Yang et al/Comparative transcriptor of Phalaenopsis flowers in white petals.This observation is consistent with a report 4-coumaroyl-CoA+malonyl-CoA that PeUFGT3 plays a critical role in red color formation in Phalaenopsis [8].On the basis of these findings,we propose the following mechanism to explain red color formation 4,2,4,6'-Tetrahydroxychalcone(yellow) in the labella(Fig.5).First,CHS,the key enzyme in the flavonoid biosynthesis pathway,catalyzes the condensa- tion of three acetate residues from malonyl-CoA with CHI 4-coumaroyl-CoA to form tetrahydroxychalcone.Next, Flavanone (colorless) tetrahydroxychalcone is stereospecifically isomerized by CHI to colorless flavanone,which is catalyzed further to 3洲 colorless dihydroquercetin by flavanone 3-hydroxylase(F3H) dihydrokaempferol and flavonoid 3-hydroxylase(F3H).Dihydroquercetin is catalyzed in turn by DFR,ANS,and UFGT3 to form an anthocyanin(cyanidin-3-glucoside),thereby generating F3H the red color of the labella [5,6].In contrast to a previous Dihydroquercetin(colorless) study [8],however,we did not identify any DEGs encoding CHS,ANS,DFR,or F3H in white petals.To explain this DFR+ANS+UFGT3 discrepancy,we suggest two possibilities.First,no method Anthocyanins(Cyanidin-3-Glucoside;red labella) is perfect;we may thus have failed to detect the expression of these genes in white petals because of technical limitations. Fig.5 Proposed pathway for synthesis of red coloration in labella Alternatively,because different Phalaenopsis plants have inherited different characteristics,an anthocyanin synthesis pathway may not have existed in our chosen white petal samples(possibly the main reason for their white color)[26] regulated by a set of WD40,MYB,and bHLH transcription We also identified several DEGs that encode key enzymes factors similar to those involved in trichome and cell shape regulating carotenoid synthesis in petals and labella,such development [9,29,30].In this study,we detected 12 MYB as phytoene synthase (PSY),phenylalanine ammonialyase genes in petals and labella,with more DEGs present and (PAL),and phytoene desaturase(PDS).In composites,floral more strongly expressed in labella(9)than in petals(3).This coloration in the orange-red to red range is determined by differential expression may be responsible for the contrasting a combination of anthocyanins and/or carotenoids [27]; coloration of labella and petals. the same observation has been made for orchids such as The results of our study should serve as a valuable genetic Disa hybrids [28].The anthocyanin pathway may thus not resource for future investigations of the molecular basis of be the only pathway controlling flower color formation in flower color and floral organ formation in Phalaenopsis,and Phalaenopsis,especially red color.Pigment biosynthesis is inform Phalaenopsis flower breeding. Acknowledgments 3.Mondragon-Palomino M,Theissen G.Why are orchid flowers so This work was supported by the National Natural Science Foundation of diverse?Reduction of evolutionary constraints by paralogues of class China(31101574),the Youth Science and Technology Innovation Fund of B floral homeotic genes.Ann Bot.2009;104(3):583-594.http://dx.doi. NJAU(KJ2011008),and the Agricultural Science and Technology Support org/10.1093/aob/mcn258 Program of Zhenjiang(NY2011015). 4.Salemme M,Sica M,Gaudio L Aceto S.Expression pattern of two paralogs of the PI/GLO-like locus during Orchis italica(Orchidaceae, Authors'contributions Orchidinae)flower development.Dev Genes Evol.2011:221(4):241- The following declarations about authors'contributions to the research 246.http:/dx.doi.org/10.1007/s00427-011-0372-6 have been made:the experimental plan was conceived and designed by CZ; 5.Tanaka Y,Sasaki N,Ohmiya A.Biosynthesis of plant pigments: participated in sample collection and RNA preparation and performed the anthocyanins,betalains and carotenoids.Plant J.2008:54(4):733-749. experiments:JW,YY;contributed to data analysis,bioinformatics analysis, htp/dx.doi.org/10.1111/.1365-313X2008.03447.x and manuscript preparation:ZM,GS;drafted and revised the manuscript: 6.Tanaka Y,Brugliera F.Kalc G,Senior M,Dyson B,Nakamura N,et IW,YY.JW and YY contributed equally to this work. al.Flower color modification by engineering of the flavonoid biosyn- thetic pathway:practical perspectives.Biosci Biotechnol Biochem. Supplementary material 2010:749):1760-1769.http:/dx.doi.org/10.1271/bbb.100358 The following supplementary material for this article is available on- 7.Fukuchi-Mizutani M.Biochemical and molecular characterization line at http://pbsociety.org.pl/journals/index.php/asbp/rt/suppFiles/ of a novel UDP-glucose:anthocyanin 3'-O-glucosyltransferase,a key asbp.2014.023/0: enzyme for blue anthocyanin biosynthesis,from gentian.Plant Physiol 1.Tab S1:unigenes mapped to KEGG pathways. 2003:132(3:1652-1663.http:/dk.doi.org/10.1104/pp.102.018242 8.Chen WH,Hsu CY,Cheng HY,Chang H,Chen HH,Ger MJ.Down References regulation of putative UDP-glucose:flavonoid 3-0-glucosyltrans- 1.Teixeira da Silva JA,Chin DP,Van PT,Mii M.Transgenic orchids ferase gene alters flower coloring in Phalaenopsis.Plant Cell Rep. Sci Hortic.2011;130(4):673-680.http:/dx.doi.org/10.1016/j 201130(6):1007-1017.htp/dx.doi..org/10.1007/s00299-011-1006-1 scienta.2011.08.025 9.Koes R,Verweij W,Quattrocchio F.Flavonoids:a colorful model for 2.Mondragon-Palomino M,Theifen G.MADS about the evolution the regulation and evolution ofbiochemical pathways.Trends Plant Sci. of orchid flowers.Trends Plant Sci.2008:13(2):51-59.http://dx.doi. 2005:10(5):236-242.http:/dx.doi..org/10.1016/j.tplants.2005.03.002 org/10.1016/j.tplants..2007.11.007 10.Harborne JB,Williams CA.Anthocyanins and other flavonoids The Author(s)2014 Published by Polish Botanical Soclety Acta Soc Bot Pol 83(3):191-199 198© The Author(s) 2014 Published by Polish Botanical Society Acta Soc Bot Pol 83(3):191–199 198 Yang et al. / Comparative transcriptome analysis of Phalaenopsis flowers Acknowledgments This work was supported by the National Natural Science Foundation of China (31101574), the Youth Science and Technology Innovation Fund of NJAU (KJ2011008), and the Agricultural Science and Technology Support Program of Zhenjiang (NY2011015). Authors’ contributions The following declarations about authors’ contributions to the research have been made: the experimental plan was conceived and designed by CZ; participated in sample collection and RNA preparation and performed the experiments: JW, YY; contributed to data analysis, bioinformatics analysis, and manuscript preparation: ZM, GS; drafted and revised the manuscript: JW, YY. JW and YY contributed equally to this work. Supplementary material The following supplementary material for this article is available on￾line at http://pbsociety.org.pl/journals/index.php/asbp/rt/suppFiles/ asbp.2014.023/0: 1. Tab S1: unigenes mapped to KEGG pathways. References 1. Teixeira da Silva JA, Chin DP, Van PT, Mii M. Transgenic orchids. Sci Hortic. 2011;130(4):673–680. http://dx.doi.org/10.1016/j. scienta.2011.08.025 2. Mondragón-Palomino M, Theißen G. MADS about the evolution of orchid flowers. Trends Plant Sci. 2008;13(2):51–59. http://dx.doi. org/10.1016/j.tplants.2007.11.007 3. Mondragon-Palomino M, Theissen G. Why are orchid flowers so diverse? Reduction of evolutionary constraints by paralogues of class B floral homeotic genes. Ann Bot. 2009;104(3):583–594. http://dx.doi. org/10.1093/aob/mcn258 4. Salemme M, Sica M, Gaudio L, Aceto S. Expression pattern of two paralogs of the PI/GLO-like locus during Orchis italica (Orchidaceae, Orchidinae) flower development. Dev Genes Evol. 2011;221(4):241– 246. http://dx.doi.org/10.1007/s00427-011-0372-6 5. Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 2008;54(4):733–749. http://dx.doi.org/10.1111/j.1365-313X.2008.03447.x 6. Tanaka Y, Brugliera F, Kalc G, Senior M, Dyson B, Nakamura N, et al. Flower color modification by engineering of the flavonoid biosyn￾thetic pathway: practical perspectives. Biosci Biotechnol Biochem. 2010;74(9):1760–1769. http://dx.doi.org/10.1271/bbb.100358 7. Fukuchi-Mizutani M. Biochemical and molecular characterization of a novel UDP-glucose:anthocyanin 3'-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from gentian. Plant Physiol. 2003;132(3):1652–1663. http://dx.doi.org/10.1104/pp.102.018242 8. Chen WH, Hsu CY, Cheng HY, Chang H, Chen HH, Ger MJ. Down￾regulation of putative UDP-glucose: flavonoid 3-O-glucosyltrans￾ferase gene alters flower coloring in Phalaenopsis. Plant Cell Rep. 2011;30(6):1007–1017. http://dx.doi.org/10.1007/s00299-011-1006-1 9. Koes R, Verweij W, Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 2005;10(5):236–242. http://dx.doi.org/10.1016/j.tplants.2005.03.002 10. Harborne JB, Williams CA. Anthocyanins and other flavonoids in white petals. This observation is consistent with a report that PeUFGT3 plays a critical role in red color formation in Phalaenopsis [8]. On the basis of these findings, we propose the following mechanism to explain red color formation in the labella (Fig. 5). First, CHS, the key enzyme in the flavonoid biosynthesis pathway, catalyzes the condensa￾tion of three acetate residues from malonyl-CoA with 4-coumaroyl-CoA to form tetrahydroxychalcone. Next, tetrahydroxychalcone is stereospecifically isomerized by CHI to colorless flavanone, which is catalyzed further to colorless dihydroquercetin by flavanone 3-hydroxylase (F3H) and flavonoid 3'-hydroxylase (F3'H). Dihydroquercetin is catalyzed in turn by DFR, ANS, and UFGT3 to form an anthocyanin (cyanidin-3-glucoside), thereby generating the red color of the labella [5,6]. In contrast to a previous study [8], however, we did not identify any DEGs encoding CHS, ANS, DFR, or F3'H in white petals. To explain this discrepancy, we suggest two possibilities. First, no method is perfect; we may thus have failed to detect the expression of these genes in white petals because of technical limitations. Alternatively, because different Phalaenopsis plants have inherited different characteristics, an anthocyanin synthesis pathway may not have existed in our chosen white petal samples (possibly the main reason for their white color) [26]. We also identified several DEGs that encode key enzymes regulating carotenoid synthesis in petals and labella, such as phytoene synthase (PSY), phenylalanine ammonialyase (PAL), and phytoene desaturase (PDS). In composites, floral coloration in the orange-red to red range is determined by a combination of anthocyanins and/or carotenoids [27]; the same observation has been made for orchids such as Disa hybrids [28]. The anthocyanin pathway may thus not be the only pathway controlling flower color formation in Phalaenopsis, especially red color. Pigment biosynthesis is regulated by a set of WD40, MYB, and bHLH transcription factors similar to those involved in trichome and cell shape development [9,29,30]. In this study, we detected 12 MYB genes in petals and labella, with more DEGs present and more strongly expressed in labella (9) than in petals (3). This differential expression may be responsible for the contrasting coloration of labella and petals. The results of our study should serve as a valuable genetic resource for future investigations of the molecular basis of flower color and floral organ formation in Phalaenopsis, and inform Phalaenopsis flower breeding. Fig. 5 Proposed pathway for synthesis of red coloration in labella