TheMYB Activator WHITE PETAL1 Associates withMtTT8 andMtWD40-1 to Regulate Carotenoid-Derived FlowerPigmentation in Medicago truncatula[OPEN]

YingyingMeng,a,1 Zuoyi Wang,a,1 YiqinWang,b ChongnanWang,a Butuo Zhu,a Huan Liu,aWenkai Ji,a Jiangqi Wen,c

Chengcai Chu,b Million Tadege,d Lifang Niu,a and Hao Lina,2

a Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, Chinab The State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,Beijing 100101, ChinacNoble Research Institute, Ardmore, Oklahoma 73401dDepartment of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401

Carotenoids are a group of natural tetraterpenoid pigments with indispensable roles in the plant life cycle and the humandiet. Although the carotenoid biosynthetic pathway has been well characterized, the regulatory mechanisms that controlcarotenoid metabolism, especially in floral organs, remain poorly understood. In this study, we identified an anthocyanin-related R2R3-MYB protein, WHITE PETAL1 (WP1), that plays a critical role in regulating floral carotenoid pigmentation inMedicago truncatula. Carotenoid analyses showed that the yellow petals of the wild-type M. truncatula contained highconcentrations of carotenoids that largely consisted of esterified lutein and that disruption of WP1 function via Tnt1 insertionled to substantially reduced lutein accumulation. WP1 mainly functions as a transcriptional activator and directly regulates theexpression of carotenoid biosynthetic genes including MtLYCe and MtLYCb through its C-terminal acidic activation motif.Further molecular and genetic analyses revealed that WP1 physically interacts with MtTT8 and MtWD40-1 proteins and thatthis interaction facilitates WP1’s function in the transcriptional activation of both carotenoid and anthocyanin biosyntheticgenes. Our findings demonstrate the molecular mechanism of WP1-mediated regulation of floral carotenoid pigmentation andsuggest that the conserved MYB-basic-helix-loop-helix-WD40 regulatory module functions in carotenoid biosynthesis inM. truncatula, with specificity imposed by the MYB partner.

INTRODUCTION

Carotenoids are important secondary metabolites widely dis-tributed in plants, algae, and certain types of bacteria and fungi(Grotewold, 2006;Nisar et al., 2015;Sunet al., 2018).Besides theircritical functions in human nutrition and health (Fraser andBramley, 2004), carotenoids play essential roles in the structureand function of the photosynthetic apparatus and provide pre-cursors for thebiosynthesis of phytohormones (Niyogi et al., 1997;Holt et al., 2005; Nambara and Marion-Poll, 2005; Al-Babili andBouwmeester, 2015). Moreover, carotenoids impart vivid colorsranging from yellow to red in flowers and fruits, and these colorsfulfill an important ecological function by attracting pollinatorvisitation and influencing reproductive success in flowering plants(Nisar et al., 2015; Sun et al., 2018).

Plant carotenoid biosynthesis has been studied extensively,and most key genes and enzymes in the carotenoid biosyntheticpathway have been well characterized (Fraser et al., 1994; Moiseet al., 2014;Nisar et al., 2015; Sunet al., 2018). The first committedstep for carotenoid biosynthesis involves the condensation of twogeranylgeranyl diphosphate (GGPP) molecules into phytoene,

catalyzed by phytoene synthase (PSY). The colorless phytoene isthen subjected to a series of desaturation and isomerization re-actions catalyzed by phytoene desaturase (PDS), z-carotenedesaturase (ZDS), and carotenoid isomerase (CRTISO), result-ing in the red pigment lycopene. Subsequent cyclization of ly-copene by lycopene b-cyclase (LYCb) alone or with lycopenee-cyclase (LYCe) produces orange b-carotene and a-carotene,respectively. These cyclized carotenes are further oxygenated byb-carotene hydroxylase (BCH), cytochromes P450 (CYP97A andCYP97C), zeaxanthin epoxidase, violaxanthin de-epoxidase, andneoxanthin synthase to yield yellow xanthophylls including lutein,zeaxanthin, antheraxanthin, violaxanthin, and neoxanthin. Furthermodification of xanthophylls and carotenes generates variousspecies-specific carotenoids in diverse plants (Giuliano, 2014,2017; Sun et al., 2018). Despite this well-established biosyntheticpathway, the regulatory control of carotenogenesis remains poorlyunderstood.Extensive studieshave indicated that thediversity of carotenoid

pigmentation in plants is largely associated with differential ex-pression of carotenoid biosynthetic genes (Moehs et al., 2001; Haet al., 2007; Chiou et al., 2010; Yamamizo et al., 2010). Recently,several families of transcription factors (TFs) have been demon-strated to directly regulate the carotenogenic genes and controlcarotenoid biosynthesis in diverse plant species. In Arabidopsis(Arabidopsis thaliana), the basic-helix-loop-helix (bHLH) TFPHYTOCHROME INTERACTING FACTOR1 (PIF1) and the basicleucine zipper TF LONG HYPOCOTYL5 (HY5) antagonisticallyregulate carotenoid accumulation by directly binding to the

1 These authors contributed equally to this work.2 Address correspondence to linhao@caas.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Hao Lin (linhao@caas.cn).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00480

The Plant Cell, Vol. 31: 2751–2767, November 2019, www.plantcell.org ã 2019 ASPB.

promoter of PSY in response to light signaling (Toledo-Ortiz et al.,2010, 2014). The APETALA2 (AP2)/ethylene-responsive elementbinding protein RAP2.2 has also been implicated in binding thecis-acting element ATCTA in the promoter of PSY (Welsch et al.,2007). Recently, CsMADS6 was found to directly activate LCYb1expression in Citrus sinensis. Overexpression of CsMADS6 sig-nificantly increased carotenoid accumulation in Citrus calli andinduced the expression of the carotenogenic genes LCYb1, PSY,and PDS (Lu et al., 2018). Although progress has been made inunderstanding the transcriptional control of carotenoid accu-mulation in diverse plants, little is known about the regulation offloral carotenoid pigmentation.TheR2R3-MYBproteins comprise oneof the largest TF families

and play essential roles in regulating primary and second me-tabolism in plants (Allan et al., 2008; Dubos et al., 2010). Previousstudies have demonstrated that the R2R3-MYB TFs fulfill a keyfunction in regulating the biosynthesis of proanthocyanidin (PA)and anthocyanin, the widely distributed flavonoid pigments thatprovide the red to blue colors in flowers and fruits, by interactingwith bHLH and WD-repeat (WDR) proteins to form a conservedtranscriptional activation complex (Baudry et al., 2004; Koes et al.,2005; Ramsay and Glover, 2005; Xu et al., 2015). R2R3-MYBTFs are emerging as modulators of carotenoid production. InCitrus reticulate, the R2R3-MYB protein CrMYB68 represses thebiosynthesis of a- and b-branch carotenoids by downregulatingCrBCH2 andCrNCED5 expression (Zhu et al., 2017). By contrast,disruption of the R2R3-MYB protein REDUCED CAROTENOIDPIGMENTATION1 (RCP1) led to downregulation of all carotenoidbiosynthetic genes and reduced carotenoid content in Mimuluslewisii flowers, indicating that RCP1 positively regulates carot-enoid biosynthesis during flower development (Sagawa et al.,2016). Recent studies in kiwifruit (Actinidia deliciosa) revealed thatAdMYB7 plays a role in modulating carotenoid accumulation byactivating AdLCY-b expression (Ampomah-Dwamena et al.,2019), indicating that there may be a variety of MYBs from un-related clades involved in carotenoid regulation in different plantspecies. Despite these enlightening reports, little is known aboutthe regulatory mechanism of R2R3-MYB TFs in the regulation ofcarotenoid metabolism.Medicago truncatula isamodel legumespecieswithvividyellow

flowers, generally assumed to be pigmented by carotenoids, withfine red veins of anthocyanin accumulation in the center of thevexillum petals (Xie et al., 2004; Jun et al., 2015). The availabilityof abundant floral pigment mutants in M. truncatula provides anideal system for investigating the regulation of flower color andfloral carotenoid pigmentation. Here, we report the identificationand characterization of white petal1 (wp1) from an M. truncatulamutant that is defective in floral pigmentation. WP1 encodesa subgroup 6 R2R3-MYB TF that positively regulates lutein and

Figure 1. Phenotype Analysis of wp1 Flowers.

(A) to (C) Flowers of the wild type (A), wp1-1 (B), and wp1-2 (C). Bars 5

1.5 mm.(D) to (F) Dissected flowers of wild type. The vexillum petal (D), the fusedalaeandkeelpetals (E), and thestamens (F)of thewild type.Bars51.5mm.(G) to (I)Dissected flowers ofwp1-1. The vexillum petal (G), the fused alaeand keel petals (H), and the stamens (I) of wp1-1. Bars 5 1.5 mm.(J) HPLC chromatograms and characteristic absorbance spectra of theextracted and saponified carotenoids from petals of the wild type and two

wp1 mutants. The peaks for lutein, lutein epoxide, and violaxanthin are

marked. Insets are spectra of indicated peaks. mAU, milli-absorbance

unit; WT, wild type.

(K)Contents of lutein, lutein epoxide, and violaxanthin in petals of the wildtype and twowp1mutants. Bars represent means6 SD of three biologicalreplicates. Asterisks indicate differences from the wild type (**P < 0.01,Student’s t test). FW, fresh weight; WT, wild type.

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anthocyanin accumulation in M. truncatula petals by directlyactivating carotenoid and anthocyanin biosynthetic genes. Ourresults demonstrate that WP1 physically interacts with theM. truncatula bHLH protein MtTT8 and WDR family memberMtWD40-1 and that this interaction facilitates WP1 function bothin floral carotenoid and anthocyanin production, suggesting thattheMYB-bHLH-WDR (MBW) regulatorymodule plays a key role incarotenogenesis that is analogous to its role in the control offlavonoid biosynthesis.

RESULTS

Identification of the wp1 Mutants of M. truncatula

Two phenotypically similar loss-of-floral pigment mutants namedwp1-1 and wp1-2 were identified by screening the M. truncatulaTnt1 insertion mutant population for visible white-flower pheno-types (Figures1A to1C).Genetic analysis revealed thatwp1-1andwp1-2 are allelic mutants (Supplemental Figure 1). In contrast tothewild-typeR108,whichhas yellowpetals, thewp1mutants lackthe typical yellow pigmentation in all vexillum, keel, and alaepetals, which appear nearly white, but no obvious pigment losswasobserved inanthers (Figures1Dto1I). Toexaminewhether thereduced petal coloration in the wp1 mutants was due to a re-duction in carotenoid accumulation, we analyzed the carotenoidprofiles of the wild-type,wp1-1, andwp1-2 petals. HPLC analysiswithorwithout saponification revealed that the yellowpetalsof thewild-type M. truncatula contained high levels of carotenoids thatlargely consisted of esterified lutein but that the levels of luteinwere drastically reduced in bothwp1-1 andwp1-2 (Figures 1J and1K; Supplemental Figures 2 and 3). In addition, the contents oflutein epoxide and violaxanthin were also decreased in the petalsofwp1mutants (Figures 1Jand1K). These results indicate that thewp1 mutants fail to accumulate lutein in petals and that WP1function is highly relevant to carotenoid biosynthesis duringM. truncatula flower development.

Molecular Cloning and Characterization of WP1

TheWP1 gene was cloned by PCR-based genotyping of flankingsequence tags (FSTs) in segregating populations (Tadege et al.,2008). Flanking sequence analysis of the Tnt1 retrotransposon inthe wp1-1 mutant by PCR-based genotyping revealed that oneflanking sequence, FST6, segregatedwith themutant phenotype.The wp1-1 mutant phenotype segregated as a single Mendelianrecessivemutation (i.e., heterozygous parents produced progenythat segregated 3:1 [71:25] for the wild-type-like and mutantplants), andallmutantplantswerehomozygous for FST6. The full-length gene sequence corresponding to this particular FST wasrecovered and designated to represent the WP1 gene. The ge-nomic sequence of WP1 contains three exons, and the Tnt1retrotransposon in wp1-1 is inserted at the middle of exon 2(Figure 2A). RT-PCR and sequencing analyses showed thatthis Tnt1 insertion led to an aberrantly splicedWP1mRNA lackingexon 2 (130 bp), which resulted in a frameshift and gener-ated a premature translation termination product (Figure 2B;Supplemental Figure 4). The transcript level of this exon-skipping

isoform ofWP1was severely decreased inwp1-1 (Figure 2B). Wefurther confirmed that in thewp1-2mutant, the presence of a Tnt1insertion in the third exon of WP1, 340 bp upstream of thetranslational stop, cosegregated with the phenotype (Figure 2A).RT-PCR analysis revealed that the full-lengthWP1 transcript wasabsent in the wp1-2 allele (Figure 2B). The identity of WP1 wasfurther confirmedbygenetic complementation.Aconstruct (WP1-GFP) including a 3.0-kb WP1 promoter region, the entire WP1genomic DNA sequence, a GFP gene fusing hemagglutinin tag,and the 1.5-kb downstream region of WP1 was introduced intowp1-1 plants by Agrobacterium tumefaciens-mediated trans-formation (Supplemental Figure 5). Phenotypic analysis showedthat the floral pigmentation phenotypes of wp1-1 were

Figure 2. Molecular Cloning and Confirmation of the WP1 Gene.

(A) Schematic representation of the gene structure of WP1 showing theTnt1 insertion sites inwp1-1 andwp1-2. Introns are represented by a line,and exons are represented by a striped box.(B)RT-PCRshowing transcript abundanceofWP1 in theflowersof thewildtype and wp1 mutants. MtActin was used as the control. WT, wild type.(C)Genetic complementation ofwp1-1. Representative flowers of the wildtype, wp1-1, and wp1-1 complemented with WP1-GFP (WP1-GFP/wp1-1). Bars 5 1.5 mm. WT, wild type.(D) Transcript levels of WP1 in petals of the wild type, wp1-1, and WP1-GFP/wp1-1. Bars represent means 6 SD of three biological replicates.Asterisks indicate differences from the wild type (**P < 0.01, Student’st test). WT, wild type.(E) Lutein concentrations in petals of the wild type,wp1-1, andWP1-GFP/wp1-1. Bars represent means 6 SD of three biological replicates. Asterisks in-dicate differences from thewild type (**P < 0.01, Student’s t test).WT, wild type.

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complemented by theWP1-GFP transgene in all three transgeniclines and comparable to those of the wild type (Figure 2C). Mo-lecularandHPLCanalyses indicated that theWP1 transcript levelsand lutein concentrationwere fully restored to thewild-type levelsin WP1-GFP complementation plants (Figures 2D and 2E). Col-lectively, these data confirm that disruption of WP1 function re-sults in thefloral carotenoid pigmentation defects ofwp1mutants.

WP1 encodes a MYB TF belonging to the R2R3 class, basedon the presence of conserved R2 and R3 MYB DNA binding do-mains (Stracke et al., 2001; Dubos et al., 2010). In a phylogeneticanalysis using the R2R3 domains, WP1 was clustered with MYBsinvolved in the regulation of anthocyanin (subgroup 6; Figure 3A;Supplemental Data Set 1). A protein BLAST search against theNational Center for Biotechnology Information (NCBI) database ofArabidopsis showed that the closest match for WP1 is AtMYB113,which controls anthocyanin regulation. WP1 was also clusteredwith subgroup 6 when analyzed using the web-based tool IT3F (AnInterspecies Transcription Factor Function Finder for Plant). Ac-cordingly, sequence analysis showed that WP1 contains theconservedbHLH-interactingmotifandtheANDVmotifwithin theR3domain immediately following the R2 domain, and the [R/K]Px[P/A/R]xx[F/Y] motif downstream of the conserved R2 and R3MYBDNAbinding domains (Supplemental Figure 6), all of whichare defining features of subgroup 6 MYBs (Stracke et al., 2001;Zimmermann et al., 2004; Dubos et al., 2010; Lin-Wang et al.,2010).

Expression Patterns of WP1 and Subcellular Localization ofthe WP1 Protein

RT-qPCR analysis of different tissues revealed that WP1 ex-pression is primarily detected in the flowers and that transcriptabundance is highest at the mature floral stage (Figures 3B and3C). Further RT-qPCR analysis of dissected floral organs showedthat WP1 is predominantly expressed in the vexillum, keel, andalae petals (Figure 3D), which is consistent with WP1 function inthe regulation of M. truncatula petal carotenoid pigmentation.

To determine the subcellular localization ofWP1, we generateda Pro35S:WP1-GFP fusion construct and coexpressed it with thenuclearmarkermonomeric redfluorescentprotein (mRFP)-AHL22(Wang et al., 2013) in Nicotiana benthamiana leaf epidermal cells.Using fluorescence microscopy, we observed that the WP1-GFPfusion protein colocalized with the nuclear marker mRFP-AHL22(Figure 3E), confirming that WP1 functions as a nuclear-localizedtranscription regulator.

WP1 Functions as a Transcriptional Activator

To gain insight into how WP1 regulates floral carotenoid pig-mentation, we performed a transactivation activity assay in yeast(Saccharomyces cerevisiae) to test the transcriptional activationactivity of WP1. The full-length WP1 was fused in-frame with theGAL4 DNA binding domain (BD) in the pGBKT7-GW vector, andthe resultant constructwas transformed into theGold yeast strain.The growth of yeast carrying pGBKT7-WP1 on selective medium(SD/2Trp/2His/2Ade) along with an a-galactosidase assay in-dicated that WP1 protein has strong transcriptional activation

Figure 3. Phylogenetic Analysis of the WP1 Protein and ExpressionPattern of WP1.

(A) Phylogenetic analysis of WP1 (marked with a red diamond) andother MYBs using R2R3 MYB domains. Subgroup (S) names are indi-cated on the right, and the subgroups are shaded.(B) to (D) RT-qPCR analysis of WP1 expression in various tissues (B),different floral stages (C), and dissected floral organs (D). Bars representmeans 6 SD of three biological replicates.(E) Subcellular localization of WP1-GFP in N. benthamianaleaf epidermal cells. AHL22 was used as a nuclear marker. Bars 5 20 mm.

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activity comparedwith the emptypGBKT7-GWvector usedas thenegative control (Figure 4A). Furthermore, fusion of an exogenousactivation domain, VP64, toWP1 enhanced its activation activity,whereas fusion of an exogenous repression module, EAR4(tetrameric repeats of EAR), to WP1 diminished this activation(Figure 4A), confirming that WP1mainly acts as a transcriptionalactivator.

To explore how the structure of WP1 influences its transcrip-tional activation activity, we divided the WP1 protein into threemajor parts based on conserved domain analysis: the R2 repeatdomain (R2; amino acids 1 to 59), R3 repeat domain (R3; aminoacids 60 to 107), andC-terminal domain (CTD; amino acids 108 to229; Figure 4B). Analyzing individual and combined domain de-letions in the yeast assay indicated that theCTD, especially aminoacids 186 to 229, is essential for the activation activity of WP1(Figure 4B).

Further examination of the activation activity in different CTDdeletion mutants revealed that a 16-amino acid activation motif,MVGEFPMDFQLEGFEA, located at the CTD and including aminoacids 186 to 201, is important for the activation activity of WP1.Complete or partial deletion of this activationmotif led to abolitionor reduction, respectively, of WP1’s transactivation activity(Figure 4B). Notably, theMVGEFPMDFQLEGFEAmotif on its ownshowed strong transcriptional activation activity, butmutations ofacidic amino acid residues in the activation motif (Glu/Asp to Alaamino acid substitutions), similar to those reported by Ikeda et al.(2009), led to abolition of transactivation activity (Figure 4C), in-dicating that the acidic amino acids are critical for activation.Taken together, these data indicate that WP1 functions asa transcriptional activator and that the C-terminal motifMVGEFPMDFQLEGFEA is essential and sufficient for its activa-tion activity in the yeast assay.

WP1 Directly Activates the Expression ofCarotenogenic Genes

To determine the mechanism by which WP1 regulates floral ca-rotenoid pigmentation inM. truncatula, we analyzed the transcriptlevels of carotenoid pathway genes in wp1 petals at the maturestage. RT-qPCR analysis showed that, with the exception ofMtPSY, most carotenoid biosynthetic genes that are involved inthe biosynthesis of lutein, including MtPDS, MtZDS, MtCRTISO,MtLYCe, MtLYCb, MtCYP97A, MtBCH, and MtCYP97C, weresignificantlydownregulated inbothwp1-1andwp1-2petals,whileexpression of these repressed genes was completely restored inWP1-complemented wp1-1 petals (Figure 5A). These data areconsistent with the significant association of decreased expres-sion of carotenoid biosynthetic genes with reduced floral luteinlevels in wp1 mutants.

Owing to the fact that WP1 acts as a transcriptional activatorand noting the downregulated expression of carotenogenesisgenes inwp1, wehypothesized thatWP1maydirectly regulate theexpression of carotenogenesis genes and thus control floral ca-rotenoid pigmentation. Plant R2R3-MYB family proteins havebeen reported to recognize and bind to the MYB-core element(C/T)NGTT(G/A) and AC-rich element ACC(A/T)(A/C/T)(A/C/T)(Gómez-Maldonado et al., 2004; Xu et al., 2015; Gao et al., 2016;Zhuetal.,2017). Sequenceanalysis using thePlantCAREprogram

Figure 4. WP1 Acts as a Transcriptional Activator.

(A) Transactivation analysis of WP1, WP1-VP64, and EAR4-WP1using a yeast assay. VP64 and EAR4 are an exogenous activationdomain and an exogenous repression module, respectively. TheGAL4 DNA BD alone was used as the negative control. Plate auxo-troph and a-galactosidase assay showing transcriptional activationof each protein. Bars represent means 6 SD of three independentexperiments. a-gal, a-galactosidase; E, EAR4; V, VP64; -W, SD/2Trp; -W-H-Ade, SD/2Trp/2His/2Ade.(B) Mapping of the transactivation motif of WP1 using a yeastassay. R2, amino acids 1 to 59; R3, amino acids 60 to 107; CTD,amino acids 108 to 229. Plate auxotroph and a-galactosidaseassay showing transcriptional activation of each protein. Bars rep-resent means 6 SD of three independent experiments. a-gal,a-galactosidase; BD, GAL4 DNA binding domain; -W, SD/2Trp; -W-H-Ade, SD/2Trp/2His/2Ade.(C) Transactivation analysis of the C-terminal motif MVGEFPMDFQLEG-FEA using a yeast assay. BD stands for the GAL4 DNA binding domain.Mutations introduced into the C-terminal motif are indicated by red font.Plate auxotrophanda-galactosidase (a-gal) assayshowing transcriptionalactivationbyeachprotein.Bars representmeans6SDof three independentexperiments. a-gal, a-galactosidase; -W, SD/2Trp; -W-H-Ade, SD/2Trp/2His/2Ade.

WP1 Regulates Carotenoid Biosynthesis 2755

Figure 5. WP1 Directly Activates the Expression of Carotenoid Biosynthesis Genes.

(A) Transcript levels of carotenoid biosynthesis genes in petals of thewild type,wp1-1,wp1-2, andWP1-GFP/wp1-1 revealed byRT-qPCR. Bars representmeans 6 SD of three biological replicates; asterisks indicate differences from the wild type (**P < 0.01, Student’s t test). WT, wild type.(B)PutativeMYBbinding sites in the promoter regions of the carotenoid biosynthesis genes. The red andblue sequences correspond to theMYB-core andAC-rich element, respectively.(C)DNAbindingassaycorresponding to theputativebindingsitesofWP1shown in (B). TheHis-TFaproteinwasusedas thenegativecontrol. Bars representmeans 6 SD of three biological replicates; asterisks indicate differences from Boxm (**P < 0.01, Student’s t test).(D)and (E)ChIPassayshowing theassociationofWP1withseveral regions in thepromotersofMtLYCe (D)andMtLYCb (E). The regions testedbyChIPassaysare indicated in theschematic representation. TheputativeMYB-coreandAC-richelements areshownby redandblue lines, respectively.MtActinwasused fornormalization. Bars represent means 6 SD of three biological replicates; asterisks indicate differences from wp1-1 (*P < 0.05, **P < 0.01, Student’s t test).(F) Schematic representation of reporter and effector constructs used in transient expression assay. CBG, carotenoid biosynthesis gene.(G)Transient expression assay inArabidopsis protoplasts showingactivationof carotenoid biosynthesis genesby theWP1effector comparedwith theGFPcontrol. Bars representmeans6 SD of three biological replicates; asterisks indicate differences from theGFP control (*P < 0.05, **P < 0.01, Student’s t test).

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(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)predicted that one or multiple typical MYB binding sites (MYB-core and/or AC-rich elements) were present in the promoterregions of the downregulated carotenoid biosynthetic genes inwp1 (Figure 5B; Supplemental Figure 7). Using an in vitro DNAbinding assay, we found that WP1 protein fused to a His andTrigger Factor tag (His-TFa-WP1)was able to bind to fragmentscontaining these predicted MYB binding sequences, whilebindingwas significantly diminished by deletingMYB-core andAC-rich elements (Figure 5C). These results indicated thatWP1activation of carotenoid biosynthetic genes may be mediatedby binding to their specific promoter regions.

The direct in vivo binding of WP1 to the promoters of thesecarotenoid biosynthetic genes was further confirmed by chro-matin immunoprecipitation (ChIP) assays. Using the WP1-GFPtransgenic complementation plants andananti-GFPantibody,wefound that several of the promoter regions of the downregulatedcarotenogenesis genes MtLYCe and MtLYCb that contain MYB-core and AC-rich elements were significantly enriched in WP1-GFP chromatin (Figures 5D and 5E), indicating that WP1 directlybinds to multiple regions on the promoters of these target genes,consistent with the in vitro DNA binding results.

To further examine whether WP1 could directly regulate thetranscription of carotenogenesis genes, a transient expressionassay was performed using the luciferase system in Arabidopsisleaf protoplasts (Figure 5F). We found that coexpression ofthe WP1 effector protein and luciferase reporter constructsdrivenby theendogenouspromoters ofMtPDS,MtZDS,MtCRTISO,MtLYCe, MtLYCb, MtBCH, and MtCYP97C, all of which aredownregulated in wp1, resulted in significantly increased lumi-nescence intensity compared to the GFP control (Figure 5G), whiledeletion of the C-terminal motif MVGEFPMDFQLEGFEA aboli-shed WP1’s activation activity and consequently failed to activatedownstream carotenogenesis genes (Supplemental Figure 8). Onthe other hand, no obvious activation was detected when the WP1effector was coexpressed with the luciferase reporter driven by thepromoter of MtPSY, whose expression was not downregulated inthe wp1 mutants (Figures 5A and 5G). These results confirm thatWP1 specifically recognizes the promoters of the downregulatedcarotenogenesis genes and can activate their expression inplanta. These data together demonstrate that WP1 functions asa transcriptional activator that modulates floral carotenoid pig-mentation by directly regulating the expression of multiple ca-rotenoid biosynthetic genes in M. truncatula.

WP1 Physically Interacts with MtTT8 and MtWD40-1

Previous studies have shown that the R2R3-MYB TFs regulatetranscription by interacting with bHLH and WDR proteins to forma conserved MBW activator complex (Baudry et al., 2004; Koeset al., 2005; Ramsay and Glover, 2005; Xu et al., 2015). Given thatWP1 protein contains the highly conserved bHLH-interactingmotif, we examined whether WP1 functions within an MBWcomplex aswell. InM. truncatula, theWDRproteinMtWD40-1andbHLH TF MtTT8 have been identified (Pang et al., 2009; Li et al.,2016) and shown to physically interact with several R2R3-MYBTFs tomodulate flavonoid biosynthesis (Liu et al., 2014; Jun et al.,2015; Li et al., 2016). Ayeast two-hybrid (Y2H)assay revealed that,

despiteWP1 andMtTT8 showing strong self-activation activities,different combinations of WP1, MtTT8, and MtWD40-1 exhibitedinteractions (Figure 6A). We also observed that WP1, MtTT8, andMtWD40-1 appear to form homodimers (Figure 6A).These interactions and homodimerization amongWP1, MtTT8,

and MtWD40-1 were verified in N. benthamiana leaves by bi-molecular fluorescence complementation (BiFC) assays usingsplit yellow fluorescent protein (YFP; Figure 6B; SupplementalFigure 9). While sharp yellow fluorescence was clearly observedwhen WP1 was fused to the N-terminal half of YFP (nYFP) andMtTT8 or MtWD40-1 was fused to the C-terminal half (cYFP), orwhen MtTT8 and MtWD40-1 were fused to the two YFP halves(Figure 6B), no YFP fluorescence signal was detected in negativecontrols in which one-half of the split YFP (nYFP or cYFP) wasfused to WP1, MtTT8, or MtWD40-1 and the other half was usedalone (Figure 6B; Supplemental Figure 9).The WP1-MtTT8-MtWD40-1 physical interaction was further

confirmed in vivo by coimmunoprecipitation (Co-IP) assays. Wetransiently coexpressed Pro35S:WP1-Myc with Pro35S:MtTT8-Flag or Pro35S:MtWD40-1-Flag in N. benthamiana leaves. Totalproteins were isolated and incubated with anti–C-MYC magneticbeads to immunoprecipitate WP1-Myc. Anti-Myc and anti-Flagantibodies were then used to detect immunoprecipitated proteinshaving the corresponding tag. MtTT8 orMtWD40-1 was detectedin the immunoprecipitated WP1 complex, but not in the negativecontrol without WP1-Myc input (Figure 6C), indicating that WP1physically associates with MtTT8 and MtWD40-1 in planta.We also transiently coexpressed Pro35S:MtTT8-Myc withPro35S:MtWD40-1-Flag in N. benthamiana leaves and detectedthe MtWD40-1-Flag protein in the immunoprecipitated MtTT8-Myc complex (Figure 6C), confirming the Y2H and BiFC results.Taken together, these data suggest that WP1 physically interactswith both MtTT8 and MtWD40-1 in vivo to potentially form anMBW complex.

MtTT8 and MtWD40-1 Are Involved in FloralCarotenoid Biosynthesis

MtTT8andMtWD40-1havebeen identifiedasessential regulatorsof anthocyanin and PA biosynthesis in seed coat and vegetativetissues (Pang et al., 2009; Li et al., 2016). Recent studies revealedthat disruption of MtTT8 function also influences flower color (Lietal., 2016).BecauseMtTT8andMtWD40-1arestrong interactorsof WP1, we investigated their roles in floral carotenoid pigmen-tation by characterizing the mttt8 and mtwd40-1 mutants.Consistent with previous findings (Pang et al., 2009; Li et al.,

2016), the Tnt1 insertional loss-of-function mutants mttt8 andmtwd40-1 (Supplemental Figure 10) showed yellowish seed coatand lacked red pigmentation in leaflets and petioles as a result ofreduced PA and anthocyanin accumulation, while seed coat andpetiole pigmentationswere normal inwp1mutants and therewereno obvious carotenoid profile changes in leaflets ofwp1-1,mttt8,andmtwd40-1 relative to the wild type (Supplemental Figure 11).Interestingly, we found that both mttt8 and mtwd40-1 showedclearly pale-yellow petals with reduced lutein content comparedwith the vivid yellow petals of the wild type (Figures 7A and 7B).Consistent with the decreased lutein production, RT-qPCRanalysis showed that transcripts of the carotenoid biosynthetic

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genes that were downregulated in wp1 (Figure 5A) were de-creased in the petals of mttt8 and mtwd40-1 mutants as well(Supplemental Figure 12). Collectively, these results indicate thatboth MtTT8 and MtWD40-1 may be involved in floral carotenoidaccumulation and affect carotenogenic gene expression ina manner similar to WP1.

The WP1-MtTT8-MtWD40-1 Complex CoordinatelyRegulates Carotenoid Pigmentation in M.truncatula Flowers

To clarify the roles of MtTT8 and MtWD40-1 with respect to WP1function in regulating floral carotenoid pigmentation, we per-formed three complementary experiments. First, we generateddouble mutants involving combinations of wp1, mttt8, andmtwd40-1 and characterized the single and double mutants toinvestigate the genetic relationship among WP1, MtTT8, andMtWD40-1 in M. truncatula floral carotenoid pigmentation. Con-sistent with the scenario that the MYB protein acts as the chiefregulator in the MBW complex, the wp1 flowers showed the mostsevere phenotype, with drastically reduced lutein relative to themttt8 and mtwd40-1 single mutants, and the wp1 mttt8 andwp1 mtwd40-1 double mutants showed white petals with abol-ished lutein accumulation similar to thewp1 single mutant (Figures7A and 7B). By contrast, both the mttt8 and mtwd40-1 singlemutants and the mttt8 mtwd40-1 double mutant showed pale-yellow petals with partially reduced lutein contents (Figures 7A and7B), indicating that the contributions of MtTT8 and MtWD40-1 tofloral carotenoid pigmentation are secondary and redundant.Second, using in vitro DNA binding assays, we tested the direct

DNA binding activity of WP1 to its putative binding sites in thepresence ofMtTT8 and/orMtWD40-1. Coexpression ofWP1withMtTT8usually resulted in ahigherDNAbindingcapacity thanWP1alone, and addition of MtWD40-1 further enhanced activation(Figure 7C). In addition, MtTT8 was able to bind all of the putativeWP1 binding sites even in the absence of WP1 and MtWD40-1,while no similar binding was detected for MtWD40-1 (Figure 7C).These results suggest that MtTT8 and MtWD40-1 facilitate thecapacity of WP1 to recognize and bind to its target promoters.Third, using a transient promoter activation assay in Arabi-

dopsis protoplasts, we found that coexpression of WP1 withMtTT8 and/or MtWD40-1 and a luciferase reporter constructdrivenby theMtLYCeorMtLYCbpromoter resulted ina significantincrease in luminescence intensity compared with WP1 alone,whileMtTT8orMtWD40-1aloneproduced luminescence from thereporter constructs comparable to that in the GFP control(Figure 7D). These data together suggest that WP1 associateswith MtTT8 and MtWD40-1 to activate floral carotenoid pro-duction and that WP1 fulfills the major function within the acti-vation complex.

WP1 Is a Positive Regulator of FloralAnthocyanin Biosynthesis

The M. truncatula vexillum petals are marked by a ray of darkerveins that radiate from the middle of the base (Figure 8A) and arethought to be caused by anthocyanin accumulation (Xie et al.,2004; Jun et al., 2015). The color of these veins was visibly di-minished in the vexillum petals of wp1 and the anthocyanincontent was reduced in wp1 petals compared to the wild type,whereas introducing the WP1 genomic sequence restoredthe vein color and anthocyanin accumulation to the wild-typelevels (Figures 8A and 8B). These data suggest thatWP1 functionmay also be relevant to anthocyanin biosynthesis during flowerdevelopment.

Figure 6. WP1 Interacts with MtTT8 and MtWD40-1 to Form an MBWComplex.

(A) Interaction between WP1, MtTT8, and MtWD40-1 in the Y2H assay.Plate auxotroph and a-galactosidase assay showing interaction of eachprotein. Bars represent means 6 SD of three independent experiments.a-gal, a-galactosidase; DDO, double dropout; pGADT7, prey plasmid;QDO, quadruple dropout.(B) Interaction between WP1, MtTT8, and MtWD40-1 in N. benthamianaleaf epidermal cells using a split YFP BiFC assay. AHL22 was used asa nuclear localization marker. Bars 5 25 mm.(C) Interaction between WP1, MtTT8, and MtWD40-1 in N. benthamianausing a Co-IP assay. Immunoblots of the total protein extracts (Input) andthe IPproductwereperformedusing theanti-Mycantibody (a-Myc) or anti-Flag antibody (a-Flag), respectively.

2758 The Plant Cell

To determine the molecular mechanism by which WP1 affectsfloral anthocyanin accumulation inM. truncatula, we analyzed thetranscript levels of anthocyanin biosynthetic pathway genes inwp1 petals. RT-qPCR analysis showed that anthocyanin bio-synthesis genes MtCHS, MtCHI, MtF3H, MtF39H, MtDFR1,MtDFR2,MtANS, andMtANR were downregulated in wp1 petals(Figure 8C). Further transient expressionassays showed thatWP1could activate luciferase reporter constructs driven by the pro-moters ofMtCHS andMtANS (Figures 8D and 8E), which containconserved MYB-core elements (Supplemental Figure 7), and thatthepresenceofMtTT8andMtWD40-1could enhance theseWP1-mediated activations (Figures 8D and 8E). Moreover, phenotypicobservation revealed that both mttt8 and mtwd40-1 petals hadveins with reduced anthocyanin content, similar to those of wp1(Supplemental Figure 13). These results together suggest thatWP1associateswithMtTT8andMtWD40-1and thereby regulatesboth carotenoid and anthocyanin production by activating theirbiosynthetic genes in M. truncatula petals.

DISCUSSION

WP1 Is Required for Both Carotenoid and AnthocyaninProduction in M. truncatula Petals

The flowers of M. truncatula contain mainly yellow pigments, al-though a fine red vein of anthocyanin accumulates in the center ofthe vexillumpetals (Xie et al., 2004; Junet al., 2015).However, untilnow, neither the pigment components that impart M. truncatulaflower color nor their regulatory controls have been clearly un-derstood. In this study,weestablished that the yellowpetals of thewild-type M. truncatula contained high levels of xanthophyll thatlargely consisted of esterified lutein (Figure 1J; SupplementalFigure3). Functional disruptionofWP1, anR2R3-MYBTF, byTnt1retrotransposon insertion resulted in loss of the vivid yellow floralpigment, which is associated with loss of lutein ester accumu-lation, aswell as reducedofanthocyanin in theveins (Figures1and8A and 8B; Supplemental Figure 3), suggesting that WP1 is re-quired for both carotenoid and anthocyanin production inM. truncatula petals. We showed thatWP1 is the central regulatorof floral carotenoid pigmentation inM. truncatula by characterizingtwo independent but allelic mutants of ecotype R108 throughforward genetics and genetic complementation (Figure 2;Supplemental Figure 1): introducing theWP1 genomic sequence

Figure 7. WP1 Associates with MtTT8 and MtWD40-1 to Modulate FloralCarotenoid Biosynthesis.

(A) Flowers of the wild type, mttt8, mtwd40-1, mttt8 mtwd40-1, wp1-1,wp1-1 mttt8, and wp1-1 mtwd40-1. Bars 5 1.5 mm. WT, wild type.(B) Lutein concentrations in the petals of the wild type, mttt8, mtwd40-1,mttt8mtwd40-1,wp1-1,wp1-1mttt8, andwp1-1mtwd40-1. Bars representmeans 6 SD of three biological replicates. FW, fresh weight; WT, wild type.

(C)DNAbinding assay showing the binding activity ofWP1 in the presenceor absence of MtTT8 and/or MtWD40-1. Box1 to Box5 indicate putativebinding sites ofWP1 shown in Figure 5B. The His-TFa protein was used asanegativecontrol. Bars representmeans6 SDof threebiological replicates;asterisks indicate differences from WP1 alone (*P < 0.05, **P < 0.01,Student’s t test).(D) Activation of MtLYCe and MtLYCb promoters in transient luciferaseassay using Arabidopsis protoplasts. Various combinations of M. trun-catula effectors (WP1, MtTT8, MtWD40-1, and GFP control) were used totransfect Arabidopsis protoplasts along with the MtLYCe or MtLYCbpromoter fused to a luciferase reporter. Bars representmeans6 SD of threebiological replicates; asterisks indicate differences from WP1 alone(*P < 0.05, **P < 0.01, Student’s t test). LUC, luciferase.

WP1 Regulates Carotenoid Biosynthesis 2759

into these mutants fully restored the yellow color and lutein ac-cumulation to the wild-type levels.

These findings are intriguing from the perspectives of bothlutein itself, which imparts the yellow color, and the R2R3-MYBregulator of the lutein biosynthetic pathway. Lutein accumulationhas been reported as a major carotenoid in other yellow flowers,including the ray petals of chrysanthemums (Kishimoto et al.,2004). However, most flower colors, including blue, red, purple,and orange in popular ornamentals such as Rosa, Petunia, andcarnation (Dianthus caryophyllus), are primarily determined byflavonoids (Forkmann,1991;Grotewold, 2006). Even thebright yellowcolors of snapdragon (Antirrhinum majus) and dahlia (Dahlia

pinnata) flowers were reported to be conferred by flavonoids (Asenet al., 1972; Schwarz-Sommer et al., 2003). Plant flavonoids andcarotenoids are produced in totally different pathways: flavonoidsare derived fromaromatic amino acids produced in the cytosol andon the endoplasmic reticulum through the shikimic acid pathway(Winkel-Shirley, 2001; Koes et al., 2005), while carotenoids arederived fromGGPPproduced in the plastids via themevalonic acidpathway (Nisar et al., 2015; Sun et al., 2018). It is thus possible thatflavonoids remain viable candidates for contributing to the yellowcolor to M. truncatula flowers.To evaluate this possibility, we analyzed six of the commonly

found major yellow flavonoids in plants including chalcone,

Figure 8. WP1 Affects Anthocyanin Accumulation in M. truncatula Petals.

(A) Anthocyanin accumulation in the vexillum petals of the wild type, wp1-1, wp1-2, and WP1-GFP/wp1-1. Red arrows indicate the anthocyanin veins.Bars 5 1.5 mm. WT, wild type.(B)Anthocyanin contents in petals of thewild type,wp1-1,wp1-2, andWP1-GFP/wp1-1. Bars representmeans6 SD of threebiological replicates; asterisksindicate differences from the wild type (*P < 0.05, Student’s t test). FW, fresh weight; WT, wild type.(C)Transcript levelsof anthocyaninbiosynthetic genes inpetalsof thewild type,wp1-1,wp1-2, andWP1-GFP/wp1-1 revealedbyRT-qPCR.Bars representmeans 6 SD of three biological replicates; asterisks indicate differences from the wild type (*P < 0.05, **P < 0.01, Student’s t test).(D)and (E)Activation ofMtCHS (D)andMtANS (E)promoters in transient luciferase assay usingArabidopsis protoplasts. Various combinationsof effectors(WP1,MtTT8,MtWD40-1, andGFP control) were used to transfect Arabidopsis protoplasts alongwith theMtCHS orMtANS promoter fused to a luciferasereporter. Bars represent means6 SD of three biological replicates; asterisks indicate significance differences (*P < 0.05, **P < 0.01, Student’s t test). LUC,luciferase.

2760 The Plant Cell

apigenin, luteolin, quercetin, and two aurones (aureusidin andsulfuretin) in thepetalsof thewild typeandwp1mutants.We foundthat while the concentrations of chalcone and luteolin were un-changed, those of apigenin and quercetin were increased in thewp1mutants comparedwith thewild type (Supplemental Table 1).Aureusidin and sulfuretin, on the other hand, were not detectablein either the wild type or thewp1mutants (Supplemental Table 1).Thus, the yellow carotenoid lutein ismost likely themajor pigmentresponsible for theyellowpetal ofM. truncatula, althoughpotentialcontributions from other flavonoids cannot be excluded.

Although anthocyanin levels were reduced (Figure 8B), those ofchalcone and luteolin were almost unchanged and those of api-genin and quercetin were increased in wp1 mutant petals com-pared with the wild type (Supplemental Table 1), even thoughexpression of several genes required for the biosynthesis of an-thocyanin and these flavonoids was repressed in the mutantpetals (Figure 8C). One possible explanation for these resultsmight involve balancing feedback, that is, the decrease of an-thocyanin inwp1 petals might be counterbalanced by an increasein other flavonoids. Consistent with this possibility, a previousstudy reported that lossof functionofMYB regulatorMtPAR led tosubstantially reduced PA in theM. truncatula seed coat, whereaslevels of anthocyanin were indistinguishable and flavonoid gly-coside content was higher (by 23.2%) in par mutants than in thewild-type controls despite the existence of a common pathwaythat generates precursors for both PA and anthocyanin bio-synthesis (Verdier et al., 2012).

WP1 belongs to subgroup 6 of the R2R3-MYB proteins(Figure 3A), which is characterized by the signature bHLH-interacting motif, the ANDV motif, and the [R/K]Px[P/A/R]xx[F/Y] motif downstream of the conserved R2 and R3 MYB DNAbinding domains (Supplemental Figure 6; Stracke et al., 2001;Zimmermann et al., 2004; Dubos et al., 2010; Lin-Wang et al.,2010). To date, all of the subgroup 6 MYBs characterized inArabidopsis, M. truncatula, and other species appear to regulateanthocyaninbiosynthesis (Liu et al., 2015;Allan andEspley, 2018).Our results showed that a member of this subgroup can alsofunction in the regulation of carotenoids, directly connecting thetwo major pigment pathways. In light of this, recent studies inM. lewisiihave reported that the subgroup21MYBproteinRCP1, thefirst TF that positively regulates carotenoid biosynthesis duringflower development, can simultaneously repress anthocyaninproduction in the petal lobe (Sagawa et al., 2016), indicating thatthere may be a variety of MYBs from different clades involvedin both carotenoid and anthocyanin regulation in different plantspecies.

The presence of conserved MYB binding elements in thepromoters of both anthocyanin and carotenoid biosynthesisgenes suggests that plants may have evolved an efficientmechanism that produces and controls carotenoid and antho-cyanin biosynthesis via conserved regulators (SupplementalFigure7). Furthermore, tissuespecificity appears tobe remarkablyprecise. Although chlorophylls and important phytohormonessuch as gibberellin and abscisic acid are derived from the sameintermediate (GGDP) as lutein, wp1-1 was indistinguishable fromthe wild type in other pigments and morphological features in-cluding lutein andanthocyanin in leaves (Supplemental Figure11),suggesting an efficient mechanism for attaining specificity.

The C-Terminal Acidic Motif Is Essential for the ActivationFunction of WP1

Although transcriptional activation domains are essential for generegulation, their intrinsic disorder and low primary sequenceconservation have made it challenging to identify the amino acidcomposition features that underlie their activity. Molecular dis-section of the transcriptional activator WP1 enabled us to gaininsight into this activationmechanism.Our results showed that theC-terminal MVGEFPMDFQLEGFEA motif, which contains multi-ple acidic amino acid residues, is critical for the activation activityof WP1 (Figures 4B and 4C). Either deletion or mutation of acidicamino acid residues of the activation motif fully abolished WP1’sactivation activity (Figures 4B and 4C; Supplemental Figure 8).Although BLASTP analysis revealed that WP1 had no similaritywith any functionally identified activationmotifs, such short acidicdomains have been reported to be important components ofMYBTFs involved in the activation of transcription. For example, themaize (Zea mays) C1 protein, the first TF identified in plants, wascharacterized as a MYB-related transcriptional activator definedby numerous acidic residues near the C terminus of the protein(Paz-Ares et al., 1987; Martin and Paz-Ares, 1997). These fore-going observations suggest that the acidic region providesa functional basis for the activation activity of MYB family TFs. Inagreement with this, a recent study in yeast reported that acidicresidues may underlie transcriptional activation domains bycreating a permissive context for a hydrophobic short linear motif(Staller et al., 2018).

WP1 Associates with MtTT8 and MtWD40-1 to ModulateFloral Carotenoid and Anthocyanin Biosynthesis

In M. truncatula, several MYB TFs including MtPAR, MtMYB14,MtMYB5, andMtLAP1 have been reported to specifically regulateanthocyanin and PA biosynthesis in the presence of the bHLHprotein MtTT8 and the WDR protein MtWD40-1 (Verdier et al.,2012; Liu et al., 2014; Li et al., 2016), establishing the so-calledMBW complex similar to those in other species (Ramsay andGlover, 2005; Xu et al., 2015). The interactions between WP1,MtTT8, and MtWD40-1 indicate that the biosynthesis of car-otenoids, anthocyanins, and PA may be regulated by differentMBW complexes that share similar components and that R2R3MYB might play a central role within the MBW complex in rec-ognizing and activating targets at a specific time and in a specifictissue.Consistentwith this hypothesis, bothMtTT8andMtWD40-1 are expressed in most tissues (Pang et al., 2009; Li et al., 2016),but their petal-specific function in carotenoid and anthocyaninregulation is brought about by the petal-specific expression ofWP1 (Figures 3B to 3D). Nevertheless, it is worth noting that thepresence of MtTT8 and MtWD40-1 enhances the WP1-mediatedactivationofbothanthocyanin andcarotenoidbiosynthesis genes(Figures 7D and 8D and 8E), although the petals of M. truncatulacontain mainly yellow xanthophyll and only a small amount ofanthocyanin accumulated in the veins in the center of the vexillumpetals.These results might be explained by two nonexclusive possi-

bilities. First, the anthocyanin biosynthesis/deposition inM. truncatulapetals might be suppressed in areas outside of the veins

WP1 Regulates Carotenoid Biosynthesis 2761

by one or more unidentified repressors epistatic to WP1. Second,although the wp1,mttt8, andmtwd40-1mutants showed reducedanthocyanin in veins, indicating that WP1, MtTT8, and MtWD40-1are each indispensable for petal anthocyanin accumulation, it ispossible that the function of WP1-dependent MBW complexesin the activation of vein-specific anthocyanin production is de-pendent on an additional partner that is present in the veins ofvexillum petals. Further identification and characterization of thesuppressors andpotential partnersofWP1areneeded to test thesepossibilities. Moreover, it is noteworthy that while lutein isseverely reduced or absent in wp1 petals, the petals ofmttt8 andmtwd40-1 showed only partial lutein reduction (Figure 7B), sug-gesting that the specific activation of carotenoid biosynthesisgenes by WP1-dependent MBW complexes may also involveadditional cofactors that remain to be characterized.

Although theMBWcomplex hasbeen extensively studied in thecontext of the anthocyanin and flavonoid pathways, the completecomplex, or members in the complex, have been co-opted indiverse plant taxa to regulate other unrelated pathways such astrichome initiation, root hair development, and seed coat differ-entiation (Stracke et al., 2001; Broun, 2005; Xu et al., 2015). Ourresults suggest that the carotenoid regulator WP1 may havebeen derived from the anthocyanin-regulating MYBs, retainingthe ability to regulate anthocyanins and using a strikingly similarmechanism involving some of the same players. Likewise, recentstudies inbeets (Beta vulgaris) reported that ananthocyaninMYB-like protein, BvMYB1, activated the betalain red pigment pathway(Hatlestad et al., 2015). These findings suggest that plants mayhave evolved complex but efficientmechanisms that produce andcontrol chemically unrelatedpigments indistinct pathways.Takentogether, our study demonstrates the molecular mechanism ofWP1-mediated regulation of floral carotenoid and anthocyaninpigmentation in M. truncatula. Our findings provide a frameworkfor comparative studies of flavonoid and carotenoid biosyntheticregulatory controls and offer mechanistic insights into the evo-lution of plant secondary metabolism.

METHODS

Plant Materials and Tnt1 Insertion Mutant Screening

Medicago truncatula ecotype R108 was used for all experimentsdescribed in this article. The wp1-1 (NF4496), wp1-2 (NF10625), mttt8(NF15995), andmtwd40-1 (NF11228)mutantswere identified fromtheTnt1retrotransposon-tagged mutant collection of M. truncatula (Tadege et al.,2008; Yarce et al., 2013). Primers used for genotyping are listed inSupplemental Table 2. Scarified M. truncatula seeds were germinatedovernight in moist Petri dishes and placed at 4°C for 1 week. Plants weregrown at 24°C day/22°C night temperature, 16-h day/8-h night photo-period, 150 to 200 mE/m2/s light intensity (full-spectrum white fluorescentlight bulbs), and 60 to 70% RH.

Molecular Cloning of WP1 and Identification of Insertion Sitesin WP1

TheTnt1FSTsofwp1mutantswereobtained fromtheM. truncatulamutantdatabase (http://medicago-mutant.noble.org/mutant/) and genotyped byPCR using Tnt1-specific and gene-specific primers. FST 6, which seg-regatedwith thewhite-flowerphenotypeofwp1-1,wasanalyzedbyBLAST

searches against theM. truncatula genome at NCBI (http://www.ncbi.nlm.nih.gov/) and Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html)to obtain the full-length sequence of WP1. PCR and RT-PCR were per-formed to amplify the WP1 genomic and coding sequences (CDS), re-spectively. The FST sequence corresponding toWP1 in thewp1-2mutantwas amplified using Tnt1- and WP1-specific primers. Tnt1 insertionfragments from PCR were further confirmed by sequencing. The primersequences are listed in Supplemental Table 2.

Plasmid Construction and Plant Transformation

To make the complementation construct, a 1.5-kb fragment downstreamof the termination site of WP1 was amplified from M. truncatula R108DNA and inserted into the PstI site of the pCAMBIA2300 vector by the In-Fusion cloning system (no. 639648, Clontech) to generate the vectorpCAMBIA2300-WP139UTR.Subsequently, the3-kbWP1promoter regionupstream of the translation start site and the 3097-bp genomic DNAfragment containing three exons and two introns were cloned intopCAMBIA2300-WP1 39UTR, at the KpnI site, to generate the destinationvector WP1-GFP. The Agrobacterium tumefaciens strain AGL1–mediatedtransformation was used to introduce the WP1-GFP construct intoM. truncatula as described previously by Tadege et al., (2011). The primersequences are listed in Supplemental Table 2.

Sequence Alignment and Phylogenetic Analysis

The amino acid sequences of WP1 and subgroup 6 MYB proteins werealignedusingClustalW (http://www.genome.jp/tools/clustalw/). Bootstrapvalues of 1000 permutations for the neighbor-joining phylogenetic treewere generated using MEGA 4.0.

mRNA Expression Analyses

Petals from fully opened flowers were collected and dissected for RNAisolation. Total RNA was isolated using TRIzol reagent (Invitrogen). cDNAwassynthesizedbyRTwithTransScript-UniOne-StepgDNARemoval andcDNA Synthesis SuperMix (no. AU311, TRAN). RT-PCR reactions wereperformed using 23Taq PCR Master Mix (no. PM604-1, UPTECH) ac-cording to the manufacturers’ instructions, and the PCR amplicons wereexamined by electrophoresis on a 1% (w/v) agarose gel. RT-qPCR wasperformed as described previously by Wang et al., (2017), with three bi-ological replicates. Gene expression was normalized using the expressionofMtActin, a housekeeping gene. Relative gene expression for each genein the mutant plants was compared with that obtained for the wild type,which was arbitrarily set to 1.0. All primers used are listed in SupplementalTable 2.

Carotenoid Analysis

Carotenoids were extracted as described previously, with minor mod-ifications (Fraser et al., 2000). Dissected petals (15 mg) from fully openedflowersor leavesat thevegetative stagewereground intoapowder in liquidnitrogen. For saponification, 200mL of methanol containing 6% (w/v) KOHwas added andmixed thoroughly and then incubated at 60°C for 1 h in thedark. After cooling to room temperature, 200 mL of 50 mM Tris-HCl buffer,pH7.5 (containing 1MNaCl), wasadded to the suspension and themixturewas incubatedon ice for 10min.Chloroform (800mL)was thenadded to themixture, which was incubated on ice for 10 min. After centrifugation at3000g for 5min at 4°C, a visible stratification formed. The lower phasewasremoved, and the aqueous phase was re-extracted with chloroform(800mL). Thepooledchloroformextractsweredriedunder a streamofnitrogen.For nonsaponification, 200 mL of methanol was added to the groundsamplesand the suspensionwasmixedby inversion for 5minat 4°C. Two

2762 The Plant Cell

hundred mL of 50 mM Tris-HCl buffer, pH 7.5 (containing 1M NaCl) wasadded to the suspension and the extraction performed as describedabove. All dried extracts were stored at 280°C under an atmosphere ofnitrogen prior to HPLC.

HPLC analysis for floral carotenoids was performed as describedpreviously by Fraser et al., (2000), with minor modifications. Briefly, a re-verse-phase C30 column (2503 4.6 mm, 5 mm; YMC) coupled toa 2034.6-mmC30guard (YMC)withmobile phases consisting ofmethanol(A),water/methanol (20/80byvolume) (B), and tert-methyl butyl ether–basedmobile phase (C) was also usedwith a 10AvpHPLCsystem (Shimadzu). Thegradient elution used with this column was 95% A, 5% B isocratically for12min; a step to80%A,5%B,15%Cat12min; followedbya linear gradientto30%A,5%B,65%Cby30min.A reverse-phaseC18HypersilODS5-mm(2003 4.6-mm) column (Thermo Fisher Scientific) coupled to a 5-mm (1034.6-mm) C18 guard column (Phenomenex) was used with same ShimadzuHPLC system for analyzing carotenoids in leaf tissues. Acetonitrile-basedmobile phases were used as described in a previous report (Holloway et al.,2000).Throughoutchromatography, theelutionwasmonitoredcontinuouslyfrom 200 to 600 nm by an online Shimadzu SPD-10Avp PDA detector.Column temperature was maintained at 25°C by a Shimadzu CTO-10ACVPcolumn oven. In all cases, flow rates of 1 mL/min were used. Carotenoidswere identified by their characteristic absorption spectra, typical retentiontime, and comparison with authentic standards. The content of eachcompoundwas calculated using the corresponding standard as an externalstandard. The carotenoid standards were neoxanthin (no. BCBZ3644,Sigma-Aldrich), violaxanthin (no. BCBZ4220, Sigma-Aldrich), lutein epoxide(no.0232,CaroteNature), lutein (no.0133,CaroteNature),antheraxanthin (no.ASB-00001885-00A, ChromaDex), and b-carotene (no. ASB-00003210-00A, ChromaDex).

Flavonoid Analysis

Theextractionandanalysisof the total content (free formandglycosides) offlavonoids were performed as described previously (Matsuda et al., 2009;Ng et al., 2015), with minor modifications. Dissected petals (10 mg) fromfully opened flowers were ground into a powder in liquid nitrogen andextracted with 1 mL of 80% (v/v) methanol/water solution. After shakingovernight at 4°C in the dark, the supernatantwas spun in amicrocentrifugeat 16,000g for 10 min and then transferred to a fresh tube. After beingevaporated in a SpeedVac centrifuge, the residue was dissolved in 400mLof 2 M HCl, heated at 80°C for 90 min to deglycosylate flavonoids andsubsequently extracted with 1 mL of ethyl acetate. Extracts (400 mL) weredried in aSpeedVac centrifuge and subsequently dissolved in 1mLof 80%(v/v) methanol. The resulting solution was filtered with a 0.22-mm syringefilter, and the sample was analyzed with an ultraperformance liquidchromatography-tandem mass spectrometry system consisting of anAgilent 1290 Infinity LC pump and a 6495 triple-quadrupole mass spec-trometer. Positive mode analysis was performed in multiple reactionmonitoring mode. The mass spectrometry parameters were optimizedusing metabolite standards. The analytical conditions were as follows: theHPLC column was an Agilent ZORBAX Extend C18 (pore size, 1.8 mm;length, 2.1 3 100 mm); the solvent system was according to a previouslydescribed method (Matsuda et al., 2009) using acetonitrile (0.1% formicacid):water (0.1% formic acid); thegradient programwas1:99 (v/v) at 0min,1:99 (v/v) at 0.1min, 99.5:0.5 at 15.5min, 99.5:0.5 at 17.0min; the flow ratewas0.4mL/min; and the temperaturewas35°C.The ionsourceparameterswere set as follows: drying gas temperature, 325°C (nitrogen); drying gasflow, 7 liters/min; nebulizer, 40 pounds per square inch; sheath gas heater,350°C; sheath gas flow, 12 liters/min; and capillary voltage, 3 kV (elec-trospray ionization). The content of each compound was calculated usingthe corresponding standard as an external standard. Flavonoid standardsusedwere apigenin (no. 42251,Sigma-Aldrich), luteolin (no. 72511,Sigma-Aldrich), quercetin (no. PHR1488, Sigma-Aldrich), chalcone (no. A14734,

Alfa Aesar), aureusidin (no. BBP05377, BioBioPha), and sulfuretin (no.B50928, Shyuanye).

Anthocyanin Analysis

Anthocyanin content was detected as described previously by Pang et al.,(2009). Ten milligrams of ground petals was added to 300 mL of 0.1% (v/v)HCl/methanol, and themixturewas rotatedovernight on a rotatingwheel at4°C in the dark. Following centrifugation at 2500g for 10 min at 4°C, thesupernatant was transferred to a fresh tube and the absorption of theextraction was recorded at 530 nm. Total anthocyanin content was cal-culated based on the molar absorbance of a cyanidin 3-O-glucosidechloride standard (no. 52976, Sigma-Aldrich).

Subcellular Localization Analysis

TheCDSofWP1wascloned intopMDC83byGatewayCloning (Invitrogen)to generate Pro35S:WP1-GFP. A plasmid expressing mRFP-AHL22protein was used as a nuclear localization marker (Wang et al., 2013).The plasmid pMDC32-GFP (Pro35S:GFP) expressing GFP protein alone,was used as the control. The A. tumefaciens strain GV2260 containingPro35S:WP1-GFP or Pro35S:GFP, and the same strain containingmRFP-AHL22, were simultaneously infiltrated into the leaves of 4-week-old Ni-cotiana benthamiana plants. P19 was used to inhibit transgenic silencing.The fluorescence signal was observed 2 to 3 d after infiltration by confocalmicroscopy (TCS SP2 microscope, Leica). The primers used for plasmidconstruction are listed in Supplemental Table 2.

Transactivation Activity Assay in Yeast

The transactivation activity assay was performed as described previouslyby Zhang et al., (2016). WP1, VP64 (encoding an exogenous activationdomain) or EAR4 (encoding an exogenous repression module) fused toWP1, different domains ofWP1, and various truncated forms ofWP1werefusedwith theGAL4DNABD in the plasmidpGBKT7-GWusing aGatewayrecombination system. These constructs were then transformed into theyeast (Saccharomyces cerevisiae) strainGold according to the instructionsfor the Frozen-EZ Yeast Transformation II kit (no. T2001, Zymo Research).Yeast colonies were patched onto SD/2Trp and SD/2Trp/2His/2Adeplates and grown at 28°C for 3 d. The a-galactosidase assay was per-formed according to the Yeast Protocols Handbook (no. PT3024-1,Clontech). The primer sequences are listed in Supplemental Table 2.

Y2H Assay

Y2H assays were performed as described previously (Niu et al., 2015). TheCDSsofWP1,MtTT8, andMtWD40-1wereamplifiedand then inserted intothe bait plasmid pGBKT7-GWor the prey plasmid pGADT7-GW. All cloneswere validated by sequencing. The bait and prey plasmids were co-transformed into yeast strain Gold using the Frozen-EZ Yeast Trans-formation II kit (no. T2001, Zymo Research). Yeast colonies were patchedonto SD/2Leu/2Trp (double dropout) and SD/2Trp/2Leu/2His/2Ade(quadruple dropout) plates and grown at 28°C for 3 d. Thea-galactosidaseassay was performed according to the Yeast Protocols Handbook (no.PT3024-1, Clontech). The primer sequences are listed in SupplementalTable 2.

BiFC Assay

BiFC assays were performed as described previously (Lu et al., 2010; Niuet al., 2015). TheCDSsofWP1,MtTT8, andMtWD40-1werecloned into thepEarleyGate 201-YN or pEarleyGate 202-YC vectors (Lu et al., 2010) usinga Gateway recombination system. The prepared vectors were introduced

WP1 Regulates Carotenoid Biosynthesis 2763

into A. tumefaciens strain GV2260. Combinations of YN and YC plasmidstogether with plasmidsmRFP-AHL22 and P19were agroinfiltrated into theleaves of 4-week-old N. benthamiana plants. Signals were observed 3 dafter infiltration by confocal microscopy (TCS SP2 microscope, Leica).Primers used for plasmid construction are listed in Supplemental Table 2.

Co-IP Assay

The Co-IP assay was performed as described previously by Meng et al.,(2013),withminormodifications.TheCDSsofWP1,MtTT8, andMtWD40-1were cloned into either the pGWB17-GW-Myc or pGWB11-GW-Flagvector, resulting in pGWB17-WP1-Myc, pGWB17-MtTT8-Myc, pGWB11-MtTT8-Flag, and pGWB11-MtWD40-1-Flag. For testing protein–proteininteractions, A. tumefaciens strain GV2260 containing pairs of theseconstructs together with P19 was co-infiltrated into the leaves of 4-week-oldN. benthamiana plants. Equal amount of samples (0.3 g) was collected3dafter infiltration,ground in liquidnitrogen,and thenhomogenized in1mLof extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA,1% Triton X-100, 0.1% Tween 20, 10% glycerol, 1 mM phenyl-methylsulfonyl fluoride, and 1 tablet/50 mL of protease inhibitor cocktail).The lysateswere incubatedat 4°C for 15min andcentrifugedat 16,000g for10 min at 4°C. The supernatant was precleared with 30 mL of DynabeadsProtein A (no. 10001D, Novex) at 4°C for 1 h. After a brief spin, the su-pernatants were incubated with 30 mL of suspensions of Pierce Anti-c-MYC magnetic beads (no. 88842, Thermo Fisher Scientific) at 4°C for 4 hand then washed four to five times with the extraction buffer. The proteinswere eluted from the beads with 30 mL of 63 Protein Loading Buffer (no.L10215, TRAN), boiled for 5min, andspun at 12,000 rpm for 10min at roomtemperature. Thesupernatantswereelectrophoretically separatedby10%SDS-PAGE and transferred to a nitrocellulosemembrane (no. A10190852,GE Healthcare). Immunoblots were performed using an anti-Myc antibody(1:4000; no. M20002, Abmart) for probing WP1-Myc or MtTT8-Myc,and an anti-Flag antibody (1:4000; no. M20008, Abmart) for probingMtTT8-Flag or MtWD40-1-Flag, sequentially. The primer sequences are listed inSupplemental Table 2.

DNA Binding Assay

TheCDSsofWP1,MtTT8, andMtWD40-1were amplified and inserted intothe EcoRI site of the pCOLD-TFa vector using the In-Fusion cloningstrategy (Clontech). The recombinant constructs were transformed intoEscherichia coli (BL21) and induced with 0.2 mM isopropyl-1-thio-D-gal-actopyranoside. Recombinant His-TFa-WP1, His-TFa-MtTT8, and His-TFa-MtWD40-1 proteins and His-TFa control protein were purified usingProfinity IMAC Ni-charged resin (no. 1560131, Bio-Rad) according to themanufacturer’s protocol and quantified by the Bio-Rad protein assay re-agent. The DNA binding assay was performed as described previously byMeng et al., (2013) and Wang et al., (2017). The putative MYB bindingfragments (Box1 to Box5) or the mutant binding fragment (Boxm) wereincubated with the His-TFa-WP1, His-TFa-MtTT8, and His-TFa-MtWD40-1 proteins, or combinations of these proteins, in the Ni-charged resin,and then the DNA binding activity (protein-bound DNA) was determinedby real-time qPCR after washing and elution. Primers used are listed inSupplemental Table 2.

Transient Luciferase Assay

The transient dual-luciferaseassaywasperformedasdescribedpreviously(Wang et al., 2017), with minor modifications. The effector plasmids wereconstructed by cloning the WP1, WP1D(186-201), MtTT8, and MtWD40-1CDSs into the pMDC32 or pEarleyGate203 vector using the GatewayCloning system (Invitrogen), resulting in vectors pMDC32-WP1, pMDC32-WP1D(186-201), pEarleyGate203-MtTT8, and pEarleyGate203-MtWD40-1,

respectively. The plasmidpMDC32-GFPwas used as the negative control.The;3-kb promoter fragments upstream of the transcription start sites ofMtPSY, MtPDS, MtZDS, MtCRTISO, MtLYCe, MtLYCb, MtBCH,MtCYP97C, MtCHS, and MtANS were cloned into pGreenII-0800-Lucusing the In-Fusion cloning strategy (Clontech) to generate the corre-sponding reporter vectors. Arabidopsis (Arabidopsis thaliana) protoplastswere cotransformedwithdifferent combinationsof plasmids, incubated for12 to 14 h in darkness, and then collected and lysed for the detection ofluciferase activity. The detection was performed according to the manu-facturer’s recommendations for the Dual-Luciferase Reporter AssaySystem (E1910,Promega).Primersusedare listed inSupplementalTable2.

ChIP-PCR Assay

TheWP1-GFP transgenicplants (T2generation)wereused forChIPassaysaccording to the previously describedmethod (Cui et al., 2016), withminormodifications. Briefly, 1 g of fully opened flowers was ground into a finepowder with liquid nitrogen, cross-linked with 1% (v/v) formaldehyde, andthenquenchedwith0.125MGly for 5minat 4°C. Thechromatin complexeswere isolated, sonicated, and then incubated with an anti-GFP antibody(no. ab290, Abcam). The washing, elution, reverse cross-linking, and DNApurification steps were performed as described previously (Bowler et al.,2004). The precipitated DNA was subjected to qPCR analysis. The ChIP-qPCR results were quantified by normalization of the immunoprecipitationsignal with the corresponding input signal and are presented as the per-centage of input (PCR signal of immunoprecipitation reaction/PCR signalof input). The primers used for the ChIP assays are listed in SupplementalTable 2.

Statistical Analysis

For statistical analysis, Student’s t testwasusedas specified in each figureand in Supplemental Data Set 2. Asterisks indicate statistical differences(*P < 0.05, **P < 0.01). Data represent mean values, and error bars are SD.

Accession Numbers

Sequence data from this article can be found in the NCBI (http://www.ncbi.nlm.nih.gov/) databases, M. truncatula Genome Database (http://www.medicagogenome.org/), or Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) under the following accession numbers: Antir-rhinum majus: ROSEA1, ABB83826; VENOSA, ABB83828. A. thaliana:AtPAP1, AT1G56650; AtPAP4, AT1G66380; AtPAP2, AT1G66390; At-MYB113, AT1G66370; AtGL1, AT3G27920; AtMYB23, AT5G40330;AtWER, AT5G14750; AtTT2, AT5G35550; AtMYB11, AT3G62610; At-MYB12, AT2G47460; AtMYB111, AT5G49330. Lotus japonicus: LjTT2a,BAG12893.Malus3 domestica: MdMYB10, ABB84753.Mimulus lewisii:NEGAN, AHJ80988; PELAN, AHJ80987. M. truncatula: WP1,Medtr0197s0010; MtLAP1, Medtr8g060940; MtLAP3, Medtr7g017260;MtLAP2, Medtr5g079290; MtLAP4, Medtr5g079220; MtTT8,Medtr1g072320; MtWD40-1, Medtr3g092840; MtPSY, Medtr5g076620;MtPDS, Medtr3g084830; MtZDS, Medtr1g081290; MtCRTISO,Medtr1g054965; MtLYCe, Medtr2g040060; MtLYCb, Medtr7g090150;MtCYP97A, Medtr7g079440; MtBCH, Medtr6g048440; MtCYP97C,Medtr1g062190; MtCHS, Medtr3g083910; MtCHI, Medtr1g115820;MtF3H, Medtr8g075890; MtF39H, Medtr3g025230; MtDFR1,Medtr1g022445; MtDFR2, Medtr1g022440; MtANS, Medtr5g011250;MtANR, Medtr4g092080. Petunia 3 hybrida: PhAN2, AF146702; PhDPL,ADW94950. Vitis vinifera: VvMYBA1, BAD18977; VvMYBPA2, ACK56131.

Supplemental Data

Supplemental Figure 1. Genetic analysis of wp1-1 and wp1-2mutants.

2764 The Plant Cell

Supplemental Figure 2. HPLC analysis of carotenoids in wild-typeR108 petals.

Supplemental Figure 3. Carotenoid profiles in wild-type, wp1-1, andwp1-2 petals.

Supplemental Figure 4. WP1 cDNA sequence and its deduced aminoacid sequence in wp1-1.

Supplemental Figure 5. Molecular characterization of WP1-GFPcomplemented transgenic plants.

Supplemental Figure 6. Sequence alignment of WP1 and subgroup6 R2R3-MYB proteins in M. truncatula and Arabidopsis.

Supplemental Figure 7. The predicted MYB binding sites in thepromoters of downregulated carotenoid and anthocyanin biosynthesisgenes in wp1.

Supplemental Figure 8. Transactivation analysis of the WP1D(186-201)

protein using transient luciferase assay.

Supplemental Figure 9. Homodimerization of WP1, MtTT8, andMtWD40-1 proteins.

Supplemental Figure 10. Molecular characterization of the mttt8 andmtwd40-1 Tnt1 insertional mutants.

Supplemental Figure 11. Phenotype analysis of the wp1-1,mttt8, andmtwd40-1 mutants.

Supplemental Figure 12. Comparison of carotenoid biosynthesisgene expression in petals of wild type, wp1-1, mttt8, andmtwd40-1.

Supplemental Figure 13. Anthocyanin accumulation in the vexillumpetals of wild type, mttt8, and mtwd40-1.

Supplemental Table 1. Quantification of flavonoids in petals of wildtype (WT), wp1-1, wp1-2, and WP1-GFP/wp1-1 plants.

Supplemental Table 2. Primers used in this study.

Supplemental Data Set 1. Alignments used to generate the phylog-eny presented in Figure 3A.

Supplemental Data Set 2. Summary of statistical tests.

ACKNOWLEDGMENTS

We thank the Metabolomics Facility of the Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences for assistanceregarding flavonoid analysis. This work was supported by the NationalTransgenic Science and Technology Program (grants 2019ZX08010-003 and 2019ZX08010-002), the National Key R&D Program of China(grants 2018YFA0901000 and 2018YFA0901003), the National NaturalScience Foundation of China (grant 31870284), the Beijing MunicipalScience and Technology Commission (grant lj201812), and the Scien-tific Research Project for Major Achievements of the AgriculturalScience and Technology Innovation Program (ASTIP; grant CAAS-ZDXT2019004).

AUTHOR CONTRIBUTIONS

Y.M., Z.W., L.N., andH. Lin designed the research. Y.M., Z.W., Y.W., C.W.,B.Z., H. Liu, and W.J. performed the experiments. Y.M., Z.W., M.T., L.N.,and H. Lin analyzed the data. J.W. and C.C. contributed analytical tools.Y.M., M.T., L.N., and H. Lin wrote the article with contributions by allauthors.

Received July 1, 2019; revised August 26, 2019; accepted September 13,2019; published September 17, 2019.

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WP1 Regulates Carotenoid Biosynthesis 2767

DOI 10.1105/tpc.19.00480; originally published online September 17, 2019; 2019;31;2751-2767Plant Cell

Wen, Chengcai Chu, Million Tadege, Lifang Niu and Hao LinYingying Meng, Zuoyi Wang, Yiqin Wang, Chongnan Wang, Butuo Zhu, Huan Liu, Wenkai Ji, Jiangqi

Medicago truncatulaCarotenoid-Derived Flower Pigmentation in The MYB Activator WHITE PETAL1 Associates with MtTT8 and MtWD40-1 to Regulate

 This information is current as of July 2, 2020

 

Supplemental Data /content/suppl/2019/09/17/tpc.19.00480.DC1.html

References /content/31/11/2751.full.html#ref-list-1

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