ABCC Transporters Mediate the Vacuolar Accumulation ofCrocins in Saffron Stigmas[OPEN]

Olivia Costantina Demurtas,a Rita de Brito Francisco,b Gianfranco Diretto,a Paola Ferrante,a Sarah Frusciante,a

Marco Pietrella,a,c Giuseppe Aprea,a Lorenzo Borghi,b Mistianne Feeney,d Lorenzo Frigerio,d Adriana Coricello,e

Giosuè Costa,e Stefano Alcaro,e Enrico Martinoia,b,1 and Giovanni Giulianoa,1

a ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, C.R. Casaccia, 00123, Rome,Italyb Department of Plant and Microbial Biology, University of Zurich, 8008 Zurich, SwitzerlandcCouncil for Agricultural Research and Economics (CREA), Research Center for Olive, Citrus and Tree Fruit, 47121 Forlì, Italyd School of Life Sciences, University of Warwick, Coventry CV4 7AL, United KingdomeDepartment of Health Sciences, Magna Græcia University of Catanzaro, 88100 Catanzaro, Italy

ORCID IDs: 0000-0002-6896-6341 (O.C.D.); 0000-0001-7728-8477 (R.d.B.F.); 0000-0002-1441-0233 (G.D.); 0000-0003-3071-2212(P.F.); 0000-0001-8161-3797 (S.F.); 0000-0002-8789-6860 (M.P.); 0000-0003-4969-2696 (G.A.); 0000-0002-9631-9694 (L.B.); 0000-0002-0057-7753 (M.F.); 0000-0003-4100-6022 (L.F.); 0000-0002-3306-9261 (A.C.); 0000-0003-0947-9479 (G.C.); 0000-0002-0437-358X (S.A.); 0000-0001-8943-7089 (E.M.); 0000-0002-2486-0510 (G.G.)

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes itsred color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression inyeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metaboliteextracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativustransporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug andtoxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the geneencoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCCfamily, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectivelyexpressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific andcooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.

INTRODUCTION

A fascinating feature of vascular plants is their capacity to produceextremelydiversespecializedmetabolites (PicherskyandLewinsohn,2011) and to accumulate some of them to extremely high concen-trations: steviol glycosides in Stevia rebaudiana leaves (Brandle andTelmer, 2007) and crocins in Crocus sativus stigmas (Bouvier et al.,2003), for instance, can make up to 10% of the tissue’s dry weight.Plants have evolved a variety of strategies for storing specializedmetabolites, such as the accumulation in specialized cell types(McConkey et al., 2000), or their sequestration in the central cellularvacuole (Martinoia et al., 2007). Well-known examples of the latterstrategyare thevacuolarsequestrationofanthocyanins ingrape (Vitisvinifera) berries (Franciscoet al., 2013), nicotine inNicotiana tabacumleaves (Morita et al., 2009), steviol glycosides inS. rebaudiana leaves(BrandleandTelmer,2007),andcrocins inC.sativusstigmas (Bouvieret al., 2003). Vacuolar sequestration has been proposed to prevent

the feedback inhibition of the biosynthetic enzymes and to reducetoxicity effects induced by high cytosolic concentrations of the finalproducts (Goodman et al., 2004).Saffron—the most expensive spice on Earth—is composed of

the dried stigmas of C. sativus (Figures 1A and 1B), which accu-mulate large amounts of crocins, apocarotenoid glycosides thatconfer the redcolor to the saffron spice (Tarantilis et al., 1995).Up to15 different crocins have been identified in mature C. sativusstigmas, consisting of both all-trans and 13-cis-crocetin esterifiedwith one to five Glc moieties (Figure 1). The proposed pathway forcrocinbiosynthesis inC. sativus stigmasstartswith the cleavage, inthe plastid, of zeaxanthin by Carotenoid Cleavage Dioxygenase2(CCD2; Frusciante et al., 2014). The cleavage product, crocetindialdehyde, migrates to the endoplasmic reticulum (ER), where it isdehydrogenated to crocetin by a membrane-associated CsALD-H3I1, and then glycosylated to crocins 1 and 29 by CsUGT74AD1,localized in the cytosol (Figures 1C and 1D; Demurtas et al., 2018).MorehighlyglycosylatedcrocinsaresynthesizedbyanunidentifiedUDP-glycosyl transferase (UGT), probably localized in the cytosol(Figure 1D). Given their polar nature, crocins synthesized in thecytosolmustbetransportedto thevacuolebyoneormoretonoplasttransporters (Figure 1D). This article describes their identification.Vacuolar transport of glycosylated metabolites, such as glyco-

sylatedflavonoidsorhormones (abscisicacidglucosylesters [ABA-GEs]), has been well documented (Gomez et al., 2009; Zhao and

1 Address correspondence to giovanni.giuliano@enea.it and enrico.martinoia@botinst.uzh.ch.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Olivia Costantina Demur-tas (olivia.demurtas@enea.it).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00193

The Plant Cell, Vol. 31: 2789–2804, November 2019, www.plantcell.org ã 2019 ASPB.

Dixon, 2009) and is effectedbybothmultidrug and toxic compoundextrusion (MATE) and ATP binding cassette C (ABCC) transporters(Martinoiaetal., 2007;deBritoFranciscoandMartinoia, 2018).ABCand MATE transporters have been characterized through expres-sion in yeast, animal, or plant cells; isolation of transporter-loadedmicrosomes; and transport assays using radiolabeled, fluorescent,or light-absorbing compounds transported into the microsomes(Marinova et al., 2007; Zhao and Dixon, 2009; Nour-Eldin et al.,2012; Burla et al., 2013; Francisco et al., 2013). This limits thenumberofcompounds forwhich transport canbestudiedaswell asthe capacity to mimic in vivo conditions, wherein a transporter isexposed to thousands of different compounds. In this work, westudy the in vitro transport of multiple metabolites from C. sativusstigma extracts, through a transportomic assay based on liquidchromatography-photodiode array-high resolution mass spectrom-etry (LC-PDA-HRMS)anditsuseto identifyandcharacterizeC.sativustonoplast transporters involved in vacuolar crocin accumulation.

RESULTS

Identification and Characterization of CandidateCrocin Transporters

A C. sativus stigma transcriptome (Supplemental Data Set 1)was searched for expressed genes belonging to the ABCCand MATE classes of tonoplast transporters. Nine ABCC and11 MATE transporters were expressed in stigmas (Figure 2A;Supplemental Table 1). The highest expressed transcript instigmas was CsMATE4, followed by CsABCC4a, CsABCC2,CsMATE1a, and CsMATE1b. Only CsABCC4a was specificallyexpressed in stigmas, while CsMATE4 was highly expressedalso in tepals and CsABCC2, CsMATE1a, and CsMATE1b weremainly expressed in other tissues. To further investigate their

possible role in crocin transport, we performed a coexpressionanalysis (see “Methods” for details) of all 20 transcriptswith totalcrocins and with the transcript encoding the first dedicatedenzyme in the crocin pathway,CsCCD2 (Frusciante et al., 2014).CsABCC4a expression correlated with both CsCCD2 and cro-cins (Pearson correlation coefficient [r] 5 0.99 and 0.95, re-spectively), followed byCsMATE4 (r5 0.84 and 0.75; Figure 2B;Supplemental Table 2). CsABCC2 displayed a good correlation(r 5 0.90) with CsCCD2, but not with crocins, while none of theremaining 17 transporters showed significant positive correlations.We decided to functionally characterize the five transporters

that were most highly expressed in stigmas (underlined inFigure 2A). The corresponding full-length transcripts were iso-lated from stigma RNA and sequenced (see “Methods”). Phy-logenetic analysis (Figure 2C) revealed thatCsABCC4a is closelyrelated to Arabidopsis (Arabidopsis thaliana) ABCC4, involvedin the vacuolar transport of folates (Klein et al., 2004), whileCsABCC2 shows high similarity to AtABCC2, which transportsABA-GEs (Burla et al., 2013), phytochelatins (Song et al., 2010),and glutathione conjugates and chlorophyll catabolites (Lu et al.,1998), and to AtABCC1, involved in the transport of phytoche-latins (Song et al., 2010) and folates (Raichaudhuri et al., 2009).CsMATE4 is closely related to the MATE1 transporter of Coptisjaponica, proposed to mediate the transport of the alkaloidberberine in rhizomes (Takanashi et al., 2017) and CsMATE1aand CsMATE1b are related to the MATE2 protein of Sorghumbicolor that mediates the vacuolar accumulation of the cyano-genic glucoside dhurrin (Darbani et al., 2016) and to Nicotianatabacum MATE1 and MATE2, which are responsible for thevacuolar accumulation of the alkaloid nicotine (Shoji et al., 2009).The typical ABC signatures (Walker A and Walker B) in the twonucleotide binding domains (nucleotide binding domain 1 andnucleotide binding domain 2) are well conserved in CsABCCtransporters, butmany differenceswere found in the transmembrane

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Figure 1. Saffron Crocin Biosynthesis and Its Compartmentation.

(A) C. sativus flower at anthesis.(B) Scanning electron microscopy of mature stigma.(C) and (D) Proposed crocin biosynthetic pathway (C) and its compartmentation (D). See Demurtas et al. (2018) for details: the CCD2 enzyme cleaveszeaxanthin in theplastid,producingcrocetindialdehyde that thenmigrates to theERand isdehydrogenated tocrocetinbyanALDH.Theglycosylation stepsare performed in the cytoplasm by UGT enzymes. Crocins are then transported into the vacuole by ABC tonoplast transporters functionally characterizedin this work.(E) Detailed crocin structures.

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Figure 2. Expression and Phylogenetic Analysis of Putative C. sativus Tonoplast Transporters.

(A)Heatmap of transcript levels for ABCC andMATE transporter genes in differentC. sativus tissues; data are expressed as log2 of reads per kilobase permillion and sorted by decreasing reads per kilobase per million values in stigma. For details, see Supplemental Data Set 1.(B)Coexpressionanalysisof genes forCsABCCsandCsMATEs (Bait)withCsCCD2andwith total crocins (Prey).Onlyr-values>0.50areshown.Fordetails,see Supplemental Data Set 1.(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 CsMATEsfunctionally characterized in this work are indicated by arrows. The trees include transporters from A. thaliana (At), C. japonica (Cj), M. truncatula (Mt),N. tabacum (Nt),S.bicolor (Sb),V.vinifera (Vv), andZ.mays (Zm).Theaccessionnumbersaredescribed in “AccessionNumbers.”Thepercentageof replicatetrees that clustered together in the bootstrap test (500 replicates) is indicated to the left of the branches. The alignment files are shown inSupplemental DataSets 3 and 4.

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domainsknowntobe responsible forsubstrate recognition/specificity(Supplemental Figure 1; Theodoulou, 2000; Jasinski et al., 2003;Wilkens, 2015).

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

vacuole (Demurtas et al., 2018). We fused the five transportergenes most expressed in stigmas to the enhanced Green Fluo-rescent Protein (eGFP) gene (Cinelli et al., 2000) and cloned them\in the pBI121 Agrobacterium transformation vector. Nicotianabenthamiana leaves were co-infiltrated with Agrobacterium

Figure 3. Subcellular Localization of C. sativus Transporters in Nicotiana Leaves.

Confocal images of GFP (green) and RFP (red) fluorescence inN. benthamiana leaves coexpressing the indicated ABCCorMATE transporter fused toGFPand gTIP (a tonoplast marker) fused to RFP. Bars 5 10 mm.

Saffron Vacuolar Crocin Transporters 2793

tumefaciens (strain C58C1) harboring constructs for expression ofdifferenteGFP fusions, and thegTIP tonoplastmarker fused to the redfluorescent protein (RFP; Figure 3; Nelson et al., 2007). At this reso-lution, the GFP signal showed colocalization with the RFP signal,suggesting that all tested transporters were predominantly tonoplastlocalized.

Establishment of a Transportomic Assay to IdentifyCrocin Transporters

Transport of intracellular metabolites by vacuolar transportersis often stereospecific (Bhatia et al., 2008; Zhou et al., 2014;

Schneider, 2015). Commercial crocin preparations are not rep-resentative of the crocin content of C. sativus stigmas, beingcomposed almost exclusively of all-trans crocin 4 (SupplementalFigure 2), while in C. sativus stigma six major and several minorcrocins, including 13-cis isomers, are present (Figure 4B;Supplemental Figure 2). Therefore, to study the transport of thenatural crocin substrates, as well as that of other glycosylatedcompounds accumulated in stigmas (picrocrocin and flavonoidglycosides), we usedaC. sativus stigmahydroalcoholic extract asa substrate for the transport reaction. Simultaneous detection ofmultiple metabolites relied on their detection by LC-PDA-HRMS(Figure 4). We expressed the five C. sativus transporters in yeastcells, isolated microsomes from those cells, and used them to

Figure 4. Transportomic Assay Using C. sativus Stigma Extract and Yeast Microsomes.

(A) Schematic representation of the assay. Microsomes overexpressing a transporter are incubated with C. sativus stigma hydroalcoholic extract in thepresenceofATP.Microsomesareseparatedby the rapidfiltration techniqueandwashed,and transportedmetabolitesareelutedandquantifiedbyLC-PDA-HRMS.(B) to (E)Representative electrospray ionization1/MSchromatogramsof the extracted accuratemass of crocetin (M1H1329.1747) generated fromcrocinfragmentation. (B)C. sativus stigma hydroalcoholic extract. (C) Import intomicrosomes isolated from yeast cells transformedwith the pNEV empty vector.(D) Import into microsomes expressing CsABCC4a incubated in the absence of ATP. (E) Import into microsomes expressing CsABCC4a incubated in thepresence of ATP. Incubationwas for 15min. Different peaks represent the following: trans-crocin 4 (retention time [RT], 10.58min); trans-crocin 3 (RT, 11.52min); trans-crocin 29 (RT, 12.54min); cis-crocin 4 (RT, 13.84min); trans-crocin 2 (RT, 14.20min); cis-crocin 3 (RT, 14.82min); trans-crocin 1 (RT, 16.16min);cis-crocin 29 (RT, 16.79 min); cis-crocin 2 (RT, 17.02 min); and cis crocin 1 (RT, 17.73 min).

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perform transportomicassays.Briefly, the transporterswerecloned inthe pNEV plasmid and expressed in a yeast strain defective in theABCC Yeast Bile Transporter1 (YBT1, ybt1 strain; Sauer and Stolz,1994; Paumi et al., 2009). Total microsomes were isolated from theyeast transformants, and their intactnesswasassessedbymeasuringthe transport of leukotriene C4 (LTC4) by LC-HRMS (see “Methods”;Supplemental Figure 3). The sensitivity of this method wascomparable to the standard radiochemical assay (Leier et al.,1994), indicating that the transportomic assay was sensitiveenoughtodetect transportofmetabolitespresentat lowconcentrations(femtomoles to picomoles). Microsomes from yeast cells transformedwith the pNEV empty vector were used as controls (Figure 4).

Microsomes expressingCsABCC4a andCsABCC2were ableto mediate the transport of different crocins with different ef-ficiency and in an ATP-dependent manner (Figures 4 and 5).CsABCC4a transported with approximately equal efficiencytrans-crocin 1,cis-crocin3, and trans-crocin29,whileCsABCC2showed lower transport efficiency on the latter crocin. In gen-eral, crocins carrying smaller Glc groups were efficientlytransported in the all-trans form, while crocins with largergentiobiose groups displayed preferential transport in the cisform. Low, but significant, levels of transport by CsABCC4a andCsABCC2were also observed for some flavonoids (Figure 5), inagreement with the broad substrate range exhibited by ABCCs(Hwang et al., 2016). Crocin transport by CsABCC4a andCsABCC2 was inhibited by probenecid, a known inhibitor ofABC-type transporters and by incubation on ice, excludingnonspecific binding to the yeast microsomes (Table 1). MATEtransporters were able to transport only flavonoid glycosides(Figure 5).We did not observe transport of picrocrocin, themostabundant glycosylatedmetabolite inC. sativus stigma, by anyofthe transporters tested.

Given the similarity of CsABCC2 to the ABA-GE transporterAtABCC2 (Burla et al., 2013), we investigated whether CsABCC2andCsABCC4a could transport ABA-GE. At concentrations up to7.5 mM, that is, much higher than its physiological intracellularconcentration (Burla et al., 2013), neither transporter was able totransport ABA-GE, while they both efficiently transported cis-crocin 3 (Table 2).

Detailed Characterization of the Stigma-Specific CsABCC4aCrocin Transporter

Although Figure 3 shows that themajority of CsABCC4a localizesto the tonoplast of N. benthamiana mesophyll cells, it does not

Table 1. Inhibition of C. sativus Stigma Metabolite Transport by CsABCC4a and CsABCC2 in the Presence of the Probenecid Inhibitor or byIncubation on Ice

CsABCC4a CsABCC2

Compound Name Probenecid 0°C Probenecid 0°C

Kaempferol 3-O-sophoroside-7- glucoside ND 19.3 6 4.2 ND 53.5 6 6.0Kaempferol 7-sophoroside 23.0 6 4.5 34.4 6 6.9 10.4 6 3.8 20.8 6 4.6Kaempferol 3-O-rutinoside ND 35.4 6 0.01 ND 2.3 6 0.3Naringenin 7-O-glucoside 27.7 6 1.2 11.1 6 0.7 16.5 6 0.6 4.4 6 0.1trans-Crocin 4 ND 20.0 6 3.4 14.3 6 2.7 NDcis-Crocin 4 ND 19.6 6 3.7 15.2 6 2.7 NDtrans-Crocin 3 ND 48.4 6 8.4 ND NDcis-Crocin 3 48.5 6 1.9 22.3 6 2.2 0.7 6 0.006 NDtrans-Crocin 2 ND 24.8 6 5.0 18.2 6 3.6 NDcis-Crocin 2 26.8 6 4.8 ND 33.6 6 4.8 0.04 6 0.005trans-Crocin 29 28.4 6 2.0 23.1 6 1.2 ND NDcis-Crocin 29 31.9 6 2.9 ND 32.9 6 5.2 NDtrans-Crocin 1 22.1 6 1.5 42.0 6 6.1 17.4 6 1.3 NDcis-Crocin 1 ND ND – –

The table shows the percentage of residual transport after treatment with probenecid or on ice, compared with control reactions performed at roomtemperature in absence of inhibitors. Data are the avg 6 SD of three independent microsome preparations. ND, not detectable (detection limit of themass spectrometer in full MS mode: 500 fg). Dashes indicate that the percentage of residual transport could not be estimated, because the CsABCC2transporter does not transport cis-crocin 1 in normal conditions.

Table 2. Transport of ABA-GE and cis-Crocin 3 by CsABCC4a andCsABCC2 Transporters

Substrate Transporter

Substrate Concentration (mM)

0.075 0.75 7.5

ABA-GE CTRL ND ND NDCsABCC4a ND ND NDCsABCC2 ND ND ND

cis-Crocin 3 CTRL ND ND NDCsABCC4a ND ND 8.8 6 0.8CsABCC2 ND ND 2.8 6 0.5

Percentage of transport at different substrate concentrations in standardassay conditions. Transport experiments were performed with purifiedABA-GE or cis-crocin 3 (7.5 mM). Results are the avg 6 SD of threeindependent microsome preparations. ND, not detectable (detection limitof the mass spectrometer in full MS mode: 500 fg).

Saffron Vacuolar Crocin Transporters 2795

completely rule out that a small fraction may also be localizedto other cellular membranes, such as the ER or the plasmamembrane, that in mesophyll cells are not well separated fromthe tonoplast. To further study the subcellular localization ofCsABCC4a:eGFP in N. benthamiana leaves, we conducted co-localization experimentswith tonoplast, ER, or plasmamembranemarkers and analyzed the Spearman correlation coefficient of thetwo signals (French et al., 2008). The CsABCC4a:GFP signalstrongly colocalized with the gTIP-RFP tonoplast marker(Figure 6A), but did not show significant colocalization with theREFP-HDEL ER marker (Figure 6B) or with the styryl dye FM4-64labeling the plasma membrane (Figure 6C). Furthermore, GFPfluorescence clearly localized to the tonoplast released afterisolation of protoplasts fromagroinfiltrated leaves and their partiallysis (Figure 6D). These data confirm that the CsABCC4a trans-porter is predominantly tonoplast localized when expressed in N.benthamiana leaves.Transient expression in N. benthamiana leaves of CsCCD2,

encoding a zeaxanthin cleavage dioxygenase leading to theproduction of crocetin dialdehyde (Frusciante et al., 2014), re-sulted in the production of low levels of both trans- and cis-crocetin and trans- and cis-crocins 1, 29, 2, and 3 (Figure 7).This suggests that N. benthamiana leaves contain endogenousaldehyde dehydrogenase (ALDH) and UGT activities able totransform the CCD2 product (crocetin dialdehyde) into crocetinand crocins (Demurtas et al., 2018). This finding allowed us totest the function of CsABCC4a in N. benthamiana leaves. WhenCsABCC4a was expressed in combination with CsCCD2, weobservedasignificant increase in theaccumulationof crocins 1, 2,and 29 and a decrease of crocetin levels (Figure 7).Transportomic assays present advantages (assaying hundreds

of metabolites at a time) but also complications (differentmetabolites can influence each other’s transport). To better in-vestigate this aspect, we studied the transport by CsABCC4a ofcis and trans isomers of crocin 3, which are very abundant inmatureC.sativusstigmasandare readily separatedbypreparativeHPLC. The two isomerswere purified fromC. sativus stigmas, andtheir purity was confirmed by LC-PDA-HRMS (SupplementalFigure 4). At a concentration comparable to that of the whole

Figure 5. CsABCC4a and CsABCC2 Transport Different Crocins in Vitro.

(A) Major glycosylated metabolites present in the C. sativus stigmahydroalcoholic extract: 1, kaempferol 3-O-sophoroside-7-glucoside;2, kaempferol 3,7,49-triglucoside; 3, kaempferol 7-sophoroside; 4, kaempferol

3-b-D-glucopyranoside; 5, kaempferol-3-O-rutinoside; 6, dihydrokaemp-

ferol 7-O-glucoside; 7, naringenin glucosides (three isomers); 8, picroc-

rocin; 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 29 (t 29); 17, cis-crocin 29 (c 29); 18,

trans-crocin 1 (t 1); and 19, cis-crocin 1 (c 1). Structures of the various

crocins are shown in Figure 1E. Values are presented as relative

abundances compared with the internal standard used (formononetin)

of mean values 6 SD of three independent extract preparations.

(B) Percentage of net transport of glycosylated metabolites by yeast mi-crosomes expressing candidate tonoplast transporters. CTRL, micro-somes form yeast cells transformed with the empty vector. CsABCC4a,CsABCC2, CsMATE4, CsMATE1a, and CsMATE1b, microsomes ex-pressing candidate tonoplast transporters. Percentage of net transportwas calculated by subtracting the peak areas in the absence of ATP fromthose in the presence of ATP after 15 min of incubation and normalizing tothe peak areas in the initial extract. Data are the avg 6 SD of three in-dependent microsome preparations.

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extract (7.5 mM), the purified cis isomer was efficiently trans-ported in a time-dependent manner by CsABCC4a, while notransport was observed for its trans isomer (Figure 8A). Sincethe trans isomer is transported in crude extracts (Figure 5), weconcluded that its transport requires a component present inthe whole extract that is lost during isomer purification. Wetested the hypothesis that this component is the cis isomer bymixing the two isomers at different concentrations and per-forming a transport assay. Transport of cis-crocin 3 wasconcentration dependent and was also enhanced by thepresence of its trans isomer (Figure 8B, left). Transport of thetrans isomer occurred only at the highest concentration andonly in the presence of the cis isomer (Figure 8B, right). Weconclude that crocin 3 transport by CsABCC4a is cooperativeand that this cooperativity allows transport of trans-crocin 3 inwhole extracts (Figure 5).

DISCUSSION

The capacity of specialized plant tissues to accumulate high levelsof specialized metabolites relies on their compartmentation ca-pacity. Plant cells contain at least two types of vacuoles: storageand lytic. While storage vacuoles of seeds are small, the lyticvacuoles,whichare themain vacuoles in the vegetative tissues, aregenerally very large and allow accumulation of large amounts ofnutrients and specialized metabolites. Lytic vacuoles also play animportant role in the detoxification of toxic compounds such asheavy metals and sodium. Additional important vacuolar functionsinclude metabolite storage as well as pH homeostasis, calciumsignaling, and the participation in guard cell movements (Martinoiaet al., 2007, 2012). Plant specializedmetabolites stored in vacuolesare usually transported into the vacuolar lumen by different classesof tonoplast-localized transporters. A single class of specialized

Figure 6. Tonoplast Localization of CsABCC4a in Nicotiana Leaves.

(A) to (C)N. benthamiana leaveswere agroinfiltratedwith the CsABCC4a:GFP construct together with the gTIP-RFP tonoplast marker (A), or with the RFP-HDEL ER marker (B), or post-stained with the FM4-64 plasma membrane marker (C), and imaged by confocal microscopy. The Spearman correlationcoefficient (Rs) between the two channels was calculated using the ImageJ analysis programwith the Pearson-Spearman correlation plug-in (French et al.,2008) to calculate colocalization and to produce scatterplots using a threshold of 40. Bars 5 5 mm.(D)Confocal images of a protoplast fromaN. benthamiana leaf expressingCsABCCa:GFP (green channel). After induced lysis of the plasmamembrane thevacuole (asterisk) is in the process of being released. Bar 5 12 mm.

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metabolites canbeasubstrate fordifferent typesof transporters, asshown for anthocyanins, which are transported by MATE-typeantiporters in grape and Medicago (Gomez et al., 2009; Zhaoand Dixon, 2009), and also by ABC-type transporters inmaize (Zeamays) and grape (Goodman et al., 2004; Francisco et al., 2013).Becausethesespecializedcompoundsexhibit anenormousvarietyof structures, it is likely that such structural modifications play animportant role in the recognition of a given substrate.

A single plant species can synthesize thousands of differentmolecules that may be transported across one or more organ-ellar membranes, which likely explains the large sizes of bothABC-type (>130) and MATE-type (>110) transporter gene fam-ilies (Hwang et al., 2016; Liu et al., 2016). Identifying the primarytransporter for a given molecule by brute force approaches istherefore extremely difficult and timeconsuming. The situation isfurther complicated by the fact that <0.01% of plant specializedmetabolites are available in purified form and even fewer asradiolabeled compounds for use in radiochemical transportexperiments. The transportomicapproachdescribedhere is ableto assess the transport of any plant natural product that can beextracted by simple methods and detected by mass spec-trometry, that is, >95% of plant metabolites. Its sensitivity iscomparable to that of radiochemical transport assays, and itallows testing of a large number of substrates—potentiallyhundreds—in a single assay without prior purification. Thisapproach was first used for the study of a human transporter(Uchida et al., 2007), and the term transportomics was coinedby Krumpochova et al. (2012) in characterization of a murinetransporter using mouse urine as substrate. To date, the max-imum number of plant substrates for which transport has beenstudied by liquid chromatography–mass spectrometry (LC-MS)is two (Schaedler et al., 2014). This article demonstrates thepossibility of studying transport in complex plant metabolitemixtures, similar to what is already possible in other organisms(Krumpochova et al., 2012).

Given the well-known cooperative nature of the transport ofseveral molecules (Liu et al., 2001), the transportomic approachallows the studyof the transport of substrates (e.g., trans-crocin 3)that might not be transported in purified form. With these ad-vantages, it is surprising thatLC-MS–based transportomicassayshave been, to date, limited to the study of animal transporters(Uchida et al., 2007; Krumpochova et al., 2012).We have previously demonstrated that the second and third

steps in crocin biosynthesis are localized in the ER and cytosol,respectively (Figure1;Demurtasetal., 2018).Sincecrocinscannotpassively diffuse through membranes (Lautenschläger et al.,2015), anactive transport system is required toallowthemtocrossthe tonoplast membrane and be accumulated within the vacuole.Combining transcriptomic data from stigma with the trans-portomic approach, we identified the ABCC transporters re-sponsible for this transport step. Both CsABCC4a and CsABCC2are highly expressed in stigmas, show high coexpression withtotal crocin levels and/orCsCCD2 inC. sativus, are predominantlylocalized to the tonoplast inN.benthamiana leaves, andareable totransport crocins in yeast microsomes. While CsABCC4a is al-most exclusively expressed in stigmas, CsABCC2 shows highlevels of expression in corms, suggesting that itmaybe involved incrocin transport in this tissue as well (Rubio-Moraga et al., 2010).CsABCC4a and CsABCC2 are related to Arabidopsis trans-

porters involved in the vacuolar transport of folates and ABA-GE,respectively (Raichaudhuri et al., 2009; Burla et al., 2013). In spite ofits similarity toAtABCC2,CsABCC2 isunable to transportABA-GE.Typical ABC motifs are well conserved in both transporters, whileseveral differences are found in the transmembrane domains,known to be involved in substrate binding (Supplemental Figure 1;Theodoulou, 2000; Jasinski et al., 2003; Wilkens, 2015). Sincecrocin biosynthesis, in the Iridaceae family, is confined to theCrocus genus, the most likely hypothesis is that crocin transportin C. sativus evolved from ancestral transporters whose originalsubstrate was different from crocins. As is frequently observed for

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 1 CsABCC4a. Data are the avg6 SD of ion peak areas ofcrocetin and crocins, normalized to the ion peak area of the internal standard formononetin (fold IS), 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 1

CsABCC4a; Student’s t test: *P < 0.05; **P < 0.01).

2798 The Plant Cell

ABCC transporters (Hwang et al., 2016), CsABCC4a andCsABCC2 are, to a certain extent, promiscuous, in thatthey transport crocins and, with lower efficiency, flavonoidglycosides.

Expression in N. benthamiana leaves of the first dedicatedenzyme in the crocin pathway, CsCCD2 (Frusciante et al., 2014),was sufficient to cause crocetin and crocin accumulation, prob-ably due to the presence of ALDH and UGT enzymes able toconvert the CsCCD2 product, crocetin dialdehyde, into down-stream compounds (Demurtas et al., 2018). CsABCC4a localizesto the tonoplast of N. benthamiana mesophyll cells, and coex-pression of it with CsCCD2 caused enhanced accumulation ofcrocins and a decrease of their precursor, crocetin. This is likelydue to increased vacuolar sequestration of crocins and an in-crease of their stability and/or decrease of feedback inhibition ofcytosolic crocinsynthesis.Unfortunately, crocin levelsobtained in

N. benthamiana leaves are too low to assess their subcellularlocalization microscopically.Crocin transport byCsABCC4a is stereospecificandcooperative

in that it shows a preference for cis-crocin 3 comparedwith its transcounterpartandthatone isomerenhancesthe transportof theother.Cooperative transport has been previously observed with wholevacuoles or AtABCC2, andglutathione and glucuronide conjugates(Lu et al., 1998; Klein et al., 2000). This cooperativity could be dueto structural changes in the large vestibule exhibited by ABCCtransporters after binding a first substrate (Johnson and Chen,2017), which could allow binding of a second substratemolecule inthe cavity. Homologymodeling and docking analysis are unlikely toprovide useful insight into the cooperative transport of trans- andcis-crocin as CsABCC4a has low similarity to ABCC transporterswhose structures have been solved (Supplemental Table 3).

Figure 8. Kinetics and Cooperativity of Purified cis- and trans-Crocin 3 Transport by CsABCC4a.

(A)Time-dependent transportofcis-crocin3 (left) or trans-crocin3 (right) at aconcentrationof7.5mMbyyeastmicrosomesexpressingCsABCC4aorcontrolmicrosomes (pNEV).(B) Cooperative transport of cis-crocin 3 (left) and trans-crocin 3 (right) in the presence of different concentrations of the other isomer. (Left) cis-Crocin 3transportwasevaluatedat twodifferent concentrations (0.75and7.5mM) in thepresenceof increasingconcentrationsof trans-crocin 3 (0, 0.75, and7.5mM).(Right) trans-Crocin 3 transport was evaluated in the same conditions and in the presence of increasing concentrations (0, 0.75, and 7.5 mM) 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 ofATP after 15 min. Data are the avg 6 SD of three independent microsome preparations.

Saffron Vacuolar Crocin Transporters 2799

Transportomic assays demonstrated that CsABCC4a andCsABCC2 transport cis-crocins 3 and 4more efficiently, while themajor crocins accumulated in C. sativus stigmas are the corre-sponding all-trans counterparts. Acidic pH conditions such asthose found in the vacuole are known to cause polyene isomer-ization (Re et al., 2001), and indeed cis-crocin 3 is convertedto its all-trans isomer at pH 5.2 (Supplemental Figure 5). Wetherefore hypothesize that crocins 3 and 4 are transported pre-dominantly in the cis form and then converted to the all-trans formonce exposed to the acidic vacuole sap. Such an isomerizationtrapping mechanism has been demonstrated, for instance, forapigenin 7-O-(6-O-malonylglucoside), a vacuolar pigment fromparsley (Petroselinum hortense) that is subjected to conforma-tionalchangesunderacidicpH (Maternetal., 1983)andsuggestedfor the conversion of fluorescent chlorophyll catabolites to non-fluorescent chlorophyll catabolites during chlorophyll breakdown(Hörtensteiner and Kräutler, 2011).

In this work, we have identified transporters for crocin inC. sativus stigmas using a transportomic approach, which hasbeen accomplished only for a few plant specializedmetabolites.Using the method developed here, identification of transportersfor other important plant specialized metabolites can be en-visaged that could, in turn, prove useful tools for increasing theaccumulation of thesemetabolites in planta. An examplemay besteviosides produced inS. rebaudiana leaves. Similar to crocins,steviosides contain an isoprenoidmoiety esterifiedwith glucosylgroups, and in both cases the synthesis starts in the plastid andproduces a glycosylated product in the cytosol that is finallytransported in the vacuole (Brandle and Telmer, 2007; Demurtaset al., 2018).

METHODS

Plant Materials and Growth Conditions

Bioinformatic Analyses

Phylogenetic analyses were conducted with MEGA version 7 (Kumaret al., 2016), using the neighbor-joining method (Saitou and Nei, 1987);the percentage of replicate trees in which the associated taxa clusteredtogether in the bootstrap test (500 replicates) is shown next to the branches(Felsenstein, 1985). The evolutionary distances were computed usingthe p-distance method (Nei and Kumar, 2000) and represent the numberof amino acid differences per site. All ambiguouspositionswere removed foreach sequence pair. Protein alignment was performed with Clustal Omegatool (https://www.ebi.ac.uk/Tools/msa/clustalo/). Coexpression analysiswasperformedasdescribedpreviouslybyComanet al., (2014) andAhrazemet al., (2018). Briefly, the pairwise Pearson correlation between eachtransporterandCCD2/crocinswascomputed,andFisher’sZ-transformationwas used to test the statistical significance of the pairwise correlations.Transmembrane domains were deduced using the TMHMM 2.0 software(http://www.cbs.dtu.dk/services/TMHMM/).

Cloning

CsABCC and CsMATE coding sequences were isolated from cDNA ob-tained by RNA extracted from C. sativus stigmas collected the day ofanthesis using the SMART PCR cDNA synthesis kit (catalog no. 634902,Clontech)and theSuperScript IIReverseTranscriptase (catalogno. 18064-014, Life Technologies). All the oligonucleotides used to isolate and clonethe genes are described in Supplemental Table 4. The CsABCC4a partial

sequence was reconstructed from contigs obtained by 454 Titanium se-quencing (Frusciante et al., 2014) and amplified from cDNA with the oli-gonucleotides 1 and 2; the amplicon (amplicon 1) was then cloned inpBluescript SK1 vector (Stratagene) digested with EcoRV restrictionenzyme. 59-Rapid amplification of cDNA ends PCR was performed toobtain the 59 sequence of the cDNA using a commercial kit (catalog no.18374-058, Life Technologies) according to the manufacturer’s in-structions.C. sativuscDNAwasamplifiedwith theoligonucleotide3.TailedcDNA was amplified using 59-rapid amplification of cDNA ends abridgedanchor primer supplied in the kit and oligonucleotide 4. The PCR product(amplicon 2) was purified using a purification kit (catalog no. 28,104;Qiagen) and cloned in pBluescript SK1 vector digested with EcoRV. AllPCR reactions were performed using Phusion High Fidelity DNA poly-merase (catalog no. M0530L, New England Biolabs). Amplicons 1 and2 were then reamplified, assembled, and cloned in the yeast (Saccharo-myces cerevisiae) expression vector pNEV-Ura (Sauer and Stolz, 1994)using Gibson assembly method (Gibson et al., 2009). Amplicon 1 wasamplified with the oligonucleotides 5 and 6, producing the fragment 1;amplicon 2 with the oligonucleotides 7 and 8, producing the fragment 2;and pNEV-Ura plasmid was digested with NotI restriction enzymes(fragment 3). The three fragments were assembled using the Gibson As-sembly Master Mix (catalog no. E2611S, New England Biolabs) accordingto the manufacturer’s instructions, thereby producing the pNEV:CsABC-C4a construct. Validation that amplicons 1 and 2 belonged to the samecoding sequence was performed by PCR on C. sativus cDNA using theoligonucleotides 9 and10 that produced the expected full-length ampliconof 4491 bp (Supplemental Figure 6).

CsABCC2 and CsMATE full-length sequences were amplified fromcDNA (Supplemental Figure 6) with the following oligonucleotides: 11 and12 forCsABCC2, 13and14 forCsMATE1a, 15and16 forCsMATE1b (theseprimers anneal on the 59 and 39 untranslated region, respectively), and17 and 18 for CsMATE4. The amplicons were then reamplified with oligo-nucleotides that insert the NotI site and subcloned in the pNEV-Uraplasmid NotI digested, producing the pNEV:CsABCC4a, pNEV:CsABCC2,pNEV:CsMATE1a, pNEV:CsMATE1b, and pNEV:CsMATE4 constructs. Alltheampliconsandconstructswereverifiedbysequencing.ThepNEV-basedconstructs were expressed in Saccharomyces cerevisiae cells as describedin “Transport Assay.”

For protein localization studies, CsABCC and CsMATE cDNAs were39 fused to the coding sequence for the eGFP using Gibson assemblymethod. The pBI:eGFPvector (Frusciante et al., 2014)was digestedwithXbaI restriction enzyme, while cDNAs were amplified from pNEV-basedconstructs, inserting the sequence encoding for Pro-Gly-Pro tripeptidebefore the eGFPcoding sequence, using the followingoligonucleotides:19 and 20 for CsABCC4a, 21 and 22 for CsABCC2, 23 and 24 forCsMATE1a, 25 and 26 for CsMATE1b, and 27 and 28 for CsMATE4.The purified PCR fragments were then assembled with the pBI:eGFPvector generating the pBI:CsABCC4a:eGFP, pBI:CsABCC2:eGFP,pBI:CsMATE1a:eGFP, pBI:CsMATE1b:eGFP, and pBI:CsMATE4:eGFPconstructs. For protein overexpression in Nicotiana benthamiana leaves,the CsCCD2 cDNA including the sequence coding for its chloroplasttransit peptide (Demurtas et al., 2018) and the CsABCC4a cDNA werecloned in the pBI121 vector by Gibson assembly method. The vectorwas digested with PacI restriction enzyme, while the cDNAs were ob-tained by PCR with the following oligonucleotides: 29 and 30 forCsCCD2 and 31 and 32 for CsABCC4a. All the plasmids were checkedby sequencing before transformation in Agrobacterium tumefaciens(strain C58C1).

Expression in N. benthamiana Leaves

Agroinfiltration of N. benthamiana leaves was performed as describedpreviously by Hamilton and Baulcombe, (1999). Leaves were co-infiltrated

2800 The Plant Cell

with C58C1 cells containing pBI121:CsABCC4a:eGFP or pBI121:CsABCC2:eGFP or pBI121:CsMATE4:eGFP or pBI121:CsMATE1a:eGFPor pBI121:CsMATE1b:eGFP and pBI:gTIP-RFP (tonoplast marker; Nelsonet al., 2007). To reduce the silencing of the transgenes, leaves were alsoinfiltrated with the silencing suppressor RK19 (Francisco et al., 2013).Leaves were also co-infiltrated with C58C1 cells containing pBI121:CsABCC4a:eGFP, pBI-RFP-HDEL (ERmarker; Leeet al., 2013), andRK19.After 3 to 6 d, leaves were analyzed using a confocal laser scanning mi-croscope (FV1000, Olympus; LSM880, Zeiss). Lasers at 488 and 543 nmwere used to detect green and red fluorescence of GFP and gTIP-RFP orRFP-HDEL (emission, 571 to 640 nm), respectively. FM4-64was excited at514 nm and detected in the range 616 to 645 nm. Images were acquiredusing a 403 oil immersion objective (numerical aperture, 1.30) with opticalzooming 13 or 33. All images were analyzed and processed with ImageJ.

For preparation ofmesophyll protoplasts, cell walls ofN. benthamianaleaves were digested for 2 h at 30°C in MCP (500 mM sorbitol, 1 mMCaCl2, and 10 mM MES-KOH, pH 5.6) supplemented with 1% (w/v)Cellulase Y-C and 0.1% (w/v) Pectolyase Y-23. Protoplasts were col-lected by centrifugation at 1200g for 5 min on a cushion constituted of100% Percoll (v/v) in 500 mM sorbitol and 20 mMMES. The supernatantwas removed, and the protoplasts were mixed with the Percoll cushion,overlaid with 25% Percoll and MCP. After centrifugation at 1200g for5 min, the purified protoplasts were collected between 25% Percoll andMCP fractions. Vacuoles were released by mixing (1:1) purified proto-plasts with lysis buffer (200mM sorbitol, 10% Ficoll, and 10mMHEPES-KOH, pH 8.0). Microscopy was performed using a TCS SP5 confocalmicroscope (Leica). For each experiment, at least three different plantswere infiltrated and observed. Each experiment was repeated at leasttwice (with different batches of plants). At least five randomly chosenregions were observed, and representative images were selected foreach experiment.

For simultaneous expression of CsCCD2 and CsABCC4a, leaveswere co-infiltrated with C58C1 cells containing pBI121:CsCCD2,pBI121:CsABCC4a, and the silencing suppressor RK19. Three in-dependent infiltration experiments (with different batches of plants) wereperformed. For each experiment, one apical leaf of four different plantswas infiltrated with the CsCCD2 construct, and the other apical leaf wasinfiltrated with the CsCCD2 1 CsABCC4a construct. At 4 days afterinoculation, the four leaves infiltrated with the same construct werecollected as a pool and stored at 280°C. The pools from the three in-dependent experiments were then analyzed by LC-PDA-HRMS. Semi-polar extracts for LC-PDA-HRMSanalysiswere prepared as described in“LC-PDA-HRMS Analyses.” Data are presented as average (avg) area ofcrocins 6 SD normalized on the area of the internal standard for-mononetin. The results obtainedwere evaluated usingStudent’s t test forthe calculation of the significance of the difference between themeans ofthe two groups (CsCCD2 and CsCCD2 1 CsABCC4a; SupplementalTable 5).

Transport Assay

The ybt1 yeast mutant (MATa; ura3D::HIS3; leu2-3, 112; his3-D200;bat1D1::URA3) was transformed by electroporation as described pre-viously (Becker and Guarente, 1991) with the pNEV-based constructs. Ascontrol, cells were transformed with the empty vector pNEV-Ura, namedpNEV for simplicity. Transformants were selected on minimal syntheticdropout medium lacking uracil. For in vitro transport studies, yeast mi-crosomes were isolated as described previously (Tommasini et al., 1996).Uptake experiments were performed using the rapid filtration technique(Tommasini et al., 1996). Briefly, 100 mL of vesicles (OD600 5 4; totalproteinscontent,;400mg)weremixedwith ice-cold transportbuffer (0.4Mglycerol, 0.1 M KCl, and 20 mM Tris-MES, pH 7.4) and freshly added with1 mM DTT, 6 or 1 mM MgSO4 (in the presence or absence of MgATP,

respectively), 100 mg/mL creatine kinase, and 10mM creatine phosphate.Substrate transportwasassayedeither in thepresenceor absenceof 4mMMgATP in a total reaction volume of 650 mL. The transport assay wasperformed at room temperature.

Intactness ofmicrosomeswas evaluated by the ability to transport thesubstrate LTC4 (Leier et al., 1994). The LTC4 substrate (catalog no.20,210; Cayman Chemical) was used at concentration of 100 mM. At twotime points (30 s and 8 min), 100 mL of the reaction mixture was im-mediately loaded on a prewetted nitrocellulose filter (0.45-mm pore size,Millipore) and rapidlywashedwith 33 2.5mLof ice-cold transport buffer.Three technical replicates (100 mL each) were performed for each con-dition and repeated for each vesicle preparation. The filter-boundvesicles were dissolved by adding 1 mL of 75% (v/v) spiked coldmethanol (0.5 mg/mL formononetin; catalog no. 47752-25MG-F, Sigma-Aldrich), and metabolites were then extracted at room temperaturethrough continuous agitation for 30min. To remove vesicle lipids, 800mLof eluted sampleswas transferred in amicrocentrifuge tube and added to400 mL of chloroform, vortexed, and shaken in a mixer mill (MM 300,Retsch) for 5min at 20-Hz frequency. Ultrapurewater (200mL) was addedto separate the phases, followed by vortexing and centrifugation at20,000g for 20 min. Finally, 800 mL of the upper phase was collected,dried with a vacuum concentrator, and resuspended in 80 mL of cold50% (v/v) methanol, and centrifuged at 20,000g for 10 min to removeprecipitates. A 2-mL sample of the supernatant was subjected to LC-MSanalysis. All solvents used for the extraction were LC-MS grade (MerckMillipore).

To study the transport of C. sativus stigma metabolites, a C. sativusstigma hydroalcoholic extract and purified crocinswere used to performuptake experiments. The C. sativus stigma hydroalcoholic extract wasprepared as follows: 3 mg of pulverized C. sativus stigmas (Castilla-LaMancha, dried 2013) was resuspended in 300 mL of cold 50% (v/v)methanol, homogenized for 40 min in a mixer mill MM 300 (Retsch) at20-Hz frequency, and centrifuged 20 min at 20,000g. The supernatantwas recovered, total crocin content was quantified by LC-DAD, anda standardized amount was used to perform the uptake experiments: ina final reaction of 650 mL, we adjusted the C. sativus stigma extractvolume in order to have a final concentration of 320 mM of total crocins.Crocin 4 analytical standard was purchased from PhytoLab (catalog no.80,391).

Crocin 3 isomers (trans and cis) were purified from C. sativus stigmahydroalcoholic extract by preparative HPLC. Briefly, 100 mg of pulver-ized stigmas were resuspended in 1 mL of cold 50% (v/v) methanol andprocessed from C. sativus stigma hydroalcoholic extract. Crocin sep-aration was performed on an LC Series 200 system (PerkinElmer) in-jecting 100 mL of extract on a C18 reverse-phase column (2503 10 mm,5 mm; Ascentis, Supelco) heated at 35°C. The mobile phases used werewater 1 0.1% (v/v) formic acid (A) and acetonitrile 1 0.1% (v/v) formicacid (B) at a total flow rate of 4 mL/min. The separation was developedusing 10% B for 1 min, a 10-min linear gradient to 20% B followed bya 20-min linear gradient to 30% B. LC conditions were kept for 10 moreminutes, before going back to the initial conditions. Detection wasperformed at 440 nm with an online UV/VIS Series 200 detector (Per-kinElmer). Two-milliliter fractions were collected, dried in a lyophilizer,and resuspended in 50 mL of 50% (v/v) methanol. Competitive assaysbetween trans- and cis-crocin 3 were performed using concentrationsranging from 0.75 to 75 mM.

All uptake experiments with C. sativus stigma extract or purified crocinswere performed using the rapid filtration technique with acetate cellulosefilters (0.45-mmporesize,Sartorius), insteadofnitrocellulose.Reactionswereperformed at room temperature in reaction mix as described for LTC4 andstopped at different time points. Inhibition assays were performed in thepresence of the inhibitor probenecid (catalog no. P8761, Sigma-Aldrich;Francisco et al., 2013). Briefly, yeast microsomes were incubated at room

Saffron Vacuolar Crocin Transporters 2801

temperature in the reaction mix containing the inhibitor (1 mM); after 10min,the C. sativus stigma extract was added and the uptake experiment wasperformedas described above. For experiments on ice, the tubescontainingthe reactionmixandC.sativusstigmaextractwereplacedon ice immediatelyafter the addition of yeast microsomes. All experiments were repeated atleast three times with independent vesicle preparations, and each experi-ment was performed with three technical replicates (three filters, eachspotted with 100 mL of reaction mix).

For ABA-GE transport studies, in vitro transport assayswere performedwith the analytical standard of ABA-GE (catalog no. 013 2781, ChemIm),using concentrations ranging from 0.075 to 7.5 mM.

The transport of each metabolite presented was evaluated byLC-PDA-HRMS.

LC-PDA-HRMS Analyses

C. sativus stigma polar extracts, standards, eluted fractions from in vitrotransport studies, and semipolar extracts from N. benthamiana agro-infiltrated leaves were analyzed with a Q-Exactive quadrupole Orbitrapmass spectrometry system (Thermo Fisher Scientific), coupled to an LCsystem equipped with a photodiode array detector (Dionex), as describedpreviously by Demurtas et al., (2018). HPLC separation was performed byinjecting 2 to 5mL of samples on aC18 Luna reverse-phase column (10032.1mm,2.5mm;Phenomenex). Themobilephasesusedwerewater10.1%(v/v) formic acid (A) and acetonitrile 1 0.1% (v/v) formic acid (B) at a totalflow rate of 250 mL/min. The separation was developed using 5% B for0.5 min, followed by a 24-min linear gradient to 75%B. The ionization wasperformed in a heated electrospray ionization sourcewith nitrogen used assheath and auxiliary gas, set to 45 and 30 units, respectively. The vaporizertemperaturewas270°C, thecapillary temperaturewas30°C, thedischargecurrent was set to 5mA, andS-lens radio frequency level was set at 50. Theacquisitionwasperformed in themass range110/1600m/zboth inpositiveand innegative ionmodewith the followingparameters: resolution, 70,000;microscan, 1; automatic gain control target, 1e6; and maximum injectiontime, 50 msec. Ultraviolet-visibile detection was continuous from 220 to700 nm. All solvents used were LC-MS grade (Merck Millipore).

Metabolite identificationwas achieved on the basis of accuratemassesandby comparisonwith authentic standards orwith literature data. The ionpeak areas were normalized to the ion peak area of the internal standard(formononetin; fold internal standard). To calculate the transport of eachmetabolite, fold internal standard values of the reactions performed in theabsence of ATPwere subtracted to those of the reactions performed in thepresence of ATP (net uptake). Data are presented as percentage of netuptake of three biological replicates (avg values6 SDs are shown in figuresand tables).

Accession Numbers

The sequences of genes reported in this article have been deposited in theGenBank database: CsABCC4a (MF966954); CsABCC2 (MF966955);CsMATE4 (MF966956); CsMATE1a (MH475368); CsMATE1b (MH475369).The sequences are also shown in the Supplemental Data Set 2. The se-quences of the transporter contigs shown in Figure 2C have likewise beendeposited with the following accession numbers: CsABCC4a (MN401321);CsABCC2 (MN401322); CsMATE4 (MN401323); CsMATE1a (MN401324);CsMATE1b (MN401325); CsABCC8 (MN380444); CsABCC3a (MN380445);CsABCC3b (MN380446); CsABCC4b (MN380447); CsABCC5 (MN380448);CsABCC15 (MN380449); CsABCC4c (MN380450); CsMATE5a (MN380451);CsMATE2e (MN380452); CsMATE5b (MN380453); CsMATE2b (MN380454);CsMATE2d (MN380455); CsMATE2c (MN380456); CsMATE2a (MN380457);CsMATE2f (MN380458).

Sequence data of ABCC and MATE transporters from Arabidopsisthaliana (At), Zea mays (Zm), Vitis vinifera (Vv), Sorghum bicolor (Sb),

Nicotiana tabacum (Nt),Medicago truncatula (Mt), andCoptis japonica (Cj)shown in Figure 2 can be found under the following accession numbers:AtABCC1 (NP_001031116) ; AtABCC2 (NP_181013) ; AtABCC3 (NP_187915);AtABCC4 (NP_182301); AtABCC5 (NP_171908); AtABCC6 (NP_187916.3);AtABCC7 (NP_187917); AtABCC8 (Q8LGU1); AtABCC9 (Q9M1C7);AtABCC10 (NP_191473); AtABCC11 (NP_174331); AtABCC12 (Q9C8H0);AtABCC13 (NP_001323940); AtABCC14 (NP_191829); ZmABCC3 (AAT37905);ZmABCC4(A7KVC2);VvABCC1(AGC23330);AtDTX35(NP_194294);AtDTX41/TT12 (NP_191462); AtDTX16 (NP_200058); AtDTX17 (NP_177511); AtDX19(NP_566730); AtDTX29 (NP_189291); AtDTX30 (NP_198619); AtDTX33(NP_175184); AtDTX40 (NP_188806); CjMATE1 (BAX73926); MtMATE1(ACX37118); MtMATE2 (ADV04045); NtJAT1 (CAQ51477); NtJAT2(BAP40098); NtMATE1 (BAF47751); NtMATE2 (BAF47752); SbMATE2(XP_021303040); VvAM1 (ACN88706); VvAM3 (ACN91542).

Supplemental Data

Supplemental Figure 1. Conserved domains and transmembranehelices of CsABCC4a and CsABCC2 and of the closely relatedAtABCC1, AtABCC2 and AtABCC4 transporters.

Supplemental Figure 2. Crocin composition of a commercial crocinstandard and of a C. sativus stigma hydroalcoholic extract.

Supplemental Figure 3. Assessment of physiological intactness ofyeast microsomes.

Supplemental Figure 4. Purity of trans and cis crocin 3 purified fromC. sativus stigma extract through preparative HPLC.

Supplemental Figure 5. Isomerization of cis crocin 3 into trans crocin3 at pH 5.2.

Supplemental Figure 6. MATE and ABCC full-length ampliconsobtained from C. sativus cDNA with the oligonucleotides describedin Methods.

Supplemental Table 1. Transcript levels of CsMATE and CsABCCtransporters in C. sativus tissues.

Supplemental Table 2. Co-expression analysis of CsABCC andCsMATE transcripts with total crocins and CsCCD2 transcript.

Supplemental Table 3. Homology of CsABCC4a with ABCC trans-porters with known structures.

Supplemental Table 4. Oligonucleotides used in this study.

Supplemental Table 5. t test result of data presented in Figure 7.

Supplemental Data Set 1. Saffron transcriptome data.

Supplemental Data Set 2. Sequences of C. sativus transporterscharacterized in this article.

Supplemental Data Set 3. Text file of the ABC alignment used for thephylogenetic analysis in Figure 2C.

Supplemental Data Set 4. Text file of the MATE alignment used forthe phylogenetic analysis in Figure 2C.

ACKNOWLEDGMENTS

We thank Barbara Bassin for preparation of yeast microsomes; ElenaRomano (Centre of Advanced Microscopy) and Patrizia Albertano (Uni-versity of Rome Tor Vergata) for help with confocal experiments; GaetanoPerrotta, Paolo Facella, Fabrizio Carbone (ENEA Trisaia Research Center)and IGA Technology Services for RNA-sequencing data; and AndreaAliboni for help with preparative HPLC. This work was supported by theEuropean Union (From discovery to products: A next generation pipeline

2802 The Plant Cell

for the sustainable generation of high-value plant products, grant no.613153 and Developing Multipurpose Nicotiana Crops for MolecularFarming using New Plant Breeding Techniques, grant no. 760331 toG.G.) and by the EuropeanCooperation in Science and Technology Action(EUROCAROTEN CA15136).

AUTHOR CONTRIBUTIONS

O.C.D., R.d.B.F., A.C., G.D., P.F., S.F., M.P., G.A., L.B., M.F., and L.F.produced data. O.C.D., R.d.B.F., M.P., L.B., L.F., M.F., E.M., A.C., G.C.,S.A., and G.G. analyzed data. E.M. and G.G. developed the experimentalstrategy.G.G.coordinated thestudy.O.C.D.,R.d.B.F.,A.C., E.M., andG.G.wrote the article. All authors reviewed the results and approved the finalversion of the article.

ReceivedMarch 22, 2019; revised June25, 2019; acceptedSeptember 12,2019; published September 23, 2019.

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2804 The Plant Cell

DOI 10.1105/tpc.19.00193; originally published online September 23, 2019; 2019;31;2789-2804Plant Cell

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

Olivia Costantina Demurtas, Rita de Brito Francisco, Gianfranco Diretto, Paola Ferrante, SarahABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas

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