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Plant Physiology and Biochemistry 112 (2017) 335e345

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Plant Physiology and Biochemistry

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Research article

Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 inDendrobium 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 andGermplasm Enhancement in Southern China, Ministry of Agriculture, Danzhou 571737, Chinab Rubber Research Institute, CATAS/Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture, Danzhou 571737, Chinac Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, Chinad 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

Article history:Received 17 November 2016Received in revised form30 December 2016Accepted 21 January 2017Available online 22 January 2017

Keywords:Anthocyanin pigmentationTranscription factorTranscriptional regulationR2R3-MYBbHLH

* Corresponding author. Tropical Crops Genetic ResAcademy of Tropical Agricultural Sciences (CATAS)/KResources and Germplasm Enhancement in Southerture, Danzhou 571737, China.** Corresponding author. Tropical Crops Genetic ResAcademy of Tropical Agricultural Sciences (CATAS)/KResources and Germplasm Enhancement in Southerture, Danzhou 571737, China.

E-mail addresses: 13976572870@163.com (G. Yan(J. Yin).

1 Contributed equally to this work.

http://dx.doi.org/10.1016/j.plaphy.2017.01.0190981-9428/© 2017 Elsevier Masson SAS. All rights res

a b s t r a c t

Dendrobium hybrids orchid are popular throughout the world. They have various floral color andpigmentation patterns that are mainly caused by anthocyanins. It is well established that anthocyaninbiosynthesis is regulated by the interplay between MYB and bHLH transcription factors (TF) in mostplants. In this study, we identified one R2R3-MYB gene, DhMYB2, and one bHLH gene, DhbHLH1, from aDendrobium hybrid. Their expression profiles were related to anthocyanin pigmentation in Dendrobiumpetals. Transient over-expression of these two TF genes showed that both DhMYB2 and DhbHLH1resulted in anthocyanin production in white petals. The interaction between the two TFs was observedin vitro. In different Dendrobium hybrids petals with various pigmentations, DhMYB2 and DhbHLH1 wereco-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 wasonly related to anthocyanin accumulation in the lips. Taken together, DhMYB2 interacted with DhbHLH1to regulate anthocyanin production in Dendrobium hybrid petals. DhbHLH1 was also responsible for thedistinct anthocyanin pigmentation in lip tissues. The functional characterization of DhMYB2 andDhbHLH1 will improve understanding of anthocyanin biosynthesis modulation in Dendrobium orchids.

© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

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

ources Institute, The Chineseey Laboratory of Crop Genen China, Ministry of Agricul-

ources Institute, The Chineseey Laboratory of Crop Genen China, Ministry of Agricul-

g), yinjunmei2011@sina.com

erved.

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

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C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345336

regulatory mechanism controlling anthocyanin biosynthesis in asingle Dendrobium orchid flower. A study on the molecular mech-anism regulating floral pigmentation patterning in Dendrobiumhybrids would benefit breeding programs that seek to developcultivars with different flower colors.

Anthocyanins are widely found in flowers, fruits, seeds, and thevegetative tissues of vascular plants. They have many biologicalfunctions, such as coloration to attract pollinators and seed dis-persers, anti-UV radiation protection, and biotic or abiotic stressresponses (Petroni and Tonelli, 2011). Anthocyanin biosynthesis, abranch of the flavonoid biosynthesis pathway, has been wellstudied and the results have suggested that anthocyanin biosyn-thesis is conserved within a wide range of plants, including theenzyme genes that are responsible for the common precursors offlavonoids producers, such as CHS, CHI, F3H, and F30H, and the latebiosynthetic genes of the anthocyanin pathway, such as DFR andANS. The anthocyanin biosynthesis process in most plants isconservatively regulated at the transcriptional level by members ofthe R2R3-MYB, basic helixeloopehelix (bHLH) and WD40 repeats(WDR) transcription factor (TF) families. Several of the late flavo-noid 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 recognizedas the main regulator of anthocyanin biosynthesis and are keyfactors that determine the various anthocyanin pigmentation pat-terns 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 Mimulusspp. (PELAN and NEGAN, Yuan et al., 2014). The anthocyanin pro-moting R2R3-MYB TFs have been shown to interact with the R/B-like bHLH from subgroup IIIf and they play important roles indetermining the spatial and temporal patterning caused by an-thocyanins biosynthesis (Xu et al., 2015). For example, maize ZmC1(R2R3-MYB) and ZmR (bHLH) interact and activate the wholeanthocyanin pathway in kernels (Dooner et al., 1991). In AsiaticHybrid Lily, LhMYB6 and LhMYB12 both interact with the LhbHLH2protein and activate anthocyanin biosynthesis in the red spots andthe pink backgrounds of petals, respectively (Yamagishi et al.,2010).

In the Orchidaceae, PsDFR and PsUMYB6 are highly expressed inthe purple flowers of Phalaenopsis schilleriana, but not in the whiteflowers of P. amabilis (Ma et al., 2009). Phalaenopsis equestrisPeMYB2, PeMYB11 and PeMYB12 control the full red colors, red spot,and venation patterns of the sepals and petals, respectively. Whenlip coloration occurs, PeMYB11 has been shown to be responsive tothe red spots in the callus, and PeMYB12 contributes to the fullpigmentation in the central lobe. These three PeMYBs may functionin cooperation with endogenous bHLHs (PebHLH1, PebHLH2, andPebHLH3) and WDR (Hsu et al., 2015). In Oncidium Gower Ramsey,OgMYB1 is specifically down-regulated in yellow lip tissue and thetransient expression of OgMYB1 confers mosaic red anthocyaninpigmentation in the yellow lip tissue. This demonstrates that thevarious anthocyanin coloration patterns on the floral organ inOncidium Gower Ramsey are mainly determined by the differentialexpression of OgMYB1 (Chiou and Yeh, 2008). In Dendrobiummoniliforme, the transcriptional control of DmF3050H is one reasonfor the lack of colors in perianths (Whang et al., 2011). In Den-drobium hybrids, several mutations in F3H and/or high expressionlevels of FLS probably cause the white floral color of hybrid ‘Sureewhite’; and the simultaneous loss of F3H, DFR, and ANS expressioncan be seen in the flower of another white hybrid ‘Jasmine white’.The dark and pale floral color of purple flowered ‘Sonia Earsakul’ iscaused by the up-regulation (the dark mutant) and down-regulation (the pale mutant) of anthocyanin genes (Kriangphanet al., 2015). However, the transcription control of anthocyanin

biosynthesis in Dendrobium spp. is not clear. Wu et al. (2003) iso-lated the cDNA fragments of 21 DwMYB genes from Dendrobiumhybrid Woo Leng. Six of these were full-length cDNA clones andtheir expression patterns were characterized. The results showedthat DwMYB4 expression was restricted to flowers and thatDwMYB9 was highly expressed in mature flowers and in-florescences, but at very low levels in young flower buds. DhMYB1has been isolated from a Dendrobium hybrid (Dendrobium bobbymessina � Dendrobium chao phraya) and characterized to controlthe conical cell shape of the epidermal cells in the flower labellum(Lau et al., 2015). Unfortunately, the transcription factors involvedin anthocyanin/flavonoid biosynthesis in Dendrobium spp. flowershave not been identified. The identification of the R2R3-MYB andbHLH genes responsible for anthocyanin biosynthesis in Den-drobium hybrids is an important step towards identifying the ge-netic control of floral pigmentation in these flowers.

The aims of this study were to clone and functionally charac-terize the TFs regulating anthocyanin biosynthesis in Dendrobiumhybrid flowers. Two cDNA clones encoding a R2R3-MYB (termedDhMYB2) and a bHLH (termed DhbHLH1) were isolated from aDendrobium hybrid, and their spatial and temporal expression, co-expression with genes in the anthocyanin biosynthetic pathway,and transient expression were investigated. We demonstrated thatDhMYB2 interacts with DhbHLH1 and that they were involved inanthocyanin biosynthesis in Dendrobium hybrid petals; DhbHLH1was also responsible for anthocyanin production in lips. This in-formation is essential if the tissue-specific anthocyanin pigmenta-tion of Dendrobium hybrid flowers is to be elucidated. Furthermore,these two genes would be ideal target genes for manipulating floralpigmentation patterns in Dendrobium breeding programs.

2. Materials and methods

2.1. Plant materials

The eleven Dendrobium hybrids used in this study were main-tained in the same containers for over 5 years by applying the samefertilization, irrigation, and disease prevention regimes undernatural light and using a 50% shade cloth between March andOctober at the Tropical Flower Resource Garden, Tropical CropsGenetic Resources Institute, Chinese Academy of Tropical Agricul-tural Sciences (Danzhou, Hainan Province, China). The 11 hybridsconsisted of four that produced purple flowers named as ‘Buranaglow’ (BG), ‘Sayuri’, ‘Red bull’ (RB), and ‘Panda’; two bluish purple-flowered hybrids named ‘Coerulea blue’ (CB) and ‘Blue sapphineNo. 3’ (BS No. 3); one pink-flowered hybrid named ‘Pink stripe’ (PS);one white-flowered hybrid called ‘Burana white dove’ (BWD); andtwo hybrids with purple lips, but white sepals and petals called‘White red tip’ (WRT) and ‘Burana charming’ (BC), and one withpurple lips, yellow sepals and petals called‘Thongchai � Pinwattana’ (T � P). Three floral development stageswere defined as described by Kriangphan et al. (2015): stage 1, earlyyoung 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 thepetals of the other hybrids were obtained midmorning duringSeptember 2015 for quantitative real-time polymerase chain reac-tion (qPCR) and the estimated content of total anthocyaninanalysis.

2.2. Total RNA extraction and cDNA synthesis

Total RNA was extracted from the floral tissues of the Den-drobium hybrids using a RNAprep pure Plant Kit (Polysaccharides&Polyphenolics-rich) (TIANGEN, Beijing, China). The cDNA was

C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345 337

prepared according to the manual for the RevertAid First StrandcDNA Synthesis Kit (Thermo Scientific, MA, USA). The cDNA wasused as the template for amplifying genes for the qPCR analysis.

2.3. Cloning and sequence analysis of the DhMYB2 and DhbHLH1genes, and the anthocyanin biosynthetic genes

A Dendrobium hybrid RNA-Seq database was obtained thatcontained a mixed RNA pool, which included the RNA extractedfrom the BS No. 3 flowers at the three different developmentalstages (stages 1e3). Library construction, sequencing, and assemblywere performed by BGI (Shenzhen, China). One MYB unigene andone 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 fulllength cDNA of DhMYB2 and DhbHLH1 and the cDNA fragmentsfrom the anthocyanin biosynthetic genes were isolated from BS No.3 flowers by PCR using primers designed according to the tran-scriptome (Table A.1).

All the amplified genes were annotated by the BLAST programsfrom NCBI Nr database and TAIR WU-BLAST. The full length of thededuced amino acid sequences for DhMYB2 and DhbHLH1 wereconstructed using the ClustalX2 and the MEGA 5.05 program (theNeighbor-Joining method with 1000 bootstrap replications) forsequence alignment and phylogenetic analysis, respectively.

2.4. qPCR analysis

The qPCR analysis was used to investigate genes expressions inDendrobium floral tissues as described by Li et al. (2016). Theprimers used to amplify the genes fragments related to anthocy-anin biosynthesis were designed based on the unigene sequencesfrom the transcriptome data (Table A.2). The amplification programwas 95 �C for 7min, 40 cycles of 95 �C for 5 s, 56 �C for 30 s, and72 �C for 30 s. The relative expressions were normalized to theexpression of 18srRNA (Kriangphan et al., 2015). Each reaction wasperformed 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 aNF333 spectrophotometer (Nippon Denshoku Industries Co. Ltd.,Tokyo, Japan) and the raw data of L* (lightness, from black towhite),a* (from green to red) and b* (from blue to yellow) were obtained.The color index for red grapes (CIRG) was calculated according toCIRG¼ (180eH)/(L*þ C), where C (color saturation)¼ (a*2þ b*2)0.5and H (color tonality) ¼ arctan (b*/a*) (Carre~no et al., 1995). Esti-mated content of total anthocyanin (ECTA) were carried out ac-cording to Li et al. (2016).

2.6. Over-expression vector construction and transient expressionusing particle bombardment

The DhMYB2 and DhbHLH1 fragments that contained full openreading frames (ORFs) were subcloned into the plant binary vectorpCXSN (T-vector) for constitutive gene expression according toChen et al. (2009). The white petal tissues were freshly detachedfrom Dendrobium hybrid ‘Burana charming’ plants. Bombardmentassay was conducted using the Helium Biolistic System (Model GJ-1000, Scientz, Ningbo, China). The particle bombardment trans-formation experiment was performed as described by Schwinnet al. (2006). The petal tissue was bombarded from a distance of55 mm at a pressure of 7000 kPawithin a vacuum of �85 kPa. After

bombardment, the tissues were incubated on MS medium at 25 �Cunder a 16 h light/8 h dark photoperiod regime. After 2 days, thetissues were observed under a dissecting microscope (Leica, Wet-zlar, Germany). The tissues transformed by empty vector pCXSNand pCAMBIA1303were used as the control. Histochemical stainingfor b-glucuronidase (GUS) activity by the tissues transformed usingempty pCAMBIA1303 was performed according to Schwinn et al.(2006).

2.7. Yeast two-hybrid assay

The yeast two-hybrid assay for investigating interactions be-tween DhMYB2 and DhbHLH1 was performed according toNakatsuka et al. (2008). The entire ORF, and one N-terminal (1e145aa) and one C-terminal (146e290 aa) fragment from DhMYB2werecloned into the pGADT7 vector (Clontech), and the ORF of DhbHLH1was cloned into the pGBKT7 vector (Clontech, Mountain View, US).All the constructs were transformed into yeast strain Y2HGold us-ing the Yeast Cell Complete Conversion Kit (GENMED, Shanghai,China). The co-transformed yeast clones were tested on syntheticdropout (SD) medium without leucine, tryptophan, histidine andadenine, but supplemented with X-a-gal (5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside) for a-galactosidase activity toconfirm the positive interactions.

3. Results

3.1. Phylogenetic relationships between the DhMYB2 and DhbHLH1TFs isolated from Dendrobium hybrid flowers

To isolate the key MYB and bHLH TFs that regulate anthocyaninbiosynthesis in the Dendrobium hybrid, a Dendrobium RNA-Seqdatabase was created, which contained a mixed RNA pool fromthe bluish purple flowers of BS No. 3 taken at the three develop-mental stages described previously. A total of 66 unigenes, whichcontained the conserved MYB domains, and 76 unigenes, whichcontained the conserved bHLH domains, were selected from thedatabase as tentative anthocyanin/flavonoid regulators accordingto the annotations given by the BLASTx in NCBI Nr and Swiss-Protprotein databases (data not shown). Among these MYB and bHLHunigenes, one unigene with a length of 1103 bp and an ORF, posi-tively matched against PeMYB2, which has been characterized asan activator of anthocyanin biosynthesis in the sepals and petals ofPhalaenopsis spp.; and one unigene with a length of 2316 bp and afull ORF was matched against a bHLH activator of anthocyaninproduction in the flowers of Phalaenopsis spp. (PebHLH1). Thisindicated that these two candidate genes may play roles in themodulation of anthocyanin biosynthesis in Dendrobium hybridflowers. The identification, based on the amino acid sequences ofthe two TFs was performed using the TAIR and NCBI database(Table A.3). The sequences of the two genes were separatelyamplified from the floral tissue of Dendrobium hybrid BS No. 3, anddesignated DhMYB2 (GenBank accession No. KY039157) andDhbHLH1 (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 regionwas highly conserved among DhMYB2, PeMYB2, OhMYB1, andZmC1 from monocot species. It shared a conserved motif “DNEI”,which distinguished it from “ANDV” in dicot plants. DhMYB2 had asimilar C-terminal-conserved motif to “KAx[K/R]C[S/T]”, which isan anthocyanin-regulating MYB in monocots (Fig. 1A). The phylo-genetic tree showed that DhMYB2was grouped into the same cladeas PeMYB2, OgMYB1, ZmC1, and AtTT2 (Fig. 1B), in which somemembers are anthocyanin activators in orchid flower tissues andsome control anthocyanidin production in the seed coat.

Fig. 1. Sequences alignments and phylogenic relationships of DhMYB2 and DhbhlH1 from Dendrobium hybrids with known R2R3-MYBs and bHLHs involved in anthocyaninbiosynthesis. (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 ofDhMYB2 with selected known anthocyanin MYB regulators from other species. Scale bar represents 0.05 substitutions per site. (C) Phylogenetic relationship of DhbHLH1 withselected 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 aminoacids. The sequence alignment of DhbHLH1 and other bHLH TFsinvolved in anthocyanin biosynthesis showed that DhbHLH1 hadthree conserved bHLH TF motifs: the MYB interaction region of theN-terminal, the bHLH domain in the C-terminal region, and thetransactivation (ACT) domain (Fig. A.1). The phylogenetic treeshowed that DhbHLH1was homologous with group IIIf members inArabidopsis bHLHs, which are involved in anthocyanin and proan-thocyanidin biosynthesis. DhbHLH1 showed a high 70.2% homol-ogy with PebHLH1 in P. equestris and had a greater homology(45.0%) with AtTT8, which regulates anthocyanin synthesis inArabidopsis seedlings, than with other anthocyanin bHLH TFs, suchas ZmLc, AtGL3, and AtEGL3 (Fig. 1C).

3.2. Isolation and sequence analysis of anthocyanin biosyntheticgenes

In addition to the TFs, a series of genes in the anthocyaninbiosynthetic pathway were identified from the transcriptome databy sequence BLAST analysis of the TAIR and NCBI database. Theseinclude genes encoding the early pathway enzymes CHS and CHI,the anthocyanin-specific pathway enzymes DFR and ANS, and theglycosyl- and acyl-transferase genes (Table A.3). Among thesegenes, DhCHI2, DhF30H2, DhF3050H2, DhGT1, DhGT2, DhGT3, andDhAT (acyl-transferase gene) were newly identified and theirfragments were isolated. The sequences of the other genes obtainedin this study showed very high homologies (data not shown) withgenes reported previously (Mudalige-Jayawickarma, 2014;Kriangphan et al., 2015).

3.3. Expression of DhMYB2 and DhbHLH1 was related toanthocyanin coloration in petals

To examine the correlation between DhMYB2 and DhbHLH1expressions and anthocyanin production, the bluish purple-flowered BS No. 3, purple RB, pink PS, and white BWD wereselected to characterize the anthocyanin accumulation and geneexpression in petals with different colors. The CIRG and the esti-mated content of total anthocyanin (ECTA) of the petals of wereassessed at developmental stage 2 (Fig. 2A). Both the CIRG valueand the ECTAwere higher in BS No. 3 purple petals than in the BWDwhite petals, and the ECTA in BWD was almost zero (Fig. 2B). Thissuggested that the amount of anthocyanin that accumulated in thepetals of the four hybrids caused the differences in colorphenotypes.

The temporal expression profiles of the DhMYB2 and DhbHLH1genes in the petals of the four hybrids were investigated by qPCR.The DhMYB2 and DhbHLH1 expression levels increased from stage 1to stage 2, which was before anthesis, and then dramaticallydecreased at stage 3 and became undetectable in the colored petalsof BS No. 3, RB, and PS. Their expression levels in the white petals ofBWD continuously reduced throughout the developmental stagesof the flower (Fig. 2C; 2D). The DhMYB2 and DhbHLH1 expressionlevels were much higher in the bluish purple petals of BS No. 3 andthe 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 antho-cyanin biosynthesis, were relatively higher in the colored petals ofBS 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 washardly detected in BWD petals during flower development (Fig. 2F;2G). These results suggested that relatively high DhMYB2 andDhbHLH1 expression levels are associated with anthocyanin accu-mulation in Dendrobium petals, which indicates that they areinvolved in the regulation of the anthocyanin biosynthesis in

Dendrobium hybrids.

3.4. DhMYB2 and DhbHLH1 activated anthocyanin biosynthesis inthe Dendrobium hybrid ‘Burana charming’ white petals by transientexpression

To confirm the functions of DhMYB2 and DhbHLH1 in anthocy-anin biosynthesis regulation, the coloration recovery ability ofthese two genes for anthocyanin production in the white petals ofDendrobium hybrid ‘Burana charming’ were tested by transientexpression through a bombardment assay. As expected, the tran-sient expression of DhMYB2 or DhbHLH1 alone resulted in purplepigment spots in the white petal tissues (Fig. 3A; 3B). Tissuestransformed with empty vector pCXSN did not have any pigmentspots (Fig. 3C) and tissues transformed with pCAMBIA1303 hadblue spots after histochemical staining for GUS activity (Fig. 3D),which demonstrated that the transient transformation was effi-cient. These results indicated that both DhMYB2 and DhbHLH1could regulate anthocyanin synthesis in petal tissues.

3.5. Proteineprotein interactions between DhMYB2 and DhbHLH1

Both DhMYB2 and DhbHLH1 were characterized as anthocyaninregulators in Dendrobium hybrid petals. The interaction mode be-tween DhMYB2 and DhbHLH1 proteins during transcriptionalregulation was further investigated by yeast two-hybrid assay. Theauto-activation of pGBK-DhMYB2, but not pGBK-DhbHLH1, wasobserved on SD/eTrp/eHis/eAde medium with X-a-Gal (Fig. A.2).Therefore, DhMYB2 was used as the prey protein and DhbHLH1 asthe bait protein in subsequent experiments. The entire ORF, one N-terminal (1e145 aa) and one C-terminal (146e290 aa) fragmentsfrom DhMYB2 were fused with the GAL4 activation domain. Thestrong protein-protein interaction between DhMYB2 and DhbHLH1was shown by the growth of colonies containing both pGAD-DhMYB2 (1e290 aa) and pGBK-DhbHLH1 vectors on SD/eLeu/eTrp/eHis/eAdeþ X-a-gal medium. Aweaker interaction betweenthe N-terminals (1e145 aa) of DhMYB2 and DhbHLH1 was alsoobserved (Fig. 4). The results showed that DhMYB2 and DhbHLH1interacted and formed a transcriptional complex, which relied onthe N eterminal structures of DhMYB2.

3.6. DhMYB2 and DhbHLH1 co-expressed with DhDFR and DhANSin petals of Dendrobium hybrids

The anthocyanin biosynthesis activation of DhMYB2 andDhbHLH1 in Dendrobium orchid petals was proved by transientbombardment assay. However, the regulatory pathways are stillunclear. In order to identify potential key enzyme genes in theanthocyanin biosynthetic pathway, which is tightly regulated byDhMYB2 and DhbHLH1, clustering analysis of all the genes andDendrobium hybrids, based on relative gene expressions in petals atdevelopmental stage 2, was performed because at that develop-mental stage, the expression levels of most genes were higher thanthat at other stages (Fig. 2C; 2D). Ten Dendrobium hybrids wereclustered separately into two major groups. The anthocyanin con-tent of these petals suggested that it accumulated in the coloredpetals (purple, bluish purple, and pink) because anthocyanins werehardly detected in the white and yellow petals (hybrids BWD, BC,and T � P) (Fig. A.2). It was interesting that the two branches of thehybrid clusteringwere distinguished by anthocyanin accumulation.This led to three hybrids (BWD, BC, and T � P) being clusteredtogether (Fig. 5).

The enzyme and TF genes involved in the anthocyanin biosyn-thesis were clearly clustered into two major groups. One includedtwo flavonoid hydroxylase genes, DhF30H1 and DhF3050H1, and

BS No.3 RB PS BWD

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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 oftotal 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 developmentalpetals 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.)

C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345340

DhGT3, and the other group consisted of two TF genes DhMYB2 andDhbHLH1, which were grouped with the other genes on theanthocyanin biosynthesis pathway (Fig. 5). DhCHS, DhCHI2, and

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

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), emptypCAMBIA1303 (D). Bright field microscopy reveals purple anthocyanin producing cells and GUS staining cells. Scale bar represents 200 mm. (For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of this article.)

pGBKT7

pGBK-DhbHLH1

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pGBK-DhbHLH1

–Leu –Trp

–Leu –Trp –His –Ade + X-α-gal

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 wasperformed to confirm positive interactions.

C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345 341

Interestingly, DhMYB2, DhbHLH1, DhDFR, and DhANS, were clearlycluster together prominently, which indicated that the expressionsof these two TF genes have major effects on the expression of latebiosynthetic genes during anthocyanin production in Dendrobiumorchid petals.

3.7. DhbHLH1 was associated with the distinct pigmentation in thelip 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 yellowsepals/petals, were selected (Fig. 6A) in order to investigate the roleof DhMYB2 and DhbHLH1 in the distinct pigmentation of lips. Theexpression profiles of DhMYB2, DhbHLH1, and two anthocyaninbiosynthetic genes, DhDFR and DhANS, in different floral tissues atthree developmental stages were assessed. The results showed thatall of the genes tested were predominately expressed at stage 1.Therefore, the data for stage 1 has been presented here. It was clearthat the purple pigmentation of the lip tissues was produced byanthocyanins (Fig. 6B). DhMYB2 transcripts were prominentlydetected in the sepals and petals of WRT and BC, whereas there

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

C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345342

were no differences in DhMYB2 levels in the three floral tissuesfrom T� P. Furthermore, DhbHLH1was expressed specifically in thepurple lips of the three hybrids (Fig. 6C; 6D). The expression profilesof the genes associated with anthocyanin biosynthesis were furtherassessed. DhDFR and DhANS were predominantly expressed in thepurple lips and had similar expression patterns to DhbHLH1(Fig. 6E; 6F). This indicated that the differential expression ofDhbHLH1 in floral tissues, but not DhMYB2, was consistent with thepurple coloration of the lips in these three Dendrobium hybrids.

4. Discussion

Dendrobium hybrids are popular tropical ornamental plants forthat have long-lived colorful flowers with diverse pigmentationpatterns. Analysis of the pigment compositions in commercialDendrobium species and hybrids has shown that anthocyanins areresponsible for various pigmentations except for the yellow, green,and white colors (Kuehnle et al., 1997). Recent research has focusedimproving floral color by increasing understanding of colorationcontrol in Dendrobium spp. (Mudalige-Jayawickrama et al., 2005;Whang et al., 2011; Pitakdantham et al., 2011; Piluk andRatanasut, 2012; Mudalige-Jayawickrama, 2014; Kriangphan et al.,2015). However, the genetic control and molecular mechanismunderlying anthocyanin biosynthesis and pigmentation patterns inDendrobium hybrid flowers is not clear.

The MYBs and bHLHs that regulate the anthocyanin biosyn-thesis have been extensively described in many ornamental plants(Yamagishi et al., 2010; Xiang et al., 2015; Hsu et al., 2015). InDendrobium hybrid Woo Leng, cDNA fragments representing 21R2R3-MYB genes were isolated and their expression profiles

investigated. The results suggested that the expression pattern ofDwMYB9 coincided with anthocyanin accumulation. The authorsinferred that DwMYB9 might play role in anthocyanin biosynthesisin Dendrobium orchids (Wu et al., 2003). However, the MYBs andbHLHs involved in anthocyanin biosynthesis have not been iden-tified so far. In this study, DhMYB2 and DhbHLH1 were confirmedas MYB and bHLH members that regulated anthocyanin biosyn-thesis in Dendrobium hybrids. Based on domain organization andsequence similarity, DhMYB2 was placed in subgroup 5 of theR2R3-MYBs, which responds to proanthocyanidins/anthocyanin inthe seed or kernel coats. DhbHLH1 was placed in the bHLH TFsubgroup IIIf, which are involved in the regulation of the flavonoidpathway and trichome development (Feller et al., 2011). Phylogenictree analysis showed that DhMYB2 was highly homologous withthe R2R3-MYBs involved in anthocyanin biosynthesis in monocotGramineae and Orchidaceae species (Paz-Ares et al., 1987; Ma et al.,2009; Chiou and Yeh, 2008; Hsu et al., 2015), and it particularlyclosely clustered with PeMYB2 from P. equestris, which determinesthe full-red pigmentation in the sepals and petals. Sequence com-parison with DhbHLH1 and related genes from other speciesshowed that DhbHLH1 shared a 70.2% identity with PebHLH1,which is an anthocyanin biosynthetic regulator in P. equestrisflowers (Hsu et al., 2015). DhbHLH1 has been categorized withknown anthocyanin biosynthesis bHLHs from other species, e.g.Arabidopsis siliques TT8 (Nesi et al., 2000), Asiatic hybrid lilyLhbHLH2 (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 thatboth were involved in producing the different anthocyanin color-ations of Dendrobium hybrid petals. This suggests that both MYB

Fig. 6. The estimated content of total anthocyanin and expression profiles of transcriptional factor genes and anthocyanin biosynthetic genes in different floral tissues of Den-drobium hybrids. (A) Three Dendrobium hybrids with distinct lip pigmentation. WRT, White red tip; BC, Burana charming; T � P, Thongchai � Pinwattana. (B) The estimated contentof 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 atdevelopmental 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'smultiple 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.)

C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345 343

and bHLH transcription factors are the limiting regulators ofanthocyanin accumulation in Dendrobium hybrids petals. This isconsistent with what has been previously observed for GtMYB3 andGtbHLH1 in gentian (Nakatsuka et al., 2008), LhMYB12 and LhbHLH2in 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 pathwayimplied that DhMYB2 and DhbHLH1 might regulate the expressionof anthocyanin biosynthetic genes in Dendrobium hybrid petals.Furthermore, transient expression of DhMYB2 or DhbHLH1 aloneinduced purple spots in the white petals of Dendrobium hybrid BC,whereas the GUS over-expression negative control did not have anypigmentation. This suggested that both DhMYB2 and DhbHLH1could induce the anthocyanin biosynthesis in the petals of Den-drobium hybrids which was in contrast to PeMYB2 and PebHLH1 in

Phalaenopsis spp. When PeMYB2 was over-expressed in the whitesepals/petals of P. aphrodite ssp. formosana, a red pigmentationwasproduced, whereas PebHLH1 over-expression did not result in anyred color (Hsu et al., 2015). The expression data together with thetransient expression results suggested that DhMYB2 and DhbHLH1acted as anthocyanin activators in Dendrobium hybrid petals.

The combination of interactions among R2R3-MYB and bHLHconservatively control anthocyanin biosynthesis in many plants,such as, PhAN2 (R2R3-MYB) and PhAN1 (bHLH) in petunia (Speltet al., 2000), ROSEA1/ROSEA2 (R2R3-MYB) and DELILA (bHLH) inAntirrhinum spp. (Schwinn et al., 2006), and GMYB10 and GMYC1(bHLH) in Gerbera hybrids (Elomaa et al., 2003). The proteinepro-tein interactions rely on aMYB protein family conserved amino acidsignature ([D/E]Lx2[R/K]x3Lx6Lx3R). In Orchidaceae, PeMYB2 fromP. equestris, and PsUMYB6 from P. schilleriana all harbor the motiffor interacting with bHLHs. Transient over-expression of PeMYB2

C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345344

together with PebHLH1 produces a more intense pigmentation thanover-expression of PeMYB2 alone (Hsu et al., 2015). However,PsUMYB6 needs ZmLc, a bHLH transcription factor from Z. mays toinduce anthocyanin pigmentation after particle bombardment (Maet al., 2009). These results suggested that there is an interactionbetween MYBs and bHLHs in the transcriptional regulation ofanthocyanin biosynthesis in orchids. However, to date, there hasnot been any direct physical interaction evidence for MYBs andbHLHs in Orchidaceae. In this study, the structure analysis showedthat DhMYB2 has the motif for interacting with bHLHs, and thatDhbHLH1 has the MYB interaction region. The proteineproteininteraction between the two TFs only exists theoretically, and yeasttwo-hybrid analysis showed that DhMYB2 interacts with DhbHLH1in vitro. This provides further evidence for that DhMYB2 is involvedin anthocyanin biosynthesis in the petals of Dendrobium hybridsthrough its interaction with DhbHLH1.

The R2R3-MYBs and bHLHs regulation of anthocyanin biosyn-thesis is ubiquitous in plants. However, the regulatory system isdifferent among species. In Arabidopsis, DFR, ANS, and UFGT areactivated by a multiple TF complex, which is mainly formed byAaMYB75 and AtMYB90 interacting with EGL3, GL3, and TT8,whereas in maize, almost all of the genes in the anthocyaninbiosynthesis pathway are activated as a single unit by the C1/Pl1(MYB) and R1/B1 (bHLH) complex (Petroni and Tonelli, 2011). Inother species, such as Chinese bayberry, the MrMYB1eMrbHLH1complex acts as activators that induce the MrCHI, MrF30H, MrDFR1,MrANS and MrUFGT promoters (Liu et al., 2013). Co-expression ofchrysanthemum CmMYB6 and CmbHLH2 activate the promoteractivity of CmDFR (Xiang et al., 2015). The LcMYB1-LcbHLH complexenhances anthocyanin accumulation by activating the transcriptionof ANS and DFR in litchi (Lai et al., 2016). In the Orchidaceae,transient over-expression of both PeMYB2 and PebHLH1 or PeMYB2alone led to the increased expression of PeF3H5, PeDFR1, andPeANS3 in P. aphrodite ssp. formosana flowers (Hsu et al., 2015), andOgMYB1 directly activated the expression of OgCHI and OgDFR inthe lips of Oncidium Gower Ramsey after particle bombardment(Chiou and Yeh, 2008). In Dendrobium hybrids, CHS, CHI1, CHI2, F3H,DFR, ANS, F3050H, and FLS were cloned and their expression profilesin purple, peach, white and greenish white flowers were investi-gated. The results suggested that CHS, CHI, F3H, DFR, and ANS weregenerally expressed in purple and peach flowers, whereas F3H, DFR,and ANSwere hardly expressed in the greenish white flowers of the‘Jasmine white’ hybrid. The results indicated that F3H, DFR, and ANScould be coordinately controlled by TFs in the ‘Jasmine white’hybrid (Kriangphan et al., 2015). However, the regulation mecha-nism controlling anthocyanin production in Dendrobium hybrids isstill unclear. In this study, gene co-expression analysis was used toinvestigate the regulatory roles of DhMYB2 and DhbHLH1 in theanthocyanin biosynthetic pathway of Dendrobium hybrids. In thehybrids investigated in this study, during the development ofpetals, strong positive correlations (Pearson correlation coefficient,r) between DhMYB2 and DhbHLH1 expressions and key anthocy-anin biosynthetic genes, particularly DhF3H, DhDFR, DhANS, andDhGT3, were observed (Table A.4). Furthermore, the hierarchicalclustering analysis of the genes related to anthocyanin biosynthesissuggested that DhMYB2 and DhbHLH1 had a close clustering rela-tionship with DhDFR and DhANS, but a very poor relationship withDhF30H, DhF3050H, and DhGT3 (Fig. 5). The results indicated that theexpression of DhDFR and DhANS appeared to be regulated byDhMYB2 and DhbHLH1 in Dendrobium hybrid petals. The findingsfrom 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 DhF30H, DhF3050H, and DhGT3might be separately regulated by other TFs. Furthermore, somegenes in the anthocyanin biosynthesis pathway are responsible for

the common precursors of flavonoids producers, DhCHS, DhCHI2,and DhF3H, and two glycosyltransferase-like genes (DhGT1 andDhGT2) might be coordinately regulated. However, the regulationmechanism needs be further investigated.

Although a strong co-expression of DhMYB2 and DhbHLH1 wasobserved in the petals with various colors (Table A.4), the expres-sion profiles of these two TF genes in the different floral organs in asingle flower were extremely variable. In the three hybrids withpurple lips, but white or yellow sepals and petals (WRT, BC, andT � P), DhbHLH1 expression was closely related to anthocyaninaccumulation and it was highly expressed in purple lips. However,in hybrids WRT and BC, the DhMYB2 expression levels in the petalsand sepals were significantly higher than in the lips, although therewas hardly any anthocyanin produced in petals/sepals. It seemsthatDhMYB2 is not responsible anthocyanin biosynthesis in the lipsof Dendrobium hybrids, whereas DhbHLH1 plays an important rolein anthocyanin production in the lips. The way in which Den-drobium hybrid MYB TFs control pigmentation in various floralorgans may be similar to that in Phalaenopsis spp. Furthermore,there may be another MYB, which is homologous with thePeMYB12 that controls full pigmentation in the lips of Phalaenopsisspp. flowers (Hsu et al., 2015), which controls anthocyaninpigmentation in the lips of Dendrobium hybrids. The recovery ofanthocyanin production in BC petals after bombardment withDhMYB2 or DhbHLH1, together with the gene expression datasuggest that the low DhMYB2 and DhbHLH1 expression levelscaused the simultaneous down-regulation of a number of genesinvolved in anthocyanin biosynthesis and resulted in white petals/sepals. Similarly, in Phalaenopsis, the white P. amabillis petals weredue to the loss of anthocyanin-specific MYB transcripts, whichsubsequently reduced DFR expression (Ma et al., 2009).

5. Conclusion

This study isolated one R2R3-MYB gene, DhMYB2, and one bHLHgene, DhbHLH1, from the Dendrobium hybrid floral RNA-seq data-base. Transient expression analysis of DhMYB2 and DhbHLH1revealed that the two TF genes were able to induce anthocyaninproduction in the white petals of Dendrobium hybrid BC. Further-more, the two genes were closely co-expressed with DhDFR andDhANS, and this correlated with anthocyanin accumulation inmulti-pigmented petals. The proteineprotein interaction betweenDhMYB2 and DhbHLH1 was confirmed by yeast two-hybrid anal-ysis. These results suggested that DhMYB2 and DhbHLH1 interactand play a role in regulating anthocyanin production in Dendrobiumhybrid petals. Furthermore, the results suggested that DhbHLH1 isalso responsible for anthocyanin biosynthesis in lips. This studyprovides valuable information on anthocyanin biosynthesis regu-lation in Dendrobium hybrids.

Formatting of funding sources

This work was supported by the National Natural ScienceFoundation of China [grant numbers 31101578; 31400579]; theGrand Special Project on Science and Technology of Hainan Prov-ince [grant number ZDZX2013012]; the National Nonprofit InstituteResearch Grant of CATAS-TCGRI [grant number 1630032015017].

Author contributions

LCH, QJ, YGS and YJM conceived the study; LCH and DL per-formed the experiments. LCH and QJ analyzed the data and wrotethe manuscript; HMZ and HSR participated in the performance ofthe experiments. All authors have read and approved the finalmanuscript.

C. Li et al. / Plant Physiology and Biochemistry 112 (2017) 335e345 345

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

We sincerely thank Prof. Jian Wang from Hainan University fortechnical assistance in particle bombardment assay.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2017.01.019.

References

Albert, N.W., Lewis, D.H., Zhang, H., Schwinn, K.E., Jameson, P.E., Davies, K.M., 2011.Members of an R2R3-MYB transcription factor family in Petunia are develop-mentally and environmentally regulated to control complex floral and vege-tative pigmentation patterning. Plant J. 65, 771e784.

Bai, Y., Pattanaik, S., Patra, B., Werkman, J.R., Xie, C.H., Yuan, L., 2011. Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco haveoriginated from two ancestors and are functionally active. Planta 234, 363e375.

Carre~no, J., Martinez, A., Almela, L., Fern�andez-L�opez, J.A., 1995. Proposal of an indexfor the objective evaluation of the colour of red table grapes. Food Res. Int. 28,373e377.

Chen, S., Songkumarn, P., Liu, J., Wang, G.L., 2009. A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiol. 150,1111e1121.

Chiou, C.Y., Yeh, K.W., 2008. Differential expression of MYB gene (OgMYB1) de-termines color patterning in floral tissue of Oncidium Gower Ramsey. Plant Mol.Biol. 66, 379e388.

da Silva, J.A.T., Jin, X., Dobr�anszki, J., Lu, J., Wang, H., Zotz, G., Cardoso, J.C., Zeng, S.,2016. Advances in Dendrobium molecular research: applications in geneticvariation, identification and breeding. Mol. Phylogenet. Evol. 95, 196e216.

Deng, W., Wang, Y., Liu, Z., Cheng, H., Xue, Y., 2014. HemI: a toolkit for illustratingheatmaps. PLoS One 9, e111988.

Dooner, H.K., Robbins, T.P., Jorgensen, R.A., 1991. Genetic and developmental controlof anthocyanin biosynthesis. Annu. Rev. Genet. 25, 173e199.

Elomaa, P., Uimari, A., Mehto, M., Albert, V.A., Laitinen, R.A.E., Teeri, T.H., 2003.Activation of anthocyanin biosynthesis in Gerbera hybrida (Asteraceae) suggestsconserved protein-protein and protein-promoter interactions between theanciently diverged monocots and edicots. Plant Physiol. 133, 1831e1842.

Feller, A., Machemer, K., Braun, E.L., Grotewold, E., 2011. Evolutionary andcomparative analysis of MYB and bHLH plant transcription factors. Plant J. 66,94e116.

Hsu, C.C., Chen, Y.Y., Tsai, W.C., Chen, W.H., Chen, H.H., 2015. Three R2R3-MYBtranscription factors regulate distinct floral pigmentation patterning in Phal-aenopsis spp. Plant Physiol. 168, 175e191.

Kriangphan, N., Vuttipongchaikij, S., Kittiwongwattana, C., Suttangkakul, A.,Pinmanee, P., Sakulsathaporn, A., Suwimon, R., Suputtitada, S.,Chanvivattana, Y., Apisitwanich, S., 2015. Effects of sequence and expression ofeight anthocyanin biosynthesis genes on floral coloration in four DendrobiumHybrids. Hortic. J. 84, 83e92.

Kuehnle, A.R., Lewis, D.H., Markham, K.R., Mitchell, K.A., Davies, K.M., Jordan, B.R.,1997. Floral flavonoids and pH in Dendrobium orchid species and hybrids.Euphytica 95, 187e194.

Lai, B., Du, L.N., Liu, R., Hu, B., Su, W.B., Qin, Y.H., Zhao, J.T., Wang, H.C., Hu, G.B., 2016.Two LcbHLH transcription factors interacting with LcMYB1 in regulating latestructural genes of anthocyanin biosynthesis in Nicotiana and Litchi chinensisduring anthocyanin accumulation. Front. Plant Sci. 7.

Lau, S.E., Schwarzacher, T., Othman, R.Y., Harikrishna, J.A., 2015. dsRNA silencing of

an R2R3-MYB transcription factor affects flower cell shape in a Dendrobiumhybrid. BMC Plant Biol. 15, 194.

Li, C., Qiu, J., Yang, G., Huang, S., Yin, J., 2016. Isolation and characterization of aR2R3-MYB transcription factor gene related to anthocyanin biosynthesis in thespathes of Anthurium andraeanum (Hort.). Plant Cell Rep. 35, 2151e2165.

Liu, X.F., Yin, X.R., Allan, A.C., Lin-Wang, K., Shi, Y.N., Huang, Y.J., Ferguson, I.B.,Xu, C.J., Chen, K.S., 2013. The role of MrbHLH1 and MrMYB1 in regulatinganthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra)during anthocyanin biosynthesis. Plant Cell Tiss. Org. 115, 285e298.

Ma, H., Pooler, M., Griesbach, R., 2009. Anthocyanin regulatory/structural geneexpression in Phalaenopsis. J. Am. Soc. Hortic. Sci. 134, 88e96.

Mudalige-Jayawickrama, R. G., 2014. Methods for expanding color palette in Den-drobium orchids. United State Patent No. 0201865 A1.

Mudalige-Jayawickrama, R.G., Champagne, M.M., Hieber, A.D., Kuehnle, A.R., 2005.Cloning and characterization of two anthocyanin biosynthetic genes fromDendrobium orchid. J. Am. Soc. Hortic. Sci. 130, 611e618.

Nakatsuka, A., Yamagishi, M., Nakano, M., Tasakia, K., Kobayashi, N., 2009. Light-induced expression of basic helixeloopehelix genes involved in anthocyaninbiosynthesis in flowers and leaves of Asiatic hybrid lily. Sci. Hortic. 121, 84e91.

Nakatsuka, T., Haruta, K.S., Pitaksutheepong, C., Abe, Y., Kakizaki, Y., Yamamoto, K.,Shimada, N., Yamamura, S., Nishihara, M., 2008. Identification and character-ization of R2R3-MYB and bHLH transcription factors regulating anthocyaninbiosynthesis in gentian flowers. Plant Cell Physiol. 49, 1818e1829.

Nesi, N., Debeaujon, I., Jond, C., Pelletier, G., Caboche, M., Lepiniec, L., 2000. The TT8gene encodes a basic Helix-Loop-Helix domain protein required for expressionof DFR and BAN genes in Arabidopsis siliques. Plant Cell 12, 1863e1878.

Paz-Ares, J., Ghosal, D., Wienand, U., Peterson, P.A., Saedler, H., 1987. The regulatoryc1 locus of Zea mays encodes a protein with homology to myb proto-oncogeneproducts and with structural similarities to transcriptional activators. EMBO J. 6,3553.

Petroni, K., Tonelli, C., 2011. Recent advances on the regulation of anthocyaninsynthesis in reproductive organs. Plant Sci. 181, 219e229.

Piluk, P., Ratanasut, K., 2012. Expression profiles of the dihydroflavonol 4-reductase(dfr) gene in the sepals and petals of Dendrobium Sonia earsakul. In: 1st MaeFah Luang University International Conference, pp. 1e8.

Pitakdantham, W., Sutabutra, T., Chiemsombat, P., Pitaksutheepong, C., 2011. Isola-tion and characterization of dihydroflavonol 4-reductase gene in Dendrobiumflowers. J. Plant Sci. 6, 88e94.

Quattrocchio, F., Wing, J., van der Woude, K., Souer, E., de Vetten, N., Mol, J., Koes, R.,1999. Molecular analysis of the anthocyanin2 gene of petunia and its role in theevolution of flower color. Plant Cell 11, 1433e1444.

Schwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E., Oyama, R., Bailey, P.,Davies, K., Martin, C., 2006. A small family of MYB-regulatory genes controlsfloral pigmentation intensity and patterning in the genus Antirrhinum. PlantCell 18, 831e851.

Spelt, C., Quattrocchio, F., Mol, J.N., Koes, R., 2000. Anthocyanin1 of petunia encodesa basic helixeloopehelix protein that directly activates transcription of struc-tural anthocyanin genes. Plant Cell 12, 1619e1632.

Whang, S.S., Um, W.S., Song, I.J., Lim, P.O., Choi, K., Park, K.W., Kang, K.W., Choi, M.S.,Koo, J.C., 2011. Molecular analysis of anthocyanin biosynthetic genes and con-trol of flower coloration by flavonoid 30 ,50-hydroxylase (F3050H) in Dendrobiummoniliforme. J. Plant Biol. 54, 209e218.

Wu, X.M., Lim, S.H., Yang, W.C., 2003. Characterization, expression and phylogeneticstudy of R2R3-MYB genes in orchid. Plant Mol. Biol. 51, 959e972.

Xiang, L.L., Liu, X.F., Li, X., Yin, X.R., Grierson, D., Li, F., Chen, K.S., 2015. A novel bHLHtranscription factor involved in regulating anthocyanin biosynthesis in chry-santhemums (Chrysanthemum morifolium Ramat.). PloS one 10, e0143892.

Xu, W., Dubos, C., Lepiniec, L., 2015. Transcriptional control of flavonoid biosyn-thesis by MYBebHLHeWDR complexes. Trends Plant Sci. 20, 176e185.

Yamagishi, M., Shimoyamada, Y., Nakatsuka, T., Masuda, K., 2010. Two R2R3-MYBgenes, homologs of petunia AN2, regulate anthocyanin biosyntheses in flowertepals, tepal spots and leaves of Asiatic hybrid lily. Plant Cell Physiol. 51,463e474.

Yuan, Y.W., Sagawa, J.M., Frost, L., Vela, J.P., Bradshaw, H.D., 2014. Transcriptionalcontrol of floral anthocyanin pigmentation in monkeyflowers (Mimulus). NewPhytol. 204, 1013e1027.

http://dx.doi.org/10.1016/j.plaphy.2017.01.019http://dx.doi.org/10.1016/j.plaphy.2017.01.019http://refhub.elsevier.com/S0981-9428(17)30031-1/sref1http://refhub.elsevier.com/S0981-9428(17)30031-1/sref1http://refhub.elsevier.com/S0981-9428(17)30031-1/sref1http://refhub.elsevier.com/S0981-9428(17)30031-1/sref1http://refhub.elsevier.com/S0981-9428(17)30031-1/sref1http://refhub.elsevier.com/S0981-9428(17)30031-1/sref2http://refhub.elsevier.com/S0981-9428(17)30031-1/sref2http://refhub.elsevier.com/S0981-9428(17)30031-1/sref2http://refhub.elsevier.com/S0981-9428(17)30031-1/sref2http://refhub.elsevier.com/S0981-9428(17)30031-1/sref3http://refhub.elsevier.com/S0981-9428(17)30031-1/sref3http://refhub.elsevier.com/S0981-9428(17)30031-1/sref3http://refhub.elsevier.com/S0981-9428(17)30031-1/sref3http://refhub.elsevier.com/S0981-9428(17)30031-1/sref3http://refhub.elsevier.com/S0981-9428(17)30031-1/sref3http://refhub.elsevier.com/S0981-9428(17)30031-1/sref3http://refhub.elsevier.com/S0981-9428(17)30031-1/sref4http://refhub.elsevier.com/S0981-9428(17)30031-1/sref4http://refhub.elsevier.com/S0981-9428(17)30031-1/sref4http://refhub.elsevier.com/S0981-9428(17)30031-1/sref4http://refhub.elsevier.com/S0981-9428(17)30031-1/sref5http://refhub.elsevier.com/S0981-9428(17)30031-1/sref5http://refhub.elsevier.com/S0981-9428(17)30031-1/sref5http://refhub.elsevier.com/S0981-9428(17)30031-1/sref5http://refhub.elsevier.com/S0981-9428(17)30031-1/sref6http://refhub.elsevier.com/S0981-9428(17)30031-1/sref6http://refhub.elsevier.com/S0981-9428(17)30031-1/sref6http://refhub.elsevier.com/S0981-9428(17)30031-1/sref6http://refhub.elsevier.com/S0981-9428(17)30031-1/sref6http://refhub.elsevier.com/S0981-9428(17)30031-1/sref7http://refhub.elsevier.com/S0981-9428(17)30031-1/sref7http://refhub.elsevier.com/S0981-9428(17)30031-1/sref8http://refhub.elsevier.com/S0981-9428(17)30031-1/sref8http://refhub.elsevier.com/S0981-9428(17)30031-1/sref8http://refhub.elsevier.com/S0981-9428(17)30031-1/sref9http://refhub.elsevier.com/S0981-9428(17)30031-1/sref9http://refhub.elsevier.com/S0981-9428(17)30031-1/sref9http://refhub.elsevier.com/S0981-9428(17)30031-1/sref9http://refhub.elsevier.com/S0981-9428(17)30031-1/sref9http://refhub.elsevier.com/S0981-9428(17)30031-1/sref10http://refhub.elsevier.com/S0981-9428(17)30031-1/sref10http://refhub.elsevier.com/S0981-9428(17)30031-1/sref10http://refhub.elsevier.com/S0981-9428(17)30031-1/sref10http://refhub.elsevier.com/S0981-9428(17)30031-1/sref11http://refhub.elsevier.com/S0981-9428(17)30031-1/sref11http://refhub.elsevier.com/S0981-9428(17)30031-1/sref11http://refhub.elsevier.com/S0981-9428(17)30031-1/sref11http://refhub.elsevier.com/S0981-9428(17)30031-1/sref12http://refhub.elsevier.com/S0981-9428(17)30031-1/sref12http://refhub.elsevier.com/S0981-9428(17)30031-1/sref12http://refhub.elsevier.com/S0981-9428(17)30031-1/sref12http://refhub.elsevier.com/S0981-9428(17)30031-1/sref12http://refhub.elsevier.com/S0981-9428(17)30031-1/sref12http://refhub.elsevier.com/S0981-9428(17)30031-1/sref13http://refhub.elsevier.com/S0981-9428(17)30031-1/sref13http://refhub.elsevier.com/S0981-9428(17)30031-1/sref13http://refhub.elsevier.com/S0981-9428(17)30031-1/sref13http://refhub.elsevier.com/S0981-9428(17)30031-1/sref14http://refhub.elsevier.com/S0981-9428(17)30031-1/sref14http://refhub.elsevier.com/S0981-9428(17)30031-1/sref14http://refhub.elsevier.com/S0981-9428(17)30031-1/sref14http://refhub.elsevier.com/S0981-9428(17)30031-1/sref15http://refhub.elsevier.com/S0981-9428(17)30031-1/sref15http://refhub.elsevier.com/S0981-9428(17)30031-1/sref15http://refhub.elsevier.com/S0981-9428(17)30031-1/sref16http://refhub.elsevier.com/S0981-9428(17)30031-1/sref16http://refhub.elsevier.com/S0981-9428(17)30031-1/sref16http://refhub.elsevier.com/S0981-9428(17)30031-1/sref16http://refhub.elsevier.com/S0981-9428(17)30031-1/sref17http://refhub.elsevier.com/S0981-9428(17)30031-1/sref17http://refhub.elsevier.com/S0981-9428(17)30031-1/sref17http://refhub.elsevier.com/S0981-9428(17)30031-1/sref17http://refhub.elsevier.com/S0981-9428(17)30031-1/sref17http://refhub.elsevier.com/S0981-9428(17)30031-1/sref18http://refhub.elsevier.com/S0981-9428(17)30031-1/sref18http://refhub.elsevier.com/S0981-9428(17)30031-1/sref18http://refhub.elsevier.com/S0981-9428(17)30031-1/sref20http://refhub.elsevier.com/S0981-9428(17)30031-1/sref20http://refhub.elsevier.com/S0981-9428(17)30031-1/sref20http://refhub.elsevier.com/S0981-9428(17)30031-1/sref20http://refhub.elsevier.com/S0981-9428(17)30031-1/sref21http://refhub.elsevier.com/S0981-9428(17)30031-1/sref21http://refhub.elsevier.com/S0981-9428(17)30031-1/sref21http://refhub.elsevier.com/S0981-9428(17)30031-1/sref21http://refhub.elsevier.com/S0981-9428(17)30031-1/sref21http://refhub.elsevier.com/S0981-9428(17)30031-1/sref21http://refhub.elsevier.com/S0981-9428(17)30031-1/sref22http://refhub.elsevier.com/S0981-9428(17)30031-1/sref22http://refhub.elsevier.com/S0981-9428(17)30031-1/sref22http://refhub.elsevier.com/S0981-9428(17)30031-1/sre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Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals1. Introduction2. Materials and methods2.1. Plant materials2.2. Total RNA extraction and cDNA synthesis2.3. Cloning and sequence analysis of the DhMYB2 and DhbHLH1 genes, and the anthocyanin biosynthetic genes2.4. qPCR analysis2.5. Petal color measurement and total anthocyanin analysis2.6. Over-expression vector construction and transient expression using particle bombardment2.7. Yeast two-hybrid assay

3. Results3.1. Phylogenetic relationships between the DhMYB2 and DhbHLH1 TFs isolated from Dendrobium hybrid flowers3.2. Isolation and sequence analysis of anthocyanin biosynthetic genes3.3. Expression of DhMYB2 and DhbHLH1 was related to anthocyanin coloration in petals3.4. DhMYB2 and DhbHLH1 activated anthocyanin biosynthesis in the Dendrobium hybrid ‘Burana charming’ white petals by transient ...3.5. Protein–protein interactions between DhMYB2 and DhbHLH13.6. DhMYB2 and DhbHLH1 co-expressed with DhDFR and DhANS in petals of Dendrobium hybrids3.7. DhbHLH1 was associated with the distinct pigmentation in the lip tissues of Dendrobium hybrids

4. Discussion5. ConclusionFormatting of funding sourcesAuthor contributionsConflict of interestAcknowledgmentsAppendix A. Supplementary dataReferences

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