# MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula

Jérôme Verdier, Jian Zhao, Ivone Torres-Jerez, Shujun Ge, Chenggang Liu, Xianzhi He, Kirankumar S Mysore, Richard A Dixon, Michael K Udvardi

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## To cite this version:

Jérôme Verdier, Jian Zhao, Ivone Torres-Jerez, Shujun Ge, Chenggang Liu, et al.. MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109 (5), pp.1766-1771. 10.1073/pnas.1120916109. hal-01601885

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Submitted on 29 May 2020

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# MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in *Medicago truncatula*

Jerome Verdier<sup>a,1</sup>, Jian Zhao<sup>a,1</sup>, Ivone Torres-Jerez<sup>a</sup>, Shujun Ge<sup>a,b</sup>, Chenggang Liu<sup>a</sup>, Xianzhi He<sup>a</sup>, Kirankumar S. Mysore<sup>a</sup>, Richard A. Dixon<sup>a,2</sup>, and Michael K. Udvardi<sup>a</sup>

<sup>a</sup>Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401; and <sup>b</sup>College of Agronomy, Agricultural University of Hebei, Baoding 071001, China

Contributed by Richard A. Dixon, December 20, 2011 (sent for review September 19, 2011)

MtPAR (*Medicago truncatula* proanthocyanidin regulator) is an MYB family transcription factor that functions as a key regulator of proanthocyanidin (PA) biosynthesis in the model legume *Medicago truncatula*. MtPAR expression is confined to the seed coat, the site of PA accumulation. Loss-of-function *par* mutants contained substantially less PA in the seed coat than the wild type, whereas levels of anthocyanin and other specialized metabolites were normal in the mutants. In contrast, massive accumulation of PAs occurred when MtPAR was expressed ectopically in transformed hairy roots of *Medicago*. Transcriptome analysis of *par* mutants and MtPAR-expressing hairy roots, coupled with yeast one-hybrid analysis, revealed that MtPAR positively regulates genes encoding enzymes of the flavonoid-PA pathway via a probable activation of WD40-1. Expression of MtPAR in the forage legume alfalfa (*Medicago sativa*) resulted in detectable levels of PA in shoots, highlighting the potential of this gene for biotechnological strategies to increase PAs in forage legumes for reduction of pasture bloat in ruminant animals.

condensed tannin | forage quality | metabolic engineering

Proanthocyanidins (PAs; also called condensed tannins) are oligomers of flavan-3-ol units and are prominent compounds in seed coats, leaves, fruits, flowers, and bark of many plant species (1). PAs and their monomeric building blocks catechin and epicatechin are antioxidants with beneficial effects on human health, including cardioprotective (2), anticancer (3), and anti-inflammatory activities (4). PAs in forage plants bind to proteins and slow their fermentation in the rumen, reducing microbial production of methane and thereby protecting the animal from potentially lethal pasture bloat (1). Moderate levels of PAs in forages also improve nitrogen nutrition, reduce urinary nitrogen excretion, and help counter intestinal parasites (5). Unfortunately, many legumes, including the world's most important forage legume, alfalfa (*Medicago sativa*), have insufficient PA levels to prohibit bloat in grazing animals. Therefore, increasing the PA content of shoots is an important target of biotechnology in alfalfa and other forage legumes.

PA biosynthesis and its regulation are quite well understood in the model nonlegume species *Arabidopsis thaliana* (6). Much of the knowledge of PA biosynthesis in *Arabidopsis* has come from the analysis of *transparent testa* (*tt*) mutants, which exhibit reduced seed pigmentation (6, 7). Twenty *TT* genes have been characterized and encode enzymes or transporters involved in PA biosynthesis and storage or proteins that regulate PA production (6, 8). The latter include a set of transcription factors (TFs): TT2, an MYB family TF; TT8, a bHLH TF; and TTG1, a WD40 protein, which together form a ternary complex that regulates transcription of anthocyanidin reductase (ANR; the enzyme producing the epicatechin building block of PAs) (9–11).

Recently, key genes/enzymes and a precursor transporter (MATE1) involved in PA biosynthesis have been isolated and characterized from the model legume *Medicago truncatula* (12–14). However, relatively little is known about the regulation of PA

biosynthesis in *M. truncatula* and other legumes; only a WD40-repeat TF has been implicated as a positive regulator of PA biosynthesis in *M. truncatula* seeds (15).

From a genome-wide study of TF and other genes that are activated during seed development in *M. truncatula*, >30 seed-induced TFs were targeted for functional characterization by using reverse genetics. One of these encodes an MYB family TF that we found regulates PA biosynthesis in *M. truncatula* seeds. Ec-topic expression of the gene in transformed hairy roots led to PA biosynthesis and accumulation. Therefore, this MYB TF has the potential to boost tannin levels for forage improvement.

## Results

**MtPAR Encodes an MYB TF with Seed Coat-Specific Expression.** We used the *M. truncatula* Gene Expression Atlas (MtGEA) (16) to select seed-induced TF genes for genetic characterization. One of these, *MtPAR*, encodes a putative MYB TF of the R2R3 class, based on the presence of highly conserved R2 and R3 MYB DNA binding domains at the N-terminal end of the protein (17). *MtPAR* (Affymetrix probe set Mtr.50541.1.S1_at) is expressed in a seed-specific manner (Fig. S1A; or see MtGEA, http://mtgea.noble.org/v2/), with maximal expression at 24 d after pollination (DAP; Fig. 1). Quantitative RT-PCR (qRT-PCR) indicated that the gene is specifically expressed in the seed coat but not in the embryo or endosperm of mature seeds (Fig. 1). Distance analysis revealed no close relationship between MtPAR and MYB TFs involved in the regulation of anthocyanin (e.g., LAP proteins from *M. truncatula* or ANTHOCYANIN1 from *Solanum lycopersicum*) (18, 19) or PA biosynthesis (e.g., TRANSPARENT TESTA2 from *A. thaliana* or MYBPA1 and MYBPA2 from *Vitis vinifera*) (refs. 9 and 20; Fig. S1B). The closest homolog of MtPAR is an MYB protein (GmMYB115; GenBank accession no. Q0PJG9) from *Glycine max* (soybean) of unknown function.

**par Mutants Are Defective in Seed Coat PA Accumulation.** We isolated four independent mutants with retrotransposon insertions in the *MtPAR* gene through a PCR screen of DNA from a *Tnt1*-insertion mutant population (21). *Tnt1* insertions were found in the second exon of *MtPAR* in mutant line NF4419 and in the third exon in lines NF2466, NF1358, and NF3308 (Fig. 2A). Homozygous

Author contributions: J.V., J.Z., R.A.D., and M.K.U. designed research; J.V., J.Z., I.T.-J., S.G., C.L., X.H., and K.S.M. performed research; J.V., J.Z., R.A.D., and M.K.U. analyzed data; and J.V., J.Z., R.A.D., and M.K.U. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: The MtPAR sequence reported in this paper has been deposited in the GenBank database (accession no. HQ337434).

¹J.V. and J.Z. contributed equally to this work.

²To whom correspondence should be addressed. E-mail: radixon@noble.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1120916109/-/DCSupplemental.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Gene expression of *MtPAR*. Expression profile of *MtPAR* through seed development (10–36 DAP) according to the MtGEA (average of biological triplicates with SD) and in seed tissues (SC, seed coat; E/Eo, embryo and endosperm) according to qRT-PCR data (average of technical triplicates with respective SD).</figcaption></figure>

insertion mutants of all four lines showed a reduction in pigmentation of mature seed compared with the wild-type (WT) control (Fig. 2B and Fig. S2A). *MtPAR* transcript levels in developing seed of the four mutants were <5% of the WT level (Fig. S1C).

4-Dimethylaminocinnamaldehyde (DMACA) staining suggested a decrease in the PA content of mature mutant seeds compared with WT (Fig. 2C). DMACA staining of developing seed revealed a similar, gradual accumulation of PA from 10 to 16 DAP in both mutant and WT. Differences between mutant and WT first became apparent at ~20–24 DAP (Fig. S2B), coinciding with maximal *MtPAR* expression in the WT. DMACA staining was confined largely to seed coats, mirroring the tissue specificity of

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Analysis of *par* mutants in *Medicago*. (A) *MtPAR* gene model with position of different *Tnt1* insertions and the names of the corresponding independent mutant lines. Introns are represented by a line, and exons are represented by a rectangle. (B) Effect of mutation on mature seed pigmentation for the *NF4419* mutant line. A similar phenotype was observed in other mutant lines (Fig. S2A). (C) DMACA staining of mature seeds from *NF2466* mutant line (similar phenotype in other mutant lines; Fig. S2A). (D) Levels of extractable PAs (soluble and insoluble) with respect to their null segregant controls. Values represent averages and SD from three biological replicates.</figcaption></figure>

*MtPAR* expression (Fig. S2C). Both soluble and insoluble PA levels in *par* seeds were significantly reduced compared with their segregant controls. Soluble PA concentration was ~50% lower and insoluble PA concentration was up to 80% lower in the mutants than in the sibling WTs (Fig. 2D). High-performance liquid chromatography (HPLC) followed by normal-phase HPLC with postcolumn derivatization with DMACA reagent indicated that mutant and WT seeds exhibited a similar size distribution of PAs (Fig. S3). Spectrophotometric analysis of anthocyanin concentration revealed no significant difference between *par* mutant and WT seeds (Fig. S2D). Thus, *MtPAR* regulates PA but not anthocyanin biosynthesis in seeds.

**Ectopic Expression of MtPAR Induces PA Biosynthesis.** To substantiate a role for MtPAR in PA biosynthesis, we transformed *M. truncatula* hairy roots with the *MtPAR* cDNA coupled to the constitutively active *CaMV-35S* promoter (22). *Agrobacterium rhizogenes* (strain ARqua 1; ref. 23) was used to transfer the *p35S::MtPAR* construct into *M. truncatula* together with a green fluorescent protein (GFP) gene that enabled identification of transformed hairy roots (Fig. 3A). Ectopic expression of *MtPAR* in *Medicago* hairy roots was checked by qRT-PCR (Fig. 3B). Initial observations of unstained hairy roots revealed a decrease in red pigmentation in transgenic roots containing the *p35S::MtPAR* construct compared with control-transformed roots containing a *p35S::GUS* (β-glucuronidase) construct (ref. 24; Fig. 3A). No differences in root growth or other morphological features were observed between roots containing *p35S::MtPAR* and controls. Subsequent staining of hairy roots with DMACA revealed a dramatic difference between *p35S::MtPAR*- and *p35S::GUS*-transformed roots; the former stained dark blue-green with DMACA, indicating the presence of PAs, whereas the controls did not (Fig. 3A).

Soluble PA levels were low in hairy roots of control transformed plants (*p35S::GUS*) but were up to 100-fold higher in some *p35S::MtPAR* lines (Fig. 3B). Moreover, a positive correlation (*r*² = 0.82) was observed between soluble PA concentration and *MtPAR* transcript levels in the different transgenic lines (Fig. 3B). In contrast, insoluble PA levels were very low in

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Ectopic expression of *MtPAR* in *Medicago* hairy roots. (A) Phenotype of *MtPAR* ectopic expression transformants in hairy roots. (Left) GFP detection as a transformation marker. (Center and Right) Unstained and DMACA-stained hairy roots. (B) Correlations between relative expression of *MtPAR* with soluble PA and anthocyanin concentrations in 14 different hairy root transformants. Cy3G, cyanidin 3-O-glucoside; Epi, (-)-epicatechin.</figcaption></figure>
control hairy roots and were not increased as a result of expression of *MtPAR* (Fig. S2E). Anthocyanin concentration was high in hairy roots of control plants but decreased with increasing soluble PA levels in *p35S::MtPAR* lines (Fig. 3B).

**MtPAR Regulates Expression of PA Biosynthesis Genes.** To determine the mechanism by which MtPAR triggers PA biosynthesis, we carried out transcriptome analysis of mutant and WT sibling seeds and of *p35S::MtPAR* and *p35S::GUS*-transformed roots, using Affymetrix *Medicago* GeneChips. Comparisons of transcript levels in seed at 20 DAP identified 49 genes that were differentially expressed (transcript ratio of <0.5 or >2; *P* < 0.05) between *par* mutants (lines NF2466, NF3308, and NF4419) and their WT siblings. Of these, 38 genes exhibited lower and 11 genes exhibited higher transcript levels in the mutants (Table S1). According to GeneBins ontology (25), 14 of the genes that were repressed in the mutants encode enzymes involved in flavonoid biosynthesis (Table S1). Some of these genes/enzymes are required for both PA and anthocyanin synthesis (e.g., chalcone synthase, CHS; flavonoid 3'-hydroxylase, F3H; and anthocyanidin synthase, ANS), whereas others act downstream of ANS and are specific to PA biosynthesis (e.g., ANR, glucosyltransferase UGT72L1). Genes that were more highly expressed in the mutants were mostly of unknown function (Table S1).

In *M. truncatula* hairy roots transformed with *p35S::MtPAR*, 171 genes were significantly altered in expression compared with *p35S::GUS*-transformed controls (transcript ratio of <0.5 or >2; *P* < 0.05; Table S2). Of these, 115 exhibited higher transcript levels in *p35S::MtPAR* roots. Eleven of the 115 genes coded for putative enzymes of flavonoid biosynthesis (e.g., CHS, F3H, and ANS).

To identify genes that might be regulated directly by MtPAR, we compared genes that were repressed in the *par* mutants with those induced in *p35S::MtPAR* lines relative to their appropriate controls. Twelve genes satisfied both criteria, and 8 of these encode enzymes involved in PA and anthocyanin biosynthesis (Table 1). Among these were genes encoding ANS (Mtr.14017.1. S1_at) and ANR (Mtr.44985.1.S1_at), which carry out the last two steps in the synthesis of epicatechin, the building block for PAs in *Medicago* (Fig. 4A). An integrated analysis of flavonoid pathway expression including all gene family members was carried out by using transcriptomic data from mutant seeds and ectopic expression in hairy roots (Fig. 4B). This analysis confirmed the regulatory effect of *MtPAR* on the expression of two central enzymes in the flavonoid pathway (i.e., CHS and F3H) and one enzyme, ANR, specifically regulating the switch between PA and anthocyanin synthesis.

To assess the impact of the *par* mutation on the flavonoid metabolic pathway, we performed metabolite profiling using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (MS). Out of 74 specialized metabolites identified in mature seeds, 19 were altered significantly in amount in *par* mutants compared with WT controls (Table S3). These metabolites belonged mainly to four groups of compounds: coumaric acid and coumarin, triterpene saponins, epicatechins, and flavonoid glycosides (two, eight, two, and seven metabolites respectively). Although the amount of individual saponins changed in the *par* mutants, the total amount of saponins was not significantly different between mutants and their WT siblings. The same was true of coumaric acid and coumarin. In contrast, total epicatechin content was substantially lower (by 45.9%) and flavonoid glycoside content was

<table><caption>**Table 1.** The 11 common genes that are down-regulated in loss-of-function mutant lines and up-regulated in ectopic expression transformant lines</caption><thead><tr><th rowspan="2">Probe sets</th><th rowspan="2">Target description</th><th rowspan="2">Representative public ID</th><th colspan="3">par mutant lines</th><th colspan="3">35S::MtPAR hairy roots</th></tr><tr><th>Ratio par/WT</th><th>P</th><th>Q</th><th>Ratio PAR/GUS</th><th>P</th><th>Q</th></tr></thead><tbody><tr><td>Mtr.20567.1.S1_at</td><td>Type III polyketide synthase; naringenin-CHS</td><td>1115.m00010</td><td>0.007</td><td><strong>2.6E-02</strong></td><td>0.0E+00</td><td>4.701</td><td><strong>1.9E-02</strong></td><td>0.0E+00</td></tr><tr><td>Mtr.36333.1.S1_at</td><td>Similar to UPIQ84JJ4 F3H</td><td>BE248436</td><td>0.027</td><td><strong>8.9E-03</strong></td><td>0.0E+00</td><td>2.296</td><td>8.9E-02</td><td>9.2E-13</td></tr><tr><td>Mtr.6517.1.S1_at</td><td>Similar to UPIQ84J65 gray pubescence F3H</td><td>BQ147749</td><td>0.049</td><td><strong>5.7E-03</strong></td><td>0.0E+00</td><td>2.637</td><td><strong>1.5E-03</strong></td><td>0.0E+00</td></tr><tr><td>Mtr.14017.1.S1_at</td><td>Weakly similar to UPILDOX_ARATH (Q96323) ANS</td><td>TC99980</td><td>0.063</td><td><strong>3.0E-02</strong></td><td>0.0E+00</td><td>3.857</td><td><strong>6.3E-04</strong></td><td>0.0E+00</td></tr><tr><td>Mtr.39897.1.S1_at</td><td>Similar to UPIP93697 CPRD12 protein</td><td>TC105988</td><td>0.080</td><td><strong>1.7E-02</strong></td><td>1.0E-231</td><td>2.063</td><td><strong>4.5E-02</strong></td><td>3.6E-22</td></tr><tr><td>Mtr.14428.1.S1_x_at</td><td>Naringenin-CHS; Type III polyketide synthase</td><td>1115.m00011</td><td>0.119</td><td>1.0E-01</td><td>0.0E+00</td><td>2.235</td><td><strong>9.3E-03</strong></td><td>2.1E-70</td></tr><tr><td>Mtr.44985.1.S1_at</td><td>ANR</td><td>TC98546</td><td>0.166</td><td><strong>2.8E-02</strong></td><td>4.2E-28</td><td>2.334</td><td>3.7E-01</td><td>2.5E-54</td></tr><tr><td>Mtr.50541.1.S1_at</td><td>MtPAR gene, Myb, DNA-binding; homeodomain-like</td><td>1054.m00009</td><td>0.197</td><td><strong>2.9E-02</strong></td><td>9.3E-16</td><td>104.692</td><td><strong>4.6E-04</strong></td><td>0.0E+00</td></tr><tr><td>Mtr.28714.1.S1_at</td><td>Homolog to PRFI1609233AI 226868I1609233A CHS 3</td><td>BI311259</td><td>0.215</td><td><strong>2.8E-02</strong></td><td>4.2E-73</td><td>2.333</td><td>9.6E-02</td><td>5.3E-181</td></tr><tr><td>Mtr.10917.1.S1_at</td><td>Similar to UPIC773 SOYBN (O48928) cytochrome P450 77A3</td><td>TC108343</td><td>0.367</td><td><strong>1.2E-02</strong></td><td>3.0E-109</td><td>4.242</td><td>1.1E-01</td><td>0.0E+00</td></tr><tr><td>Mtr.26465.1.S1_s_at</td><td>Similar to UPIPEAM SPIOL (Q9M571) phosphoethanolamine N-methyltransferase</td><td>1520.m00027</td><td>0.421</td><td><strong>3.4E-04</strong></td><td>0.0E+00</td><td>2.180</td><td>1.1E-01</td><td>4.8E-04</td></tr><tr><td>Mtr.37221.1.S1_at</td><td>Homolog to UPIQ43437 photosystem II type I chlorophyll a/b-binding protein precursor</td><td>TC100154</td><td>0.477</td><td><strong>3.8E-02</strong></td><td>5.7E-07</td><td>3.033</td><td>3.7E-01</td><td>6.1E-150</td></tr></tbody></table>

Affymetrix ID, putative annotation, TC, expression ratios between *par* mutant vs. WT or overexpressing lines vs. control with their respective *P* and *Q* values indicated. Significant *P* values are indicated in bold.
higher (by 23.2%) in *par* mutants than in WT controls (Fig. 4C). The reduced amount of epicatechin in the *par* mutants mirrored the reduced level of soluble PAs (Fig. 2D).

Together, the results of genetic, transcriptomic and metabolic analyses indicate that MtPAR functions as a positive regulator of PA biosynthesis in *M. truncatula*.

**MtPAR Acts Upstream of WD40-1.** Previously, a WD40 repeat protein, orthologous to *Arabidopsis* TTG1, was identified in *M. truncatula* and called MtWD40-1 (15). *M. truncatula* *wd40-1* mutants display a drastic decrease of soluble and insoluble PA (15). However, overexpression of *MtWD40-1* in *M. truncatula* hairy roots results in an increase of anthocyanin concentration without affecting PA concentration. We compared published transcriptome data of *wd40-1* mutants (15) to those of *par* mutants. Out of the 38 genes that were down-regulated in 20 DAP seeds of *par* mutants compared with WT controls, 16 were also down-regulated in *wd40-1* mutant seeds collected at 16 DAP (Table S4). Moreover, 14 out of the 16 are related to flavonoid biosynthesis according to Genesin ontology (see complete list and annotations in Table S4). To test whether MtPAR and MtWD40-1 act via a common regulatory pathway to induce target genes, we measured *WD40-1* gene

<figure><img src="image_6.png" /></figure>

**Fig. 4.** Role of MtPAR in regulation of the flavonoid pathway. (A) Schematic representation of the flavonoid biosynthetic pathway leading to PAs and anthocyanins. (*) represents enzymes for which transcript levels are significantly affected in both mutant lines and overexpressing transformants. PAL, L-phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHI, chalcone isomerase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; OMT, O-methyltransferase; UFGT, UDP flavonoid glucosyl transferase; RT, rhamnosyl transferase. (B) Cumulative expression values of different probe sets encoding putative genes involved in flavonoid biosynthesis in *par* mutant seeds at 20 DAP and in hairy roots overexpressing *MtPAR*. Averages of the three biological replicates are indicated with their respective SD. *PAL* gene expression is constituted by cumulative expression of 8 different probe sets encoding for putative PAL enzyme; *4CL* by 17 probe sets; *CHS*, 31 probe sets; *CHI*, 10 probe sets; *F3H*, 12 probe sets; *FLS*, 3 probe sets; *DFR*, 4 probe sets; *LAR*, 1 probe set; *ANS*, 3 probe sets; *ANR*, 2 probe sets; and *GT* (*UGT72L1*), 6 probe sets. Different probe set IDs for each gene are indicated in *SI Materials and Methods*. Expression values for WT were normalized against respective control lines and adjusted to 1. (C) Statistically significant changes in flavonoid content in mature *par* seeds expressed relative to the concentration in segregant WT lines. All other flavonoid content changes are indicated in Table S3. Averages of the three biological replicates are indicated with their respective SD.

expression in *par* mutants by qRT-PCR. Significantly, *WD40-1* transcript levels were between 15 and 50 times lower in the different *par* mutant seeds than in WT sibling seed controls at 20 DAP (Fig. 5A). In contrast, *PAR* transcript levels were unaffected by mutations in the *WD40-1* gene in 16 DAP seeds (15). Furthermore, *MtPAR* overexpression induced *MtWD40-1* expression in *Medicago* hairy roots (Fig. 5B). Direct interactions between *PAR* and *WD40-1* and *ANR* promoters were investigated by using yeast one-hybrid assay (Fig. 5C). MtPAR activated transcription from the *WD40-1* promoter, but not the *ANR* promoter (Fig. 5C).

**Ectopic Expression of MtPAR Induces PA Accumulation in Alfalfa Leaves.** To investigate whether *MtPAR* can be used to engineer PA production in alfalfa leaves, we generated >300 transgenic alfalfa lines overexpressing *MtPAR*. Driven by the constitutive 35S promoter, *MtPAR* expression levels in leaves and stems were significantly higher in almost half of the tested transgenic lines than in the controls. Analysis of PAs from extracts of leaves and stems with the DMACA-based assay indicated that low levels of PAs accumulated in these transgenic lines. PA production was positively correlated with *MtPAR* expression level (Fig. S4 A–D). The presence of PAs was further confirmed by using tandem MS. MS ion peak signals for epicatechin (M-H+, 289) and epicatechin hexoside (M-H+, 450.8), as well as epicatechin dimer (M-H+, 577), were present in extracts from transgenic alfalfa leaves ec-topically expressing *MtPAR*, whereas no signals were detected in extracts from WT alfalfa (Fig. S4E).

Discussion

**MtPAR Is a Positive Regulator of PA Biosynthesis.** We have characterized *MtPAR*, a member of the MYB R2R3 TF family, in *M. truncatula*. Its closest known homolog is an MYB protein of soybean that remains uncharacterized. Our data indicate that MtPAR plays a specific role in the regulation of PA biosynthesis in *Medicago* seeds. First, *MtPAR* gene expression is confined to the seed coat, the site of PA accumulation in developing seed. Second, loss-of-function (*Tnt1* insertion) *par* mutants accumulate

<figure><img src="image_7.png" /></figure>

**Fig. 5.** PAR activates *WD40-1* expression. (A and B) Transcript levels of *MtWD40-1* in seeds (20 DAP) from different *par* lines (A) and in hairy root transformants (B). Relative expression was calculated from qRT-PCR data with respect to the geometric average of transcript levels of two house-keeping genes, *MSC27* and *PDF2*. (C) Yeast one-hybrid analysis. (Top) The reporter-expressing cassette used in yeast one-hybrid assays. The promoters of *MtWD40-1* (27 to –725) and *MtANR* (–1 to –643) were cloned in front of the reporter gene *AUR1-C*, an antibiotic resistance gene that confers Auroeobasidin A (AbA) resistance in yeast. (Middle) No basal expression of *WD40-1* promoter and *ANR* promoter was detected in yeast. (Bottom) Yeast growth assays after the Y1H reporter strains were transformed with plasmids carrying cassettes constitutively expressing PAR effector.
substantially less PA in the seed coat than do WT controls. Third, anthocyanin levels remain normal in *par* mutant seed despite the existence of a common pathway that generates precursors for PA and anthocyanin biosynthesis. Fourth, no aberrant phenotype apart from pale seed color is evident in any organ of *par* mutants. Fifth, ectopic expression of *MtPAR* in roots leads to production of soluble PAs in an organ that normally does not accumulate PAs. Sixth, genes that are repressed in the seed of *par* mutants and induced in *MtPAR* overexpressing roots are largely involved in flavonoid and PA biosynthesis. These putative target genes of MtPAR protein activity include three *CHS* genes, two *F3H* genes, and one *ANS* gene, which are required for both PA and anthocyanin production, as well as the *ANR* gene, which is required for PA production alone.

It is intriguing that anthocyanin levels were unaffected, whereas PA levels were substantially reduced in *par* mutant seed compared with the WT, given that many of the genes required for both PA and anthocyanin biosynthesis were repressed in the mutant. Several hypotheses could explain these results. First, if *MtPAR* is not expressed in cells accumulating anthocyanins, the decrease of PA could be counterbalanced by the increase of flavonoid glycosides and not anthocyanins. Another explanation could involve metabolic channeling. If *ANS* and *ANR* are physically coupled, the product of *ANS* activity, 3-OH-anthocyanidin, would be converted preferentially to epicatechin (and ultimately PA) by *ANR*, rather than being glycosylated for anthocyanin production; theoretical decoupling of *ANS* and *ANR* could result in a relative increase in anthocyanin over epicatechin.

**MtPAR Activates MtWD40 in the Regulatory Network Controlling PA Biosynthesis.** Transcriptional regulation of flavonoid biosynthesis is poorly understood in legumes. In the nonlegume *Arabidopsis*, six loci are known to have regulatory functions in PA biosynthesis, *TT1*, *TT2*, *TT8*, *TT16*, *TTG1*, and *TTG2* (6). *TT1* and *TT16* encode a zinc finger and a MADS box protein, respectively, and are essential for seed pigmentation (26, 27). *TTG2* encodes a WRKY TF, which acts downstream of *TTG1* (28). *TT2*, *TT8*, and *TTG1* encode a MYB (9), a bHLH (29), and a WD40 protein (30), respectively, which interact to form a ternary TF complex. Mutation in any one of these TFs affects PA concentrations in seeds via down-regulation of flavonoid biosynthetic genes (8). In the *Medicago par* mutants, we also observed down-regulation of key genes of the flavonoid pathway.

Little is known about regulation of PA biosynthetic genes in *Medicago*. A single WD40-repeat TF, MtWD40-1, orthologous to *Arabidopsis* AtTTG1, was identified as a positive regulator of PA biosynthesis in *M. truncatula* seeds (15). We compared the action of *MtWD40-1* and *MtPAR* in *M. truncatula*, and it appears that both genes may belong to the same regulatory network. Both mutants exhibit a substantial decrease of PA levels in seed (Fig. 2D; ref. 15). Furthermore, transcriptomic analysis revealed that a common set of genes was down-regulated in mutants defective in these genes. Gene expression analysis also revealed a decrease of *MtWD40-1* gene expression in *par* mutant lines, suggesting that MtPAR regulates *MtWD40-1* expression. The converse was not the case, as *MtPAR* expression was not affected in *wd40-1* mutants (15). A yeast one-hybrid assay revealed a direct interaction between PAR and the *WD40-1*-promoter in yeast, but no direct interaction with the *ANR* promoter, which implies that, in planta, PAR regulates genes encoding enzymes of the flavonoid-PA pathway via its direct role on *WD40-1* expression. This finding may explain why ectopic expression of *MtPAR*, but not of *MtWD40-1*, resulted in PA biosynthesis in roots (Fig. 3; ref. 15). If a complex of TFs is required to induce flavonoid biosynthesis genes in *Medicago*, as is the case in *Arabidopsis* (11), then ectopic expression of *MtPAR*, and consequent induction of *MtWD40-1*, may have provided the requisite TFs for PA biosynthesis in roots. In contrast, ectopic expression of *MtWD40-1* would not induce

*MtPAR* expression and would, therefore, fail to induce PA biosynthesis.

**MtPAR as a Tool for Bioengineering PA Content in Legumes.** Bio-technological solutions are being sought to enhance PA levels in legume shoots to reduce pasture bloat. TFs are attractive in this respect because they have the potential to activate whole metabolic pathways. For example, ectopic expression of *PAP1* led to anthocyanin production in tobacco and *Arabidopsis* (31, 32), as did *LAP1* in alfalfa (18). However, similar approaches to increase PA levels in legume foliage have not yet succeeded.

*MtPAR* offers promise for bioengineering of PA content in crop and pasture species. Indeed, ectopic overexpression of *MtPAR* led to massive accumulation of PAs in *M. truncatula* hairy roots, as a result of increases in the expression of multiple genes involved in PA/anthocyanin biosynthesis, including *CHS*, *F3H*, *ANS*, and *ANR*. This key result shows that *MtPAR* expression is sufficient to induce PA accumulation in organs other than the seed where it functions normally, although the TF activity of *MtPAR* may require additional proteins, such as MtWD40-1. Stable, constitutive overexpression of *MtPAR* in alfalfa resulted in accumulation of PA in shoots, albeit to a level substantially below that needed to provide effective bloat protection (~20 mg/g dry weight). Nonetheless, this finding represents an advance in PA pathway engineering in legumes that may lead to improvement of alfalfa to prevent pasture bloat in the future.

## Materials and Methods

**Gene Identification and Distance Analysis.** The *MtPAR* gene was identified by its seed-specific expression profile (probe set ID Mtr.50541.1.51_at) by using the MtGEA Web server (http://mtgea.noble.org/v2/; refs. 16 and 33). Alignment of the deduced amino acid sequences of MtPAR and other proteins of the MYB R2R3 family was carried out by using ClustalW in the Geneious software suite (Biomatters). The tree based on distance was built by using a neighbor-joining algorithm with 100 bootstrap replicates. The R2R3 domain of each MYB factor was identified by using the PFAM protein family database (34).

**Insertional Mutant Screening.** Generation of the *M. truncatula* *Tnt1* insertional mutant population and growth of R1 seeds were done as described (21). Reverse genetic screening for *Tnt1* retrotransposon insertions in *MtPAR* was performed by using a nested PCR approach (35). PCR products from target mutant lines were purified with QIAquick PCR purification kit (Qiagen) and sequenced by using *Tnt1* primers to confirm insertions in *MtPAR*.

**Analysis of PAs, Anthocyanins, and Flavonoids.** DMACA staining was used to evaluate qualitative changes in the PA contents of mature seeds. Seeds were stained overnight and destained in ethanol for observation. Extraction and analysis of flavonoids from seeds and hairy roots of *M. truncatula* by UV spectroscopy, DMACA staining, and reverse- or normal-phase HPLC coupled to postcolumn DMACA derivatization, UV diode array detection, or MS are described in *SI Materials and Methods*.

**Ectopic Expression of PAR in *Medicago* Hairy Roots and Alfalfa Plants.** Cloning of MtPAR and expression in transgenic hairy roots of *M. truncatula* and plants of alfalfa were performed as described in *SI Materials and Methods*.

**RNA Extraction, Microarray, and qRT-PCR Analysis.** Total RNA was isolated from developing seeds (20 DAP) by using a modified cetyltrimethylammonium bromide method (36) and from hairy roots and alfalfa shoots by using TRIzol reagent, according to the manufacturer's instructions (Invitrogen). Ten micrograms of total RNA from each sample was DNase treated (Turbo DNase; Ambion) and purified (RNeasy MinElute Cleanup kit; Qiagen), according to manufacturer's instructions. Microarray and qRT-PCR analyses were as described in *SI Materials and Methods*. PCR primers are given in Table S5.

**Yeast One-Hybrid Assay.** Yeast one-hybrid assay was performed by using the Matchmaker Gold Yeast One-Hybrid System (Clontech), as described in *SI Materials and Methods*.
**ACKNOWLEDGMENTS.** We thank Yuhong Tang and Stacy Allen for assistance with Affymetrix data analysis; David Huhman and Lloyd Sumner for UPLC-MS analysis; Pascal Ratet and Million Tadege for the construction of the *Tnt1* mutant population; and Jiangqi Wen and Xiaofei Cheng for the screening of this population for *par* mutants. This work was supported by US

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