ORIGINAL PAPER
Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco ?owers
Takashi Nakatsuka ?Eri Yamada ?Misa Saito ?
Kohei Fujita ?Masahiro Nishihara
Received:12July 2013/Revised:28August 2013/Accepted:29August 2013/Published online:14September 2013óSpringer-Verlag Berlin Heidelberg 2013
Abstract
Key message Single-repeat MYB transcription factors,GtMYB1R1and GtMYB1R9,were isolated from gentian.Overexpression of these genes reduced anthocyanin accumulation in tobacco ?owers,demonstrating their applicability to modi?cation of ?ower color .
Abstract RNA interference (RNAi)has recently been used to successfully modify ?ower color intensity in sev-eral plant species.In most ?oricultural plants,this tech-nique requires prior isolation of target ?avonoid biosynthetic genes from the same or closely related species.To overcome this limitation,we developed a simple and ef?cient method for reducing ?oral anthocyanin accumu-lation based on genetic engineering using novel transcrip-tion factor genes isolated from Japanese gentians.We identi?ed two single-repeat MYB genes—GtMYB1R and GtMYB1R9—predominantly expressed in gentian petals.Transgenic tobacco plants expressing these genes were produced,and their ?owers were analyzed for ?avonoid components and expression of ?avonoid biosynthetic genes.Transgenic tobacco plants expressing GtMYB1R1or GtMYB1R9exhibited signi?cant reductions in ?oral anthocyanin accumulation,resulting in white-?owered phenotypes.Expression levels of chalcone isomerase
(CHI ),dihydro?avonol 4-reductase (DFR ),and anthocy-anidin synthase (ANS )genes were preferentially sup-pressed in these transgenic tobacco ?owers.A yeast two-hybrid assay demonstrated that both GtMYB1R1and GtMYB1R9proteins interacted with the GtbHLH1protein,previously identi?ed as an anthocyanin biosynthesis reg-ulator in gentian ?owers.In addition,a transient expres-sion assay indicated that activation of the gentian GtDFR promoter by the GtMYB3-GtbHLH1complex was partly canceled by addition of GtMYB1R1or GtMYB1R9.These results suggest that GtMYB1R1and GtMYB1R9act as antagonistic transcription factors of anthocyanin biosyn-thesis in gentian ?owers.These genes should consequently be useful for manipulating anthocyanin accumulation via genetic engineering in ?owers of other ?oricultural plant species.
Keywords Antagonistic transcription factor áAnthocyanin biosynthesis áFloral pigmentation áJapanese gentian áMYB1R
Abbreviations 4CL 4-Coumatate:CoA-ligase ANS Anthocyanidin synthase bHLH Basic helix-loop-helix C4H Cinnamate 4-hydroxylase CaMV Cauli?ower mosaic virus CHI Chalcone isomerase CHS Chalcone synthase EAR ERF-associated amphiphilic repression F3H Flavonoid 3-hydroxylase F30H Flavonoid 30-hydroxylase FLS Flavonol synthase DFR Dihydro?avonol 4-reductase PAL Phenylalanine ammonia lyase
Communicated by K.Toriyama.
T.Nakatsuka
Department of Biological and Environmental Science,Graduate School of Agriculture,Shizuoka University,836Ohya Suruga-ku,Shizuoka 422-8529,Japan E.Yamada áM.Saito áK.Fujita áM.Nishihara (&)Iwate Biotechnology Research Center,22-174-4Narita,Kitakami,Iwate 024-0003,Japan e-mail:mnishiha@ibrc.or.jp
Plant Cell Rep (2013)32:1925–1937DOI 10.1007/s00299-013-1504-4
qRT-PCR Quantitative reverse-transcription polymerase chain reaction
RNAi RNA interference
Introduction
Flower color is one of the most important traits in?oricul-tural plants.Molecular breeding by genetic transformation has consequently been used to alter this trait in various plant species(Davies2009;Nishihara and Nakatsuka2011;Ta-naka et al.2010).To obtain desired colors through such manipulation of?oral pigmentation,the silencing of endogenous genes is often required to reduce or redirect the metabolic?ow of intermediates.Although techniques for inducing suppression of speci?c genes of interest in?owers are proven strategies,including transcriptional or post-transcriptional gene-silencing technologies such as anti-sense,co-suppression,and RNA interference(RNAi) methods(Tsuda et al.2004;Nakamura et al.2006),they usually require the isolation of target genes from individual ?oricultural plants.Genomic or expressed sequence tag analyses have still not been performed for most?oricultural plant species,and even when sequence information is available,cloning and vector construction is a time-con-suming process.To solve this problem,some researchers have applied dominant-negative mutant-based methods.For example,a dominant-negative chalcone synthase(CHS), developed by mutating a165th-residue cysteine,essential for catalytic activity,and a138th-residue methionine pro-truding into the adjoining CHS monomer,has been reported to modulate?ower color intensity in transgenic petunias (Hanumappa et al.2007).Similarly,constitutive expression of a mutated allele of c1,which encodes a transcription factor for anthocyanin biosynthesis in maize kernels,has been found to signi?cantly reduce anthocyanin accumulation in transgenic tobacco petals(Chen et al.2004).Chimeric repressor gene-silencing technology(CRES-T)has also been developed as an ef?cient silencing system.In this system,a transcription factor is converted from an activator into a repressor by fusion to the ERF-associated amphiphilic repression(EAR)motif;this chimeric repressor dominantly suppresses expression of target genes of the transcription factor(Hiratsu et al.2003;Mitsuda et al.2006).CRES-T has been found to be useful for suppressing anthocyanin bio-synthetic genes regulated by certain transcription factors, with color intensity reduction and color pattern design suc-cessfully achieved in Japanese gentian(Nakatsuka et al. 2011)and torenia(Shikata et al.2011)?owers.Transcription factor genes are thus promising targets for regulating?ower color intensity via these genetic engineering techniques.
The MYB family is one of the largest transcription factor families in higher plants and has acquired diverse biological functions over the course of its evolution(Dubos et al.2010; Feller et al.2011;Martin and Paz-Ares1997;Stracke et al. 2001).Some MYB transcription factors have been charac-terized as regulators of secondary metabolite synthesis(e.g., lignins,?avonoids,and volatile compounds)and cell mor-phological development(e.g.,trichome,hairy root,epider-mal cell,and pollen development).Of these factors,those controlling?avonoid biosynthesis have been well studied in Arabidopsis(Arabidopsis thaliana),maize(Zea mays), snapdragon(Antirrhinum majus),and petunia(Petunia hybrida).Flavonoids,including anthocyanins,?avonols,?avones,and proanthocyanins,are secondary metabolites playing important biological roles in plants(Winkel2006), with anthocyanins the most visible of these in?ower and fruit pigmentation.Anthocyanin and proanthocyanidin biosyn-theses are regulated by a transcriptional activation complex (MYB-bHLH-WDR)consisting of R2R3-MYB,basic helix-loop-helix(bHLH),and WD-repeat(WDR)proteins,which activates transcription of?avonoid biosynthetic genes (Baudry et al.2004;Xu et al.2013).In petunia?owers, ANTHOCYANIN2(AN2,R2R3-MYB)interacts with bHLH proteins,either ANTHOCYANIN1(AN1)or JAF13, and the resulting complexes activate transcription of the dihydro?avonol4-reductase(DFR)gene(Quattrocchio et al. 1999;Spelt et al.2000).In Arabidopsis,PRODUCTION OF ANTHOCYANIN PIGMENT1(PAP1)and PAP2,which belong to R2R3-MYB subgroup6,control anthocyanin biosynthesis in vegetative tissues(Borevitz et al.2000), while TRANSPARENT TESTA2(TT2),belonging to subgroup2,controls proanthocyanidins in the seed coat (Baudry et al.2004).The[D/E]Lx2[R/K]x3Lx6Lx3R signa-ture motif,involved in the interaction with bHLH proteins (Zimmermann et al.2004),is present in the R3repeat region of MYB proteins responsible for regulation of trichome patterning,root hair formation,and proanthocyanin,antho-cyanin,and sinapate ester biosynthesis in Arabidopsis(Du-bos et al.2010).We have previously demonstrated that biosynthesis of polyacylated anthocyanin(gentiodelphin)in Japanese gentian(Gentiana tri?ora)?owers is controlled by a complex between GtMYB3and GtbHLH1transcription factors(Nakatsuka et al.2008).
In contrast to the above examples,some?avonoid bio-synthesis pathways are controlled by R2R3-MYB tran-scription factors independently of bHLH proteins (Grotewold et al.2000).For example,R2R3-MYBs At-MYB11,AtMYB12,and AtMYB111,classi?ed into sub-group7,control?avonol biosynthesis in all Arabidopsis plant tissues(Mehrtens et al.2005;Stracke et al.2007).In addition,maize P1and P2control C-glycosyl?avone mysine and3-deoxyanthocyanidin phlobaphene biosyn-theses(Cocciolone et al.2005;Grotewold et al.1994).
More recently,we reported that GtMYBP3and GtMYBP4 from Japanese gentian activate transcription of early?a-vonoid biosynthetic genes(Nakatsuka et al.2012).
In addition to the transcription activators for?avonoid biosynthesis described above,transcription repressors have also been reported in several plants.Arabidopsis At-MYBL2,strawberry FaMYB1have the features of MYBR1and R2R3-MYB,respectively,and contain C-terminal motifs such as EAR or TLLLFR(L2R)(Aha-roni et al.2001;Dubos et al.2008;Matsui et al.2008). Arabidopsis MYB4,categorized into R2R3-MYB sub-group4,controls sinapate ester biosynthesis in a UV-dependent manner and suppresses cinnamate4-hydroxy-lase(C4H)gene expression(Jin et al.2000;Hemm et al. 2001).The AtMYB4protein also contains an EAR motif at the C-terminal.Petunia PhMYB27was also an R2R3-MYB subgroup4repressor and involved in the light-induced anthocyanin accumulations in vegetative tissues(Albert et al.2011).In addition to MYB transcription factors with repression motifs,Arabidopsis CAPRICE(AtCPC),a MYB1R transcription factor,is not only involved in root hair differentiation and trichome initiation(Wada et al. 1997),but is also a negative regulator of anthocyanin biosynthesis(Zhu et al.2009).The amino acid sequence of AtCPC also contains a bHLH interaction motif.The At-CPC protein is thus believed to negatively control antho-cyanin biosynthesis by competing with anthocyanin biosynthetic R2R3-MYB(PAP1/2,in the case of Arabi-dopsis)for complexation with bHLH transcription factors (GLABRA3[GL3]/ENHANCER OF GLABRA3[EGL3]) (Zhu et al.2009).More recently,an MYB1R repressor, ROSE INTENSITY1(ROI1),was identi?ed as a major quantitative trait locus(QTL)regulating anthocyanin concentration in Mimulus(Yuan et al.2013).
In this study,we identi?ed and characterized two novel MYB1R transcription factors,GtMYB1R1and GtMYB1R9,from a Japanese gentian petal cDNA library. Overexpression of GtMYB1R1and GtMYB1R9in tobacco plants induced a decrease in?oral anthocyanin accumula-tion,indicating that these two genes had anthocyanin suppression abilities in planta.Detailed molecular biolog-ical analysis suggested that they act as competitive repressors in anthocyanin biosynthesis.These genes should therefore serve as useful biotechnological tools for gener-ation of?owers having different anthocyanin levels. Materials and methods
Construction of a cDNA library from gentian petals
Total RNAs were isolated from petal samples of Japanese gentian(G.tri?ora‘Maciry’)using RNAiso Plus and Fruit-Mate for RNA Puri?cation(Takara-bio,Otsu,Japan). mRNAs were puri?ed using a polyATract mRNA isolation system(Promega,Madison,WI,USA)and normalized using a TRIMMER cDNA normalization kit(Evrogen, Moscow,Russia).Normalized gentian petal cDNAs were subjected to sequencing on a Roche FLX genome sequencer,with the resulting cDNA contigs assembled by Genaris(Yokohama,Japan).
Isolation of single MYB transcription factors
from gentian petals
We searched the assembled contigs from the normalized gentian petal cDNA library for MYB transcription factor candidates using BLAST and InterProScan with the Blas-t2GO program(Conesa et al.2005).To obtain full-length cDNAs of GtMYB1R1and GtMYB1R9,30-and50-rapid ampli?cation of cDNA ends(RACE)technology was performed using a GeneRacer kit(Invitrogen,Carlsbad, CA,USA).Ampli?ed fragments were subcloned into a pCR4TA TOPO cloning vector(Invitrogen)and sequenced using a Big Dye terminator cycle sequencing kit version 1.1on an ABI PRISM3130DNA sequencer (Applied Biosystems,Foster City,CA,USA).Nucleotide sequences were conceptually translated into amino acid sequences using GENETYX-MAC version12(GEN-ETYX,Tokyo,Japan)and compared using the BLAST network service from the National Center for Biotechnol-ogy Information(NCBI).A phylogenetic tree was con-structed by the neighbor-joining method with1,000 bootstrap replicates using MEGA version5(Tamura et al. 2011).
Expression analysis of GtMYBs and GtbHLH1 transcription factors
Gene expression analysis was carried out using quantitative real-time PCR(qRT-PCR).Total RNA was isolated from petals at several?oral developmental stages(as de?ned by Nakatsuka et al.2005),leaves,and stems of Japanese gentian cultivar Maciry.qRT-PCR was performed on a StepOne Plus system(Applied Biosystems)using SYBR GreenER qPCR Super Mix(Invitrogen).After removal of genomic DNAs,cDNAs were synthesized from total RNAs using a PrimeScript RT reagent kit with gDNA eraser (Takara).Reaction mixtures(10l l)included the following components:19Master Mix,0.2l M of each primer,and 1l l cDNA.Cycling conditions were95°C for20s, followed by40cycles of95°C for1s and60°C for20s. The following primers are used:GtMYB1R1,50-GC GAAGGAAATAATATCCACCA-30and50-CCAAATA GAACGATCAATGCAA-30;GtMYB1R9,50-AAGATT ACCTGGACGGAGTGAA-30and50-TTGATGCTTTCT
GAATTTCCAA-30;GtMYB3,50-TGCACAAAATGACGA TAATACCCT-30and50-CCCCCGCTACTTTGAAAGT G-30;GtbHLH1,50-TCTCTTACTTTTTCCCTCCGGC-30 and50-CCGGACTACCAGGAAGGGCATACGC-30.Indi-vidual gene expression levels were calibrated using ubiq-uitin(GtUBQ)gene expression as a reference.
Protein-to-protein interaction assay
A yeast two-hybrid assay was performed using a Match-maker Yeast Two-Hybrid System3(Clontech,Mountain View,CA,USA)as described previously(Nakatsuka et al. 2008).Brie?y,open reading frame(ORF)sequences of GtMYB1R1and GtMYB1R9were cloned into pGAD-T7 vectors.We also used pGAD-GtMYB3and pGBK-GtbHLH1constructs as described in Nakatsuka et al. (2008).A quantitative assay of b-galactosidase(b-Gal) activity was performed using o-nitrophenyl b-D-galacto-pyranoside(ONPG)as a substrate.
Production of transgenic tobacco plants
pSKan-35SpGtMYB1R1and pSKan-35SpGtMYB1R9 were constructed from binary vectors harboring a pSMAB704backbone.Their constructs were transformed into Agrobacterium tumefaciens strain EHA101by elec-troporation.Tobacco plants(Nicotiana tabacum‘SR-1’), aseptically grown from seeds for about a month,were transformed via an A.tumefaciens-mediated leaf disc pro-cedure(Horsch et al.1985)and selected using200mg l-1 of kanamycin.After rooting and acclimatization,regener-ated plants were grown in a greenhouse to set seeds by self-pollination.T1transgenic plant lines were used for further analyses.Concentrations of anthocyanin and?avonol pig-ments in petals of transgenic tobacco plants were measured as described by Nakatsuka et al.(2007).
Expression analysis of endogenous?avonoid biosynthetic genes in transgenic tobacco plants
Total RNAs of transgenic tobacco were isolated from their petals at?oral developmental stage3using a FastRNA Green kit(Q-Bio,Irvine,CA,USA).qRT-PCR analysis was performed as described above.Primer sets for tobacco ?avonoid biosynthetic genes are given in Nakatsuka et al. (2012).
Transient expression assay using tobacco BY2cells
To evaluate whether GtMYB1R1and GtMYB1R9are responsible for regulation of?avonoid biosynthesis,tran-sient expression assays were performed using protoplasts from tobacco BY2suspension cells as described in Na-katsuka et al.(2012).We constructed the reporter vector GtDFRpro-LUC,which contained the?re?y luciferase (LUC)gene under the control of the Japanese gentian DFR promoter.ORFs of GtMYB1R1and GtMYB1R9were inserted into a p35Spro expression vector under the control of the CaMV35S promoter and NOS terminator,resulting in p35Spro-GtMYB1R1and p35Spro-GtMYB1R9, respectively.pBI221(Clontech)was used as a negative control vector.p35Spro-RLUC,the Renilla luciferase (RLUC)gene under the control of the CaMV35S promoter, was used as a transformation control.Protoplast isolation and PEG-transfection experiments were performed as described by Hartmann et al.(1998).Fire?y and Renilla luciferase activities were measured using a Dual-Glo luciferase assay system(Promega)and a Luminescencer JNR II(ATTO,Tokyo,Japan).To demonstrate reproduc-ibility,at least?ve independent transfections were per-formed for each plasmid combination.
Results
Isolation of MYB1R transcription factors
A normalized cDNA library was synthesized from Japa-nese gentian petals and subjected to sequencing analysis using GS FLX454pyrosequencing.After clustering and assembly,a total of701,124reads were incorporated into 16,534contigs with an average length of952.7bp.
Fig.1Phylogenetic tree and alignment of MYB proteins.a Results of phylogenetic analysis of MYB transcription factors based on R3 repeat sequences.The phylogenetic tree was created by neighbor-joining using MEGA5software(Tamura et al.2011).GenBank accession numbers for analyzed transcription factors are as follows: Japanese gentian(Gentiana tri?ora)GtMYB1R1(AB779612,this study),GtMYB1R9(AB779613,this study),GtMYB3(AB289445), GtMYBP3(AB733016),and GtMYBP4(AB289446);Arabidopsis CPC(NM_130205),ETC2(NM_179814),GL1(NM_113708), MYB2(NM_130287),MYB4(NM_120023),MYB12(DQ224277), MYB24(NM_123399),MYB32(NM_119665),MYBL2 (NM_105772),PAP1(NM_104541),TRY(NM_124699),TT2 (NM_122946),and WER(NM_121479);maize C1(MZEMYBAA) and P1(M73028);snapdragon MIXTA(X79108);petunia AN2 (AF146702),PH4(AY973324),mybPh1(Z13996)and mybPh2 (Z13997);potato mybSt1(S74753);strawberry FaMYB1 (AF401220),FaMYB5(JQ989280),FaMYB9(JQ989281),FaM-YB10(EU155162),and FaMYB11(JQ989282),and Mimulus lewisii MlROI1(JX992854).Closed circles indicate MYB1R transcription factor genes.Numbers at branches correspond to bootstrap values from1,000replicates,and the bar below the phylogenetic tree indicates genetic distance.b Amino acid sequence alignments of GtMYB1R1,GtMYB1R9,Arabidopsis AtMYBL2,AtCPC,and Mimulus MlROI1.The thick underline indicates the R3repeat region. The bHLH1interaction motif([D/E]Lx2[R/K]x3Lx6Lx3R),EAR-like repressor motif,and L2R motif are boxed
c
In addition to previously characterized MYB genes (Nakatsuka et al.2008,2012),14candidate MYB tran-scription factor genes,including partial fragments,were newly identi?ed from the Japanese gentian cDNA library using BLAST analysis.Of these,two MYB1R genes,termed GtMYB1R1and GtMYB1R9,showed high simi-larities to Arabidopsis anthocyanin biosynthetic negative regulator MYBL2(Dubos et al.2008;Matsui et al.2008;Fig.1).The GtMYB1R1cDNA sequence (GenBank accession number AB779612)was 908bp long and encoded a protein of 188amino acid residues,whereas the GtMYB1R9cDNA (accession no.AB779613)was 735bp and encoded 197amino acid residues.The deduced amino acid sequence of GtMYB1R1showed 90.6%identity with that of GtMYB1R9in the R3repeat motif and 67.7%identity overall (Fig.1b).The bHLH interaction motif [D/E]Lx 2[R/K]x 3Lx 6Lx 3R (Zimmermann et al.2004)was well conserved within the R3repeat domain of both GtMYB1R1and GtMYB1R9;however,neither EAR nor L2R motifs (Fig.1b),the repression motifs characterized in AtMYBL2(Matsui et al.2008)and FaMYB1(Aharoni et al.2001)were found in GtMYB1R1or GtMYB1R9.
Gene expression pro?les of GtMYB1R1and GtMYB1R9in gentian plants
To investigate temporal and spatial expression of GtMYB1R1and GtMYB1R9genes,we performed qRT-PCR analysis (Fig.2).Transcripts of both GtMYB1R1and GtMYB1R9were abundant in petal tissues,with maximum expression detected at stages 2and 3before anthesis.Slight GtMYB1R1expression was also detected in stems,pistils,and especially leaves,whereas the GtMYB1R9transcript was barely detected in any tissues except for petals.GtMYB1R1and GtMYB1R9gene expression pro?les were similar to those of the GtMYB3gene,a transcription activator for anthocyanin biosynthesis (Fig.2c).On the other hand,expression of GtbHLH1,a counterpart interacting with GtMYB3,was detected throughout the entire plant (Fig.2d).Effects of GtMYB1R overexpression in tobacco plants To investigate whether GtMYB1R1and GtMYB1R9are involved in anthocyanin biosynthesis in petals,we produced and analyzed transgenic tobacco plants overexpressing GtMYB1R1and GtMYB1R9.Two independent T 1
transgenic
Fig.2Temporal and spatial expression analysis of
GtMYB1R1,GtMYB1R9,and anthocyanin biosynthetic transcription factor genes in gentian.Expressions of
a GtMYB1R1,
b GtMYB1R9,
c GtMYB3,an
d d GtbHLH1wer
e investigated by
quantitative real-time PCR
(qRT-PCR)analysis of samples of petals at four different ?oral development stages,stamens,pistils,leaves,and stems of G.tri?ora ‘Maciry’.Each value was normalized relative to GtUBQ expression and is indicated as the
average ±standard deviation (SD)of ?ve biological replicates
lines from each construct were selected based on transgene expression levels (Fig.3a,b).After growing in a closed greenhouse,almost all of the ?owers of GtMYB1R1-or GtMYB1R9-expressing transgenic tobacco plants were white,compared with the pink ?owers of wild-type tobacco (Fig.3c).Anthocyanin levels in transgenic tobacco petals were 4.4–11.0%of wild-type petals (Fig.3d).Total ?avo-nol accumulations in transgenic tobacco petals were also signi?cantly decreased,showing 40–70%reductions com-pared with the wild type (Fig.3e).In transgenic tobacco ?owers,levels of quercetin derivatives were more reduced than those of kaempferol.Based on HPLC analyses,how-ever,no signi?cantly different peak related to anthocyanin and ?avonol compounds was observed between wild-type and transgenic ?owers (data not shown).
We then carried out an expression analysis of endoge-nous genes involved in the ?avonoid biosynthetic pathway in transgenic tobacco petals.These genes were phenylala-nine ammonia lyase (NtPAL ),NtC4H ,and 4-coumarate:CoA-ligase (Nt4CL )from phenylpropanoid
biosynthesis,chalcone synthase (NtCHS ),chalcone isom-erase (NtCHI ),?avanone 3-hydroxylase (F3H ),?avonol 30-hydroxylase (NtF30H ),and ?avonol synthase (NtFLS )from ?avonol biosynthesis,and NtDFR and anthocyanidin syn-thase (NtANS )from anthocyanin biosynthesis.Of these ten structural genes,the expressions of three,NtCHI ,NtDFR ,and NtANS ,were consistently downregulated in transgenic tobacco petals compared with wild type (Fig.4).In par-ticular,NtDFR and NtANS transcripts in both GtMYB1R1and GtMYB1R9transgenic tobacco showed remarkable reductions,3.2–37.5and 1.6–22.6%,respectively,com-pared with wild type.The expression levels of these two endogenous genes correlated with accumulated anthocya-nin amounts in petals (Fig.3d).NtCHI transcripts were also signi?cantly suppressed in transgenic plants,with a 46.4–54.2%reduction compared with wild-type petals (Fig.4).Expression levels of other phenylpropanoid bio-synthetic genes did not differ between wild-type
and
Fig.3Typical phenotypes and ?avonoid amounts of
GtMYB1R1-and GtMYB1R9-expressed tobacco ?owers.a Expression levels of the GtMYB1R1transgene in wild type (WT)and two lines of GtMYB1R1-overexpressed transgenic plants
(GtMYB1R1ox).b Expression levels of the GtMYB1R9
transgene in WT and two lines of GtMYB1R9-overexpressed transgenic plants
(GtMYB1R9ox).c Typical ?oral phenotypes of WT,
GtMYB1R1ox (Nos.1and 2),and GtMYB1R9ox (Nos.1and 2)plants.d Relative
anthocyanin concentrations of WT and transgenic petals.Anthocyanins were extracted with methanol containing 1%(v/v)hydrochloric acid,and the absorbance of the solution was then measured at 530nm.Replicates consisted of at least ?ve ?owers per line.e Flavonol concentrations of WT and transgenic petals.Flavonols were extracted with 80%methanol and converted to aglycon by hydrolysis
treatment.They were analyzed by HPLC as described in ‘‘Materials and methods ’’.Replicates consisted of at least ?ve ?owers per line
transgenic tobacco ?owers.NtAN2is a transcription factor that regulates late ?avonoid biosynthesis in tobacco plants (Pattanaik et al.2010).Although slight variations in NtAN2expression were observed between wild-type and transgenic petals (Fig.4),reduced anthocyanin accumula-tions observed in transgenic tobacco petals appear to be due to suppression of ANS and DFR transcription levels rather than to that of NtAN2
.
Fig.4Expression analysis of endogenous ?avonoid biosynthetic genes in transgenic tobacco ?owers.The effects of GtMYB1R1-and GtMYB1R9-overexpression on endogenous phenylpropanoid,?avo-nol,and anthocyanin biosynthetic genes were investigated by qRT-PCR analysis of petals of wild-type (WT)and transgenic plants immediately before anthesis,as de?ned by Nishihara et al.(2005).
The following plants,shown in Fig.3,were analyzed:WT,GtMYB1R1ox no.1(1–1)and no.2(1–2),and GtMYB1R9ox no.1(9–1)and no.2(9–2).Asterisks indicate statistically signi?cant differences between means of WT and transgenic lines,as judged by Student’s t test (*P \0.05;**P \0.01)
Protein-to-protein interaction between GtMYB1Rs and GtbHLH1
Because both GtMYB1R1and GtMYB1R9contained a bHLH interaction motif in the R3repeat region (Fig.1b),
we performed a yeast two-hybrid analysis to con?rm their interaction with the GtbHLH1protein.Yeasts harboring either pGAD-GtMYB1R1/pGBD-GtbHLH1or pGAD-GtMYB1R9/pGBD-GtbHLH1were able to survive on quadruple-dropout medium (data not shown),showing that GtMYB1R1and GtMYB1R9proteins formed heterodimers with the GtbHLH1protein.Interaction intensity between the three MYBs (GtMYB1R1,GtMYB1R9,and GtMYB3)and GtbHLH1was determined based on b -Gal activity (Fig.5).The strongest interaction with GtbHLH1was shown by the GtMYB3protein,followed by GtMYB1R1and then GtMYB1R9;in particular,GtMYB3exhibited 2.3-and 3.8-fold stronger b -Gal activity than GtMYB1R1and GtMYB1R9,respectively.
Promoter activation assay by transient expression of GtMYB1Rs and GtbHLH1
To investigate whether GtMYB1R1and GtMYB1R9had transcription suppression ability against anthocyanin bio-synthetic genes,we employed a transient expression assay using tobacco BY2cells and a GtDFR pro-LUC reporter vector (Fig.6).As observed previously (Nakatsuka et al.2008),the combination of GtMYB3and GtbHLH1enhanced GtDFR promoter activity 9.5-fold.In contrast,neither GtMYB1R1nor GtMYB1R9alone affected GtDFR promoter activity.Activation of the GtDFR promoter by coexpression of GtMYB3and GtbHLH1(100%)was partly canceled,however,by the addition of GtMYB1R1(47.3%)or GtMYB1R9(67.3%)(Fig.6).These levels of competitive suppression by GtMYB1R1and GtMYB1R9correlated with GtbHLH1protein interaction intensities observed using the yeast two-hybrid assay (Fig.5).
Discussion
To establish an alternative method for suppression of ?oral pigmentation by genetic transformation,we identi?ed two novel negative regulator genes involved in anthocyanin biosynthesis from Japanese gentian petals and analyzed the effects of heterologous expression in transgenic tobacco plants.
GtMYB1R1and GtMYB1R9possess the features of MYB1R transcription factors and exhibit high similarity (Fig.1)with the N-terminal R3repeat motif of Arabidopsis MYBL2,a known negative regulator of ?avonoid biosyn-thesis (Dubos et al.2008;Matsui et al.2008).Arabidopsis AtMYBL2and strawberry FaMYB1,both negative regu-lators of anthocyanin biosynthesis,contain EAR and L2R repressor motifs in their C-terminal regions (Aharoni et al.2001;Matsui et al.2008).In contrast,the C-terminal regions of GtMYB1R1and GtMYB1R9do not include
any
Fig.5Protein-to-protein interaction analysis using a yeast two-hybrid assay.The GtbHLH1protein was fused to the GAL4DNA-binding domain (BD)and assayed for its ability to bind with GtMYB1R1,GtMYB1R9,and GtMYB3fused to the GAL4activa-tion domain (AD).Interaction intensity between each protein is shown by yeast b -galactosidase activity.pGBK-T7and pGAD-T7are the negative controls for bait and prey,
respectively
Fig.6Effect of GtMYB1R1and GtMYB1R9on promoter activities of the gentian DFR gene.Transient expression assays were performed by transfecting reporter and effector plasmid DNA into protoplasts from tobacco BY2cells.GtDFRpro-LUC was used as the reporter,and p35Spro-GtMYB1R1,p35Spro-GtMYB1R9,p35Spro-GtMYB3/p35Spro-GtbHLH1,or pBI221(negative control)was used as the effector.Our previous study discovered that the combination of p35Spro-GtMYB3and p35Spro-GtbHLH1can activate transcripts of anthocyanin biosynthetic genes (Nakatsuka et al.2008).p35Spro-RLUC was also used as a transformation control.Promoter activation activities are indicated as relative values compared with that of the negative control
obvious repression motifs(Fig.1b).These features of GtMYB1R1and GtMYB1R9are similar to AtCPC rather than AtMYBL2.Consequently,although GtMYB1R1and GtMYB1R9exhibit high similarities with Arabidopsis MYBL2,they would be considered putative orthologues of AtMYBL2.
Tobacco plants overexpressing GtMYB1R1and GtMYB1R9had signi?cantly reduced levels of anthocyanin pigmentation in their petals,resulting in white?owers (Fig.3).In these transgenic petals,slight decreases in?a-vonol accumulation were also observed.Expression anal-yses con?rmed that three endogenous?avonoid biosynthetic genes,NtCHI,NtDFR,and NtANS,were sig-ni?cantly downregulated in transgenic tobacco plants,with NtDFR and NtANS transcripts notably suppressed(Fig.4). These results indicate that suppression of speci?c?avonoid biosynthetic genes induced the reduction in?oral pig-mentation.The transcription factor NtAN2activates anthocyanin biosynthesis in tobacco plants and regulates DFR and ANS gene expression(Pattanaik et al.2010).No correlated changes in NtAN2expression levels and?oral pigmentation were observed,however,between wild-type and transgenic?owers(Fig.4).Consequently,the antho-cyanin reduction in transgenic tobacco?owers was not due to changes in the expression level of the endogenous NtAN2transcription factor gene.These results suggest that GtMYB1R1and GtMYB1R9act as repressors of antho-cyanin biosynthesis in?owers and may have a redundant function.NtCHI gene downregulation appears to be responsible for the apparent reduction in?avonol concen-tration in transgenic tobacco?owers(Fig.4).In another study,suppression of NtAN2by RNAi did not affect expression levels of the early biosynthetic genes NtCHS and NtCHI in tobacco?owers(Pattanaik et al.2010).The downregulation of NtCHI in GtMYB1R1-and GtMYB1R9-transgenic tobacco?owers thus seems to be caused by a different mechanism than that responsible for NtDFR and NtANS gene suppression.When apple anthocyanidin reductase(MdANR)was overexpressed in tobacco plants, endogenous NtCHI,NtDFR,and NtLAR genes were sig-ni?cantly suppressed in transgenic?owers compared with those of the wild type(Han et al.2012).The transgenic tobacco plants had suppressed proanthocyanin and antho-cyanin biosyntheses,resulting in white-?owered pheno-types.We thus believe that decreases in anthocyanin accumulation induced feedback suppression of NtCHI transcripts in our transgenic tobacco plants.
The yeast two-hybrid assay demonstrated that both GtMYB1R1and GtMYB1R9interact with the GtbHLH1 protein(Fig.5).As in Arabidopsis AtMYBL2and AtCPC (Zimmermann et al.2004),GtMYB1R1and GtMYB1R9 also contain a motif involved in interaction with the bHLH protein in the R3repeat region(Fig.1b).Their interactions with GtbHLH1were weaker than that of GtMYB3,how-ever,with interaction intensities42.9%(GtMYB1R1-GtbHLH1)and26.1%(GtMYB1R9-GtbHLH1)of that detected between GtMYB3and GtbHLH1(Fig.5).In the transient expression assay with tobacco BY2cell protop-lasts,activation of the GtDFR promoter by coexpression of GtMYB3and GtbHLH1was signi?cantly suppressed by the addition of GtMYB1R1or GtMYB1R9(Fig.6).This result suggests that these proteins regulate the?avonoid biosynthetic pathway by acting as antagonistic transcrip-tion factors.Their competitive suppression abilities seemed to correlate well with observed interaction intensity with the GtbHLH1protein(Fig.5).
Suppression of?ower color intensity by heterologous expression of transcription factor genes has been reported previously(Chen et al.2004;Zhang et al.2009).For example,heterologous expression of AtCPC gene has been found to induce suppression of anthocyanin accumulation in tobacco?owers,and to alter the density of trichomes and hairy roots(Zhang et al.2009);this is consistent with the fact that AtCPC is a regulator not only of anthocyanin biosynthesis,but also of trichome and hairy root develop-mental programs(Wada et al.1997;Zhu et al.2009).In another case,the overexpression of chrysanthemum CmMYB1,an R2R3-MYB categorized into subgroup4,not only resulted in reduced?avonoid levels,but also altered lignin composition in transgenic Arabidopsis(Zhu et al. 2013).
Finally,we consider the biological function of GtMYB1R1and GtMYB1R9in Japanese gentian?owers. The repressors(GtMYB1R1and GtMYB1R9)and activator (GtMYB3)of anthocyanin biosynthesis were expressed simultaneously in gentian petals(Fig.2).In strawberry fruits,FaMYB1and FaMYB10are the R2R3-MYB repressor and activator,respectively,of anthocyanin bio-synthesis(Aharoni et al.2001;Lin-Wang et al.2012). More recently,Salvatierra et al.(2013)revealed that FcMYB1,which is an ortholog of FaMYB1,regulates the branching point of anthocyanin and proanthocyanin bio-synthesis in fruits of white Chilean strawberry.In one study,the expression pro?le of FaMYB1was similar to that of FaMYB10,with the highest transcription taking place at the ripe fruit stage.A40,000-fold increase in FaMYB10relative transcript levels was detected,while FaMYB1expression showed little change(Lin-Wang et al. 2012).In our study,GtMYB3relative expression increased 20-fold,with GtMYB1R1and GtMYB1R9experiencing only 4.0-and 6.1-fold increases,respectively(Fig.2). Anthocyanin biosynthesis in gentian petals is therefore most likely controlled by expressional balance of the GtMYB3activator and GtMYB1R1/9repressors,as seen in strawberry fruit tissues.Consequently,one possible bio-logical role for GtMYB1R1and GtMYB1R9in gentian
petals is to serve as a brake on anthocyanin biosynthesis in petals,as previously proposed in petunia(PhMYBx) researches(Koes et al.2005;Quattrocchio et al.2006; Albert et al.2011).PhMYBx was expressed in petals and anthocyanin-induced vegetative tissue(Albert et al.2011). PhMYBx protein can interact with bHLH proteins,PhAN1 and PhJAF13(Koes et al.2005;Quattrocchio et al.2006). PhMYBx has been proposed to act as an inhibitor of anthocyanin biosynthesis by sequestrating PhAN1in inactive complexes.Constitutive expression of PhMYBx accordingly suppresses anthocyanin biosynthesis in petunia ?owers(Quattrocchio et al.2006).Constitutive expressions of the maize c1mutated allele and Arabidopsis CPC sig-ni?cantly reduce anthocyanin accumulation in transgenic tobacco petals(Chen et al.2004;Zhang et al.2009).In Mimulus,ROI1,categorized into MYB1R,has been iden-ti?ed as a major QTL for petal anthocyanin concentration (Yuan et al.2013).The anthocyanin concentrations of ROI1-suppressed transgenic lines were*2-fold higher than that of wild type.This result has supported that MYB1R with bHLH interaction motif might play a role as feedback repressor of anthocyanin biosynthesis.In Arabi-dopsis,the MYB-bHLH-WDR complex can also regulate the expression of TT8,which encodes a bHLH protein,in a positive feedback loop that ensures high accumulation of proanthocyanin in the endothelium(Baudry et al.2006). Because PhMYBx mRNA expression is partially down-regulated in an1and an11petals;it may be part of an auto-regulatory loop that modulates PhAN1activity(Koes et al. 2005).In our study,the interaction between GtMYB3and GtbHLH1was stronger than interactions of GtMYB1R1/9 with GtbHLH1(Fig.5).The combinatorial interaction of MYB and bHLH proteins in?uences epidermal cell identity and it is the MYB proteins that ultimately determine which cell fate is adopted(Ramsay and Glover2005).Therefore, GtMYB1R1,GtMYB1R9and GtMYB3would exhibit their biological functions when the expression of each MYB overlaps with that of GtbHLH1(Fig.2).Indeed,these three MYB genes indicate similar expression pro?les(Fig.2). This implies that GtMYB1R1and GtMYB1R9compete with GtMYB3for binding to a limited supply of GtbHLH1 protein.Consequently,GtMYB1R1and GtMYB1R9may participate in an anthocyanin biosynthesis braking activa-tion loop by disrupting the complex between GtMYB3and GtbHLH1.
In summary,we identi?ed two novel single-repeat R3 MYB transcription factors from Japanese gentian—GtMYB1R1and GtMYB1R9—that appear to act as antagonistic transcription factors in anthocyanin biosyn-thesis in gentian petals.To date,negative regulation of anthocyanin biosynthesis in?owers is largely unknown; our?ndings thus provide new insights into regulatory mechanisms of?oral pigmentation.In addition,because GtMYB1R1and GtMYB1R9are dominant-negative reg-ulators of anthocyanin biosynthesis—as demonstrated in heterologous tobacco plants—they should be useful for genetically engineering pure-and pale-white?ower colors in other important?oricultural plants.
Acknowledgments We thank Ms.Akiko Kubota,Iwate Biotech-nology Research Center,for technical support.This work was ?nancially supported by Grants-in-Aid for Scienti?c Research from the Japan Society for the Promotion of Science(No.24380024)and by the Iwate prefectural government.
Con?ict of interest The authors declare that they have no con?ict of interest.
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