The R2R3-MYB, bHLH, WD40, and related transcription factors in

ORIGINAL PAPER

The R2R3-MYB,bHLH,WD40,and related transcription factors in flavonoid biosynthesis

Lei Zhao &Liping Gao &Hongxue Wang &

Xiaotian Chen &Yunsheng Wang &Hua Yang &Chaoling Wei &Xiaochun Wan &Tao Xia

Received:29July 2012/Revised:24October 2012/Accepted:29October 2012/Published online:27November 2012#Springer-Verlag Berlin Heidelberg 2012

Abstract R2R3-MYB,bHLH,and WD40proteins have been shown to control multiple enzymatic steps in the bio-synthetic pathway responsible for the production of flavo-noids,important secondary metabolites in Camellia sinensis .Few related transcription factor genes have been documented.The presence of R2R3-MYB ,bHLH ,and WD40were statistically and bioinformatically analyzed on 127,094C .sinensis transcriptome unigenes,resulting in identification of 73,49,and 134genes,respectively.C .sinensis phylogenetic trees were constructed for R2R3-MYB and bHLH proteins using previous Arabidopsis data and further divided into 27subgroups (Sg)and 32subfami-lies.Motifs in some R2R3-MYB subgroups were redefined.Furthermore,Sg26and Sg27were expanded compared to Arabidopsis data,and bHLH proteins in C .sinensis were grouped into nine subfamilies.According to the functional annotation of Arabidopsis ,flavonoid biosynthesis in C .sinensis was predicted to include R2R3-MYB genes in Sg4(6),Sg5(2),and Sg7(1),as well as bHLH genes in subfamily 2(2)and subfamily 24(5).The wide evolutionary gap prevented phylogenetic analysis of WD40s;however,a single gene,CsWD40-1,was observed to share 80.4%se-quence homogeny with AtTTG1.Analysis of CsMYB4-1,

CsMYB4-2,CsMYB4-3,CsMYB4-4,CsMYB5-1,and CsMYB5-2revealed the interaction motif [DE]Lx2[RK]x3Lx6Lx3R,potentially contributing to the specificity of the bHLH partner in the stable MYB –bHLH complex.Full-length end-to-end polymerase chain reaction (PCR)and quantitative reverse transcriptase (qRT)-PCR were used to validate select-ed genes and generate relative expression ratio profiles in C .sinensis leaves by developmental stage and treatment condi-tions,including hormone and wound treatments.Potential target binding sites were predicted.

Keywords Camellia sinensis .MYB .bHLH .

Bioinformatic analysis .Flavonoid biosynthetic pathway

Introduction

Flavonoids are important secondary metabolites that accu-mulate in most vascular plants (Hichri et al.2011).The involvement of these compounds in a wide range of vital functions in plants,including seed germination (Debeaujon et al.2003),UV-B protection (Albert et al.2009),and pathogen and biotic stress defenses (Punyasiri et al.2004),is well documented.In addition to their numerous physio-logical roles,flavonoids also exhibit beneficial bioactive properties in humans,contributing to prevention of disease and oxidative stress (Khan et al.2010;Ross and Kasum 2002).

Chalcones,flavones,flavonols,flavandiols,anthocya-nins,condensed tannins,and/or proanthocyanidins (PAs)are the primary subgroups of flavonoids,though other spe-cialized flavonoids have also been identified (Winkel-Shirley 2001).Overall,catechins account for approximately 70–80%of all identified flavonoids.Catechins,the most abundant flavonoid in Camellia sinensis ,are flavan-3-ol monomers that form the initiation and elongation unit of

Electronic supplementary material The online version of this article (doi:10.1007/s10142-012-0301-4)contains supplementary material,which is available to authorized users.

L.Zhao :H.Wang :H.Yang :C.Wei :X.Wan :T.Xia (*)Key Laboratory of Tea Biochemistry &Biotechnology,Ministry of Agriculture &Ministry of Education,Anhui Agricultural University,Hefei,Anhui 230036,China

e-mail:xiatao62@https://www.360docs.net/doc/993954669.html,

L.Gao (*):X.Chen :Y .Wang

College of Life Science,Anhui Agricultural University,Hefei,Anhui 230036,China e-mail:gaolp62@https://www.360docs.net/doc/993954669.html,

Funct Integr Genomics (2013)13:75–98DOI 10.1007/s10142-012-0301-4

PAs.These compounds possess a basic2-phenylchromone structure composed of three phenolic rings(A,B,and C rings)characterized by di-or tri-hydroxyl group substitu-tions of the B ring,2,3-position isomerisms of the C ring, and a3-postion galloyl group of the C ring.Catechins share the same PA biosynthetic pathways found in the model plant Arabidopsis thaliana(Arabidopsis)and certain grapevine species.Functional genes involved in the biosynthetic path-ways of PAs,anthocyanins,and flavonols have been exten-sively studied and documented(Gutha et al.2010;Petroni and Tonelli2011).

The flavonoid biosynthesis pathway is highly organized. Key structural genes and numerous transcription factors act as regulatory protein modulators of gene expression through sequence-specific DNA binding at the transcriptional level or by protein–protein interactions during chromatin remod-eling(Hichri et al.2011).To date,at least six distinct transcription factors(MYB,bHLH,WD40,WRKY,Zinc finger,and MADS box proteins)have been identified in flavonoid biosynthesis(Terrier et al.2009).Transcription factors like R2R3-MYB,bHLH,and WD40can work indi-vidually or orchestrate with others in controlling the multi-ple enzymatic steps involved in the flavonoid biosynthetic pathways of various species(Mano et al.2007).Early biosynthesis genes(EBGs),for example,are mainly activat-ed by independent R2R3-MYBs,leading to the production of flavonols.Conversely,the activation of late biosynthesis genes(LBGs)leading to the production of PAs generally requires a ternary complex of transcription factors(MYB–bHLH–WD40,MBW),as demonstrated in Arabidopsis. Notably,MYB commonly plays a central role in distinguish-ing the target gene in these pathways(Nesi et al.2001; Baudry et al.2004).

MYB proteins,a large and functionally diverse super-family of regulatory transcription factors,are involved in plant developmental and defense processes(Xie et al.2006). MYBs are classified into three major groups according to the conserved N-terminal MYB domain,consisting of one to three imperfect repeat sequences(R1,R2,and R3;Martin and Paz-Ares1997).R2R3-MYBs,a type of MYB protein, participate in the regulation of secondary metabolism in plants(V om Endt et al.2002),contributing to positive and negative regulation of biosynthetic enzymes required for the production of phenylpropanoids(Bomal et al.2008),flavo-noids(Taylor and Grotewold2005),and benzenoids (Verdonk et al.2005).In Arabidopsis,126R2R3-MYBs have been identified and further classified into22subgroups according to the presence of conserved C-terminal amino acid motifs in the region outside the MYB domain(except for Sg2;Kranz et al.1998).In a recently extended dataset, Stracke et al.(2001)re-evaluated these motifs,confirming most of the previously identified subgroups and extending the analysis to include three new subgroups.This data further revealed that most MYB genes sharing similar func-tions cluster in the same phylogenetic clades in this species, a trend likely to be observed in other species(Matus et al. 2008).

Another widespread transcription factor is the basic helix-loop-helix(bHLH)proteins.These bHLH proteins occur in a range of organisms,from simple yeasts to com-plex multicellular organisms like humans,and are recog-nized for their role in regulation of cell proliferation and differentiation pathways in numerous species(Massari and Murre2000).In plants,bHLH transcription factors have broad functions such as regulation of floral organ develop-ment(Heisler et al.2001;Sorensen et al.2003),photomor-phogenesis(Leivar et al.2008),epidermal cell differentiation (Morohashi et al.2007;Zhang et al.2003;Bou-Torrent et al. 2008),hormonal responses(Bou-Torrent et al.2008;Li et al. 2007),and flavonoids biosynthesis(Ohno et al.2011).In Arabidopsis,162bHLH proteins have been identified,and 133bHLHs of them were initially classified into12subgroups (I–XII)(Heim et al.2003).Additional studies have since isolated638distinct bHLH gene families in Arabidopsis, poplar,rice,moss,and algae,resulting in the extension of the original phylogenetic analysis to include32subfamilies (Carretero-Paulet et al.2010).

WD40proteins have been increasingly recognized as key regulators in many eukaryotic cellular processes,including cell division,vesicle formation and trafficking,signal trans-duction,and RNA processing(Van Nocker and Ludwig 2003).These proteins are generally characterized by a peptide motif of44–60amino acids,typically delimited by the GH dipeptide(Gly-His)at the N-terminus and the WD dipeptide (Trp-Asp)at the C-terminus(Van Nocker and Ludwig2003). Notably,WD40s participate in chromatin remodeling through histone protein modification,thus exerting influence on tran-scription processes(Suganuma et al.2008).

The interaction between MYB and bHLH proteins has been extensively investigated in recent years,and flavonoid biosynthesis is probably the best-studied ex-ample.WD40proteins are not thought to have any catalytic activity,but rather seem to be a docking plat-form in regulating the anthocyanin and PA biosynthetic pathways.For instance,in Arabidopsis,TTG1acts mainly through a bHLH partner in the TT2-TT8-TTG1 regulation of ban expression.Investigation of the inter-action between MYB and bHLH proteins has been prominent in recent decades,especially prolific in the flavonoid biosynthetic pathway(Feller et al.2011). Though WD40proteins are not thought to exhibit cata-lytic activity,it has been hypothesized that these pro-teins act as docking platforms involved in the regulation of both anthocyanin and PA biosynthetic pathways.In Arabidopsis,TTG1acts primarily through a bHLH part-ner in the TT2-TT8-TTG1regulation of ban expression

(Nesi et al.2001;Baudry et al.2004).In other species, the interactions between MYB and bHLH proteins are less well known.

Some cDNA libraries for C.sinensis have been char-acterized using conventional Sanger sequencing methods, including the EST sequences from the young root cDNA library(Shi and Wan2009),the subtractive cDNA li-brary special for young leaves(Park et al.2004),the young leaf cDNA library(Chen et al.2005),the light-induced suppression subtractive hybridization(SSH) cDNA libraries of Calli tea(Wang et al.2012),and the drought-stressed root SSH cDNA https://www.360docs.net/doc/993954669.html,pared with the model plant,few C.sinensis transcription factor genes have been documented in the National Center for Biotechnology Information(NCBI).The C.sinensis tran-scriptome dataset obtained by high-throughput Illumina for large-scale RNA sequencing helps us to fill this deficiency in C.sinensis transcription factor gene docu-mentation.The C.sinensis transcriptome represents ap-proximately tenfold more genes than all C.sinensis genes deposited in GenBank,and approximately20-fold times more genes than those recorded in the existing C. sinensis cDNA libraries(Shi et al.2011).

The current study investigated all R2R3-MYB,bHLH,and WD40genes from a total of127,094unigenes of C.sinensis transcriptome by confirming their conserved domains.The transcription factor genes of C.sinensis were divided into subgroups by phylogenetic analysis and amino acids align-ment,allowing classification and further study of candidate genes in the flavonoid biosynthetic pathway.Additional end-to-end polymerase chain reaction(PCR)and real-time quan-titative reverse transcriptase(qRT)-PCR were used to validate selected genes and to analyze developmental and organ-specific expression.Relative expression ratios of candidate genes were profiled by treatment condition,including hor-mone and wound treatments,resulting in data directly pertain-ing to the involvement of transcription factors in the flavonoid synthesis pathway of C.sinensis.

Materials and methods

Homologues search and phylogenetic tree construction

R2R3-MYB,bHLH,and WD40genes were isolated from a total of127,094C.sinensis unigenes and translated using the NCBI ORF finder(Center for Biotechnology Information, USA).The conserved domain of each predicted protein was identified by the search tool ScanProsite(Expasy;SIB Swiss Institute of Bioinformatics,Switzerland).Sequences of all126 A.thaliana R2R3-MYB proteins were retrieved from The Arabidopsis Information Resource(TAIR)Arabidopsis Genome Annotation version7.0released in April2007(Carnegie Institution for Science Department of Plant Biology,USA).Additionally,151AtbHLH sequences were retrieved from the UniProt Database(UniProt,Switzerland).

Sequence alignments were performed using the ClustalW algorithm-based AlignX mode in Molecular Evolutionary Genetics Analysis version5(MEGA5; MegaSoftware,USA)(Tamura et al.2011).A phyloge-netic tree was subsequently constructed according to the neighbor-joining(NJ)statistical method(Saitou and Nei 1987).Tree nodes were evaluated by the Bootstrap method for1,000replicates(Felsenstein1985),and branches corresponding to partitions reproduced in less than50%of bootstrap replicates were condensed into single branches.Evolutionary distances were computed using the p-distance method and expressed in units of amino acid differences per site.All positions containing gaps and missing data were eliminated prior to con-struction of the phylogenetic trees for R2R3-MYB and WD40genes.Similarly,all ambiguous positions for each sequence pair were removed prior to construction of phylogenetic trees for bHLH genes.Amino acids of R2R3-MYBs,bHLHs,and WD40s in C.sinensis and other species were aligned by ClustalX.The conserved motifs of each R2R3-MYB subgroup were predicted by MEME(Bailey et al.2009).

Plant materials and RNA isolation

C.sinensis(L.)O.Kuntze cv.“Shuchazao”was obtained from the experimental tea garden of Anhui Agricultural University in Anhui,China(latitude31.86N,longitude 117.27E,altitude20m above mean sea level).Specimens were selected based on the visible maturity of leaves;buds; first,second,third,and fourth leaves;young stems;and tender roots.Selected leaves and organs were subjected to real-time qRT-PCR analysis and full rapid amplification of cDNA ends PCR(RACE-PCR).The qRT-PCR specimens were subjected to variant treatments.The third leaves were evenly covered with hormone-treated100μmol/L abscisic acid(ABA)solution,100μmol/L gibberellic acid(GA) solution,or distilled water(control)applied with a fine-bristled brush.Six damage regions were created on the same leaf using a hemostat(wound treatment groups),and leaves were smudged with the hormone treatment every12h. Untreated leaves were used as a control for wound treat-ment.Treated leaves and control leaves were sampled at1 and48h,respectively.All samples were immediately frozen in liquid nitrogen and stored at?80°C prior to experimental analysis.Total RNA was isolated from C.sinensis organs with RNAiso-mate for Plant Tissue(Takara,DaLian,China; Code:D325A)and RNAiso Plus(Takara,DaLian,China; Code:D9108B),according to the instructions provided by the manufacturer.

RACE-PCR analysis

The cDNA strands at the3′and5′ends were subjected to RACE-PCR and synthesized with SMARTer?RACE cDNA Amplification Kit(Clontech,USA;Cat.Nos.634923and 634924),according to the instructions provided by the manu-facturer.The five candidate genes identified from the C.sinen-sis transcriptome and CsMYB4-5from an EST sequence (CsSSHR54)in the NCBI database were subjected to standard RACE-PCR reactions.The3′-and5′-RACE primers for CsMYB4-5,CsMYB4-6(tie_GLEAN_10017628),CsMYB5-2 (tie_GLEAN_10028252),CsbHLH2-1(cam_GLEAN_ 10012690),CsbHLH2-2(tie_GLEAN_10005499),and CsWD40-1(tie_GLEAN_10026686)were designed using Primer Premier5.0software(Premier,Vancouver,BC, Canada)and synthesized(Invitrogen,Shanghai,China; Table1).PCR products were gel purified using the MiniBEST Agarose Gel Extraction Kit(Takara,DaLian, China),ligated into a pMD18-T vector,and transformed into strain DH5αcompetent cells for sequencing analysis.

The results were assembled using Dnaman7software (Lynnon,Canada).End-to-end PCR was additionally per-formed to validate the assembled full-length and ORF of candidate genes using the primers listed in Table1.The cDNA strands for end-to-end PCR were synthesized with Phusion?High-Fidelity DNA Polymerase(NEB,USA). Briefly,end-to-end PCR was performed under the following conditions:98°C for30s,30cycles at98°C for30s,57°C for10s,72°C for40s,and a final extension at72°C for 10min.

Expression difference by qRT-PCR

RNA and cDNA were prepared as previously described.All primers were blasted against the NCBI database to guaran-tee specificity(Table1).The cDNA strands for qRT-PCR were synthesized with PrimeScript?RT reagent Kit(Takara, DaLian,China;Code:DRR037A).The PCR mixture contained cDNA template(approximately0.01μg/μL), 10μL SYBR Green PCR Master Mix(Takara),and 200nmolL?1of each gene-specific primer in a final volume of20μL.Real-time PCRs were performed using a CFX96?optical reaction module(Bio-Rad,USA).The PCRs were performed with the following program:95°C for30s,followed by40cycles at95°C for5s,and60°C for30s(58°C for30s for root)in96-well optical reaction plates.The specificity of amplicons was verified by melting curve analysis(55–95°C).Gene expression was examined in various experimental groups,including those of different organs,growth stages(fourth leaf),and wound treatments, as well as the control groups.The control check(CK)was used to calibrate experiments.The resultant data was expressed as the mean value of four replicates,and these values were normalized against expression levels of the housekeeping gene glyceraldehyde-3-phosphate dehydroge-nase(GAPDH).The relative expression values were calcu-lated by the2àΔΔC t method,as follows:

ΔC

T?C T;targetàC T;GAPDHàΔΔC T

?àΔC T;targetàΔC T;4th leaf

àá

where C T,target and C T,GAPDH represent the threshold cycles of target and housekeeping(GAPDH)genes, respectively.

Results

R2R3-MYB,bHLH,and WD40in the C.sinensis transcriptome

Annotation of the C.sinensis unigenes revealed77MYB genes in Anhui No.1and63MYB genes in Tieguanyin. Further analysis revealed24bHLH genes in Anhui No.1 and28bHLH genes in Tieguanyin.A total of93WD40 genes were revealed in Anhui No.1,and102WD40genes were revealed in Tieguanyin.Translation and conserved domain scanning revealed45R2R3-MYBs in Anhui No.1 and28MYB genes in Tieguanyin,23bHLH genes in Anhui No.1and26bHLH genes in Tieguanyin,and70WD40 genes in Anhui No.1and64WD40genes in Tieguanyin. Each of these genes was verified in the C.sinensis tran-scriptome dataset.

Motif and subgroup classification of R2R3-MYBs

in the flavonoid biosynthetic pathway

A total of73C.sinensis R2R3-MYBs and126 Arabidopsis R2R3-MYBs were used in phylogenetic tree construction(Fig.1).C.sinensis MYBs were integrated with AtMYBs in clustered phylogenetic clades or sub-clades,divided into27MY

B subgroups overall.The resulting tree exhibited the same basic subgroups observed by Matus et al.(2008),further confirming the validity of the proposed tree.According to Matus,most same-subgroup MYBs share motifs outside of the MYB do-main.The conserved motifs predicted by MEME are listed in Table2,where the genes in each subgroup were renamed accordingly.

Consistent motifs were generally observed between Arabidopsis and C.sinensis,including Sg7(GR[T/V] SRSx[M/A]K),Sg9(AQWESARxxAExRLxRES), Sg19(PxLxFSEW),and Sg24(IQMExDPxTH).Some motifs were redefined,including the motif in Sg3 (WFKHLESELGLEExDNQQQ)that was redefined as

Table1Primers for RACE and

qRT-PCR Gene names Primer sequences(5′-3′)

Housekeeping gene

GADPH qRT-PCR-F:TTGGCATCGTTGAGGGTCT

qRT-PCR-R:CAGTGGGAACACGGAAAGC

Sg4

CsMYB4-1(cam_GLEAN_10020745)End-to-end F:ATGAGAAAACCTTGTTGTGA

End-to-end R:TCAGTTCATAGCTAAGGAC

qRT-PCR-F:ATCCACAACGATAAACCG

qRT-PCR-R:CGACATAGTCAGCCCAGA

CsMYB4-2(cam_GLEAN_10022034)End-to-end F:ATGAGGAAGCCA TGCTGTGAA

End-to-end R:TCA TCTAAAGAGAACAAGGGTG

qRT-PCR-F:TTCTTAGCAGGGACCAGA

qRT-PCR-R:AGGCGTACTCAACATTC

CsMYB4-3(cam_GLEAN_10033998)End-to-end F:ATGGGACGGTCTCCGTGCTGT

End-to-end R:TCA TTTCA TCTCCAAGCTTCTGTAA

qRT-PCR-F:AGATGAGGAAGAGGGCAGAG

qRT-PCR-R:CCATTCGAGAAACACCAC

CsMYB4-4(tie_GLEAN_10016351)End-to-end F:ATGAGGAAGCCTTGCTGTGACA

End-to-end R:TCACTTCTCTCCCTCGAGCAC

qRT-PCR-F:ATTGAACCAAACACTTCCTCG

qRT-PCR-R:GACGGAATGGCAATGGAG

CsMYB4-5(CsMYB3)3′-GSP1:TGCCAGGAAGAACAGACAATGA

3′-GSP2:CCACGAAATCTCCACACCAACC

5′-GSP1:GGTTGTAGGTGTGTGGTTGAGCGGG

5′-GSP2:GGACCATTTATTTCCAATCAAGCC

End-to-end F:CAAGTTCCATGGGCAGATC

End-to-end R:TGTTAGTAGTTGGA TTCA TTTCAGTA

qRT-PCR-F:ATAAGTCTCCCAAATCCACCTC

qRT-PCR-R:ATGATGCCCCGAAGAGC

CsMYB4-6(tie_GLEAN_10017628)3′-GSP1:CGGTCCGAGAATGCTGTCC

3′-GSP2:AGCAGCAACACTGGTTATGATTTC

5′-GSP1:AGAGACCGCCAGCAGCCTTCGCCGTG

5′-GSP2:TTCTTTGGTTCATCCTCCTTTG

End-to-end F:ATGGGAAGGTCACCTTGCTGTG

End-to-end R:GCTTCTGTAA TCCAAAACACCACTT

qRT-PCR-F:GAATACAAAACAGCGAAGAGTGC

qRT-PCR-R:CAACCCTAAGAAA TCA TAACCAGTG

Sg5

CsMYB5-1(cam_GLEAN_10020350)End-to-end F:ATGGGGAGGAGTCCATGCTGCTC

End-to-end R:TCA TGGCCAGTCCTCAGAA TCAAG

qRT-PCR-F:GCTGTCATAACTTTGAACTCCAC

qRT-PCR-R:GAATCAAGAAAGGATGCCAA

CsMYB5-2(tie_GLEAN_10028252)3′-GSP1:TGGGCAACCGATGGTCTCTTAT

3′-GSP2:ATTCCGACCCAACCTACA

5′-GSP1:GTGGGCA TTGGGTTCTGTTCCTTGTCTT

5′-GSP2:TAGCCTTCCAGCGATAAGAGAC

End-to-end F:A TGGGAAGGGCTCCTTGTTGTTCTAA

End-to-end R:TCAGA TCAACAAAGA TTCAGCAAAGG

qRT-PCR-F:GAATACAAAACAGCGAAGAGTGC

qRT-PCR-R:CAACCCTAAGAAA TCA TAACCAGTG

Sg7

CsMYB7-1(cam_GLEAN_10034384)End-to-end F:ATGGAGAAGTCTCATGGAGGGC

End-to-end R:CTACTTGGCAGATGCTATCATGC

qRT-PCR-F:CCACCATTGTGAACTTGC

qRT-PCR-R:CACCATCATTGGGGATT

LE[N/S]ELGL—D[I/P]D[F/I]W[D/S][F/M][L/I]D,the motif in Sg5(DExWRLxxT)that was redefined as IRTKA[I/L]RC,and the motif in Sg11(PRLDLLD)that was redefined as LxNPExLRLAxxL[L/F].Several other exceptions were observed,including the notable absence of C.sinensis genes in the Arabidopsis subclades Sg6, Sg12,and Sg15.Sg12(“glucosinolate”clade)is comprised of AtMYB28,AtMYB29,and AtMYB76,and it functions in regulation of aliphatic glucosinolate biosynthesis(Rosso et al.2008;Matus et al.2008).Interestingly,no rice and/

Table1(continued)

Gene names Primer sequences(5′-3′)

bHLH genes

Subfamily2

CsbHLH2-1(cam_GLEAN_10012690) (tie_GLEAN_10001220)3′-GSP1:TCCCCCGAACCCTAATC

3′-GSP2:CATTGTCTGAATCGGGGGC

5′-GSP1:CACAATCGTAAAACTGC

5′-GSP2:GTCCATTGTTGGTGGAA

End-to-end F:ATGGCTGAGATTATCTCTTCC End-to-end R:TTAAGAAATCTCCAACAATACCT qRT-PCR-F:CCTATTCAACAACGCCTCC

qRT-PCR-R:AGCGGTCATTGCTGTCTTC

CsbHLH2-2(cam_GLEAN_10006335) (tie_GLEAN_10005499)3′-GSP1:GAATCCCAATCCCACAAGGC

3′-GSP2:GCGGATGCGGAAGTGGAG

5′-GSP1:ATCAATTTTGAATTTGAG

5′-GSP2:GGAGGGAAAGATTCCGGTTC End-to-end F:ATGGCTGAGATTATCTCTTCC End-to-end R:TTAAATCTGAAACCTGCTAA qRT-PCR-F:CTCCTAGACTTTGGTGTTCC qRT-PCR-R:GCCCCTGATTCAGATGAT

Subfamily24

CsbHLH24-1(tie_GLEAN_10024436)End-to-end F:ATGCCTTTCTCAGAGTTTT

End-to-end R:TCAGCTACTGGCATCAGCTT

qRT-PCR-F:CTGTTGGAATGGGAATGAGGA

qRT-PCR-R:CGTGGGGTAGGGAAAAGTGT

CsbHLH24-2(tie_GLEAN_10022821) (cam_GLEAN_10020333)End-to-end F:ATGCTCCTCCGCTCATCAT End-to-end R:TCAGCACAGCTGTAAACTTAC qRT-PCR-F:TATGTGCCTACCTGGAGTCTTGC qRT-PCR-R:GGGTATTTGTCGGAGTTTCTTG

CsbHLH24-3(cam_GLEAN_10025059)End-to-end F:ATGGCGGATCTGTACGGA

End-to-end R:TTAAAGTAA TGTGA TTACCTTTGAA

qRT-PCR-F:AGTGCTGAATACAAACGGAGGA

qRT-PCR-R:TGGTGTTAGGAGATTTGGCTGA CsbHLH24-4(tie_GLEAN_10042126)End-to-end F:ATGAAAGGGGAAACAGACCG

End-to-end R:TTAACCCCCTTGAACATGGC

qRT-PCR-F:CAGGCACAGGCAACTTCAGA

qRT-PCR-R:GAACATGGCTTGATGGCAAA

CsbHLH24-5(cam_GLEAN_10023958) (tie_GLEAN_10006711)End-to-end F:ATGATAGTAATGGCGGATATGT End-to-end R:TCACATTGTCGAAGCGTAAGCA qRT-PCR-F:TCTCCCAGAAAACCCAAACC qRT-PCR-R:CCAGAGCTTCAAAACCCTCC

WD40

CsWD40-1(tie_GLEAN_10026686)3′-GSP1:GAGATAAGGAACACTCTAC

3′-GSP2:GGCTCCCCAGAGTCATCGCC

5′-GSP1:ATTTGCGGAGGGAGGCAGAG

5′-GSP2:GTATTCCTCTATGAAGC

End-to-end F:ATGGAGAATTCGAGCCAAG

End-to-end R:TCAAACTTTCAGAAGCTGCATT

qRT-PCR-F:CGGCAGCTTCATAGAGG

qRT-PCR-R:TGGGAGGGTAAGGGTGT

or grape genes were grouped within this clade,which may be a result of β-type duplication that evolved inde-pendently in Brassicales as an adaptive response to her-bivory (Matus et al.2008).Several genes clustered in the functional subclades still lacked the motif,including CsMYB5-2that lacks the IRTKA [I/L]RC motif but exhibits high homology to DkMYB4in Sg5.Also,the two functional clades Sg26and Sg27were expanded due to shared motifs SSS[S/T]ST and KRxxxSP [T/I]SSxSNC[S/C]S[S/A]S,respectively.

MYBs in Sg4,Sg5,Sg6,Sg7,and Sg15are reported regulators of anthocyanin and PA biosynthesis (Hichri et al.2011).There were six C .sinensis genes in Sg4,two in Sg5,and one in Sg7,though no MYB genes were found in either Sg6or Sg15.Amino acid sequence alignment,evolu-tionary analysis,and conserved motifs of C .sinensis R2R3-MYB genes and their homologous genes in Arabidopsis are shown in Fig.2.Sg4in Arabidopsis ,also called the C2repressor motif clade,has C-terminal C1and C2motifs that participate in bHLH interactions and promoter repression (Stracke et al.2001),respectively.Among the Sg4members (Fig.2b ),CsMYB4-1,CsMYB4-2,and CsMYB4-3were shown to be closely related,clustering in an independent sub-clade.CsMYB4-4and CsMYB4-6shared 60.44%and 71.88%identity with AtMYB4(AEE86955),and 79.8%and 80.54%identity with AmMYB308(P81393).CsMYB4-5showed clos-est homology to AtMYB7(AEC06531),PtMYB14(ABD60279),and PgMYB16(ACN12958).Furthermore,the amino acids of CsMYB4-1,CsMYB4-2,CsMYB4-3,and CsMYB4-4possessed the [DE]Lx2[RK]x3Lx6Lx3R motif be-ginning at positions 75or 76,indicating potential interaction with a bHLH partner to form a MYB –bHLH complex (Lin-Wang et al.2010;Zimmermann et al.2004).

CsMYB5-1and CsMYB5-2were shown to belong to Sg5(Fig.2b ),the subgroup alternatively known as the epidermal cell fate clade and including AtMYB123(TT2,Q9FJA2),VvMYB5a (AAS68190),GhMYB38(AAK19618),and DkMYB4(BAI49721)(Deluc et al.2006;Akagi et al.2009b ).CsMYB5-1showed closest homology to GhMYB38.CsMYB5-2shared 75.09%identity with DkMYB4.Although GhMYB38and DkMYB4are well documented in Sg5,neither exhibited the motif IRTKA [I/L]RC.Notably,CsMYB5-1and CsMYB5-2also have an interaction motif that contributes to the MYB –bHLH complex.CsMYB7-1,together

with

Fig.1Evolutionary

relationships of R2R3-MYBs.Full-length amino acid sequen-ces of R2R3-MYBs in Camellia sinensis transcriptome dataset and Arabidopsis genome were firstly aligned by ClustalW of MEGA5.The phylogenetic tree was constructed according to the neighbor-joining method.Branches corresponding to par-titions reproduced in less than 50%bootstrap replicates were collapsed.The evolutionary distances were computed using the p -distance method.All am-biguous positions were re-moved for each sequence pair.Evolutionary analyses were conducted in MEGA5

Table2Subgroups of R2R3-MYB in Camellia sinensis and motifs

Subgroups MYB gene name in C.sinensis a Putative function clade and gene function b

No.c Motif d

1TYASSTENI[A/S][R/K]LL—EKWL[F/L]CsMYB1-1(cam_GLEAN_10007705)Stomatal aperture(AtMYB60)

Hypersensitive response,cooperates with BES1to

regulate brassinosteroid-induced gene expression

AtMYB30(Froidure et al.2010;Yin et al.2009)

2DESFW—DDxMDFW CsMYB2-1(tie_GLEAN_10020149)Freezing tolerance

CsMYB2-2(cam_GLEAN_10018304)AtMYB15(Dong et al.2010)

CsMYB2-3(cam_GLEAN_10018153)Shoot apex morphogenesis(AtMYB13)

CsMYB2-4(tie_GLEAN_10018260)

DESFW—CsMYB2-5(cam_GLEAN_10025700)

3LE[N/S]ELGL—D[I/P]D[F/I]W[D/S][F/M][L/I]D CsMYB3-1(cam_GLEAN_10020032)AtMYB58,AtMYB63

CsMYB3-2(CsMYB6)

4DLNLEL—[E/N]CS CsMYB4-1(cam_GLEAN_10020745)C2repressor motif clade(Soreq et al.2008;Wixon

and Ashurst2003)

CsMYB4-2(cam_GLEAN_10022034)Anther development

CsMYB4-3(cam_GLEAN_10033998)Cinnamic acid and sinapate ester synthesis

CsMYB4-4(tie_GLEAN_10016351)AtMYB3,AtMYB7,AtMYB32

CsMYB4-5(CsMYB3)Regulate the C4H gene

CsMYB4-6(tie_GLEAN_10017628)AtMYB4(Jin et al.2000)

5IRTKA[I/L]RC CsMYB5-1(cam_GLEAN_10020350)Epidermal cell fate clade

Seed pigmentation(PAs)

TT2(Nesi et al.2001)

CsMYB5-2tie_GLEAN_10028252

6KPRPR[S/T]F–Epidermal cell fate clade

Anthocyanin-related subclade

PAP gene(Borevitz et al.2000)

AtMYB75/PAP1

AtMYB90/PAP2

7GR[T/V]SRSx[M/A]K CsMYB7-1(cam_GLEAN_10034384)Flavonol glycosides clade(Kumar and Maiti2008) 9AQWESARxxAExRLxRES CsMYB9-1(tie_GLEAN_10029256)Cell and petal morphogenesis(MIXTA)

AtMYB16,AtMYB106(Jakoby et al.2008)

10QxxAAAxxN//KxQLxHxMxQ//DDxxSDSxWK–

11LxNPExLRLAxxL[L/F]CsMYB11-1(tie_GLEAN_10041273)Wounding response

CsMYB11-2(cam_GLEAN_10024047)AtMYB102(De V os et al.2006)

CsMYB11-3(tie_GLEAN_10017628)Cell expansion,response to osmotic stress

AtMYB41(Lippold et al.2009),AtMYB74

12[L/F]LN[K/R]VA–Glucosinolate clade(Sethi et al.2008;Glinsky2008) 13D[L/V]F[N/S]KDLQR[I/M]AxxxGQ CsMYB13-1(tie_GLEAN_10015754)Seed mucilage deposition,stomatal closure

CsMYB13-2(cam_GLEAN_10026284)AtMYB61(Liang et al.2005)

14NxxPYWP[E/K]L CsMYB14-1(cam_GLEAN_10036976)Rax1,2,3axillary meristems clade

CsMYB14-2(tie_GLEAN_10046752)

CsMYB14-3(cam_GLEAN_10033620)

CsMYB14-4(cam_GLEAN_10029524)

CsMYB14-5(tie_GLEAN_10038768)

CsMYB14-6(cam_GLEAN_10013375)

CsMYB14-7(cam_GLEAN_10021482)

15WVxxDxFELSxL–Epidermal cell fate specification

Trichome subclade

AtMYB23(Kang et al.2009)

16PxLxFSEW CsMYB16-1(cam_GLEAN_10035542)Photomorphogenesis

AtMYB18,AtMYB19

18GLP[L/V]YP CsMYB18-1(cam_GLEAN_10036108)GAMYB-like genes

ABA/miR regulate

CsMYB18-2(tie_GLEAN_10047941)Anther development

Table2(continued)

Subgroups MYB gene name in C.sinensis a Putative function clade and gene function b

No.c Motif d

AtMYB65,AtMYB33,AtMYB101(Gocal et al.2001;

Flowers et al.2009)

19PxLxFSEW CsMYB19-1(cam_GLEAN_10018317)Anther development

CsMYB19-2(cam_GLEAN_10037103)AtMYB21(Lesne and Benecke2008;Yang et al.

2007),AtMYB24

20WxPRL CsMYB20-1(tie_GLEAN_10044994)ABA-dep gene expression,response to drought

CsMYB20-2(cam_GLEAN_10035632)AtMYB2(Guo and Gan2011)

CsMYB20-3(cam_GLEAN_10032957)Biotic/abiotic stress-related AtMYB108

CsMYB20-4(cam_GLEAN_10031476)

CsMYB20-5(cam_GLEAN_10008153)

CsMYB20-6(CsMYB1)

CsMYB20-7(cam_GLEAN_10034617)

21FxDFL CsMYB21-1(tie_GLEAN_10014553)Root quiescent center expression

CsMYB21-2(cam_GLEAN_10028723)AtMYB56

CsMYB21-3(cam_GLEAN_10012398)Fruit development AtMYB117(Gomez et al.2011)

CsMYB21-4(tie_GLEAN_10018271)ABA hypersensitivity and drought tolerance

CsMYB21-5(cam_GLEAN_10006316)AtMYB52(Park et al.2011)

22TGLYMSPxSP,GxFMxV VQEMLxxEVRSYM CsMYB22-1(cam_GLEAN_10015419)Auxin signaling pathway(AtMYB77)

CsMYB22-2(tie_GLEAN_10008899)Stomatal closure

Induce EIN2expression

CsMYB22-3(tie_GLEAN_10014947)AtMYB44(Liu et al.2011)

23AS[S/P]EE[T/H]L CsMYB23-1(cam_GLEAN_10028165)AtMYB1,AtMYB25,AtMYB109

CsMYB23-2(tie_GLEAN_10036963)

24IQMExDPxTH CsMYB24-1(cam_GLEAN_10024535)AtMYB53,AtMYB92,AtMYB93

CsMYB24-2(tie_GLEAN_10004571)

25LxxYIxx[I/V]N[N/D]CsMYB25-1(tie_GLEAN_10020427)Embryogenesis

CsMYB25-2(tie_GLEAN_10028528)AtMYB118(Zhang et al.2009)

26SSS[S/T]ST CsMYB26-1(cam_GLEAN_10023039)AtMYB99,AtMYB42,AtMYB43,AtMYB85,

AtMYB20

27KRxxxSP[T/I]SSxSNC[S/C]S[S/A]S CsMYB27-1(cam_GLEAN_10013986)Alternative splicing/non-canonical intron subgroup

AtMYB48

Root growth and cell cycle progression

AtMYB59(Mu et al.2009)

Other–cam_GLEAN_10012902AtMYB40

tie_GLEAN_10026681

Other–cam_GLEAN_10028088Anther development

tie_GLEAN_10030857AtMYB103(Parekh et al.2008)

Other–cam_GLEAN_10033790Anther development

tie_GLEAN_10041353AtMYB26(Samollow2008)

Other–cam_GLEAN_10022275AtMYB83

Other–tie_GLEAN_10040319AtMYB121

Other–cam_GLEAN_10019657Tepet layer,trichome development

AtMYB35,AtMYB80

Other–cam_GLEAN_10039398Sperm cell formation

tie_GLEAN_10046608AtMYB125

Other–cam_GLEAN_10011661AS1leaf morphogenesis(polarity specificity)and

plant immune response AtMYB91

cam_GLEAN_10018270

Other–cam_GLEAN_10032232Guard cell division restriction clade AtMYB124,

AtMYB88

a Number of R2R3-MYB genes in C.sinensis transcriptome

b Functional annotations of Arabidopsis

c Subgroup numbers from1to27were those describe

d by Strack

e et al.(2001),26and27were newly named to the subgroups who hold significant moti

f by MEME

d Motifs in tea transcriptom

e have been confirmed in most o

f these subgroups usin

g MEME(Bailey et al.2009)

0.02

Sg7

Sg4

Sg5

a

c

Fig.2Sg4,Sg5,and Sg7R2R3-MYBs'alignment of Camellia sinensis and Arabidopsis and phylogenetic tree.a R2R3-MYB amino acid sequences of C.sinensis and Arabidopsis in subgroups 4,5,and7were aligned by ClustalX,respectively.Particle align-ment was shown,and the conserved motifs were detected by MEME and were framed with black box.b Phylogenetic tree of R2R3-MYBs of C.sinensis and other plants in subgroups4,5,and 7was constructed by MEGA5(option selected:neighbor-joining method,p-distance,complete deletion,1,000bootstrap replicates).c Conserved motifs were detected by MEME and were listed behind each subgroup.Conserved motifs of subgroups4,5,and7:LNL [E/D]L,IRTKA[I/L]RC,and GR[T/V]SRSx[M/A]K.Gen-Bank accession numbers were Sg4:AtMYB3(AEE30263),AtMYB4(AEE86955),AtMYB7(AEC06531),PtMYB14 (ABD60279),DvMYB2(BAJ33514),HlMYB1(CAI46244), PhMYB4(ADX33331),EgMYB1(CAE09058),GmMYBZ2 (ABI73970),GhMYB9(AAS92347),AmMYB308(P81393), AmMYB330(P81395),GmMYB48(ABH02823),PtMYB057 (EEF00115),ZmMYB42(CAJ42204),PgMYB16(ACN12958), and PgMYB16(ACN12958);Sg5:GhMYB38(AAK19618), AtMYB123(Q9FJA2),OsMYB3(BAA23339),ZmC1 (AAA33482),VvMYB5a(AAS68190),DkMYB4(BAI49721); and Sg7:AtMYB11(XP_002876680),AtMYB12(O22264), AtMYB111(XP_002865729),VvMYBF1(ACV81697),GhMYB1 (CAD87007),LjMYB12(BAF74782),MdMYB22(AAZ20438), and ZmP(P27898)

AtMYB11(XP_002876680),AtMYB12(O22264),and AtMYB111(XP_002865729),shares the motif GR[T/V] SRSx[M/A]K(Fig.2b).Sg7may participate in the regulation of flavonol biosynthesis(Stracke et al.2010).CsMYB7-1 shared41.9%and35.42%identity with VvMYBF1 (ACV81697)and ZmP1(P27898).

Subfamilies of bHLHs and members involved

in the flavonoid biosynthetic pathway

Based on conserved short motifs identified outside of the DNA binding domain,Heim et al.(2003)classified the113 observed bHLH proteins into12groups from I to XII.In this system,members of IIId,IIIe,and IIIf function as TF regulating genes in flavonoid metabolism(Heim et al. 2003).Carretero-Paulet et al.presented a comprehensive classification as well as structural and evolutionary analy-sis of the bHLH gene family in plants,classifying638 observed bHLH genes from Arabidopsis,poplar,rice, mosses,and algae species into32subfamilies.Notably, subfamilies2,5,and24were identified as regulators in flavonoid or anthocyanin metabolism(Carretero-Paulet et al.2010).Using the same methods,49C.sinensis bHLHs and151AtbHLHs were constructed into a phylogenetic tree and divided into12groups from I to XII(inside number)and1–32subfamilies(outside number;Fig.3).

C.sinensis bHLHs were gathered and dispersed in nine subfamilies,including the subfamilies1,2,3,15,24,25, 26,28,and31.

The number of C.sinensis bHLHs association with flavo-noid biosynthesis was less than expected,with two C.sinensis bHLHs in subfamily2(cam_GLEAN_10012690and cam_GLEAN_10006335;renamed CsbHLH2-1and CsbHLH2-2)and five C.sinensis bHLHs in subfamily24 (tie_GLEAN_10024436,tie_GLEAN_10022821, cam_GLEAN_10025059,tie_GLEAN_10042126,and cam_GLEAN_10023958;renamed CsbHLH24-1to CsbHLH24-5).The two classification systems were consistent (e.g.,subgroup IIId members are included in subfamily2, subgroup IIIf members are equivalent to subfamily5,and subgroup VII members are equivalent to subfamily24). Surprisingly,no C.sinensis bHLHs clustered in subfamily5 were homologous to TT8.

CsbHLH2-1and CsbHLH2-2clustered in a single sub-clade,closely related to VvMYC4(XP_002279973)and HbMYC4(AEG74014)as shown in Fig.4b.CsbHLH24-2 and CsbHLH24-5were closely related to Pp_SPATUAlike (ADG56590),AtbHLH24(Q9FUA4),and CrMYC5 (ACM41588).CsbHLH24-1,CsbHLH24-3,and CsbHLH24-4shared71.95%,72.48%,and71.3%identity with AtbHLH8(O80536),respectively.

The HLH domain commonly provides information on specific DNA-binding ability due to the amphipathic affinity of its N-terminal.This is especially apparent in the critical His-Glu-Arg(H-E-R)located at positions5,9,and13.These HER sites have been shown to bind to a variation of the E-box hexanucleotide sequence(E-box:5′-CANNTG-3′,variation G-box:5′-CACGTG-3′)(Buck and Atchley2003).The HLH region is composed of two hydrophobicα-helices linked by a divergent loop,marked by the alignment of amino acids of subfamilies2and24in Fig.4a.The activation or repression domains,which are outside of the DNA binding domain,are important in gene expression regulation.These sequences usually contain short motifs that are conserved between relat-ed proteins in different species.In most cases,the protein architecture is remarkably conserved within specific subfami-lies,providing further support for phylogenetic analysis based on bHLH domains.

According to50motifs of variable length(8–80amino acids)summarized from the638bHLHs examined by Carretero-Paulet et al.(2010),conserved motif architecture structures in subfamilies2and24were11-14-8-20-12-2-1-7-4 and22-48-2-1-39-15,respectively(Fig.4c).The relative amino acid sequence motifs of C.sinensis observed in the current study were consistent with these previous findings.Motifs1 and2were commonly found in almost every bHLH protein and were identified as the helix2and helix1regions of the bHLH domain,respectively.No single motif was detected in either the basic or loop regions.All bHLH subgroups and subfamilies are listed in Table3along with the architecture of conserved motifs identified by MEME and gene function predicted by Arabidopsis data.

Involvement of a WD40gene in the flavonoid biosynthetic pathway

A total of195WD genes were isolated from the C.sinensis transcriptome dataset.Of these,134showed clear homology with WDR proteins in other eukaryotes.In contrast to Arabidopsis WDR proteins containing four or more recog-nizable copies of the motif in237of269WD40s,64of134 C.sinensis WD40s contained four or more WD repeats. Copy numbers of each WD40protein were shown in Online Resource1.A variety of parameters were selected in the MEGA5software in order to explore the relationship between the70WD40s in Anhui No.1,the64WD40s in Tieguanyin,and the237WDRs in Arabidopsis,attempting to form one cohesive phylogenetic tree.In this analysis, some paralogs significantly diverged outside of the WDR region,and were too diverged to allow them to be compiled unambiguously.Thus,the phylogenetic trees of Anhui No.1 and Tieguanyin WD40s were constructed separately from those of Arabidopsis WDRs.The phylogenetic trees were supplied as additional data shown in Online Resource2.

The tie_GLEAN_10026686(renamed CsWD40-1)was the only gene involved in the flavonoid biosynthesis pathway.

Sequence alignment of WDRs of known function was performed with ClustalX,and results are shown in Fig.5.CsWD40-1showed 80.4%identity with AtTTG1(CAB45372),81.1%identity with Medicago truncatula WD40-1(ABW08112),and 85.42%with Malus domes-tica TTG1(ADI58760).

Full-length validation and expression features

The 3′and 5′ends of CsMYB4-5,CsMYB4-6,CsMYB5-2,CsbHLH2-1,CsbHLH2-2,and CsWD40-1were amplified and assembled by 3′-and 5′-RACE-PCR.End-to-end PCR was performed,and the PCR products were purified,cloned,and sequenced.All products,with the exception of several bases on the 3′terminal of CsMYB7-1and CsbHLH24-3,were confirmed to be consistent with expected results.There were eight and five nucleotides inconsistent with the original CsMYB7-1and CsbHLH24-3of transcriptome dataset which coded for three and two different amino acids,respectively.Relative expression was profiled using qRT-PCR in C .sinensis leaves at different developmental stages and treat-ment conditions,including the two experimental hormone smudges (ABA and GA)and the six experimental wound treatment groups.Notably,the transcription factor genes exhibited different expression patterns (Fig.6).In tender roots,CsMYB4-2and CsMYB4-3exhibited a characteristic low expression,but CsMYB4-5and CsMYB4-6expression levels remained high.High levels of CsMYB5-1were expressed in mature second leaves,while CsMYB5-2in stems.Moderate CsMYB7-1expression was observed in all different organs examined.Hormone stimulation revealed that CsMYB4-1expression was reduced by more than twofold upon GA stimulation,but CsMYB4-4expression was increased more than twofold upon ABA stimulation.Expression of CsMYB5-2was inhibited by more than twofold upon GA stimulation and similarly enhanced by ABA stimu-lation.Furthermore,wound treatment was shown to enhance CsMYB7-1expression significantly.

High expression levels of CsbHLH2-1and CsbHLH2-2were found in roots.CsbHLH24-3and CsbHLH24-5were highly expressed in leaves,and CsbHLH24-1expression was highly expressed in stems.The expression levels of CsbHLH2-1,CsbHLH2-2,CsbHLH24-1,and CsbHLH24-5were increased by more than twofold upon ABA stimulation.The expression levels of CsbHLH24-2and CsbHLH24-3were inhibited by twofold upon GA stimulation and

similarly

Fig.3Evolutionary

relationships of bHLHs.Full-length amino acid sequences of bHLHs in Camellia sinensis transcriptome dataset and Ara-bidopsis genome were aligned by ClustalW of MEGA5.The phylogenetic tree was con-structed according to the neighbor-joining method.

Branches corresponding to par-titions reproduced in less than 50%bootstrap replicates were collapsed.The evolutionary distances were computed using the p -distance method.All positions containing gaps and missing data were eliminated.Evolutionary analyses were conducted in MEGA5.The in-ner subgroups from I to XII were according to Heim,and the outside subfamilies from 1to 32were marked based on Carretero-Paulet et al.

Table 3Subfamilies of bHLHs in Camellia sinensis and motifs

Subgroup No.a Subfamily No.b bHLH gene name in Arabidopsis c bHLH gene name in C .sinensis d

Conserved motifs in subfamilies e

Relative gene functions f

Ia

10

AtbHLH6748-28-2-26-1-39-7-4-19

Stomata differentiation:FAMA (AtbHLH97)

AtbHLH97AtbHLH70AtbHLH71AtbHLH57AtbHLH99AtbHLH96AtbHLH94AtbHLH98AtbHLH45

Ib

12

AtbHLH952-1-37

Stomata differentiation:MUTE (AtbHLH45),SPEECHLESS (AtbHLH98)

AtbHLH55Iron homeostasis:ORG2(AtbHLH38),ORG3(AtbHLH39)

AtbHLH125AtbHLH126AtbHLH118AtbHLH120AtbHLH36AtbHLH100AtbHLH101AtbHLH38AtbHLH39

II

9

AtbHLH9148-45-22-2-1-7-29AtbHLH10AtbHLH89

IIIa

1

AtbHLH2211-2-1-47-4

Regulation of iron uptake:FIT (AtbHLH29)

AtbHLH29AtbHLH21

Anther development:AMS (AtbHLH21),DYT1(AtbHLH22)IIIb

AtbHLH93Stomata development and cold acclimatization

response and freezing tolerance:ICE1(AtbHLH116),SCRM2(AtbHLH33)

AtbHLH61AtbHLH33AtbHLH116

IIIc

AtbHLH90AtbHLH35AtbHLH27

IIId

2

AtbHLH3cam_GLEAN_10006335(tie_GLEAN_10005499)11-14-8-20-12-2-1-7-4AtbHLH13tie_GLEAN_10001220

(cam_GLEAN_10012690)

AtbHLH17AtbHLH14

IIIe

AtbHLH6Tryptophan biosynthesis:dominant mutation of ATR2(AtbHLH5);ABA,JA,and light signaling pathway:MYC2(AtbHLH6)

AtbHLH4AtbHLH5AtbHLH28

IIIf

5

AtbHLH42/TT811-2-1-12-47-29

Anthocyanin biosynthesis (GL3,EGL3,TT8)AtbHLH12/MYC1Seed coat differentiation (GL3,EGL3,TT8,MYC1)AtbHLH1/GL3Proanthocyanidin biosynthesis (TT8)AtbHLH2/EGL3

Trichome formation (MYC1,GL3,EGL3)

Iva

3

AtbHLH20tie_GLEAN_1004209127-2-1-7-4Endoplasmic reticulum body formation:NAI1(AtbHLH20)

AtbHLH19tie_GLEAN_10006869AtbHLH18tie_GLEAN_10029282AtbHLH25

cam_GLEAN_10018241cam_GLEAN_10018117

IVb

4

AtbHLH4731-2-1-9-15

Metal homeostasis regulation,response to auxin stimulus,stress response,seed development

AtbHLH11

Subgroup No.a

Subfamily No.b

bHLH gene name in Arabidopsis c bHLH gene name in C .sinensis d

Conserved motifs in subfamilies e

Relative gene functions f

AtbHLH121

IVc

AtbHLH105Metal homeostasis,auxin-conjugate metabolism:ILR3(AtbHLH105)

AtbHLH115AtbHLH34AtbHLH104

IVd 7AtbHLH412-1-44-29AtbHLH92Va 14AtbHLH1022-1-50-4Brassinosteroid signaling

AtbHLH46Vb

13

AtbHLH13135-2-1-25-35-22

AtbHLH30AtbHLH32AtbHLH107AtbHLH106AtbHLH51

17

AtbHLH15126-2-1Unidimensional cell growth

AtbHLH144AtbHLH143AtbHLH142AtbHLH145

VI Orphans (32)AtbHLH10948-48-2-48-1-21-4Embryonic development ending in seed dormancy AtbHLH108Early embryo development:MEE8(AtbHLH108)VIIa

24

AtbHLH65tie_GLEAN_10024436

10-16-3-2-21-1-23Light and gibberellin signaling:PIF1(AtbHLH105),

PIL5(AtbHLH65),PIF3(AtbHLH8),PIF4(AtbHLH9)

AtbHLH9AtbHLH8AtbHLH56AtbHLH127AtbHLH119AtbHLH23AtbHLH15AtbHLH124AtbHLH132

VIIb

AtbHLH26tie_GLEAN_10042126Regulation of anthocyanin,regulation of seed

germination,gibberellic acid signaling,regulation of chlorophyll metabolism,negative gravitropism AtbHLH72AtbHLH16cam_GLEAN_10025059AtbHLH73cam_GLEAN_10023958AtbHLH24

tie_GLEAN_10006711Fertilization process:UNE10(AtbHLH16)tie_GLEAN_10022821Fruit dehiscence:ALC (AtbHLH73)

cam_GLEAN_10020333Phytochrome and cytochrome signaling:HFR1(AtbHLH26),carpel margin development

Mediator of germination responses to light and

temperature:SPT (AtbHLH24),female gametophyte development

VIIIa 30

AtbHLH1172-1-15

AtbHLH52AtbHLH53VIIIb 31AtbHLH87cam_GLEAN_1002730518-10-21-45

Flower and fruit development,initiation/maintenance of axillary meristems

AtbHLH88tie_GLEAN_10015094AtbHLH37tie_GLEAN_10033927Transmitting tract and stigma development:HEC1

(AtbHLH88),HEC2(AtbHLH37),HEC3(AtbHLH43)

AtbHLH43tie_GLEAN_10038431AtbHLH40cam_GLEAN_10028044AtbHLH140

cam_GLEAN_10018105VIIIc 28AtbHLH54tie_GLEAN_100212143-2-1Root hair,rhizoid,and caulonemata development

AtbHLH84cam_GLEAN_10006896Root hair formation:RHD6(AtbHLH83),RSL1(AtbHLH86)AtbHLH85tie_GLEAN_10031011Root hair development:RSL4(AtbHLH54),RSL3

(AtbHLH84),RSL2(AtbHLH85),SL5(AtbHLH135)

AtbHLH83

tie_GLEAN_10011950

Subgroup No.a Subfamily

No.b

bHLH gene name

in Arabidopsis c

bHLH gene name

in C.sinensis d

Conserved motifs

in subfamilies e

Relative gene functions f

AtbHLH86cam_GLEAN_10032416

AtbHLH139

IX27AtbHLH1303-2-1-24

AtbHLH128

AtbHLH129

AtbHLH122

AtbHLH81

AtbHLH80

X15AtbHLH113tie_GLEAN_1002732348-45-2-1-13Response to ethylene stimulus

AtbHLH123

AtbHLH68

AtbHLH133

AtbHLH110

AtbHLH103

AtbHLH114

AtbHLH112

AtbHLH111

XI26AtbHLH69cam_GLEAN_1003229222-22-22-3-2-43-1Female gametophyte development,response to phosphate

deficiency stress

AtbHLH66tie_GLEAN_10019021

AtbHLH82tie_GLEAN_10032589Root hair development:AbHLH66,AbHLH69,AbHLH82

AtbHLH7cam_GLEAN_10033481Fertilization forming zygote and endosperm:

UNE12(AbHLH59)

AtbHLH59cam_GLEAN_10010677

XII25AtbHLH75tie_GLEAN_1003188022-3-2-1-30-5Brassinosteroid and abscisic acid signaling,floral

transition,petal morphogenesis

AtbHLH50tie_GLEAN_10040329

AtbHLH44cam_GLEAN_10015063

AtbHLH63cam_GLEAN_10023216

AtbHLH49tie_GLEAN_10016811

AtbHLH76cam_GLEAN_10025896

AtbHLH78tie_GLEAN_10035514

AtbHLH62cam_GLEAN_10036734

AtbHLH74tie_GLEAN_10046947

AtbHLH48cam_GLEAN_10026427

AtbHLH60(tie_GLEAN_10028643)

AtbHLH31tie_GLEAN_10023068

AtbHLH79cam_GLEAN_10018573

AtbHLH58tie_GLEAN_10022404

AtbHLH64cam_GLEAN_10012772

AtbHLH137tie_GLEAN_10026434

16AtbHLH1342-1Gibberellic acid-light and gibberellic acid signaling AtbHLH135

AtbHLH136

19AtbHLH14727-2-1

AtbHLH148

AtbHLH149

AtbHLH150

21AtbHLH1462-1Light and auxin signaling,shade avoidance

a Subgroup numbers from I a to XII were those described by Heim et al.(2003)

b Subfamily numbers from1to32were those described by Carretero-Paulet et al.(2010)

c Number of bHLH genes in C.sinensis transcriptome

d Architectur

e conserved motifs described by Carretero-Paulet et al.(2010)

e Functional annotations mainly with reference to Arabidopsis(Feller et al.2011;Carretero-Paulet et al.2010)

f Short conserved motifs summarized from the638bHLHs with50motifs of variable length(Carretero-Paulet et al.2010)

enhanced by ABA stimulation.Notably,CsbHLH2-2and CsbHLH24-4expressions were enhanced by GA stimulation. Wound treatment was shown to downregulated the expression of CsbHLH2-1.The expression of CsWD40-1was highest in the first and fourth leaves,demonstrating suppression upon wound stimulation.

Subfamily2

Subfamily24

a

c b

Fig.4Subfamilies2and24bHLHs'alignment of Camellia sinensis and phylogenetic tree.a Amino acid sequences of bHLHs in subfami-lies2and24of C.sinensis and Arabidopsis were aligned by ClustalX. The basic helix-loop-helix(bHLH)domain,which always provides the specific DNA-binding ability,was shown.The His5-Glu9-Arg13(H-E-R)motif could be easily found.b Phylogenetic tree of bHLHs of C. sinensis and other plants in subfamilies2and24was constructed by MEGA5(option selected:neighbor-joining method,p-distance,pair-wise deletion,1,000bootstrap replicates).c Architectures of conserved motifs of bHLHs in subfamilies2and24in bHLH genes:11-14-8-20-12-2-1-7-4and22-48-2-1-39-15,respectively.GenBank accession numbers were subfamily2:HbMYC4(AEG74014),Rc_02518914(XP_002518914),Pt_002299425(XP_002299425),VvMYC4 (XP_002279973),GmMYC2-like(XP_003528771),AtbHLH3 (O23487),AtbHLH14(O23090),AtbHLH17(Q9ZPY8),and AtbHLH26(Q9FE22);subfamily24:VvPIF3-like(XP_002276198), Md_AEX32796(AEX32796),VITISV_042277(CAN83346), Pp_SPATULA-like(ADG56590),Rc_002514702(XP_002514702), CrMYC5(ACM41588),AtbHLH24(Q9FUA4),AtbHLH8 (O80536),AtbHLH16(Q8GZ38),AtbHLH72(Q570R7),AtbHLH119 (Q8GT73),AtbHLH127(Q7XHI7),AtbHLH23(Q9SVU6), AtbHLH15(Q8GZM7),AtbHLH9(Q8W2F3),AtbHLH65 (Q8GZM7),AtbHLH124(Q84LH8),and AtbHLH132(Q8L5W7)

Discussion

Characteristics and function prediction of candidate R2R3-MYB genes in Sg4,Sg5,and Sg7

Arabidopsis is a model plant useful for the study of flavonoid biosynthesis due to its rich variety of mutant types,the availability of comprehensive genome data,and its relatively short growth https://www.360docs.net/doc/993954669.html,pared with Arabidopsis ,the study of regulation networks involved in polyphenol biosynthesis in more complex plants,such as C .sinensis ,is considerably more difficult.The C .sinensis transcriptome dataset applied in the current study contains a wealth of genetic information,consist-ing of over 127,094unigenes.Transcription factor gene identification is necessary to improve the general under-standing of polyphenol metabolism regulation in the commercially important plant C .sinensis .

Target sites in the flavonoid biosynthetic pathway in C .sinensis are likely regulated by specific transcription factors,as shown in Fig.7.Genes in the same phylogenetic clades generally exhibit similar functions,while genes in different clades usually are involved with variant biological processes with distinct regulatory characteristics.For example,Sg3R2R3-MYBs (MYB58,MYB63,and MYB85)are involved in stimulating the synthesis of lignin.Sg5R2R3-MYBs can activate LBGs in PA biosynthesis pathways,usually requir-ing a ternary complex (TT2/TT8/TTG1)of transcription factors (Hichri et al.2011).R2R3-MYBs of Sg6(PAP1,PAP2,MYB113,and MYB114)are known to combine with bHLHs (TT8,GL3,and EGL3)and WD40(TTG1)to form a ternary complex that activates LBGs and transfers protein genes,such as ANS ,DFR ,F3′H ,LDOX ,UGT78D2,and UGT75C1,in Arabidopsis (Gonzalez et al.2008).As a result,these compounds regulate the synthesis of anthocya-nins in these plants.R2R3-MYBs of Sg7generally activate EBG promoters,such as CHS ,CHI ,F3H ,and FLS1,in-volved in control of the flavonol branch.Notably,this occurs without combining with bHLH proteins,while R2R3-MYBs of Sg4(MYB4,MYB3,MYB7,and MYB32)downregulate polyphenol biosynthesis by interact-ing with bHLH proteins.

Phylogenetic and motif analysis can be an effective meth-od for predicting the function of transcription factors in tea plants,such as C .sinensis .In most tea plants,high levels of polyphenol compounds are generally present,ranging from 18%to 30%of the dry weight of C .sinensis ,but only nine R2R3-MYB genes have been predicted to participate in flavonoid biosynthesis.Notably,no distribution of C .sinen-sis genes was observed in Sg15.This observation may be explained by the relationship between the Sg15subgroup and epidermal cell fate specification or trichome morpho-genesis and initiation (e.g.,AtMYB23).Similarly,no C .sinensis genes were observed in the Sg6subgroup,widely known as the anthocyanin-related subclade.This lack of Sg6genes may be a result of low anthocyanin contents in tea plants,normally accounting for only 2–3%of the dry weight.

Members of the Sg4subgroup have been shown to be representative factors for phenolic acid metabolism and lignin biosynthesis.For example,AtMYB4has been shown to be a transcriptional repressor involved in the inhibition of upstream gene expression,such as the cinnamate 4-hydroxylase gene (C4H )(Jin et al.2000).Furthermore,enhanced levels of sinapate esters have been identified in the AtMYB4mutant,a characteristic presumed to be associ-ated with enhanced expression of C4H (Jin et al.2000).AmMYB308significantly downregulates the expression of C4H ,4-coumaroyl-CoA ligase gene (4CL ),and the cin-namyl alcohol dehydrogenase gene (CAD )in the phenyl-propanoid metabolism of transgenic tobacco (Tamagnone et al.1998).CsMYB4-4and CsMYB4-6showed

close

Fig.5Alignment of WD40s from Camellia sinensis and other species.Full-length amino acid sequences of WD40s in C .sinensis and other species aligned by ClustalX.GenBank accession numbers were MdTTG1(ADI58760),PhAN11(AAC18914),InWDR (BAE94407),PFWD (BAB58883),AtTTG1(CAB45372),and ZmPAC1(AAM76742)

homology with AtMYB4and AmMYB308,and were thus predicted to possess the similar functions.Similarly, CsMYB4-5was closely related to PtMYB14,a tissue-preferential promoter for CAD that is involved in the accu-mulation of sesquiterpene in conifers(Bedon et al.2010). Although C.sinensis genes were often clustered in the same subgroups,their expression patterns varied widely(Fig.6). For example,CsMYB4-5and CsMYB4-6were highly expressed in roots,but low expression of CsMYB4-2and CsMYB4-3were observed in roots.The expression of CsMYB4-1was significantly reduced by GA stimulation, while the expression of CsMYB4-4was increased more than twofold by ABA stimulation.In fact,different flavonoids were shown to accumulate differently in various organs of C.sinensis.For instance,lower levels of catechins(flavan-3-ols monomers)and flavonols,and higher levels of PAs (polymerized flavan-3-ols)were observed in roots compared with those observed in leaves.Further evaluations by trans-genic studies are being performed to validate the function of these genes.

CsMYB4-1

Fig.6Relative expression levels of17R2R3-MYB,bHLH,and WD40genes in different Camellia sinensis organs and different treat-ments by real qRT-PCR.The gene expression was determined by qRT-PCR experiment,and the relative expression values are calculated by the2àΔΔC t method.Values were normalized against the expression level of housekeeping gene GAPDH,and relative to fourth leaf and CK as a calibrator.The left part is the relative expression levels in C.sinensis organs,and the right part is the relative expression levels in third leaves treated by hormones(ABA,GA)and wound.ΔC T?C T;targetàC T;GAPDH;àΔΔC T?àΔC T;targetàΔC T;fourth leaf

àá

;and àΔΔC T?àΔC T;targetàΔC T;CK leaf1h

àá

,where C T,target and C T, GAPDH

are the threshold cycles of targets and housekeeping gene GAPDH,respectively.Ordinate is the value of2àΔΔCt

R2R3-MYBs of subgroup 5have been reported to regu-late PA biosynthesis.Due to the high content of flavan-3-ols in C .sinensis ,Sg5may be the most worthy research sub-group.R2R3-MYBs in Sg5are known to combine with bHLHs (TT8,GL3,and EGL3)and WD40s (TTG1)to form a ternary complex (MBW)that activates LBGs for PA in Arabidopsis (Nesi et al.2001).The functions of many known R2R3-MYBs in Sg5have already been investigated,including TT2,VvMYB5a,and DkMYB4.It has been sug-gested that the stringent expression of BAN promoting PA accumulation is partially determined by TT2(Nesi et al.2001;Baudry et al.2004).Nesi et al.(2001)predicted that TT2is required for normal expression of the DFR gene in the immature siliques of Arabidopsis ,demonstrating that it has a relatively high expression in the early globular to globular stages.It has also been demonstrated that overexpression of VvMYB5a in tobacco leads to high accu-mulation of anthocyanins and PAs,especially in flowering plants (Deluc et al.2006).CsMYB5-2was shown to be closely related with DkMYB4,proven to regulate PA bio-synthesis in persimmon fruit.The suppression of DkMyb4in persimmon calluses also caused a substantial downregula-tion of the PA pathway genes and PA biosynthesis (Akagi et al.2009a ).The expression levels of CsMYB5-1and CsMYB5-2in different organs varied (Fig.6).Surprisingly,both of them were minimally expressed in roots,suggesting that they may be involved in other metabolic pathways as well,offering a potential explanation for the inconsistency of their expression patterns with PA accumulation.

R2R3-MYBs of subgroup 7are known for their roles in flavonol biosynthesis regulation.Flavonol and flavonol gly-coside account for 3–4%of the dry weight of C .sinensis .In

ANR Sg5+bHLH2+WD

40

Sg5+bHLH24+WD40

Sg7, bHLH24

Sg7

Sg7Sg4,Sg7,bHLH24Sg4Sg4Sg4

CHI 1-phenylalanine PAL

Cinnamic acid C4H

4-coumarate

4CL

4-coumaroyl-coA

CHS Naringenin Chalcones

Delphinidin 3-O-glucoside LAR

ANS

2,3-trans-flavan-3-ol ((+)catechin,C)

Anthocyanidin

ANR

Cyanidin 3-O-glucoside

LAR

2,3-trans-flavan-3-ol ((+)gallocatechin,GC)

ANS

Anthodelphinidin UFGT

UFGT

2,3-cis-flavan-3-ol ((-) Epicatechin, EC)

(-)Epicatechin-3-gallate

(ECG)

2,3-cis-flavan-3-ol ((-) Epigallocatechin,EGC)

(-) Epigallocatechin-3-gallate

(EGCG)Galloyl

acid

UGGT

β-glucogallin

(βG)

ECGT

ECGT

GCH

GCH

Naringenin

F3’H

F3’5’H

F3H

2,3-dihydroquercetin

DFR leucocyanidin

2,3-dihydromyricetin

DFR

leucodelphinidin

Pentahydroxyflavanone

Eriodictyol F3H

F3’H

F3’5’H

Dihydrokaempferol F3H

Flavonoids

FLS FLS FLS

Fig.7The flavonoid biosynthesis pathway in Camellia sinensis and the predicted transcription factors.Transcription factors that may be combining with the target genes in the flavonoid biosynthesis pathway in C .sinensis were in italics and bold type .Genes and products were shown in accordance with the order of the arrow .P AL

phenylalanine ammonia lyase,C4H cinnamate 4-hydroxylase,4CL 4-coumaroyl-CoA ligase,CHS chalcone synthase,CHI chalcone isomerase,F3H flava-none 3-hydroxylase,F3′H fla-vonoid 3′-hydroxylase,F3′5′H flavonoid 3′,5′-hydroxylase,FLS flavonol synthase,DFR dihydroflavonol 4-reductase,LAR leucoanthocyanidin reduc-tase,ANS anthocyanidin syn-thase,ANR anthocyanidin reductase,UGGT UDP-glucose:galloyl-1-O -β-D -glu-cosyltransferase,ECGT epica-techins:1-O -galloyl-β-D -glucose O -galloyltransferase,GCH galloylated catechins hydrolase

Arabidopsis,MYB11,MYB12(PFG2),and MYB111 (PFG3)control the flavonol branch of flavonoid biosynthe-sis,differentially influencing the spatial accumulation of specific flavonol derivatives(Stracke et al.2010). Promoters of the early flavonoid biosynthetic genes CHS, CHI,F3H,and FLS1are activated by independent MYB12, without a bHLH partner.Mechanistically,it has been sug-gested that this compound binds similar cis-element(s)in the promoters of different target genes(Stracke et al.2007). CsMYB7-1is closely related to VvMYBF1and ZmP1 clades.The expression of VvMYBF1facilitates the accumu-lation of flavonols,significantly enhancing the expression of VvFLS1(Czemmel et al.2009).ZmP1is also a Sg7member that activates a subset of the flavonoid pathway genes re-quired for biosynthesis of phlobaphene pigments and3-deoxy-flavonoids.Notably,this is accomplished without binding a bHLH protein(Grotewold et al.1994).Sg7R2R3-MYBs were also shown to control additional target genes, including the UDP-glycosyltransferases genes UGT91A1and UGT84A1(Czemmel et al.2009).Moderate CsMYB7-1ex-pression was observed in different organs(Fig.6). Characteristics and function prediction of candidate bHLH genes in subfamilies2and24

Evolutionary analysis of bHLH reveals both evolutionary relationships as well as predictions of gene https://www.360docs.net/doc/993954669.html,ing this technique,it can be simply confirmed that members of a single clade are generally involved in similar biological processes.For example,members in subgroup IIIf have been extensively documented for their involvement in an-thocyanin biosynthesis(Spelt et al.2000),seed coat differ-entiation(Nesi et al.2000),trichome formation,and root hair formation(Dixon et al.2005).BHLHs in this subgroup were reported to combine with MYBs harbored[DE] Lx2[RK]x3Lx6Lx3R motif.The C.sinensis bHLHs did not cluster in subgroup IIIf.The fact that fewer bHLH genes were identified in our transcriptome analysis than expected may be a result of the high genetic heterozygosity of C. sinensis,making sequence assembly difficult.As sequenc-ing techniques improve,future studies will be likely to detect more bHLH genes in C.sinensis.

Previous classifications relied on bHLH signature domains, which are composed of approximately60amino acids ar-ranged according to their bifunctional structure.Because bHLH proteins usually contain short conserved motifs outside of the bHLH domain,additional functional properties may be better characterized by subfamily-specific motifs(Carretero-Paulet et al.2010).For instance,motif14is commonly found in subfamilies2,5,and23,where it plays a functionally important role in the expression of several downstream genes acting in the Trp biosynthesis pathway(Smolen and Bender 2002).Motif6is shared by the members of subfamily23and has been characterized in AtLHW as necessary for homodi-merization(Ohashi-Ito and Bergmann2007).

Based on these methods,two bHLH genes of subfamily 2and five bHLH genes of subfamily24have been pre-dicted to be involved in flavonoid metabolism.In subfam-ily24,AtbHLH8(PIF3)positively regulates anthocyanin biosynthesis by binding anthocyanin biosynthetic gene promoters containing G-box elements such as CHS, F3H,DFR,and LDOX(Shin et al.2007).From Fig.6, the expressions of both CsbHLH2-1and CsbHLH2-2were significantly increased by ABA stimulation,predicting that they may at least participate in ABA signaling. Meanwhile,CsbHLH2-1might be involved in wound responses as well.GA treatment was shown to upregulate the expression of CsbHLH24-4,and downregulate the expression of CsbHLH24-2and CsbHLH24-3,indicating their regulation in GA signaling.These are consistent with functions of subfamilies2and24reported in other species (Carretero-Paulet et al.2010).

In most cases,proteins within a single subgroup share conserved motifs and have similar,though not necessarily identical,functions.Most bHLH proteins interact with R3 repeat domains of MYB proteins at the N-terminal acidic region to form the MYB–bHLH complex involved in flavo-noid biosynthetic pathway regulation.Another important interaction domain in bHLH proteins is the ACT-like dimer-ization domain located on the C-terminal of the bHLH motif,where it plays a role in homodimeric bHLH–bHLH formation(Feller et al.2011).

Characteristics and function prediction of CsWD40-1 Analysis of the transcription factors R2R3-MYB,bHLH, and WD40revealed that only the WD40s of C.sinensis could not be combined with those of Arabidopsis to construct a phylogenetic tree.This can be explained by the relatively large evolutionary gap of WD40be-tween Anhui No.1and Tieguanyin.An experimental study showed that MdTTG1was capable of fully replacing AtTTG1for AtBAN promoter activation in cooperation with TT2and TT8in a co-transfection system(Brueggemann et al.2010).AtTTG1plays an important role in the regulation of DFR,LDOX,and ANR expression by interacting with a bHLH transcrip-tion factor(GL3,EGL3,or TT8)and a MYB transcrip-tion factor(PAP1,PAP2,MYB113,or MYB114)in MBW form.This ternary complex always affects PA accumulation in seeds and anthocyanin accumulation in plant tissues(Walker et al.1999;Hichri et al.2011; Petroni and Tonelli2011;Appelhagen et al.2011).Pang et al.(2009)found that the deficiency of MtWD40-1 expression strongly suppressed flavonoid structural genes thus blocked accumulation of a range of phenolic