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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus</title><author><name sortKey="Cao, Hongbo" sort="Cao, Hongbo" uniqKey="Cao H" first="Hongbo" last="Cao">Hongbo Cao</name></author><author><name sortKey="Zhang, Jiancheng" sort="Zhang, Jiancheng" uniqKey="Zhang J" first="Jiancheng" last="Zhang">Jiancheng Zhang</name></author><author><name sortKey="Xu, Jidi" sort="Xu, Jidi" uniqKey="Xu J" first="Jidi" last="Xu">Jidi Xu</name></author><author><name sortKey="Ye, Junli" sort="Ye, Junli" uniqKey="Ye J" first="Junli" last="Ye">Junli Ye</name></author><author><name sortKey="Yun, Ze" sort="Yun, Ze" uniqKey="Yun Z" first="Ze" last="Yun">Ze Yun</name></author><author><name sortKey="Xu, Qiang" sort="Xu, Qiang" uniqKey="Xu Q" first="Qiang" last="Xu">Qiang Xu</name></author><author><name sortKey="Xu, Juan" sort="Xu, Juan" uniqKey="Xu J" first="Juan" last="Xu">Juan Xu</name></author><author><name sortKey="Deng, Xiuxin" sort="Deng, Xiuxin" uniqKey="Deng X" first="Xiuxin" last="Deng">Xiuxin Deng</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22611233</idno><idno type="pmc">3421982</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421982</idno><idno type="RBID">PMC:3421982</idno><idno type="doi">10.1093/jxb/ers115</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">001031</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus</title><author><name sortKey="Cao, Hongbo" sort="Cao, Hongbo" uniqKey="Cao H" first="Hongbo" last="Cao">Hongbo Cao</name></author><author><name sortKey="Zhang, Jiancheng" sort="Zhang, Jiancheng" uniqKey="Zhang J" first="Jiancheng" last="Zhang">Jiancheng Zhang</name></author><author><name sortKey="Xu, Jidi" sort="Xu, Jidi" uniqKey="Xu J" first="Jidi" last="Xu">Jidi Xu</name></author><author><name sortKey="Ye, Junli" sort="Ye, Junli" uniqKey="Ye J" first="Junli" last="Ye">Junli Ye</name></author><author><name sortKey="Yun, Ze" sort="Yun, Ze" uniqKey="Yun Z" first="Ze" last="Yun">Ze Yun</name></author><author><name sortKey="Xu, Qiang" sort="Xu, Qiang" uniqKey="Xu Q" first="Qiang" last="Xu">Qiang Xu</name></author><author><name sortKey="Xu, Juan" sort="Xu, Juan" uniqKey="Xu J" first="Juan" last="Xu">Juan Xu</name></author><author><name sortKey="Deng, Xiuxin" sort="Deng, Xiuxin" uniqKey="Deng X" first="Xiuxin" last="Deng">Xiuxin Deng</name></author></analytic><series><title level="j">Journal of Experimental Botany</title><idno type="ISSN">0022-0957</idno><idno type="eISSN">1460-2431</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (<italic>CrtB</italic>). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the β-carotene hydroxylase gene (<italic>HYD</italic>), which was suppressed in the calli. Co-expression of <italic>CrtB</italic> and <italic>HYD</italic> in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of β-carotene. In addition, a model is proposed to interpret the common appearance of a favoured β,β-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.</p></div></front><back><div1 type="bibliography"><listBibl><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="Bai, L" uniqKey="Bai L">L Bai</name></author><author><name sortKey="Kim, E" uniqKey="Kim E">E Kim</name></author><author><name sortKey="Dellapenna, D" uniqKey="Dellapenna D">D DellaPenna</name></author><author><name sortKey="Brutnell, T" uniqKey="Brutnell T">T Brutnell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Barsan, C" 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Exp Bot</journal-id><journal-id journal-id-type="iso-abbrev">J. Exp. Bot</journal-id><journal-id journal-id-type="hwp">jexbot</journal-id><journal-id journal-id-type="publisher-id">exbotj</journal-id><journal-title-group><journal-title>Journal of Experimental Botany</journal-title></journal-title-group><issn pub-type="ppub">0022-0957</issn><issn pub-type="epub">1460-2431</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22611233</article-id><article-id pub-id-type="pmc">3421982</article-id><article-id pub-id-type="doi">10.1093/jxb/ers115</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Paper</subject></subj-group></article-categories><title-group><article-title>Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Cao</surname><given-names>Hongbo</given-names></name><xref ref-type="author-notes" rid="fn1">*</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Jiancheng</given-names></name><xref ref-type="author-notes" rid="fn1">*</xref><xref ref-type="author-notes" rid="fn2">†</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Xu</surname><given-names>Jidi</given-names></name></contrib><contrib contrib-type="author"><name><surname>Ye</surname><given-names>Junli</given-names></name></contrib><contrib contrib-type="author"><name><surname>Yun</surname><given-names>Ze</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Xu</surname><given-names>Qiang</given-names></name></contrib><contrib contrib-type="author"><name><surname>Xu</surname><given-names>Juan</given-names></name><xref ref-type="corresp" rid="cor1">‡</xref></contrib><contrib contrib-type="author"><name><surname>Deng</surname><given-names>Xiuxin</given-names></name><xref ref-type="corresp" rid="cor1">‡</xref></contrib></contrib-group><aff>Key Laboratory of Horticultural Plant Biology (Ministry of Education), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, China</aff><author-notes><corresp id="cor1"><label>‡</label>To whom correspondence should be addressed. E-mail: <email>xujuan@mail.hzau.edu.cn</email>; <email>xxdeng@mail.hzau.edu.cn</email></corresp><fn id="fn1"><label>*</label><p>These authors contributed equally to this study.</p></fn><fn id="fn2"><label>†</label><p>Present address: Shanxi Agricultural University, Taigu, Shanxi 030801, China.</p></fn></author-notes><pub-date pub-type="ppub"><month>7</month><year>2012</year></pub-date><pub-date pub-type="epub"><day>18</day><month>5</month><year>2012</year></pub-date><pub-date pub-type="pmc-release"><day>18</day><month>5</month><year>2012</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>63</volume><issue>12</issue><fpage>4403</fpage><lpage>4417</lpage><history><date date-type="received"><day>6</day><month>1</month><year>2012</year></date><date date-type="rev-recd"><day>21</day><month>3</month><year>2012</year></date><date date-type="accepted"><day>23</day><month>3</month><year>2012</year></date></history><permissions><copyright-statement>© 2012 The Author(s).</copyright-statement><copyright-year>2012</copyright-year><license license-type="open-access"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0">http://creativecommons.org/licenses/by-nc/3.0</ext-link>), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p><license-p>This paper is available online free of all access charges (see <ext-link ext-link-type="uri" xlink:href="http://jxb.oxfordjournals.org/open_access.html">http://jxb.oxfordjournals.org/open_access.html</ext-link> for further details)</license-p></license></permissions><abstract><p>Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (<italic>CrtB</italic>). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the β-carotene hydroxylase gene (<italic>HYD</italic>), which was suppressed in the calli. Co-expression of <italic>CrtB</italic> and <italic>HYD</italic> in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of β-carotene. In addition, a model is proposed to interpret the common appearance of a favoured β,β-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.</p></abstract><kwd-group><kwd>Amyloplast</kwd><kwd>carotenogenesis</kwd><kwd>chromoplast</kwd><kwd>citrus</kwd><kwd><italic>CrtB</italic></kwd><kwd>crystal</kwd><kwd>engineered cell model</kwd><kwd>flavedo</kwd><kwd>phytoene synthase</kwd></kwd-group><counts><page-count count="15"></page-count></counts></article-meta></front><body><sec><title>Introduction</title><p>Carotenoids and their derivatives play essential physiological and ecological roles in plant growth, development, and reproduction (<xref ref-type="bibr" rid="bib6">Cazzonelli and Pogson, 2010</xref>). They are also essential health-protecting compounds involved in human vision, immunity, embryonic development, and reproduction (<xref ref-type="bibr" rid="bib52">von Lintig, 2010</xref>). Carotenoids in nature originate from phytoene biosynthesis via the condensation of geranylgeranyl pyrophosphate (GGPP), which is the crucial rate-controlling step mediated by phytoene synthase (PSY) (<xref ref-type="bibr" rid="bib53">Welsch <italic>et al.</italic>, 2010</xref>) (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S1</ext-link> at <italic>JXB</italic> online). Enzymatic desaturation and isomerization, as well as a light-mediated photoisomerization, convert non-coloured phytoene into coloured lycopene (<xref ref-type="bibr" rid="bib8">Chen <italic>et al.</italic>, 2010</xref>). The lycopene flux then branches into two pathways via cyclization reaction. Lycopene β-cyclase (LCYB) adds two β-rings to the ends of lycopene molecule to form β-carotene, while the dual action of lycopene ε-cyclase (LCYE) and LCYB results in the formation of α-carotene with one β-ring and one ε-ring (<xref ref-type="bibr" rid="bib2">Bai <italic>et al.</italic>, 2009</xref>). Subsequently, carotenes are converted into various xanthophylls, which is mediated by carotene hydroxylases and epoxidase as well as de-epoxidase (<xref ref-type="bibr" rid="bib51">Vallabhaneni <italic>et al.</italic>, 2009</xref>).</p><p>Carotenoids are assembled and accumulated in nearly all types of plastids (<xref ref-type="bibr" rid="bib28">Howitt and Pogson, 2006</xref>), while the deposition of massive carotenoids requires plastid modification for enhancing the storage capacity. Chromoplasts have been postulated to be the most important plastid type for striking carotenoid sequestration in higher plants (<xref ref-type="bibr" rid="bib32">Kim <italic>et al.</italic>, 2010</xref>). They are generally derived from pre-existing plastids like chloroplasts and amyloplasts during the ripening of carotenoid-containing organs such as fruits and flowers (<xref ref-type="bibr" rid="bib16">Egea <italic>et al.</italic>, 2010</xref>). For example, amyloplast to chromoplast conversion is observed in developing ornamental tobacco floral nectaries (<xref ref-type="bibr" rid="bib25">Horner <italic>et al.</italic>, 2007</xref>). Amyloplasts are starch-storing plastids, and are distinguished by the starch-granule-filled profile (<xref ref-type="bibr" rid="bib54">Wise, 2006</xref>). During amyloplast conversion, starch breakdown begins, the starch granules diminish then disappear, while carotenoid sequestering structures such as plastoglobules and carotenoid crystals form and chromoplasts appear (<xref ref-type="bibr" rid="bib25">Horner <italic>et al.</italic>, 2007</xref>). Genes including <italic>fibrillin</italic>, <italic>CHRC</italic>, and <italic>OR</italic> have been identified to participate in the regulation of chromoplast development and carotenoid sequestration (<xref ref-type="bibr" rid="bib35">Li and Van Eck, 2007</xref>).</p><p>In recent decades, genetic manipulation has been successfully used to modify carotenogenesis in many plants (<xref ref-type="bibr" rid="bib21">Giuliano <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="bib19">Fraser <italic>et al.</italic>, 2009</xref>; <xref ref-type="bibr" rid="bib18">Farré <italic>et al.</italic>, 2011</xref>). In particular, over-expression of <italic>PSY</italic> leads to a higher carotenoid generation in carotenoid-poor organs, such as canola seeds (<xref ref-type="bibr" rid="bib49">Shewmaker <italic>et al.</italic>, 1999</xref>), potato tubers (<xref ref-type="bibr" rid="bib15">Ducreux <italic>et al.</italic>, 2005</xref>), white carrot root (<xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic>, 2009</xref>), and cassava root (<xref ref-type="bibr" rid="bib53">Welsch <italic>et al.</italic>, 2010</xref>). These engineered crops have exhibited an effective approach to human health promotion, especially the alleviation of vitamin A deficiency. However, many physiological and biochemical alterations associated with modification of the carotenoid pathway that occur in genetically engineered plants remain unexplainable. For example, an altered carotenoid profile, particularly an increased β,β-carotenoid flux, is commonly observed (<xref ref-type="bibr" rid="bib15">Ducreux <italic>et al.</italic>, 2005</xref>; <xref ref-type="bibr" rid="bib42">Paine <italic>et al.</italic>, 2005</xref>; <xref ref-type="bibr" rid="bib47">Schaub <italic>et al.</italic>, 2005</xref>; <xref ref-type="bibr" rid="bib59">Zhu <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic>, 2009</xref>; <xref ref-type="bibr" rid="bib12">Diretto <italic>et al.</italic>, 2010</xref>). In addition, it is difficult to obtain the desired phenotypes for some important carotenoids, such as lycopene (<xref ref-type="bibr" rid="bib47">Schaub <italic>et al.</italic>, 2005</xref>). Moreover, engineered plants with enhanced carotenoid biosynthesis require sink formation for effective carotenoid storage (<xref ref-type="bibr" rid="bib35">Li and Van Eck, 2007</xref>). Morphological changes in plastids have been observed in <italic>PSY</italic> over-expressed tomato fruits and canola endosperm (<xref ref-type="bibr" rid="bib49">Shewmaker <italic>et al.</italic>, 1999</xref>; <xref ref-type="bibr" rid="bib20">Fraser <italic>et al.</italic>, 2007</xref>), which implies the possibility of plastid modification for carotenoid deposition. Over-expression of <italic>PSY</italic> can also drive crystalline-type carotenoid sequestering in <italic>Arabidopsis</italic> callus (<xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic>, 2009</xref>). However, the crucial mechanism governing carotenoid metabolic sink formation is still unclear, and needs to be further clarified.</p><p>Genetic manipulation of carotenogenesis in higher plants has primarily been focused on fruits (tomato), seeds (rice, maize, and canola), and roots or tubers (potato, carrot, and cassava). However, inherent deficiencies in these systems, including time-consuming cultivation and management due to long growth cycles and poorly controlled growth conditions, challenge the further study on carotenogenesis. Plant cell culture offers an attractive option for producing natural active compounds. Despite the fact that plant cells <italic>in vitro</italic> often exhibit an inconsistent yield of natural products owing to the dedifferentiation process, it could be circumvented partially through selection of the genotype and explant source for deriving cell lines (<xref ref-type="bibr" rid="bib17">Engelmann <italic>et al.</italic>, 2010</xref>; <xref ref-type="bibr" rid="bib33">Lee <italic>et al.</italic>, 2010</xref>), as well as through metabolic engineering (<xref ref-type="bibr" rid="bib24">Hellwig <italic>et al.</italic>, 2004</xref>). Interestingly, the carotenoid accumulation patterns in <italic>OR</italic> mutant cauliflower curd (<xref ref-type="bibr" rid="bib34">Li <italic>et al.</italic>, 2006</xref>), engineered potato tuber (<xref ref-type="bibr" rid="bib11">Diretto <italic>et al.</italic>, 2007</xref>, <xref ref-type="bibr" rid="bib12">2010</xref>), and carrot root (<xref ref-type="bibr" rid="bib30">Jayaraj <italic>et al.</italic>, 2008</xref>) are similar to those in the homologous cell lines <italic>in vitro</italic>. Besides, maize callus provides an efficient model for identifying the most productive <italic>PSY</italic> gene from different plant species (<xref ref-type="bibr" rid="bib42">Paine <italic>et al.</italic>, 2005</xref>). <italic>Arabidopsis</italic> callus has been used as a non-green system in investigating carotenoid metabolic sink formation driven by the over-expression of <italic>AtPSY</italic> (<xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic>, 2009</xref>). Thus, it is particularly important to construct a plant cell model cultured <italic>in vitro</italic> that could facilitate the study on the molecular, biochemical and cytological processes related to carotenoid accumulation.</p><p>Citrus is one of the most important and widely grown fruit crops with great economic significance and value for humans (<xref ref-type="bibr" rid="bib50">Talon and Gmitter, 2008</xref>). Citrus fruits have diverse carotenoid patterns, highly regulated by the co-ordinated expression of carotenogenic genes (<xref ref-type="bibr" rid="bib31">Kato <italic>et al.</italic>, 2004</xref>). Over the years, citrus fruits have served as a unique system to provide new insights into the understanding of carotenogenesis. A novel chromoplast-specific <italic>LCYB</italic> gene has been found to be associated with lycopene accumulation in the fruits of Star Ruby grapefruit (<italic>C. paradise</italic> Macf.) (<xref ref-type="bibr" rid="bib1">Alquezar <italic>et al.</italic>, 2009</xref>). Recent studies using comparative ‘omics’ revealed some processes, such as post-transcriptional regulation, partial impairment of lycopene downstream flux, enhanced photosynthesis, and miRNA-directed molecular process, in conferring the red-fleshed phenotype in the ‘Honganliu’ sweet orange (<xref ref-type="bibr" rid="bib43">Pan <italic>et al.</italic>, 2009</xref>; <xref ref-type="bibr" rid="bib56">Xu <italic>et al.</italic>, 2009</xref>, <xref ref-type="bibr" rid="bib55">2010</xref>). In addition, an <italic>in vitro</italic> culture system of citrus juice sacs provides a new approach to the comprehension of carotenogenesis in response to controlled environmental stimuli (<xref ref-type="bibr" rid="bib60">Zhang <italic>et al.</italic>, 2012</xref>).</p><p>By applying genetic manipulation of carotenoid accumulation as well as using carotenoid-rich citrus fruit, it is possible to understand the basis of carotenoid biosynthesis further. Therefore, engineered cell models (ECMs) were established by over-expressing CrtB (phytoene synthase from <italic>Erwinia herbicola</italic>) or co-expressing CrtB and DSM2 (a non-haem-diiron β-carotene hydroxylase from rice) in various non-coloured citrus embryogenic callus cells. To compare with the ECMs, the ripe flavedos from four genotypes were offered as natural carotenoid accumulation systems. Further analysis using comparative biochemistry, molecular biology, and cytology contributed to a better understanding of crystalline β-carotene accumulation in the amyloplasts of the ECM cells.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>Plant materials</title><p>Abortive ovule embryogenic calli from four citrus genotypes were used in this study. They were derived from Star Ruby grapefruit (<italic>C. paradise</italic> Macf.), Pink Marsh grapefruit (<italic>C. paradise</italic> Macf.), Cara Cara navel orange [<italic>C. sinensis</italic> (L.) Osb.], and Sunburst mandarin [<italic>Citrus reticulata</italic> Blanco×(<italic>C. paradisi</italic> Macf.×<italic>C. reticulata</italic>)], designated as RB, M, HQC, and SBT, respectively. The specimens were supplied by the National Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University (HZAU), Wuhan, China.</p><p>Fruits were obtained from citrus trees grown at the National Center for Citrus Breeding, Huazhong Agricultural University. Marsh grapefruit (M) (<italic>C. paradise</italic> Macf.) was selected instead of Pink Marsh grapefruit because Pink Marsh grapefruit is a periclinal chimera whose abortive ovules and flavedos are identical to those of the Marsh grapefruit in terms of their genotype (<xref ref-type="bibr" rid="bib5">Cameron <italic>et al.</italic>, 1964</xref>). Ripe fruits were collected at the same time in November. Flavedos of the fruits were separated with scalpels. A part of flavedo tissues were used for cytology, and others were immediately frozen in liquid nitrogen, and stored at –80 °C until analysis. The samples used for carotenoid extraction were lyophilized at –55 °C in a Lyolab 3000 (Heto, Denmark) and then stored at –80 °C.</p></sec><sec><title>Plasmid construction and transformation</title><p>The <italic>CrtB</italic> gene (a bacterial phytoene synthase gene, GenBank No. M90698) from <italic>Erwinia herbicola</italic> was PCR-amplified from plasmid vector PSL525, which was kindly provided by Professor Shih-Tung Liu (Taiwan, China). Pea rbcS transit peptide cDNA (GenBank No. X00806) was amplified by RT-PCR from the pea leaf pool. To create the chimeric gene (tp–rbcS–CrtB) construct, the PCR products were gel-purified, digested with <italic>Xho</italic>I/<italic>Bam</italic>HI, and ligated into the corresponding sites in the PMV vector with a kanamycin resistance marker and the CaMV 35S promoter (modified pBI121) (<xref ref-type="bibr" rid="bib58">Zhang <italic>et al.</italic>, 2009</xref>). The primer pairs used were as listed in <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Table S1</ext-link> at <italic>JXB</italic> online. The plasmid pCB2004H with <italic>DSM2</italic> [a non-haem-diiron β-carotene hydroxylase gene isolated from rice (<xref ref-type="bibr" rid="bib13">Du <italic>et al.</italic>, 2010</xref>)] over-expression construct was kindly provided by Professor Lizhong Xiong, Huazhong Agricultural University, China. These constructs and empty vectors were electroporated into an <italic>Agrobacterium tumefaciens</italic> strain (EHA105).</p><p>Explant preparation and transformation were performed according to <xref ref-type="bibr" rid="bib14">Duan <italic>et al.</italic> (2007)</xref>. To reduce the occurrence of chimeric calli, a very small piece of each recovered callus, as an independent cell line, was transferred onto selective culture media and subcultured for several cycles. To generate <italic>CrtB/DMS2</italic> double transgetic lines, the <italic>DSM2</italic> over-expression construct was introduced into a stable <italic>CrtB</italic> transformed callus line (M-33) and the transgenic calli were selected with hygromycin (50 mg l<sup>−1</sup>). Each independent line was propagated in the dark or under 35 μmol m<sup>−2</sup> s<sup>−1</sup> illumination for 16 h daily and kept at 25±1 °C.</p><p>Twenty-day-old calli were harvested for cellular analysis or stored at –80 °C for molecular analysis. The calli used for carotenoids extraction were lyophilized and stored at –80 °C until use.</p></sec><sec><title>DNA analysis</title><p>Genomic DNA for PCR and Southern blot analysis was extracted from stored calli according to <xref ref-type="bibr" rid="bib9">Cheng <italic>et al.</italic> (2003)</xref>. A PCR-derived <italic>CrtB</italic> fragment (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Table S1</ext-link> at <italic>JXB</italic> online) was labelled with a Dig-DNA labelling kit and used as a probe (Roche Diagnostics GmbH). Prehybridization, hybridization, and membrane washing and detection were conducted according to the manufacturer’s instructions (Roche Diagnostics GmbH).</p></sec><sec><title>Quantitative analysis of gene expression</title><p>First-strand cDNA was synthesized from 1 μg of total RNA isolated from calli and flavedos using the RevertAid M-MuLV KIT (MBI, Lithuania) as previously described by <xref ref-type="bibr" rid="bib37">Liu <italic>et al.</italic> (2006)</xref>. The primer pairs used were as listed (<xref ref-type="bibr" rid="bib36">Liu <italic>et al.</italic>, 2007</xref>) or designed using the Primer Express software (Applied Biosystems, Foster City, CA, USA) ((see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Table S2</ext-link> at <italic>JXB</italic> online)). Actin was used as an endogenous control to normalize expression in different samples (<xref ref-type="bibr" rid="bib36">Liu <italic>et al.</italic>, 2007</xref>). Quantitative real-time PCR was performed using ABI 7500 Real Time System (PE Applied Biosystems; Foster City, CA, USA) according to <xref ref-type="bibr" rid="bib36">Liu <italic>et al.</italic> (2007)</xref>.</p></sec><sec><title>Generation of anti-CrtB antibodies and western blot analysis</title><p>The sequence of <italic>CrtB</italic> encoding 117 amino acids in the C-terminus was amplified, cloned into pGEX-4T, and transformed into <italic>E. coli</italic> strain BL21 (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Table S1</ext-link> at <italic>JXB</italic> online). After induction and purification, inclusion bodies were dissolved in SDS sample buffer and separated by SDS-PAGE. Gel slices containing the peptide were used to immunize rabbits to generate anti-CrtB antibodies.</p><p>Total callus and flavedo proteins were prepared as described previously (<xref ref-type="bibr" rid="bib29">Isaacson <italic>et al.</italic>, 2006</xref>; <xref ref-type="bibr" rid="bib43">Pan <italic>et al.</italic>, 2009</xref>), and quantified using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) based on the Lowry method using bovine serum albumin (BSA) as standard. Protein samples (50 μg) were separated by SDS-PAGE and blotted onto PVDF membranes (Millipore, USA). Membranes were blocked with a TBST solution, containing 5% (w/v) not-fat dry milk power overnight at 4 °C and were incubated with anti-CrtB antibodies (1:2500) in the same solution for 2 h at room temperature. After rinsing five times for 10 min each with TBST solution, the membranes were incubated with secondary antibodies (peroxidase-conjugated immunopure goat anti-rabbit IgG [H+L], Pierce) at 1:20 000 in TBST solution, containing 5% (w/v) not-fat dry milk power and rinsed six times for 10 min each with TBST solution. The signal was detected using SuperSignal West Pico system (Thermo Scientific, USA) according to the manufacturer’s instructions. Two rabbit polyclonal antibodies (Agrisera@, Sweden) against the Rubisco large subunit (RubcL, 53 kDa) and PsbA protein (D1 protein of Photosystem II, 32 kDa) were used to detect the plastid marker proteins.</p></sec><sec><title>Carotenoid profile analysis</title><p>Carotenoid extraction and analysis using reversed-phase high-performance liquid chromatography (RP-HPLC) was conducted as previously described (<xref ref-type="bibr" rid="bib42">Paine <italic>et al.</italic>, 2005</xref>; <xref ref-type="bibr" rid="bib36">Liu <italic>et al.</italic>, 2007</xref>). For the calli, a 0.2 g homogeneous lyophilized sample was extracted. Because of the abundant carotenoid esters in citrus fruits, the extracts from 0.3 g of lyophilized flavedos were saponified with 15% (w/v) KOH:methanol. The RP-HPLC analysis was performed in a Waters liquid chromatography system equipped with a model 600E solvent delivery system, a model 2996 photodiode array detection (PAD) system, a model 717 plus autosampler and an Empower chromatography manager. A C30 carotenoid column (150×4.6 mm; YMC, Japan) was used to elute the carotenoids. The carotenoids were identified by their characteristic absorption spectra and typical retention time based on the literature and standards from CaroNature Co. (Bern, Switzerland). The quantification of the carotenoids was achieved using calibration curves for standards including violaxanthin, lutein, phytoene, α-carotene, β-carotene, β-cryptoxanthin, and lycopene; phytofluene was quantified as phytoene, and α-cryptoxanthin was quantified as β-cryptoxanthin.</p></sec><sec><title>Microscopy analysis</title><p>Protoplasts from the calli were isolated as described by <xref ref-type="bibr" rid="bib22">Grosser and Gmitter (1990)</xref>, after which protoplast suspensions were plated onto microscope slides to observe modes of carotenoid deposition. Microcellular light microscopy of fresh flavedo cells were performed using a frozen sectioning technique with a Leica CM1900 (Leica, Germany). More detailed inspections of the carotenoid accumulative structures <italic>in vitro</italic> were performed based on a protocol for isolating chromoplasts from fresh tissues (<xref ref-type="bibr" rid="bib38">Lopez <italic>et al.</italic>, 2008</xref>). An optical microscope (BX61, Olympus) equipped with a DP70 camera was used in tandem with differential interference contrast (DIC) technique.</p><p>The same callus and flavedo samples were also selected for transmission electron microscopy (TEM) analysis. Samples were prepared using a normal fixation process with 2.5% glutaraldehyde adjusted to pH 7.4, and 0.1 M phosphate buffer with 2% OsO<sub>4</sub>. They were dehydrated and embedded in epoxy resin (<xref ref-type="bibr" rid="bib39">Luft, 1961</xref>) and SPI-812, respectively. Ultrathin sections obtained with a Leica UC6 ultramicrotome were stained with uranyl acetate and subsequently with lead citrate. Recording of the images was performed with a HITACHI H-7650 transmission electron microscope at 80 kV and a Gatan 832 CCD camera. Images were obtained at various magnifications along with a corresponding a waffle-pattern diffraction grafting replica with line spacing being 0.463 μm (Product No. 607, Ted Pella, Inc., Redding. CA).</p></sec><sec><title>Statistical analysis</title><p>The data presented in this study were analysed using SAS statistical software. Analysis of variance (ANOVA) was used to determine Pearson correlation (<italic>r</italic>) and to compare the statistical difference based on Student–Newman–Keuls’ multiple range test at the significance levels of <italic>P</italic> <0.05 (*), <italic>P</italic> <0.01 (**), and <italic>P</italic> <0.001 (***), respectively.</p></sec></sec><sec sec-type="results"><title>Results</title><sec><title>Construction of carotenoid engineered cell models (ECMs)</title><p><italic>CrtB</italic> was first transformed into embryogenic calli from four citrus genotypes. Positive transformants of 58 independent lines were recovered, including five lines from Star Ruby grapefruit (RB), five lines from Sunburst mandarin (SBT), six lines from Cara Cara navel orange (HQC), and 32 lines from Pink Marsh grapefruit (M). Most of the transgenic lines had a yellow or orange-red colour that was not observed in the empty-vector controls and wild types. Coloured transgenic callus lines were selected as engineered cell models (ECMs). Four engineered genotypes presented distinctive colour characteristics where RB ECMs appeared red and the others orange or yellow (<xref ref-type="fig" rid="fig1">Fig. 1A</xref>).</p><fig id="fig1" position="float"><label>Fig. 1.</label><caption><p>Various ECMs and ripe citrus fruits used in this study. (A) The representative ECMs from four genotypes (from left to right: RB-4, M-33, HQC-2, and SBT-6). (B) Various M lines [wild type (WT) is in the middle]. (C) Ripe fruits [from left to right: Marsh grapefruit (M), Ruby Star grapefruit (RB), Cara Cara navel orange (HQC), and Sunburst mandarin (SBT)].</p></caption><graphic xlink:href="jexboters115f01_3c"></graphic></fig><p>Southern blot analysis confirmed the independent transformation events in the representative M ECMs (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S2</ext-link> at <italic>JXB</italic> online). However, no correlation was observed between transgene copy number and carotenoid content (data not shown). Western blot analysis using anti-CrtB antibodies indicated the presence of CrtB proteins in the representative ECMs but an absence in the wild type and negative control line (<xref ref-type="fig" rid="fig2">Fig. 2A, B</xref>). Furthermore, correlation between CrtB protein levels and carotenoid contents was observed in seven M ECMs (<xref ref-type="fig" rid="fig2">Fig. 2B</xref>). The ECMs showed normal growth, and their pigment expression was found to be stable in subcultures for at least three years.</p><fig id="fig2" position="float"><label>Fig. 2.</label><caption><p>CrtB protein levels and their correlation with carotenoid contents in various ECMs. (A) CrtB protein levels and carotenoid contents in the representative ECMs from four genotypes. (B) Positive correlation between CrtB levels and carotenoid contents in various M lines, including the ECMs, wild type (WT), and negative line (NC). CS, Coomassie blue staining for loading control. Values and bars represent the means and ±SD (<italic>n</italic>=3 replicate experiments), respectively.</p></caption><graphic xlink:href="jexboters115f02_ht"></graphic></fig></sec><sec><title>Carotenoid accumulation patterns in the ECMs</title><p>The carotenoid profile was investigated in various callus lines from dark-grown culture using HPLC. Low amount of carotenoids was observed in wild-type embryogenic calli (<xref ref-type="table" rid="tbl1">Table 1</xref>). They mainly consisted of violaxanthin, lutein, β-carotene, and phytoene, and their accumulation patterns were markedly diverse among the genotypes (<xref ref-type="fig" rid="fig3">Fig. 3</xref>). In particular, RB contained predominantly lutein, and SBT had significantly suppressed lutein biosynthesis. Other important carotenoids, such as β-cryptoxanthin, lycopene, and esterified xanthophylls, were all at negligible levels or undetected in these calli.</p><table-wrap id="tbl1" position="float"><label>Table 1.</label><caption><p>Carotenoid content and composition in wild-type callus lines, representative ECMs and flavedos</p><p>Vio, violaxanthin; α-Cry, α-cryptoxanthin, β-Cry. β-cryptoxanthin; Phy, phytoene; Phytof, phytofluene; α-Car, α-carotene; β-Car, β-carotene; Lyc, lycopene; β/ε ratio represents the β-cycle to ε-cycle derivatives ratio. Xan./Tol. represents the xanthophylls to total carotenoids ratio. (–) Not detected or trace; (– –) infinite. Values are means ±SD from three replicate experiments and are expressed as μg g<sup>−1</sup> dry weight.</p></caption><table frame="hsides" rules="groups"><thead><tr><td rowspan="1" colspan="1">Event</td><td rowspan="1" colspan="1">Vio</td><td rowspan="1" colspan="1">Lutein</td><td rowspan="1" colspan="1">α-Cry</td><td rowspan="1" colspan="1">β-Cry</td><td rowspan="1" colspan="1">Phy</td><td rowspan="1" colspan="1">Phytof</td><td rowspan="1" colspan="1">α-Car</td><td rowspan="1" colspan="1">β-Car</td><td rowspan="1" colspan="1">Lyc</td><td rowspan="1" colspan="1">Total</td><td rowspan="1" colspan="1">β/ε ratio</td><td rowspan="1" colspan="1">Xan./Tol.</td></tr></thead><tbody><tr><td colspan="13" rowspan="1"><bold>Wild-type callus lines from dark-grown culture (non-saponified samples)</bold></td></tr><tr><td rowspan="1" colspan="1">M</td><td align="char" char="plusmn" rowspan="1" colspan="1">13.78±0.46</td><td align="char" char="plusmn" rowspan="1" colspan="1">12.05±0.56</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">9.41±0.89</td><td align="char" char="plusmn" rowspan="1" colspan="1">4.32±0.48</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.98±0.042</td><td align="char" char="plusmn" rowspan="1" colspan="1">13.14±1.26</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">53.68±3.13</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.07±0.086</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.48±0.019</td></tr><tr><td rowspan="1" colspan="1">RB</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.16±0.14</td><td align="char" char="plusmn" rowspan="1" colspan="1">9.17±0.45</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.98±0.82</td><td align="char" char="plusmn" rowspan="1" colspan="1">1.52±0.17</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.88±0.008</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.27±0.17</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">17.98±0.94</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.24±0.015</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.63±0.03</td></tr><tr><td rowspan="1" colspan="1">HQC</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.48±0.14</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.63±0.19</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">9.80±0.57</td><td align="char" char="plusmn" rowspan="1" colspan="1">5.41±0.45</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.99±0.079</td><td align="char" char="plusmn" rowspan="1" colspan="1">5.06±2.26</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">27.37±2.18</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.36±0.55</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.22±0.024</td></tr><tr><td rowspan="1" colspan="1">SBT</td><td align="char" char="plusmn" rowspan="1" colspan="1">7.75±0.45</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">9.48±0.14</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.26±0.18</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.88±0.007</td><td align="char" char="plusmn" rowspan="1" colspan="1">5.73±0.31</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">27.09±0.39</td><td align="char" char="plusmn" rowspan="1" colspan="1">15.32±0.47</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.29±0.016</td></tr><tr><td colspan="13" rowspan="1"><bold>ECMs from dark-grown culture (non-saponified samples)</bold></td></tr><tr><td rowspan="1" colspan="1">M-33</td><td align="char" char="plusmn" rowspan="1" colspan="1">31.97±0.89</td><td align="char" char="plusmn" rowspan="1" colspan="1">65.14±0.73</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.49±0.027</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.13±0.14</td><td align="char" char="plusmn" rowspan="1" colspan="1">138.51±2.16</td><td align="char" char="plusmn" rowspan="1" colspan="1">70.79±0.95</td><td align="char" char="plusmn" rowspan="1" colspan="1">26.76±0.32</td><td align="char" char="plusmn" rowspan="1" colspan="1">897.82±16.2</td><td align="char" char="plusmn" rowspan="1" colspan="1">19.73±0.54</td><td align="char" char="plusmn" rowspan="1" colspan="1">1257.34±19.64</td><td align="char" char="plusmn" rowspan="1" colspan="1">9.78±0.056</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.083±0.001</td></tr><tr><td rowspan="1" colspan="1">RB-4</td><td align="char" char="plusmn" rowspan="1" colspan="1">14.84±1.81</td><td align="char" char="plusmn" rowspan="1" colspan="1">89.73±5.63</td><td align="char" char="plusmn" rowspan="1" colspan="1">15.34±1.48</td><td align="char" char="plusmn" rowspan="1" colspan="1">9.51±0.70</td><td align="char" char="plusmn" rowspan="1" colspan="1">1204.39±72.54</td><td align="char" char="plusmn" rowspan="1" colspan="1">644.66±37.75</td><td align="char" char="plusmn" rowspan="1" colspan="1">132.53±6.97</td><td align="char" char="plusmn" rowspan="1" colspan="1">2732.07±78.20</td><td align="char" char="plusmn" rowspan="1" colspan="1">62.22±4.47</td><td align="char" char="plusmn" rowspan="1" colspan="1">4935.30±204.85</td><td align="char" char="plusmn" rowspan="1" colspan="1">11.60±0.39</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.026±0.0009</td></tr><tr><td rowspan="1" colspan="1">HQC-2</td><td align="char" char="plusmn" rowspan="1" colspan="1">14.29±0.59</td><td align="char" char="plusmn" rowspan="1" colspan="1">11.27±0.44</td><td align="char" char="plusmn" rowspan="1" colspan="1">5.69±0.79</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.89±0.72</td><td align="char" char="plusmn" rowspan="1" colspan="1">1108.63±32.79</td><td align="char" char="plusmn" rowspan="1" colspan="1">523.00±8.98</td><td align="char" char="plusmn" rowspan="1" colspan="1">15.26±0.37</td><td align="char" char="plusmn" rowspan="1" colspan="1">1179.28±31.11</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">2861.30±73.02</td><td align="char" char="plusmn" rowspan="1" colspan="1">37.16±1.09</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.012±0.002</td></tr><tr><td rowspan="1" colspan="1">SBT-6</td><td align="char" char="plusmn" rowspan="1" colspan="1">18.10±0.98</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.59±0.21</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.26±0.46</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.94±0.44</td><td align="char" char="plusmn" rowspan="1" colspan="1">298.47±15.03</td><td align="char" char="plusmn" rowspan="1" colspan="1">118.21±6.24</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.75±0.18</td><td align="char" char="plusmn" rowspan="1" colspan="1">472.10±29.39</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">918.42±51.55</td><td align="char" char="plusmn" rowspan="1" colspan="1">65.02±4.96</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.029±0.001</td></tr><tr><td colspan="13" rowspan="1"><bold>Flavedos (saponified samples)</bold></td></tr><tr><td rowspan="1" colspan="1">M</td><td align="char" char="plusmn" rowspan="1" colspan="1">10.37±1.29</td><td align="char" char="plusmn" rowspan="1" colspan="1">1.64±0.22</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.76±0.074</td><td align="char" char="plusmn" rowspan="1" colspan="1">8.77±1.06</td><td align="char" char="plusmn" rowspan="1" colspan="1">6.12±0.68</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.32±0.01</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.31±0.38</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">30.28±3.62</td><td align="char" char="plusmn" rowspan="1" colspan="1">6.89±0.23</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.42±0.009</td></tr><tr><td rowspan="1" colspan="1">RB</td><td align="char" char="plusmn" rowspan="1" colspan="1">9.86±0.78</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.19±0.37</td><td align="char" char="plusmn" rowspan="1" colspan="1">1.62±0.036</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">167.75±7.92</td><td align="char" char="plusmn" rowspan="1" colspan="1">177.38±9.09</td><td align="char" char="plusmn" rowspan="1" colspan="1">2.23±0.13</td><td align="char" char="plusmn" rowspan="1" colspan="1">177.19±13.32</td><td align="char" char="plusmn" rowspan="1" colspan="1">225.71±13.86</td><td align="char" char="plusmn" rowspan="1" colspan="1">763.92±45.22</td><td align="char" char="plusmn" rowspan="1" colspan="1">30.99±0.63</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.018±0.0005</td></tr><tr><td rowspan="1" colspan="1">HQC</td><td align="char" char="plusmn" rowspan="1" colspan="1">97.49±5.91</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">3.66±0.17</td><td align="char" char="plusmn" rowspan="1" colspan="1">72.23±5.03</td><td align="char" char="plusmn" rowspan="1" colspan="1">25.87±1.35</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.60±0.11</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">199.86±10.09</td><td rowspan="1" colspan="1">– –</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.51±0.017</td></tr><tr><td rowspan="1" colspan="1">SBT</td><td align="char" char="plusmn" rowspan="1" colspan="1">163.46±13.03</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">24.20±2.38</td><td align="char" char="plusmn" rowspan="1" colspan="1">79.95±7.85</td><td align="char" char="plusmn" rowspan="1" colspan="1">101.78±6.89</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">7.04±1.02</td><td rowspan="1" colspan="1">–</td><td align="char" char="plusmn" rowspan="1" colspan="1">376.44±28.77</td><td rowspan="1" colspan="1">– –</td><td align="char" char="plusmn" rowspan="1" colspan="1">0.50±0.011</td></tr></tbody></table></table-wrap><fig id="fig3" position="float"><label>Fig. 3.</label><caption><p>HPLC chromatograms of carotenoids (non-saponified) in the wild types and representative ECMs (35S::<italic>CrtB</italic>) monitored at 450 nm. The peaks are numbered according to the elution time as detailed in <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Table S3</ext-link> at <italic>JXB</italic> online. Inserted tables show the proportion (%) and ±SD (<italic>n</italic>=3 replicate experiments) of the representative carotenoids relative to total carotenoid amount.</p></caption><graphic xlink:href="jexboters115f03_lw"></graphic></fig><p>By contrast, the levels of total carotenoids markedly increased in all ECMs compared with the wild types (<xref ref-type="table" rid="tbl1">Table 1</xref> shows the carotenoid profiles in four representative ECMs). Phenotypes with a darker orange colour in the RB ECMs were associated with a larger amount of carotenoids, ranging from 3052 to 4880 μg g<sup>−1</sup> dry weight. Although ECMs’ carotenoid patterns were diverse among genotypes, up-regulation of the β,β-pathway was favoured (<xref ref-type="table" rid="tbl1">Table 1</xref>; <xref ref-type="fig" rid="fig3">Fig. 3</xref>). For example, in wild-type RB and M, the proportion of β-carotene in the total carotenoids was only 1.51% and 24.48%, respectively, and lutein accounted for 50.99% and 22.45%; but in the corresponding ECMs, β-carotene rose to 55.98% and 71.78% while lutein decreased to 1.84% and 5.21%, respectively (<xref ref-type="fig" rid="fig3">Fig. 3</xref>). It was also noted that violaxanthin increased slightly, so its proportion decreased in the ECMs. To characterize the altered balance of carotenoid patterns in the ECMs further, lutein levels were analysed in 32 M callus lines, including the wild type, empty-vector control, negative control, and 29 ECMs (35S::<italic>CrtB</italic>). Statistical analysis showed that the content/proportion change of lutein relative to lycopene flux followed a power function (<xref ref-type="fig" rid="fig4">Fig. 4</xref>). Herein, lycopene flux represented the total content of violaxanthin, lutein, α-carotene, β-carotene, and lycopene. This power functional relationship showed the dynamic change of branch flux, suggesting that the β,ε-branch was less competitive when the upstream flux become strong. Noticeably, a novel ECM (M-30) was found not to correlate well with the power function (<xref ref-type="fig" rid="fig4">Fig. 4</xref>). M-30 showed a dark yellow colour and contained a higher lutein proportion (19.7%) in relation to other ECMs (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S3A</ext-link> at <italic>JXB</italic> online).</p><fig id="fig4" position="float"><label>Fig. 4.</label><caption><p>Correlation between lutein content (A) or lutein proportion (B) and lycopene flux. 32 M callus lines, including wild type, empty-vector control, negative control, and 29 ECMs, are used for analysis. Lycopene flux is represented by the total content of violaxanthin, lutein, α-carotene, β-carotene, and lycopene, and lutein proportion (%) is relative to this total content. Pearson correlation (<italic>r</italic>) and statistical significance (<italic>P</italic>) are determined using SAS statistical software. The arrows show a significantly deviating line (M-30).</p></caption><graphic xlink:href="jexboters115f04_lw"></graphic></fig><p>In addition, a large number of carotenoids under the detection threshold in wild types were detected in the ECMs (<xref ref-type="fig" rid="fig3">Fig. 3</xref>; see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S4</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Table S3</ext-link> at <italic>JXB</italic> online). In particular, a low level of lycopene was observed in the ECMs of RB and M. Furthermore, identical HPLC chromatography profiles for saponified and unsaponified ECM samples demonstrated that non-esterified carotenoids were predominant in the ECMs (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S5A</ext-link> at <italic>JXB</italic> online).</p></sec><sec><title>Effect of illumination on carotenoid accumulation in the ECMs</title><p>To determine the carotenoid accumulation in the ECMs from light-grown culture, illumination (35 μmol m<sup>−2</sup> s<sup>−1</sup> illumination for 16 h daily) treatments were performed. As shown in <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S6</ext-link> at <italic>JXB</italic> online, after excitation with 35 μmol m<sup>−2</sup> s<sup>−1</sup> irradiance (light/dark, 16/8 h), the visual inspection of the calli revealed that M-33 and RB-4 were both yellow instead of dark orange. HPLC analysis showed that the levels of carotenoids except lutein decreased under illumination. This was especially the case for β-carotene, which showed a 56% and an 85% reduction in M-33 and RB-4, respectively.</p></sec><sec><title>Carotenoid accumulation patterns in the ripe flavedos from four genotypes</title><p>To compare the ECMs with the natural carotenoid-rich system, carotenoid profiles in the ripe flavedos from four corresponding genotypes were characterized (<xref ref-type="fig" rid="fig1">Fig. 1C</xref>; <xref ref-type="table" rid="tbl1">Table 1</xref>). The flavedos of SBT and HQC chiefly contained violaxanthin, cryptoxanthin, phytoene, and phytofluene. RB accumulated high level of lycopene as well as β-carotene, phytoene, and phytofluene in the flavedo. There was a low level of carotenoids consisting predominantly of violaxanthin, phytoene, and phytofluene in the flavedo of Marsh grapefruit (M). In addition, carotenoid esterification is a typical character of carotenogenesis in the ripe flavedos (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S5B</ext-link> at <italic>JXB</italic> online).</p></sec><sec><title>Light microscopic observations of carotenoid storage structures</title><p>Significant carotenoid accumulation is accommodated by chromoplast development. To examine the formation of carotenoid sequestering structures in the ECMs, light microscopy of the protoplasts was performed. Unlike wild-type controls, visible red-orange carotenoid sequestering structures were found in the ECM cells (<xref ref-type="fig" rid="fig5">Fig. 5A, B, E, F</xref>), and polarization microscopy confirmed their crystal nature (<xref ref-type="fig" rid="fig5">Fig. 5C, D, G, H</xref>). Our further observation showed that the crystals were formed in a circular pattern localizing to the periphery of the amyloplasts (<xref ref-type="fig" rid="fig5">Fig. 5I</xref>). Some orange conglomerates, which were undergoing Brownian motion, were observed inside the vacuoles of the ECM cells (<xref ref-type="fig" rid="fig5">Fig. 5J</xref>). The conglomerates exhibited birefringence (<xref ref-type="fig" rid="fig5">Fig. 5K</xref>), indicating their crystal nature and the likelihood of containing carotenoids. According to a protocol for isolating plastids (<xref ref-type="bibr" rid="bib38">Lopez <italic>et al.</italic>, 2008</xref>), abundant carotenoid crystals with the size from 1 μm to 10 μm were detected free in the isotonic buffer (Fig. 5L), and no surrounding membrane envelope was observed. Coloured plastid structures containing plastoglobules peripherally were observed in abundance in isotonic buffer (Fig. 5M). Noticeably, compared with the wild types (Fig. 5N-a), these structures in the ECMs loaded more carotenoids (Fig. 5N-b).</p><fig id="fig5" position="float"><label>Fig. 5.</label><caption><p>Light microscopic inspection. (A) Protoplasts from wild-type RB under normal light field microscope. (B) Protoplasts from wild-type M under normal light field microscope. (C, D) Polarization micrographs for (A) and (B), respectively. (E, F) Protoplasts exhibiting the orange-coloured structures in the ECM cells of RB and M, respectively. (G, H) Carotenoid crystals confirmed by polarization microscopy in the ECM cells of RB and M, respectively. (I) Crystals located around the amyloplasts. (J) Coloured conglomerate (shown with arrows) observed in the vacuole of the ECM cells. (K) Conglomerate in the vacuole containing carotenoid crystals. (L) Flattened sheet (carotenoid crystals) in the lysate sample of the ECM cells. (M) Coloured plastid structure containing plastoglobules peripherally. (N) Coloured plastid structures from the wild-type cells (a) and ECM cells (b). (O) Inspection of the flavedo cells from Marsh; arrows show the chromoplasts. (P) Inspection of the flavedo cells from Ruby Star; arrows show the chromoplasts. (Q) Globular chromoplast (shown with arrows) in the lysate samples of the flavedo cells from Marsh. (R) Polarization microscopy for (Q). (S) Chromoplasts in the lysate samples of the flavedo cells from RB. (T) Polarization microscopy for (S), confirming the carotenoid crystals inside the chromoplasts of RB. The bar in each figure represents 10 μm.</p></caption><graphic xlink:href="jexboters115f05_3c"></graphic></fig><p>Carotenoid sequestering structures in the flavedo cells were also inspected using frozen sectioning and plastid isolation techniques. As shown in Fig. 5O, Q, and R (and <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S7</ext-link> at <italic>JXB</italic> online), the flavedo cells of ripe fruits from all four genotypes contained abundant globular chromoplasts. Interestingly, the globular chromoplasts in the RB flavedo cells showed a distinct morphology characterized by visible carotenoid crystals inside (Fig. 5P, S, T). The crystals exhibited red colour, implicating the association with the accumulation of lycopene and β-carotene in the RB flavodo cells. This type of chromoplast has not been reported in citrus, while its morphology is similar to the crystalline chromoplast in tomato fruit and carrot root (<xref ref-type="bibr" rid="bib4">Ben-Shaul and Naftali, 1969</xref>; <xref ref-type="bibr" rid="bib32">Kim <italic>et al.</italic>, 2010</xref>).</p></sec><sec><title>Transmission electron microscopic observations of carotenoid storage structures</title><p>Transmission electron microscopy (TEM) was used to study the carotenoid sequestering structures of our callus and flavedo specimens further. Amyloplasts were the main plastid type observed in all callus cells (<xref ref-type="fig" rid="fig6">Fig. 6A, B</xref>). By comparison, there were slightly more plastoglobules in the amyloplasts of the ECM cells, which also had remnant carotenoid crystals and characteristic internal membranes exclusively, showing chromoplast-like structures (<xref ref-type="fig" rid="fig6">Fig. 6A</xref>). Conglomerate structures were noted in the vacuoles of the ECM cells under the TEM field (<xref ref-type="fig" rid="fig6">Fig. 6C</xref>), and high resolution image demonstrated that the conglomerate consisted of carotenoid plastoglobules and crystals without a surrounding membrane structure (<xref ref-type="fig" rid="fig6">Fig. 6D</xref>). The results of extensive TEM investigation indicated the frequent appearance of conglomerate structure in the vacuoles (data not shown), which provided further confirmation of the vacuolar location of carotenoid sequestering structures.</p><fig id="fig6" position="float"><label>Fig. 6.</label><caption><p>Cellular ultrastructure of the callus and flavedo. (A) Ultrastructure of ECM cells (M) containing conspicuous amyloplasts (showed with arrows). Inserts in the lower corner showed the detail of amyloplasts, which contained a small number of plastoglobules, carotenoid crystal remnant, and characteristic internal membrane. (B) Ultrastructure of wild-type callus cells. (C) Conglomerate in the vacuole of the ECM cell. (D) A detailed image of the conglomerate containing plastoglobules and crystals without a surrounding membrane structure. (E, F) Aspects of the young chromoplasts in the flavedo cells from ripe fruits of Marsh (M) and Ruby Star (RB). Crystal structure is observed in the chromoplast of RB (F). Remnant thylakoids are always observed in all chromoplasts. S, starch granules; P, plastoglobules; M, mitochondrion; C, crystal; V, vacuole; Co, conglomerate; T, remnant thylakoids.</p></caption><graphic xlink:href="jexboters115f06_ht"></graphic></fig><p>In contrast to the ECM cells, plastids containing massive plastoglobules and remnant thylakoid membranes were detected in the flavedo cells of all four genotypes (<xref ref-type="fig" rid="fig6">Fig. 6E, F</xref>; see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S7</ext-link> at <italic>JXB</italic> online), which defined the young chromoplasts derived from chloroplasts. Moreover, the chromoplasts in the flavedo cells of RB contained crystals and were almost devoid of starch granules (<xref ref-type="fig" rid="fig6">Fig. 6F</xref>).</p></sec><sec><title>Western blot analysis of two plastid proteins</title><p>Comparative microscopy indicated distinctive plastid formation in flavedos and calli. To characterize the plastids further, Western blot analysis was performed to detect two plastid proteins, a Rubisco large subunit and PsbA/D1, both of which are photosynthetic proteins and typical of chloroplasts, and are also detected in the chromoplasts arising from chloroplasts (<xref ref-type="bibr" rid="bib3">Barsan <italic>et al.</italic>, 2010</xref>). The result revealed that both proteins were missing in all calli, both light-grown and dark-grown cultures, although they were expressed actively in all flavedos (<xref ref-type="fig" rid="fig7">Fig. 7</xref>).</p><fig id="fig7" position="float"><label>Fig. 7.</label><caption><p>Western blot analysis of two plastid proteins: a Rubisco large subunit and PsbA/D1 protein. Western blot result of only one sample in each genotype was demonstrated here, representing the results of wild type and ECMs cultured in the light and dark, as they all show the same blot response. Total proteins from all samples were loaded in 50 μg, and separated by SDS-PAGE.</p></caption><graphic xlink:href="jexboters115f07_ht"></graphic></fig></sec><sec><title>Transcript profiles of isoprenoid and carotenoid metabolism in the ECMs and flavedos</title><p>Expression data showed that most genes encoding enzymes for isoprenoid and carotenoid metabolism were expressed at a similar level in all callus lines, including four wild types and four representative ECMs (M-33, RB-4, SBT-6, HQC-2) (<xref ref-type="fig" rid="fig8">Fig. 8</xref>; see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S8</ext-link> at <italic>JXB</italic> online). However, some exceptions were observed. The gene encoding phytoene desaturase (PDS) was expressed approximately 6-fold higher in HQC and RB compared with M. <italic>LCYE</italic> was expressed 7-fold higher and approximately 15-fold lower in RB and SBT, respectively, compared with M. Transcriptional analysis of all tested M callus lines showed that M-30 exhibited the highest transcript level of <italic>LCYE</italic> gene (<italic>P</italic> <0.001) (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S3B</ext-link> at <italic>JXB</italic> online).</p><fig id="fig8" position="float"><label>Fig. 8.</label><caption><p>Transcriptional analysis of genes encoding enzymes for isoprenoid and carotenoid metabolism. Transcript levels are expressed relative to M wild type. For one genotype at least five ECMs were analysed, and data of only one ECM was shown due to their similar expression trend. All data are presented as means ±SD from three replicate experiments.</p></caption><graphic xlink:href="jexboters115f08_ht"></graphic></fig><p>Furthermore, comparison of the expression patterns of the calli and the corresponding flavedos, it was possible to group these genes into three categories. The first group exhibited a flavedo-specific expression pattern, including the genes encoding DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase), HDS (hydroxymethylbutenyl 4-diphosphate synthase), HDR (hydroxymethylbutenyl 4-diphosphate reductase), PSY, LCYB2 (lycopene β-cyclase 2), HYD (β-carotene hydroxylase), NCEDs (9-<italic>cis</italic>-epoxycarotenoid dioxygenases), and CCDs (carotenoid cleavage dioxygenases). In particular, <italic>HYD</italic> was the most flavedo-specific gene, reflected by the striking up-regulation of its expression in the flavedo of almost all four genotypes whereas suppression in all calli (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S8B</ext-link> at <italic>JXB</italic> online). The second group showed a constitutive expression pattern in the calli and corresponding flavedos, including the genes encoding IPI (isopentenyl diphosphate isomerase), GGPPS (geranylgeranyl diphosphate synthase), PDS, and CRTISO (carotene isomerase). Other genes were assigned to the third category due to their unique expression patterns. In particular, <italic>DXS</italic> (encoding 1-deoxy-D-xylulose 5-phosphate synthase) and <italic>ZDS</italic> (encoding ζ-carotene desaturase) were expressed at higher levels in the flavedos of RB as compared to other lines, while <italic>LCYE</italic> was expressed at unusually low levels only in the callus of SBT.</p></sec><sec><title>Over-expression of CrtB in combination with DSM2 in callus tissue</title><p>Transcriptional analysis suggested HYD plays a key role in regulating the preferred carotenoid patterns between the ECMs and flavedos. To prove this, one non-haem-diiron β-carotene hydroxylase gene (<italic>DMS2</italic>, also named as <italic>HYD3</italic>) from rice (<xref ref-type="bibr" rid="bib13">Du <italic>et al.</italic>, 2010</xref>) was introduced into an ECM of M. This co-transformation system was also expected to be used to examine whether β-carotene was the major component of the crystals in the ECMs.</p><p>As shown in <xref ref-type="fig" rid="fig9">Fig. 9</xref>, co-transformed lines showed an altered phenotype from orange to gold. HPLC analysis revealed an 80.4% reduction of β-carotene on average and a 67.7% reduction of α-carotene in the co-transformed lines. As expected, xanthophyll content was noticeably increased in the co-transformed lines, particularly violaxanthin content, which exhibited a 2.4-fold increase. However, unlike flavedos, the co-transformed lines accumulated unesterified xanthophylls. Furthermore, carotenoid crystals became smaller in size, as shown by small dots attaching to the amyloplasts of co-transformed cells, or even being invisible (<xref ref-type="fig" rid="fig9">Fig. 9A, B</xref>). Subsequently, the ultrastructural inspection of the amyloplasts in the co-transformed cells showed an increased number of plastoglobules (<xref ref-type="fig" rid="fig9">Fig. 9C</xref>).</p><fig id="fig9" position="float"><label>Fig. 9.</label><caption><p>Carotenoid analysis and cellular inspection of co-transformed lines (35S::<italic>CrtB/DSM2</italic>) and M-33 (35S::<italic>CrtB</italic>). Via., violaxanthin; Zea., zeaxanthin; Lut., lutein; Phy., phytoene; α-Car., α-carotene; β-Car., β-carotene. Values and bars represent the means and ±SD (<italic>n</italic>=3 replicate experiments), respectively. ** Indicates that the values are significantly different compared with M-33 at the significance level of <italic>P</italic> <0.01. (A) Normal light microscopy. (B) Polarization microscopy (arrows show the dot-crystals attaching to the amyloplasts). (C) Ultrastructure of amyloplast (arrow shows the electron-transparent plastoglobules; S, starch granule). The bar in picture (A) represents 10 μm.</p></caption><graphic xlink:href="jexboters115f09_3c"></graphic></fig></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>Comparative analysis of various ECMs with the natural carotenoid-rich system (flavedo) provides a powerful tool for gaining insight into the key regulatory patterns and metabolic sink formation, particularly those with regard to β-carotene accumulation. More importantly, the ECMs from dark-grown culture were found to be excellent accumulators of carotenoids consisting predominantly of β-carotene. In a representative RB ECM, the carotenoid content, with about 56% β-carotene, was even higher than those of carotenoid-rich crops, such as carrot roots (2000–4000 μg g<sup>−1</sup> dry weight) and tomatoes (2000 μg g<sup>−1</sup> dry weight) (<xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic>, 2009</xref>). Therefore, an improved understanding of the mechanisms underlying β-carotene accumulation in the ECMs could promote effective carotenoid engineering in non-green tissues, which represent major food sources for humans (<xref ref-type="bibr" rid="bib28">Howitt and Pogson, 2006</xref>).</p><sec><title>ECMs possess a favoured β,β-pathway</title><p>CrtB created a considerable carotenogenic flux in the callus tissues of various genotypes, including a red-fruit variety (RB) which accumulated lycopene in ripe fruits. However, in the ECMs, no abundant lycopene accumulation was found, except for a low level in that of RB and M (<xref ref-type="table" rid="tbl1">Table 1</xref>; <xref ref-type="fig" rid="fig3">Fig. 3</xref>). Supposedly, lycopene accumulation in fruits is a tissue-specific developmental process that is difficult to assess from non-fruit tissues, conforming to the report that a unique chromoplast-specific lycopene β-cyclase gene (<italic>LCYB2</italic>) determines the lycopene accumulation in the RB fruits (<xref ref-type="bibr" rid="bib1">Alquezar <italic>et al.</italic>, 2009</xref>). The ECMs contained a substantial favoured β,β-branch pathway, consistent with the observations in other engineered plants, such as canola seeds (<xref ref-type="bibr" rid="bib49">Shewmaker <italic>et al.</italic>, 1999</xref>), golden rice (<xref ref-type="bibr" rid="bib47">Schaub <italic>et al.</italic>, 2005</xref>), and golden potatoes (<xref ref-type="bibr" rid="bib12">Diretto <italic>et al.</italic>, 2010</xref>).</p><p>However, in most cases, the β,β-branch pathway is not predominant in wild-type tissues (or organs), this is also confirmed by the result of our experiment in which β-carotene only accounted for 1.5% of total carotenoids in wild-type RB calli, while lutein took up a substantial proportion (51.1%). Thus, the balance of carotenoid accumulation was altered as a result of genetic manipulation. It is noted that, in the present study, a suppressed transcript level for <italic>LCYE</italic> could explain the scarce accumulation of lutein in SBT callus, and M-30, which probably contained additional variation, exhibited increased <italic>LCYE</italic> transcription, yielding high lutein accumulation (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S3</ext-link> at <italic>JXB</italic> online). Based on these data and the power function of the content/proportion change of lutein relative to lycopene flux (<xref ref-type="fig" rid="fig4">Fig. 4</xref>), as well as a suggestion of metabolic channelling from LCYE to LCYB (<xref ref-type="bibr" rid="bib2">Bai <italic>et al.</italic>, 2009</xref>), a model was established to explain the favouring of the β,β-pathway in the ECMs here (<xref ref-type="fig" rid="fig10">Fig. 10</xref>) and is predicted to be valid in other carotenoid engineered plants where the phenomena is observed. In the wild-type calli of the model, a small amount of synthesized lycopene was sufficiently captured by LCYE and converted to δ-carotene, the substrate for LCYB, and formed a high proportion of the β,ε-carotenoids, such as lutein. However, in this model, LCYE was regarded as a cyclase with limited activity relative to LCYB, based on the fact that LCYB is the effective bi-cyclic cyclase, in the absence of which, lycopene accumulates mainly in plant tissues, whereas LCYE is generally the mono-cyclic cyclase, and less lycopene accumulates when LCYE is absent (<xref ref-type="bibr" rid="bib44">Pogson <italic>et al.</italic>, 1996</xref>; <xref ref-type="bibr" rid="bib1">Alquezar <italic>et al.</italic>, 2009</xref>; <xref ref-type="bibr" rid="bib2">Bai <italic>et al.</italic>, 2009</xref>). Accordingly, in the ECMs abundant lycopene molecules that were not captured by LCYE diffused to LCYB and were converted to β-carotene. Subsequently, β-carotene was predominantly accumulated, but less leaked to xanthophylls, which implicated the low carotenoid hydroxylase activity in the ECMs.</p><fig id="fig10" position="float"><label>Fig. 10.</label><caption><p>A hypothetical schematic model of carotenoid metabolic channels toward the β-carotene branch point in callus tissues. In wild-type callus, most of lycopene molecules can be captured by LCYE because of low carotenoid metabolism flux. In the ECMs, exogenous CrtB engineers a striking carotenoid metabolism flux. The activity of LCYE is unaltered. A mass of lycopene molecules diffuses to LCYB and is converted into β-carotene.</p></caption><graphic xlink:href="jexboters115f10_3c"></graphic></fig><p>Therefore, this model suggests that LCYE has a bottleneck role in the presence of abundant lycopene substrates leading to an altered carotenoid composition. This is supported by the reports that diverse LCYE enzymatic activity determines alternative β-carotene and lutein compositions in maize and wheat (<xref ref-type="bibr" rid="bib23">Harjes <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="bib27">Howitt <italic>et al.</italic>, 2009</xref>), as well as by the fact that over-expression of CrtI, a bacterial phytoene desaturase that converts phytoene to lycopene, leads to substantially increased β,β-carotenoid accumulation in tobacco leaves (<xref ref-type="bibr" rid="bib41">Misawa <italic>et al.</italic>, 1993</xref>) and tomato fruits (<xref ref-type="bibr" rid="bib45">Romer <italic>et al.</italic>, 2000</xref>).</p><p>In addition, a β- and ε-cyclase complex has once been proposed to explain the branch control of carotenoid pathway (<xref ref-type="bibr" rid="bib2">Bai <italic>et al.</italic>, 2009</xref>). In this complex, it is speculated that there is competition for substrate between LCYB and LCYE, probably relying on their different cyclase affinity to substrate. This proposed system suggested a feedback regulation mechanism, mediated by the altered lycopene substrate preference by cyclase, which may be acting to influence the proportion of β,β-carotenoids and β,ε-carotenoids (<xref ref-type="bibr" rid="bib6">Cazzonelli and Pogson, 2010</xref>).</p></sec><sec><title>Amyloplasts serve as crystalline-type β-carotene sequestering sink in the ECMs</title><p>As revealed by cellular inspection and Western blot analysis, plastid types differed in the callus and flavedo cells. In flavedo cells, chromoplasts transformed from chloroplasts served as a carotenoid metabolic sink. By comparison, almost all the observed carotenoids in the ECM cells were sequestered within amyloplasts; despite the fact that amyloplasts showed chromoplast-like profiles owing to visible crystals and plastoglobules inside, starch granules were still the most predominant (<xref ref-type="fig" rid="fig6">Fig. 6A</xref>). β-Carotene content was correlated with crystal development in <italic>CrtB/DSM2</italic> co-transformed lines, which reveals that the carotenoid crystals predominantly contained β-carotene. Other carotenoids, such as xanthophylls, might be deposited in amyloplasts without crystallization, which was suggested by the multiplication of plastoglobules observed in the amyloplasts (<xref ref-type="fig" rid="fig6">Figs 6A</xref>, <xref ref-type="fig" rid="fig9">9C</xref>), and also by the coloured plastid structures (Fig. 5M), which were probably the broken amyloplasts losing most starch granules, as previously observed in potato tubers (<xref ref-type="bibr" rid="bib38">Lopez <italic>et al.</italic>, 2008</xref>). In agreement with the previous description of crystal formation in proplastids driven by over-expression of <italic>PSY</italic> (<xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic>, 2009</xref>), the observations in the present study demonstrate that carotenoid sequestering sink can also be formed in the amyloplasts.</p><p>Chromoplast development requires the controlled changes of the pre-existing plastids, such as the internal membrane remodelling (<xref ref-type="bibr" rid="bib16">Egea <italic>et al.</italic>, 2010</xref>), which may be mediated by some crucial factors. For example, OR functions in directing the transition from non-coloured plastids to chromoplasts, over-expression of <italic>OR</italic> drives β-carotene crystal formation in potato tubers and cauliflowers (<xref ref-type="bibr" rid="bib38">Lopez <italic>et al.</italic>, 2008</xref>). In addition, metabolite-induced plastid transition is found in the engineered tomato fruits over-expressing <italic>PSY</italic> (<xref ref-type="bibr" rid="bib20">Fraser <italic>et al.</italic>, 2007</xref>). A previous study of tobacco floral nectaries has clearly shown that amyloplasts could be converted into chromoplasts (<xref ref-type="bibr" rid="bib25">Horner <italic>et al.</italic>, 2007</xref>). However, amyloplasts are sustained in the ECMs, whether cultured in the light or in the dark. Unlike <italic>Arabidopsis</italic>, citrus has a chromoplast developmental programme (<xref ref-type="bibr" rid="bib1">Alquezar <italic>et al.</italic>, 2009</xref>; <xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic>, 2009</xref>). Probably, such a programme in the cell models is suppressed by the predominant starch accumulation, or the cell models lack some factors that initiate this progress. Further investigation is needed to find out the causes repressing the transition of amyloplasts to chromoplasts, particularly the impact of starch metabolism on chromoplast development and carotenogenesis.</p></sec><sec><title>Hypothetical carotenoid degradation in the ECMs</title><p>In the present study, abundant carotenoid crystals without surrounding membranes were found in the isotonic buffer (Fig. 5L), suggesting that the crystals were not firmly attached to the amyloplast membranes. As previously discussed by <xref ref-type="bibr" rid="bib40">Maass <italic>et al.</italic> (2009)</xref>, carotenoid crystals are free from the lipophilic phase of the plastid membrane, and the crystalline form may protect carotenoids from enzymatic catabolism. However, crystallization seems not to protect β-carotene from photo-oxidative degradation in the ECMs. It is noted that the ECMs under irradiation showed a marked decrease in β-carotene level (see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S6</ext-link> at <italic>JXB</italic> online). This result is not due to a conversion of β-carotene to β,β-xanthophylls as in the cyanobacterium (<xref ref-type="bibr" rid="bib46">Schäfer <italic>et al.</italic>, 2006</xref>), because violaxanthin levels decreased in the light-grown ECMs. Light can generate oxidative stress, which is also confirmed by our experiment in the citrus callus (data not shown). Thus, it is possible that the lost carotenoids in the light-grown ECMs are involved in an oxidative reaction. Micrographs of the ECM cells revealed carotenoid sequestering structures in the vacuoles, specially plastoglobules and crystals without a membrane structure (<xref ref-type="fig" rid="fig5 fig6">Figs 5J, K, 6C, D</xref>). The structures appear to be similar to those of starch granules without an intact amyloplast membrane found inside the vacuoles of cultured rice cells during sucrose starvation through vacuolar phagocytosis (Chen. <italic>et al.</italic>, 1994). This provides evidence of an additional site for carotenoids and implies that there is an underlying catabolism mechanism that governs carotenoids in the vacuoles. Further studies are required to discover new pathways related to carotenoids degradation.</p></sec><sec><title>HYD transcription predominantly determines the variation of carotenoid accumulation in calli and flavedos</title><p>Transcriptional analysis confirmed the crucial rate-controlling role of PSY in the ECMs, given an over-expressed <italic>CrtB</italic> driving the abundant accumulation of carotenoid. However, the major accumulation pattern of β-carotene in the ECMs is very different from that of esterified β,β-xanthophylls in common fruits (<xref ref-type="table" rid="tbl1">Table 1</xref>). In the ECMs, the proportion of violaxanthin was lower than in the wild types (<xref ref-type="fig" rid="fig3">Fig. 3</xref>). This phenomenon seemed like a result of feedback inhibition from striking metabolic flux. Furthermore, in the flavedos of the present study, a co-ordinated transcriptional up-regulation of most genes encoding enzymes for isoprenoid and carotenoid metabolism, especially the <italic>HYD</italic> gene, reflects a fruit-specific regulation of carotenogenesis (<xref ref-type="fig" rid="fig8">Fig. 8</xref>; see <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Fig. S8</ext-link> at <italic>JXB</italic> online) (<xref ref-type="bibr" rid="bib31">Kato <italic>et al.</italic>, 2004</xref>). As reported previously, HYD is associated with β-carotene accumulation in the endosperm of maize (<xref ref-type="bibr" rid="bib51">Vallabhaneni <italic>et al.</italic>, 2009</xref>; <xref ref-type="bibr" rid="bib57">Yan <italic>et al.</italic>, 2010</xref>). Therefore, the over-expression of an <italic>HYD</italic> gene from rice was performed in the ECM, which resulted in a significantly decreased β-carotene content and increased violaxanthin and other xanthophylls (<xref ref-type="fig" rid="fig9">Fig. 9</xref>). This study identifies the crucial role of HYD in determining the diversified carotenoid accumulation patterns between the ECMs and flavedos, and also suggests that HYD can be a key target for β-carotene metabolic engineering in citrus fruits. However, it is noteworthy that the β,β-xanthophylls in <italic>CrtB/DSM2</italic> co-transformed lines were all non-esterified forms, which is presumed to be associated with an inactive fatty acid synthesis in the amyloplasts of the ECM cells. In general, esterification of xanthophylls with fatty acids is a tissue-specific behaviour which is more likely to occur in ripe fruits (<xref ref-type="bibr" rid="bib26">Hornero-Mendez and Minguez-Mosquera, 2000</xref>; <xref ref-type="bibr" rid="bib10">D’Ambrosio <italic>et al.</italic>, 2011</xref>).</p></sec></sec><sec sec-type="conclusion"><title>Conclusion</title><p>In summary, this paper reports diverse ECMs depending on the genotypes of citrus used for the construction. However, the carotenoid pattern in the ECMs of one genotype seems not to correlate with that in its flavedo. Comparative analysis of various ECMs with flavedos probes into the β-carotene accumulation mechanism with new insights. The present study correlates striking β-carotene accumulation with the suppressed HYD transcription and a channelling control of alternative lycopene flux in the ECMs. It also shows that, in the ECM cells, β-carotene is synthesized and sequestered in the amyloplasts in crystalline form. Furthermore, carotenoid crystals consisting mainly of β-carotene are confirmed. The likelihood of carotenoid degradation mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is also proposed. Utilization of the ECMs is not limited to comprehension of β-carotene accumulation. The ECMs may facilitate the investigations into more potential gene targets associated with carotenogenesis and into the regulation metabolism of carotenoid accumulation in response to various environmental stimuli.</p></sec><sec sec-type="supplementary-material"><title>Supplementary data</title><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary data</ext-link> can be found at <italic>JXB</italic> online.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Table S1</bold></ext-link><bold>.</bold> Primers used in this study.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Table S2.</bold></ext-link> Specific primers used in real-time reverse transcriptase-PCR.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Table S3.</bold></ext-link> Analysis of carotenoid profiles detected in the wild-type and ECM lines.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S1.</bold></ext-link> Carotenoid biosythesis pathway in higher plants.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S2.</bold></ext-link> A DNA gel blot analysis indicating inserted patterns of T-DNA in the representative lines.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S3.</bold></ext-link> A novel 35S::<italic>CrtB</italic> ECM (M-30) containing a high proportion of lutein due to the highest expression of the <italic>LCYE</italic> gene.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S4.</bold></ext-link> Absorbance spectra of the peaks detected in ECMs (peak identification in <ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1">Supplementary Table S3</ext-link>).</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S5.</bold></ext-link> HPLC chromatograms of carotenoids extracted from the RB ECM (A) and HQC flavedo (B) at 450 nm.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S6.</bold></ext-link> Comparative analysis of carotenoids contents in light/dark-grown engineered lines.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S7.</bold></ext-link> Additional photographs of the visual inspection of the flavedo cells and plastid isolation.</p><p><ext-link ext-link-type="uri" xlink:href="http://www.jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ers115/-/DC1"><bold>Supplementary Fig. S8.</bold></ext-link> Comparative transcriptional analysis of genes encoding enzymes for isoprenoid and carotenoid metabolism among wild-type calli, ECMs, and flavedos.</p><supplementary-material id="PMC_1" content-type="local-data"><caption><title>Supplementary Data</title></caption><media mimetype="text" mime-subtype="html" xlink:href="supp_63_12_4403__index.html"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="supp_ers115_00075879-file001.pdf"></media></supplementary-material></sec></body><back><ack><p>This research was supported by the National Basic Research Program of China (No. 2011CB100600) and NSFC (30830078, 30921002). We thank Professor Shih-Tung Liu (Taiwan, China) for providing the <italic>Erwinia herbicola CrtB</italic> gene, and Professor Lizhong Xiong and Hao Du (Huazhong Agricultural University, China) for the gift of the <italic>DSM2</italic> over-expressing construct. We thank Professor Yuanhuai Han (Shanxi Agricultural University, China), Professor Jihong Liu (Huazhong Agricultural University, China), Professor Hanhui Kuang (Huazhong Agricultural University, China), Zhiyong Pan and Jinzhi Zhang (Huazhong Agricultural University, China) for their critical reading of this paper. 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