1 Running head: Medicago saponin inducing bHLH factors 2 · 81 the cytosolic mevalonate (MVA)...

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Running head: Medicago saponin inducing bHLH factors 1

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Corresponding author: 3

Alain Goossens 4

Department of Plant Systems Biology 5

VIB-Ghent University 6

Technologiepark 927 7

B-9052 Gent (Belgium) 8

Tel.: +32 9 3313851; Fax: +32 9 3313809; E-mail: [email protected] 9

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Plant Physiology Preview. Published on November 20, 2015, as DOI:10.1104/pp.15.01645

Copyright 2015 by the American Society of Plant Biologists

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The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin 13

Biosynthesis in Medicago truncatula1[OPEN] 14

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Jan Mertens, Jacob Pollier, Robin Vanden Bossche, Irene Lopez-Vidriero, José Manuel 16

Franco-Zorrilla and Alain Goossens* 17

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Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (J.M., J.P., R.V.B, 19

A.G.); Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 20

Ghent, Belgium (J.M., J.P., R.V.B, A.G.); Genomics Unit, Centro Nacional de Biotecnología-21

CSIC, 28049 Madrid, Spain (I.L-V, J.M.F-Z.) 22

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ORCID ID: 0000-0002-1599-551X (A.G.) 24

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One-sentence summary: Basic helix-loop-helix family transcription factors from subclade 26

IVa specifically regulate the biosynthesis of the triterpene saponins in the model legume 27

Medicago truncatula. 28

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1 This work was supported by the Special Research Funds from Ghent University through the 30

project BOF01J14813 . J.M. and J.P. are pre- and postdoctoral fellows of the Research 31

Foundation Flanders, respectively. 32 * Address correspondence to [email protected]. 33

The author responsible for distribution of materials integral to the findings presented in this 34

article in accordance with the policy described in the Instructions for Authors 35

(www.plantphysiol.org) is: Alain Goossens ([email protected]). 36 [OPEN] Articles can be viewed without a subscription. 37

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Plants respond to stresses by producing a broad spectrum of bioactive specialized 39

metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit 40

leading to the concerted activation of specific metabolic pathways. However, for many 41

specialized metabolic pathways, the transcription factors involved remain unknown. 42

Here, we report on two homologous jasmonate-inducible transcription factors of the 43

basic helix-loop-helix family, Triterpene Saponin biosynthesis Activating Regulator 44

(TSAR) 1 and 2, which direct triterpene saponin biosynthesis in Medicago truncatula. 45

TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-46

hydroxy-3-methylglutaryl-CoA 1 reductase (HMGR1) and Makibishi1, the rate-limiting 47

enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 48

levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box 49

in the promoter of HMGR1. In transient expression assays in tobacco protoplasts, 50

TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream 51

triterpene saponin biosynthetic genes hinting at distinct functionalities within regulation 52

of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula 53

hairy roots resulted in elevated transcript levels of known triterpene saponin 54

biosynthetic genes and strongly increased accumulation of triterpene saponins. TSAR2 55

overexpression specifically boosted haemolytic saponin biosynthesis, whereas TSAR1 56

overexpression primarily stimulated non-haemolytic soyasaponin biosynthesis. Both 57

TSARs also activated all genes of the precursor mevalonate pathway, but did not affect 58

sterol biosynthetic genes, pointing to their specific role as regulators of specialized 59

triterpene metabolism in M. truncatula. 60

61

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5

Plants are frequently confronted with various sorts of biotic and abiotic stress 63

situations. This triggers defense responses such as the production of bioactive specialized 64

metabolites. These compounds are often family-, genus- or even species-specific and thereby 65

constitute a distinct metabolic fingerprint. A specific group of defense compounds are the 66

saponins, a structurally diverse class of amphipathic glycosides with a lipophilic triterpenoid, 67

steroid, or steroidal alkaloid aglycone backbone, also called sapogenin, which is covalently 68

linked to one or more hydrophilic sugar chains via a glycosidic bond (Augustin et al., 2011; 69

Osbourn et al., 2011; Gholami et al., 2014). The structural and functional diversity of the 70

saponins is reflected by their broad spectrum of biological activities that encompass, among 71

others, antimicrobial, anti-insect, allelopathic, anti-carcinogenic, cholesterol-lowering, anti-72

inflammatory and hepatoprotective activities (Avato et al., 2006; Vincken et al., 2007; 73

Augustin et al., 2011; Pollier and Goossens, 2012; Moses et al., 2013). The model legume 74

Medicago truncatula (barrel medic), a member of the Fabaceae plant family, provides a rich 75

source of pentacyclic, oleanane-type triterpene saponins (TSs) and has been widely used to 76

study TS biosynthesis (Gholami et al., 2014). 77

The TS-specific biosynthesis starts with the cyclization of 2,3-oxidosqualene 78

(Supplemental Fig. S1). This is a precursor shared with the phytosterol synthesis route and is 79

a condensation product of six isopentenyl pyrophosphate (IPP) units. IPP is generated through 80

the cytosolic mevalonate (MVA) pathway. The key rate-limiting enzyme of this pathway is 3-81

hydroxy-3-methylglutaryl-CoA reductase (HMGR) that catalyzes the formation of MVA, and 82

of which five isoforms have been characterized in M. truncatula (Kevei et al., 2007). The 83

cyclization of 2,3-oxidosqualene forms the branch point between primary phytosterol and 84

secondary TS metabolism. During primary sterol metabolism, 2,3-oxidosqualene is cyclized 85

to cycloartenol by cycloartenol synthase (CAS) (Corey et al., 1993), whereas during TS 86

biosynthesis, 2,3-oxidosqualene is cyclized to the pentacyclic aglycone β-amyrin by β-amyrin 87

synthase (BAS) (Suzuki et al., 2002; Iturbe-Ormaetxe et al., 2003). Subsequently, the 88

competitive action of two cytochrome P450-dependent mono-oxygenases (P450s) causes 89

another branching of the TS biosynthetic pathway in M. truncatula, and consequently the M. 90

truncatula TS are divided into two distinct classes: haemolytic and non-haemolytic TS. The 91

first committed step for the production of haemolytic TSs is carried out by CYP716A12 that 92

performs three consecutive oxidations at position C-28 of β-amyrin to yield oleanolic acid 93

(Carelli et al., 2011; Fukushima et al., 2011). Subsequently, additional oxidations frequently 94

occur at C-2 and C-23 and are carried out by the P450 enzymes CYP72A67 and 95

CYP72A68v2 (Fukushima et al., 2013; Biazzi et al., 2015). The non-haemolytic soyasaponins 96



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are distinguished by hydroxylation of β-amyrin at positon C-24 catalyzed by CYP93E2 97

(Fukushima et al., 2013). In planta, this modification precludes oxidation at C-28 (Tava et al., 98

2011) and is followed by oxidation at the C-22 position by CYP72A61v2 to yield 99

soyasapogenol B (Fukushima et al., 2013). Additional decorations of the backbone by P450s 100

and the covalent attachment of sugar moieties by UDP-dependent glycosyltransferases 101

(UGTs) further diversify the TS compendium in Medicago (Achnine et al., 2005; Naoumkina 102

et al., 2010; Gholami et al., 2014). 103

All M. truncatula organs appear to accumulate TSs, more particularly as tissue-104

specific mixes of tens of different TSs. Besides this constitutive accumulation, induced TS 105

biosynthesis is often observed in response to herbivore feeding or pathogen attack (Gholami 106

et al., 2014). Inducible TS biosynthesis under stress conditions is mediated by concerted 107

transcriptional activation of the TS pathway (Broeckling et al., 2005; Suzuki et al., 2005; 108

Pollier et al., 2013a), a molecular process in which jasmonates (JAs) play a crucial role. JAs 109

are oxylipin-derived phytohormones that mediate the reprogramming of many metabolic 110

pathways in response to different environmental and developmental cues (Pauwels et al., 111

2009; De Geyter et al., 2012). Accordingly, TS production is strongly enhanced in M. 112

truncatula cell suspension cultures treated exogenously with JAs (Broeckling et al., 2005; 113

Suzuki et al., 2005). To date, little is known about the regulators involved. Post-translational 114

regulation of TS biosynthesis has been shown to be imposed by Makibishi1 (MKB1), a RING 115

membrane-anchor (RMA)-like E3 ubiquitin ligase that monitors TS production by targeting 116

HMGR for endoplasmic reticulum-associated degradation (ERAD) by the 26S proteasome 117

(Pollier et al., 2013a). However, the transcription factors (TFs) triggering the concerted 118

transcriptional activation of TS biosynthetic genes following e.g. JA perception have 119

remained elusive. In fact, only few TFs specifically modulating plant terpene biosynthesis 120

have been identified in general. The basic helix-loop-helix (bHLH) TF MYC2, also known as 121

a primary player in the JA signaling cascade (Kazan and Manners, 2013), and its homologs 122

have been shown to play a role in the regulation of the biosynthesis of sesquiterpenes in 123

Arabidopsis thaliana, Solanum lycopersicum (tomato) and Artemisia annua (Hong et al., 124

2012; Ji et al., 2014; Spyropoulou et al., 2014). Very recently, two other bHLH TFs, Bl (bitter 125

leaf) and Bt (bitter fruit), not related to MYC2, have been found to regulate the accumulation 126

of cucurbitacin triterpenes in Cucumis sativus (cucumber) (Shang et al., 2014). Likewise, 127

another bHLH TF not related to MYC2, i.e. bHLH iridoid synthesis 1 (BIS1), has been shown 128

to control the monoterpene (iridoid) branch of the monoterpene indole alkaloid (MIA) 129

pathway in Catharanthus roseus (Van Moerkercke et al., 2015). 130



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In this study, we examined transcriptomics datasets from M. truncatula and through 131

coexpression analyses, we identified two highly specialized bHLH TFs that are involved in 132

the regulation of the different branches of TS metabolism in M. truncatula. 133

134

RESULTS 135

Coexpression Analyses Reveal Candidate Regulators for TS Metabolism in M. 136

truncatula 137

Previously, we observed a strikingly strong coexpression of HMGR1 and MKB1 in M. 138

truncatula roots and suspension cells under various stress conditions, and/or treated with 139

phytohormones such as JAs (Pollier et al., 2013a). TFs regulating specialized metabolite 140

pathways are often also coexpressed with the target genes encoding the pathway enzymes (De 141

Geyter et al., 2012). Hence, in order to identify candidate regulators of the MVA and/or TS 142

biosynthesis pathways in M. truncatula, we mined the M. truncatula Gene Expression Atlas 143

(MtGEA; http://bioinfo.noble.org/gene-atlas/) (He et al., 2009) for TF-encoding genes with 144

expression profiles that strongly overlap with those of the HMGR1 and MKB1 genes in the 145

tissues and conditions mentioned above. This allowed constituting a shortlist of six TFs that 146

were coexpressed with HMGR1 and MKB1 with a Pearson’s correlation coefficient higher 147

than 0.6 (Table 1, Fig. 1A and Supplemental Fig. S2). This list comprised genes encoding 148

four bHLH proteins, one MYB protein, and one homeodomain-leucine zipper (HD-ZIP) 149

protein. By subsequent BLAST analysis with these TF sequences against the M. truncatula 150

genome, we identified a seventh TF-encoding gene, Medtr4g066460, a homolog of the bHLH 151

Medtr7g080780, which was also JA-inducible and followed a similar trend under some of the 152

selected expression conditions, and therefore was also selected for further functional analysis. 153

154

Two Subclade IVa bHLH TFs Transactivate the Promoters of HMGR1 and MKB1 155

To test whether the putative regulators are able to transactivate the promoters of TS 156

biosynthetic genes, we launched a transient expression assay (TEA) screen in Nicotiana 157

tabacum protoplasts (De Sutter et al., 2005; Vanden Bossche et al., 2013). To this end, we 158

cloned the 1000-bp region upstream of the start codon of HMGR1 (ProHMGR1) and fused it 159

to the Firefly luciferase (fLUC) gene to create a reporter construct. 160

This promoter construct was cotransformed in tobacco protoplasts with the candidate 161

TFs driven by the Cauliflower Mosaic Virus (CaMV) 35S promoter, revealing that two bHLH 162

TFs strongly induced the luciferase activity by 9- and 28-fold, respectively, compared to the 163

fLUC activity in protoplasts cotransfected with a GUS control (Fig. 1B). These two TFs, 164



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which we named ‘Triterpene Saponin Activation Regulator’ (TSAR) 1 and 2, correspond to 165

the homologous genes Medtr7g080780 and Medtr4g066460, respectively. Since the 166

remaining five TFs did not have an effect on ProHMGR1 transactivation comparable with that 167

of the two TSARs (Supplemental Fig. S3), we focused on TSAR1 and TSAR2. We first 168

examined whether they could modulate MKB1 expression in a TEA using the 1000-bp 169

promoter region upstream of the MKB1 transcriptional start site (ProMKB1). This region was 170

defined as such because the MKB1 open reading frame (ORF) is preceded by a 252-bp 5’ 171

untranscribed region (UTR) containing an intron of 1106 bp. Both TSAR1 and TSAR2 172

transactivated ProMKB1 with strengths comparable with those of ProHMGR1 (Fig. 1B), 173

indicating they represent potential general regulators of M. truncatula TS biosynthesis. 174

Members of the bHLH family possess an overall low sequence homology, but are 175

defined by their bHLH signature domain that spans about 50 amino acids (Heim et al., 2003; 176

Toledo-Ortiz et al., 2003; Carretero-Paulet et al., 2010; Pires and Dolan, 2010). The N-177

terminal basic region of ~15 amino acids is responsible for DNA binding and specificity. The 178

C-terminal HLH region of ~45 amino acids contains two amphiphatic α-helices separated by a 179

loop region and promotes the formation of homo- or heterodimeric protein complexes. To 180

determine which phylogenetic clade of the bHLH family harbors TSAR1 and TSAR2, we 181

constructed a neighbor-joining tree based on the alignment of their bHLH domains including 182

all bHLH proteins from A. thaliana clades I, III and IV and the bHLH-type regulators of 183

triterpene synthesis in cucumber. Both TSAR1 and TSAR2 confine in subclade IVa of the 184

bHLH family as defined by Heim et al. (2003) (Supplemental Figs. S4 and S5), whereas for 185

instance the well-known MYC2-type and the recently identified cucumber triterpene 186

regulating bHLH proteins belong to subclades IIIe and Ib, respectively. Notably, BIS1, the 187

recently identified positive regulator of the iridoid branch of MIA biosynthesis in C. roseus, 188

also belongs to subclade IVa of the bHLH proteins (Van Moerkercke et al. 2015) and was 189

therefore included in our phylogenetic analysis for further comparison (Supplemental Figs. S4 190

and S5). 191

192

TSAR1 and TSAR2 Mediate HMGR Transactivation by Binding the N-box in the 193

HMGR Promoter 194

Many studied bHLH proteins, including MYC2 and the cucumber Bl and Bt, act 195

through recognition of an E-box hexanucleotide sequence in the promoter region, which has a 196

consensus sequence 5’-CANNTG-3’ (Carretero-Paulet et al., 2010; Kazan and Manners, 197

2013; Shang et al., 2014). However, it has been shown that bHLHs have different affinities 198



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for variations of this consensus sequence (Fernández-Calvo et al., 2011). As several potential 199

E-box sequences are present in the ProHMGR1, we pinpointed the promoter elements that are 200

essential for TSAR1- and TSAR2-mediated activation by performing protoplast assays using 201

promoter deletion constructs. First, two promoter constructs were generated that span the 202

regions -1 to -500 (ProHMGR1[-1, -500]) and -101 to -281 ProHMGR1[-101, -281], relative 203

to the start of the ORF. In the latter construct, the first 101 bp upstream of the ORF that 204

represent the 5’UTR were omitted. In TEAs in tobacco protoplasts, TSAR1 and TSAR2 205

transactivated both promoters (Fig. 2A). Upon examining ProHMGR1[-101, -281] we 206

identified one E-box-like motif (CACGAG-3’), also referred to as an N-box (Pires and Dolan, 207

2010), at position -246 (see also Supplemental Table S1). To assess the importance of this 208

box, we replaced it by the sequence TGAATT to create a mutated version (ProHMGR1[-101, 209

-281] mut). This replacement completely impeded transactivation by both TSAR1 and 210

TSAR2, indicating that the presence of this box is necessary for TSAR1/2 activity (Fig. 2A). 211

To assess whether TSAR1 and TSAR2 bind directly to the N-box, a yeast one-hybrid 212

(Y1H) assay was carried out. To this end, a yeast strain was generated that contains a 213

synthetic promoter construct in which the ProHMGR1 N-box was repeated in triplicate 214

(3xCACGAG[HMGR1]). Yeast growth on selective medium was observed for yeast expressing 215

TSAR2, but, unexpectedly, not for yeast expressing TSAR1 (Fig. 2B). The latter could be due 216

to impaired folding and/or functionality of TSAR1 in yeast. 217

To further investigate the DNA binding properties of TSAR1 and TSAR2, we used a 218

protein binding microarray, an in vitro system that allows screening for 11-mer nucleotide 219

sequences targeted by TFs (Godoy et al., 2011). To this end, both TSAR TFs were fused with 220

a maltose-binding protein (MBP) allowing detection by anti-MBP antibodies. Enrichment 221

scores (E-scores) represent binding affinities per 8-mer motifs (Fig. 2C). In this assay, both 222

TSAR1 and TSAR2 exhibit strongest affinity for the G-box (CACGTG), similar to 223

Arabidopsis MYC2 (Fig. 2c). TSAR1, TSAR2 and MYC2 also show similar affinities to the 224

N-box CACGAG, but lower as compared to the G-box. Unlike MYC2 however, TSAR1 and 225

TSAR2 show no to low affinity for the G-box like motifs CATGTG and AACGTG. 226

Conversely, as compared to MYC2, TSAR1 and TSAR2 show higher affinity for the box 227

variant CACGCG. Together, our data demonstrate that the TSAR proteins preferably bind to 228

the motif 5’-CACGHG-3’, in which H may be T, A and C, and supports therefore that this 229

element is necessary and sufficient for the TSAR proteins to exert their activity. 230

231

TSAR1 and TSAR2 Transactivate TS Biosynthesis Gene Promoters 232



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To assess the regulatory range of the TSARs, we cloned the promoter sequences of the 233

genes encoding two non-haemolytic TS P450s (ProCYP93E2 and ProCYP72A61v2), two 234

haemolytic TS P450 promoters (ProCYP716A12 and ProCYP72A67), as well as two UGT 235

promoters (ProUGT73K1 and ProUGT73F3) to make reporter constructs containing the 236

1000-bp regions upstream of the start codon of all mentioned genes to carry out 237

transactivation assays in tobacco protoplasts. 238

TSAR1 was able to transactivate the promoters of the non-haemolytic TS P450s, 239

CYP93E2 and CYP72A61v2, by 39- and 14-fold respectively, whereas TSAR2 could only 240

transactivate these constructs by 8- and 3-fold respectively (Fig. 3A). We identified two N-241

boxes, at position -252 and -210, within the promoter sequence of CYP93E2 (ProCYP93E2) 242

(Supplemental Table S1). To assess the necessity of these elements for transactivation of 243

ProCYP93E2, we generated a reporter construct containing a promoter fragment spanning the 244

region from -160 to -300 that encompasses both motifs. In parallel, we created a second 245

construct, spanning the same promoter region but in which the N-box at -252 and -210 were 246

mutated. As expected, this small promoter fragment was sufficient to mediate TSAR1 247

transactivation, whereas this was completely abolished in the mutant version (Fig. 3B), further 248

supporting that the presence of an N-box is necessary and sufficient to enable TSAR1 activity. 249

Neither TSAR1 nor TSAR2 transactivated the 1000-bp promoters of the haemolytic 250

TS P450s by more than 1.5-fold (Fig. 3A). This was a puzzling observation but we reasoned 251

that by spanning only 1000 bp of the promoter region we could have missed important 252

elements for transactivation. Indeed, ProCYP72A67 contained N-box sequences within the 253

1500-bp region of the start codon (Supplemental Table S1). The corresponding 1500-bp 254

promoter fragment could successfully be cloned and used for TEAs. Using this reporter 255

construct, clear transactivation by TSAR2 (9-fold) could be observed (Fig. 3B), further 256

pointing to the importance of the N-box sequence, also for TSAR2 activity. Remarkably, this 257

long ProCYP72A67 reporter construct was hardly activated by TSAR1 (only 2-fold; Fig. 3B). 258

Taking into account the fact that TSAR1 appeared more efficient in transactivating non-259

haemolytic TS P450 gene promoters, this may point to specificities for TSAR1 and TSAR2 in 260

the distinct TS pathway branches. No N-box like sequences could be detected in the available 261

sequence upstream of the cloned ProCYP716A12 and unfortunately we did not manage to 262

clone larger promoter fragments of CYP716A12 either, based on the available genome 263

sequence Mt4.0. Hence, transactivation of ProCYP716A12 was not further assessed. 264

Finally, TSAR1 strongly induced luciferase activity using ProUGT73K1, compared to 265

TSAR2 (i.e. 31- vs 5-fold), whereas ProUGT73F3 was strongly transactivated by both 266



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TSAR1 and TSAR2 (i.e. by 24- and 48-fold) (Fig. 3A). Together, this indicates that, at least 267

in TEAs in tobacco protoplasts, both TSAR1 and TSAR2 encompass the whole TS pathway, 268

albeit possibly with distinct specificities for the two branches. 269

270

Overexpression of TSAR1 Increases the Biosynthesis of Soyasaponins in M. truncatula 271

Hairy Roots 272

To assess the role and specificity of TSAR1 in planta, we generated three independent 273

stably transformed M. truncatula hairy root lines overexpressing TSAR1 (TSAR1OE). For 274

controls, we made three independent lines expressing the GUS gene. Quantitative RT-PCR 275

(qPCR) analysis confirmed overexpression of TSAR1 by ca. 3- to 5-fold (Fig. 4A). All three 276

TSAR1OE lines exhibited a significant but modest increase in HMGR1 and MKB1 transcripts, 277

between 1.5- and 2-fold, and a significant elevation of BAS, CYP93E2, UGT73F3 and 278

UGT73K1 transcript levels, ranging from 3- to 8-fold (Fig. 4A). Finally, in two of the three 279

TSAR1OE lines, we observed a modest increase in TSAR2 and CYP716A12 transcript levels, 280

by ca. 1.75- and 3-fold, respectively (Fig. 4A). Overall, this indicates that TSAR1 281

overexpression has a pathway-encompassing effect on the expression of TS genes. 282

To investigate the effect of TSAR1 overexpression on the M. truncatula metabolome, 283

we performed untargeted metabolite profiling of hairy root extracts by liquid chromatography 284

mass spectrometry (LC-MS). Five technical replicates of three TSAR1OE and three control 285

lines were profiled, yielding a total of 2,813 m/z peaks. To identify the peaks that are different 286

between the control and TSAR1OE lines, a partial least squares discriminant analysis (PLS-287

DA) model that separates the TSAR1OE and control hairy roots was generated (Fig. 4B). This 288

PLS-DA model was subsequently used to generate an S-plot of the correlation and covariance 289

of all m/z peaks. Peaks with an absolute covariance value above 0.03 and an absolute 290

correlation value above 0.8 were considered as significantly different. As such, only peaks 291

that were higher in the TSAR1OE roots contributed to the observed differences (Fig. 4C). The 292

metabolites corresponding to these peaks were elucidated based on their MSn spectra, thereby 293

revealing that TSAR1 overexpression leads to higher levels of specific TSs (Fig. 4D and 294

Supplemental Table S2). In particular, we observed a significant elevation, by 3- to 9-fold 295

relative to control lines, of non-haemolytic soyasaponins, such as Soyasaponin I, Rha-Gal-296

GlcA-Soyasapogenol E, and dHex-Hex-HexA-dHex-Soyasapogenol B (Fig. 4D). Taken 297

together, these data indicate that TSAR1 overexpression has a strong effect on the 298

accumulation of non-haemolytic TSs and support its role as regulator of non-haemolytic TS 299

biosynthesis. 300



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In the qPCR analysis, we also observed an increase in CYP716A12 expression in two 301

of the three TSAR1OE lines. CYP716A12 is the P450 that oxidizes β-amyrin at the C-28 302

position, thereby directing TS biosynthesis toward the haemolytic TS (Carelli et al., 2011). 303

Hence, we probed the effect of TSAR1OE on the accumulation of haemolytic TSs and in 304

accordance with the qPCR analysis, we observed an increase of certain haemolytic TSs, only 305

in the two TSAR1OE lines with a higher expression of CYP716A12 (Fig. 4D and Supplemental 306

Table S2). 307

308

Overexpression of TSAR2 Boosts the Biosynthesis of Haemolytic TSs in M. truncatula 309

Hairy Roots 310

To evaluate the TSAR2 function, we generated three independent M. truncatula 311

TSAR2 overexpression hairy root lines (TSAR2OE). The extent of TSAR2 overexpression was 312

variable across the three independent lines, ranging between 5- to 23-fold, but effectuated in 313

all cases a clear rise in HMGR1, BAS and UGT73F3 transcript levels, ranging between 2- to 314

18-fold (Fig. 5A). Like TSAR1OE roots, TSAR2OE roots exhibited a significant but modest 315

increase in MKB1 transcript levels, between 1.5- and 2-fold (Fig. 5A). In contrast to the 316

TSAR1OE lines however, the transcript levels of CYP93E2 and UGT73K1 were not altered. 317

Furthermore, the transcript level of CYP716A12 increased spectacularly, with more than 150-318

fold in the strongest TSAR2OE line, pointing to a specificity of TSAR2 in the regulation of the 319

haemolytic TSs. 320

To substantiate this, we investigated the changes on the metabolome of TSAR2OE lines 321

by LC-MS, which yielded a total of 2,993 m/z peaks. Like for the TSAR1OE lines, a PLS-DA 322

model separating the TSAR2OE and control lines was generated (Fig. 5B) and used to create 323

an S-plot and depict the significantly different peaks (Fig. 5C). Analogous to the TSAR1OE 324

lines, only peaks that were higher in the TSAR2OE roots contributed to the observed 325

differences with the control roots. Identification of the corresponding metabolites revealed 326

that haemolytic TSs overaccumulated in the TSAR2OE lines (Fig. 5D and Supplemental Table 327

S3). This was exemplified by over 10-fold increases of 3-Glc-Malonyl-28-Glc-Medicagenic 328

acid, Hex-Hex-HexA-Hederagenin and Hex-Hex-Hex-Medicagenic acid (Fig. 5D). 329

The qPCR analysis of the TSAR2OE lines indicated a specific effect of TSAR2 on the 330

haemolytic TSs as the expression of CYP93E2 remained unaltered (Fig. 5A). Accordingly, no 331

soyasaponins were labeled as accumulating significantly different in the TSAR2OE lines 332

(Supplemental Table S3). To ascertain this, we verified the accumulation pattern of the 333

soyasaponins that were most altered in the TSAR1OE lines (Fig. 4D and Supplemental Table 334



13

S3). This confirmed that the effect of TSAR2 overexpression is indeed restricted to the 335

haemolytic TSs (Fig. 5D) and support its role as regulator of haemolytic TS biosynthesis. The 336

specificity of TSAR2 in the regulation of haemolytic TSs could also explain the observed 337

small increase in the production of haemolytic TSs in two of the three TSAR1OE lines, which 338

was correlated with increased TSAR2 expression levels. In this regard, it is noteworthy to 339

mention that none of the TSAR1OE lines actually showed this increased expression of TSAR2 340

and CYP716A12, nor any increase in the accumulation of haemolytic TSs early after the 341

generation of the root lines (Supplemental Fig. S6). Only following the repeated subculturing 342

of the TSAR1OE lines, necessary for the upscaling for the metabolome and transcriptome 343

profiling (see also below), two of the three TSAR1OE lines started to show a modest increase 344

in the expression of these two genes and, accordingly, in the accumulation of haemolytic TSs. 345

Therefore, we consider that TSAR1 and TSAR2 primarily and specifically activate non-346

haemolytic and haemolytic TS biosynthesis, respectively. Long-term culturing of TSAR1OE 347

lines may eventually cause feedback leading to the induction of TSAR2 expression and 348

consequent haemolytic TS biosynthesis. Such feedback may be caused by TS pathway 349

intermediates and/or end products, and may be reminiscent of the feedback repression on the 350

TS pathway genes in the MKB1KD line that overaccumulates monoglycosylated TS (Pollier et 351

al., 2013). 352

353

TSAR1 Knockdown Results in Decreased TS Gene Expression 354

Examining the TSAR expression levels by mining MtGEA and in-house generated 355

RNA-Seq data (see also Table 2 and Supplemental Tables S4 and S5), we noticed that TSAR1 356

exhibited several-fold higher expression levels than TSAR2 in (hairy) roots under ‘standard’, 357

i.e. non-stressed, culturing conditions. Therefore, assessing the physiological role in planta of 358

the TSAR TFs through a gene silencing approach seemed most practical for TSAR1. Indeed, 359

we successfully managed to create three independent TSAR1 knockdown lines (TSAR1KD), 360

with less than 25% of the wild-type TSAR1 transcript levels remaining (Fig. 6A). In contrast, 361

we did not manage to generate TSAR2 knockdown lines, despite repeated transformation 362

rounds, hence we focused further on the TSAR1KD lines. Across those three TSAR1KD lines, 363

we observed a significant decrease in MKB1, HMGR1, CYP93E2, UGT73F3 and UGT73K1 364

transcript levels, varying between 20 and 70% of the control transcript levels (Fig. 6A). No 365

decrease in BAS and CYP716A12 expression levels was apparent in the TSAR1KD lines. 366

Together, this supports the importance of TSAR1 for expression of the genes involved in the 367

biosynthesis of non-haemolytic TSs. 368



14

As for the TSAR overexpression lines, we conducted metabolite profiling of the 369

TSAR1KD root lines by LC-MS (Fig. 6, B-D). Using the same analytical criteria, we could not 370

detect significant changes in TS accumulation (Fig. 6D), hence the decrease in TS synthesis 371

transcripts did not lead to significantly decreased TS metabolite levels. The PLS-DA analysis 372

did not indicate increased abundances of any metabolite, but did reveal a decreased 373

accumulation of 137 metabolites (Fig. 6C), although the fold decrease was modest (less than 374

1.5-fold). The mass data and fragmentation patterns from the FT-MS analysis did not allow 375

identification or tentative annotation of these metabolites. 376

377

RNA-Seq Analyses Confirm and Reveal Downstream Targets of TSAR1 and TSAR2 378

To assess the specificity and range of TSAR1 and TSAR2, we performed a genome-379

wide transcript profiling study by RNA-Seq. To this end, the three independent M. truncatula 380

TSAR1OE, TSAR2OE and control root lines were subjected to RNA sequencing using the 381

Illumina HiSeq2500 platform. A total of 353,231,400 single-end reads of 50 bp were obtained 382

(Supplemental Table S4) and mapped on the M. truncatula genome v4.0 (Tang et al., 2014). 383

Genes with significant differential expression levels between the control lines and the 384

TSAR1OE or TSAR2OE lines were selected using the Cuffdiff algorithm (Trapnell et al., 2010) 385

(Table 2 and Supplemental Table S5). Respectively 859 and 845 genes showed differential 386

expression levels in TSAR1OE and TSAR2OE roots. Between these two gene pools, there was 387

an overlap of 356 genes (Supplemental Fig. S7 and Supplemental Table S5). 388

First, the strong overexpression of TSAR1 and TSAR2 in their respective OE lines was 389

confirmed. Among the differentially expressed genes, we next checked for the known or 390

suggested TS biosynthesis pathway genes (see also Supplemental Fig. S1). In accordance with 391

our qPCR analysis, we found that TSAR1 overexpression strongly enhanced the expression of 392

BAS, CYP93E2, UGT73F3 and UGT73K1, while TSAR2 overexpression instigated increments 393

of BAS, CYP716A12 and UGT73F3 transcripts (Fig. 7 and Supplemental Table S5). In 394

addition, the increased expression of CYP716A12 and TSAR2 in two of the three TSAR1OE 395

lines was confirmed. In these and in the TSAR2OE lines, this was accompanied by increased 396

transcript levels of the CYP72A68v2 and CYP72A67 genes. CYP72A68v2 catalyzes the C-23 397

oxidation of the TS backbone in the synthesis of haemolytic TSs. CYP72A67 has recently 398

been shown to hydroxylate C-2 in the haemolytic TS biosynthesis branch (Fukushima et al., 399

2013; Biazzi et al., 2015). In TSAR1OE lines, we observed a strong increase in CYP72A61v2 400

transcripts encoding the P450 that catalyzes the C-22 oxidation of the TS backbone in the 401

synthesis of soyasaponins. These data further corroborate the specific effect of the TSAR1 402



15

and TSAR2 TFs on the soyasaponin and haemolytic branches of the TS biosynthesis pathway, 403

respectively. 404

To verify TSAR specificity for TS biosynthesis and to examine whether they could 405

also stimulate precursor pathways in an encompassing way, we looked whether genes 406

encoding enzymes involved in the MVA and sterol pathways were among the differentially 407

expressed genes in our analysis. We retrieved the putative MVA and sterol pathway synthesis 408

genes from the Medicago truncatula Pathway Database 2.0 (http://mediccyc.noble.org/) 409

(Urbanczyk-Wochniak and Sumner, 2007). When more than one homolog existed, we 410

identified the closest homolog of the corresponding pathway genes from A. thaliana. Nearly 411

all MVA pathway genes were significantly upregulated in both the TSAR1OE and TSAR2OE 412

roots, albeit usually less pronounced than the TS-specific genes (Fig. 7 and Table 2). The 413

strongest upregulation was observed for HMGR1, the key rate-limiting enzyme of the 414

pathway. None of the sterol synthesis genes were affected by TSAR1 or TSAR2 (Table 2). 415

A few genes annotated as encoding enzymes involved in flavonoid biosynthesis, such 416

as isoflavone synthases and chalcone synthases, appeared differentially expressed, but the 417

FPKM values were not consistent for the three lines per construct (Supplemental Table S5). 418

No other genes potentially involved in the synthesis of phenolic compounds were induced in 419

any of the TSAROE lines (Supplemental Table S5). Hence, no concerted effect of TSAR 420

overexpression on flavonoid biosynthetic gene expression was observed, thus further 421

underscoring the specific activity of TSAR1 and TSAR2 for TS biosynthesis within M. 422

truncatula specialized metabolism. Notably, overexpression of TSAR1, but not of TSAR2, also 423

stimulates expression of genes encoding 9S-lipoxygenases, leginsulins (also annotated as 424

albumins) and Kunitz-type protease inhibitors (Supplemental Table S5), suggesting that 425

additional roles, beyond regulation of specialized metabolism, may exist for TSAR1. 426

427

DISCUSSION 428

Upon predation, plants produce a plethora of specialized metabolites to safeguard their 429

integrity and survival. The phytohormone JA plays a pivotal role herein. TFs at the top of the 430

JA signaling hierarchy, such as MYC2, have been extensively studied in several plant species. 431

However, less is known about the downstream TFs that boost the flow through specific 432

biosynthesis pipelines in plant specialized metabolism. 433

434

Members of Clade IVa of the bHLH Family Activate Different Branches of TS Synthesis 435

in M. truncatula 436



16

Limited data are available about TFs implicated in the activation of specialized terpene 437

metabolite production. In cotton, a WRKY TF has been shown to directly bind the promoter 438

of a sesquiterpene synthase-encoding gene essential for the biosynthesis of sesquiterpene 439

phytoalexins (Xu et al., 2004). In Artemisia annua, two JA-responsive AP2 family TFs and a 440

MYC-type bHLH TF have been identified as activators of the expression of genes encoding 441

the sesquiterpene synthase and P450 essential for the production of the anti-malaria 442

compound artemisinin (Yu et al., 2012; Ji et al., 2014). In A. thaliana, it has been observed 443

that the bHLH TF MYC2 directly binds sesquiterpene synthase gene promoters, resulting in 444

an elevated release of volatile sesquiterpenes (Hong et al., 2012). Likewise, in tomato, a 445

MYC-type bHLH and a WRKY TF have been described to bind the promoter of a 446

sesquiterpene synthase gene (Spyropoulou et al., 2014). Finally, the bHLH TFs Bl (bitter leaf) 447

and Bt (bitter fruit) from cucumber and BIS1 from C. roseus have very recently been 448

identified as activators of the production of cucurbitane-type triterpenes and iridoid-type 449

monoterpenes, respectively (Shang et al., 2014; Van Moerkercke et al., 2015). Here, we 450

identified two other bHLH TFs, TSAR1 and TSAR2 that activate TS biosynthesis in the 451

model legume M. truncatula. 452

Notably, TSAR1 and TSAR2 have distinct preferences in steering fluxes to the two 453

branches of the TS biosynthesis pathway, implying the existence of distinct control 454

mechanisms for the different classes of TSs that accumulate in M. truncatula and that may 455

have a distinct biological function or activity. TSAR1 primarily drives the expression of BAS 456

and all genes encoding the known non-haemolytic or soyasaponin-specific P450s, such as 457

CYP93E2 and CYP72A61v2, as well as the UGTs UGT73K1 and UGT73F3. Upon 458

overexpression in hairy roots, this led to a clear rise in non-haemolytic TS accumulation. 459

Conversely, TSAR2 drives the expression of the BAS, CYP716A12, CYP72A68v2 and 460

UGT73F3 genes, which upon overexpression in hairy roots led to major increases in 461

haemolytic TS levels. Non-haemolytic TS metabolism remained unaffected in TSAR2OE 462

lines. Notably, TSAR2 overexpression did not mediate changes in UGT73K1 levels, 463

suggesting that despite their ability to glucosylate sapogenin skeletons of both TS types in 464

vitro (Achnine et al., 2005), they might only glucosylate non-haemolytic sapogenins in 465

planta. UGT71G1 was not induced by either TSAR1 or TSAR2 overexpression, suggesting that 466

the corresponding UGT might not be implicated in TS biosynthesis and that its activity might 467

be restricted to the glucosylation of flavonoids, for which the recombinant UGT71G1 468

possesses high catalytic activity (Achnine et al., 2005). Consequently, we believe that further 469



17

mining of the RNA-Seq data from TSAR1 and TSAR2 overexpression lines will lead to the 470

discovery of the missing enzymes in M. truncatula TS biosynthesis. 471

Despite the observation that also the expression of the MVA precursor pathway genes 472

is controlled by both TSAR TFs, their specific commitment to the TS pathway was 473

demonstrated by the lack of impact on the sterol biosynthesis pathway. In this regard, the 474

TSAR TFs have a similar pathway-encompassing effect as the MKB1 E3 ubiquitin ligase 475

(Pollier et al. 2013) and it is striking that the TSARs also drive MKB1 expression. However, 476

contrary to M. truncatula hairy root lines with a loss-of-function of MKB1, in which 477

overaccumulation of monoglycosylated TSs is associated with perturbed root development 478

and integrity (Pollier et al. 2013), the pronounced increases in the accumulation of TSs did not 479

result in any phenotypical change in any of the TSAR overexpression lines (Supplemental Fig. 480

S8). Likewise, although we observed significantly lower MKB1 transcript levels in the 481

TSAR1KD roots, this did not result in any phenotypical change and the TS levels remained 482

unaltered. Considering that also mutants defective in CYP716A12 and UGT73F3 suffer from 483

growth retardation (Naoumkina et al., 2010; Carelli et al., 2011), it seems important for 484

normal plant growth and development that the flux through the TS pathway proceeds to the 485

multihydroxylated and multiglycosylated end products. Strict and concerted regulation of TS 486

biosynthetic genes by the TSARs may provide the necessary safety mechanism to achieve 487

this. 488

489

TSAR1 and TSAR2 Are Integrated in a JA Signaling Cascade that Steers Defense 490

Responses 491

bHLHs comprise a major family of TFs and are widely spread across the three 492

eukaryotic kingdoms (Heim et al., 2003; Toledo-Ortiz et al., 2003; Carretero-Paulet et al., 493

2010; Pires and Dolan, 2010). Notably, TSAR1 and TSAR2 belong to subclade IVa of the 494

bHLH family, distinguishing them from the MYC2-type TFs that reside in subclade IIIe and 495

the cucumber Bl and Bt TFs that sort in subclade Ib (Heim et al., 2003). Besides divergent 496

bHLH domains, clade IVa bHLHs comprise an ORF that is about half the size of that of 497

MYC-type TFs. In addition, MYC-type TFs carry a defined JAZ interaction domain (JID) that 498

is lacking in IVa bHLHs. The JID domain is responsible for the interaction of MYCs with 499

Jasmonate ZIM-Domain (JAZ) repressor proteins that thereby block the activity of the MYCs 500

and the transactivation of their target genes. Upon JA perception, JAZ proteins are targeted 501

for degradation by the 26S proteasome (Chini et al., 2007; Thines et al., 2007; Fernández-502

Calvo et al., 2011; Pauwels and Goossens, 2011). As the MYC genes, also TSAR1 and TSAR2 503



18

are JA-inducible. Hence, they seem to constitute an integrated element in the JA signaling 504

cascade. The determination of their exact position in this cascade, relative to the primary 505

signaling module composed of COI1-JAZ-MYC-NINJA (Cuéllar Pérez and Goossens, 2013; 506

Wasternack and Hause, 2013) will be subject of further study. 507

JAs trigger a signaling cascade that mediates broad-scale defense responses resulting 508

in a strictly regulated production of various defense products in response to various biotic and 509

abiotic stresses (Wasternack and Hause, 2013). Our current data suggest that TSAR2 510

specifically drives haemolytic TS production, since only those specialized metabolites were 511

induced in the TSAR2OE lines, and a RNA-Seq analysis did not readily indicated any 512

concerted modulation of other defense or developmental processes. TSAR1, in contrast, 513

clearly also stimulates the production of at least two other types of defense molecules besides 514

soyasaponin TS, namely leginsulins and Kunitz-type protease inhibitors (Supplemental Table 515

S5). Likewise, TSAR1OE roots, but not TSAR2OE roots, showed a strong elevation of several 516

linoleate 9S-lipoxygenase transcripts (Supplemental Table S5). Mining of public expression 517

data on MtGEA indicated that most of the abovementioned genes are JA-inducible, as are the 518

TS biosynthesis genes. Leginsulins (also annotated as albumins) are cysteine-rich peptides 519

that are prevalent within the plant family of Fabaceae (Louis et al., 2004). These 35- to 40-520

kDa peptides contain three disulfide bridges which render them highly resistant to degradation 521

during digestion (Le Gall et al., 2005). In addition, they exhibit anti-insect and hormonal 522

functions in plants and disturb blood glucose levels in mice (Watanabe et al., 1994; Dun et al., 523

2007). Kunitz-type protease inhibitors, are widely distributed across the plant kingdom (Luiza 524

Vilela Oliva et al., 2011). Frequently, they consist of one polypeptide chain that harbors two 525

disulfide bridges. Among others, they inhibit proteases that activate digestive enzymes, 526

leading to the perturbation of digestion, an impaired uptake of amino acids and reduced 527

growth, which is considered to be particularly important as a defense against insect feeding 528

(Howe and Jander, 2008; Oliva et al., 2010). Finally, 9-lipoxygenases are enzymes at the base 529

of the committed biosynthesis of specific oxylipins that act in defense against microbial 530

pathogens (Vicente et al., 2012). 531

532

Conserved Subclade IVa bHLH TFs Regulate Plant Terpene Biosynthesis 533

In A. thaliana, it has been shown that homologous TFs can instigate different effects 534

on jasmonate-responsive genes and cooperate with other TFs, of which many are still 535

unknown, to set off the full complement of jasmonate responses (Fernández-Calvo et al., 536

2011; De Geyter et al., 2012). 537



19

In plants, it is generally assumed that IPP used for the generation of tri- and 538

sesquiterpenes is derived from the MVA pathway, whereas synthesis of other terpenes 539

consumes IPP from the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway 540

(Moses et al., 2013). Overexpression of the C. roseus bHLH TF BIS1 was recently shown to 541

specifically mediate elevated expression of the biosynthesis genes required to yield the MEP-542

pathway dependent monoterpene (iridoid) loganic acid in cell suspension cultures and hairy 543

roots (Van Moerkercke et al., 2015). Interestingly, C. roseus is also a source of non-544

glycosylated pentacyclic triterpenes, including oleanolic acid, but overexpression of BIS1 545

does not affect the expression of MVA or triterpene pathway genes (Van Moerkercke et al., 546

2015). BIS1, like TSAR1 and TSAR2, belongs to the subclade IVa of bHLH proteins 547

(Supplemental Figs. S4 and S5). Remarkably however, in M. truncatula, the TSARs drive 548

MVA and TS pathway gene expression, without altering MEP pathway gene expression 549

(Table 1 & Supplemental Table S5). In C. roseus, the genes required for MIA production are 550

induced by JAs but this is not case for the MVA and TS pathway genes (Van Moerkercke et 551

al., 2013). This differs from the situation in M. truncatula where MVA pathway and TS 552

synthesis genes are JA responsive (Broeckling et al., 2005; Suzuki et al., 2005; Pollier et al., 553

2013). 554

C. roseus and M. truncatula belong to the Apocynaceae and Fabaceae, respectively. 555

Both are dicot plant families, but are representative of the two different clades within that 556

group, the Asterids and Rosids, respectively. Hence, our results show that clade IVa bHLH 557

TFs in two distantly related dicot species exert a similar function in regulating terpene 558

biosynthesis, but are capable of acting on different classes of terpenes depending on the 559

species. Moreover they act not only on the species-specific specialized metabolite pathway 560

branches, but also on the respective primary precursor pathways, i.e. the MEP pathway for 561

MIA synthesis in C. roseus and the MVA pathway for TS biosynthesis in M. truncatula. As 562

well as nearly all genes encoding the enzymes involved in all the above mentioned pathways, 563

the genes encoding the clade IVa bHLH TFs are JA-responsive themselves. This indicates 564

that they are essential elements in the universal capacity of JA to elicit specific specialized 565

terpene pathways across the plant kingdom. 566

567

MATERIALS AND METHODS 568

DNA Constructs 569

Sequences of the full-length ORFs of all TFs were retrieved from the M. truncatula 570

genome v4.0 (Tang et al., 2014) and were cloned using the Gateway technology (Invitrogen). 571



20

Full-length coding sequences were PCR-amplified (for primers, see Supplemental Table S6) 572

and recombined in the donor vector pDONR221. Following sequence verification, the entry 573

clones were recombined with the destination vector p2GW7 for protoplast assays (Vanden 574

Bossche et al., 2013). For overexpression in M. truncatula hairy roots, TSAR1 and TSAR2 575

entry clones were recombined with the binary overexpression vector pK7WG2D (Karimi et 576

al., 2002). A construct to silence TSAR1 through hairpin RNA interference was generated by 577

amplifying a 231-bp fragment from the 3’ UTR from the TSAR1 mRNA. After insertion into 578

pDONR221, the construct was recombined into the binary vector pK7GWIWG2D(II) (Karimi 579

et al., 2002). 580

The promoter regions of HMGR1, CYP93E2, CYP716A12, CYP72A61v2, CYP72A67, 581

UGT73K1 and UGT73F3 were determined using the M. truncatula genome v4.0 (Tang et al., 582

2014). This version of the genome did not contain MKB1, but we were able to trace MKB1 583

and its promoter in another version of the M. truncatula genome, namely Mt20120830-LIPM 584

(Roux et al., 2014). 1000-bp regions upstream of the respective translational start sites were 585

PCR-amplified (Supplemental Table S1) except for MKB1 for which 1000 bp upstream of the 586

transcriptional start were amplified (Supplemental Table S1). In addition, a longer fragment 587

of 1500 bp, upstream of the ORF of CYP72A67, was generated (Supplemental Table S1). 588

Likewise, we amplified shorter fragments of the HMGR1 promoter. A promoter fragment in 589

which the hexanucleotide CACGAG was substituted by TGAATT was generated by overlap 590

extension PCR. A shorter fragment was also generated for ProCYP93E2. A fragment, of 591

which the two N-boxes were substituted with 5’-TGAATT-3’ and 5’-CTATTA-3’, was 592

constructed by overlap extension PCR. All promoter sequences were successively recombined 593

into pDONR221 and sequence-verified entry clones were recombined with the pGWL7 594

plasmid to generate promoter:fLUC reporter constructs (Vanden Bossche et al., 2013). A Y1H 595

bait fragment was generated by overlap extension PCR. Three identical (CACGAG) motifs 596

with their 10 flanking nucleotides from the HMGR1 promoter were fused using two linker 597

sequences (Supplemental Fig. S9). This construct was cloned in the reporter plasmid pMW#2 598

(Deplancke et al., 2006). 599

600

Transient Expression Assays in Tobacco Protoplasts 601

Transient expression assays in N. tabacum Bright Yellow-2 protoplasts were carried 602

out as described previously (De Sutter et al., 2005; Vanden Bossche et al., 2013). Briefly, 603

protoplasts were transfected with a reporter, an effector and a normalizer plasmid. The 604

reporter plasmid consists of a fusion between the promoter fragment of interest and fLUC 605



21

gene. The effector plasmid contains the selected TF driven by the CaMV 35S promoter. The 606

normalizer plasmid, harboring the Renilla luciferase (rLUC), is under control of the CaMV 607

35S promoter. Protoplasts were incubated overnight and lyzed. fLUC and rLUC readouts 608

were collected using the Dual-Luciferase® Reporter Assay System (Promega). Each assay 609

incorporated eight or four biological repeats. Promoter activities were normalized by dividing 610

the fLUC values with the corresponding rLUC values. The average of the normalized fLUC 611

values was calculated and set out relatively to the control fLUC values; i.e. protoplasts 612

transfected with an effector plasmid carrying a GUS gene. 613

614

Phylogenetic Analysis 615

The bHLH domain amino acid sequences of A. thaliana, C. sativus and M. truncatula 616

were defined as by Heim et al. (2003). All members of A. thaliana from subclade I, III and IV 617

were used for the assembly. We incorporated M. truncatula TSAR1 and TSAR2, C. roseus 618

BIS1, and C. sativus Bl and Bt. All sequences were aligned with the ClustalW tool of 619

BioEdit7. Gaps, which are prevalent in the loop region, were dealt with by complete deletion 620

of the corresponding sites. A neighbor-joining tree was constructed in MEGA5 using the 621

Jones, Taylor, and Thorton (JTT) amino acid substitution model (Jones et al., 1992; Tamura et 622

al., 2011). A bootstrap analysis was carried out with 1,000 replicates and an unrooted tree was 623

generated. 624

625

Protein Binding Microarrays 626

N-terminal fusions of TSAR1 and TSAR2 with a HIS-MBP-tag were generated by 627

cloning into the plasmid pDEST-HisMBP (Nallamsetty et al., 2005). The plasmids were 628

introduced in E.coli One Shot® BL21 Star™ (DE3) cells (Thermo Scientific™). Protein 629

expression, purification and DNA-binding in the PBM were carried out according to 630

Godoy et al. (2011). 631

632

Generation of Medicago truncatula Hairy Roots 633

Sterilization of M. truncatula seeds (ecotype Jemalong J5), transformation of seedlings 634

by Agrobacterium rhizogenes (strain LBA 9402/12) and the subsequent generation of hairy 635

roots was carried out as described previously (Pollier et al., 2011). Hairy roots were cultivated 636

for 21 days in liquid medium to provide proper amounts to be used for RNA and metabolite 637

extraction. 638

639



22

Quantitative RT-PCR 640

Frozen roots of three independent transgenic lines were ground under liquid nitrogen. 641

The material was used to prepare total RNA and first-strand cDNA with the RNeasy mini kit 642

(Qiagen) and the iScript cDNA synthesis kit (Biorad), respectively, according to the 643

manufacturer’s instructions. qPCR primers for TSAR1 and TSAR2 were designed using 644

Beacon Designer 4 (Premier Biosoft International). The M. truncatula 40S ribosomal protein 645

S8 and translation elongation factor 1α were used as reference genes. All qRT-primers used 646

are listed in Supplemental Table S6. The qPCR was carried out with a Lightcycler 480 647

(Roche) and the Lightcycler 480 SYBR Green I Master kit (Roche) according to the 648

manufacturer’s guidelines. Three replicates were made for each reaction. Relative expression 649

levels using multiple reference genes were calculated using qBase (Hellemans et al., 2007). 650

651

Yeast One-Hybrid (Y1H) 652

The Y1H reporter strain was made as described (Deplancke et al., 2006). TSAR1 and 653

TSAR2 full-length ORFs were cloned into pDEST22 (Invitrogen), thereby creating a fusion 654

with the GAL4 activation domain. Empty pDEST22 served as a negative control. The yeast 655

reporter strain was transformed with the pDEST22 preys followed by assessment of growth 656

on SD-His plates with and without 20 mM 3-aminotriazole after an incubation period of 4 657

days at 30˚C. 658

659

RNA-Seq Analysis 660

Total RNA of three independent transformant lines per construct was submitted to 661

GATC Biotech (http://www.gatc-biotech.com/) for Illumina HiSeq2500 RNA sequencing (50 662

bp, single-end read). As described (Pollier et al., 2013b) and using default parameters, the raw 663

RNA-Seq reads were quality-trimmed and mapped on the M. truncatula genome v4.0 (Tang 664

et al., 2014) with TOPHAT v2.0.6. Uniquely mapped reads were counted and FPKM values 665

were determined with CUFFLINKS version v2.2.1. (Trapnell et al., 2010; Kim et al., 2013). 666

Differential expression analyses were performed using Cuffdiff (Trapnell et al., 2010). 667

668

Metabolite Profiling of Transformed M. truncatula Hairy Roots 669

Upon harvest, M. truncatula hairy roots were rinsed with purified water, frozen and 670

ground in liquid nitrogen. 400 mg of the ground material was used for metabolite extractions 671

as described previously (Pollier et al., 2011). LC–ESI–MS analysis was performed using an 672

Acquity UPLC BEH C18 column (150 x 2.1 mm, 1.7 μm; Waters) mounted on an Acquity 673



23

UPLC system (Waters). The LC system was coupled to an LTQ XL™ linear ion trap mass 674

spectrometer (IT-MS) (Thermo Electron Corporation) or an LTQ FT Ultra (Thermo Electron 675

Corporation) via an electrospray ionization source operated in the negative mode. The 676

following gradient was run using acidified (0.1% formic acid) water/acetonitrile (99:1, v/v) 677

(solvent A) and acetonitrile/water (99:1, v/v) (solvent B): time 0 min, 5% B; 30 min, 55% B; 678

35 min, 100% B. The injection volume was 10 μL, the flow rate 300 μL/min, and the column 679

temperature 40°C. Negative ionization was obtained with a capillary temperature of 150°C, 680

sheath gas of 25 (arbitrary units), aux. gas of 3 (arbitrary units), and a spray voltage 4.5 kV. 681

Full MS spectra between m/z 120–1400 were recorded. For identification, full MS spectra 682

were interchanged with a dependent MS2 scan event in which the most abundant ion in the 683

previous full MS scan was fragmented, and two dependent MS3 scan events in which the two 684

most abundant daughter ions were fragmented. The collision energy was set at 35%. All 685

samples were analyzed on the LTQ XL™ linear ion trap system and for identification, 686

representative samples were re-analyzed on the LTQ FT Ultra system. The resulting 687

chromatograms were integrated and aligned using the Progenesis QI software (Waters). The 688

PLS-DA analysis was performed with the SIMCA-P 11 software package (Umetrics AB) with 689

Pareto-scaled MS data. Peaks with an absolute covariance value above 0.03 and an absolute 690

correlation value above 0.8 were considered as significantly different. 691

692

Data Deposition 693

The GenBank EMBL/DDBJ accession numbers for TSAR1 and TSAR2 are 694

KM409647 and KR349466, respectively. The raw RNA-Seq reads reported in this paper data 695

are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession 696

number E-MTAB-3532. 697

698

Supplemental Data 699

The following supplemental materials are available. 700

Supplemental Figure S1. Schematic overview of the triterpene saponin and sterol 701

biosynthesis pathways in Medicago truncatula. 702

Supplemental Figure S2. Coexpression Analysis of HMGR1, MKB1 and TF Genes. 703

Supplemental Figure S3. Relative Transactivation of ProHMGR1 by the Seven Selected 704

TF Candidates (in Gray) for Involvement in TS Metabolism. 705

Supplemental Figure S4. Alignment of bHLH Domains. 706



24

Supplemental Figure S5. Phylogenetic Analysis of TSAR1 and TSAR2. 707

Supplemental Figure S6. Overexpresssion of TSAR1 Primarily Activates Non-Haemolytic 708

TS Biosynthesis in M. truncatula Hairy Roots. 709

Supplemental Figure S7. Number of Differentially Expressed Genes in TSAR-710

Overexpressing M. truncatula Hairy Roots. 711

Supplemental Figure S8. TSAR1OE, TSAR2OE and TSAR1KD Roots Exhibit No 712

Phenotypical Changes Compared to Control Roots. 713

Supplemental Figure S9. Nucleotide Sequence of 3xCACGAG[HMGR1]. 714

715



25

Supplemental Table S1. TS promoter sequences. 716

Supplemental Table S2. Differential Peaks Identified by LC-MS Analysis in TSAR1OE 717

M. truncatula Hairy Roots. 718

Supplemental Table S3. Differential Peaks Identified by LC-MS Analysis in TSAR2OE 719

M. truncatula Hairy Roots. 720

Supplemental Table S4. Number of Sequenced Reads in the RNA-Seq Analysis of TSAR-721


Supplemental Table S5. FPKM Values of All Differentially Expressed Genes in TSAR-723


Supplemental Table S6. Primers Used. 725

726

ACKNOWLEDGMENTS 727

We thank Jennifer Fiallos-Jurado for technical assistance, Frederik Coppens for support with 728

the RNA-Seq analysis, Geert Goeminne for his aid with the data processing of the metabolite 729

profiling and Annick Bleys for help with preparing the article. 730

731

AUTHOR CONTRIBUTIONS 732

J.M., J.P., and A.G. designed the research. J.M., J.P., R.V.B. and I.L-V performed the 733

research. J.M., J.P., J.M.F-Z., and A.G. analyzed the data. J.M., J.P., and A.G. wrote the 734

article. 735

736

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931

932



32

TABLES 933

934

Table 1. Selected TFs that Display Highly Overlapping Expression Profiles with HMGR1 or 935

MKB1. 936

Coexpression was analyzed using the MtGEA tool (He et al., 2009). All conditions shown in 937

Figure 1A were taken into account for the analyses. Pearson correlation coefficients were 938

calculated to measure the degree of coexpression. Probe set sequences were blasted against 939

the M. truncatula genome v4.0 to retrieve the corresponding M. truncatula gene IDs (Tang et 940

al., 2014). Medtr4g066460 is a homolog of Medtr7g080780 that displays a similar trend under 941

some of the conditions based on visual inspection. 942

Pearsons correlation

coefficient

Probeset Gene ID TF Family HMGR1 MKB1

Mtr.10397.1.S1_at Medtr5g026500/HMGR - 1.0 0.7983

Mtr.43815.1.S1_at *MKB1 - 0.7983 1.0

Mtr.28568.1.S1_at Medtr7g117670 bHLH 0.8027 <0.6

Mtr.38762.1.S1_at Medtr8g027495 bHLH 0.6495 0.7246

Mtr.51379.1.S1_at Medtr2g038040 bHLH 0.7742 0.6379

Mtr.43316.1.S1_at Medtr7g080780/TSAR

1

bHLH 0.7527 0.646

Mtr.38413.1.S1_at Medtr3g065440 MYB 0.6685 <0.6

Mtr.18769.1.S1_at Medtr8g026960 HD-Zip 0.8204 0.6084

Mtr.9397.1.S1_at Medtr4g066460/TSAR

2

bHLH <0.6 <0.6

*The MKB1 gene is not present on the M. truncatula genome v4.0 (Tang et al., 2014). 943

944

945



33

Table 2. Fragments per kilobase of exon per million fragments mapped (FPKM) values of 946

triterpene genes in transformed M. truncatula hairy roots. Log base 2 values of fold-changes 947

in bold designate genes that show significant differential expression. AVG, average of the 948

three independent control (CTR) and TSAR-overexpressing lines. 949

Gene ID Name CTR TSAR1 OE TSAR2 OE AVG FPKM

AVG FPKM

log2 (fold change)

AVG FPKM

log2 (fold change)

Medtr7g080780 TSAR1 27.84 143.41 2.30 27.06 -0.29 Medtr4g066460 TSAR 2 4.89 7.80 0.64 89.70 4.21Medtr5g026500 HMGR1 6.69 27.02 2.11 56.89 3.23Medtr4g005190 BAS 6.80 32.15 2.32 67.44 3.44Medtr7g056103 CYP93E2 67.24 571.25 3.13 52.73 -0.29 Medtr8g100135 CYP716A12 5.41 17.18 1.63 580.16 6.75Medtr2g035020 UGT73F3 17.06 94.34 2.59 143.81 3.23Medtr4g031800 UGT73K1 54.46 359.68 2.84 49.75 0.04 Medtr2g023680 CYP72A67 2.21 6.20 1.48 197.26 6.50Medtr2g055470 CYP72A68 3.58 9.37 1.44 193.89 5.81Medtr4g031820 CYP72A61 32.30 178.60 2.50 34.70 0.17 Medtr5g098310 acetyl-CoA

acetyltransferase 37.10 44.87 0.37 68.61 1.03

Medtr5g011040 hydroxymethylglutaryl-CoA synthase

42.71 72.86 0.79 133.29 1.68

Medtr7g113660 mevalonate kinase 14.04 16.99 0.32 21.86 0.71Medtr3g091190 Phosphomevalonate

kinase 8.51 12.86 0.63 34.19 2.08

Medtr1g112230 mevalonate diphosphate decarboxylase

45.39 64.45 0.61 102.97 1.33

Medtr7g080060 isopentenyl-diphosphate Delta-isomerase

104.87 222.62 1.14 268.16 1.44

Medtr2g027300 farnesyl pyrophosphate synthase

50.93 87.97 0.81 212.82 2.12

Medtr4g071520 squalene synthase 62.26 107.75 0.85 276.92 2.25Medtr1g017270 squalene epoxidase 71.87 123.42 0.87 406.71 2.63Medtr5g008810 cycloartenol synthase

(CAS) 36.45 36.84 0.09 31.27 -0.10

Medtr3g032530 C-24 methyltransferase (C24MT)

91.87 104.74 0.22 89.97 0.05

Medtr8g006450 C-14 demethylase (CYP51G1)

45.32 46.65 0.01 55.40 0.31

Medtr1g061240 C-14 reductase (Fackel) 8.80 9.04 0.00 9.05 0.03 Medtr6g084920 C-8,7 isomerase (Hydra1) 18.40 19.39 0.03 17.57 -0.06 Medtr3g114780 C-24 methyltransferase

(CVP1) 88.83 92.57 0.17 90.98 0.18

Medtr2g019640 C-22 desaturase (CYP710A15)

19.26 19.68 0.13 18.35 0.07

Medtr5g070090 UGT71G1 5.08 3.93 -0.38 4.25 -0.24

950

951



34

FIGURE LEGENDS 952

953

Figure 1. TSAR1 and TSAR2 are coexpressed with and transactivate HMGR1 and MKB1. A, 954

Coexpression profiles of HMGR1 (black; Mtr.10397), MKB1 (orange; Mtr.43815), TSAR1 955

(blue; Mtr43316) and TSAR2 (green; Mtr.9397) in M. truncatula roots under various culturing 956

conditions, generated with the MtGEA tool (He et al., 2009). Values in the y-axis reflect 957

transcript levels determined by microarray analysis (He et al., 2009). Arrows depict values in 958

methyl jasmonate (MJ)-treated cell suspension cultures. B, Transactivation of ProHMGR1 959

and ProMKB1 by TSAR1 (blue) and TSAR2 (green) in transfected N. tabacum protoplasts. 960

Values in the y-axis are normalized fold-changes relative to protoplasts cotransfected with the 961

reporter constructs and a pCaMV35S:GUS (GUS) control plasmid (black). For the 962

normalization procedure, see Methods. ProHMGR1and ProMKB1 span the 1000-bp region 963

upstream of the translational start site of the HMGR1 and MKB1 gene promoter, respectively. 964

The error bars designate SE of the mean (n=8). Statistical significance was determined by a 965

Student’s t-test (*P<0.05, **P<0.01, ***P<0.001). 966

967

Figure 2. TSARs interact with the N-box in the HMGR promoter. A, Transactivation of 968

HMGR1 promoter fragments by TSAR1 (blue) and TSAR2 (green). Promoter fragments 969

comprise the indicated regions relative to the start codon (between brackets). In the third 970

fragment (mut), the N-box (CACGAG motif) was substituted by TGAATT. Values in the y-971

axis are normalized fold-changes relative to protoplasts cotransfected with the reporter 972

constructs and a pCaMV35S:GUS (GUS) control plasmid (in black). The error bars designate 973

SE of the mean (n=8). Statistical significance was determined by a Student’s t-test (*P<0.05, 974

**P<0.01, ***P<0.001). B, Y1H analysis of the binding of TSAR1 and TSAR2 to a 975

3xCACGAG[HMGR1] promoter element. The TSAR1 and TSAR2 ORFs fused to GAL4AD or the 976

empty vector control (pDEST22) were expressed in reporter strains harboring the HIS3 gene 977

under control of a synthetic promoter element consisting of a triple repeat of the CACGAG 978

element with 10 flanking nucleotides as found in the HMGR1 promoter (3xCACGAG[HMGR1]). 979

Transformed yeast cultures dropped in serial dilutions (10- and 100-fold) were grown for six 980

days on selective medium (minus histidine and plus 3-amino-1,2,4-triazole (3-AT)). C, 981

Identification of TSAR-binding motifs in vitro by PBM analysis. Shown are the box plots of 982

E-values of G-box and N-box variants. The line in the box indicates the median (quartile 983

50%). Boxes indicate the quartiles from 25% to 75% of the distribution. Bars represent the 984



35

quartiles from 1-25% and from 75-100%. Dots represent outliers. For comparison we 985

included Arabidopsis MYC2 (Godoy et al., 2011). 986

987

Figure 3. TSARs transactivate triterpene saponin biosynthesis gene promoters. A, 988

Transactivation of ProCYP93E2, ProCYP72A61v2, ProCYP716A12, ProCYP72A67, 989

ProUGT73K1 and ProUGT73F3 by TSAR1 (blue) and TSAR2 (green) in transfected N. 990

tabacum protoplasts. Values in the y-axis are normalized fold-changes relative to protoplasts 991

cotransfected with the reporter constructs and a pCaMV35S:GUS (GUS) control plasmid 992

(black). The error bars designate SE of the mean (n=4). Statistical significance was 993

determined by a Student’s t-test (*P<0.05, **P<0.01, ***P<0.001). All promoters comprise 994

the 1000-bp region upstream of their respective translational start sites. B, TSAR1 and 995

TSAR2 depend on the N-boxes in ProCYP93E2 and ProCYP72A67 [-1, -1500], respectively. 996

Promoter fragments comprise the indicated regions relative to the start codon (between 997

brackets). In the mutant (mut) version of ProCYP93E2, the two N-boxes (see Supplemental 998

Table S1) were substituted with TGAATT and CTATTA. Values on the y-axis are normalized 999

fold-changes relative to protoplasts cotransfected with the reporter constructs and a 1000

pCaMV35S:GUS (GUS) control plasmid (in black). The error bars designate SE of the mean 1001

(n=4). Statistical significance was determined by a Student’s t-test (*P<0.05, **P<0.01, 1002

***P<0.001). 1003

1004

Figure 4. Overexpresssion of TSAR1 boosts non-haemolytic TS biosynthesis in M. truncatula 1005

hairy roots. A, qPCR analysis of TS biosynthetic genes in three independent control (CTR) 1006

and three independent TSAR1OE M. truncatula hairy root lines. Control lines were 1007

transformed with a pCaMV35S:GUS (GUS) construct. Expression ratios were plotted 1008

relatively to the normalized CTR-1. The error bars designate SE of the mean (n=3). B, Partial 1009

least squares discriminant analysis (PLS-DA) of samples from TSAR1OE (red) and CTR 1010

(black) roots. C, S-plot for correlation (p(corr)[1]) and covariance (w*c[1]) derived from 1011

PLS-DA. Metabolites in the bottom left and top right quadrants (marked by dotted red lines) 1012

are significantly higher and lower, respectively, in abundance in the TSAR1OE samples. D, 1013

Average total ion current (TIC) of peaks corresponding to TS. The error bars designate SE of 1014

the mean (n=5). Statistical significances were calculated by a Student’s t-test (*P<0.05, 1015

**P<0.01, ***P<0.001). 1016



36

1017

Figure 5. Overexpresssion of TSAR2 boosts haemolytic TS biosynthesis in M. truncatula 1018

hairy roots. A, qPCR analysis of TS biosynthetic genes in three independent control (CTR) 1019

and three independent TSAR2OE M. truncatula hairy root lines. Expression ratios were plotted 1020

relatively to the normalized CTR-1. The error bars designate SE of the mean (n=3). B, PLS-1021

DA of samples from TSAR2OE (red) and CTR (black) roots. C, S-plot for correlation 1022

(p(corr)[1]) and covariance (w*c[1]) derived from PLS-DA. Metabolites in the bottom left 1023

and top right quadrants (marked by dotted red lines) are significantly higher and lower, 1024

respectively, in abundance in the TSAR2OE samples. D, Average total ion current (TIC) of 1025

peaks corresponding to TS. The error bars designate SE of the mean (n=5). Statistical 1026

significances were calculated by a Student’s t-test (*P<0.05, **P<0.01, ***P<0.001). 1027

1028

Figure 6. Knockdown of TSAR1 lowers TS biosynthesis gene expression. A, qPCR analysis 1029

of TS biosynthetic genes in three independent control (CTR) and TSAR1KD M. truncatula 1030

hairy root lines. Expression ratios were plotted relatively to the normalized CTR-1. The error 1031

bars designate SE of the mean (n=3). B, PLS-DA of samples from TSAR1KD (red) and CTR 1032

(black) roots. C, S-plot for correlation (p(corr)[1]) and covariance (w*c[1]) derived from 1033

PLS-DA. Metabolites in the bottom left and top right quadrants (marked by dotted red lines) 1034

are significantly higher and lower in abundance in the TSAR1KD samples, respectively. D, 1035

Average total ion current (TIC) of peaks corresponding to TS. The error bars designate SE of 1036

the mean (n=5). Statistical significances were calculated by a Student’s t-test (*P<0.05, 1037

**P<0.01, ***P<0.001). 1038

1039

Figure 7. TSAR1 and TSAR2 overexpression specifically modulates the TS biosynthesis 1040

pathway in Medicago truncatula. Log base 2 values of the fold upregulation of biosynthetic 1041

genes in TSAR1OE and TSAR2OE roots relative to the control roots were calculated using 1042

Cufflinks (Trapnell et al., 2010), and are depicted by color codes (left and right for TSAR1 1043

and TSAR2, respectively). 1044

1045

















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Biazzi E, Carelli M, Tava A, Abruscato P, Losini I, Avato P, Scotti C, Calderini O (2015) CYP72A67 catalyzes a key oxidative step inMedicago truncatula hemolytic saponin biosynthesis. Mol Plant: epub ahead of print


Broeckling CD, Huhman DV, Farag MA, Smith JT, May GD, Mendes P, Dixon RA, Sumner LW (2005) Metabolic profiling of Medicagotruncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. J Exp Bot 56: 323-336


Carelli M, Biazzi E, Panara F, Tava A, Scaramelli L, Porceddu A, Graham N, Odoardi M, Piano E, Arcioni S, et al (2011) Medicagotruncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins. Plant Cell 23: 3070-3081


Carretero-Paulet L, Galstyan A, Roig-Villanova I, Martínez-García JF, Bilbao-Castro JR, Robertson DL (2010) Genome-wideclassification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae.Plant Physiol 153: 1398-1412


Chini A, Fonseca S, Fernández G, Adie B, Chico JM, Lorenzo O, García-Casado G, López-Vidriero I, Lozano FM, Ponce MR, et al(2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448: 666-671


Corey EJ, Matsuda SPT, Bartel B (1993) Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functionalexpression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen. Proc Natl Acad Sci USA 90:11628-11632


Cuéllar Pérez A, Goossens A (2013) Jasmonate signalling: a copycat of auxin signalling? Plant Cell Environ 36: 2071-2084Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

De Geyter N, Gholami A, Goormachtig S, Goossens A (2012) Transcriptional machineries in jasmonate-elicited plant secondarymetabolism. Trends Plant Sci 17: 349-359


De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inzé D, Goossens A, Hilson P (2005) Exploration ofjasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J 44: 1065-1076


Deplancke B, Vermeirssen V, Arda HE, Martinez NJ, Walhout AJM (2006) Gateway-compatible yeast one-hybrid screens. ColdSpring Harb Protoc 2006: pdb.prot4590

Pubmed: Author and TitleCrossRef: Author and Title


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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Science%5BTitle%5D%29 AND 346%5BVolume%5D AND 1084%5BPagination%5D AND Shang Y%2C Ma Y%2C Zhou Y%2C Zhang H%2C Duan L%2C Chen H%2C Zeng J%2C Zhou Q%2C Wang S%2C Gu W%2C et al%5BAuthor%5D&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28RNA sequencing on Solanum lycopersicum trichomes identifies transcription factors that activate terpene synthase promoters%2E%5BTitle%5D%29 AND Spyropoulou EA%2C Haring MA%2C Schuurink RC%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=RNA sequencing on Solanum lycopersicum trichomes identifies transcription factors that activate terpene synthase promoters.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Spyropoulou&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant J%5BTitle%5D%29 AND 32%5BVolume%5D AND 1033%5BPagination%5D AND Suzuki H%2C Achnine L%2C Xu R%2C Matsuda SPT%2C Dixon RA%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Suzuki&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Planta%5BTitle%5D%29 AND 220%5BVolume%5D AND 696%5BPagination%5D AND Suzuki H%2C Reddy MSS%2C Naoumkina M%2C Aziz N%2C May GD%2C Huhman DV%2C Sumner LW%2C Blount JW%2C Mendes P%2C Dixon RA%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic re-programming in cell suspension cultures of the model legume Medicago truncatula&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Suzuki&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28MEGA%5BTitle%5D%29 AND 5%5BVolume%5D AND molecular evolutionary genetics analysis using maximum likelihood%2C evolutionary distance%2C and maximum parsimony methods%2E Mol Biol Evol 28%3A 2731%5BPagination%5D AND Tamura K%2C Peterson D%2C Peterson N%2C Stecher G%2C Nei M%2C Kumar S%5BAuthor%5D&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Phytochem Rev%5BTitle%5D%29 AND 10%5BVolume%5D AND 459%5BPagination%5D AND Tava A%2C Scotti C%2C Avato P%5BAuthor%5D&dopt=abstract

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http://search.crossref.org/?page=1&rows=2&q=Toledo-Ortiz G, Huq E, Quail PH (2003) The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15: 1749-1770

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Toledo-Ortiz&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nat Biotechnol%5BTitle%5D%29 AND 28%5BVolume%5D AND 511%5BPagination%5D AND Trapnell C%2C Williams BA%2C Pertea G%2C Mortazavi A%2C Kwan G%2C van Baren MJ%2C Salzberg SL%2C Wold BJ%2C Pachter L%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28: 511-515

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Trapnell&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Transient expression assays in tobacco protoplasts%2E In L Pauwels%2C A Goossens%2C eds%2C Jasmonate Signaling%2C Ed%5BTitle%5D%29 AND 1%5BVolume%5D AND 227%5BPagination%5D AND Vanden Bossche R%2C Demedts B%2C Vanderhaeghen R%2C Goossens A%5BAuthor%5D&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Proc Natl Acad Sci USA%5BTitle%5D%29 AND 112%5BVolume%5D AND 8130%5BPagination%5D AND Van Moerkercke A%2C Steensma P%2C Schweizer F%2C Pollier J%2C Gariboldi I%2C Payne R%2C Vanden Bossche R%2C Miettinen K%2C Espoz J%2C Purnama PC%2C Kellner F%2C Sepp�nen%2DLaakso T%2C O%27Connor SE%2C Rischer H%2C Memelink J%2C Goossens A%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Van Moerkercke A, Steensma P, Schweizer F, Pollier J, Gariboldi I, Payne R, Vanden Bossche R, Miettinen K, Espoz J, Purnama PC, Kellner F, Sepp�nen-Laakso T, O'Connor SE, Rischer H, Memelink J, Goossens A (2015) The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus. Proc Natl Acad Sci USA 112: 8130-8135

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http://scholar.google.com/scholar?as_q=The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Van&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Mol Plant%5BTitle%5D%29 AND 5%5BVolume%5D AND 914%5BPagination%5D AND Vicente J%2C Casc�n T%2C Vicedo B%2C Garc�a%2DAgust�n P%2C Hamberg M%2C Castresana C%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Vicente J, Casc�n T, Vicedo B, Garc�a-Agust�n P, Hamberg M, Castresana C (2012) Role of 9-lipoxygenase and a-dioxygenase oxylipin pathways as modulators of local and systemic defense. Mol Plant 5: 914-928

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Phytochemistry%5BTitle%5D%29 AND 68%5BVolume%5D AND 275%5BPagination%5D AND Vincken J%2DP%2C Heng L%2C de Groot A%2C Gruppen H%5BAuthor%5D&dopt=abstract

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1 Running head: Medicago saponin inducing bHLH factors 2 · 81 the cytosolic mevalonate (MVA)...

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