Yang et al/Comparative transcriptome analysis of Phalaenopsis flowers Tab.2 Some unigenes mapped to Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways. KEGG pathways Sub-pathways of KEGG pathway Branches of sub-pathway PN PNU PL PLU Metabolism Energy metabolism Photosynthesis-antenna proteins 11 0 11 11 Photosynthesis 12 2 12 10 Carbon fixation in photosynthetic organisms 14 0 14 14 Oxidative phosphorylation 32 3 32 29 Biosynthesis of other secondary Tropane,piperidine and pyridine alkaloid 1 0 1 metabolites biosynthesis Flavonoid biosynthesis 2 6 4 Flavone and flavonol biosynthesis 0 2 Stilbenoid,diarylheptanoid and gingerol 2 biosynthesis Betalain biosynthesis 0 0 Isoquinoline alkaloid biosynthesis 2 Lipid metabolism Glycerolipid metabolism 2 2 Glycerophospholipid metabolism 3 PN-number of mapped unigenes from petals;PNU-number of mapped up-regulated unigenes from petals;PL-number of mapped unigenes from labella;PLU-number of mapped up-regulated unigenes from labella. With respect to metabolic processes (Tab.2),the dis- between labella and petals.In addition,several DEGs related tributions of petal and labella unigenes involved in energy to flower color and floral differentiation were found in labella metabolism were significantly different.As 67 unigenes and petals. from labella were participants in this pathway compared with 10 unigenes from petals,more DEGs in labella were Genes related to floral differentiation accordingly related to this KEGG sub-pathway.These results MADS-box genes have long been noted for their roles in suggest that the labella is associated with a more complex floral organ identity.In this study,we detected 16 genes dif- energy metabolism mechanism than are petals;the labella ferentially expressed between petals and labella that mapped may thus not only be an energy sink,but may also serve as to 29 MADS-like genes.All 16 of these DEGs were expressed an energy source for its own function or supply energy to in petals,with 9 found to be up-regulated.Only 12 of the 16 other floral organs. DEGs were expressed in labella,with 7 up-regulated in that floral organ.One of the latter seven DEGs was a MADS4- Genes related to floral differentiation and color like unigene,which is specifically expressed in the labella Transcription factors control genomic expression and and can be ectopically expressed in the labella-like petals play vital roles in all aspects of higher plant life cycles.The of a peloric mutant [14].Another identified unigene was distribution of transcription factors associated with floral homologous to AGL9-like genes,which may function at the differentiation-including MADS(16 DEGs),MYB,ARE, top of a proposed regulatory hierarchy of MADS-box genes NAC,and TCP-was similar between petals and labella,but controlling floral development [15].The TCP family is an their expression levels were significantly different(Tab.3). ancient group of plant developmental transcription factors Orchids produce flowers of a variety of colors ranging that regulate cell division in vegetative and reproductive from greenish,white,and yellow to intense red,blue,and structures and that are essential to the establishment of flower brown.Several anthocyanin biosynthetic DEGs,such as zygomorphy[16].Three unigenes associated with TCP were CHI,CHS,UDP-glucose:flavonoid-3-O-glucosyltransferase identified as expressed in both petals and labella,although (UFGT3),DFR,and ANS,were uncovered in this study all three had much higher expression levels in labella than in (Tab.4). petals.Studies have shown that TCP may have played a role in the diversification and radiation of Orchidaceae[17].The elevated expression of these genes in labella imply that TCP Discussion has a more prominent role in patterning of labella than of petals.MYB transcription factors,which can affect overall In this study,we obtained 37 723 and 34 020 unigenes petal morphology by influencing cell shape [18],were more from petals and labella,respectively.A total of 2736 DEGs numerous and more highly expressed in labella(9)than were identified,many of which were functionally annotated in petals(3).These genes may therefore also contribute to by BLAST searches against several public databases.We differences in differentiation between the labella and petals. mapped 837 DEGs(432 up-regulated in petals and 405 We detected 10 unigenes that encode auxin response factor up-regulated in labella)to 102 KEGG pathways.Almost (ARF);although all of them were expressed in both labella all of these pathways were found in both flower parts:102 and petals,more were up-regulated in labella(7 unigenes) pathways in petals and 99 in labella.DEGs involved in en- than in petals(3 unigenes).Auxin signaling plays key roles ergy metabolism were significantly differentially distributed in many plant growth and developmental processes from The Author(s)2014 Published by Polish Botanical Soclety Acta Soc Bot Pol 83(3):191-199 196© The Author(s) 2014 Published by Polish Botanical Society Acta Soc Bot Pol 83(3):191–199 196 Yang et al. / Comparative transcriptome analysis of Phalaenopsis flowers With respect to metabolic processes (Tab. 2), the dis￾tributions of petal and labella unigenes involved in energy metabolism were significantly different. As 67 unigenes from labella were participants in this pathway compared with 10 unigenes from petals, more DEGs in labella were accordingly related to this KEGG sub-pathway. These results suggest that the labella is associated with a more complex energy metabolism mechanism than are petals; the labella may thus not only be an energy sink, but may also serve as an energy source for its own function or supply energy to other floral organs. Genes related to floral differentiation and color Transcription factors control genomic expression and play vital roles in all aspects of higher plant life cycles. The distribution of transcription factors associated with floral differentiation – including MADS (16 DEGs), MYB, ARF, NAC, and TCP – was similar between petals and labella, but their expression levels were significantly different (Tab. 3). Orchids produce flowers of a variety of colors ranging from greenish, white, and yellow to intense red, blue, and brown. Several anthocyanin biosynthetic DEGs, such as CHI, CHS, UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT3), DFR, and ANS, were uncovered in this study (Tab. 4). Discussion In this study, we obtained 37 723 and 34 020 unigenes from petals and labella, respectively. A total of 2736 DEGs were identified, many of which were functionally annotated by BLAST searches against several public databases. We mapped 837 DEGs (432 up-regulated in petals and 405 up-regulated in labella) to 102 KEGG pathways. Almost all of these pathways were found in both flower parts: 102 pathways in petals and 99 in labella. DEGs involved in en￾ergy metabolism were significantly differentially distributed between labella and petals. In addition, several DEGs related to flower color and floral differentiation were found in labella and petals. Genes related to floral differentiation MADS-box genes have long been noted for their roles in floral organ identity. In this study, we detected 16 genes dif￾ferentially expressed between petals and labella that mapped to 29 MADS-like genes. All 16 of these DEGs were expressed in petals, with 9 found to be up-regulated. Only 12 of the 16 DEGs were expressed in labella, with 7 up-regulated in that floral organ. One of the latter seven DEGs was a MADS4- like unigene, which is specifically expressed in the labella and can be ectopically expressed in the labella-like petals of a peloric mutant [14]. Another identified unigene was homologous to AGL9-like genes, which may function at the top of a proposed regulatory hierarchy of MADS-box genes controlling floral development [15]. The TCP family is an ancient group of plant developmental transcription factors that regulate cell division in vegetative and reproductive structures and that are essential to the establishment of flower zygomorphy [16]. Three unigenes associated with TCP were identified as expressed in both petals and labella, although all three had much higher expression levels in labella than in petals. Studies have shown that TCP may have played a role in the diversification and radiation of Orchidaceae [17]. The elevated expression of these genes in labella imply that TCP has a more prominent role in patterning of labella than of petals. MYB transcription factors, which can affect overall petal morphology by influencing cell shape [18], were more numerous and more highly expressed in labella (9) than in petals (3). These genes may therefore also contribute to differences in differentiation between the labella and petals. We detected 10 unigenes that encode auxin response factor (ARF); although all of them were expressed in both labella and petals, more were up-regulated in labella (7 unigenes) than in petals (3 unigenes). Auxin signaling plays key roles in many plant growth and developmental processes from KEGG pathways Sub-pathways of KEGG pathway Branches of sub-pathway PN PNU PL PLU Metabolism Energy metabolism Photosynthesis–antenna proteins 11 0 11 11 Photosynthesis 12 2 12 10 Carbon fixation in photosynthetic organisms 14 0 14 14 Oxidative phosphorylation 32 3 32 29 Biosynthesis of other secondary metabolites Tropane, piperidine and pyridine alkaloid biosynthesis 1 0 1 1 Flavonoid biosynthesis 3 2 6 4 Flavone and flavonol biosynthesis 1 0 2 2 Stilbenoid, diarylheptanoid and gingerol biosynthesis 2 1 2 1 Betalain biosynthesis 1 1 0 0 Isoquinoline alkaloid biosynthesis 2 1 1 1 Lipid metabolism Glycerolipid metabolism 3 1 2 2 Glycerophospholipid metabolism 4 1 3 3 Tab. 2 Some unigenes mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. PN – number of mapped unigenes from petals; PNU – number of mapped up-regulated unigenes from petals; PL – number of mapped unigenes from labella; PLU – number of mapped up-regulated unigenes from labella