1

RESEARCH ARTICLE 1 2

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron 3

Stigmas 4

5 Olivia Costantina Demurtas

1, Rita de Brito Francisco

2, Gianfranco Diretto

1, Paola Ferrante

1, Sarah 6

Frusciante1, Marco Pietrella

1,3, Giuseppe Aprea

1, Lorenzo Borghi

2, Mistianne Feeney

4, Lorenzo Frigerio

4, 7

Adriana Coricello5, Giosuè Costa

5, Stefano Alcaro

5, Enrico Martinoia

2*, Giovanni Giuliano

1* 8

9 1ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, C.R. 10 Casaccia, 00123, Rome, Italy 11 2Department of Plant and Microbial Biology, University of Zurich, 8008 Zurich, Switzerland 12 3Council for Agricultural Research and Economics (CREA), Research Center for Olive, Citrus and Tree Fruit, 13 47121 Forlì, Italy 14 4School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom 15 5Department of Health Sciences, Magna Græcia University of Catanzaro, 88100 Catanzaro, Italy 16

17 *Corresponding authors: 18 giovanni.giuliano@enea.it 19 enrico.martinoia@botinst.uzh.ch 20

21 Short title: Saffron vacuolar crocin transporters 22 One-sentence summary: A ‘transportomics’ approach allows identification of crocin vacuolar transporters 23 from saffron stigmas. 24

25

The author(s) responsible for distribution of materials integral to the findings presented in this article in 26 accordance with the policy described in the Instructions for Authors (www.plantcell.org) is Olivia Costantina 27 Demurtas (olivia.demurtas@enea.it) 28

29 ABSTRACT 30

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron 31 spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in 32 intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on 33 co-expression analysis, heterologous expression in yeast, and in vitro “transportomic” assays using yeast 34 microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, 35 and used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three 36 transporters, belonging to both the Multidrug And Toxic Compound Extrusion (MATE) and ATP Binding 37 Cassette C (ABCC) families, were co-expressed with crocins and/or with the gene encoding the first dedicated 38 enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to 39 mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed 40 in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and 41 cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana 42 leaves. 43

44 Keywords: Crocus sativus, stigma, ATP Binding Cassette (ABC) transporters, Multidrug And Toxic Compound 45 Extrusion (MATE) transporters, crocins, in vitro transport assay, microsomes, confocal microscopy. 46

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Plant Cell Advance Publication. Published on September 23, 2019, doi:10.1105/tpc.19.00193

©2019 American Society of Plant Biologists. All Rights Reserved

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3

INTRODUCTION 50

A fascinating feature of vascular plants is their capacity to produce extremely diverse specialized 51

metabolites (Pichersky and Lewinsohn, 2011) and to accumulate some of them to extremely high 52

concentrations: steviol glycosides in Stevia rebaudiana leaves (Brandle and Telmer, 2007) and crocins 53

in Crocus sativus stigmas (Bouvier et al., 2003), for instance, can make up to 10% of the tissue’s dry 54

weight. Plants have evolved a variety of strategies for storing specialized metabolites, such as the 55

accumulation in specialized cell types (McConkey et al., 2000), or their sequestration in the central 56

cellular vacuole (Martinoia et al., 2007). Well-known examples of the latter strategy are the vacuolar 57

sequestration of anthocyanins in Vitis vinifera berries (Francisco et al., 2013), of nicotine in Nicotiana 58

tabacum leaves (Morita et al., 2009), of steviol glycosides in Stevia rebaudiana leaves (Brandle and 59

Telmer, 2007) and of crocins in Crocus sativus stigmas (Bouvier et al., 2003). Vacuolar sequestration 60

has been proposed to prevent the feedback inhibition of the biosynthetic enzymes, and to reduce 61

toxicity effects induced by high cytosolic concentrations of the final products (Goodman et al., 2004). 62

63

Saffron - the most expensive spice on Earth - is composed of the dried stigmas of Crocus sativus 64

(Figure 1A-B), which accumulate large amounts of crocins, apocarotenoid glycosides that confer the 65

red color to the saffron spice (Tarantilis et al., 1995). Up to 15 different crocins have been identified in 66

mature C. sativus stigmas, consisting of both all-trans and 13-cis crocetin esterified with 1 to 5 glucose 67

moieties (Figure 1). The proposed pathway for crocin biosynthesis in C. sativus stigmas starts with the 68

cleavage, in the plastid, of zeaxanthin by Carotenoid Cleavage Dioxygenase 2 (CCD2) (Frusciante et 69

al., 2014). The cleavage product, crocetin dialdehyde, migrates to the endoplasmic reticulum (ER), 70

where it is dehydrogenated to crocetin by a membrane-associated CsALDH3I1, and then glycosylated 71

to crocins 1 and 2ʹ by CsUGT74AD1, localized in the cytosol (Figure 1C-D) (Demurtas et al., 2018). 72

More highly glycosylated crocins are synthesized by an unidentified UDP-glycosyl transferase (UGT), 73

probably localized in the cytosol (Figure 1D). Given their polar nature, crocins synthesized in the 74

cytosol must be transported to the vacuole by one or more tonoplast transporters (Figure 1D). This 75

paper describes their identification. 76

77

Vacuolar transport of glycosylated metabolites, such as glycosylated flavonoids or hormones (ABA 78

glucosyl esters), has been well documented (Gomez et al., 2009; Zhao and Dixon, 2009) and is effected 79

by both Multidrug And Toxic Compound Extrusion (MATE) and ATP Binding Cassette C (ABCC) 80

4

transporters (Martinoia et al., 2007; Francisco and Martinoia, 2018). ABC and MATE transporters have 81

been characterized through expression in yeast, animal or plant cells, isolation of transporter-loaded 82

microsomes, and transport assays using radiolabeled, fluorescent or light-absorbing compounds 83

transported into the microsomes (Marinova et al., 2007; Zhao and Dixon, 2009; Nour-Eldin et al., 84

2012; Burla et al., 2013; Francisco et al., 2013). This limits the number of compounds whose transport 85

can be studied, as well as the capacity to mimic in vivo conditions, where a transporter is exposed to 86

thousands of different compounds. In this work, we study the in vitro transport of multiple metabolites 87

from C. sativus stigma extracts, through a transportomic assay based on liquid chromatography-88

photodiode array-high resolution mass spectrometry (LC-PDA-HRMS), and its use to identify and 89

characterize C. sativus tonoplast transporters involved in vacuolar crocin accumulation. 90

91

RESULTS 92

Identification and characterization of candidate crocin transporters 93

94

A C. sativus stigma transcriptome (Supplemental Data set 1), was searched for expressed genes 95

belonging to the ABCC and MATE classes of tonoplast transporters. Nine ABCC and 11 MATE 96

transporters were expressed in stigmas (Figure 2A, Supplemental Table 1). The highest expressed 97

transcript in stigmas was CsMATE4, followed by CsABCC4a, CsABCC2, CsMATE1a and CsMATE1b. 98

Only CsABCC4a was specifically expressed in stigmas, while CsMATE4 was highly expressed also in 99

tepals and CsABCC2, CsMATE1a and CsMATE1b were mainly expressed in other tissues. To further 100

investigate their possible role in crocin transport, we performed a co-expression analysis (see Methods 101

for more details) of all 20 transcripts with total crocins and with the transcript encoding the first 102

dedicated enzyme in the crocin pathway, CsCCD2 (Frusciante et al., 2014). CsABCC4a expression 103

correlated with both CsCCD2 and crocins (Pearson correlation coefficient (ρ) = 0.99 and 0.95, 104

respectively), followed by CsMATE4 (ρ = 0.84 and 0.75) (Figure 2B, Supplemental Table 2). 105

CsABCC2 displayed a good correlation (ρ= 0.90) with CsCCD2, but not with crocins, while none of the 106

remaining 17 transporters showed significant positive correlations. 107

108

We decided to functionally characterize the five transporters that were most highly expressed in 109

stigmas (underlined in Figure 2A). The corresponding full-length transcripts were isolated from stigma 110

RNA and sequenced (see Methods). Phylogenetic analysis (Figure 2C) revealed that CsABCC4a is 111

5

closely related to Arabidopsis thaliana (At) ABCC4, involved in the vacuolar transport of folates 112

(Klein et al., 2004), while CsABCC2 shows high similarity to AtABCC2, which transports ABA 113

glucosyl esters (ABA-GEs) (Burla et al., 2013), phytochelatins (Song et al., 2010), glutathione 114

conjugates and chlorophyll catabolites (Lu et al., 1998), and to AtABCC1, involved in the transport of 115

phytochelatins (Song et al., 2010) and folates (Raichaudhuri et al., 2009). CsMATE4 is closely related 116

to the MATE1 transporter of Coptis japonica, proposed to mediate the transport of the alkaloid 117

berberine in rhizomes (Takanashi et al., 2017) and CsMATE1a and CsMATE1b are related to the 118

MATE2 protein of Sorghum bicolor that mediates the vacuolar accumulation of the cyanogenic 119

glucoside dhurrin (Darbani et al., 2016), and to Nicotiana tabacum MATE1 and MATE2, which are 120

responsible for the vacuolar accumulation of the alkaloid nicotine (Shoji et al., 2009). The typical ABC 121

signatures (Walker A and Walker B) in the two nucleotide binding domains (NBD1 and NBD2) are 122

well conserved in CsABCC transporters, but many differences were found in the transmembrane 123

domains known to be responsible for substrate recognition/specificity (Theodoulou, 2000; Jasinski et 124

al., 2003; Wilkens, 2015) (Supplemental Figure 1). 125

126

Crocin transporters must be localized in the plant tonoplast, since crocins are synthesized in the cytosol 127

and accumulate in the vacuole (Demurtas et al., 2018). We fused the five transporter genes most 128

expressed in stigmas to the enhanced Green Fluorescent Protein (eGFP) gene (Cinelli et al., 2000) and 129

cloned them in the pBI121 Agrobacterium transformation vector. Nicotiana benthamiana leaves were 130

co-infiltrated with Agrobacterium tumefaciens (strain C58C1) harboring constructs for expression of 131

different eGFP fusions, and the γTIP tonoplast marker fused to the red fluorescent protein (RFP) 132

(Nelson et al., 2007) (Figure 3). At this resolution, the GFP signal showed co-localization with the RFP 133

signal, suggesting that all tested transporters were predominantly tonoplast localized. 134

135

Establishment of a transportomic assay to identify crocin transporters 136

137

Transport of intracellular metabolites by vacuolar transporters is often stereospecific (Bhatia et al., 138

2008; Zhou et al., 2014; Schneider, 2015). Commercial crocin preparations are not representative of the 139

crocin content of C. sativus stigmas, being composed almost exclusively of all-trans crocin 4 140

(Supplemental Figure 2), while in C. sativus stigma six major and several minor crocins, including 13-141

cis isomers, are present (Supplemental Figure 2 and Figure 4B). Therefore, to study the transport of the 142

6

natural crocin substrates, as well as that of other glycosylated compounds accumulated in stigmas 143

(picrocrocin and flavonoid glycosides), we used a C. sativus stigma hydroalcoholic extract as a 144

substrate for the transport reaction. Simultaneous detection of multiple metabolites relied on their 145

detection by LC-PDA-HRMS (Figure 4). We expressed the five C. sativus transporters in yeast cells, 146

isolated microsomes from those cells, and used them to perform transportomic assays. Briefly, the 147

transporters were cloned in the pNEV plasmid and expressed in a yeast strain defective in the ABCC 148

Yeast Bile Transporter1 (YBT1) (ybt1 strain) (Sauer and Stolz, 1994; Paumi et al., 2009). Total 149

microsomes were isolated from the yeast transformants and their intactness was assessed by measuring 150

the transport of leukotriene C4 (LTC4) by LC-HRMS (see Methods) (Supplemental Figure 3). The 151

sensitivity of this method was comparable to the standard radiochemical assay (Leier et al., 1994), 152

indicating that the transportomic assay was sensitive enough to detect transport of metabolites present 153

at low concentrations (fmol to pmol). Microsomes from yeast cells transformed with the pNEV empty 154

vector were used as controls (Figure 4). 155

156

Microsomes expressing CsABCC4a and CsABCC2 were able to mediate the transport of different 157

crocins with different efficiency and in an ATP-dependent manner (Figures 4 and 5). CsABCC4a 158

transported with approximately equal efficiency trans crocin 1, cis crocin 3 and trans crocin 2ʹ, while 159

CsABCC2 showed lower transport efficiency on the latter crocin. In general, crocins carrying smaller 160

glucose groups were efficiently transported in the all-trans form, while crocins with larger gentiobiose 161

groups displayed preferential transport in the cis form. Low, but significant, levels of transport by 162

CsABCC4a and CsABCC2 were also observed for some flavonoids (Figure 5), in agreement with the 163

broad substrate range exhibited by ABCCs (Hwang et al., 2016). Crocin transport by CsABCC4a and 164

CsABCC2 was inhibited by probenecid, a known inhibitor of ABC-type transporters and by incubation 165

on ice, excluding non-specific binding to the yeast microsomes (Table 1). MATE transporters were 166

able to transport only flavonoid glycosides (Figure 5). We did not observe transport of picrocrocin, the 167

most abundant glycosylated metabolite in C. sativus stigma, by any of the transporters tested. 168

169

Given the similarity of CsABCC2 to the ABA-GE transporter AtABCC2 (Burla et al., 2013), we 170

investigated whether CsABCC2 and CsABCC4a could transport ABA-GE. At concentrations up to 7.5 171

µM, i.e. much higher than its physiological intracellular concentration (Burla et al., 2013), neither 172

transporter was able to transport ABA-GE, while they both efficiently transported cis crocin 3 (Table 173

2). 174

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175

Detailed characterization of the stigma-specific CsABCC4a crocin transporter 176

Although Figure 3 shows that the majority of CsABCC4a localizes to the tonoplast of N. benthamiana 177

mesophyll cells, it does not completely rule out that a small fraction may also be localized to other 178

cellular membranes, such as the ER or the plasma membrane, that in mesophyll cells are not well 179

separated from the tonoplast. To further study the subcellular localization of CsABCC4a:eGFP in N. 180

benthamiana leaves, we conducted co-localization experiments with tonoplast, ER or plasma 181

membrane markers and analyzed the Spearman correlation coefficient (Rs) of the two signals (French 182

et al., 2008). The CsABCC4a:GFP signal strongly co-localized with the γTIP:RFP tonoplast marker 183

(Figure 6A), but did not show significant co-localization with the REFP:HDEL ER marker (Figure 6B) 184

or with the styryl dye FM4-64 labelling the plasma membrane (Figure 6C). Furthermore, GFP 185

fluorescence clearly localized to the tonoplast released after isolation of protoplasts from agroinfiltrated 186

leaves and their partial lysis (Figure 6D). These data confirm that the CsABCC4a transporter is 187

predominantly tonoplast localized when expressed in N. benthamiana leaves. 188

189

Transient expression in N. benthamiana leaves of CsCCD2, encoding a zeaxanthin cleavage 190

dioxygenase leading to the production of crocetin dialdehyde (Frusciante et al., 2014) resulted in the 191

production of low levels of both trans and cis crocetin and trans and cis crocins 1, 2ʹ, 2 and 3 (Figure 192

7). This suggests that N. benthamiana leaves contain endogenous aldehyde dehydrogenase (ALDH) 193

and UGT activities able to transform the CCD2 product (crocetin dialdehyde) into crocetin and crocins 194

(Demurtas et al., 2018). This finding allowed us to test the function of CsABCC4a in N. benthamiana 195

leaves. When CsABCC4a was expressed in combination with CsCCD2, we observed a significant 196

increase in the accumulation of crocins 1, 2 and 2ʹ, and a decrease of crocetin levels (Figure 7). 197

Transportomic assays present advantages (assaying hundreds of metabolites at a time) but also 198

complications (different metabolites can influence each other’s transport). To better investigate this 199

aspect, we studied the transport by CsABCC4a of cis and trans isomers of crocin 3, which are very 200

abundant in mature C. sativus stigmas and are readily separated by preparative HPLC. The two isomers 201

were purified from C. sativus stigmas and their purity was confirmed by LC-PDA-HRMS 202

(Supplemental Figure 4). At a concentration comparable to that of the whole extract (7.5 µM), the 203

purified cis isomer was efficiently transported in a time-dependent manner by CsABCC4a, while no 204

transport was observed for its trans isomer (Figure 8A). Since the trans isomer is transported in crude 205

8

extracts (Figure 5) we concluded that its transport requires a component present in the whole extract 206

that is lost during isomer purification. We tested the hypothesis that this component is the cis isomer by 207

mixing the two isomers at different concentrations and performing a transport assay. Transport of cis 208

crocin 3 was concentration-dependent and was also enhanced by the presence of its trans isomer 209

(Figure 8B, left panel). Transport of the trans isomer occurred only at the highest concentration, and 210

only in the presence of the cis one (Figure 8B, right panel). We conclude that crocin 3 transport by 211

CsABCC4a is cooperative and that this cooperativity allows transport of trans crocin 3 in whole 212

extracts (Figure 5). 213

214

215

Discussion 216

The capacity of specialized plant tissues to accumulate high levels of specialized metabolites relies on 217

their compartmentation capacity. Plant cells contain at least two types of vacuoles, storage and lytic. 218

While storage vacuoles of seeds are small, the lytic vacuoles, which are the main vacuoles in the 219

vegetative tissues, are generally very large and allow accumulation of large amounts of nutrients and 220

specialized metabolites. Lytic vacuoles also play an important role in the detoxification of toxic 221

compounds such as heavy metals and sodium. Additional important vacuolar functions include 222

metabolite storage as well as pH homeostasis, calcium signaling, and the participation in guard cell 223

movements (Martinoia et al., 2007; Martinoia et al., 2012). Plant specialized metabolites stored in 224

vacuoles are usually transported into the vacuolar lumen by different classes of tonoplast-localized 225

transporters. A single class of specialized metabolites can be a substrate for different types of 226

transporters, as shown for anthocyanins, which are transported by MATE-type antiporters in grape and 227

Medicago (Gomez et al., 2009; Zhao and Dixon, 2009), and also by ABC-type transporters in maize 228

and grape (Goodman et al., 2004; Francisco et al., 2013). Due to the fact that specialized compounds 229

exhibit an enormous variety of structures, it is likely that such structural modifications play an 230

important role in the recognition of a given substrate. 231

A single plant species can synthesize thousands of different molecules that may be transported across 232

one or more organellar membranes, which likely explains the large sizes of both ABC-type (>130) and 233

MATE-type (>110) transporter gene families (Hwang et al., 2016; Liu et al., 2016). Identifying the 234

primary transporter for a given molecule by brute force approaches is, therefore, extremely difficult and 235

9

time consuming. The situation is further complicated by the fact that <0.01% of plant specialized 236

metabolites are available in purified form, and even fewer as radiolabeled compounds for use in 237

radiochemical transport experiments. The transportomic approach described here is able to assess the 238

transport of any plant natural product that can be extracted by simple methods and detected by mass 239

spectrometry, i.e. >95% of plant metabolites. Its sensitivity is comparable to radiochemical transport 240

assays and it allows testing of a large number of substrates – potentially hundreds - in a single assay 241

without prior purification. This approach was first used for the study of a human transporter (Uchida et 242

al., 2007) and the term transportomics was coined by (Krumpochova et al., 2012) in characterization of 243

a murine transporter using mouse urine as substrate. Up to now, the maximum number of plant 244

substrates whose transport has been studied by LC-MS is two (Schaedler et al., 2014). This paper 245

demonstrates the possibility of studying transport in complex plant metabolite mixtures, similar to what 246

is already possible in other organisms (Krumpochova et al., 2012). 247

Given the well-known cooperative nature of the transport of several molecules (Liu et al., 2001), the 248

transportomic approach allows the study of the transport of substrates (e.g. trans crocin 3) that might 249

not be transported in purified form. With these advantages, it is surprising that LC-MS based 250

transportomic assays have been, to date, limited to the study of animal transporters (Uchida et al., 2007; 251

Krumpochova et al., 2012). 252

We have previously demonstrated that the second and third steps in crocin biosynthesis are localized in 253

the ER and cytosol, respectively (Demurtas et al., 2018) (Figure 1). Since crocins cannot passively 254

diffuse through membranes (Lautenschlager et al., 2015), an active transport system is required to 255

allow them to cross the tonoplast membrane and be accumulated within the vacuole. Combining 256

transcriptomic data from stigma with the transportomic approach, we identified the ABCC transporters 257

responsible for this transport step. Both CsABCC4a and CsABCC2 are highly expressed in stigmas, 258

show high co-expression with total crocin levels and/or CsCCD2 in C. sativus, predominantly localized 259

to the tonoplast in N. benthamiana leaves, and able to transport crocins in yeast microsomes. While 260

CsABCC4a is almost exclusively expressed in stigmas, CsABCC2 shows high levels of expression in 261

corms, suggesting that it may be involved in crocin transport in this tissue as well (Rubio-Moraga et al., 262

2010). 263

CsABCC4a and CsABCC2 are related to Arabidopsis transporters involved in the vacuolar transport of 264

folates and ABA-GE, respectively (Raichaudhuri et al., 2009; Burla et al., 2013). In spite of its 265

10

similarity to AtABCC2, CsABCC2 is unable to transport ABA-GE. Typical ABC motifs are well 266

conserved in both transporters, while several differences are found in the transmembrane domains, 267

known to be involved in substrate binding (Theodoulou, 2000; Jasinski et al., 2003; Wilkens, 2015) 268

(Supplemental Figure 1). Since crocin biosynthesis, in the Iridaceae family, is confined to the Crocus 269

genus, the most likely hypothesis is that crocin transport in C. sativus evolved from ancestral 270

transporters whose original substrate was different from crocins. As is frequently observed for ABCC 271

transporters (Hwang et al., 2016), CsABCC4a and CsABCC2 are, to a certain extent, promiscuous, in 272

that they transport crocins and, with lower efficiency, flavonoid glycosides. 273

Expression in N. benthamiana leaves of the first dedicated enzyme in the crocin pathway, CsCCD2 274

(Frusciante et al., 2014), was sufficient to cause crocetin and crocin accumulation, probably due to the 275

presence of ALDH and UGT enzymes able to convert the CsCCD2 product, crocetin dialdehyde, into 276

downstream compounds (Demurtas et al., 2018). CsABCC4a localizes to the tonoplast of N. 277

benthamiana mesophyll cells, and co-expression of it with CsCCD2 caused enhanced accumulation of 278

crocins and a decrease of their precursor, crocetin. This is likely due to increased vacuolar 279

sequestration of crocins and an increase of their stability and/or decrease of feedback inhibition of 280

cytosolic crocin synthesis. Unfortunately, crocin levels obtained in N. benthamiana leaves are too low 281

to assess their subcellular localization microscopically. 282

Crocin transport by CsABCC4a is stereospecific and cooperative, in that it shows a preference for cis 283

crocin 3 compared to its trans counterpart, and that one isomer enhances the transport of the other. 284

Cooperative transport has been previously observed with whole vacuoles or AtABCC2, and glutathione 285

and glucuronide conjugates (Lu et al., 1998; Klein et al., 2000). This cooperativity could be due to 286

structural changes in the large vestibule exhibited by ABCC transporters after binding a first substrate 287

(Johnson and Chen, 2017), which could allow binding of a second substrate molecule in the cavity. 288

Homology modeling and docking analysis is unlikely to provide useful insight into the cooperative 289

transport of trans and cis crocin as CsABCC4a has low similarity to ABCC transporters whose 290

structures have been solved (Supplemental Table 3). 291

Transportomic assays demonstrated that CsABCC4a and CsABCC2 transport cis crocins 3 and 4 more 292

efficiently, while the major crocins accumulated in C. sativus stigmas are the corresponding all-trans 293

counterparts. Acidic pH conditions like those found in the vacuole are known to cause polyene 294

isomerization (Re et al., 2001), and indeed cis crocin 3 is converted to its all-trans isomer at pH 5.2 295

11

(Supplemental Figure 5). We therefore hypothesize that crocins 3 and 4 are transported predominantly 296

in the cis form, and then converted to the all-trans one once exposed to the acidic vacuole sap. Such an 297

“isomerization trapping” mechanism has been demonstrated for instance for apigenin 7 (6-O-malonyl) 298

glucoside, a vacuolar pigment from Petroselinum hortense (parsley) that is subjected to conformational 299

changes under acidic pH (Matern et al., 1983) and suggested for the conversion of FCCs (fluorescent 300

Chl catabolites) to NCCs (non-fluorescent chlorophyll catabolites) during chlorophyll breakdown 301

(Hortensteiner and Krautler, 2011). 302

In this work, we have identified transporters for crocin in C. sativus stigmas using a transportomic 303

approach, which has been accomplished only for a few plant specialized metabolites. Using the method 304

developed here, identification of transporters for other important plant specialized metabolites can be 305

envisaged, which could in turn prove useful tools for increasing the accumulation of these metabolites 306

in planta. An example may be steviosides produced in Stevia rebaudiana leaves. Similar to crocins, 307

steviosides contain an isoprenoid moiety esterified with glucosyl groups, and in both cases the 308

synthesis starts in the plastid and produces a glycosylated product in the cytosol, which is finally 309

transported in the vacuole (Brandle and Telmer, 2007; Demurtas et al., 2018). 310

12

METHODS 311

Plant materials and growth conditions 312

Bioinformatic analyses 313

Phylogenetic analyses were conducted with MEGA version 7 (Kumar et al., 2016), using the neighbor-314

joining method (Saitou and Nei, 1987); the percentage of replicate trees in which the associated taxa 315

clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 316

1985). The evolutionary distances were computed using the p-distance method (Nei and Kumar, 2000) 317

represent number of amino acid differences per site. All ambiguous positions were removed for each 318

sequence pair. Protein alignment was performed with Clustal Omega tool 319

(https://www.ebi.ac.uk/Tools/msa/clustalo/). Co-expression analysis was performed as previously 320

described (Coman et al., 2014; Ahrazem et al., 2018). Briefly, the pairwise Pearson correlation between 321

each transporter and CCD2/crocins was computed, and Fisher’s Z–transformation was used to test the 322

statistical significance of the pairwise correlations. Transmembrane domains were deduced using the 323

TMHMM 2.0 software (http://www.cbs.dtu.dk/services/TMHMM/). 324

325

Cloning 326

CsABCC and CsMATE CDSs were isolated from cDNA obtained by RNA extracted from C. sativus 327

stigma collected the day of anthesis using the SMART PCR cDNA synthesis kit (Clontech; Cat. No. 328

634902) and the Super-ScriptII reverse transcriptase (Life Technologies, Cat No. 18064-014). All the 329

oligonucleotides used to isolate and clone the genes are described in Supplemental Table 4. The 330

CsABCC4a partial sequence was reconstructed from contigs obtained by 454 Titanium sequencing 331

(Frusciante et al., 2014) and amplified from cDNA with the oligonucleotides 1 and 2; the amplicon 332

(amplicon 1) was then cloned in pBlueScript SK (+) vector (Stratagene) digested with EcoRV 333

restriction enzyme. 5ʹ RACE PCR was performed to obtain the 5ʹ sequence of the cDNA using a 334

commercial kit (Life Technologies, Cat No. 18374-058) according to the manufacturer’s instructions. 335

C. sativus cDNA was amplified with the oligonucleotide 3. Tailed cDNA was amplified using 5′-336

RACE abridged anchor primer supplied in the kit and oligonucleotide 4. The PCR product (amplicon 2) 337

was purified using a purification kit (Qiagen, Cat No. 28104) and cloned in pBlueScript SK (+) vector 338

digested with EcoRV. All PCR reactions were performed using Phusion High Fidelity DNA 339

polymerase (New England Biolabs, Cat No. M0530L). Amplicon 1 and 2 were then re-amplified, 340

assembled and cloned in the yeast expression vector pNEV-Ura (Sauer and Stolz, 1994) using Gibson 341

13

assembly method (Gibson et al., 2009). Amplicon 1 was amplified with the oligonucleotides 5 and 6, 342

producing the fragment 1; amplicon 2 with the oligonucleotides 7 and 8, producing the fragment 2; 343

pNEV-Ura plasmid was digested with NotI restriction enzymes (fragment 3). The three fragments were 344

assembled using the Gibson Assembly® Master Mix (New England Biolabs, Cat No. E2611S) 345

according to the manufacturer’s instructions thereby producing the pNEV:CsABCC4a construct. 346

Validation that amplicon 1 and 2 belonged to the same coding sequence was performed by PCR on C. 347

sativus cDNA using the oligonucleotides 9 and 10, that produced the expected full-length amplicon of 348

4491 bp (Supplemental Figure 6). 349

CsABCC2 and CsMATE full-length sequences were amplified from cDNA (Supplemental Figure 6) 350

with the following oligonucleotides: 11 and 12 for CsABCC2; 13 and 14 for CsMATE1a; 15 and 16 for 351

CsMATE1b (these primers anneal on the 5ʹ and 3ʹ UTR, respectively); 17 and 18 for CsMATE4. The 352

amplicons were then re-amplified with oligonucleotides that insert the NotI site and subcloned in the 353

pNEV-Ura plasmid NotI-digested, producing the pNEV:CsABCC4a, pNEV:CsABCC2, 354

pNEV:CsMATE1a, pNEV:CsMATE1b and pNEV:CsMATE4 constructs. All the amplicons and 355

constructs were verified by sequencing. The pNEV-based constructs were expressed in Saccharomyces 356

cerevisae cells as described in the “Transport assay” section. 357

358

For protein localization studies, CsABCC and CsMATE cDNAs were 3ʹ-fused to the coding sequence 359

for the enhanced Green Fluorescent Protein (eGFP) using Gibson assembly method. The pBI:eGFP 360

vector (Frusciante et al., 2014) was digested with XbaI restriction enzyme while cDNAs were 361

amplified from pNEV-based constructs, inserting the sequence encoding for Pro-Gly-Pro tripeptide 362

before the eGFP CDS, using the following oligonucleotides: 19 and 20 for CsABCC4a; 21 and 22 for 363

CsABCC2; 23 and 24 for CsMATE1a; 25 and 26 for CsMATE1b; 27 and 28 for CsMATE4. The purified 364

PCR fragments were then assembled with the pBI:eGFP vector generating the pBI:CsABCC4a:eGFP, 365

pBI:CsABCC2:eGFP, pBI:CsMATE1a:eGFP, pBI:CsMATE1b:eGFP and pBI:CsMATE4:eGFP 366

constructs. For protein overexpression in N. benthamiana leaves, the CsCCD2 cDNA including the 367

sequence coding for its chloroplast transit peptide (Demurtas et al., 2018) and the CsABCC4a cDNA 368

were cloned in the pBI121 vector by Gibson assembly method. The vector was digested with PacI 369

restriction enzyme while the cDNAs were obtained by PCR with the following oligonucleotides: 29 370

and 30 for CsCCD2; 31 and 32 for CsABCC4a. All the plasmids were checked by sequencing before 371

transformation in Agrobacterium tumefaciens strain C58C1. 372

373

14

Expression in Nicotiana benthamiana leaves 374

Agroinfiltration of N. benthamiana leaves was performed as described (Hamilton and Baulcombe, 375

1999). Leaves were co-infiltrated with C58C1 cells containing pBI121:CsABCC4a:eGFP or 376

pBI121:CsABCC2:eGFP or pBI121:CsMATE4:eGFP or pBI121:CsMATE1a:eGFP or 377

pBI121:CsMATE1b:eGFP and pBI:γ-TIP-RFP (tonoplast marker, (Nelson et al., 2007)). To reduce the 378

silencing of the transgenes, leaves were also infiltrated with the silencing suppressor RK19 (Francisco 379

et al., 2013). Leaves were also co-infiltrated with C58C1 cells containing pBI121:CsABCC4a:eGFP, 380

pBI-RFP-HDEL (ER marker (Lee et al., 2013)) and RK19. After 3-6 days, leaves were analyzed using 381

a confocal laser scanning microscope (Olympus FV1000 and Zeiss LSM880). Lasers at 488 nm and 382

543 nm were used to detect green and red fluorescence of GFP and γTIP-RFP or RFP-HDEL (emission 383

571-640 nm), respectively. FM4-64 was excited at 514 nm and detected in the range 616 nm – 645 nm.384

Images were acquired using a 40x oil immersion objective (N.A. 1,30) with optical zooming 1x or 3x. 385

All images were analyzed and processed with ImageJ. 386

For preparation of mesophyll protoplasts, cell walls of N. benthamiana leaves were digested for 2 h at 387

30°C in MCP (500 mM Sorbitol, 1 mM CaCl2, 10 mM MES-KOH, pH 5.6) supplemented with 1% 388

(w/v) Cellulase Y-C, 0.1% (w/v) Pectolyase Y-23. Protoplasts were collected by centrifugation at 1,200 389

x g for 5 min on a cushion constituted of 100% Percoll (v/v) in 500 mM Sorbitol, 20 mM MES. The 390

supernatant was removed and the protoplasts were mixed with the Percoll cushion, overlaid with 25% 391

Percoll and MCP. After centrifugation at 1,200 x g for 5 min the purified protoplasts were collected 392

between 25% Percoll and MCP fractions. Vacuoles were released by mixing (1:1) purified protoplasts 393

with lysis buffer (200 mM Sorbitol, 10% Ficoll, 10 mM HEPES-KOH, pH 8.0). Microscopy was 394

performed using a Leica TCS SP5 confocal microscope. For each experiment, at least three different 395

plants were infiltrated and observed. Each experiment was repeated at least twice (with different 396

batches of plants). At least 5 randomly chosen regions were observed, and representative images were 397

selected for each experiment. 398

For simultaneous expression of CsCCD2 and CsABCC4a, leaves were co-infiltrated with C58C1 cells 399

containing pBI121:CsCCD2, pBI121:CsABCC4a and the silencing suppressor RK19. Three 400

independent infiltration experiments (with different batches of plants) were performed. For each 401

experiment, one apical leaf of four different plants was infiltrated with the CsCCD2 construct, and the 402

other apical leaf with the CsCCD2 + CsABCC4a construct. At four d.p.i., the four leaves infiltrated 403

15

with the same construct were collected as a pool and stored at -80°C. The pools from the three 404

independent experiments were then analyzed by LC-PDA-HRMS. Semi-polar extracts for LC-PDA-405

HRMS analysis were prepared as described in “LC-PDA-HRMS analyses”. Data are presented as 406

average (avg) area of crocins ± standard deviation (std) normalized on the area of the internal standard 407

formononetin (fold IS). The results obtained were evaluated using Student's t-test for the calculation of 408

the significance of the difference between the means of the two groups (CsCCD2 and CsCCD2+ 409

CsABCC4a) (Supplemental Table 5). 410

411

Transport assay 412

The ybt1 yeast mutant (MATa; ura3D::HIS3; leu2-3, 112; his3-D200; bat1D1::URA3) was transformed 413

by electroporation as described (Becker and Guarente, 1991) with the pNEV-based constructs. As 414

control, cells were transformed with the empty vector pNEV-Ura, named pNEV for simplicity. 415

Transformants were selected on minimal synthetic dropout medium lacking uracil. For in vitro 416

transport studies, yeast microsomes were isolated as described (Tommasini et al., 1996). Uptake 417

experiments were performed using the rapid filtration technique (Tommasini et al., 1996). Briefly, 100 418

µL of vesicles (OD600= 4; total proteins content about 400 µg) were mixed with ice-cold transport 419

buffer (0.4 M glycerol, 0.1 M KCl, and 20 mM Tris-MES, pH 7.4) and freshly added with 1 mM DTT, 420

6 mM or 1 mM MgSO4 (in the presence or absence of MgATP, respectively), 100 μg/mL creatine 421

kinase, and 10 mM creatine phosphate. Substrate transport was assayed either in the presence or 422

absence of 4 mM MgATP in a total reaction volume of 650 µL. The transport assay was performed at 423

room temperature. 424

Intactness of microsomes was evaluated by the ability to transport the substrate leukotriene C4 (LTC4) 425

(Leier et al., 1994). The LTC4 substrate (Cayman Chemicals, Cat N. 20210) was used at concentration 426

of 100 µM. At two time points (30 sec and 8 min), 100 μl of the reaction mixture were immediately 427

loaded on a pre-wetted nitrocellulose filter (0.45µm pore size; Millipore) and rapidly washed with 3 x 428

2.5 mL of ice-cold transport buffer. Three technical replicates (100 μl each) were performed for each 429

condition and repeated for each vesicle preparation. The filter-bound vesicles were dissolved by adding 430

1 ml of 75% (v/v) spiked cold methanol (0.5 µg/ml formononetin; Sigma-Aldrich, Cat. No. 47752-431

25MG-F), and metabolites were then extracted at room temperature through continuous agitation for 30 432

min. To remove vesicle lipids, 800 µl of eluted samples was transferred in a microcentrifuge tube and 433

added to 400 µl of chloroform, vortexed and shaken in a Mixer Mill 300 (MM, Retsch) for 5 min at 20 434

16

Hz frequency. Ultrapure water (200 µl) was added to separate the phases, followed by vortexing and 435

centrifugation at 20,000 g for 20 min. Finally, 800 µl of the upper phase was collected, dried with a 436

vacuum concentrator and resuspended in 80 µl of cold 50% (v/v) methanol, and centrifuged at 20,000 g 437

for 10 min to remove precipitates. A 2 µl sample of the supernatant was subjected to LC-MS analysis. 438

All solvents used for the extraction were LC-MS grade (Merck Millipore). 439

To study the transport of C. sativus stigma metabolites, a C. sativus stigma hydroalcoholic extract and 440

purified crocins were used to perform uptake experiments. The C. sativus stigma hydroalcoholic extract 441

was prepared as following: 3 mg of pulverized C. sativus stigmas (Castilla-La Mancha, dried 2013) 442

was resuspended in 300 µl of cold 50% (v/v) methanol, homogenized for 40 min in a mixer mill MM 443

300 (Retsch GmbH, Haan, Germany) at 20 Hz frequency, and centrifuged 20 min at 20,000 g. The 444

supernatant was recovered, total crocin content quantified by LC-DAD, and a standardized amount 445

used to perform the uptake experiments: in a final reaction of 650 µl, we adjusted the C. sativus stigma 446

extract volume in order to have a final concentration of 320 µM of total crocins. Crocin 4 analytical 447

standard was purchased from PhytoLab (Cat. No. 80391). 448

Crocin 3 isomers (trans and cis) were purified from C. sativus stigma hydroalcoholic extract by 449

preparative HPLC. Briefly, 100 mg of pulverized stigmas were resuspended in 1 ml of cold 50% (v/v) 450

methanol and processed as described above. Crocin separation was performed on a LC system (Series 451

200, Perkin Elmer) injecting 100 µl of extract on a C18 reverse-phase column (250 x 10 mm, 5 µm; 452

Ascentis, Supelco) heated at 35 °C. The mobile phases used were water + 0.1% formic acid (A) and 453

acetonitrile + 0.1% formic acid (B) at a total flow rate of 4 ml/min. The separation was developed 454

using 10% B for 1 min, a 10 min linear gradient to 20% B followed by a 20 min linear gradient to 30% 455

B. LC conditions were kept for 10 more minutes, before going back to the initial conditions. Detection456

was performed at 440 nm with an online UV/VIS Detector (Series 200, Perkin Elmer). Two ml 457

fractions were collected, dried in a lyophilizer and resuspended in 50 µl of 50% (v/v) methanol. 458

Competitive assays between trans and cis crocin 3 were performed using concentrations ranging from 459

0.75 µM to 75 µM. 460

All uptake experiments with C. sativus stigma extract or purified crocins were performed using the 461

rapid filtration technique with acetate cellulose filters (0.45 µm pore size; Sartorius), instead of 462

nitrocellulose. Reactions were performed at room temperature in reaction mix as described for LTC4 463

and stopped at different time points. Inhibition assays were performed in the presence of the inhibitor 464

probenecid (SIGMA, Cat. No. P8761) (Francisco et al., 2013). Briefly, yeast microsomes were 465

incubated at room temperature in the reaction mix containing the inhibitor (1 mM); after 10 min, the C. 466

17

sativus stigma extract was added and the uptake experiment performed as described above. For 467

experiments on ice, the tubes containing the reaction mix and C. sativus stigma extract were placed on 468

ice immediately after the addition of yeast microsomes. All experiments were repeated at least three 469

times with independent vesicle preparations, and each experiment was performed with three technical 470

replicates (3 filters, each spotted with 100 µl of reaction mix). 471

For ABA-GE transport studies, in vitro transport assays were performed with the analytical standard of 472

ABA-GE (ChemIm Ltd, Cat. No. 013 2781), using concentrations ranging from 0.075 µM to 7.5 µM. 473

The transport of each metabolite presented was evaluated by LC-PDA-HRMS. 474

475

LC-PDA-HRMS analyses 476

C. sativus stigma polar extracts, standards, eluted fractions from in vitro transport studies and semi-477

polar extracts from N. benthamiana agroinfiltrated leaves were analyzed with a Q-Exactive quadrupole 478

Orbitrap mass spectrometry system (ThermoFisher Scientific), coupled to an LC system equipped with 479

a photodiode array detector (Dionex), as previously described (Demurtas et al., 2018). HPLC 480

separation was performed by injecting 2–5 µl of samples on a C18 Luna reverse-phase column (100 x 481

2.1 mm, 2.5µm; Phenomenex). The mobile phases used were water + 0.1% formic acid (A) and 482

acetonitrile + 0.1% formic acid (B) at a total flow rate of 250 µl/min. The separation was developed 483

using 5% B for 0.5 min, followed by a 24 min linear gradient to 75% B. The ionization was performed 484

in a heated electrospray ionization (HESI) source with nitrogen used as sheath and auxiliary gas, set to 485

45 and 30 units, respectively. The vaporizer temperature was 270 °C, the capillary temperature was 30 486

°C, the discharge current was set to 5 μA and S-lens RF level set at 50. The acquisition was performed 487

in the mass range 110/1600 m/z both in positive and in negative ion mode with the following 488

parameters: resolution 70000, microscan 1, AGC target 1e6, maximum injection time 50. UV-VIS489

detection was continuous from 220 to 700 nm. All solvents used were LC-MS grade (Merck Millipore). 490

Metabolite identification was achieved on the basis of accurate masses and by comparison with 491

authentic standards or with literature data. The ion peak areas were normalized to the ion peak area of 492

the internal standard (formononetin) (fold internal standard). To calculate the transport of each 493

metabolite, fold internal standard values of the reactions performed in the absence of ATP were 494

subtracted to those of the reactions performed in the presence of ATP (net uptake). Data are presented 495

as percentage of net uptake of three biological replicates (average values ± standard deviations are 496

shown in figures and tables). 497

498

18

Accession numbers 499

The sequences of genes reported in this paper have been deposited in the GenBank database: 500

CsABCC4a, accession no. MF966954; CsABCC2, accession no. MF966955; CsMATE4, accession no. 501

MF966956; CsMATE1a, accession no. MH475368; CsMATE1b, accession no. MH475369. The 502

sequences are also shown in the Supplemental Data set 2. The sequences of the transporter contigs 503

shown in Fig. 2C have likewise been deposited with the following accessions: CsABCC4a, accession 504

no. MN401321; CsABCC2, accession no. MN401322; CsMATE4, accession no. MN401323; 505

CsMATE1a, accession no. MN401324; CsMATE1b, accession no. MN401325; CsABCC8, accession 506

no. MN380444; CsABCC3a, accession no. MN380445; CsABCC3b, accession no. MN380446; 507

CsABCC4b, accession no. MN380447; CsABCC5, accession no. MN380448; CsABCC15, accession 508

no. MN380449; CsABCC4c, accession no. MN380450; CsMATE5a, accession no. MN380451; 509

CsMATE2e, accession no. MN380452; CsMATE5b, accession no. MN380453; CsMATE2b, accession 510

no. MN380454; CsMATE2d, accession no. MN380455; CsMATE2c, accession no. MN380456; 511

CsMATE2a, accession no. MN380457; CsMATE2f, accession no. MN380458. 512

Sequence data of ABCC and MATE transporters from Arabidopsis thaliana (At), Zea mays (Zm), Vitis 513

vinifera (Vv), Sorghum bicolor (Sb), Nicotiana tabacum (Nt), Medicago truncatula (Mt) and Coptis 514

japonica (Cj) shown in Figure 2 can be found under the following accession numbers: AtABCC1, 515

NP_181013; AtABCC2, NP_001031116; AtABCC3, NP_187915; AtABCC4, NP_182301; AtABCC5, 516

NP_171908; AtABCC6, NP_187916.3; AtABCC7, NP_187917; AtABCC8, Q8LGU1; AtABCC9, 517

Q9M1C7; AtABCC10, NP_191473; AtABCC11, NP_174331; AtABCC12, Q9C8H0; AtABCC13, 518

NP_001323940; AtABCC14, NP_191829; ZmABCC3, AAT37905; ZmABCC4, A7KVC2; 519

VvABCC1, AGC23330; AtDTX35, NP_194294; AtDTX41/TT12, NP_191462; AtDTX16, 520

NP_200058; AtDTX17, NP_177511; AtDX19, NP_566730; AtDTX29,NP_189291; AtDTX30, 521

NP_198619; AtDTX33, NP_175184; AtDTX40, NP_188806; CjMATE1, BAX73926; MtMATE1, 522

ACX37118; MtMATE2, ADV04045; NtJAT1, CAQ51477; NtJAT2, BAP40098; NtMATE1, 523

BAF47751; NtMATE2, BAF47752; SbMATE2, XP_021303040; VvAM1, ACN88706; VvAM3, 524

ACN91542. 525

526

Supplemental Data 527

Supplemental Figure 1. Conserved domains and transmembrane helices of CsABCC4a and 528

CsABCC2 and of the closely related AtABCC1, AtABCC2 and AtABCC4 transporters. 529

19

Supplemental Figure 2. Crocin composition of a commercial crocin standard and of a C. sativus 530

stigma hydroalcoholic extract. 531

Supplemental Figure 3. Assessment of physiological intactness of yeast microsomes. 532

Supplemental Figure 4. Purity of trans and cis crocin 3 purified from C. sativus stigma extract 533

through preparative HPLC. 534

Supplemental Figure 5. Isomerization of cis crocin 3 into trans crocin 3 at pH 5.2. 535

Supplemental Figure 6. MATE and ABCC full-length amplicons obtained from C. sativus cDNA with 536

the oligonucleotides described in Methods. 537

Supplemental Table 1. Transcript levels of CsMATE and CsABCC transporters in C. sativus tissues. 538

Supplemental Table 2. Co-expression analysis of CsABCC and CsMATE transcripts with total 539

crocins and CsCCD2 transcript. 540

Supplemental Table 3. Homology of CsABCC4a with ABCC transporters with known structures. 541

Supplemental Table 4. Oligonucleotides used in this study. 542

Supplemental Table 5. T-test result of data presented in Figure 7. 543

Supplemental Data set 1. Saffron transcriptome data 544

Supplemental Data set 2. Sequences of C. sativus transporters characterized in this paper. 545

Supplemental Data set 3. Text file of the ABC alignment used for the phylogenetic analysis in Figure 546

2C. 547

Supplemental Data set 4. Text file of the MATE alignment used for the phylogenetic analysis in 548

Figure 2C. 549

550

Author contributions 551

OCD, RBF, AC, GD, PF, SF, MP, GA, LB, MF and LF produced data. OCD, RBF, MP, LB, LF, MF, 552

EM, AC, GC, SA, and GG analyzed data. EM and GG developed the experimental strategy. GG 553

coordinated the study. OCD, RBF, AC, EM and GG wrote the manuscript. All authors reviewed the 554

results and approved the final version of the manuscript. The authors declare no conflict of interest. 555

556

Acknowledgments 557

We thank Barbara Bassin for the preparation of yeast microsomes, Elena Romano at the Centre of 558

Advanced Microscopy “Patrizia Albertano” of University of Rome Tor Vergata for help with confocal 559

20

experiments, Gaetano Perrotta, Paolo Facella, Fabrizio Carbone at the ENEA Trisaia Research Center 560

and IGA Technology Services for RNA-Seq data, and Andrea Aliboni for help with preparative HPLC. 561

This work was supported by grants from the European Union to GG [“From discovery to products: A 562

next generation pipeline for the sustainable generation of high-value plant products”, Contract 613153 563

and “Developing Multipurpose Nicotiana Crops for Molecular Farming using New Plant Breeding 564

Techniques”, Contract 760331] and by COST (European Cooperation in Science and Technology) 565

Action CA15136 “EUROCAROTEN”. 566

Figure legends 567

Figure 1. Saffron crocin biosynthesis and its compartmentation 568

(A) C. sativus flower at anthesis. (B) Scanning electron microscopy of mature stigma. (C) Proposed569

crocin biosynthetic pathway and (D) its compartmentation (Demurtas et al, 2018): the CCD2 enzyme 570

cleaves zeaxanthin in the plastid, producing crocetin dialdehyde, which then migrates to the 571

endoplasmic reticulum (ER) and is dehydrogenated to crocetin by an aldehyde dehydrogenase 572

(ALDH); the glycosylation steps are performed in the cytoplasm by UDP-glycosyltransferase (UGT) 573

enzymes; crocins are then transported into the vacuole by ABC tonoplast transporters functionally 574

characterized in this work. (E) Detailed crocin structures. 575

Figure 2. Expression and phylogenetic analysis of putative C. sativus tonoplast transporters. 576

(A) Heat map of transcript levels for ABCC and MATE transporter genes in different C. sativus577

tissues; data are expressed as log2 of reads per kilobase per million (RPKM) and sorted by decreasing 578

RPKM values in stigma. For further details, see Supplemental Data set 1. (B) Co-expression analysis of 579

genes for CsABCCs and CsMATEs (Bait) with CsCCD2 and with total crocins (Prey). Only ρ values 580

>0.50 are shown. For further details, see Supplemental Data set 1. (C) Phylogenetic relationships of581

ABCC (left) and MATE (right) transporters expressed in C. sativus (Cs) stigma (underlined) inferred 582

using the neighbor-joining method. Colored dots indicate the class of transported substrates for the 583

functionally characterized transporters. The CsABCCs and CsMATEs functionally characterized in this 584

work are indicated by arrows. The trees include transporters from Arabidopsis thaliana (At), Coptis 585

japonica (Cj), Medicago truncatula (Mt), Nicotiana tabacum (Nt), Sorghum bicolor (Sb), Vitis vinifera 586

(Vv) and Zea mays (Zm). The accession numbers are described in the Accession numbers section. The 587

percentage of replicate trees that clustered together in the bootstrap test (500 replicates) is indicated to 588

the left of the branches. The alignment files are shown in the Supplemental Data Sets 3 and 4. 589

21

Figure 3. Subcellular localization of C. sativus transporters in Nicotiana leaves. 590

Confocal images of GFP (green) and RFP (red) fluorescence in N. benthamiana leaves co-expressing 591

the indicated ABCC or MATE transporter fused to GFP and γTIP (a tonoplast marker) fused to RFP. 592

Scale bars, 10 µm. 593

Figure 4. Transportomic assay using C. sativus stigma extract and yeast microsomes. 594

(A) Schematic representation of the assay. Microsomes overexpressing a transporter are incubated with595

C. sativus stigma hydroalcoholic extract in the presence of ATP. Microsomes are separated by the rapid596

filtration technique and washed, and transported metabolites are eluted and quantified by LC-PDA-597

HRMS. 598

(B-E) Representative ESI+/MS chromatograms of the extracted accurate mass of crocetin (M+H+

599

329.1747) generated from crocin fragmentation. (B) C. sativus stigma hydroalcoholic extract; (C) 600

import into microsomes isolated from yeast cells transformed with the pNEV empty vector; (D) import 601

into microsomes expressing CsABCC4a incubated in the absence of ATP; (E) import into microsomes 602

expressing CsABCC4a incubated in the presence of ATP. Incubation was for 15 min. Different peaks 603

represent: trans crocin 4 (RT 10.58); trans crocin 3 (RT 11.52); trans crocin 2ʹ (RT 12.54); cis crocin 4 604

(RT 13.84); trans crocin 2 (RT 14.20); cis crocin 3 (RT 14.82); trans crocin 1 (RT 16.16); cis crocin 2ʹ 605

(RT 16.79); cis crocin 2 (RT 17.02); cis crocin 1 (RT 17.73). 606

Figure 5. CsABCC4a and CsABCC2 transport different crocins in vitro 607

(A) Major glycosylated metabolites present in the C. sativus stigma hydroalcoholic extract. 1:608

kaempferol 3-O-sophoroside-7-glucoside; 2: kaempferol 3,7,4ʹ-triglucoside; 3: kaempferol 7-609

sophoroside; 4: kaempferol 3-β-D-glucopyranoside; 5: kaempferol‐3‐O‐rutinoside; 6: 610

dihydrokaempferol 7-O-glucoside; 7: naringenin glucosides (3 isomers); 8: picrocrocin; 9: trans crocin 611

5 (t 5); 10: trans crocin 4 (t 4); 11: cis crocin 4 (c 4); 12: trans crocin 3 (t 3); 13: cis crocin 3 (c 3); 14: 612

trans crocin 2 (t 2); 15: cis crocin 2 (c 2); 16: trans crocin 2ʹ (t 2ʹ); 17: cis crocin 2ʹ (c 2ʹ); 18: trans 613

crocin 1 (t 1); 19: cis crocin 1 (c 1). Structures of the various crocins are shown in Figure 1E. Values 614

are presented as relative abundances compared to the internal standard used (formononetin) of mean 615

values ± stdev of three independent extract preparations. 616

(B) % net transport of glycosylated metabolites by yeast microsomes expressing candidate tonoplast617

transporters. CTRL: microsomes form yeast cells transformed with the empty vector. CsABCC4a, 618

22

CsABCC2, CsMATE4, CsMATE1a, CsMATE1b: microsomes expressing candidate tonoplast 619

transporters. % net transport was calculated by subtracting the peak areas in the absence of ATP from 620

those in the presence of ATP after 15 min of incubation, and normalizing to the peak areas in the initial 621

extract. Data are the avg ± stdev of three independent microsome preparations. 622

Figure 6. Tonoplast localization of CsABCC4a in Nicotiana leaves. 623

(A-C) N. benthamiana leaves were agroinfiltrated with the indicated constructs and imaged by confocal 624

microscopy. The Spearman correlation coefficient (Rs) between the two channels was calculated using 625

the ImageJ analysis program with the Pearson-Spearman correlation plug-in (French et al., 2008) to 626

calculate co-localization and to produce scatterplots using a threshold of 40. Scale bars, 5 µm. (D) 627

Confocal images of a protoplast from a N. benthamiana leaf expressing CsABCCa:GFP (green 628

channel). After induced lysis of the plasma membrane the vacuole (asterisk) is in the process of being 629

released. Scale bar, 12 µm. 630

Figure 7. Functional assay of the C. sativus ABCC4a transporter in N. benthamiana leaves. 631

Quantification of crocetin and crocins in leaves agroinfiltrated to express CsCCD2 or 632

CsCCD2+CsABCC4a. Data are the avg ± stdev of ion peak areas of crocetin and crocins, normalized to 633

the ion peak area of the internal standard formononetin (fold internal standard), in three independent 634

pools of agroinfiltrated leaves (see Methods for details). Asterisks indicate statistical significance of the 635

difference between the means of the two groups (CsCCD2 and CsCCD2+ CsABCC4a) (Student’s t-636

test; * p-value<0.05; ** p-value<0.01). 637

Figure 8. Kinetics and cooperativity of purified cis and trans crocin 3 transport by CsABCC4a 638

(A) Time-dependent transport of cis (left) or trans (right) crocin 3 at a concentration of 7.5 µM by639

yeast microsomes expressing CsABCC4a or control microsomes (pNEV). (B) Cooperative transport of 640

cis (left) and trans (right) crocin 3 in the presence of different concentrations of the other isomer. Left: 641

cis crocin 3 transport was evaluated at two different concentrations (0.75 µM and 7.5µM) in the 642

presence of increasing concentrations of trans crocin 3 (0, 0.75, 7.5 µM). Right: trans crocin 3 643

transport was evaluated in the same conditions and in the presence of increasing concentrations (0, 644

0.75, 7.5 µM) of cis crocin 3. Results are presented as net transport, calculated by subtracting the 645

values measured in the absence of ATP from the values measured in the presence of ATP after 15 min. 646

Data are the avg ± stdev of three independent microsome preparations. 647

23

Table 1. Inhibition of C. sativus stigma metabolite transport by CsABCC4a and CsABCC2 in the 648

presence of the probenecid inhibitor or by incubation on ice. 649

The table shows the percent residual transport after treatment with probenecid or on ice, compared to 650

control reactions performed at room temperature in absence of inhibitors. Data are the avg ± stdev of 651

three independent microsome preparations. n.d.= not detectable (detection limit of the mass 652

spectrometer in full MS mode: 500 fg). Since the CsABCC2 transporter doesn’t transport cis crocin 1 653

in normal conditions, the percent residual transport is indicated as “-”. 654

CsABCC4a CsABCC2

Compound name Probenecid 0°C Probenecid 0°C

kaempferol 3-O-

sophoroside-7-

glucoside

n.d. 19.3 ± 4.2 n.d. 53.5 ± 6.0

kaempferol 7-

sophoroside 23.0 ± 4.5 34.4 ± 6.9 10.4 ± 3.8 20.8 ± 4.6

Kaempferol 3‐O‐

rutinoside n.d. 35.4 ± 0.01 n.d. 2.3 ± 0.3

Naringenin 7-O-

glucoside 27.7 ± 1.2 11.1 ± 0.7 16.5 ± 0.6 4.4 ± 0.1

trans crocin 4 n.d. 20.0 ± 3.4 14.3 ± 2.7 n.d.

cis crocin 4 n.d. 19.6 ± 3.7 15.2 ± 2.7 n.d.

trans crocin 3 n.d. 48.4 ± 8.4 n.d. n.d.

cis crocin 3 48.5 ± 1.9 22.3 ± 2.2 0.7 ± 0.006 n.d.

trans crocin 2 n.d. 24.8 ± 5.0 18.2 ± 3.6 n.d.

cis crocin 2 26.8 ± 4.8 n.d. 33.6 ± 4.8 0.04 ± 0.005

trans crocin 2’ 28.4 ± 2.0 23.1 ± 1.2 n.d. n.d.

cis crocin 2’ 31.9 ± 2.9 n.d. 32.9 ± 5.2 n.d.

trans crocin 1 22.1 ± 1.5 42.0 ± 6.1 17.4 ± 1.3 n.d.

cis crocin 1 n.d. n.d. - -

655

Table 2. Transport of ABA-GE and cis crocin 3 by CsABCC4a and CsABCC2 transporters. 656

% transport at different substrate concentrations in standard assay conditions. Transport experiments 657

were performed with purified ABA-GE or cis crocin 3 (7.5 µM). Results are the avg ± stdev of three 658

24

independent microsome preparations. n.d.= not detectable (detection limit of the mass spectrometer in 659

full MS mode: 500 fg) 660

Substrate Transporter Substrate concentration (µM)

0.075 0.75 7.5

ABA-GE

CTRL n.d. n.d. n.d.

CsABCC4a n.d. n.d. n.d.

CsABCC2 n.d. n.d. n.d.

cis crocin 3

CTRL n.d. n.d. n.d.

CsABCC4a n.d. n.d. 8.8 ± 0.8

CsABCC2 n.d. n.d. 2.8 ± 0.5

661

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818

Figure 1. Saffron crocin biosynthesis and its compartmentation

(A) Saffron flower at anthesis. (B) Scanning electron microscopy of mature stigma. (C) Proposed crocin biosynthetic

pathway and (D) its compartmentation (Demurtas et al, 2018): the CCD2 enzyme cleaves zeaxanthin in the plastid,

producing crocetin dialdehyde, which then migrates to the endoplasmic reticulum (ER) and is dehydrogenated to

crocetin by an aldehyde dehydrogenase (ALDH); the glycosylation steps are performed in the cytoplasm by UDP-

glycosyltransferase (UGT) enzymes; crocins are then transported into the vacuole by ABC tonoplast transporters

functionally characterized in this work. (E) Detailed crocin structures.

C

A B

500 µm

ChromoplastZeaxanthin V

ac

uo

le

Crocetin

dialdehyde

ER

Crocins

Cytoplasm

Tonoplast

transporters?

500 nm

UGTs

ALDH

CCD2

Crocetin

D

zeaxanthin OH

HO

picrocrocin

safranal

O

crocetin dialdehyde3-OH-β-cyclocitral

OHO

O

R1OO

O

UGT?

?

CCD2

ALDH3I1

OO

UGTsHO

OH

OO

crocetin

crocins

R1O

R2O

E

Figure 2. Expression and phylogenetic analysis of putative C. sativus tonoplast transporters.

(A) Heat map of transcript levels for ABCC and MATE transporter genes in different C. sativus tissues; data are

expressed as log2 of reads per kilobase per million (RPKM) and sorted by decreasing RPKM values in stigma. (B) Co-

expression analysis of genes for CsABCCs and CsMATEs (Bait) with CsCCD2 and with total crocins (Prey). Only ρ

values >0.50 are shown. (C) Phylogenetic relationships of ABCC (left) and MATE (right) transporters expressed in C.

sativus (Cs) stigma (underlined) inferred using the neighbor-joining method. Colored dots indicate the class of

transported substrates for the functionally characterized transporters. The CsABCCs and CsMATEs functionally

characterized in this work are indicated by arrows. The trees include transporters from Arabidopsis thaliana (At), Coptis

japonica (Cj), Medicago truncatula (Mt), Nicotiana tabacum (Nt), Sorghum bicolor (Sb), Vitis vinifera (Vv) and Zea mays

(Zm). The accession numbers are described in Methodsthe Accession numbers section. The percentage of replicate

trees that clustered together in the bootstrap test (500 replicates) is indicated to the left of the branches.

Acorm leaf stamen tepal stigma

CsABCC4aCsABCC2

CsABCC4bCsABCC8CsABCC4cCsABCC5CsABCC15

CsABCC3b

CsABCC3a

CsMATE4

CsMATE1aCsMATE1bCsMATE2aCsMATE2f

CsMATE2dCsMATE5bCsMATE2b

CsMATE5aCsMATE2e

CsMATE2c

B Bait Pearson’s

ρ

Prey

CsCCD2 0.96 Total crocins

CsABCC4a 0.99 Total crocins

CsMATE4 0.84 Total crocins

CsABCC4a 0.95 CsCCD2

CsABCC2 0.90 CsCCD2

CsMATE4 0.75 CsCCD2

AtABCC5

ZmABCC4

AtABCC2

AtABCC1AtABCC1

AtABCC2

CsABCC2

AtABCC11

AtABCC12

AtABCC13

AtABCC4

AtABCC14

CsABCC4a

CsABCC4b

CsABCC4c

CsABCC8

AtABCC8

ZmABCC3

AtABCC10

VvABCC1

AtABCC9

AtABCC15

CsABCC15

CsABCC3a

CsABCC3b

AtABCC3

AtABCC6

AtABCC7

ZmABCC4

CsABCC5

AtABCC5

CsABCC3a

CsABCC3b

AtABCC10

AtABCC3

ZmABCC3

CsABCC4b

CsABCC2

AtABCC9

AtABCC11

AtABCC12

AtABCC13

AtABCC4

AtABCC14

CsABCC4a

CsABCC4c

CsABCC8

AtABCC8

VvABCC1

AtABCC15

CsABCC15

AtABCC7

CsABCC5

AtABCC6

CsMATE2e

AtDTX35

NtJAT1

AtDTX33

CsMATE5b

SbMATE2

NtMATE1

CsMATE1b

CsMATE1a

VvAM3

VvAM1

NtJAT2

AtDTX40AtDTX40

SbMATE2

CsMATE1a

CsMATE1b

NtMATE1

NtMATE2

AtDTX41/TT12

MtMATE1

AtDX19

NtJAT1

CsMATE5a

CsMATE5b

AtDTX16

AtDTX17

CsMATE2c

CsMATE2b

CsMATE2a

MtMATE2

AtDTX35

NtJAT2

VvAM1

VvAM3

CsMATE2d

AtDTX33

CJMATE1

CsMATE4

AtDTX29

AtDTX30

CsMATE2e

CsMATE2f

NtMATE2

AtDTX41/TT12

MtMATE1

AtDTX19

CsMATE5a

AtDTX16

AtDTX17

CsMATE2c

CsMATE2b

CsMATE2a

MtMATE2

CsMATE2d

CjMATE1

CsMATE4

AtDTX29

AtDTX30

CsMATE2f

Substrates

flavonoids

chlorophyll catabolites

folates

phytate

ABA-GE

phytochelatins

glutathione conjugates

alkaloids

cyanogenic glycosides

C

Figure 3. Subcellular localization of C. sativus transporters in Nicotiana leaves.

Confocal images of GFP (green) and RFP (red) fluorescence in N. benthamiana leaves co-expressing the indicated

ABCC or MATE transporter fused to GFP and γTIP (a tonoplast marker) fused to RFP. Scale bars, 10 µm.

CsA

BC

C4

a:G

FP

CsA

BC

C2

:GF

P

γTIP:RFP mergeGFP

CsM

AT

E4

:GF

PC

sM

AT

E1

a:G

FP

CsM

AT

E1

b:G

FP

Figure 4. Transportomics assay using C. sativus stigma extract and yeast microsomes.

(A) Schematic representation of the assay. Microsomes overexpressing a transporter are incubated with

saffron hydroalcoholic extract in the presence of ATP. Microsomes are separated by the rapid filtration technique

and washed, and transported metabolites are eluted and quantified by LC-PDA-HRMS.

(B-E) Representative ESI+/MS chromatograms of the extracted accurate mass of crocetin (M+H+ 329.1747)

generated from crocin fragmentation. (B) C. sativus stigma hydroalcoholic extract; (C) import into microsomes

isolated from yeast cells transformed with the pNEV empty vector; (D) import into microsomes expressing

CsABCC4a incubated in the absence of ATP; (E) import into microsomes expressing CsABCC4a incubated in the

presence of ATP. Incubation was for 15 min. Different peak represent: trans crocin 4 (RT 10.58); trans crocin 3 (RT

11.52); trans crocin 2ʹ (RT 12.54); cis crocin 4 (RT 13.84); trans crocin 2 (RT 14.20); cis crocin 3 (RT 14.82); trans

crocin 1 (RT 16.16); cis crocin 2ʹ (RT 16.79); cis crocin 2 (RT 17.02); cis crocin 1 (RT 17.73).

ATP

ATP

ATP

Yeast cells

Saffron extract

In vitro transport assay

Microsome

recovery

Analysis of uptake

by LC-PDA-HRMS

A

0

0

0

trans

4

106

Ion

in

ten

sit

y

106

Ion

in

ten

sit

y

107

Saffron extract

CsABCC4a - ATP

CsABCC4a + ATP

Ion

in

ten

sit

y

B

0

106

Ion

in

ten

sit

y

205 10 15 25 30

CTRL + ATP

trans

3

trans2’

cis

4 trans

2cis

3tr

ans

1cis2’

cis

2

trans

4

trans

3tr

ans

3tr

ans

3tr

ans

4tr

ans 4

trans2’

trans2’

trans2’

cis

4cis

4tr

ans

2cis

3cis

3

trans

2tr

ans 2

cis

4

cis

3

trans

1cis2’

cis

1

D

C

E

Figure 5. CsABCC4a and CsABCC2 transport

different crocins in vitro

(A) Major glycosylated metabolites present in the C.

sativus stigma hydroalcoholic extract. 1: kaempferol 3-

O-sophoroside-7-glucoside; 2: kaempferol 3,7,4ʹ-

triglucoside; 3: kaempferol 7-sophoroside; 4:

kaempferol 3-β-D-glucopyranoside; 5:

kaempferol‐3‐O‐rutinoside; 6: dihydrokaempferol 7-O-

glucoside; 7: naringenin glucosides (3 isomers); 8:

picrocrocin; 9: trans crocin 5 (t 5); 10: trans crocin 4 (t

4); 11: cis crocin 4 (c 4); 12: trans crocin 3 (t 3); 13: cis

crocin 3 (c 3); 14: trans crocin 2 (t 2); 15: cis crocin 2

(c 2); 16: trans crocin 2ʹ (t 2’); 17: cis crocin 2ʹ (c 2’);

18: trans crocin 1 (t 1); 19: cis crocin 1 (c 1). Structures

of the various crocins are shown in Figure 1E. Values

are presented as relative abundances compared to the

internal standard used (formononetin) of mean values

± stdev of three independent extract preparations.

(B) % net transport of glycosylated metabolites by

yeast microsomes expressing candidate tonoplast

transporters. CTRL: microsomes form yeast cells

transformed with the empty vector. CsABCC4a,

CsABCC2, CsMATE4, CsMATE1a, CsMATE1b:

microsomes expressing candicate tonoplast

transposters. % net transport was calculated by

subtracting the peak areas in the absence of ATP from

those in the presence of ATP after 15 min of

incubation, and normalizing to the peak areas in the

initial extract. Data are the avg ± stdev of three

independent microsome preparations.

B

0

2

4

6

8

10

% t

ran

sp

ort

t4 t

3c4

c3

t2

c2

t2’

c2’

t1

c1

CsABCC4a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

0.2

0.4

0.6

0.8

1Saffron extract

t 4

t 5

t3

c4

c3 t2

c 2 t2’

c2’

t1

c1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Flavonoids Crocins

Rela

tive a

bu

nd

an

ce

0

2

4

6

8

10CsMATE4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

2

4

6

8

10

t 4 t3

c4

c3

t2

c2

t2’ c2’

t1

% t

ran

sp

ort

CsABCC2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

2

4

6

8

10

% t

ran

sp

ort

CTRL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

2

4

6

8

10CsMATE1a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

2

4

6

8

10CsMATE1b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

% t

ran

sp

ort

% t

ran

sp

ort

% t

ran

sp

ort

Figure 6. Tonoplast localization of CsABCC4a in Nicotiana leaves.

(A-C) N. benthamiana leaves were agroinfiltrated with the indicated constructs and imaged by confocal microscopy.

The Spearman correlation coefficient (Rs) between the two channels was calculated using the ImageJ analysis

program with the Pearson-Spearman correlation plug-in (French et al., 2008), to calculate co-localization and to

produce scatterplots using a threshold of 40. Scale bars, 5 µm. (D) Confocal images of a protoplast from a N.

benthamiana leaf expressing CsABCCa:GFP (green channel). After induced lysis of the plasma membrane the

vacuole (asterisk) is in the process of being released. Scale bar, 12 µm.

* **

CsABCC4a:GFP

mergebright field CsABCC4a:GFP chlorophyll

D

RFP-HDEL

merge

CsABCC4a:GFP

20 µm12 µm

Rs = -0.05

FM4-64

γTIP-RFP merge

CsABCC4a:GFP merge

Rs = + 0.75

Rs = - 0.05

colocalization

A

B

C

colocalization

colocalization

Figure 7. Functional assay of the C. sativus ABCC4a transporter in N. benthamiana leaves.

Quantification of crocetin and crocins in leaves agroinfiltrated to express CsCCD2 or CsCCD2+CsABCC4a. Data are

the avg ± stdev of ion peak areas of crocetin and crocins, normalized to the ion peak area of the internal standard

formononetin (fold internal standard), in three independent pools of agroinfiltrated leaves (see Methods for details).

Asterisks indicate statistical significance of the difference between the means of the two groups (CsCCD2 and

CsCCD2+ CsABCC4a) (Student’s t-test; * p-value<0.05; ** p-value<0.01).

0

2

4

6

8

Fold

IS

CCD2 CCD2+ABCC4

**

** **

*

CsCCD2 + CsABCC4aCsCCD2**

**

Figure 8. Kinetics and cooperativity of purified cis and trans-crocin 3 transport by CsABCC4a

(A) Time-dependent transport of cis (left) or trans (right) crocin 3 at a concentration of 7.5 µM by yeast microsomes

expressing CsABCC4a or control microsomes (pNEV). (B) Cooperative transport of cis (left) and trans (right) crocin 3

in the presence of different concentrations of the other isomer. Left: cis crocin 3 transport was evaluated at two different

concentrations (0.75 µM and 7.5µM) in the presence of increasing concentrations of trans crocin 3 (0, 0.75, 7.5 µM).

Right: trans crocin 3 transport was evaluated in the same conditions and in the presence of increasing concentrations

(0, 0.75, 7.5 µM) of cis crocin 3. Results are presented as net transport, calculated by subtracting the values measured

in the absence of ATP from the values measured in the presence of ATP after 15 min. Data are the avg ± stdev of three

independent microsome preparations.

0

20

40

60

80

100

120

140

0 5 10 15 20 25

ng

cis

cro

cin

3/m

g p

rote

in

CsABCC4a

pNEV

cis 7.5 μM

A

B

0

50

100

150

200

250

300

trans 0 trans 0.75 trans 7.5

ng

cis

cro

cin

3/m

g p

rote

in

0

50

100

150

200

250

300

cis 0 cis 0.75 cis 7.5

ng

tra

ns

cro

cin

3/m

g p

rote

in

0

20

40

60

80

100

120

140

0 5 10 15 20 25

ng

tra

ns

cro

cin

3/m

g p

rote

in

CsABCC4a

pNEV

trans 7.5 μM

cis trans

0.75 μM

7.5 μM

0.75 μM

7.5 μM

DOI 10.1105/tpc.19.00193; originally published online September 23, 2019;Plant Cell

Giosuè Costa, Stefano Alcaro, Enrico Martinoia and Giovanni GiulianoPietrella, Giuseppe Aprea, Lorenzo Borghi, Mistianne Feeney, Lorenzo Frigerio, Adriana Coricello,

Olivia Costantina Demurtas, Rita Francisco, Gianfranco Diretto, Paola Ferrante, Sarah Frusciante, MarcoABCC transporters mediate the vacuolar accumulation of crocins in saffron stigmas

 This information is current as of October 24, 2020

 

Supplemental Data /content/suppl/2019/09/23/tpc.19.00193.DC1.html

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