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Identification and Functional Analysis of Anthocyanin Biosynthesis Genes in Phalaenopsis Hybrids

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Identification and Functional Analysis of Anthocyanin Biosynthesis Genes in Phalaenopsis Hybrids DOI: 10.1007/s10535-017-0763-2 BIOLOGIA PLANTARUM 62 (1): 45-54, 2018 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 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. 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: [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) proteins (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
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