DOI: 10.1007/s10535-017-0763-2 BIOLOGIA PLANTARUM 62 (1): 45-54, 2018

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Identification and functional analysis of anthocyanin biosynthesis genes in Phalaenopsis hybrids L.M. WANG, J. ZHANG, X.Y. DONG, Z.Z. FU, H. JIANG, and H.C. ZHANG* Horticulture Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou, 450002, P.R. China Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.

Additional key words: comparative genomics, gene expression pattern, petunia, regulatory genes. Introduction Phalaenopsis species are popular ornamental plants worldwide because of their long-lived and elegant flowers. Their flowers present various colours and pigmentation patterns. The colourful appearance of Phalaenopsis flowers reflects a very complicated mechanism of pigment accumulation. There are three main classes of pigments for coloration that have been identified in plants: anthocyanins, betalains, and carotenoids (Tanaka et al. 2008). Of these, anthocyanins are responsible for the widest array of colours (Winkel-Shirley 2001, Sasaki et al. 2014). Therefore, a comprehensive understanding of anthocyanin synthesis is

important for understanding the flower colours of Phalaenopsis species. The pathways of anthocyanin biosynthesis have been well studied, and the corresponding genes have been characterized in various plants including Arabidopsis, maize, and petunia (Consonni et al. 1993, 1997, Koes et al. 2005, Saito et al. 2013). Based on their functions, these genes can be classified into three families: anthocyanin synthesis (AS) structural genes, AS modification and transfer genes, and AS regulatory genes. Anthocyanin biosynthesis begins with phenylalanine formed via the general phenylpropanoid pathway.

Submitted 30 November 2016, last revision 25 June 2017, accepted 26 June 2017. Abbreviations: AN - anthocyanin; AS - anthocyanin synthesis; AT - acyltransferase; bHLH - basic helix-loop-helix; CHI - chalcone isomerise; CHS - chalcone synthase; DFR - dihydroflavonol 4-reductase; DHK - dihydrokaempferol; DHM - dihydromyricetin; DHQ - dihydroquercetin; F3H - flavanone 3-hydroxylase; F3H - flavonoid 3-hydroxylase; F35H - flavonoid 3,5-hydroxylase; FLS - flavonols by flavonol synthase; GT - glycosyltransferase; LDOX/ANS - leuco-anthocyanidin dioxygenase/anthocyanidin synthase; MT - methyltransferase; p35S - the promoter of CAMV 35S; R2R3-MYB - R2R3 repeat myeloblastosis protein; RT-qPCR - reverse-transcription quantitative PCR; SD - synthetic dropout medium; TFs - transcription factors; TT - transparent testa; TTG - transparent testa glabra; WD40 - beta-transducin repeat protein. Acknowledgments: This research was supported by the National Natural Science Foundation of China (U1504320) and the Science-Technology Foundation for Outstanding Young Scientists of Henan Academy of Agricultural Sciences (2016YQ10). * Corresponding author; e-mail: zhanghechen@hnagri.org.cn

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Chalcone synthase (CHS), the first enzyme in the anthocyanin biosynthesis process, catalyzes the synthesis of a chalcone which is consecutively catalyzed by chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H) to yield the dihydrokaempferol (DHK). DHK can be catalyzed by flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H) to form two types of dihydroflavonols, dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively (Koes et al. 2005, Tanaka et al. 2008, Carletti et al. 2014). The activities of the F3H, F3′H, and F3′5′H determine the structures of anthocyanins and thus play an important role in the coloration of flowers (Tanaka and Brugliera 2013). Dihydroflavonols are further catalyzed to produce anthocyanidin by the action of dihydroflavonol 4-reductase (DFR) and leuco-anthocyanin dioxygenase/anthocyanidin synthase (LDOX/ANS), or flavonols by flavonol synthase (FLS) (Owens et al. 2008). Anthocyanidins are unstable and require stabilization by glycosylation, acylation, or methylation to form great diversity of anthocyanins (Buer et al. 2010, Fournier-Level et al. 2011, Yonekura-Sakakibara et al. 2012, Sasaki et al. 2014). Grapefruit and petunia flower petals produce anthocyanins with methyl groups or with a single sugar modification (Brouillard et al. 2003, Fournier-Level et al. 2011, Provenzano et al. 2014), whereas Arabidopsis seeds accumulate antho-cyanins with sugar or acyl moieties but no methyl groups (Saito et al. 2013). After synthesis, anthocyanins are transferred into the vacuoles by glutathione S-trans-ferase and multi-drug toxic efflux (MATE) transporters (Arabidopsis TT12 and TT19, petunia AN9, and maize ZmBz2) (Marrs et al. 1995, Mueller et al. 2000, Debeaujon et al. 2001, Kitamura et al. 2004, Marinova et al. 2007, Pourcel et al. 2010, Dixon et al. 2013). In plants, AS is mainly controlled by AS structural genes. These genes are regulated by transcription factors (TFs), including R2R3 repeat myeloblastosis (R2R3-MYB), basic helix-loop-helix (bHLH), and beta-transducin repeat (WD40), that determine their temporal or spatial expression in specific tissues or cells (Ramsay and Glover 2005, Koes et al. 2005). Yeast two-hybrid assays indicate that these TFs can form a ternary MBW

protein complex (M: R2R3-MYB, B: bHLH, W: WD40). Genes associated with this complex have been identified in all plants studied to date (Koes et al. 2005, Xu et al. 2015). Thus, the temporal or spatial expression patterns of AS structural genes are determined by different MBW complexes (De Vetten et al. 1997, Quattrocchio et al. 1999, Spelt et al. 2000, 2002). Besides the MBW complex, anthocyanin biosynthesis can also be regulated by other TFs with MADS box, Zn-finger, WRKY, or LOB domains (Johnson et al. 2002, Nesi et al. 2002, Sagasser et al. 2002, Takeda 2006, Matsumura et al. 2009, Rubin et al. 2009). Recently, it has been found that anthocyanin production is also modulated by other mechanisms, including posttranslational modification (Maier et al. 2013), chromatin remodeling (Hernandez et al. 2007), and repression of MBW complex activities by repressor proteins (Matsui et al. 2008, Yuan et al. 2013). In Arabidopsis and petunia, R3-MYB members (CPC, MYBL2 in Arabidopsis; MYBx in petunia) have been demonstrated to act as repressors and limiters of anthocyanin production (Matsui et al. 2008, Zhang et al. 2009, Albert et al. 2011, 2014). They are thought to assert a repressive function through competition for a bHLH partner with R2R3-MYB factors (Koes et al. 2005, Zhang et al. 2009). Apart from all of the abovementioned genes (enzymes), flower colour can also be influenced by vacuolar pH, co-pigmentation, and metal ions (Verweij et al. 2008, Nishihara and Nakatsuka 2011, Miyahara et al. 2013, Yoshida and Negishi 2013, Faraco et al. 2014) (Fig. 1 Suppl.). Phalaenopsis species are excellent model plants for studying the molecular mechanisms of anthocyanin biosynthesis because of their natural variation in flower colour. The complete sequenced genome of Phalaenopsis equestris and some orchid transcriptome sequencing data make easy to identify candidate genes. The aim of this work was to obtain comprehensive information about the AS genes in Phalaenopsis. These data can be important not only for functional dissection of the anthocyanin synthesis network, but can also provide foundation for future genetic engineering the flower colour in Phalaenopsis.

Materials and methods Phalaenopsis genome database resources: To identify and analyze the AS-related genes in Phalaenopsis, their sequences were searched in the Phalaenopsis database (http://orchidbase.itps.ncku.edu.tw/est/Phalaenopsis_Genome.aspx) using BLAST (Cai et al. 2015). To remove non-target genes, we scanned all of the candidate sequences in the SMART (http://smart.embl-heidelberg.de/), PROSITE (http://prosite.expasy.org/), and NCBI (http://www.ncbi.nlm.nih.gov/) databases to filter them based on the functional annotations of the

closest homologue genes. In addition, to confirm the gene sequences and analyze their transcript patterns, we did also BLAST search of them against the Orchidstra database (http://orchidstra.abrc.sinica.edu.tw/none/). Phylogenetic and alignment analysis of sequences: A phylogenetic tree of the AS genes was generated using the MEGA 6.0 software with the maximum likelihood method based on multiple alignments of their protein amino acid sequences. The AS genes of Arabidopsis were

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obtained from the TAIR database (http://www. arabidopsis.org/) and the other plant genes were obtained from NCBI (accession numbers are listed in Table 1 Suppl.). An alignment of the DNA sequences was generated using the DNAMAN software. Reverse transcription (RT)-PCR and real-time quantitative (q)PCR: We analyzed expression profiles of AS genes by RT-PCR and real-time qPCR in Phalaenopsis species. The primers for each PCR were designed by Primer 6.0 software (Tables 2 and 3 Suppl.). Phalaenopsis cv. Big Chili with full red flowers, cv. Fuller’s Sunset with yellow sepals/petals and a red lip (labellum), cv. Sogo Yukidian V3 with white flowers, cv. Sogo Passat with mottled flowers and red spots, cv. Sogo Lit-Sunny with white sepals/petals and a red lip, and cv. Wedding Promenade with pink flowers were used for expression pattern analysis (Fig. 2 Suppl.). The gene expression patterns at different flower development stages (Fig. 3 Suppl.) and in different floral organs were also analyzed (Fig. 4 Suppl.). RT-PCR analysis was performed following our previous paper (Zhang et al. 2008). The Actin gene (PEQU_10749) was used as an expression control. The PCR cycle numbers were optimized for the amplification. To confirm some of the gene expression profiles, real-time qPCR was also performed using the Applied Biosystems® QuantStudio® 3 system with a SYBR-Green PCR Master Mix kit (Applied Biosystems, Foster City, USA). The relative expressions were calculated using the 2-ΔΔCt method (Pfaffl 2001) with the Actin gene as an internal control. The data were statistically analyzed using the SPSS software. Differences among samples were analyzed by one-way ANOVA using the LSD test at P < 0.05.

Generation of constructs: All constructs were generated by the Gateway vector system. The Phalaenopsis F3′5′H, MYB2, 11, and 12 cDNAs were first cloned into the pDONR207 vector by BP reaction to make entry constructs. The entry vectors were then used to make the destination vectors with pK2GW2.0/rfa (over-expression), pGBKT7/GW (BD, for yeast two-hybrid) or pGADT7/GW (AD, for yeast two-hybrid) by LR reaction. The numbers of constructs used in this study are given in Table 4 Suppl. Petunia transformation and genotype analysis: Stable transformation of petunia (Petunia × hybrida, M1 × R27) plants harboring p35S::F3′5′H, p35S::VwF3′5′H, and p35S::MYB2, 11 and 12 was performed by Agrobacterium mediated leaf-disc transformation following the protocol of Conner et al. (2009). The surviving plants were confirmed by PCR with DNA and RT-PCR with cDNA. The primers for checking the transgenic plants are shown in Table 5 Suppl. Yeast two-hybrid analysis: Firstly, we transformed the BD or AD constructs into the yeast strain AH109 by the lithium acetate method before spreading the yeast on SD-Leu (for AD) or SD-Trp (for BD) media plates to grow for 2 - 3 d. The transformants were then used for co-transformation of specific pairs of AD or BD constructs and spread on SD-Leu/Trp media plates. After growing for 2 - 3 d, the transformants were streaked on selective SD-Leu/Trp/His/Ade medium to indicate protein-protein interactions. Because all transformants containing BD::MYB displayed self-activation on the selective medium, we only show here the results of AD::MYB and corresponding BD construct interactions.

Results Based on homology analysis of the P. equestris genome, a total of 49 anthocyanin synthesis-related genes were identified, including 14 structural genes, 9 modification and transfer genes, and 26 regulatory genes (Table 6 Suppl.). Comparison with members in Arabidopsis and petunia suggested that PeCHS, PeF3′H, PeMT, and PebHLH in P. equestris had been duplicated during evolution, resulting in multiple copies. Additionally, some genes had only one copy, e.g., FLS, while for some modification genes, e.g., anthocyanin acyltransferase genes, we did not find any homologue. Phalaenopsis clearly contained more paralogues of AS genes than Arabidopsis. The Phalaenopsis genome had seven gene pairs, including PeCHS-3/4, PeF3′H-2/3, PeMT-3/4, PeMYB1a/b, PeMYB11a/b, PeWD40-3/3, and PeMYBx-1/2, four of which were located in the same scaffold and may have followed species-specific evolutionary paths with gene duplication events (Table 7 Suppl. and Figs. 5 - 11 Suppl.). Furthermore, because the

P-type H+-ATPases PH1 and PH5 in petunia determine the vacuolar pH and affect the flower colour, we BLAST searched them against the P. equestris genome and the Orchidstra database. We uncovered six P-type H+-ATPase genes, but no PH1 or PH5 homologues were identified (Fig. 12 Suppl.). To predict the functions of the AS structural genes, firstly we analyzed their expression patterns in the red cv. Big Chili at different flower development stages (Fig. 1A). Most of the structural genes were expressed strongly during the very early flower development stage, which was associated with the colour of the corolla. These genes were down-regulated during bud develop-ment and reached expression minimums at stage 5, the fully open bud stage. However, the F3′5′H gene was an exception; it was expressed very weakly at stages 1 - 3. The other three exceptions were F3′H-2 and 3, and FLS, which were expressed very weakly at all bud development stages (Fig. 13 Suppl.).

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Fig. 1. Comparative expression pattern analysis of anthocyanin synthesis structural genes in Phalaenopsis cv. Big Chili at different flower development stages (A) and in different Phalaenopsis cvs. (B). Means SDs, n = 3, * - significant differences at P < 0.05 according to the LSD test.

Fig. 2. Expression patterns of structural genes in different organs of Phalaenopsis cvs. Big Chili (1), Sogo Yukidian V3 (2), Sogo Lit-Sunny (3), and Fuller′s Sunset (4). Structural genes: A - CHS-1, B - F3H, C - F3’H-1, D - DFR. Means SDs, n = 3, * - significant differences at P < 0.05 according to the LSD test.

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We also analyzed the expression profiles of the structural genes in Phalaenopsis species with different petal colours. The results indicated that CHS and CHI, the genes in the upstream step of the anthocyanin biosynthesis pathway, were expressed in all of the cultivars examined, regardless of whether they were white or coloured. However, the genes in the downstream pathway, including F3H, F3′H, and DFR, were expressed more strongly in the coloured species (Fig. 1B and Fig. 14 Suppl.). This suggested that the genes in the downstream pathway play key roles in anthocyanin biosynthesis. To confirm this deduction, we further analyzed the expression patterns in cvs. Fuller’s Sunset and Sogo Lit-Sunny, which have different colours in their sepals/petals and lips (labellum). We used the solid colour species with entirely red and white flowers, Big Chili and Sogo Yukidian V3, as controls. Interestingly, CHS-1 expression did not show obvious differences in any flower organs, whereas F3H, F3′H-1, and DFR showed very high transcript accumulation in the red coloured flower parts (Fig. 2 and Fig. 14B Suppl.). This was consistent with the above deduction and indicated that F3H, F3′H, and DFR were located at key nodes of the anthocyanin biosynthesis pathway and might control anthocyanin synthesis as rate-limiting enzymes. To date, breeders have not managed to produce orchid cultivars with blue flowers using traditional breeding methods, even though the Phalaenopsis genome contains the F3′5′H gene, which is the key gene for violet/blue colour. We speculated that this gene might lack the function to change dihydrokaempferol to dihydro-myricetin. Phylogenetic analysis showed that the F3′5′H gene in Phalaenopsis (PeF3′5′H) had a relatively large evolutionary distance to other plant F3′5′Hs (Fig. 15 Suppl.) and shared only approximately 50 % identity with them. To confirm this, we cloned the F3′5′H gene from cv. Big Chili (PhF3′5′H) and continuously expressed it in petunia hybrid M1 × R27, which contains a mutated endogenous F3′5′H (HF1). Thirty independent over-expression lines (OE) were generated and assessed by PCR (Fig. 16 Suppl.). However, none of them showed the violet or purple flowers (Fig. 3). Compared with the PhF3′5′H transgenic lines, the Viola × hybrida F3′5′H over-expression transgenic lines (VhF3′5′H) showed an obvious phenotype of violet corollas, which was similar to the phenotype of petunia hybrid M1 × V30 plants with normal HF1. It has been shown that some TF genes play important roles in regulating anthocyanin production. Here, we identified 26 members in the Phalaenopsis genome based on previously reported TF genes (Table. 6 Suppl. and Fig. 17 Suppl.). These genes were relatively conserved, with the exception of the Zn-finger family member TT1, of which we did not find a homologue. For the R2R3-MYB family, we identified 11 members that may regulate anthocyanin biosynthesis, including 8 members that have been characterized (Hsu et al. 2015). We followed the

existing naming system and analyzed their evolutionary relationships (Fig. 4). Interestingly, there were two copies of PeMYB1 and PeMYB11 in the genome that shared very high identities in their base sequences (Figs. 5 and 6 Suppl.). Phylogenetic analysis showed PeMYB2, 11, and 12 were located in the same subgroup as ZmC1, ZmPL, and AtTT2, which play important roles in regulating anthocyanin biosynthesis. PeMYB1, 13, and 14 were close to the clade containing Arabidopsis MYB5 and petunia PH4, which were found to regulate vacuolar pH and influence flower colour (Stracke et al. 2001, Quattrocchio et al. 2006). PeMYB3 was clustered with AtMYB11, 12, and 111, and was located in a clade of flavonol biosynthesis genes. Besides these R2R3-MYBs, we identified another two members, PeMYB17 and 18, that may be also involved in regulating anthocyanin synthesis, although only partial CDSs were obtained by bioinformatic analysis.

Fig. 3. Flower pigmentation of petunia plants. A - Wild type petunia M1 × R27. B - Petunia over-expressing F3’5’H from Phalaenopsis. C - Wild type petunia M1 × V30. D - Petunia of over-expressing F3’5’H from Viola. Expression analysis of the regulatory genes in different floral organs of cvs. Sogo Lit-Sunny, Big Chili, and Sogo Yukidian V3 showed that even though we identified homologous genes of WD40, bHLH, LBD, COP, and RIF, no obvious differences in expression patterns were found in the organs of these Phalaenopsis cultivars (Fig. 18 Suppl.). Interestingly, some of the MYB gene members showed distinct expression patterns in different organs of the three Phalaenopsis species. For example, PeMYB1 was only expressed in the upper sepal of Big Chili and PeMYB17 was only expressed in the labellum in all three species. PeMYB2 was strongly expressed in both the upper and lower sepals, while PeMYB12 was strongly expressed in lateral petals and labellum. For the repressors among the TFs, we found that PeMYBx-1 was expressed strongly and consistently in the different floral parts of the red cv. Big Chili. However, it expressed very differently in the organs of

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the white cvs. Sogo Lit-Sunny and Sogo Yukidian V3. It has been shown that R2R3-MYB gene family members play a key role in regulating anthocyanin biosynthesis. To confirm their functions, we cloned MYB2, 11, and 12 from Phalaenopsis cv. Big Chili and over-expressed them in petunia hybrid M1 × R27. Sixty independent OE lines were generated, including 21 MYB2 lines, 15 MYB11 lines and 24 MYB12 lines. We checked these transgenic lines using PCR to identify whether the MYB genes were induced and expressed (Fig. 19 Suppl.). The results showed that 17 of the MYB2 transgenic lines

had significantly increased petal pigmentation with strong violet coloration patterns, even at very early bud development stages. Furthermore, other floral organs including the pistil and stamen, and even the stem, were pigmented in some MYB2 transgenic lines (Fig. 5). However, none of the MYB11 transgenic plants showed a change in flower colour; the MYB11 gene only specifically induced stem pigmentation. MYB12 also had an effect on anthocyanin biosynthesis, but it was very weak; only 6 of 24 OE lines showed a small amount of pigmentation in their lower stems.

Fig. 4. Phylogenetic relationships of MYB transcription factors involved in the anthocyanin synthesis regulatory pathway. The genes of Arabidopsis and other plants were obtained from TAIR and GenBank database referred to the accession numbers shown in Table 1 Suppl. The tree was constructed using MEGA 6.0 software with the maximum likelihood method based on the multiple alignments oftheir protein amino acid sequences. The yeast two-hybrid results indicated that all three MYB members, MYB2, 11, and 12, could interact with the petunia anthocyanins (AN)1 and AN11 (Fig. 6A).

This suggests that the interaction mechanism of the MBW complex in Phalaenopsis and petunia is somewhat conserved. Because anthocyanin biosynthesis is

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controlled by a series of AS structural genes, we examined the expression patterns of the genes involved in the anthocyanin biosynthesis pathway in petunia to determine which genes were regulated by R2R3-MYB

Fig. 5. Flower pigmentation of petunia plants:. A, C, E, andG - flowers of wild type petunia M1 × R27; B, D, F, andH - flowers of transgenic petunia overexpressing MYB2 fromPhalaenopsis. proteins (Fig. 6B). The result showed that over-expression of MYB2 in petunia significantly up-regulated the structural genes CHSa, CHI, F3H, and DFR in transgenic flower petals. The gene expressionals were consistent with the phenotype. For example, the stems of the MYB2 and MYB11 lines accumulated more anthocyanins and, correspondingly, the anthocyanin biosynthesis structural genes showed higher expression than MYB12 lines.

Fig. 6. Phalaenopsis MYB2, MYB11 and MYB12 up-regulate the expression of anthocyanin synthesis genes by forming MBW complex. A - Interaction of MYB with petunia AN1 and AN11 by using a yeast two-hybrid assay. B - Effect of the MYB on the expression pattern of petunia anthocyanin synthesis genes (actin was used as expression control). BD - pGBKT7, AD - pGADT7, SD-Leu/Trp - synthetic dropout medium without leucine and tryptophan, SD-Leu/Trp/His/Ade - synthetic dropout medium without leucine, tryptophan, histidine and adenine, OE - over-expressing lines.

Discussion In this study, we present a comparative analysis of the anthocyanin synthesis-related genes in the Phalaenopsis genome and a total of 49 members were identified here. Genome-wide analysis of petunia, Arabidopsis and Phalaenopsis suggested that the AS genes have followed species-specific evolutionary paths with gene duplication events. For example, the PeCHS-3/4, PeF3′H-2/3, PeMT-3/4, PeMYB11a/b, and PeMYBx-1/2 gene pairs showed very high identities and were located in the same scaffold, which strongly suggests that they arose from duplication events in Phalaenopsis genome. The CHS, F3′H and MT genes expanded in Phalaenopsis, whereas

the FLS gene specifically expanded in Arabidopsis. In addition to the diversity in gene family numbers, the gene identities vary between these species. The genes encoding structural enzymes were much better conserved than those encoding AS modification proteins. The AS modification genes showed low identities in amino acid sequences of glycosyltransferase (GT) and methyl-transferase (MT) or no homologues were found in acyltransferase (AT). This suggests that anthocyanin modifications are very complicated. Because GT, AT, and MT genes form a large super family, there must be many genes involved in the anthocyanin modification

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processes. To uncover their functions, a gene-by-gene study needs to be conducted. Although the AS structural genes are well conserved, the timing, level, and spatial distribution of anthocyanin formation are determined by TFs. Among them, R2R3-MYB, bHLH, and WD40 proteins can interact with each other, forming a MBW complex, which play an important role in regulating anthocyanin biosynthesis. In this study, some homologous AS regulatory genes were also identified. From their expression patterns in different Phalaenopsis species, we can deduce that the regulation of anthocyanin biosynthesis is very complicated, but we cannot conclude which regulatory gene had a decisive effect on floral colour. In a paper by Hsu et al. (2015), PeMYB2 was proposed to regulate full red pigmentation, PeMYB11 to control the red spots in the callus of the lip, and PeMYB12 to be the major factor for pigmentation in the central lobe of the lip. However, in our study, we found that PeMYB2 was expressed strongly in the sepals of all three species, regardless of whether they were red or white. MYB12 was not expressed in the white petals of cv. Sogo Yukidian V3 but was expressed in another white cultivar Sogo Lit-Sunny. This indicates that these MYB genes might have different regulatory modes in different genetic backgrounds. It also shows that although phylogenetic and expression pattern analyses could give clues to functions, the functions need to be established on a gene-by-gene basis for different species (D′Auria 2006, Luo et al. 2007). In addition, data from transgenic petunia OE lines showed that although MYB2 had strong transcriptional activity, it induced an uneven pigment distribution in the transgenic petals. MYB11 did not induce red pigment spots in petals, while MYB12 had a weak effect on anthocyanin biosynthesis. These results

suggest that even though MYB2, 11, and 12 are clustered together phylogenetically, the TF binding sites in the cis-regulatory regions of their target genes may be different. Therefore, more direct evidence is needed to determine how these regulatory genes specifically affect floral colour. In addition, Phalaenopsis has two copies of MYB11, four of WD40, and three of bHLH proteins, so many forms of MBW complex may exist. Thus, the specific functions of each MYB would also be influenced by its partners. Furthermore, the genomes of some Phalaenopsis species have undergone polyploidization and many of the AS genes are present in multiple copies. We deduced that the combined expression of regulatory genes and the formation of various protein complexes may result in various pigmentation patterns. Flower colour is one of the most important attributes of Phalaenopsis and many different colours except blue have been reached using traditional breeding methods. Our research showed that the Phalaenopsis genome contains the F3′5′H gene, the key gene for blue flowers. However, our data suggest that it lost the function to change dihydrokaempferol to dihydromyricetin during evolution. This explains why breeders have not produced cultivars with blue flowers. Of course, the vacuolar pH and metal ion chelation also influence flower appearance. Much research is needed to generate orchid cultivars with blue flowers, but the most important work is to determine whether it is possible to create transgenic plants containing exogenous F3′5′H. In summary, this study improves our understanding of the anthocyanin biosynthesis pathway and provides important clues for molecular breeding of Phalaenopsis cultivars with flower colour modulation.

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