Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals

Plant Physiology and Biochemistry 112 (2017) 335e345

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Research article

Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals Chonghui Li a, d, 1, Jian Qiu b, 1, Ling Ding a, c, Mingzhong Huang a, d, Surong Huang a, d, Guangsui Yang a, d, **, Junmei Yin a, d, * a Tropical Crops Genetic Resources Institute, The Chinese Academy of Tropical Agricultural Sciences (CATAS)/Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture, Danzhou 571737, China b Rubber Research Institute, CATAS/Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture, Danzhou 571737, China c Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China d The Engineering Technology Research Center of Tropical Ornamental Plant Germplasm Innovation and Utilization, Hainan Province, Danzhou 571737, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 November 2016 Received in revised form 30 December 2016 Accepted 21 January 2017 Available online 22 January 2017

Dendrobium hybrids orchid are popular throughout the world. They have various floral color and pigmentation patterns that are mainly caused by anthocyanins. It is well established that anthocyanin biosynthesis is regulated by the interplay between MYB and bHLH transcription factors (TF) in most plants. In this study, we identified one R2R3-MYB gene, DhMYB2, and one bHLH gene, DhbHLH1, from a Dendrobium hybrid. Their expression profiles were related to anthocyanin pigmentation in Dendrobium petals. Transient over-expression of these two TF genes showed that both DhMYB2 and DhbHLH1 resulted in anthocyanin production in white petals. The interaction between the two TFs was observed in vitro. In different Dendrobium hybrids petals with various pigmentations, DhMYB2 and DhbHLH1 were co-expressed with DhDFR and DhANS, which are regarded as potential regulatory targets of the two TFs. In flowers with distinct purple lips but white or yellow petals/sepals, the expression of DhbHLH1 was only related to anthocyanin accumulation in the lips. Taken together, DhMYB2 interacted with DhbHLH1 to regulate anthocyanin production in Dendrobium hybrid petals. DhbHLH1 was also responsible for the distinct anthocyanin pigmentation in lip tissues. The functional characterization of DhMYB2 and DhbHLH1 will improve understanding of anthocyanin biosynthesis modulation in Dendrobium orchids. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Anthocyanin pigmentation Transcription factor Transcriptional regulation R2R3-MYB bHLH

1. Introduction The genus Dendrobium is a member of the Orchidaceae, which contains more than 1500 species and man-made hybrids, and mostly grow in tropical and subtropical Asia and eastern Australia (da Silva et al., 2016). Dendrobium hybrids have become popular

* Corresponding author. Tropical Crops Genetic Resources Institute, The Chinese Academy of Tropical Agricultural Sciences (CATAS)/Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture, Danzhou 571737, China. ** Corresponding author. Tropical Crops Genetic Resources Institute, The Chinese Academy of Tropical Agricultural Sciences (CATAS)/Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture, Danzhou 571737, China. E-mail addresses: [email protected] (G. Yang), [email protected] (J. Yin). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2017.01.019 0981-9428/© 2017 Elsevier Masson SAS. All rights reserved.

worldwide as commercial cut flowers and potted plants due to their valuable traits, including floral colors, forms, specific fragrance, and a long vase life. The color and shape of the petal, sepal, and the lip which is modified by the third petal, are considered to be the major ornamental traits of Dendrobium flowers. The floral colors are dark red, purple, pink, yellow, green, white etc. and flowers show different colorations with various pigmentation patterns, such as full pigmentation and/or veins of the whole corolla, specific pigmentation in the lips, or a combination of these. The purple, red, peach, or pink colorations in Dendrobium flowers are produced by anthocyanins, which are water soluble flavonoid pigments found in the vacuoles (Kuehnle et al., 1997). The flower color of Dendrobium hybrids is an important consumer consideration. Breeding flowers with altered colors, hues, and patterns is an important research area. The pigmentation patterns usually differ between the petals/sepals and the lip of Dendrobium orchid flowers, which indicates that there is a complex

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regulatory mechanism controlling anthocyanin biosynthesis in a single Dendrobium orchid flower. A study on the molecular mechanism regulating floral pigmentation patterning in Dendrobium hybrids would benefit breeding programs that seek to develop cultivars with different flower colors. Anthocyanins are widely found in flowers, fruits, seeds, and the vegetative tissues of vascular plants. They have many biological functions, such as coloration to attract pollinators and seed dispersers, anti-UV radiation protection, and biotic or abiotic stress responses (Petroni and Tonelli, 2011). Anthocyanin biosynthesis, a branch of the flavonoid biosynthesis pathway, has been well studied and the results have suggested that anthocyanin biosynthesis is conserved within a wide range of plants, including the enzyme genes that are responsible for the common precursors of flavonoids producers, such as CHS, CHI, F3H, and F30 H, and the late biosynthetic genes of the anthocyanin pathway, such as DFR and ANS. The anthocyanin biosynthesis process in most plants is conservatively regulated at the transcriptional level by members of the R2R3-MYB, basic helixeloopehelix (bHLH) and WD40 repeats (WDR) transcription factor (TF) families. Several of the late flavonoid biosynthetic genes are activated by the MYB-bHLH-WD40 (MBW) ternary transcriptional complex (Petroni and Tonelli, 2011; Xu et al., 2015). The R2R3-MYB family has been recognized as the main regulator of anthocyanin biosynthesis and are key factors that determine the various anthocyanin pigmentation patterns of flowers in plant species such as Antirrhinum spp. (ROSEA1, ROSEA2 and VENOSA, Schwinn et al., 2006), Petunia spp. (AN2, DPL, and PHZ, Quattrocchio et al., 1999; Albert et al., 2011), and Mimulus spp. (PELAN and NEGAN, Yuan et al., 2014). The anthocyanin promoting R2R3-MYB TFs have been shown to interact with the R/Blike bHLH from subgroup IIIf and they play important roles in determining the spatial and temporal patterning caused by anthocyanins biosynthesis (Xu et al., 2015). For example, maize ZmC1 (R2R3-MYB) and ZmR (bHLH) interact and activate the whole anthocyanin pathway in kernels (Dooner et al., 1991). In Asiatic Hybrid Lily, LhMYB6 and LhMYB12 both interact with the LhbHLH2 protein and activate anthocyanin biosynthesis in the red spots and the pink backgrounds of petals, respectively (Yamagishi et al., 2010). In the Orchidaceae, PsDFR and PsUMYB6 are highly expressed in the purple flowers of Phalaenopsis schilleriana, but not in the white flowers of P. amabilis (Ma et al., 2009). Phalaenopsis equestris PeMYB2, PeMYB11 and PeMYB12 control the full red colors, red spot, and venation patterns of the sepals and petals, respectively. When lip coloration occurs, PeMYB11 has been shown to be responsive to the red spots in the callus, and PeMYB12 contributes to the full pigmentation in the central lobe. These three PeMYBs may function in cooperation with endogenous bHLHs (PebHLH1, PebHLH2, and PebHLH3) and WDR (Hsu et al., 2015). In Oncidium Gower Ramsey, OgMYB1 is specifically down-regulated in yellow lip tissue and the transient expression of OgMYB1 confers mosaic red anthocyanin pigmentation in the yellow lip tissue. This demonstrates that the various anthocyanin coloration patterns on the floral organ in Oncidium Gower Ramsey are mainly determined by the differential expression of OgMYB1 (Chiou and Yeh, 2008). In Dendrobium moniliforme, the transcriptional control of DmF30 50 H is one reason for the lack of colors in perianths (Whang et al., 2011). In Dendrobium hybrids, several mutations in F3H and/or high expression levels of FLS probably cause the white floral color of hybrid ‘Suree white’; and the simultaneous loss of F3H, DFR, and ANS expression can be seen in the flower of another white hybrid ‘Jasmine white’. The dark and pale floral color of purple flowered ‘Sonia Earsakul’ is caused by the up-regulation (the dark mutant) and downregulation (the pale mutant) of anthocyanin genes (Kriangphan et al., 2015). However, the transcription control of anthocyanin

biosynthesis in Dendrobium spp. is not clear. Wu et al. (2003) isolated the cDNA fragments of 21 DwMYB genes from Dendrobium hybrid Woo Leng. Six of these were full-length cDNA clones and their expression patterns were characterized. The results showed that DwMYB4 expression was restricted to flowers and that DwMYB9 was highly expressed in mature flowers and inflorescences, but at very low levels in young flower buds. DhMYB1 has been isolated from a Dendrobium hybrid (Dendrobium bobby messina  Dendrobium chao phraya) and characterized to control the conical cell shape of the epidermal cells in the flower labellum (Lau et al., 2015). Unfortunately, the transcription factors involved in anthocyanin/flavonoid biosynthesis in Dendrobium spp. flowers have not been identified. The identification of the R2R3-MYB and bHLH genes responsible for anthocyanin biosynthesis in Dendrobium hybrids is an important step towards identifying the genetic control of floral pigmentation in these flowers. The aims of this study were to clone and functionally characterize the TFs regulating anthocyanin biosynthesis in Dendrobium hybrid flowers. Two cDNA clones encoding a R2R3-MYB (termed DhMYB2) and a bHLH (termed DhbHLH1) were isolated from a Dendrobium hybrid, and their spatial and temporal expression, coexpression with genes in the anthocyanin biosynthetic pathway, and transient expression were investigated. We demonstrated that DhMYB2 interacts with DhbHLH1 and that they were involved in anthocyanin biosynthesis in Dendrobium hybrid petals; DhbHLH1 was also responsible for anthocyanin production in lips. This information is essential if the tissue-specific anthocyanin pigmentation of Dendrobium hybrid flowers is to be elucidated. Furthermore, these two genes would be ideal target genes for manipulating floral pigmentation patterns in Dendrobium breeding programs. 2. Materials and methods 2.1. Plant materials The eleven Dendrobium hybrids used in this study were maintained in the same containers for over 5 years by applying the same fertilization, irrigation, and disease prevention regimes under natural light and using a 50% shade cloth between March and October at the Tropical Flower Resource Garden, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences (Danzhou, Hainan Province, China). The 11 hybrids consisted of four that produced purple flowers named as ‘Burana glow’ (BG), ‘Sayuri’, ‘Red bull’ (RB), and ‘Panda’; two bluish purpleflowered hybrids named ‘Coerulea blue’ (CB) and ‘Blue sapphine No. 3’ (BS No. 3); one pink-flowered hybrid named ‘Pink stripe’ (PS); one white-flowered hybrid called ‘Burana white dove’ (BWD); and two hybrids with purple lips, but white sepals and petals called ‘White red tip’ (WRT) and ‘Burana charming’ (BC), and one with purple lips, yellow sepals and petals called ‘Thongchai  Pinwattana’ (T  P). Three floral development stages were defined as described by Kriangphan et al. (2015): stage 1, early young bud (~0.5  1.0e1.5 cm: width  height), stage 2, mature bud (~1.5  1.5e2.0 cm), and stage 3, fully open flower. The sepal, petal, and lip tissues of the three hybrids WRT, BC, and T  P, and the petals of the other hybrids were obtained midmorning during September 2015 for quantitative real-time polymerase chain reaction (qPCR) and the estimated content of total anthocyanin analysis. 2.2. Total RNA extraction and cDNA synthesis Total RNA was extracted from the floral tissues of the Dendrobium hybrids using a RNAprep pure Plant Kit (Polysaccharides & Polyphenolics-rich) (TIANGEN, Beijing, China). The cDNA was

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prepared according to the manual for the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA). The cDNA was used as the template for amplifying genes for the qPCR analysis. 2.3. Cloning and sequence analysis of the DhMYB2 and DhbHLH1 genes, and the anthocyanin biosynthetic genes A Dendrobium hybrid RNA-Seq database was obtained that contained a mixed RNA pool, which included the RNA extracted from the BS No. 3 flowers at the three different developmental stages (stages 1e3). Library construction, sequencing, and assembly were performed by BGI (Shenzhen, China). One MYB unigene and one bHLH unigene were selected from the RNA-seq database, which annotated the possible anthocyanin biosynthesis regulators. The unigenes were designated as DhMYB2 and DhbHLH1. The full length cDNA of DhMYB2 and DhbHLH1 and the cDNA fragments from the anthocyanin biosynthetic genes were isolated from BS No. 3 flowers by PCR using primers designed according to the transcriptome (Table A.1). All the amplified genes were annotated by the BLAST programs from NCBI Nr database and TAIR WU-BLAST. The full length of the deduced amino acid sequences for DhMYB2 and DhbHLH1 were constructed using the ClustalX2 and the MEGA 5.05 program (the Neighbor-Joining method with 1000 bootstrap replications) for sequence alignment and phylogenetic analysis, respectively.

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bombardment, the tissues were incubated on MS medium at 25  C under a 16 h light/8 h dark photoperiod regime. After 2 days, the tissues were observed under a dissecting microscope (Leica, Wetzlar, Germany). The tissues transformed by empty vector pCXSN and pCAMBIA1303 were used as the control. Histochemical staining for b-glucuronidase (GUS) activity by the tissues transformed using empty pCAMBIA1303 was performed according to Schwinn et al. (2006). 2.7. Yeast two-hybrid assay The yeast two-hybrid assay for investigating interactions between DhMYB2 and DhbHLH1 was performed according to Nakatsuka et al. (2008). The entire ORF, and one N-terminal (1e145 aa) and one C-terminal (146e290 aa) fragment from DhMYB2 were cloned into the pGADT7 vector (Clontech), and the ORF of DhbHLH1 was cloned into the pGBKT7 vector (Clontech, Mountain View, US). All the constructs were transformed into yeast strain Y2HGold using the Yeast Cell Complete Conversion Kit (GENMED, Shanghai, China). The co-transformed yeast clones were tested on synthetic dropout (SD) medium without leucine, tryptophan, histidine and adenine, but supplemented with X-a-gal (5-bromo-4-chloro-3indolyl-a-D-galactopyranoside) for a-galactosidase activity to confirm the positive interactions. 3. Results

2.4. qPCR analysis The qPCR analysis was used to investigate genes expressions in Dendrobium floral tissues as described by Li et al. (2016). The primers used to amplify the genes fragments related to anthocyanin biosynthesis were designed based on the unigene sequences from the transcriptome data (Table A.2). The amplification program was 95  C for 7min, 40 cycles of 95  C for 5 s, 56  C for 30 s, and 72  C for 30 s. The relative expressions were normalized to the expression of 18srRNA (Kriangphan et al., 2015). Each reaction was performed in triplicate and the data were analyzed using the 2eDCt method. Heat maps were prepared by HemI software (Deng et al., 2014). 2.5. Petal color measurement and total anthocyanin analysis Petal color at developmental stage 2 was measured using a NF333 spectrophotometer (Nippon Denshoku Industries Co. Ltd., Tokyo, Japan) and the raw data of L* (lightness, from black to white), a* (from green to red) and b* (from blue to yellow) were obtained. The color index for red grapes (CIRG) was calculated according to CIRG ¼ (180 e H)/(L* þ C), where C (color saturation) ¼ (a*2 þ b*2)0.5 ~ o et al., 1995). Estiand H (color tonality) ¼ arctan (b*/a*) (Carren mated content of total anthocyanin (ECTA) were carried out according to Li et al. (2016). 2.6. Over-expression vector construction and transient expression using particle bombardment The DhMYB2 and DhbHLH1 fragments that contained full open reading frames (ORFs) were subcloned into the plant binary vector pCXSN (T-vector) for constitutive gene expression according to Chen et al. (2009). The white petal tissues were freshly detached from Dendrobium hybrid ‘Burana charming’ plants. Bombardment assay was conducted using the Helium Biolistic System (Model GJ1000, Scientz, Ningbo, China). The particle bombardment transformation experiment was performed as described by Schwinn et al. (2006). The petal tissue was bombarded from a distance of 55 mm at a pressure of 7000 kPa within a vacuum of 85 kPa. After

3.1. Phylogenetic relationships between the DhMYB2 and DhbHLH1 TFs isolated from Dendrobium hybrid flowers To isolate the key MYB and bHLH TFs that regulate anthocyanin biosynthesis in the Dendrobium hybrid, a Dendrobium RNA-Seq database was created, which contained a mixed RNA pool from the bluish purple flowers of BS No. 3 taken at the three developmental stages described previously. A total of 66 unigenes, which contained the conserved MYB domains, and 76 unigenes, which contained the conserved bHLH domains, were selected from the database as tentative anthocyanin/flavonoid regulators according to the annotations given by the BLASTx in NCBI Nr and Swiss-Prot protein databases (data not shown). Among these MYB and bHLH unigenes, one unigene with a length of 1103 bp and an ORF, positively matched against PeMYB2, which has been characterized as an activator of anthocyanin biosynthesis in the sepals and petals of Phalaenopsis spp.; and one unigene with a length of 2316 bp and a full ORF was matched against a bHLH activator of anthocyanin production in the flowers of Phalaenopsis spp. (PebHLH1). This indicated that these two candidate genes may play roles in the modulation of anthocyanin biosynthesis in Dendrobium hybrid flowers. The identification, based on the amino acid sequences of the two TFs was performed using the TAIR and NCBI database (Table A.3). The sequences of the two genes were separately amplified from the floral tissue of Dendrobium hybrid BS No. 3, and designated DhMYB2 (GenBank accession No. KY039157) and DhbHLH1 (GenBank accession No. KY039158), respectively. DhMYB2 had an 870 bp long ORF that encoded 290 amino acids. The amino acid alignment showed that the R2R3 repeats region was highly conserved among DhMYB2, PeMYB2, OhMYB1, and ZmC1 from monocot species. It shared a conserved motif “DNEI”, which distinguished it from “ANDV” in dicot plants. DhMYB2 had a similar C-terminal-conserved motif to “KAx[K/R]C[S/T]”, which is an anthocyanin-regulating MYB in monocots (Fig. 1A). The phylogenetic tree showed that DhMYB2 was grouped into the same clade as PeMYB2, OgMYB1, ZmC1, and AtTT2 (Fig. 1B), in which some members are anthocyanin activators in orchid flower tissues and some control anthocyanidin production in the seed coat.

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Fig. 1. Sequences alignments and phylogenic relationships of DhMYB2 and DhbhlH1 from Dendrobium hybrids with known R2R3-MYBs and bHLHs involved in anthocyanin biosynthesis. (A) Alignments of the full-length deduced amino acid sequences of DhMYB2 with other R2R3-MYBs. The R2 and R3 domains are shown; a the bHLH interacting motif; b indicates the different conserved motif “DNEI” for monocots and “ANDV” for dicots in the R2R3 domain of anthocyanin-promoting MYBs; c a C-terminal-conserved motif KAx[K/R] C[S/T] for anthocyanin-regulating MYBs of monocots. Identical nucleotides are shown on a black background, and gaps are indicated by dashes. (B) Phylogenetic relationship of DhMYB2 with selected known anthocyanin MYB regulators from other species. Scale bar represents 0.05 substitutions per site. (C) Phylogenetic relationship of DhbHLH1 with selected known anthocyanin bHLH regulators from other species. Scale bar represents 0.1 substitutions per site. MYB names and GenBank accession numbers are as follows: AmROSEA1, AKB94073, AmROSEA2, ABB83827, AmVENOSA, ABB83828 (Antirrhinum majus); NtAN2, ACO52472 (Nicotiana tabacum); PhAN2, BAP28593 (Petunia  hybrida); AtMYB113, NP_176811, AtMYB114, NP_176812, AtMYB75, NP_176057, AtMYB90, NP_176813, AtTT2, CAC40021 (Arabidopsis thaliana); LhMYB12, BAJ05398, LhMYB6, BAJ05399 (Lilium hybrid division I); ZmC1, 1613412E, ZmP, AAA19821 (Zea mays); PeMYB2, AIS35919, PeMYB11, AIS35928, PeMYB12, AIS35929 (Phalaenopsis equestris); OgMYB1, ABS58501 (Oncidium hybrid); OsMYB1, CAA75509 (Oryza sativa); PsUMYB6, ACH95792 (Phalaenopsis schilleriana). bHLH names and GenBank accession numbers are as follows: NtAn1a, AEE99257, NtAn1b, AEE99258 (Nicotiana tabacum); PhAN1, AAG25927 (Petunia  hybrida); ElbHLH3, AII25878 (Erythranthe lewisii); AtTT8, AEE82802; AtGL3, AED94664; AtEGL3, AEE34125 (Arabidopsis thaliana); LhbHLH2, BAE20058 (Lilium hybrid division I); PebHLH1, AIS35934 (Phalaenopsis equestris); ZmLc, NP_001105339 (Zea mays); SmbHLH1, AIP93872 (Solanum melongena); AmDELILA, AAA32663.1 (Antirrhinum majus); VvMYCA1, NP_001267954 (Vitis vinifera).

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The ORF of DhbHLH1 encoded proteins contained 665 amino acids. The sequence alignment of DhbHLH1 and other bHLH TFs involved in anthocyanin biosynthesis showed that DhbHLH1 had three conserved bHLH TF motifs: the MYB interaction region of the N-terminal, the bHLH domain in the C-terminal region, and the transactivation (ACT) domain (Fig. A.1). The phylogenetic tree showed that DhbHLH1 was homologous with group IIIf members in Arabidopsis bHLHs, which are involved in anthocyanin and proanthocyanidin biosynthesis. DhbHLH1 showed a high 70.2% homology with PebHLH1 in P. equestris and had a greater homology (45.0%) with AtTT8, which regulates anthocyanin synthesis in Arabidopsis seedlings, than with other anthocyanin bHLH TFs, such as ZmLc, AtGL3, and AtEGL3 (Fig. 1C). 3.2. Isolation and sequence analysis of anthocyanin biosynthetic genes In addition to the TFs, a series of genes in the anthocyanin biosynthetic pathway were identified from the transcriptome data by sequence BLAST analysis of the TAIR and NCBI database. These include genes encoding the early pathway enzymes CHS and CHI, the anthocyanin-specific pathway enzymes DFR and ANS, and the glycosyl- and acyl-transferase genes (Table A.3). Among these genes, DhCHI2, DhF30 H2, DhF30 50 H2, DhGT1, DhGT2, DhGT3, and DhAT (acyl-transferase gene) were newly identified and their fragments were isolated. The sequences of the other genes obtained in this study showed very high homologies (data not shown) with genes reported previously (Mudalige-Jayawickarma, 2014; Kriangphan et al., 2015). 3.3. Expression of DhMYB2 and DhbHLH1 was related to anthocyanin coloration in petals To examine the correlation between DhMYB2 and DhbHLH1 expressions and anthocyanin production, the bluish purpleflowered BS No. 3, purple RB, pink PS, and white BWD were selected to characterize the anthocyanin accumulation and gene expression in petals with different colors. The CIRG and the estimated content of total anthocyanin (ECTA) of the petals of were assessed at developmental stage 2 (Fig. 2A). Both the CIRG value and the ECTA were higher in BS No. 3 purple petals than in the BWD white petals, and the ECTA in BWD was almost zero (Fig. 2B). This suggested that the amount of anthocyanin that accumulated in the petals of the four hybrids caused the differences in color phenotypes. The temporal expression profiles of the DhMYB2 and DhbHLH1 genes in the petals of the four hybrids were investigated by qPCR. The DhMYB2 and DhbHLH1 expression levels increased from stage 1 to stage 2, which was before anthesis, and then dramatically decreased at stage 3 and became undetectable in the colored petals of BS No. 3, RB, and PS. Their expression levels in the white petals of BWD continuously reduced throughout the developmental stages of the flower (Fig. 2C; 2D). The DhMYB2 and DhbHLH1 expression levels were much higher in the bluish purple petals of BS No. 3 and the purple petals of RB than in the pink and white petals (Fig. 2C; 2D). In addition to the TF genes, the expression profiles of DhF3H, DhDFR, and DhANS, which were directly activated during anthocyanin biosynthesis, were relatively higher in the colored petals of BS No. 3, RB, and PS than in the white petals of BWD (Fig. 2E; 2F; 2G). It should be noted that DhDFR and DhANS expression was hardly detected in BWD petals during flower development (Fig. 2F; 2G). These results suggested that relatively high DhMYB2 and DhbHLH1 expression levels are associated with anthocyanin accumulation in Dendrobium petals, which indicates that they are involved in the regulation of the anthocyanin biosynthesis in

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Dendrobium hybrids. 3.4. DhMYB2 and DhbHLH1 activated anthocyanin biosynthesis in the Dendrobium hybrid ‘Burana charming’ white petals by transient expression To confirm the functions of DhMYB2 and DhbHLH1 in anthocyanin biosynthesis regulation, the coloration recovery ability of these two genes for anthocyanin production in the white petals of Dendrobium hybrid ‘Burana charming’ were tested by transient expression through a bombardment assay. As expected, the transient expression of DhMYB2 or DhbHLH1 alone resulted in purple pigment spots in the white petal tissues (Fig. 3A; 3B). Tissues transformed with empty vector pCXSN did not have any pigment spots (Fig. 3C) and tissues transformed with pCAMBIA1303 had blue spots after histochemical staining for GUS activity (Fig. 3D), which demonstrated that the transient transformation was efficient. These results indicated that both DhMYB2 and DhbHLH1 could regulate anthocyanin synthesis in petal tissues. 3.5. Proteineprotein interactions between DhMYB2 and DhbHLH1 Both DhMYB2 and DhbHLH1 were characterized as anthocyanin regulators in Dendrobium hybrid petals. The interaction mode between DhMYB2 and DhbHLH1 proteins during transcriptional regulation was further investigated by yeast two-hybrid assay. The auto-activation of pGBK-DhMYB2, but not pGBK-DhbHLH1, was observed on SD/eTrp/eHis/eAde medium with X-a-Gal (Fig. A.2). Therefore, DhMYB2 was used as the prey protein and DhbHLH1 as the bait protein in subsequent experiments. The entire ORF, one Nterminal (1e145 aa) and one C-terminal (146e290 aa) fragments from DhMYB2 were fused with the GAL4 activation domain. The strong protein-protein interaction between DhMYB2 and DhbHLH1 was shown by the growth of colonies containing both pGADDhMYB2 (1e290 aa) and pGBK-DhbHLH1 vectors on SD/eLeu/ eTrp/eHis/eAde þ X-a-gal medium. A weaker interaction between the N-terminals (1e145 aa) of DhMYB2 and DhbHLH1 was also observed (Fig. 4). The results showed that DhMYB2 and DhbHLH1 interacted and formed a transcriptional complex, which relied on the N eterminal structures of DhMYB2. 3.6. DhMYB2 and DhbHLH1 co-expressed with DhDFR and DhANS in petals of Dendrobium hybrids The anthocyanin biosynthesis activation of DhMYB2 and DhbHLH1 in Dendrobium orchid petals was proved by transient bombardment assay. However, the regulatory pathways are still unclear. In order to identify potential key enzyme genes in the anthocyanin biosynthetic pathway, which is tightly regulated by DhMYB2 and DhbHLH1, clustering analysis of all the genes and Dendrobium hybrids, based on relative gene expressions in petals at developmental stage 2, was performed because at that developmental stage, the expression levels of most genes were higher than that at other stages (Fig. 2C; 2D). Ten Dendrobium hybrids were clustered separately into two major groups. The anthocyanin content of these petals suggested that it accumulated in the colored petals (purple, bluish purple, and pink) because anthocyanins were hardly detected in the white and yellow petals (hybrids BWD, BC, and T  P) (Fig. A.2). It was interesting that the two branches of the hybrid clustering were distinguished by anthocyanin accumulation. This led to three hybrids (BWD, BC, and T  P) being clustered together (Fig. 5). The enzyme and TF genes involved in the anthocyanin biosynthesis were clearly clustered into two major groups. One included two flavonoid hydroxylase genes, DhF30 H1 and DhF30 50 H1, and

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C Relative expression

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f

fff BWD

Fig. 2. Estimated content of total anthocyanin, CIRG value, and expression profiles of transcriptional factor genes and anthocyanin biosynthetic genes in Dendrobium hybrids petals. (A) Three developmental of stages of four Dendrobium hybrids petals. BS No.3, Blue sapphine No. 3; RB, Red bull; PS, Pink stripe; BWD, Burana white dove. (B) Estimated content of total anthocyanin (ECTA) and CIRG value of petals at developmental stage 2. The expression of DhMYB2 (C), DhbHLH1 (D), DhF3H (E), DhDFR (F) and DhANS (G) in developmental petals of four hybrids. The data were presented as the mean ± SD (n ¼ 3). Values with different letters are significantly different according to Duncan's multiple range tests at the 5% level. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

DhGT3, and the other group consisted of two TF genes DhMYB2 and DhbHLH1, which were grouped with the other genes on the anthocyanin biosynthesis pathway (Fig. 5). DhCHS, DhCHI2, and

DhF3H, which appear early on the anthocyanin biosynthesis pathway, and DhDFR and DhANS, which appear later in the anthocyanin pathway, were grouped into independent subgroups.

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Fig. 3. Macro view of white petal tissues of Dendrobium hybrid ‘Burana charming’ following biolistic transformation with DhMYB2 (A), DhbHLH1 (B), empty pCXSN vector (C), empty pCAMBIA1303 (D). Bright field microscopy reveals purple anthocyanin producing cells and GUS staining cells. Scale bar represents 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

pGBKT7 –Leu –Trp

pGBK-DhbHLH1

pGBKT7 –Leu –Trp –His –Ade + X-α-gal pGBK-DhbHLH1 Fig. 4. Interactions between DhMYB2 and DhbHLH1 detected through the yeast two-hybrid assay. Y2HGold yeast cells containing plasmids pGADT7 þ pGBKT7, pGAD-DhMYB2 (1e290 aa)þ pGBKT7, pGAD-DhMYB2 (1e145 aa) þ pGBKT7, pGAD-DhMYB2 (146e290 aa)þ pGBKT7, pGADT7þ pGBK-DhbHLH1, pGAD-DhMYB2 (1e290 aa) þ pGBK-DhbHLH1, pGAD-DhMYB2 (1e145 aa) þ pGBK-DhbHLH1 or pGAD-DhMYB2 (146e290 aa) þ pGBK-DhbHLH1 were grown on double- and quadruple-selection media. The X-a-gal assay was performed to confirm positive interactions.

Interestingly, DhMYB2, DhbHLH1, DhDFR, and DhANS, were clearly cluster together prominently, which indicated that the expressions of these two TF genes have major effects on the expression of late biosynthetic genes during anthocyanin production in Dendrobium orchid petals.

3.7. DhbHLH1 was associated with the distinct pigmentation in the lip tissues of Dendrobium hybrids As the pigmentation patterns usually differ between the petals/ sepals and the lip in a single Dendrobium orchid flower, three

hybrids: WRT, BC and T  P with purple lips, but white or yellow sepals/petals, were selected (Fig. 6A) in order to investigate the role of DhMYB2 and DhbHLH1 in the distinct pigmentation of lips. The expression profiles of DhMYB2, DhbHLH1, and two anthocyanin biosynthetic genes, DhDFR and DhANS, in different floral tissues at three developmental stages were assessed. The results showed that all of the genes tested were predominately expressed at stage 1. Therefore, the data for stage 1 has been presented here. It was clear that the purple pigmentation of the lip tissues was produced by anthocyanins (Fig. 6B). DhMYB2 transcripts were prominently detected in the sepals and petals of WRT and BC, whereas there

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Fig. 5. Clustering analysis of Dendrobium hybrids and all genes in anthocyanin biosynthesis pathway based on relative expression. Color bar: Log10. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

were no differences in DhMYB2 levels in the three floral tissues from T  P. Furthermore, DhbHLH1 was expressed specifically in the purple lips of the three hybrids (Fig. 6C; 6D). The expression profiles of the genes associated with anthocyanin biosynthesis were further assessed. DhDFR and DhANS were predominantly expressed in the purple lips and had similar expression patterns to DhbHLH1 (Fig. 6E; 6F). This indicated that the differential expression of DhbHLH1 in floral tissues, but not DhMYB2, was consistent with the purple coloration of the lips in these three Dendrobium hybrids. 4. Discussion Dendrobium hybrids are popular tropical ornamental plants for that have long-lived colorful flowers with diverse pigmentation patterns. Analysis of the pigment compositions in commercial Dendrobium species and hybrids has shown that anthocyanins are responsible for various pigmentations except for the yellow, green, and white colors (Kuehnle et al., 1997). Recent research has focused improving floral color by increasing understanding of coloration control in Dendrobium spp. (Mudalige-Jayawickrama et al., 2005; Whang et al., 2011; Pitakdantham et al., 2011; Piluk and Ratanasut, 2012; Mudalige-Jayawickrama, 2014; Kriangphan et al., 2015). However, the genetic control and molecular mechanism underlying anthocyanin biosynthesis and pigmentation patterns in Dendrobium hybrid flowers is not clear. The MYBs and bHLHs that regulate the anthocyanin biosynthesis have been extensively described in many ornamental plants (Yamagishi et al., 2010; Xiang et al., 2015; Hsu et al., 2015). In Dendrobium hybrid Woo Leng, cDNA fragments representing 21 R2R3-MYB genes were isolated and their expression profiles

investigated. The results suggested that the expression pattern of DwMYB9 coincided with anthocyanin accumulation. The authors inferred that DwMYB9 might play role in anthocyanin biosynthesis in Dendrobium orchids (Wu et al., 2003). However, the MYBs and bHLHs involved in anthocyanin biosynthesis have not been identified so far. In this study, DhMYB2 and DhbHLH1 were confirmed as MYB and bHLH members that regulated anthocyanin biosynthesis in Dendrobium hybrids. Based on domain organization and sequence similarity, DhMYB2 was placed in subgroup 5 of the R2R3-MYBs, which responds to proanthocyanidins/anthocyanin in the seed or kernel coats. DhbHLH1 was placed in the bHLH TF subgroup IIIf, which are involved in the regulation of the flavonoid pathway and trichome development (Feller et al., 2011). Phylogenic tree analysis showed that DhMYB2 was highly homologous with the R2R3-MYBs involved in anthocyanin biosynthesis in monocot Gramineae and Orchidaceae species (Paz-Ares et al., 1987; Ma et al., 2009; Chiou and Yeh, 2008; Hsu et al., 2015), and it particularly closely clustered with PeMYB2 from P. equestris, which determines the full-red pigmentation in the sepals and petals. Sequence comparison with DhbHLH1 and related genes from other species showed that DhbHLH1 shared a 70.2% identity with PebHLH1, which is an anthocyanin biosynthetic regulator in P. equestris flowers (Hsu et al., 2015). DhbHLH1 has been categorized with known anthocyanin biosynthesis bHLHs from other species, e.g. Arabidopsis siliques TT8 (Nesi et al., 2000), Asiatic hybrid lily LhbHLH2 (Nakatsuka et al., 2009), and Nicotiana tabacum NtAn1 (Bai et al., 2011) in the same clade as group IIIf TFs. The expression analysis of DhMYB2 and DhbHLH1 showed that both were involved in producing the different anthocyanin colorations of Dendrobium hybrid petals. This suggests that both MYB

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Fig. 6. The estimated content of total anthocyanin and expression profiles of transcriptional factor genes and anthocyanin biosynthetic genes in different floral tissues of Dendrobium hybrids. (A) Three Dendrobium hybrids with distinct lip pigmentation. WRT, White red tip; BC, Burana charming; T  P, Thongchai  Pinwattana. (B) The estimated content of total anthocyanin in different floral tissues at developmental stage 1. The expression of DhMYB2 (C), DhbHLH1 (D), DhDFR (E), and DhANS (F) in different floral tissues at developmental stage 1. Se, sepal; Pe, petal; Li, lip. The data were presented as the mean ± SD (n ¼ 3). Values with different letters are significantly different according to Duncan's multiple range tests at the 5% level. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and bHLH transcription factors are the limiting regulators of anthocyanin accumulation in Dendrobium hybrids petals. This is consistent with what has been previously observed for GtMYB3 and GtbHLH1 in gentian (Nakatsuka et al., 2008), LhMYB12 and LhbHLH2 in Asiatic hybrid lily (Nakatsuka et al., 2009; Yamagishi et al., 2010), and CmMYB6 and CmbHLH2 in chrysanthemum (Xiang et al., 2015). The temporal and spatial expression analysis of DhMYB2, DhbHLH1, and key enzyme genes in the anthocyanin biosynthetic pathway implied that DhMYB2 and DhbHLH1 might regulate the expression of anthocyanin biosynthetic genes in Dendrobium hybrid petals. Furthermore, transient expression of DhMYB2 or DhbHLH1 alone induced purple spots in the white petals of Dendrobium hybrid BC, whereas the GUS over-expression negative control did not have any pigmentation. This suggested that both DhMYB2 and DhbHLH1 could induce the anthocyanin biosynthesis in the petals of Dendrobium hybrids which was in contrast to PeMYB2 and PebHLH1 in

Phalaenopsis spp. When PeMYB2 was over-expressed in the white sepals/petals of P. aphrodite ssp. formosana, a red pigmentation was produced, whereas PebHLH1 over-expression did not result in any red color (Hsu et al., 2015). The expression data together with the transient expression results suggested that DhMYB2 and DhbHLH1 acted as anthocyanin activators in Dendrobium hybrid petals. The combination of interactions among R2R3-MYB and bHLH conservatively control anthocyanin biosynthesis in many plants, such as, PhAN2 (R2R3-MYB) and PhAN1 (bHLH) in petunia (Spelt et al., 2000), ROSEA1/ROSEA2 (R2R3-MYB) and DELILA (bHLH) in Antirrhinum spp. (Schwinn et al., 2006), and GMYB10 and GMYC1 (bHLH) in Gerbera hybrids (Elomaa et al., 2003). The proteineprotein interactions rely on a MYB protein family conserved amino acid signature ([D/E]Lx2[R/K]x3Lx6Lx3R). In Orchidaceae, PeMYB2 from P. equestris, and PsUMYB6 from P. schilleriana all harbor the motif for interacting with bHLHs. Transient over-expression of PeMYB2

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together with PebHLH1 produces a more intense pigmentation than over-expression of PeMYB2 alone (Hsu et al., 2015). However, PsUMYB6 needs ZmLc, a bHLH transcription factor from Z. mays to induce anthocyanin pigmentation after particle bombardment (Ma et al., 2009). These results suggested that there is an interaction between MYBs and bHLHs in the transcriptional regulation of anthocyanin biosynthesis in orchids. However, to date, there has not been any direct physical interaction evidence for MYBs and bHLHs in Orchidaceae. In this study, the structure analysis showed that DhMYB2 has the motif for interacting with bHLHs, and that DhbHLH1 has the MYB interaction region. The proteineprotein interaction between the two TFs only exists theoretically, and yeast two-hybrid analysis showed that DhMYB2 interacts with DhbHLH1 in vitro. This provides further evidence for that DhMYB2 is involved in anthocyanin biosynthesis in the petals of Dendrobium hybrids through its interaction with DhbHLH1. The R2R3-MYBs and bHLHs regulation of anthocyanin biosynthesis is ubiquitous in plants. However, the regulatory system is different among species. In Arabidopsis, DFR, ANS, and UFGT are activated by a multiple TF complex, which is mainly formed by AaMYB75 and AtMYB90 interacting with EGL3, GL3, and TT8, whereas in maize, almost all of the genes in the anthocyanin biosynthesis pathway are activated as a single unit by the C1/Pl1 (MYB) and R1/B1 (bHLH) complex (Petroni and Tonelli, 2011). In other species, such as Chinese bayberry, the MrMYB1eMrbHLH1 complex acts as activators that induce the MrCHI, MrF30 H, MrDFR1, MrANS and MrUFGT promoters (Liu et al., 2013). Co-expression of chrysanthemum CmMYB6 and CmbHLH2 activate the promoter activity of CmDFR (Xiang et al., 2015). The LcMYB1-LcbHLH complex enhances anthocyanin accumulation by activating the transcription of ANS and DFR in litchi (Lai et al., 2016). In the Orchidaceae, transient over-expression of both PeMYB2 and PebHLH1 or PeMYB2 alone led to the increased expression of PeF3H5, PeDFR1, and PeANS3 in P. aphrodite ssp. formosana flowers (Hsu et al., 2015), and OgMYB1 directly activated the expression of OgCHI and OgDFR in the lips of Oncidium Gower Ramsey after particle bombardment (Chiou and Yeh, 2008). In Dendrobium hybrids, CHS, CHI1, CHI2, F3H, DFR, ANS, F30 50 H, and FLS were cloned and their expression profiles in purple, peach, white and greenish white flowers were investigated. The results suggested that CHS, CHI, F3H, DFR, and ANS were generally expressed in purple and peach flowers, whereas F3H, DFR, and ANS were hardly expressed in the greenish white flowers of the ‘Jasmine white’ hybrid. The results indicated that F3H, DFR, and ANS could be coordinately controlled by TFs in the ‘Jasmine white’ hybrid (Kriangphan et al., 2015). However, the regulation mechanism controlling anthocyanin production in Dendrobium hybrids is still unclear. In this study, gene co-expression analysis was used to investigate the regulatory roles of DhMYB2 and DhbHLH1 in the anthocyanin biosynthetic pathway of Dendrobium hybrids. In the hybrids investigated in this study, during the development of petals, strong positive correlations (Pearson correlation coefficient, r) between DhMYB2 and DhbHLH1 expressions and key anthocyanin biosynthetic genes, particularly DhF3H, DhDFR, DhANS, and DhGT3, were observed (Table A.4). Furthermore, the hierarchical clustering analysis of the genes related to anthocyanin biosynthesis suggested that DhMYB2 and DhbHLH1 had a close clustering relationship with DhDFR and DhANS, but a very poor relationship with DhF30 H, DhF30 50 H, and DhGT3 (Fig. 5). The results indicated that the expression of DhDFR and DhANS appeared to be regulated by DhMYB2 and DhbHLH1 in Dendrobium hybrid petals. The findings from this study partially support the proposed idea that F3H, DFR, and ANS could be coordinately controlled in hybrid ‘Jasmine white’ (Kriangphan et al., 2015), whereas DhF30 H, DhF30 50 H, and DhGT3 might be separately regulated by other TFs. Furthermore, some genes in the anthocyanin biosynthesis pathway are responsible for

the common precursors of flavonoids producers, DhCHS, DhCHI2, and DhF3H, and two glycosyltransferase-like genes (DhGT1 and DhGT2) might be coordinately regulated. However, the regulation mechanism needs be further investigated. Although a strong co-expression of DhMYB2 and DhbHLH1 was observed in the petals with various colors (Table A.4), the expression profiles of these two TF genes in the different floral organs in a single flower were extremely variable. In the three hybrids with purple lips, but white or yellow sepals and petals (WRT, BC, and T  P), DhbHLH1 expression was closely related to anthocyanin accumulation and it was highly expressed in purple lips. However, in hybrids WRT and BC, the DhMYB2 expression levels in the petals and sepals were significantly higher than in the lips, although there was hardly any anthocyanin produced in petals/sepals. It seems that DhMYB2 is not responsible anthocyanin biosynthesis in the lips of Dendrobium hybrids, whereas DhbHLH1 plays an important role in anthocyanin production in the lips. The way in which Dendrobium hybrid MYB TFs control pigmentation in various floral organs may be similar to that in Phalaenopsis spp. Furthermore, there may be another MYB, which is homologous with the PeMYB12 that controls full pigmentation in the lips of Phalaenopsis spp. flowers (Hsu et al., 2015), which controls anthocyanin pigmentation in the lips of Dendrobium hybrids. The recovery of anthocyanin production in BC petals after bombardment with DhMYB2 or DhbHLH1, together with the gene expression data suggest that the low DhMYB2 and DhbHLH1 expression levels caused the simultaneous down-regulation of a number of genes involved in anthocyanin biosynthesis and resulted in white petals/ sepals. Similarly, in Phalaenopsis, the white P. amabillis petals were due to the loss of anthocyanin-specific MYB transcripts, which subsequently reduced DFR expression (Ma et al., 2009). 5. Conclusion This study isolated one R2R3-MYB gene, DhMYB2, and one bHLH gene, DhbHLH1, from the Dendrobium hybrid floral RNA-seq database. Transient expression analysis of DhMYB2 and DhbHLH1 revealed that the two TF genes were able to induce anthocyanin production in the white petals of Dendrobium hybrid BC. Furthermore, the two genes were closely co-expressed with DhDFR and DhANS, and this correlated with anthocyanin accumulation in multi-pigmented petals. The proteineprotein interaction between DhMYB2 and DhbHLH1 was confirmed by yeast two-hybrid analysis. These results suggested that DhMYB2 and DhbHLH1 interact and play a role in regulating anthocyanin production in Dendrobium hybrid petals. Furthermore, the results suggested that DhbHLH1 is also responsible for anthocyanin biosynthesis in lips. This study provides valuable information on anthocyanin biosynthesis regulation in Dendrobium hybrids. Formatting of funding sources This work was supported by the National Natural Science Foundation of China [grant numbers 31101578; 31400579]; the Grand Special Project on Science and Technology of Hainan Province [grant number ZDZX2013012]; the National Nonprofit Institute Research Grant of CATAS-TCGRI [grant number 1630032015017]. Author contributions LCH, QJ, YGS and YJM conceived the study; LCH and DL performed the experiments. LCH and QJ analyzed the data and wrote the manuscript; HMZ and HSR participated in the performance of the experiments. All authors have read and approved the final manuscript.

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