniqKey="Rodriguez Illal N A">A Rodríguez‐Villalón</name></author><author><name sortKey="Gas, E" uniqKey="Gas E">E Gas</name></author><author><name sortKey="Rodriguez Oncepci N, M" uniqKey="Rodriguez Oncepci N M">M Rodríguez‐Concepción</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Welsch, R" uniqKey="Welsch R">R Welsch</name></author><author><name sortKey="Arango, J" uniqKey="Arango J">J Arango</name></author><author><name sortKey="B R, C" uniqKey="B R C">C Bär</name></author><author><name sortKey="Salazar, B" uniqKey="Salazar B">B Salazar</name></author><author><name sortKey="Al Babili, S" uniqKey="Al Babili S">S Al-Babili</name></author><author><name sortKey="Beltran, J" uniqKey="Beltran J">J Beltrán</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ronen, G" uniqKey="Ronen G">G Ronen</name></author><author><name sortKey="Carmel Goren, L" uniqKey="Carmel Goren L">L Carmel-Goren</name></author><author><name sortKey="Zamir, D" uniqKey="Zamir D">D Zamir</name></author><author><name sortKey="Hirschberg, J" uniqKey="Hirschberg J">J Hirschberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Davison, P" uniqKey="Davison P">P Davison</name></author><author><name sortKey="Hunter, C" uniqKey="Hunter C">C Hunter</name></author><author><name sortKey="Horton, P" uniqKey="Horton P">P Horton</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Yu, Q" uniqKey="Yu Q">Q Yu</name></author><author><name sortKey="Schaub, P" uniqKey="Schaub P">P Schaub</name></author><author><name sortKey="Ghisla, S" uniqKey="Ghisla S">S Ghisla</name></author><author><name sortKey="Al Babili, S" uniqKey="Al Babili S">S Al-Babili</name></author><author><name sortKey="Krieger Liszkay, A" uniqKey="Krieger Liszkay A">A Krieger-Liszkay</name></author><author><name sortKey="Beyer, P" uniqKey="Beyer P">P Beyer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Emanuelsson, O" uniqKey="Emanuelsson O">O Emanuelsson</name></author><author><name sortKey="Nielsen, H" uniqKey="Nielsen H">H Nielsen</name></author><author><name sortKey="Heijne, Gv" uniqKey="Heijne G">GV Heijne</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Saitou, N" uniqKey="Saitou N">N Saitou</name></author><author><name sortKey="Nei, M" uniqKey="Nei M">M Nei</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thompson, Jd" uniqKey="Thompson J">JD Thompson</name></author><author><name sortKey="Higgins, Dg" uniqKey="Higgins D">DG Higgins</name></author><author><name sortKey="Gibson, Tj" uniqKey="Gibson T">TJ Gibson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fu, D" uniqKey="Fu D">D Fu</name></author><author><name sortKey="Uauy, C" uniqKey="Uauy C">C Uauy</name></author><author><name sortKey="Blechl, A" uniqKey="Blechl A">A Blechl</name></author><author><name sortKey="Dubcovsky, J" uniqKey="Dubcovsky J">J Dubcovsky</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sparks, C" uniqKey="Sparks C">C Sparks</name></author><author><name sortKey="Jones, H" uniqKey="Jones H">H Jones</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sander, Lc" uniqKey="Sander L">LC Sander</name></author><author><name sortKey="Sharpless, Ke" uniqKey="Sharpless K">KE Sharpless</name></author><author><name sortKey="Pursch, M" uniqKey="Pursch M">M Pursch</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fraser, Pd" uniqKey="Fraser P">PD Fraser</name></author><author><name sortKey="Pinto, Mes" uniqKey="Pinto M">MES Pinto</name></author><author><name sortKey="Holloway, De" uniqKey="Holloway D">DE Holloway</name></author><author><name sortKey="Bramley, Pm" uniqKey="Bramley P">PM Bramley</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Liu, Q" uniqKey="Liu Q">Q Liu</name></author><author><name sortKey="Xu, J" uniqKey="Xu J">J Xu</name></author><author><name sortKey="Liu, Y" uniqKey="Liu Y">Y Liu</name></author><author><name sortKey="Zhao, X" uniqKey="Zhao X">X Zhao</name></author><author><name sortKey="Deng, X" uniqKey="Deng X">X Deng</name></author><author><name sortKey="Guo, L" uniqKey="Guo L">L Guo</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">BMC Plant Biol</journal-id><journal-id journal-id-type="iso-abbrev">BMC Plant Biol</journal-id><journal-title-group><journal-title>BMC Plant Biology</journal-title></journal-title-group><issn pub-type="epub">1471-2229</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25943989</article-id><article-id pub-id-type="pmc">4433027</article-id><article-id pub-id-type="publisher-id">514</article-id><article-id pub-id-type="doi">10.1186/s12870-015-0514-5</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>The lycopene β-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Zeng</surname><given-names>Jian</given-names></name><address><email>zengjian@hust.edu.cn</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Wang</surname><given-names>Cheng</given-names></name><address><email>wcsuai@163.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Xi</given-names></name><address><email>529121755@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Zang</surname><given-names>Mingli</given-names></name><address><email>931293801@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Cuihong</given-names></name><address><email>524965239@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Xiatian</given-names></name><address><email>wangxiatiancwz@163.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Qiong</given-names></name><address><email>754631109@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Miao</given-names></name><address><email>waterheart405@126.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Xiaoyan</given-names></name><address><email>417068352@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Ling</given-names></name><address><email>15238065@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Kexiu</given-names></name><address><email>651380086@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Chang</surname><given-names>Junli</given-names></name><address><email>cjl@hust.edu.cn</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Yuesheng</given-names></name><address><email>wysh@hust.edu.cn</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Yang</surname><given-names>Guangxiao</given-names></name><address><email>ygx@hust.edu.cn</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>He</surname><given-names>Guangyuan</given-names></name><address><email>hegy@hust.edu.cn</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><aff id="Aff1">The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</aff></contrib-group><pub-date pub-type="epub"><day>7</day><month>5</month><year>2015</year></pub-date><pub-date pub-type="pmc-release"><day>7</day><month>5</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>15</volume><elocation-id>112</elocation-id><history><date date-type="received"><day>16</day><month>1</month><year>2015</year></date><date date-type="accepted"><day>29</day><month>4</month><year>2015</year></date></history><permissions><copyright-statement>© Zeng et al.; licensee BioMed Central. 2015</copyright-statement><license license-type="open-access"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0">http://creativecommons.org/licenses/by/4.0</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>) applies to the data made available in this article, unless otherwise stated.</license-p></license></permissions><abstract id="Abs1"><sec><title>Background</title><p>Lycopene β-cyclase (LCYB) is a key enzyme catalyzing the biosynthesis of β-carotene, the main source of provitamin A. However, there is no documented research about this key cyclase gene’s function and relationship with β-carotene content in wheat. Therefore, the objectives of this study were to clone <italic>TaLCYB</italic> and characterize its function and relationship with β-carotene biosynthesis in wheat grains. We also aimed to obtain more information about the endogenous carotenoid biosynthetic pathway and thus provide experimental support for carotenoid metabolic engineering in wheat.</p></sec><sec><title>Results</title><p>In the present study, a lycopene β-cyclase gene, designated <italic>TaLCYB</italic>, was cloned from the hexaploid wheat cultivar Chinese Spring. The cyclization activity of the encoded protein was demonstrated by heterologous complementation analysis. The <italic>TaLCYB</italic> gene was expressed differentially in different tissues of wheat. Although <italic>TaLCYB</italic> had a higher expression level in the later stages of grain development, the β-carotene content still showed a decreasing tendency. The expression of <italic>TaLCYB</italic> in leaves was dramatically induced by strong light and the β-carotene content variation corresponded with changes of <italic>TaLCYB</italic> expression. A post-transcriptional gene silencing strategy was used to down-regulate the expression of <italic>TaLCYB</italic> in transgenic wheat, resulting in a decrease in the content of β-carotene and lutein, accompanied by the accumulation of lycopene to partly compensate for the total carotenoid content. In addition, changes in <italic>TaLCYB</italic> expression also affected the expression of several endogenous carotenogenic genes to varying degrees.</p></sec><sec><title>Conclusion</title><p>Our results suggest that <italic>TaLCYB</italic> is a genuine lycopene cyclase gene and plays a crucial role in β-carotene biosynthesis in wheat. Our attempt to silence it not only contributes to elucidating the mechanism of carotenoid accumulation in wheat but may also help in breeding wheat varieties with high provitamin A content through RNA interference (RNAi) to block specific carotenogenic genes in the wheat endosperm.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1186/s12870-015-0514-5) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Lycopene</kwd><kwd>Lycopene β-cyclase</kwd><kwd>β-carotene</kwd><kwd>Provitamin A</kwd><kwd>RNA interference</kwd><kwd>Wheat</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2015</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1" sec-type="introduction"><title>Background</title><p>Carotenoids are important natural isoprenoid pigments synthesized in plants that have essential roles in protecting against excess light energy and oxidative damage, and in light-harvesting [<xref ref-type="bibr" rid="CR1">1</xref>,<xref ref-type="bibr" rid="CR2">2</xref>]. Their provitamin A activity and antioxidant properties are their most attractive functions. β-carotene is the major and most effective vitamin A precursor among carotenoids, and plays a crucial role in human health, protecting against age-related degenerative diseases, cardiovascular disease, certain cancers and vitamin A deficiency (VAD) [<xref ref-type="bibr" rid="CR3">3</xref>-<xref ref-type="bibr" rid="CR5">5</xref>]. Generally, β-carotene is the most attractive target product for metabolic engineering.</p><p>In higher plants, although the main pathway of carotenoid biosynthesis has been studied extensively [<xref ref-type="bibr" rid="CR6">6</xref>-<xref ref-type="bibr" rid="CR8">8</xref>], the regulatory mechanisms of carotenoid biosynthesis are still not well known. Lycopene cyclization is the first branch point of the carotenoid biosynthetic pathway, and is hypothesized to regulate the proportion of carotenes through two competing lycopene cyclases, LCYB and lycopene ε-cyclase (LCYE). In general, lycopene is cyclized by LCYE and LCYB to introduce ε and β-ionone end groups and produce α- and β-carotene, respectively (Figure <xref rid="Fig1" ref-type="fig">1</xref>). Only a small number of species such as <italic>Lactuca sativa</italic> produce ε,ε-carotene [<xref ref-type="bibr" rid="CR9">9</xref>]. Because of the special position of lycopene cyclization, researchers have focused on the function of LCYB and its relationship with carotenoid accumulation in plants [<xref ref-type="bibr" rid="CR10">10</xref>-<xref ref-type="bibr" rid="CR13">13</xref>]. Plant LCYBs share similar and highly conserved functional domains, which are involved in many reactions in β-ionone catalysis [<xref ref-type="bibr" rid="CR10">10</xref>,<xref ref-type="bibr" rid="CR14">14</xref>].<fig id="Fig1"><label>Figure 1</label><caption><p>Carotenoid biosynthetic pathway in wheat. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, zeta-carotene desaturase; CRTISO, carotene isomerase; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; HYDB, β-carotene hydroxylase; CYP, carotenoid ε-hydroxylase (cytochrome P450 type).</p></caption><graphic xlink:href="12870_2015_514_Fig1_HTML" id="MO1"></graphic></fig></p><p>Through the deeper understanding of the benefits of carotenoids for human health, scientists have been prompted to explore effective methods to increase the carotenoid composition and content in plants, especially in staple crops. However, precise carotenoid metabolic engineering in crop plants has been hindered by limited data about the endogenous regulation of carotenogenic genes despite recent progress in staple crops [<xref ref-type="bibr" rid="CR15">15</xref>-<xref ref-type="bibr" rid="CR17">17</xref>]. Thus, the first step to understanding how carotenoids are biosynthesized is to identify the related key enzymes and clone the relevant genes.</p><p>Wheat is one of the most important cereal crops in the world [<xref ref-type="bibr" rid="CR18">18</xref>]. Given the huge daily consumption of wheat-based products in populations worldwide, increasing the β-carotene content in wheat grains could significantly impact VAD. Although carotenoids are one of the major pigments that affect the nutritional value of wheat [<xref ref-type="bibr" rid="CR19">19</xref>], wheat grains have a very low carotenoid content and mainly accumulate lutein, which lacks provitamin A activity. To improve the carotenoid or provitamin A content in wheat, the detailed regulation of carotenoid biosynthesis must be clarified. So far, the polyploid characteristics and huge size of the wheat genome have been substantial barriers to identifying and cloning key carotenoid biosynthetic genes. Only a few carotenoid biosynthetic enzyme genes such as phytoene synthase (<italic>PSY</italic>) and <italic>LCYE</italic> have been identified [<xref ref-type="bibr" rid="CR20">20</xref>-<xref ref-type="bibr" rid="CR23">23</xref>]. Therefore, identifying and cloning more genes in the wheat carotenoid biosynthetic pathway will provide more information about carotenoid biosynthesis and its regulatory mechanism. According to the latest research, about 50% of the genome of hexaploid wheat has now been sequenced [<xref ref-type="bibr" rid="CR24">24</xref>]. Although gene cloning will become easier and more precise after sequencing is completed in the future, there is still plenty of work to be done and many difficulties to be overcome. Recently, we found that endogenous <italic>LCYB</italic> was up-regulated by the co-expression of <italic>CrtB</italic> and <italic>CrtI</italic> in transgenic wheat, which resulted in an increase in the total carotenoid and provitamin A contents [<xref ref-type="bibr" rid="CR25">25</xref>]. However, there is no documented research about this key cyclase gene’s function and its relationship with β-carotene content in wheat. Therefore, the objectives of this study were to clone <italic>TaLCYB</italic> and characterize its function and relationship with β-carotene biosynthesis in the wheat grain. We also aimed to obtain more information about the endogenous carotenoid biosynthetic pathway and thus provide experimental support for carotenoid metabolic engineering in wheat.</p></sec><sec id="Sec2" sec-type="results"><title>Results</title><sec id="Sec3"><title>Cloning and sequence analysis of <italic>TaLCYB</italic></title><p>A 1,455 bp full-length cDNA of <italic>LCYB</italic> from common wheat was isolated through an <italic>in silico</italic> cloning strategy. The full-length cDNA of the <italic>LCYB</italic> gene was designated <italic>TaLCYB</italic> (GenBank Accession No.: JN622196.1). Comparison of the obtained cDNA sequence with the gDNA sequences of wheat revealed an intronless structure. Based on the latest database of the International Wheat Genome Sequencing Consortium, <italic>TaLCYB</italic> was localized on 6AS and 6DS (<ext-link ext-link-type="uri" xlink:href="https://urgi.versailles.inra.fr/blast/blast.php">https://urgi.versailles.inra.fr/blast/blast.php</ext-link>). The ORF encoded a polypeptide of 484 amino acid residues with a predicted relative molecular mass of 53.3 kDa containing a predicted plastid transit peptide of 30 amino acids. Multiple alignment showed that TaLCYB shared a significant degree of sequence identify with other LCYB proteins in monocots (86.3% sequence identity with OsLCYB from <italic>O. sativa</italic>, 86.2% with ZmLCYB from <italic>Z. mays</italic>), and relatively lower homology with LCYB proteins from dicot species, such as <italic>C. annuum</italic>, <italic>A. thaliana</italic>, <italic>S. lycopersicum</italic> (67.4%, 67.1% and 66.5% respectively). Conserved motifs analysis (Figure <xref rid="Fig2" ref-type="fig">2</xref>A) showed a conserved β-LCY region, a dinucleotide-binding signature, a LCY-specific motif, cyclase motifs I and II, a charged region, two predicted TM helices and three β-LCY CAD regions (Catalytic Activity Domain). These domains were shown to be essential for LCYB activity [<xref ref-type="bibr" rid="CR10">10</xref>,<xref ref-type="bibr" rid="CR26">26</xref>,<xref ref-type="bibr" rid="CR27">27</xref>]. A phylogenetic tree was constructed based on the amino acid sequence alignment of TaLCYB and five other plant LCYBs from GenBank (Figure <xref rid="Fig2" ref-type="fig">2</xref>B). These results suggested that TaLCYB isolated from wheat was a genuine member of the plant lycopene β-cyclase family.<fig id="Fig2"><label>Figure 2</label><caption><p>Comparative alignment and phylogenetic tree of lycopene β-cyclase. <bold>(A)</bold> The alignment was created using ClustalW. The amino acid residues which are identical in all sequences are shown in white text on a black background, whereas different residues are shown in black text on a white background. Characteristic regions of plant LCYBs are indicated under the LCYB sequence: Conserved LCYB region, Di-nucleotide binding site, LCY’s specific motif, Cyclase motifs (CM) I and II, Predicted TM helices, Charged region and β-LCY CAD (Catalytic Activity Domain). AtLCYB: <italic>A. thaliana</italic> lycopene β-cyclase; CaLCYB: <italic>C. annuum</italic> lycopene β-cyclase; SlLCYB: <italic>S. lycopersicum</italic> lycopene β-cyclase; ZmLCYB: <italic>Z. mays</italic> lycopene β-cyclase; OsLCYB: <italic>O. sativa</italic> lycopene β-cyclase. <bold>(B)</bold> The multiple alignments were generated by ClustalW and the phylogenetic tree was constructed with MEGA4.0 using a bootstrap test of phylogeny with minimum evolution test and default parameters.</p></caption><graphic xlink:href="12870_2015_514_Fig2_HTML" id="MO2"></graphic></fig></p></sec><sec id="Sec4"><title>Functional characterization of TaLCYB in <italic>E. coli</italic></title><p>While the LCYB conserved motifs gave an indication of the enzymatic function of the encoded protein, they could not fully determine or reflect its cyclic function <italic>in vivo</italic>. To investigate the function of TaLCYB, an <italic>in vivo</italic> analysis using <italic>E. coli</italic> BL21 was conducted. <italic>TaLCYB</italic> was cloned into pET32α(+), which was then designated pET32-LCYB. <italic>E. coli</italic> strain BL21 was co-transformed with pAC-LYC, which contains genes for lycopene biosynthesis, and pET32-LCYB. Carotenoids were extracted from the bacterial cells and analyzed by High Performance Liquid Chromatography (HPLC). As shown in Figure <xref rid="Fig3" ref-type="fig">3</xref>, HPLC analysis of BL21 extracts showed that the strains containing pAC-LYC or pAC-LYC + pET32α(+) exhibited a single peak, whose retention time and absorbance spectrum corresponded to lycopene, and the cultures appeared pink. In contrast, extracts from pAC-LYC + pET32-LCYB cells mainly accumulated β-carotene, the cultures turned yellow with an undefined peak (maybe an isomer of β-carotene), and lycopene was virtually undetectable. These results demonstrated that TaLCYB was a functional β-cyclase in the carotenoid biosynthetic pathway in <italic>E. coli</italic>, which could convert lycopene to β-carotene.<fig id="Fig3"><label>Figure 3</label><caption><p>Reverse phase HPLC analysis of carotenoids accumulated in <italic>E. coli</italic> BL21 strain complemented with <italic>TaLCYB</italic>. Carotenoids were extracted from suspension cultures of cells with different plasmids <bold>(A)</bold> plasmids pAC-LYC; <bold>(B)</bold> pAC-LYC + pET32-LCYB; <bold>(C)</bold> pAC-LYC + pET32.</p></caption><graphic xlink:href="12870_2015_514_Fig3_HTML" id="MO3"></graphic></fig></p></sec><sec id="Sec5"><title><italic>TaLCYB</italic> is expressed in different tissues including developing grains of common wheat</title><p>To assess the spatial and temporal expression patterns of <italic>TaLCYB</italic> in different wheat tissues, quantitative PCR (qPCR) was carried out with RNA extracted from leaves, stems, roots, pistils, stamens and five developmental stages of grains: Grain 1 (4–10 days after pollination (DAP)), Grain 2 (10–16 DAP), Grain 3 (16–20 DAP), Grain 4 (20–25 DAP) and Grain 5 (25–35 DAP). As shown in Figure <xref rid="Fig4" ref-type="fig">4</xref>, <italic>TaLCYB</italic> was expressed in all of these tissues. The highest expression of the <italic>TaLCYB</italic> gene was observed in the leaf followed by the stamen, pistil, stem and root. In developing grains, it was interesting that the expression of <italic>TaLCYB</italic> always remained at a relatively high level, particularly at the later stages. <italic>TaLCYB</italic> expression peaked (15-fold) at 20–25 DAP (Grain 4) in common wheat and then decreased, but still remained at 4.1-fold in Grain 5 when compared with Grain 1 (Figure <xref rid="Fig4" ref-type="fig">4</xref>B).<fig id="Fig4"><label>Figure 4</label><caption><p>Expression patterns of wheat <italic>TaLCYB</italic> revealed by qRT-PCR analysis. <bold>(A)</bold> Expression patterns of <italic>TaLCYB</italic> in different tissues. <bold>(B)</bold> Expression levels of the endogenous carotenoid biosynthetic genes in developing grains of common wheat. Poly(A)<sup>+</sup> mRNA of 200 ng was subjected to reverse transcription, and served as the qPCR template. qPCR results for each gene were performed in three biological replicates with three technical repeats each and all data are shown as Mean ± SEM. Single asterisk indicates significant differences in the expression levels between controls at <italic>P</italic> = 0.05 probability level.</p></caption><graphic xlink:href="12870_2015_514_Fig4_HTML" id="MO4"></graphic></fig></p></sec><sec id="Sec6"><title>Common wheat carotenoid accumulation presents a decreasing tendency in developing grains</title><p>The carotenoid composition of wheat grains at different developmental stages was determined by HPLC analysis. As shown in Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S1, detailed HPLC analysis revealed a decreasing tendency in total carotenoid content during grain development. The β-carotene content in the wheat grains also showed a decreasing tendency, despite <italic>TaLCYB</italic> expression remaining at a high level during later developmental stages. Carotenoid pigments, encompassing lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene, were measured and their concentrations all decreased progressively during grain development. At the last stage, the main carotenoids were lutein, zeaxanthin and β-carotene (Table <xref rid="Tab1" ref-type="table">1</xref>).<table-wrap id="Tab1"><label>Table 1</label><caption><p><bold>Cartenoids content and compositions in developing grains</bold></p></caption><table frame="hsides" rules="groups"><thead><tr valign="top"><th rowspan="2"><bold>Grain</bold></th><th><bold>Lutein</bold></th><th><bold>Zeaxanthin</bold></th><th><bold>β-cryptoxanthin</bold></th><th><bold>α-carotene</bold></th><th><bold>β-carotene</bold></th><th><bold>Total carotenoid</bold></th></tr><tr valign="top"><th><bold>(μ</bold><bold>g g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th></tr></thead><tbody><tr valign="top"><td>1</td><td>5.52 ± 0.61a (60%)</td><td>0.60 ± 0.09a (7%)</td><td>0.27 ± 0.03a (3%)</td><td>0.36 ± 0.05a (4%)</td><td>2.35 ± 0.31a (26%)</td><td char="±" align="char">9.10 ± 1.82a</td></tr><tr valign="top"><td>2</td><td>4.70 ± 0.42b (59%)</td><td>0.56 ± 0.08a (7%)</td><td>0.23 ± 0.02a (3%)</td><td>0.33 ± 0.04a (4%)</td><td>2.12 ± 0.29b (27%)</td><td char="±" align="char">7.94 ± 0.95b</td></tr><tr valign="top"><td>3</td><td>3.80 ± 0.88c (62%)</td><td>0.40 ± 0.06b (6%)</td><td>0.17 ± 0.02b (3%)</td><td>0.32 ± 0.04a (5%)</td><td>1.47 ± 0.21c (24%)</td><td char="±" align="char">6.16 ± 0.80c</td></tr><tr valign="top"><td>4</td><td>1.93 ± 0.29d (50%)</td><td>0.22 ± 0.03c (6%)</td><td>0.08 ± 0.01c (2%)</td><td>0.31 ± 0.03a (8%)</td><td>1.32 ± 0.15d (34%)</td><td char="±" align="char">3.86 ± 0.54d</td></tr><tr valign="top"><td>5</td><td>0.52 ± 0.08e (53%)</td><td>0.17 ± 0.03c (17%)</td><td>ND</td><td>ND</td><td>0.29 ± 0.03e (30%)</td><td char="±" align="char">0.98 ± 0.16e</td></tr></tbody></table><table-wrap-foot><p>Data represent the average carotenoid content (±SEM) of grains from five individual ears per line. Different letters indicate significant differences (<italic>P</italic> = 0.05) in carotenoid pigment content were determined by Tukey’s HSD test. Values in parentheses represent the percentages of each carotenoid composition relative to the total content. ND = not detected.</p></table-wrap-foot></table-wrap></p><p>In parallel with the carotenoid content analysis, the expression of carotenogenic genes in developing wheat grains was also analyzed. As shown in Figure <xref rid="Fig4" ref-type="fig">4</xref>B, <italic>TaPSY</italic>, <italic>TaPDS</italic>, <italic>TaZDS</italic> and <italic>TaHYD2</italic> showed similar expression patterns in developing grains, presenting a declining tendency. The expression pattern of <italic>TaLCYE</italic> was similar to that of <italic>TaLCYB</italic>; both maintained a relatively high expression level in all development stages (Grains 1–5). <italic>TaHYD1</italic> expression showed a decline in Grains 1–3, but was up-regulated in Grains 4–5. In the last two stages, carotenogenic gene expression was dramatically reduced in comparison with the early stages, except that <italic>TaLCYE</italic> and <italic>TaLCYB</italic> were slightly down-regulated and <italic>TaHYD1</italic> was up-regulated. Overall, the carotenogenic genes had relatively stable expression levels in the early stages (Grains 1–3). This suggested that carotenoids were synthesized at a stable rate during the early stages.</p></sec><sec id="Sec7"><title>Expression patterns of <italic>TaLCYB</italic> under different abiotic stresses and their effects on β-carotene accumulation</title><p>qPCR was performed to analyze the expression level of <italic>TaLCYB</italic> under different abiotic stresses, such as strong light, darkness and cold. As shown in Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S2, <italic>TaLCYB</italic> transcripts were up-regulated by strong light and cold, and inhibited by darkness. Under strong light conditions, dramatic and fast changes in <italic>TaLCYB</italic> expression were observed. The expression of <italic>TaLCYB</italic> reached a peak (about 8.5-fold) after 4 h under strong light treatment. By contrast, changes in <italic>TaLCYB</italic> expression were more gradual under cold, with the highest expression (1.4-fold) at 8 h after treatment. As the expression of <italic>TaLCYB</italic> was dramatically induced by strong light, the carotenoid profiles of leaves at different treatment times were analyzed by HPLC (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S3). Notably, the β-carotene content variation seemed concurrent with changes of <italic>TaLCYB</italic> expression; the highest expression level corresponded to the maximum β-carotene content (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S2B). The expression of other upstream genes in the pathway such as <italic>TaPSY</italic>, <italic>TaPDS</italic>, <italic>TaZDS</italic> and <italic>TaLCYE</italic> was also up-regulated by strong light (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S4). These results suggested a correlation between <italic>TaLCYB</italic> expression and β-carotene content in wheat.</p></sec><sec id="Sec8"><title><italic>TaLCYB</italic> RNAi increases lycopene by decreasing β-carotene accumulation in the seeds</title><p>To explore the function of <italic>TaLCYB</italic> in the wheat carotenoid biosynthetic pathway, an RNAi vector was constructed and transformed into wheat (cv. Chinese Spring). After herbicide-selective regeneration, positive transgenic wheat lines were screened out in the T<sub>0</sub> generation by specific PCR-amplification of both the <italic>bar</italic> gene sequence and vector (sense-intron) sequence. Three transgenic lines and several lines only transformed with the pAHC25 plasmid were obtained; the latter lines were regarded as vector control lines (VC). Self-pollination of the PCR-positive transgenic plants in subsequent generations led to the identification of non-segregant RNAi transgenic lines. HPLC analysis of carotenoids showed no distinction in the carotenoid composition between the VC and wild-type. The carotenoid profiles and total carotenoid content of the transgenic lines differed from the wild-type. However, transgenic line BI-2 did not show any changes in carotenoid content or profile compared with the wild-type. Several novel carotenoids were observed in the transgenic wheat lines including lycopene, β-cryptoxanthin and α-carotene (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Table S1). To further analyze the carotenoid profiles, detailed HPLC analysis was carried out on the T<sub>3</sub> generation, which showed significant differences in carotenoid content and composition in seeds between transgenic and control lines, implying profound changes in the carotenoid biosynthetic pathways of the transgenic lines (Figure <xref rid="Fig5" ref-type="fig">5</xref>). The total carotenoid content slightly decreased to 0.84 μg g<sup>−1</sup> seed dry weight in BI-6 and 0.75 μg g<sup>−1</sup> seed dry weight in BI-9 compared with the wild-type (0.96 μg g<sup>−1</sup>). In these two lines, consistent with the hypothesized silencing of <italic>TaLCYB</italic> genes, the β-carotene content decreased to 0.16 μg g<sup>−1</sup> and 0.09 μg g<sup>−1</sup> compared with the wild-type (0.22 μg g<sup>−1</sup>). Lycopene is the immediate precursor of lycopene β-cyclase, and was accumulated to 0.22 μg g<sup>−1</sup> and 0.39 μg g<sup>−1</sup> in BI-6 and BI-9, respectively. Because LCYB participates in the biosynthesis of lutein, the lutein content was also decreased to 0.22 μg g<sup>−1</sup> and 0.18 μg g<sup>−1</sup> in BI-6 and BI-9, respectively, compared with 0.59 μg g<sup>−1</sup> in the wild-type (Table <xref rid="Tab2" ref-type="table">2</xref>).<fig id="Fig5"><label>Figure 5</label><caption><p>HPLC characterization of carotenoids extracted from grains of T<sub>3</sub> transgenic and control wheat. <bold>(A)</bold> BI-9; <bold>(B)</bold> BI-6; <bold>(C)</bold> BI-2; <bold>(D)</bold> VC-10 (transgenic vector control); <bold>(E)</bold> Chinese Spring (wild-type); Peak 1, lutein; Peak 2, zeaxanthin; Peak 3, β-cryptoxanthin; Peak 4, α-carotene; Peak 5, <italic>trans</italic>-β-carotene; Peak 6, 9-<italic>cis</italic>-β-carotene; Peak 7, undefined carotene; Peak 8,9, <italic>cis</italic>-lycopene; Peak 10, <italic>trans</italic>-lycopene.</p></caption><graphic xlink:href="12870_2015_514_Fig5_HTML" id="MO5"></graphic></fig><table-wrap id="Tab2"><label>Table 2</label><caption><p><bold>Cartenoids content and compositions in grains from transgenic wheat</bold></p></caption><table frame="hsides" rules="groups"><thead><tr valign="top"><th rowspan="2"></th><th><bold>Lutein</bold></th><th><bold>Zeaxanthin</bold></th><th><bold>Lycopene</bold></th><th><bold>α-carotene</bold></th><th><bold>β-carotene</bold></th><th><bold>β-cryptoxanthin</bold></th></tr><tr valign="top"><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th><th><bold>(</bold><bold>μg g</bold><sup><bold>−1</bold></sup><bold>)</bold></th></tr></thead><tbody><tr valign="top"><td>Bobwhite</td><td>0.59 ± 0.07a</td><td>0.15 ± 0.017a</td><td>ND</td><td>ND</td><td>0.22 ± 0.025a</td><td>ND</td></tr><tr valign="top"><td>VC</td><td>0.57 ± 0.08a</td><td>0.17 ± 0.015a</td><td>ND</td><td>ND</td><td>0.24 ± 0.03a</td><td>ND</td></tr><tr valign="top"><td>BI-2-9</td><td>0.51 ± 0.06a</td><td>0.18 ± 0.02a</td><td>ND</td><td>ND</td><td>0.26 ± 0.03a</td><td>ND</td></tr><tr valign="top"><td>BI-6-1</td><td>0.22 ± 0.02b</td><td>0.13 ± 0.018a</td><td>0.22 ± 0.028b</td><td>0.06 ± 0.016a</td><td>0.16 ± 0.017b</td><td>0.04 ± 0.015</td></tr><tr valign="top"><td>BI-9-5</td><td>0.18 ± 0.015c</td><td>0.05 ± 0.014b</td><td>0.39 ± 0.06a</td><td>0.05 ± 0.016a</td><td>0.09 ± 0.022c</td><td>ND</td></tr></tbody></table><table-wrap-foot><p>Carotenoid composition in wheat grains from transgenic and control lines in T<sub>3</sub> generation. Average of each carotenoid species are determined from five individual plants ears per line. Data represent the average carotenoid content (±SEM) of grains from five individual ears per line. Different letters indicate significant differences (<italic>P</italic> = 0.05) in carotenoid pigment content were determined by Tukey’s HSD test. ND = not detected.</p></table-wrap-foot></table-wrap></p></sec><sec id="Sec9"><title>Decreased β-carotene content in transgenic wheat was due to down-regulation of <italic>TaLCYB</italic></title><p>Transcriptional regulation of carotenogenic genes is a crucial regulatory mechanism of carotenoid accumulation in plants. A post-transcriptional gene silencing strategy was used to verify the function of <italic>TaLCYB</italic>. The β-carotene and lutein contents were demonstrated to be reduced through HPLC analysis. The expression levels of the endogenous carotenogenic genes were thus analyzed in both endosperms and leaves from transgenic and control lines to investigate whether the decrease of β-carotene and lutein content in transgenic lines was related with carotenogenic gene expression. As shown in Figure <xref rid="Fig6" ref-type="fig">6</xref>, transgenic line BI-2, VC and the wild-type showed similar expression levels for all carotenogenic genes in the endosperm. In transgenic lines BI-6 and BI-9, the expression of <italic>TaLCYB</italic> showed a 70% and 84% reduction, respectively. This was consistent with the HPLC results that lower expression of <italic>TaLCYB</italic> accompanied decreased β-carotene content. In these transgenic lines, the expression levels of <italic>TaZDS</italic>, <italic>TaLCYE</italic> and <italic>TaHYD1</italic> were up-regulated. The expression of <italic>TaPSY</italic> showed slight suppression, which was possibly correlated with the decrease in total carotenoids. The remaining carotenogenic genes such as <italic>TaPDS</italic> and <italic>TaHYD2</italic> appeared to be unaffected by the reduced <italic>TaLCYB</italic> expression.<fig id="Fig6"><label>Figure 6</label><caption><p>Expression levels of the endogenous carotenoid biosynthetic genes in endosperms and leaf from transgenic and control wheat lines. Gene expression levels were measured by qPCR and are determined relative to the transcript levels of the constitutively expressed β-actin gene in the same samples. Expression levels of these genes for the transformed lines are given as expression levels relative to the values for non-transformed control line Chinese Spring (CS). qPCR results for each gene were performed in three biological replicates with three technical repeats each and all data are shown as Mean ± SEM. Single asterisk and double asterisk indicate significant differences in the expression levels between control CS and transgenic lines at <italic>P =</italic> 0.05 or <italic>P =</italic> 0.01 probability level, respectively.</p></caption><graphic xlink:href="12870_2015_514_Fig6_HTML" id="MO6"></graphic></fig></p><p>Endogenous carotenogenic genes from the transgenic lines were much less affected in leaves than in the endosperm. In transgenic lines BI-6 and BI-9, the expression of <italic>TaLCYB</italic> was down-regulated, the other cyclase <italic>TaLCYE</italic> was up-regulated, and <italic>TaHYD2</italic> was slightly down-regulated. In the leaves of transgenic line BI-2, the expression of all carotenogenic genes showed the same transcriptional levels as the VC line and wild-type (Figure <xref rid="Fig6" ref-type="fig">6</xref>B). The carotenoid composition and content in the leaves of transgenic and control lines were also analyzed by HPLC; there was no distinction between the transgenic lines and control lines (data not shown).</p></sec></sec><sec id="Sec10" sec-type="discussion"><title>Discussion</title><p>Because of the nutritional value and health benefits of carotenoids, there have been many attempts to improve the carotenoid content in staple crops by metabolic engineering, especially the β-carotene content. For example, the transgenic cereal ‘Golden Rice 2’ was developed because of the deficiency of β-carotene in rice grains [<xref ref-type="bibr" rid="CR28">28</xref>], in which the endogenous LCYB plays a crucial role in β-carotene biosynthesis. However, owing to the complexity of the wheat genome, there are limited reports on carotenoid metabolic engineering to improve the carotenoid content in wheat. One of the major limitations to metabolic engineering in wheat is the lack of a fully elucidated carotenoid biosynthetic pathway [<xref ref-type="bibr" rid="CR25">25</xref>,<xref ref-type="bibr" rid="CR28">28</xref>-<xref ref-type="bibr" rid="CR30">30</xref>]. Additionally, only a few related genes have been cloned and characterized in wheat because of its complicated and huge genome, which seriously hinders the understanding of carotenoid biosynthesis in wheat. Therefore, cloning and analyzing carotenoid biosynthetic genes in wheat is very important to elucidate the carotenoid biosynthesis pathway and to improve its nutritional value by metabolic engineering. In this study, a novel wheat gene, <italic>TaLCYB</italic>, was identified and characterized to function as a lycopene β-cyclase. Its relationship to carotenoid biosynthesis was also investigated, in particular to β-carotene biosynthesis.</p><sec id="Sec11"><title>TaLCYB has β-lycopene cyclase function according to bioinformatics analysis and heterologous complementation in <italic>E. coli</italic></title><p>To provide more information about <italic>TaLCYB</italic>, the TaLCYB protein was analyzed by comparing its amino acid sequence with other LCYBs from monocot (OsLCYB and ZmLCYB) and dicot species (CaLCYB, AtLCYB and SlLCYB) [<xref ref-type="bibr" rid="CR11">11</xref>,<xref ref-type="bibr" rid="CR31">31</xref>-<xref ref-type="bibr" rid="CR33">33</xref>]. The amino acid sequence analysis revealed that TaLCYB contains all the conserved domains of plant LCYBs (Figure <xref rid="Fig2" ref-type="fig">2</xref>A). For instance, β-LCY CAD regions, which have been reported as crucial to LCYB catalytic activity, were found in TaLCYB. The “Conserved region β-LCY” was also found in TaLCYB, which is regarded as a crucial factor for the association of LCYB with membrane components and for its catalytic activity. These conserved motifs showed a high degree of conservation in amino acids between monocot and dicot species (Figure <xref rid="Fig2" ref-type="fig">2</xref>B) [<xref ref-type="bibr" rid="CR10">10</xref>,<xref ref-type="bibr" rid="CR26">26</xref>,<xref ref-type="bibr" rid="CR27">27</xref>]. However, the sequence analysis only gave a preliminary indication of the function of TaLCYB. Therefore, a heterologous complementation system was used to verify its function <italic>in vivo</italic>. This method has been proved to be efficient for functional characterization of carotenoid biosynthetic genes [<xref ref-type="bibr" rid="CR8">8</xref>]. Consistent with the results of sequence analysis, TaLCYB was demonstrated to be a functional β-cyclase enzyme <italic>in vivo</italic>, converting lycopene to β-carotene (Figure <xref rid="Fig3" ref-type="fig">3</xref>).</p></sec><sec id="Sec12"><title>The endogenous expression level of <italic>TaLCYB</italic> is not positively correlated with β-carotene accumulation in developing grains</title><p>The tissue specificity of gene expression usually mirrors the function of the corresponding gene products in plant development. Maximal expression in leaves should be associated with the photosynthetic system and photo-protective function where carotenoids are a key component [<xref ref-type="bibr" rid="CR34">34</xref>,<xref ref-type="bibr" rid="CR35">35</xref>]. In developing grains, it was found that <italic>TaLCYB</italic> reached its highest expression at 20–25 DAP (Grain 4), suggesting that at later developmental stages grains may also have high ability to synthesize β-carotene compared with the early stages. The decreasing tendency of β-carotene content meant that increased β-ring cyclization capacity did not present as a large amount of β-carotene accumulation in developing wheat grains (from 2.35 μg g<sup>−1</sup> to 0.29 μg g<sup>−1</sup>). This phenomenon might be explained by the β-carotene produced by increased synthesis being rapidly transformed into downstream compounds, resulting in a net decrease in β-carotene content. The total carotenoid content in developing wheat grains also showed a decreasing trend, which was the same as in Qin <italic>et al</italic>. [<xref ref-type="bibr" rid="CR20">20</xref>]. The high expression of <italic>TaHYD1</italic> in later stages also suggested high downstream synthetic capacity to some degree, which is more preferred to the β,β-branch [<xref ref-type="bibr" rid="CR25">25</xref>]. In addition, the accumulation of carotenoids is inversely determined by the rate of carotenoid turnover, in which the activities of various carotenoid cleavage dioxygenases (<italic>CCDs</italic>) play a crucial role [<xref ref-type="bibr" rid="CR36">36</xref>]. The CCD family catabolizes the turnover of different carotenoids to apo-carotenoids in various crops, such as rice and maize [<xref ref-type="bibr" rid="CR37">37</xref>]. Experimental evidence from the expression of carotenoid cleavage dioxygenase 1 and the carotenoid content in maize endosperm demonstrates that high expression of <italic>CCD1</italic> accompanies lower carotenoid accumulation [<xref ref-type="bibr" rid="CR37">37</xref>,<xref ref-type="bibr" rid="CR38">38</xref>]. LCYB is supposed to be a key step for β,ε- and β,β-branch biosynthesis [<xref ref-type="bibr" rid="CR10">10</xref>]. In kiwifruit and papaya, the major carotenoid was controlled by the expression level of <italic>LCYB</italic> [<xref ref-type="bibr" rid="CR12">12</xref>,<xref ref-type="bibr" rid="CR13">13</xref>]. In transgenic wheat with introduced <italic>CrtB</italic> and <italic>CrtI</italic>, constant lutein content in mature grains was still maintained despite high expression of <italic>TaLCYB</italic>. The relatively higher expression of <italic>TaLCYB</italic> and <italic>TaLCYE</italic> in developing grains did not translate into accumulation of the corresponding carotenoids. All of these phenomena suggest that coordination between <italic>TaLCYB</italic> and <italic>TaLCYE</italic> expression would regulate carbon flux through different branches in the wheat carotenoid pathway. They also show that there is a mechanism resulting in a net decrease due to more carotenoid compounds entering into turnover relative to the biosynthetic capacity.</p></sec><sec id="Sec13"><title><italic>TaLCYB</italic> plays a crucial role in the β-carotene biosynthesis of wheat</title><p><italic>LCYB</italic> and <italic>LCYE</italic> determine the flux towards the β,β- and β,ε-carotenoid branches. Thus, a strategy of modulating the levels of these two competing cyclases should enable the control of carotenoid composition [<xref ref-type="bibr" rid="CR39">39</xref>]. Our results showed that the <italic>TaLCYB</italic> transcript could be regulated by silencing, which resulted in decreased β-carotene content and lycopene accumulation. In the transgenic lines BI-6 and BI-9, the β-carotene content showed obvious reduction (Table <xref rid="Tab2" ref-type="table">2</xref>). The most dramatic down-regulation of the endogenous <italic>TaLCYB</italic> gene was coincident with the lowest metabolic flux into the β,β-carotene branch in BI-9. The same results were also observed in transgenic carrot with <italic>DcLCYB1</italic> silencing, while the β-carotene content was increased by over-expression of <italic>DcLCYB1</italic> [<xref ref-type="bibr" rid="CR40">40</xref>]. Under strong light treatment, qPCR and HPLC analysis results showed that the expression level of <italic>TaLCYB</italic> and the β-carotene content in leaves presented the same change tendency. The increased biosynthesis of β-carotene was due mainly to the combined effects of <italic>TaLCYB</italic> and <italic>TaPSY</italic> up-regulation. In the present study, lycopene and α-carotene were the carotenoid composition in the silenced transgenic lines, but these were undetectable in the VC and wild-type. Lycopene and α-carotene are direct substrates or products of LCYB, which indicates that the silencing of <italic>TaLCYB</italic> simultaneously affects its upstream and downstream products. TaLCYB is also required for lutein synthesis; thus, one of the possible reasons for the decreased lutein content could be that TaLCYB is the key enzyme of lutein biosynthesis in wheat seeds. Additionally, it is also possible that changing the pigment composition could regulate the expression of enzymes. For example, the expression of <italic>TaHYD1</italic>, which is more related with β,β-branch synthesis, was up-regulated in the transgenic lines while the expression of <italic>TaHYD2</italic>, which is more related with β,ε-branch synthesis, maintained stable expression. In addition, cyclases and hydroxylases are thought to form a protein complex to function; thus, the down-regulation of <italic>TaLCYB</italic> may impair protein complex formation and influence the biosynthesis of lutein or other carotenoids [<xref ref-type="bibr" rid="CR41">41</xref>]. The accumulation of lycopene partly compensated for the decrease of β-carotene and lutein in the total carotenoid content, and also showed that carotenoid flux was a whole, opening the possibility for the metabolic engineering of compounds in the carotenoid pathway through an appropriate strategy to modulate the expression of carotenogenic genes in the carotenoid biosynthetic pathway. Generally, all these results suggested that TaLCYB acts as the key enzyme in the downstream carotenoid biosynthetic pathway and determines the β-carotene synthesis capacity.</p></sec><sec id="Sec14"><title>Down-regulation of <italic>TaLCYB</italic> transcripts affects the expression of other genes in the carotenoid biosynthetic pathway of wheat</title><p>The expression levels of related endogenous carotenogenic genes are often altered when introducing exogenous genes, and simultaneously alter the levels of carotenoids in the biosynthetic pathway. This phenomenon has been documented in tomato leaves, potato tubers and maize kernels [<xref ref-type="bibr" rid="CR29">29</xref>,<xref ref-type="bibr" rid="CR30">30</xref>,<xref ref-type="bibr" rid="CR42">42</xref>]. In the present study, the expression of endogenous carotenogenic genes was analyzed in endosperms and leaves from transgenic and control lines. In endosperms, the expression level of <italic>TaLCYB</italic> was reduced, <italic>TaHYD1</italic> and <italic>TaLCYE</italic> were up-regulated, <italic>TaHYD2</italic> maintained stable expression, and <italic>TaPSY</italic> was slightly down-regulated (Figure <xref rid="Fig6" ref-type="fig">6</xref>A). In previous research, <italic>TaHYD1</italic> was shown to be more related with β,β-branch synthesis, while <italic>TaHYD2</italic> was more related to β,ε-branch synthesis. This implies that the down-regulation of <italic>TaLCYB</italic> led to more flux to the β,ε-branch, accompanied by the occurrence of α-carotene and the accumulation of lycopene. Other intermediates such as phytoene, phytofluene or ζ-carotene upstream of the carotenoid pathway were not detected, which was probably due mainly to the expression of <italic>TaZDS</italic> and <italic>TaPDS</italic> without significantly altering compared with the wild-type. Because of insufficient TaLCYB, the expression of <italic>TaHYD2</italic> in the transgenic lines was stable. Although <italic>TaHYD1</italic> and <italic>TaLCYE</italic> were up-regulated, the repression of <italic>TaLCYB</italic> could explain the reduction of zeaxanthin and lutein. Since ubiquitous feedback or forward regulation exists in the carotenoid biosynthetic pathway and <italic>PSY</italic> has frequently been reported as the rate-limiting gene in non-green plant tissues [<xref ref-type="bibr" rid="CR28">28</xref>,<xref ref-type="bibr" rid="CR43">43</xref>-<xref ref-type="bibr" rid="CR45">45</xref>], the accumulation of lycopene may lead to feedback regulation to suppress <italic>TaPSY</italic> accompanied by a decrease in total carotenoid content, which might suggest an attenuated phytoene synthesis capacity as a consequence of <italic>TaLCYB</italic> down-regulation. These results are consistent with a previous report on the endosperm of transgenic lines that were transformed with the exogenous genes <italic>CRTB</italic> or/and <italic>CRTI</italic> [<xref ref-type="bibr" rid="CR25">25</xref>]. However, in the leaves of our transgenic lines (Figure <xref rid="Fig6" ref-type="fig">6</xref>B), carotenogenic gene transcripts and carotenoid content and composition did not show dramatic changes. There are four possible explanations for this phenomenon. (1) It is possible that the silencing efficiency was too low or that hexaploid wheat has other <italic>LCYB</italic> gene copies that might not be totally silenced by <italic>LCYB</italic> RNAi, and which might have compensated for β-ring formation. For example, there are two types of LCYBs expressed differently in the diploid species tomato; LCYB1 is active in green tissues, while LCYB2/CYCB functions only in chromoplast-containing tissues such as ripening fruit [<xref ref-type="bibr" rid="CR46">46</xref>]. The same phenomenon has been observed with the other wheat carotenoid biosynthetic genes, such as the different expression patterns of <italic>TaHYD1</italic> and <italic>TaHYD2</italic> in vegetative tissues and developing grains of wheat. Additionally, our results suggest that <italic>TaLCYB</italic> may be located on 6AS and 6DS according to the latest sequencing of hexaploid wheat [<xref ref-type="bibr" rid="CR24">24</xref>]; these results indicate that <italic>TaLCYB</italic> might have other copies in hexaploid wheat. (2) The regulatory mechanism of carotenoid biosynthesis in leaves is more stringent to prevent disruption of photosynthesis, because carotenoids are an important part of the photosynthetic apparatus [<xref ref-type="bibr" rid="CR35">35</xref>,<xref ref-type="bibr" rid="CR47">47</xref>]. (3) The enzyme activity of LCYB is sufficient for product synthesis to maintain normal photosynthesis in leaves, so incomplete silencing might not affect carotenoid biosynthesis in leaves. (4) The effects in leaves are very weak, which correlates with the low reduction in co-suppressed <italic>TaLCYB</italic>. Thus, it also appears likely that the lines with stronger co-suppression of <italic>TaLCYB</italic> than those we obtained were not viable as a critical level of photosynthetically relevant carotenoids could not be attained.</p></sec></sec><sec id="Sec15" sec-type="conclusion"><title>Conclusions</title><p>In summary, this study demonstrates that <italic>TaLCYB</italic> is a genuine carotenoid biosynthetic gene. The silencing of <italic>TaLCYB</italic> led to a decrease of β-carotene content and altered the carotenoid profile and accumulation, accompanied by changes in the expression of endogenous carotenogenic genes to varying degrees. This provides new ideas and means for improving the total carotenoid content or specific carotenoid products by metabolic engineering in wheat. For example, the combination of RNAi-induced gene silencing with overexpression of upstream synthetic genes constitutes a strategy to improve specific carotenoid products in wheat. Generally, our data demonstrate that LCYB is a key enzyme of β-carotene biosynthesis and plays an essential role in the regulation of provitamin A biosynthesis in wheat, controlling flux to the downstream carotenogenic pathway. Although the precise regulatory mechanisms of carotenoid biosynthesis in wheat need to be investigated in future, these findings increase our knowledge of carotenoid biosynthesis in wheat and provide novel implications for wheat carotenoid bioengineering.</p></sec><sec id="Sec16" sec-type="materials|methods"><title>Methods</title><sec id="Sec17"><title>Plant materials and treatments</title><p>Wheat (<italic>Triticum aestivum</italic> L. cv. Chinese Spring) plants were grown in the experimental field of Huazhong University of Science and Technology in Wuhan, China. Developing grains were collected between 5 and 35 DAP at 5-day intervals. Leave, stem, root, stamen and pistil tissues were collected from wheat plants in the field. Abiotic stresses including cold, darkness and strong light (800 μmol · m<sup>−2</sup> · s<sup>−1</sup>) were used to examine their effect on the expression of <italic>TaLCYB</italic>. The 10-day-old seedlings were transferred respectively into a dark box, cold room (4°C) or in a growth chamber at constant temperature of 25°C as a control. Strong light stress was imposed by increasing light intensity to 800 μmol · m<sup>−2</sup> · s<sup>−1</sup> photosynthetic photon flux density.</p></sec><sec id="Sec18"><title>RNA and genomic DNA isolation</title><p>Total RNA was extracted from different wheat tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA concentration and purity were analyzed by Nanodrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE). The integrity of RNA sample was assessed by a non-denaturing agarose gel analysis. Genomic DNA was isolated from wheat leaves by cetyltrimethyl ammonium bromide (CTAB) extraction method [<xref ref-type="bibr" rid="CR48">48</xref>].</p></sec><sec id="Sec19"><title>Cloning and bioinformatics analysis of <italic>TaLCYB</italic></title><p>Total RNA extracted from wheat seedlings was used to synthesize cDNAs using RevertAid™ first-strand cDNA synthesis kit (Fermentas, Lithuania). In order to identify putative lycopene cyclase genes in wheat, a BLASTN search was performed with the sequences of <italic>OsLCYB</italic> from <italic>O. sativa</italic> (GenBank Accession No.: AP005849) [<xref ref-type="bibr" rid="CR49">49</xref>], and <italic>ZmLCYB</italic> from <italic>Z. mays</italic> to identify the putative <italic>LCYB</italic> (GenBank Accession No.: AAO18661) [<xref ref-type="bibr" rid="CR11">11</xref>]. A <italic>Triticum aestivum</italic> cDNA clone (WT009_F16) from cultivar Chinese Spring showed high identity with <italic>OsLCYB</italic> and <italic>ZmLCYB</italic>. Sequence analysis by ORF Finder showed that WT009_F16 contained the full-length ORF and amplified from wheat cDNA by PCR using specific primer pairs (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Table S2). Cycling parameters for RT-PCR were: 94°C for 3 min, 30 cycles of 94/58/72°C for 30/30/90 s, respectively, and 72°C for 10 min. Purified PCR products were cloned into pMD18-T simple vector (Takara, Dalian, China) then sequenced.</p><p>Prediction of transit peptide of TaLCYB was performed using ChloroP 1.1 Prediction Server program [<xref ref-type="bibr" rid="CR50">50</xref>]. LCYBs sequences were searched at the NCBI (Bethesda, USA) and five amino acid sequences of LCYB were used for phylogenetic analysis. A phylogenetic tree was constructed by the Neighbor–Joining method [<xref ref-type="bibr" rid="CR51">51</xref>] included in the ClustalW program [<xref ref-type="bibr" rid="CR52">52</xref>] and bootstrap re-sampling analysis (1000 replicates) was performed.</p></sec><sec id="Sec20"><title>Functional characterization of wheat TaLCYB in <italic>E. coli</italic></title><p>Full-length of the <italic>TaLCYB</italic> cDNA was cloned into pET-32a+. The plasmid pET-LCYB with pAC-LYC was used to transform <italic>E. coli</italic> strain. Plasmid pAC-LYC is a pACYC184 derived vector including several carotenoid biosynthesis genes, such as geranylgeranyl pyrophosphate synthase (<italic>CrtE</italic>), phytoene synthase (<italic>CrtB</italic>), and phytoene desaturase (<italic>CrtI</italic>) [<xref ref-type="bibr" rid="CR8">8</xref>]. <italic>E. coli</italic> colonies containing pAC-LYC accumulate lycopene and appear pink. The co-transformants of plasmid pET-LCYB with pAC-LYC were plated onto LB agar medium added with chloramphenicol (50 μg ml<sup>−1</sup>) and ampicillin (100 μg ml<sup>−1</sup>). Colonies were incubated for 24 h at 37°C.</p></sec><sec id="Sec21"><title>Plasmid constructs</title><p>PCR primers were designed on the sequence of <italic>TaLCYB</italic> using the Primer 5. The pAHC25 containing the maize <italic>ubi-1</italic> promoter and the nopaline synthase terminator were used to construct RNAi vector. Fragments of 204 bp corresponding to <italic>TaLCYB</italic> were isolated by RT-PCR using specific primers with incorporated restriction sites (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Table S2). The selected fragments followed the selection strategies for RNAi in wheat [<xref ref-type="bibr" rid="CR53">53</xref>]. Cycling parameters for PCR amplification were: 35 cycles of 94/62/72°C for 30/45/30 s, respectively, and 72°C for 10 min. The amplified fragments were then subcloned into pBluescript SK plus and sequenced. The fragments recovered by <italic>Sma</italic>I and <italic>Not</italic>I digestion were cloned in the same restriction site of plasmid pAHC25. RNAi construct contained a cDNA fragment derived from <italic>TaLCYB</italic> and oriented in the sense and antisense directions at the 3’ and 5’ ends of the construct separated by an intron sequence, respectively, and the resulting plasmid was named pAHC25-LCYB-RNAi (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S5). The intron was derived from the wheat <italic>TAK14</italic> gene (AF325198).</p></sec><sec id="Sec22"><title>Wheat transformation and plant regeneration</title><p>Wheat genetic transformation was according to the bombardment method reported by Sparks [<xref ref-type="bibr" rid="CR54">54</xref>]. Wheat immature scutella (14 DAP) from Chinese Spring were transformed with the plasmids of pAHC25-LCYB-RNAi or pAHC25 as a control. The regenerated plants were screened by the herbicide phosphinotricin medium (3 mg L<sup>−1</sup>). The surviving plants were transferred to soil and grown to maturity under growth chamber conditions (22°C/16°C day/night, 16/8-h light/dark cycle and 300 μmol · m<sup>−2</sup> · s<sup>−1</sup> photosynthetic photon flux density). The regenerated plants were continued to screen by PCR amplification using gene-specific primers (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Table S2). The PCR-positive transgenic plants were self pollinated and the non-segregant lines were selected to analyze the carotenoid profiles and expression levels of carotenoid biosynthetic genes (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S6).</p></sec><sec id="Sec23"><title>qPCR analysis</title><p>The qPCR analysis was performed with the Realtime System (Bio-Rad, CFX Connect Optics Module, USA) using SuperReal PreMix Plus (SYBR Green) (FP205, Tiangen, Beijing, China). The amplification was performed with the following programme: 40 cycles of 95°C for 15 s, and 60°C for 60 s. Fluorescence was acquired at 60°C. The specificity of the unique amplification product was determined by a melt curve analysis from 55-99°C. Data were analyzed using the Lightcycler software version 4 and normalized to the expression of wheat β<italic>-actin</italic> gene as its relatively constitutive expression levels throughout wheat developmental process. The quality of the cDNA templates and PCR amplifications were verified by the analysis of negative controls without template and no-reverse transcription for each primer pair. Dissociation curve analysis was performed following qPCR and a single peak was observed for each primer pair. A portion of qPCR products was separated on agarose gels and single band at expected sizes were detected.</p></sec><sec id="Sec24"><title>Analysis of carotenoid composition by HPLC</title><p>Carotenoids in the mature wheat seeds were extracted according to Wang <italic>et al.</italic> [<xref ref-type="bibr" rid="CR25">25</xref>] with some modifications. Seed samples were ground into fine powder. One gram powder was added with 15 mL extracting solvent (hexane: acetone: ethanol, 50:25:25, v/v/v) containing 0.01% (w/v) 2,6-di-tert-butyl-methylphenol (BHT, Sigma, Shanghai, China) following sonication for 30 min (SB-5200DTN, Scientz, China), and centrifuged for 10 min at 10,000 g under 4°C (CR-21G, Himac, Japan). The colored supernatant was collected, and the residue was re-extracted several times with extraction operation until colorless. The combined supernatant was then washed three times with saturated NaCl solution until neutral, and the aqueous phase was discarded. The solvent was evaporated under nitrogen stream, the pigments were redissolved in 0.3 mL methyltert-butyl ether (MTBE) containing 0.01% (w/v) BHT. After centrifuging at 12,000 rpm under 4°C for 30 min, the sample was filtered through a 0.22 μm filter before HPLC analysis. For quantitative purpose, β<italic>-</italic>apo-8’-carotenal was added to each sample as an internal standard prior to extraction (10 μg g<sup>−1</sup> of freeze-dried sample). Carotenoids in the <italic>E. coli</italic> cells were extracted according to Alquezar [<xref ref-type="bibr" rid="CR27">27</xref>].</p><p>The sample was injected into the HPLC system. The HPLC system included a model 2996 photodiode array detection (DAD) system, a 1525 solvent delivery system, and a Breeze2 Chromatography Manager (Waters Corpora-tion, Milford, MA. Carotenoids were separated by an YMC C30 carotenoid column (150 × 4.6 mm, packing 3 μm) (Wilmington, NC, USA) at 25°C. All the eluate was under 200 to 700 nm monitoring. The solvent A was acetonitrile/methanol (3:1, v/v), containing 0.01% BHT and 0.05% triethylamine (TEA, Sigma, Shanghai, China), and solvent B was 100% MTBE, containing 0.01% BHT. The parameters of mobile-phase gradient were programmed as follows: 0–10 min, A-B (95:5); 10–19 min, A-B (86:14); 19–29 min, A-B (75:25); 29–54 min, A-B (50:50); 54–66 min, A-B (26:74) and back to the initial condition for re-equilibration. All solvents were HPLC grade (J.T. Baker, Phillipsburg, USA). Carotenoid standards for lutein, zeaxanthin, β-cryptoxanthin, α-carotene, <italic>trans</italic>-β-carotene, β-apo-8’-carotenal, <italic>trans</italic>-lycopene calibration were purchased from Sigma-Aldrich (Shanghai, China); <italic>9</italic>-<italic>cis</italic>-β-carotene was purchased from Carotenature (Lupsingen, Switzerland). These standards, and the β-apo-8’-carotenal internal standard, were used to generate standard calibration curves. Carotenoids and chlorophylls were identified by comparing the retention time and spectra with published data and then quantified from their peak areas [<xref ref-type="bibr" rid="CR55">55</xref>-<xref ref-type="bibr" rid="CR58">58</xref>].</p></sec></sec></body><back><app-group><app id="App1"><sec id="Sec25"><title>Additional file</title><p><media position="anchor" xlink:href="12870_2015_514_MOESM1_ESM.pdf" id="MOESM1"><label>Additional file 1: Figure S1.</label><caption><p>HPLC characterization of carotenoids extracted from developing grains. <bold>Figure S2.</bold> Expression patterns of wheat <italic>TaLCYB</italic> in leaves with different treatments and β-carotene content varied with strong light. <bold>Figure S3.</bold> HPLC characterization of wheat leaf’s carotenoids composition under strong light treatment. <bold>Figure S4.</bold> Expression levels of the endogenous carotenoid biosynthetic genes in leaves with strong light treatment. <bold>Figure S5.</bold> The structures of transformation plasmids (pAHC25-LCYB-RNAi and pAHC25) used in this study. <bold>Figure S6.</bold> Propagation of transgenic wheat and selection of non-segregant lines of <italic>TaLCYB</italic> silencing. <bold>Table S1.</bold> Cartenoids content and compositions in T<sub>2</sub> seeds from the transgenic and control wheat plants. <bold>Table S2</bold>. Primer sequences used in this study.</p></caption></media></p></sec></app></app-group><glossary><title>Abbreviations</title><def-list><def-item><term>BHT</term><def><p>2,6-di-tert-butyl-methylphenol</p></def></def-item><def-item><term>β-LCY CAD region</term><def><p>β-LCY Catalytic Activity Domain region</p></def></def-item><def-item><term>CCDs</term><def><p>Carotenoid cleavage dioxygenases</p></def></def-item><def-item><term>CrtB</term><def><p>Phytoene synthase</p></def></def-item><def-item><term>CrtE</term><def><p>Geranylgeranyl pyrophosphate synthase</p></def></def-item><def-item><term>CrtI</term><def><p>Phytoene desaturase</p></def></def-item><def-item><term>CTAB</term><def><p>Cetyltrimethyl ammonium bromide</p></def></def-item><def-item><term>DAD</term><def><p>Photodiode array detection</p></def></def-item><def-item><term>DAP</term><def><p>Days after pollination</p></def></def-item><def-item><term>HPLC</term><def><p>High Performance Liquid Chromatography</p></def></def-item><def-item><term>HYD</term><def><p>β-carotene hydroxylase</p></def></def-item><def-item><term>LCYB</term><def><p>Lycopene β-cyclase</p></def></def-item><def-item><term>LCYE</term><def><p>Lycopene ε-cyclase</p></def></def-item><def-item><term>MTBE</term><def><p>Methyl tert-butyl ether</p></def></def-item><def-item><term>PDS</term><def><p>Phytoene desaturase</p></def></def-item><def-item><term>PSY</term><def><p>Phytoene synthase</p></def></def-item><def-item><term>qPCR</term><def><p>Quantitative PCR</p></def></def-item><def-item><term>RNAi</term><def><p>RNA interference</p></def></def-item><def-item><term>TEA</term><def><p>Triethylamine</p></def></def-item><def-item><term>VAD</term><def><p>Vitamin A deficiency</p></def></def-item><def-item><term>VC</term><def><p>Vector control</p></def></def-item><def-item><term>ZDS</term><def><p>Zeta-carotene desaturase.</p></def></def-item></def-list></glossary><fn-group><fn><p>Jian Zeng and Cheng Wang contributed equally to this work.</p></fn><fn><p><bold>Competing interests</bold></p><p>The authors declare that they have no competing interests.</p></fn><fn><p><bold>Authors’ contributions</bold></p><p>GYH and GXY conceived the study. XC, MLZ, CHY, XTW, QW, ML, XYL and LC performed the experiments. KXL, JLC and YSW carried out the analysis. JZ, CW, GYH and GXY designed the experiments and wrote the manuscript. All authors read and approved this submitted manuscript.</p></fn><fn><p><bold>Authors’ information</bold></p><p>The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China</p></fn></fn-group><ack><title>Acknowledgements</title><p>We thank Dr. F. X. Cunningham, University of Maryland, for providing the complementation plasmid pAC-LCY. This work was supported by International S & T Cooperation Key Projects of MoST (Grant no. 2009DFB30340), National Genetically Modified New Varieties of Major Projects of China (Grant no. 2013ZX08002004-007 and 2014ZX08010004), the National Natural Science Foundation of China (no. 31071403 and no. 31371614), Research Fund for the Doctoral Program of Higher Education of China (Grant no. 2012014211075) and Open Research Fund of State Key Laboratory of Hybrid Rice in Wuhan University (Grant no. KF201302).</p></ack><ref-list id="Bib1"><title>Reference</title><ref id="CR1"><label>1.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bramley</surname><given-names>PM</given-names></name></person-group><article-title>Regulation of carotenoid formation during tomato fruit ripening and development</article-title><source>J Exp Bot</source><year>2002</year><volume>53</volume><issue>377</issue><fpage>2107</fpage><lpage>13</lpage><pub-id pub-id-type="doi">10.1093/jxb/erf059</pub-id><pub-id pub-id-type="pmid">12324534</pub-id></element-citation></ref><ref id="CR2"><label>2.</label><mixed-citation publication-type="other">Britton G. Functions of intact carotenoids. In: <italic>carotenoids.</italic> Springer. 2008. 189–212.</mixed-citation></ref><ref id="CR3"><label>3.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fraser</surname><given-names>PD</given-names></name><name><surname>Bramley</surname><given-names>PM</given-names></name></person-group><article-title>The biosynthesis and nutritional uses of carotenoids</article-title><source>Prog Lipid Res</source><year>2004</year><volume>43</volume><issue>3</issue><fpage>228</fpage><lpage>65</lpage><pub-id pub-id-type="doi">10.1016/j.plipres.2003.10.002</pub-id><pub-id pub-id-type="pmid">15003396</pub-id></element-citation></ref><ref id="CR4"><label>4.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Clagett-Dame</surname><given-names>M</given-names></name><name><surname>Knutson</surname><given-names>D</given-names></name></person-group><article-title>Vitamin A in reproduction and development</article-title><source>Nutrients</source><year>2011</year><volume>3</volume><issue>4</issue><fpage>385</fpage><lpage>428</lpage><pub-id pub-id-type="doi">10.3390/nu3040385</pub-id><pub-id pub-id-type="pmid">22254103</pub-id></element-citation></ref><ref id="CR5"><label>5.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Abdel-Aal</surname><given-names>E-SM</given-names></name><name><surname>Akhtar</surname><given-names>H</given-names></name><name><surname>Zaheer</surname><given-names>K</given-names></name><name><surname>Ali</surname><given-names>R</given-names></name></person-group><article-title>Dietary sources of lutein and zeaxanthin carotenoids and their role in eye health</article-title><source>Nutrients</source><year>2013</year><volume>5</volume><issue>4</issue><fpage>1169</fpage><lpage>85</lpage><pub-id pub-id-type="doi">10.3390/nu5041169</pub-id><pub-id pub-id-type="pmid">23571649</pub-id></element-citation></ref><ref id="CR6"><label>6.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hirschberg</surname><given-names>J</given-names></name></person-group><article-title>Carotenoid biosynthesis in flowering plants</article-title><source>Curr Opin Plant Biol</source><year>2001</year><volume>4</volume><issue>3</issue><fpage>210</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1016/S1369-5266(00)00163-1</pub-id><pub-id pub-id-type="pmid">11312131</pub-id></element-citation></ref><ref id="CR7"><label>7.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ruiz-Sola</surname><given-names>MA</given-names></name><name><surname>Rodriguez-Concepcion</surname><given-names>M</given-names></name></person-group><article-title>Carotenoid biosynthesis in Arabidopsis: a colorful pathway</article-title><source>Arabidopsis Book</source><year>2012</year><volume>10</volume><pub-id pub-id-type="doi">10.1199/tab.0158</pub-id><pub-id pub-id-type="pmid">22582030</pub-id></element-citation></ref><ref id="CR8"><label>8.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cunningham</surname><given-names>FX</given-names></name><name><surname>Sun</surname><given-names>Z</given-names></name><name><surname>Chamovitz</surname><given-names>D</given-names></name><name><surname>Hirschberg</surname><given-names>J</given-names></name><name><surname>Gantt</surname><given-names>E</given-names></name></person-group><article-title>Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942</article-title><source>The Plant Cell Online</source><year>1994</year><volume>6</volume><issue>8</issue><fpage>1107</fpage><lpage>21</lpage><pub-id pub-id-type="doi">10.1105/tpc.6.8.1107</pub-id></element-citation></ref><ref id="CR9"><label>9.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cunningham</surname><given-names>FX</given-names></name><name><surname>Gantt</surname><given-names>E</given-names></name></person-group><article-title>One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases</article-title><source>Proc Natl Acad Sci</source><year>2001</year><volume>98</volume><issue>5</issue><fpage>2905</fpage><lpage>10</lpage><pub-id pub-id-type="doi">10.1073/pnas.051618398</pub-id><pub-id pub-id-type="pmid">11226339</pub-id></element-citation></ref><ref id="CR10"><label>10.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cunningham</surname><given-names>FX</given-names></name><name><surname>Pogson</surname><given-names>B</given-names></name><name><surname>Sun</surname><given-names>Z</given-names></name><name><surname>McDonald</surname><given-names>KA</given-names></name><name><surname>DellaPenna</surname><given-names>D</given-names></name><name><surname>Gantt</surname><given-names>E</given-names></name></person-group><article-title>Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation</article-title><source>The Plant Cell Online</source><year>1996</year><volume>8</volume><issue>9</issue><fpage>1613</fpage><lpage>26</lpage></element-citation></ref><ref id="CR11"><label>11.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Singh</surname><given-names>M</given-names></name><name><surname>Lewis</surname><given-names>PE</given-names></name><name><surname>Hardeman</surname><given-names>K</given-names></name><name><surname>Bai</surname><given-names>L</given-names></name><name><surname>Rose</surname><given-names>JK</given-names></name><name><surname>Mazourek</surname><given-names>M</given-names></name><etal></etal></person-group><article-title>Activator mutagenesis of the pink scutellum1/viviparous7 locus of maize</article-title><source>The Plant Cell Online</source><year>2003</year><volume>15</volume><issue>4</issue><fpage>874</fpage><lpage>84</lpage><pub-id pub-id-type="doi">10.1105/tpc.010249</pub-id></element-citation></ref><ref id="CR12"><label>12.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ampomah-Dwamena</surname><given-names>C</given-names></name><name><surname>McGhie</surname><given-names>T</given-names></name><name><surname>Wibisono</surname><given-names>R</given-names></name><name><surname>Montefiori</surname><given-names>M</given-names></name><name><surname>Hellens</surname><given-names>RP</given-names></name><name><surname>Allan</surname><given-names>AC</given-names></name></person-group><article-title>The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit</article-title><source>J Exp Bot</source><year>2009</year><volume>60</volume><issue>13</issue><fpage>3765</fpage><lpage>79</lpage><pub-id pub-id-type="doi">10.1093/jxb/erp218</pub-id><pub-id pub-id-type="pmid">19574250</pub-id></element-citation></ref><ref id="CR13"><label>13.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Devitt</surname><given-names>LC</given-names></name><name><surname>Fanning</surname><given-names>K</given-names></name><name><surname>Dietzgen</surname><given-names>RG</given-names></name><name><surname>Holton</surname><given-names>TA</given-names></name></person-group><article-title>Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.)</article-title><source>J Exp Bot</source><year>2010</year><volume>61</volume><issue>1</issue><fpage>33</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1093/jxb/erp284</pub-id><pub-id pub-id-type="pmid">19887502</pub-id></element-citation></ref><ref id="CR14"><label>14.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bouvier</surname><given-names>F</given-names></name><name><surname>d'Harlingue</surname><given-names>A</given-names></name><name><surname>Hugueney</surname><given-names>P</given-names></name><name><surname>Marin</surname><given-names>E</given-names></name><name><surname>Marion-Poll</surname><given-names>A</given-names></name><name><surname>Camara</surname><given-names>B</given-names></name></person-group><article-title>Xanthophyll biosynthesis cloning, expression, functional reconstitution, and regulation of β-cyclohexenyl carotenoid epoxidase from pepper (Capsicum annuum)</article-title><source>J Biol Chem</source><year>1996</year><volume>271</volume><issue>46</issue><fpage>28861</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1074/jbc.271.46.28861</pub-id><pub-id pub-id-type="pmid">8910532</pub-id></element-citation></ref><ref id="CR15"><label>15.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sandmann</surname><given-names>G</given-names></name><name><surname>Römer</surname><given-names>S</given-names></name><name><surname>Fraser</surname><given-names>PD</given-names></name></person-group><article-title>Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants</article-title><source>Metab Eng</source><year>2006</year><volume>8</volume><issue>4</issue><fpage>291</fpage><lpage>302</lpage><pub-id pub-id-type="doi">10.1016/j.ymben.2006.01.005</pub-id><pub-id pub-id-type="pmid">16621640</pub-id></element-citation></ref><ref id="CR16"><label>16.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhu</surname><given-names>C</given-names></name><name><surname>Naqvi</surname><given-names>S</given-names></name><name><surname>Gomez-Galera</surname><given-names>S</given-names></name><name><surname>Pelacho</surname><given-names>AM</given-names></name><name><surname>Capell</surname><given-names>T</given-names></name><name><surname>Christou</surname><given-names>P</given-names></name></person-group><article-title>Transgenic strategies for the nutritional enhancement of plants</article-title><source>Trends Plant Sci</source><year>2007</year><volume>12</volume><issue>12</issue><fpage>548</fpage><lpage>55</lpage><pub-id pub-id-type="doi">10.1016/j.tplants.2007.09.007</pub-id><pub-id pub-id-type="pmid">18006362</pub-id></element-citation></ref><ref id="CR17"><label>17.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fraser</surname><given-names>PD</given-names></name><name><surname>Enfissi</surname><given-names>E</given-names></name><name><surname>Bramley</surname><given-names>PM</given-names></name></person-group><article-title>Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches</article-title><source>Arch Biochem Biophys</source><year>2009</year><volume>483</volume><issue>2</issue><fpage>196</fpage><lpage>204</lpage><pub-id pub-id-type="doi">10.1016/j.abb.2008.10.009</pub-id><pub-id pub-id-type="pmid">18948075</pub-id></element-citation></ref><ref id="CR18"><label>18.</label><mixed-citation publication-type="other">Shewry P. Wheat. <italic>J Exp Bot</italic> 2009. 60(6):1537–1553.</mixed-citation></ref><ref id="CR19"><label>19.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mares</surname><given-names>DJ</given-names></name><name><surname>Campbell</surname><given-names>A</given-names></name></person-group><article-title>Mapping components of flour and noodle colour in Australian wheat</article-title><source>Crop and Pasture Science</source><year>2001</year><volume>52</volume><issue>12</issue><fpage>1297</fpage><lpage>309</lpage><pub-id pub-id-type="doi">10.1071/AR01048</pub-id></element-citation></ref><ref id="CR20"><label>20.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Qin</surname><given-names>X</given-names></name><name><surname>Zhang</surname><given-names>W</given-names></name><name><surname>Dubcovsky</surname><given-names>J</given-names></name><name><surname>Tian</surname><given-names>L</given-names></name></person-group><article-title>Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains</article-title><source>Plant Mol Biol</source><year>2012</year><volume>80</volume><issue>6</issue><fpage>631</fpage><lpage>46</lpage><pub-id pub-id-type="doi">10.1007/s11103-012-9972-4</pub-id><pub-id pub-id-type="pmid">23015203</pub-id></element-citation></ref><ref id="CR21"><label>21.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cong</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Li</surname><given-names>ZQ</given-names></name><name><surname>Chen</surname><given-names>L</given-names></name><name><surname>Yang</surname><given-names>GX</given-names></name><name><surname>Wang</surname><given-names>YS</given-names></name><etal></etal></person-group><article-title>cDNA cloning and expression analysis of wheat (Triticum aestivum L.) phytoene and zeta-carotene desaturase genes</article-title><source>Mol Biol Rep</source><year>2010</year><volume>37</volume><issue>7</issue><fpage>3351</fpage><lpage>61</lpage><pub-id pub-id-type="doi">10.1007/s11033-009-9922-7</pub-id><pub-id pub-id-type="pmid">20013057</pub-id></element-citation></ref><ref id="CR22"><label>22.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>He</surname><given-names>X</given-names></name><name><surname>He</surname><given-names>Z</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Xia</surname><given-names>X</given-names></name></person-group><article-title>Cloning and phylogenetic analysis of phytoene synthase 1 (Psy1) genes in common wheat and related species</article-title><source>Hereditas</source><year>2009</year><volume>146</volume><issue>5</issue><fpage>208</fpage><lpage>56</lpage><pub-id pub-id-type="doi">10.1111/j.1601-5223.2009.02132.x</pub-id><pub-id pub-id-type="pmid">19891741</pub-id></element-citation></ref><ref id="CR23"><label>23.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Howitt</surname><given-names>CA</given-names></name><name><surname>Cavanagh</surname><given-names>CR</given-names></name><name><surname>Bowerman</surname><given-names>AF</given-names></name><name><surname>Cazzonelli</surname><given-names>C</given-names></name><name><surname>Rampling</surname><given-names>L</given-names></name><name><surname>Mimica</surname><given-names>JL</given-names></name><etal></etal></person-group><article-title>Alternative splicing, activation of cryptic exons and amino acid substitutions in carotenoid biosynthetic genes are associated with lutein accumulation in wheat endosperm</article-title><source>Funct Integr Genomics</source><year>2009</year><volume>9</volume><issue>3</issue><fpage>363</fpage><lpage>76</lpage><pub-id pub-id-type="doi">10.1007/s10142-009-0121-3</pub-id><pub-id pub-id-type="pmid">19330366</pub-id></element-citation></ref><ref id="CR24"><label>24.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mayer</surname><given-names>KF</given-names></name><name><surname>Rogers</surname><given-names>J</given-names></name><name><surname>Doležel</surname><given-names>J</given-names></name><name><surname>Pozniak</surname><given-names>C</given-names></name><name><surname>Eversole</surname><given-names>K</given-names></name><name><surname>Feuillet</surname><given-names>C</given-names></name><etal></etal></person-group><article-title>A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome</article-title><source>Sci</source><year>2014</year><volume>345</volume><issue>6194</issue><fpage>1251788</fpage><pub-id pub-id-type="doi">10.1126/science.1251788</pub-id></element-citation></ref><ref id="CR25"><label>25.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Zeng</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Hu</surname><given-names>W</given-names></name><name><surname>Chen</surname><given-names>L</given-names></name><name><surname>Miao</surname><given-names>Y</given-names></name><etal></etal></person-group><article-title>Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI</article-title><source>J Exp Bot</source><year>2014</year><volume>65</volume><issue>9</issue><fpage>2545</fpage><lpage>56</lpage><pub-id pub-id-type="doi">10.1093/jxb/eru138</pub-id><pub-id pub-id-type="pmid">24692648</pub-id></element-citation></ref><ref id="CR26"><label>26.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hugueney</surname><given-names>P</given-names></name><name><surname>Badillo</surname><given-names>A</given-names></name><name><surname>Chen</surname><given-names>H-C</given-names></name><name><surname>Klein</surname><given-names>A</given-names></name><name><surname>Hirschberg</surname><given-names>J</given-names></name><name><surname>Camara</surname><given-names>B</given-names></name><etal></etal></person-group><article-title>Metabolism of cyclic carotenoids: A model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts</article-title><source>Plant J</source><year>1995</year><volume>8</volume><issue>3</issue><fpage>417</fpage><lpage>24</lpage><pub-id pub-id-type="doi">10.1046/j.1365-313X.1995.08030417.x</pub-id><pub-id pub-id-type="pmid">7550379</pub-id></element-citation></ref><ref id="CR27"><label>27.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alquezar</surname><given-names>B</given-names></name><name><surname>Zacarias</surname><given-names>L</given-names></name><name><surname>Rodrigo</surname><given-names>MJ</given-names></name></person-group><article-title>Molecular and functional characterization of a novel chromoplast-specific lycopene beta-cyclase from Citrus and its relation to lycopene accumulation</article-title><source>J Exp Bot</source><year>2009</year><volume>60</volume><issue>6</issue><fpage>1783</fpage><lpage>97</lpage><pub-id pub-id-type="doi">10.1093/jxb/erp048</pub-id><pub-id pub-id-type="pmid">19325166</pub-id></element-citation></ref><ref id="CR28"><label>28.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Paine</surname><given-names>JA</given-names></name><name><surname>Shipton</surname><given-names>CA</given-names></name><name><surname>Chaggar</surname><given-names>S</given-names></name><name><surname>Howells</surname><given-names>RM</given-names></name><name><surname>Kennedy</surname><given-names>MJ</given-names></name><name><surname>Vernon</surname><given-names>G</given-names></name><etal></etal></person-group><article-title>Improving the nutritional value of Golden Rice through increased pro-vitamin A content</article-title><source>Nat Biotechnol</source><year>2005</year><volume>23</volume><issue>4</issue><fpage>482</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1038/nbt1082</pub-id><pub-id pub-id-type="pmid">15793573</pub-id></element-citation></ref><ref id="CR29"><label>29.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aluru</surname><given-names>M</given-names></name><name><surname>Xu</surname><given-names>Y</given-names></name><name><surname>Guo</surname><given-names>R</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Li</surname><given-names>S</given-names></name><name><surname>White</surname><given-names>W</given-names></name><etal></etal></person-group><article-title>Generation of transgenic maize with enhanced provitamin A content</article-title><source>J Exp Bot</source><year>2008</year><volume>59</volume><issue>13</issue><fpage>3551</fpage><lpage>62</lpage><pub-id pub-id-type="doi">10.1093/jxb/ern212</pub-id><pub-id pub-id-type="pmid">18723758</pub-id></element-citation></ref><ref id="CR30"><label>30.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Diretto</surname><given-names>G</given-names></name><name><surname>Al-Babili</surname><given-names>S</given-names></name><name><surname>Tavazza</surname><given-names>R</given-names></name><name><surname>Papacchioli</surname><given-names>V</given-names></name><name><surname>Beyer</surname><given-names>P</given-names></name><name><surname>Giuliano</surname><given-names>G</given-names></name></person-group><article-title>Metabolic Engineering of Potato Carotenoid Content through Tuber-Specific Overexpression of a Bacterial Mini-Pathway</article-title><source>PLoS One</source><year>2007</year><volume>2</volume><issue>4</issue><pub-id pub-id-type="doi">10.1371/journal.pone.0000350</pub-id><pub-id pub-id-type="pmid">17406674</pub-id></element-citation></ref><ref id="CR31"><label>31.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Scolnik</surname><given-names>PA</given-names></name><name><surname>Bartley</surname><given-names>G</given-names></name></person-group><article-title>Nucleotide sequence of Lycopene Cyclase (Genbank L40176) from Arabidopsis</article-title><source>Plant Physiol</source><year>1995</year><volume>108</volume><fpage>1342</fpage></element-citation></ref><ref id="CR32"><label>32.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Guzman</surname><given-names>I</given-names></name><name><surname>Hamby</surname><given-names>S</given-names></name><name><surname>Romero</surname><given-names>J</given-names></name><name><surname>Bosland</surname><given-names>PW</given-names></name><name><surname>O’Connell</surname><given-names>MA</given-names></name></person-group><article-title>Variability of carotenoid biosynthesis in orange colored <italic>Capsicum</italic> spp</article-title><source>Plant Sci</source><year>2010</year><volume>179</volume><issue>1</issue><fpage>49</fpage><lpage>59</lpage><pub-id pub-id-type="doi">10.1016/j.plantsci.2010.04.014</pub-id><pub-id pub-id-type="pmid">20582146</pub-id></element-citation></ref><ref id="CR33"><label>33.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pecker</surname><given-names>I</given-names></name><name><surname>Gabbay</surname><given-names>R</given-names></name><name><surname>Cunningham</surname><given-names>FX</given-names><suffix>Jr</suffix></name><name><surname>Hirschberg</surname><given-names>J</given-names></name></person-group><article-title>Cloning and characterization of the cDNA for lycopene beta-cyclase from tomato reveals decrease in its expression during fruit ripening</article-title><source>Plant Mol Biol</source><year>1996</year><volume>30</volume><issue>4</issue><fpage>807</fpage><lpage>19</lpage><pub-id pub-id-type="doi">10.1007/BF00019013</pub-id><pub-id pub-id-type="pmid">8624411</pub-id></element-citation></ref><ref id="CR34"><label>34.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Demmig-Adams</surname><given-names>B</given-names></name><name><surname>Gilmore</surname><given-names>AM</given-names></name><name><surname>Adams</surname><given-names>WW</given-names><suffix>III</suffix></name></person-group><article-title>In vivo functions of carotenoids in higher plants</article-title><source>FASEB J</source><year>1996</year><volume>10</volume><issue>4</issue><fpage>403</fpage><lpage>12</lpage><pub-id pub-id-type="pmid">8647339</pub-id></element-citation></ref><ref id="CR35"><label>35.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dall'Osto</surname><given-names>L</given-names></name><name><surname>Fiore</surname><given-names>A</given-names></name><name><surname>Cazzaniga</surname><given-names>S</given-names></name><name><surname>Giuliano</surname><given-names>G</given-names></name><name><surname>Bassi</surname><given-names>R</given-names></name></person-group><article-title>Different roles of α-and β-branch xanthophylls in photosystem assembly and photoprotection</article-title><source>J Biol Chem</source><year>2007</year><volume>282</volume><issue>48</issue><fpage>35056</fpage><lpage>68</lpage><pub-id pub-id-type="doi">10.1074/jbc.M704729200</pub-id><pub-id pub-id-type="pmid">17913714</pub-id></element-citation></ref><ref id="CR36"><label>36.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nisar</surname><given-names>N</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Lu</surname><given-names>S</given-names></name><name><surname>Khin</surname><given-names>NC</given-names></name><name><surname>Pogson</surname><given-names>BJ</given-names></name></person-group><article-title>Carotenoid Metabolism in Plants</article-title><source>Mol Plant</source><year>2015</year><volume>8</volume><issue>1</issue><fpage>68</fpage><lpage>82</lpage><pub-id pub-id-type="doi">10.1016/j.molp.2014.12.007</pub-id><pub-id pub-id-type="pmid">25578273</pub-id></element-citation></ref><ref id="CR37"><label>37.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vallabhaneni</surname><given-names>R</given-names></name><name><surname>Bradbury</surname><given-names>LM</given-names></name><name><surname>Wurtzel</surname><given-names>ET</given-names></name></person-group><article-title>The carotenoid dioxygenase gene family in maize, sorghum, and rice</article-title><source>Arch Biochem Biophys</source><year>2010</year><volume>504</volume><issue>1</issue><fpage>104</fpage><lpage>11</lpage><pub-id pub-id-type="doi">10.1016/j.abb.2010.07.019</pub-id><pub-id pub-id-type="pmid">20670614</pub-id></element-citation></ref><ref id="CR38"><label>38.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>da Silva</surname><given-names>MR</given-names></name></person-group><article-title>Galli V, dos Anjos e Silva SD, Rombaldi CV: Carotenoid Biosynthetic and Catabolic Pathways: Gene Expression and Carotenoid Content in Grains of Maize Landraces</article-title><source>Nutrients</source><year>2014</year><volume>6</volume><issue>2</issue><fpage>546</fpage><lpage>63</lpage><pub-id pub-id-type="doi">10.3390/nu6020546</pub-id><pub-id pub-id-type="pmid">24476639</pub-id></element-citation></ref><ref id="CR39"><label>39.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Diretto</surname><given-names>G</given-names></name><name><surname>Tavazza</surname><given-names>R</given-names></name><name><surname>Welsch</surname><given-names>R</given-names></name><name><surname>Pizzichini</surname><given-names>D</given-names></name><name><surname>Mourgues</surname><given-names>F</given-names></name><name><surname>Papacchioli</surname><given-names>V</given-names></name><etal></etal></person-group><article-title>Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase</article-title><source>BMC Plant Biol</source><year>2006</year><volume>6</volume><fpage>13</fpage><pub-id pub-id-type="doi">10.1186/1471-2229-6-13</pub-id><pub-id pub-id-type="pmid">16800876</pub-id></element-citation></ref><ref id="CR40"><label>40.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Moreno</surname><given-names>JC</given-names></name><name><surname>Pizarro</surname><given-names>L</given-names></name><name><surname>Fuentes</surname><given-names>P</given-names></name><name><surname>Handford</surname><given-names>M</given-names></name><name><surname>Cifuentes</surname><given-names>V</given-names></name><name><surname>Stange</surname><given-names>C</given-names></name></person-group><article-title>Levels of Lycopene β-Cyclase 1 Modulate Carotenoid Gene Expression and Accumulation in Daucus carota</article-title><source>PLoS One</source><year>2013</year><volume>8</volume><issue>3</issue><pub-id pub-id-type="doi">10.1371/journal.pone.0058144</pub-id><pub-id pub-id-type="pmid">23555569</pub-id></element-citation></ref><ref id="CR41"><label>41.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cunningham</surname><given-names>FX</given-names></name><name><surname>Gantt</surname><given-names>E</given-names></name></person-group><article-title>Genes and enzymes of carotenoid biosynthesis in plants</article-title><source>Annu Rev Plant Physiol Plant Mol Biol</source><year>1998</year><volume>49</volume><fpage>557</fpage><lpage>83</lpage><pub-id pub-id-type="doi">10.1146/annurev.arplant.49.1.557</pub-id><pub-id pub-id-type="pmid">15012246</pub-id></element-citation></ref><ref id="CR42"><label>42.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rosati</surname><given-names>C</given-names></name><name><surname>Aquilani</surname><given-names>R</given-names></name><name><surname>Dharmapuri</surname><given-names>S</given-names></name><name><surname>Pallara</surname><given-names>P</given-names></name><name><surname>Marusic</surname><given-names>C</given-names></name><name><surname>Tavazza</surname><given-names>R</given-names></name><etal></etal></person-group><article-title>Metabolic engineering of beta-carotene and lycopene content in tomato fruit</article-title><source>Plant J</source><year>2000</year><volume>24</volume><issue>3</issue><fpage>413</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1046/j.1365-313x.2000.00880.x</pub-id><pub-id pub-id-type="pmid">11069713</pub-id></element-citation></ref><ref id="CR43"><label>43.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fraser</surname><given-names>PD</given-names></name><name><surname>Romer</surname><given-names>S</given-names></name><name><surname>Shipton</surname><given-names>CA</given-names></name><name><surname>Mills</surname><given-names>PB</given-names></name><name><surname>Kiano</surname><given-names>JW</given-names></name><name><surname>Misawa</surname><given-names>N</given-names></name><etal></etal></person-group><article-title>Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner</article-title><source>Proc Natl Acad Sci U S A</source><year>2002</year><volume>99</volume><issue>2</issue><fpage>1092</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1073/pnas.241374598</pub-id><pub-id pub-id-type="pmid">11805345</pub-id></element-citation></ref><ref id="CR44"><label>44.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rodríguez‐Villalón</surname><given-names>A</given-names></name><name><surname>Gas</surname><given-names>E</given-names></name><name><surname>Rodríguez‐Concepción</surname><given-names>M</given-names></name></person-group><article-title>Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in dark‐grown Arabidopsis seedlings</article-title><source>PlJ</source><year>2009</year><volume>60</volume><issue>3</issue><fpage>424</fpage><lpage>35</lpage></element-citation></ref><ref id="CR45"><label>45.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Welsch</surname><given-names>R</given-names></name><name><surname>Arango</surname><given-names>J</given-names></name><name><surname>Bär</surname><given-names>C</given-names></name><name><surname>Salazar</surname><given-names>B</given-names></name><name><surname>Al-Babili</surname><given-names>S</given-names></name><name><surname>Beltrán</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene</article-title><source>The Plant Cell Online</source><year>2010</year><volume>22</volume><issue>10</issue><fpage>3348</fpage><lpage>56</lpage><pub-id pub-id-type="doi">10.1105/tpc.110.077560</pub-id></element-citation></ref><ref id="CR46"><label>46.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ronen</surname><given-names>G</given-names></name><name><surname>Carmel-Goren</surname><given-names>L</given-names></name><name><surname>Zamir</surname><given-names>D</given-names></name><name><surname>Hirschberg</surname><given-names>J</given-names></name></person-group><article-title>An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato</article-title><source>Proc Natl Acad Sci</source><year>2000</year><volume>97</volume><issue>20</issue><fpage>11102</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1073/pnas.190177497</pub-id><pub-id pub-id-type="pmid">10995464</pub-id></element-citation></ref><ref id="CR47"><label>47.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Davison</surname><given-names>P</given-names></name><name><surname>Hunter</surname><given-names>C</given-names></name><name><surname>Horton</surname><given-names>P</given-names></name></person-group><article-title>Overexpression of β-carotene hydroxylase enhances stress tolerance in Arabidopsis</article-title><source>Nature</source><year>2002</year><volume>418</volume><issue>6894</issue><fpage>203</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1038/nature00861</pub-id><pub-id pub-id-type="pmid">12110893</pub-id></element-citation></ref><ref id="CR48"><label>48.</label><mixed-citation publication-type="other">Sambrook J, Fritsch E. Maniatis. 1989. Molecular cloning: a laboratory manual. In<italic>.</italic>: Cold Spring Harbor Laboratory, Cold Spring Harbor. NY. 1997.</mixed-citation></ref><ref id="CR49"><label>49.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname><given-names>Q</given-names></name><name><surname>Schaub</surname><given-names>P</given-names></name><name><surname>Ghisla</surname><given-names>S</given-names></name><name><surname>Al-Babili</surname><given-names>S</given-names></name><name><surname>Krieger-Liszkay</surname><given-names>A</given-names></name><name><surname>Beyer</surname><given-names>P</given-names></name></person-group><article-title>The lycopene cyclase CrtY from Pantoea ananatis (formerly Erwinia uredovora) catalyzes an FADred-dependent non-redox reaction</article-title><source>J Biol Chem</source><year>2010</year><volume>285</volume><issue>16</issue><fpage>12109</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1074/jbc.M109.091843</pub-id><pub-id pub-id-type="pmid">20178989</pub-id></element-citation></ref><ref id="CR50"><label>50.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Emanuelsson</surname><given-names>O</given-names></name><name><surname>Nielsen</surname><given-names>H</given-names></name><name><surname>Heijne</surname><given-names>GV</given-names></name></person-group><article-title>ChloroP, a neural network‐based method for predicting chloroplast transit peptides and their cleavage sites</article-title><source>Protein Sci</source><year>1999</year><volume>8</volume><issue>5</issue><fpage>978</fpage><lpage>84</lpage><pub-id pub-id-type="doi">10.1110/ps.8.5.978</pub-id><pub-id pub-id-type="pmid">10338008</pub-id></element-citation></ref><ref id="CR51"><label>51.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Saitou</surname><given-names>N</given-names></name><name><surname>Nei</surname><given-names>M</given-names></name></person-group><article-title>The neighbor-joining method: a new method for reconstructing phylogenetic trees</article-title><source>Mol Biol Evol</source><year>1987</year><volume>4</volume><issue>4</issue><fpage>406</fpage><lpage>25</lpage><pub-id pub-id-type="pmid">3447015</pub-id></element-citation></ref><ref id="CR52"><label>52.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thompson</surname><given-names>JD</given-names></name><name><surname>Higgins</surname><given-names>DG</given-names></name><name><surname>Gibson</surname><given-names>TJ</given-names></name></person-group><article-title>CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice</article-title><source>Nucleic Acids Res</source><year>1994</year><volume>22</volume><issue>22</issue><fpage>4673</fpage><lpage>80</lpage><pub-id pub-id-type="doi">10.1093/nar/22.22.4673</pub-id><pub-id pub-id-type="pmid">7984417</pub-id></element-citation></ref><ref id="CR53"><label>53.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fu</surname><given-names>D</given-names></name><name><surname>Uauy</surname><given-names>C</given-names></name><name><surname>Blechl</surname><given-names>A</given-names></name><name><surname>Dubcovsky</surname><given-names>J</given-names></name></person-group><article-title>RNA interference for wheat functional gene analysis</article-title><source>Transgenic Res</source><year>2007</year><volume>16</volume><issue>6</issue><fpage>689</fpage><lpage>701</lpage><pub-id pub-id-type="doi">10.1007/s11248-007-9150-7</pub-id><pub-id pub-id-type="pmid">17952622</pub-id></element-citation></ref><ref id="CR54"><label>54.</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Sparks</surname><given-names>C</given-names></name><name><surname>Jones</surname><given-names>H</given-names></name></person-group><source>Transformation of wheat by biolistics</source><year>2004</year><publisher-loc>In</publisher-loc><publisher-name>Transgenic Crops of the World. Springer</publisher-name><fpage>19</fpage><lpage>34</lpage></element-citation></ref><ref id="CR55"><label>55.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sander</surname><given-names>LC</given-names></name><name><surname>Sharpless</surname><given-names>KE</given-names></name><name><surname>Pursch</surname><given-names>M</given-names></name></person-group><article-title>C < sub > 30 Stationary phases for the analysis of food by liquid chromatography</article-title><source>J Chromatogr A</source><year>2000</year><volume>880</volume><issue>1</issue><fpage>189</fpage><lpage>202</lpage><pub-id pub-id-type="doi">10.1016/S0021-9673(00)00121-7</pub-id><pub-id pub-id-type="pmid">10890520</pub-id></element-citation></ref><ref id="CR56"><label>56.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fraser</surname><given-names>PD</given-names></name><name><surname>Pinto</surname><given-names>MES</given-names></name><name><surname>Holloway</surname><given-names>DE</given-names></name><name><surname>Bramley</surname><given-names>PM</given-names></name></person-group><article-title>Application of high‐performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids</article-title><source>PlJ</source><year>2000</year><volume>24</volume><issue>4</issue><fpage>551</fpage><lpage>8</lpage></element-citation></ref><ref id="CR57"><label>57.</label><mixed-citation publication-type="other">Rodriguez-Amaya D, Kimura M: Harvest plus handbook for carotenoid analysis. international Food Policy Research institute and international Center for Tropical agriculture. 2004.</mixed-citation></ref><ref id="CR58"><label>58.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>Q</given-names></name><name><surname>Xu</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Zhao</surname><given-names>X</given-names></name><name><surname>Deng</surname><given-names>X</given-names></name><name><surname>Guo</surname><given-names>L</given-names></name><etal></etal></person-group><article-title>A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck)</article-title><source>J Exp Bot</source><year>2007</year><volume>58</volume><issue>15–16</issue><fpage>4161</fpage><lpage>71</lpage><pub-id pub-id-type="doi">10.1093/jxb/erm273</pub-id><pub-id pub-id-type="pmid">18182424</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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