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

Identification and functional analysis of anthocyanin biosynthesis 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 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 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, pattern, petunia, regulatory genes.

Introduction

Phalaenopsis species are popular ornamental plants important for understanding the flower colours of worldwide because of their long-lived and elegant Phalaenopsis species. flowers. Their flowers present various colours and The pathways of anthocyanin biosynthesis have been pigmentation patterns. The colourful appearance of well studied, and the corresponding genes have been Phalaenopsis flowers reflects a very complicated characterized in various plants including Arabidopsis, mechanism of pigment accumulation. There are three maize, and petunia (Consonni et al. 1993, 1997, Koes main classes of pigments for coloration that have been et al. 2005, Saito et al. 2013). Based on their functions, identified in plants: anthocyanins, betalains, and these genes can be classified into three families: carotenoids (Tanaka et al. 2008). Of these, anthocyanins anthocyanin synthesis (AS) structural genes, AS are responsible for the widest array of colours (Winkel- modification and transfer genes, and AS regulatory genes. Shirley 2001, Sasaki et al. 2014). Therefore, a Anthocyanin biosynthesis begins with phenylalanine comprehensive understanding of anthocyanin synthesis is formed via the general phenylpropanoid pathway.

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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; F3H - flavonoid 3-hydroxylase; F35H - 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: [email protected]

45 L.M. WANG et al.

Chalcone synthase (CHS), the first enzyme in the protein complex (M: R2R3-MYB, B: bHLH, W: WD40). anthocyanin biosynthesis process, catalyzes the synthesis Genes associated with this complex have been identified of a chalcone which is consecutively catalyzed by in all plants studied to date (Koes et al. 2005, Xu et al. chalcone isomerase (CHI) and flavanone 2015). Thus, the temporal or spatial expression patterns 3-hydroxylase (F3H) to yield the dihydrokaempferol of AS structural genes are determined by different MBW (DHK). DHK can be catalyzed by flavonoid complexes (De Vetten et al. 1997, Quattrocchio et al. 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase 1999, Spelt et al. 2000, 2002). Besides the MBW (F3′5′H) to form two types of dihydroflavonols, complex, anthocyanin biosynthesis can also be regulated dihydroquercetin (DHQ) and dihydromyricetin (DHM), by other TFs with MADS box, Zn-finger, WRKY, or respectively (Koes et al. 2005, Tanaka et al. 2008, LOB domains (Johnson et al. 2002, Nesi et al. 2002, Carletti et al. 2014). The activities of the F3H, F3′H, and Sagasser et al. 2002, Takeda 2006, Matsumura et al. F3′5′H determine the structures of anthocyanins and thus 2009, Rubin et al. 2009). play an important role in the coloration of flowers Recently, it has been found that anthocyanin (Tanaka and Brugliera 2013). Dihydroflavonols are production is also modulated by other mechanisms, further catalyzed to produce anthocyanidin by the action including posttranslational modification (Maier et al. of dihydroflavonol 4-reductase (DFR) and leuco- 2013), chromatin remodeling (Hernandez et al. 2007), anthocyanin dioxygenase/anthocyanidin synthase and repression of MBW complex activities by repressor (LDOX/ANS), or flavonols by flavonol synthase (FLS) (Matsui et al. 2008, Yuan et al. 2013). In (Owens et al. 2008). Anthocyanidins are unstable and Arabidopsis and petunia, R3-MYB members (CPC, require stabilization by glycosylation, acylation, or MYBL2 in Arabidopsis; MYBx in petunia) have been methylation to form great diversity of anthocyanins (Buer demonstrated to act as repressors and limiters of et al. 2010, Fournier-Level et al. 2011, Yonekura- anthocyanin production (Matsui et al. 2008, Zhang Sakakibara et al. 2012, Sasaki et al. 2014). Grapefruit and et al. 2009, Albert et al. 2011, 2014). They are thought to petunia flower petals produce anthocyanins with methyl assert a repressive function through competition for a groups or with a single sugar modification (Brouillard bHLH partner with R2R3-MYB factors (Koes et al. 2005, et al. 2003, Fournier-Level et al. 2011, Provenzano et al. Zhang et al. 2009). Apart from all of the abovementioned 2014), whereas Arabidopsis seeds accumulate antho- genes (enzymes), flower colour can also be influenced by cyanins with sugar or acyl moieties but no methyl groups vacuolar pH, co-pigmentation, and metal ions (Verweij (Saito et al. 2013). After synthesis, anthocyanins are et al. 2008, Nishihara and Nakatsuka 2011, Miyahara transferred into the vacuoles by glutathione S-trans-ferase et al. 2013, Yoshida and Negishi 2013, Faraco et al. and multi-drug toxic efflux (MATE) transporters 2014) (Fig. 1 Suppl.). (Arabidopsis TT12 and TT19, petunia AN9, and maize Phalaenopsis species are excellent model plants for ZmBz2) (Marrs et al. 1995, Mueller et al. 2000, studying the molecular mechanisms of anthocyanin Debeaujon et al. 2001, Kitamura et al. 2004, Marinova biosynthesis because of their natural variation in flower et al. 2007, Pourcel et al. 2010, Dixon et al. 2013). colour. The complete sequenced genome of Phalaenopsis In plants, AS is mainly controlled by AS structural equestris and some orchid transcriptome sequencing data genes. These genes are regulated by transcription factors make easy to identify candidate genes. The aim of this (TFs), including R2R3 repeat myeloblastosis (R2R3- work was to obtain comprehensive information about the MYB), basic helix-loop-helix (bHLH), and beta- AS genes in Phalaenopsis. These data can be important transducin repeat (WD40), that determine their temporal not only for functional dissection of the anthocyanin or spatial expression in specific tissues or cells (Ramsay synthesis network, but can also provide foundation for and Glover 2005, Koes et al. 2005). Yeast two-hybrid future genetic engineering the flower colour in assays indicate that these TFs can form a ternary MBW Phalaenopsis.

Materials and methods

Phalaenopsis genome database resources: To identify closest homologue genes. In addition, to confirm the gene and analyze the AS-related genes in Phalaenopsis, their sequences and analyze their transcript patterns, we did sequences were searched in the Phalaenopsis database also BLAST search of them against the Orchidstra (http://orchidbase.itps.ncku.edu.tw/est/Phalaenopsis_Gen database (http://orchidstra.abrc.sinica.edu.tw/none/). ome.aspx) using BLAST (Cai et al. 2015). To remove non-target genes, we scanned all of the candidate Phylogenetic and alignment analysis of sequences: A sequences in the SMART (http://smart.embl- phylogenetic tree of the AS genes was generated using heidelberg.de/), PROSITE (http://prosite.expasy.org/), the MEGA 6.0 software with the maximum likelihood and NCBI (http://www.ncbi.nlm.nih.gov/) databases to method based on multiple alignments of their protein filter them based on the functional annotations of the amino acid sequences. The AS genes of Arabidopsis were

46 ANTHOCYANIDIN BIOSYNTHESIS GENES IN PHALAENOPSIS obtained from the TAIR database (http://www. Generation of constructs: All constructs were generated arabidopsis.org/) and the other plant genes were obtained by the Gateway vector system. The Phalaenopsis F3′5′H, from NCBI (accession numbers are listed in Table 1 MYB2, 11, and 12 cDNAs were first cloned into the Suppl.). An alignment of the DNA sequences was pDONR207 vector by BP reaction to make entry generated using the DNAMAN software. constructs. The entry vectors were then used to make the destination vectors with pK2GW2.0/rfa (over- Reverse transcription (RT)-PCR and real-time expression), pGBKT7/GW (BD, for yeast two-hybrid) or quantitative (q)PCR: We analyzed expression profiles pGADT7/GW (AD, for yeast two-hybrid) by LR reaction. of AS genes by RT-PCR and real-time qPCR in The numbers of constructs used in this study are given in Phalaenopsis species. The primers for each PCR were Table 4 Suppl. designed by Primer 6.0 software (Tables 2 and 3 Suppl.). Phalaenopsis cv. Big Chili with full red flowers, Petunia transformation and genotype analysis: Stable cv. Fuller’s Sunset with yellow sepals/petals and a red lip transformation of petunia (Petunia × hybrida, M1 × R27) (labellum), cv. Sogo Yukidian V3 with white flowers, plants harboring p35S::F3′5′H, p35S::VwF3′5′H, and cv. Sogo Passat with mottled flowers and red spots, p35S::MYB2, 11 and 12 was performed by cv. Sogo Lit-Sunny with white sepals/petals and a red lip, Agrobacterium mediated leaf-disc transformation and cv. Wedding Promenade with pink flowers were used following the protocol of Conner et al. (2009). The for expression pattern analysis (Fig. 2 Suppl.). The gene surviving plants were confirmed by PCR with DNA and expression patterns at different flower development RT-PCR with cDNA. The primers for checking the stages (Fig. 3 Suppl.) and in different floral organs were transgenic plants are shown in Table 5 Suppl. also analyzed (Fig. 4 Suppl.). RT-PCR analysis was performed following our previous paper (Zhang et al. Yeast two-hybrid analysis: Firstly, we transformed the 2008). The Actin gene (PEQU_10749) was used as an BD or AD constructs into the yeast strain AH109 by the expression control. The PCR cycle numbers were lithium acetate method before spreading the yeast on optimized for the amplification. To confirm some of the SD-Leu (for AD) or SD-Trp (for BD) media plates to gene expression profiles, real-time qPCR was also grow for 2 - 3 d. The transformants were then used for performed using the Applied Biosystems® QuantStudio® co-transformation of specific pairs of AD or BD 3 system with a SYBR-Green PCR Master Mix kit constructs and spread on SD-Leu/Trp media plates. After (Applied Biosystems, Foster City, USA). The relative growing for 2 - 3 d, the transformants were streaked on expressions were calculated using the 2-ΔΔCt method selective SD-Leu/Trp/His/Ade medium to indicate (Pfaffl 2001) with the Actin gene as an internal control. protein-protein interactions. Because all transformants The data were statistically analyzed using the SPSS containing BD::MYB displayed self-activation on the software. Differences among samples were analyzed by selective medium, we only show here the results of one-way ANOVA using the LSD test at P < 0.05. AD::MYB and corresponding BD construct interactions.

Results

Based on homology analysis of the P. equestris genome, P-type H+-ATPases PH1 and PH5 in petunia determine a total of 49 anthocyanin synthesis-related genes were the vacuolar pH and affect the flower colour, we BLAST identified, including 14 structural genes, 9 modification searched them against the P. equestris genome and the and transfer genes, and 26 regulatory genes (Table 6 Orchidstra database. We uncovered six P-type Suppl.). Comparison with members in Arabidopsis and H+-ATPase genes, but no PH1 or PH5 homologues were petunia suggested that PeCHS, PeF3′H, PeMT, and identified (Fig. 12 Suppl.). PebHLH in P. equestris had been duplicated during To predict the functions of the AS structural genes, evolution, resulting in multiple copies. Additionally, firstly we analyzed their expression patterns in the red some genes had only one copy, e.g., FLS, while for some cv. Big Chili at different flower development stages modification genes, e.g., anthocyanin acyltransferase (Fig. 1A). Most of the structural genes were expressed genes, we did not find any homologue. Phalaenopsis strongly during the very early flower development stage, clearly contained more paralogues of AS genes than which was associated with the colour of the corolla. Arabidopsis. The Phalaenopsis genome had seven gene These genes were down-regulated during bud develop- pairs, including PeCHS-3/4, PeF3′H-2/3, PeMT-3/4, ment and reached expression minimums at stage 5, the PeMYB1a/b, PeMYB11a/b, PeWD40-3/3, and fully open bud stage. However, the F3′5′H gene was an PeMYBx-1/2, four of which were located in the same exception; it was expressed very weakly at stages 1 - 3. scaffold and may have followed species-specific The other three exceptions were F3′H-2 and 3, and FLS, evolutionary paths with gene duplication events (Table 7 which were expressed very weakly at all bud Suppl. and Figs. 5 - 11 Suppl.). Furthermore, because the development stages (Fig. 13 Suppl.).

47 L.M. WANG et al.

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.

48 ANTHOCYANIDIN BIOSYNTHESIS GENES IN PHALAENOPSIS

We also analyzed the expression profiles of the existing naming system and analyzed their evolutionary structural genes in Phalaenopsis species with different relationships (Fig. 4). Interestingly, there were two copies petal colours. The results indicated that CHS and CHI, the of PeMYB1 and PeMYB11 in the genome that shared very genes in the upstream step of the anthocyanin high identities in their base sequences (Figs. 5 and biosynthesis pathway, were expressed in all of the 6 Suppl.). Phylogenetic analysis showed PeMYB2, 11, cultivars examined, regardless of whether they were and 12 were located in the same subgroup as ZmC1, white or coloured. However, the genes in the downstream ZmPL, and AtTT2, which play important roles in pathway, including F3H, F3′H, and DFR, were expressed regulating anthocyanin biosynthesis. PeMYB1, 13, and 14 more strongly in the coloured species (Fig. 1B and were close to the clade containing Arabidopsis MYB5 and Fig. 14 Suppl.). This suggested that the genes in the petunia PH4, which were found to regulate vacuolar pH downstream pathway play key roles in anthocyanin and influence flower colour (Stracke et al. 2001, biosynthesis. To confirm this deduction, we further Quattrocchio et al. 2006). PeMYB3 was clustered with analyzed the expression patterns in cvs. Fuller’s Sunset AtMYB11, 12, and 111, and was located in a clade of and Sogo Lit-Sunny, which have different colours in their flavonol biosynthesis genes. Besides these R2R3-MYBs, sepals/petals and lips (labellum). We used the solid we identified another two members, PeMYB17 and 18, colour species with entirely red and white flowers, Big that may be also involved in regulating anthocyanin Chili and Sogo Yukidian V3, as controls. Interestingly, synthesis, although only partial CDSs were obtained by CHS-1 expression did not show obvious differences in bioinformatic analysis. 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 Fig. 3. Flower pigmentation of petunia plants. A - Wild type them. To confirm this, we cloned the F3′5′H gene from petunia M1 × R27. B - Petunia over-expressing F3’5’H from cv. Big Chili (PhF3′5′H) and continuously expressed it in Phalaenopsis. C - Wild type petunia M1 × V30. D - Petunia of petunia hybrid M1 × R27, which contains a mutated over-expressing F3’5’H from Viola. endogenous F3′5′H (HF1). Thirty independent over- expression lines (OE) were generated and assessed by Expression analysis of the regulatory genes in PCR (Fig. 16 Suppl.). However, none of them showed the different floral organs of cvs. Sogo Lit-Sunny, Big Chili, violet or purple flowers (Fig. 3). Compared with the and Sogo Yukidian V3 showed that even though we PhF3′5′H transgenic lines, the Viola × hybrida F3′5′H identified homologous genes of WD40, bHLH, LBD, over-expression transgenic lines (VhF3′5′H) showed an COP, and RIF, no obvious differences in expression obvious phenotype of violet corollas, which was similar patterns were found in the organs of these Phalaenopsis to the phenotype of petunia hybrid M1 × V30 plants with cultivars (Fig. 18 Suppl.). Interestingly, some of the MYB normal HF1. gene members showed distinct expression patterns in It has been shown that some TF genes play important different organs of the three Phalaenopsis species. For roles in regulating anthocyanin production. Here, we example, PeMYB1 was only expressed in the upper sepal identified 26 members in the Phalaenopsis genome based of Big Chili and PeMYB17 was only expressed in the on previously reported TF genes (Table. 6 Suppl. and labellum in all three species. PeMYB2 was strongly Fig. 17 Suppl.). These genes were relatively conserved, expressed in both the upper and lower sepals, while with the exception of the Zn-finger family member TT1, PeMYB12 was strongly expressed in lateral petals and of which we did not find a homologue. For the R2R3- labellum. For the repressors among the TFs, we found MYB family, we identified 11 members that may regulate that PeMYBx-1 was expressed strongly and consistently anthocyanin biosynthesis, including 8 members that have in the different floral parts of the red cv. Big Chili. been characterized (Hsu et al. 2015). We followed the However, it expressed very differently in the organs of

49 L.M. WANG et al. the white cvs. Sogo Lit-Sunny and Sogo Yukidian V3. had significantly increased petal pigmentation with strong It has been shown that R2R3-MYB gene family violet coloration patterns, even at very early bud members play a key role in regulating anthocyanin development stages. Furthermore, other floral organs biosynthesis. To confirm their functions, we cloned including the pistil and stamen, and even the stem, were MYB2, 11, and 12 from Phalaenopsis cv. Big Chili and pigmented in some MYB2 transgenic lines (Fig. 5). over-expressed them in petunia hybrid M1 × R27. Sixty However, none of the MYB11 transgenic plants showed a independent OE lines were generated, including 21 MYB2 change in flower colour; the MYB11 gene only lines, 15 MYB11 lines and 24 MYB12 lines. We checked specifically induced stem pigmentation. MYB12 also had these transgenic lines using PCR to identify whether the an effect on anthocyanin biosynthesis, but it was very MYB genes were induced and expressed (Fig. 19 Suppl.). weak; only 6 of 24 OE lines showed a small amount of The results showed that 17 of the MYB2 transgenic lines 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 of their protein amino acid sequences.

The yeast two-hybrid results indicated that all three This suggests that the interaction mechanism of the MYB members, MYB2, 11, and 12, could interact with MBW complex in Phalaenopsis and petunia is somewhat the petunia anthocyanins (AN)1 and AN11 (Fig. 6A). conserved. Because anthocyanin biosynthesis is

50 ANTHOCYANIDIN BIOSYNTHESIS GENES IN PHALAENOPSIS 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, and G - flowers of wild type petunia M1 × R27; B, D, F, and H - flowers of transgenic petunia overexpressing MYB2 from Phalaenopsis.

Fig. 6. Phalaenopsis MYB2, MYB11 and MYB12 up-regulate the proteins (Fig. 6B). The result showed that over- expression of anthocyanin synthesis genes by forming MBW expression of MYB2 in petunia significantly up-regulated complex. A - Interaction of MYB with petunia AN1 and AN11 the structural genes CHSa, CHI, F3H, and DFR in by using a yeast two-hybrid assay. B - Effect of the MYB on the transgenic flower petals. The gene expressionals were expression pattern of petunia anthocyanin synthesis genes (actin consistent with the phenotype. For example, the stems of was used as expression control). BD - pGBKT7, the MYB2 and MYB11 lines accumulated more AD - pGADT7, SD-Leu/Trp - synthetic dropout medium anthocyanins and, correspondingly, the anthocyanin without leucine and tryptophan, SD-Leu/Trp/His/Ade - biosynthesis structural genes showed higher expression synthetic dropout medium without leucine, tryptophan, histidine and adenine, OE - over-expressing lines. than MYB12 lines.

Discussion

In this study, we present a comparative analysis of the the FLS gene specifically expanded in Arabidopsis. In anthocyanin synthesis-related genes in the Phalaenopsis addition to the diversity in gene family numbers, the gene genome and a total of 49 members were identified here. identities vary between these species. The genes encoding Genome-wide analysis of petunia, Arabidopsis and structural enzymes were much better conserved than Phalaenopsis suggested that the AS genes have followed those encoding AS modification proteins. The AS species-specific evolutionary paths with gene duplication modification genes showed low identities in amino acid events. For example, the PeCHS-3/4, PeF3′H-2/3, sequences of glycosyltransferase (GT) and methyl- PeMT-3/4, PeMYB11a/b, and PeMYBx-1/2 gene pairs transferase (MT) or no homologues were found in showed very high identities and were located in the same acyltransferase (AT). This suggests that anthocyanin scaffold, which strongly suggests that they arose from modifications are very complicated. Because GT, AT, duplication events in Phalaenopsis genome. The CHS, and MT genes form a large super family, there must be F3′H and MT genes expanded in Phalaenopsis, whereas many genes involved in the anthocyanin modification

51 L.M. WANG et al. processes. To uncover their functions, a gene-by-gene suggest that even though MYB2, 11, and 12 are clustered study needs to be conducted. together phylogenetically, the TF binding sites in the cis- Although the AS structural genes are well conserved, regulatory regions of their target genes may be different. the timing, level, and spatial distribution of anthocyanin Therefore, more direct evidence is needed to determine formation are determined by TFs. Among them, R2R3- how these regulatory genes specifically affect floral MYB, bHLH, and WD40 proteins can interact with each colour. In addition, Phalaenopsis has two copies of other, forming a MBW complex, which play an important MYB11, four of WD40, and three of bHLH proteins, so role in regulating anthocyanin biosynthesis. In this study, many forms of MBW complex may exist. Thus, the some homologous AS regulatory genes were also specific functions of each MYB would also be influenced identified. From their expression patterns in different by its partners. Furthermore, the genomes of some Phalaenopsis species, we can deduce that the regulation Phalaenopsis species have undergone polyploidization of anthocyanin biosynthesis is very complicated, but we and many of the AS genes are present in multiple copies. cannot conclude which regulatory gene had a decisive We deduced that the combined expression of regulatory effect on floral colour. In a paper by Hsu et al. (2015), genes and the formation of various protein complexes PeMYB2 was proposed to regulate full red pigmentation, may result in various pigmentation patterns. PeMYB11 to control the red spots in the callus of the lip, Flower colour is one of the most important attributes and PeMYB12 to be the major factor for pigmentation in of Phalaenopsis and many different colours except blue the central lobe of the lip. However, in our study, we have been reached using traditional breeding methods. found that PeMYB2 was expressed strongly in the sepals Our research showed that the Phalaenopsis genome of all three species, regardless of whether they were red contains the F3′5′H gene, the key gene for blue flowers. or white. MYB12 was not expressed in the white petals of However, our data suggest that it lost the function to cv. Sogo Yukidian V3 but was expressed in another white change dihydrokaempferol to dihydromyricetin during cultivar Sogo Lit-Sunny. This indicates that these MYB evolution. This explains why breeders have not produced genes might have different regulatory modes in different cultivars with blue flowers. Of course, the vacuolar pH genetic backgrounds. It also shows that although and metal ion chelation also influence flower appearance. phylogenetic and expression pattern analyses could give Much research is needed to generate orchid cultivars with clues to functions, the functions need to be established on blue flowers, but the most important work is to determine a gene-by-gene basis for different species (D′Auria 2006, whether it is possible to create transgenic plants Luo et al. 2007). In addition, data from transgenic petunia containing exogenous F3′5′H. OE lines showed that although MYB2 had strong In summary, this study improves our understanding of transcriptional activity, it induced an uneven pigment the anthocyanin biosynthesis pathway and provides distribution in the transgenic petals. MYB11 did not important clues for molecular breeding of Phalaenopsis induce red pigment spots in petals, while MYB12 had a cultivars with flower colour modulation. weak effect on anthocyanin biosynthesis. These results

References

Albert, N.W., Davies, K.M., Lewis, D.H., Zhang, H., Y.C., Chuang, Y.C., Dievart, A., Dufayard, J.F., Xu, X., Montefiori, M., Brendolise, C., Boase, M.R., Ngo, H., Wang, J.Y., Wang, J., Xiao, X.J., Zhao, X.M., Du, R., Jameson, P.E., Schwinn, K.E.: A conserved network of Zhang, G.Q., Wang, M., Su, Y.Y., Xie, G.C., Liu, G.H., Li, transcriptional activators and repressors regulates L.Q., Huang, L.Q., Luo, Y.B., Chen, H.H., Van de, Peer, Y., anthocyanin pigmentation in eudicots. - Plant Cell 26: 962- Liu, Z.J.: The genome sequence of the orchid Phalaenopsis 980, 2014. equestris.- Nat. Genet. 47: 65-72, 2015. Albert, N.W., Lewis, D.H., Zhang, H., Schwinn, K.E., Jameson, Carletti, G., Nervo, G., Cattivelli, L.: Flavonoids and melanins: P.E., Davies, K.M.: Members of an R2R3-MYB a common strategy across two kingdoms. - Int. J. biol. Sci. family in petunia are developmentally 10: 1159-1170, 2014. and environmentally regulated to control complex floral and Conner, A.J., Albert, N.W., Deroles, S.C.: Transformation and vegetative pigmentation patterning. - Plant J. 65: 771-784, regeneration of petunia. - In: Greats, T., Strommer, J. (ed.): 2011. Evolutionary, Developmental and Physiological Genetics. Brouillard, R., Chassaing. S., Fougerousse. A.: Why are Pp. 395-409. Springer, New York 2009. grape/fresh wine anthocyanins so simple and why is it that Consonni, G., Geuna, F., Gavazzi, G., Tonelli, C.: Molecular red wine color lasts so long? - Phytochemistry 64: 1179- homology among members of the R gene family in maize. - 1186, 2003. Plant J. 3: 335-346, 1993. Buer, C.S., Imin, N., Djordjevic, M.A.: Flavonoids: new roles Consonni, G., Ronchi, A., Pilu, R., Gavazzi, G., Dellaporta, for old molecules. - J. integr. Plant Biol. 52: 98-111, 2010. S.L., Tonelli, C.: Ectopic anthocyanin pigmentation in Cai, J., Liu, X., Vanneste, K., Proost, S., Tsai, W.C., Liu, K.W., maize as a tool for defining interactions between Chen, L.J., He, Y., Xu, Q., Bian, C., Zheng, Z., Sun, F., Liu, homologous regulatory factors. - Mol. gen. Genet. 256: 265- W., Hsiao, Y.Y., Pan, Z.J., Hsu, C.C., Yang, Y.P., Hsu, 276, 1997.

52 ANTHOCYANIDIN BIOSYNTHESIS GENES IN PHALAENOPSIS

D'Auria, J.C.: Acyltransferases in plants: a good time to be glutathione S-transferase involved in vacuolar transfer BAHD. - Curr. Opin. Plant Biol. 9: 331-340, 2006. encoded by the maize gene Bronze-2. - Nature 375: 397- Debeaujon, I., Peeters, A.J., Léon-Kloosterziel, K.M., 400, 1995. Koornneef, M.: The TRANSPARENT TESTA12 gene of Matsui, K., Umemura, Y., Ohme-Takagi, M.: AtMYBL2, a Arabidopsis encodes a multidrug secondary transporter-like protein with a single MYB domain, acts as a negative protein required for flavonoid sequestration in vacuoles of regulator of anthocyanin biosynthesis in Arabidopsis. - the seed coat endothelium. - Plant Cell 13: 853-871, 2001. Plant J. 55: 954-967, 2008. De Vetten, N., Quattrocchio, F., Mol, J., Koes, R.: The an11 Matsumura, Y., Iwakawa, H., Machida, Y., Machida, C.: locus controlling flower pigmentation in petunia encodes a Characterization of genes in the ASYMMETRIC novel WD-repeat protein conserved in yeast, plants, and LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) animals. - Gene Dev. 1: 1422-1434, 1997. family in Arabidopsis thaliana, and functional and Dixon, R.A., Liu, C., Jun, J.H.: Metabolic engineering of molecular comparisons between AS2 and other family anthocyanins and condensed tannins in plants. - Curr. Opin. members. - Plant J. 58: 525-537, 2009. Biotechnol. 24: 329-335, 2013. Miyahara, T., Sakiyama, R., Ozeki, Y., Sasaki, N.: Acyl- Faraco, M., Spelt, C., Bliek, M., Verweij, W., Hoshino, A., glucose-dependent glucosyltransferase catalyzes the final Espen, L., Prinsi, B., Jaarsma, R., Tarhan, E., De Boer, step of anthocyanin formation in Arabidopsis. - J. Plant A.H., Di Sansebastiano, G.P., Koes, R., Quattrocchio F.M.: Physiol. 170: 619-624, 2013. Hyperacidification of vacuoles by the combined action of Mueller, L.A., Goodman, C.D., Silady, R.A., Walbot, V.: AN9, two different P-ATPases in the tonoplast determines flower a petunia glutathione S-transferase required for anthocyanin color. - Cell Rep. 6: 32-43, 2014. sequestration, is a flavonoid-binding protein. - Plant Fournier-Level, A., Hugueney, P., Verriès, C., This, P., Physiol. 123: 1561-1570, 2000. Ageorges, A.: Genetic mechanisms underlying the Nesi, N., Debeaujon, I., Jond, C., Stewart, A.J., Jenkins, G.I., methylation level of anthocyanins in grape (Vitis vinifera Caboche, M., Lepiniec, L.: The TRANSPARENT TESTA16 L.). - BMC. Plant Biol. 11: 179, 2011. locus encodes the ARABIDOPSIS BSISTER MADS Hernandez, J.M., Feller, A., Morohashi, K., Frame, K., domain protein and is required for proper development and Grotewold, E.: The basic helix loop helix domain of maize pigmentation of the seed coat. - Plant Cell 14: 2463-2479, R links transcriptional regulation and histone modifications 2002. by recruitment of an EMSY-related factor. - Proc. nat. Nishihara, M., Nakatsuka, T.: Genetic engineering of flavonoid Acad. Sci. USA 104: 17222-17227, 2007. pigments to modify flower color in floricultural plants. - Hsu, C.C., Chen, Y.Y., Tsai, W.C., Chen, W.H., Chen, H.H.: Biotechnol. Lett. 33: 433-441, 2011. Three R2R3-MYB transcription factors regulate distinct Owens, D.K., Crosby, K.C., Runac, J., Howard, B.A., Winkel, floral pigmentation patterning in Phalaenopsis spp. - Plant B.S.J.: Biochemical and genetic characterization of Physiol. 168: 175-191, 2015. Arabidopsis flavanone 3b-hydroxylase. - Plant Physiol. Johnson, C.S., Kolevski, B., Smyth, D.R.: TRANSPARENT Biochem. 46: 833-843, 2008. TESTA GLABRA2, a trichome and seed coat development Pfaffl, M.W.: A new mathematical model for relative gene of Arabidopsis, encodes a WRKY transcription factor. quantification in real-time RT-PCR. - Nucl. Acids Res. 29: - Plant Cell 14: 1359-1375, 2002. e45, 2001. Kitamura, S., Shikazono, N., Tanaka, A.: TRANSPARENT Pourcel, L., Irani, N.G., Lu, Y., Riedl, K., Schwartz, S., TESTA 19 is involved in the accumulation of both Grotewold, E.: The formation of anthocyanic vacuolar anthocyanins and proanthocyanidins in Arabidopsis. - Plant inclusions in Arabidopsis thaliana and implications for the J. 37: 104-114, 2004. sequestration of anthocyanin pigments. - Mol. Plants 3: 78- Koes, R., Verweij, W., Quattrocchio, F.: Flavonoids: a colorful 90, 2010. model for the regulation and evolution of biochemical Provenzano, S., Spelt, C., Hosokawa, S., Nakamura, N., pathways. - Trends Plant Sci. 10: 236-242, 2005. Brugliera, F., Demelis, L., Geerke, D.P., Schubert, A., Luo, J., Nishiyama, Y., Fuell, C., Taguchi, G., Elliott, K., Hill, Tanaka, Y., Quattrocchio, F., Koes, R.: Genetic control and L., Tanaka, Y., Kitayama, M., Yamazaki, M., Bailey, P., evolution of anthocyanin methylation. - Plant Physiol. 165: Parr, A., Michael, A.J., Saito, K., Martin, C.: Convergent 962-977, 2014. evolution in the BAHD family of acyl transferases: Quattrocchio, F., Verweij, W., Kroon, A., Spelt, C., Mol, J., identification and characterization of anthocyaninacyl Koes, R.: PH4 of Petunia is an R2R3-MYB protein that transferases from Arabidopsis thaliana. - Plant J. 50: 678- activates vacuolar acidification through interactions with 695, 2007. basic-helix-loop-helix transcription factors of the Maier, A., Schrader, A., Kokkelink, L., Falke, C., Welter, B., anthocyanin pathway. - Plant Cell 18: 1274-1291, 2006. Iniesto, E., Rubio, V., Uhrig, J.F., Hülskamp, M., Hoecker, Quattrocchio, F., Wing, J., Van der, Woude, K., Souer, E., De U.: Light and the E3 ubiquitin ligase COP1/SPA control the Vetten, N., Mol, J., Koes, R.: Molecular analysis of the protein stability of the MYB transcription factors PAP1 and anthocyanin2 gene of Petunia and its role in the evolution PAP2 involved in anthocyanin accumulation in Arabidopsis. of flower color. - Plant Cell 11: 1433-1444, 1999. - Plant J. 74: 638-651, 2013. Ramsay, N.A., Glover, B.J.: MYB–bHLH–WD40 protein Marinova, K., Pourcel, L., Weder, B., Schwarz, M., Barron, D., complex and the evolution of cellular diversity. - Trends Routaboul, J.M., Debeaujon, I., Klein, M.: The Arabidopsis Plant Sci. 10: 63-70, 2005. MATE transporter TT12 acts as a vacuolar flavonoid/H+- Rubin, G., Tohge, T., Matsuda, F., Saito, K., Scheible, W.R.: antiporter active in proanthocyanidin-accumulating cells of Members of the LBD family of transcription factors repress the seed coat. - Plant Cell 19: 2023-2038, 2007. anthocyanin synthesis and affect additional nitrogen Marrs, K.A., Alfenito, M.R., Lloyd, A.M., Walbot, V.: A responses in Arabidopsis. - Plant Cell 21: 3567-3584, 2009.

53 L.M. WANG et al.

Sagasser, M., Lu, G.H., Hahlbrock, K., Weisshaar, B.: A. Reale, L., Ferranti, F., Koes, R., Quattrocchio, F.: An H+ P- thaliana TRANSPARENT TESTA 1 is involved in seed coat ATPase on the tonoplast determines vacuolar pH and flower development and defines the WIP subfamily of plant zinc colour. - Nat. cell. Biol. 10: 1456-62, 2008. finger proteins. - Gene Dev. 16: 138-149, 2002. Winkel-Shirley, B.: Flavonoid biosynthesis. A colorful model Saito, K., Yonekura-Sakakibara, K., Nakabayashi, R., Higashi, for genetics, biochemistry, cell biology, and biotechnology. Y., Yamazaki, M., Tohge T, Fernie, A.R.: The flavonoid - Plant Physiol. 126: 485-493, 2001. biosynthetic pathway in Arabidopsis: structural and genetic Xu, W., Dubos, C., Lepiniec, L.: Transcriptional control of diversity. - Plant Physiol. Biochem. 72: 21-34, 2013. flavonoid biosynthesis by MYB-bHLH-WDR complexes. - Sasaki, N., Nishizaki, Y., Ozeki, Y., Miyahara, T.: The role of Trends Plant Sci. 20: 176-185, 2015. acyl-glucose in anthocyanin modification. - Molecules 19: Yonekura-Sakakibara, K., Fukushima, A., Nakabayashi, R., 18747-18766, 2014. Hanada, K., Matsuda, F., Sugawara, S., Inoue, E., Spelt, C., Quattrocchio, F., Mol, J.N., Koes, R.: Kuromori, T., Ito, T., Shinozaki, K., Wangwattana, ANTHOCYANIN1 of petunia encodes a basic helix-loop- B.,Yamazaki, M., Saito, K.: Two glycosyltransferases helix protein that directly activates transcription of involved in anthocyanin modification delineated by structural anthocyanin genes. - Plant Cell 12: 1619-1632, transcriptome independent component analysis in 2000. Arabidopsis thaliana. - Plant J. 69: 154-167, 2012. Spelt, C., Quattrocchio, F., Mol, J.N., Koes, R.: Yoshida, K., Negishi, T.: The identification of a vacuolar iron ANTHOCYANIN1 of petunia controls pigment synthesis, transporter involved in the blue coloration of cornflower vacuolar pH, and seed coat development by genetically petals. - Phytochemistry 94: 60-67, 2013. distinct mechanisms. - Plant Cell 14: 2121-2135, 2002. Yuan, Y.W., Sagawa, J.M., Young, R.C., Christensen, B.J., Stracke, R., Werber, M., Weisshaar, B.: The R2R3-MYB gene Bradshaw, H.D., Jr.: Genetic dissection of a major family in Arabidopsis thaliana. - Curr. Opin. Plant Biol. 4: anthocyanin QTL contributing to pollinator-mediated 447-456, 2001. reproductive isolation between sister species of Mimulus. - Takeda, K.: Blue metal complex pigments involved in blue Genetics 194: 255-263, 2013. flower color. - Proc. jap. Acad. Ser. B. phys. biol. Sci. 82: Zhang, H., Yin, W., Xia, X.: Calcineurin B-like family in 142-154, 2006. Populus: comparative genome analysis and expression Tanaka, Y., Brugliera, F.: Flower colour and cytochromes P450. pattern under cold, drought and salt stress treatment. - Plant - Phil. Trans. roy. Soc. London B. biol. Sci. 368: 20120432, Growth Regul. 56: 129-140, 2008. 2013. Zhang, W., Ning, G., Lv, H., Liao, L., Bao, M.: Single MYB- Tanaka, T., Sasaki, N., Ohmiya, A.: Biosynthesis of plant type transcription factor AtCAPRICE: a new efficient tool pigments: anthocyanins, betalains and carotenoids. - Plant J. to engineer the production of anthocyanin in tobacco. - 54: 733-749, 2008. Biochem. biophys. Res. Commun. 388: 742-747, 2009. Verweij, W., Spelt C, Di San Sebastiano, G.P., Vermeer, J.,

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