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GATA and Phytochrome Interacting Factor TranscriptionFactors Regulate Light-Induced Vindoline Biosynthesis inCatharanthus roseus1[OPEN]

Yongliang Liu,a,b,2 Barunava Patra,b,2 Sitakanta Pattanaik,b,3 Ying Wang,a and Ling Yuana,b,3,4

aKey Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South ChinaBotanical Garden, Chinese Academy of Sciences, Guangzhou, China 510650bDepartment of Plant and Soil Sciences and Kentucky Tobacco Research and Development Center, Universityof Kentucky, Lexington, Kentucky 40546

ORCID IDs: 0000-0001-7702-8341 (S.P.); 0000-0003-4767-5761 (L.Y.).

Catharanthus roseus is the exclusive source of an array of terpenoid indole alkaloids including the anticancer drugs vincristine andvinblastine, derived from the coupling of catharanthine and vindoline. Leaf-synthesized vindoline is regulated by light. A seven-step enzymatic process is involved in the sequential conversion of tabersonine to vindoline; however, the regulatory mechanismcontrolling the expression of genes encoding these enzymes has not been elucidated. Here, we identified CrGATA1, an Leu-Leu-Met domain GATA transcription factor that regulates light-induced vindoline biosynthesis in C. roseus seedlings. Expression ofCrGATA1 and the vindoline pathway genes T16H2, T3O, T3R, D4H, and DAT was significantly induced by light. In addition,CrGATA1 activated the promoters of five light-responsive vindoline pathway genes in plant cells. Two GATC motifs in the D4Hpromoter were critical for CrGATA1-mediated transactivation. Transient overexpression of CrGATA1 in C. roseus seedlingsresulted in up-regulation of vindoline pathway genes and increased vindoline accumulation. Conversely, virus-induced genesilencing of CrGATA1 in young C. roseus leaves significantly repressed key vindoline pathway genes and reduced vindolineaccumulation. Furthermore, we showed that a C. roseus Phytochrome Interacting Factor, CrPIF1, is a repressor of CrGATA1 andvindoline biosynthesis. Transient overexpression or virus-induced gene silencing of CrPIF1 in C. roseus seedlings alteredCrGATA1 and vindoline pathway gene expression in the dark. CrPIF1 repressed CrGATA1 and DAT promoter activity bybinding to G/E-box/PBE elements. Our findings reveal a regulatory module involving Phytochrome Interacting Factor-GATA that governs light-mediated biosynthesis of specialized metabolites.

Catharanthus roseus (Madagascar periwinkle) isthe unique source of more than 100 terpenoid indolealkaloids (TIAs), including the two anticancer drugs,vincristine and vinblastine. Biosynthesis of TIAs(Supplemental Fig. S1) starts with the production ofstrictosidine, which is formed by the condensation ofthe terpenoid precursor secologanin and the indoleprecursor tryptamine (Courdavault et al., 2014; Panet al., 2016; Thamm et al., 2016). Strictosidine serves

as a universal precursor for various TIAs, includingajmalicine, serpentine, catharanthine, and taber-sonine. Tabersonine is sequentially converted tovindoline by a seven-step enzymatic process, andgenes encoding the seven enzymes have been char-acterized (Vazquez-Flota et al., 1997; St-Pierre et al.,1998; Levac et al., 2008; Liscombe et al., 2010; Besseauet al., 2013; Qu et al., 2015). Vincristine and vinblas-tine are derived from the coupling of vindoline andcatharanthine. Expression of TIA biosynthetic path-way genes in four different cell types is elicited by en-vironmental cues and phytohormones (Courdavaultet al., 2014).

In C. roseus, a number of transcription factors (TFs)regulate TIA biosynthesis. These TFs include theApetala2/Ethylene Response Factors (AP2/ERFs)ORCA2/ORCA3/ORCA4/ORCA5 and CR1 (Menkeet al., 1999; van der Fits and Memelink, 2000; Panet al., 2012; Li et al., 2013; Liu et al., 2017; Paul et al.,2017), basic helix-loop-helix (bHLH) TFs CrMYC2,BIS1/BIS2, and RMT1 (Zhang et al., 2011; VanMoerkercke et al., 2015, 2016; Patra et al., 2018),Cys2/His2-type zinc finger proteins ZCT1/ZCT2/ZCT3 (Pauw et al., 2004), MYB-like factor CrBPF1 (vander Fits et al., 2000; Li et al., 2015), G-box-binding

1This work was supported in part by the Harold R. Burton En-dowed Professorship to L.Y. and by the National Science Foundation(cooperative agreement no. 1355438 to L.Y.).

2These authors contributed equally to the article.3Senior authors.4Author for contact: [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ling Yuan ([email protected]).

L.Y., B.P., S.P., and Y.W. designed the research; Y.L., B.P., and S.P.performed experiments; Y.L. and B.P. analyzed data; Y.L., B.P., S.P.,Y.W., and L.Y. wrote the article.

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factors CrGBF1 and CrGBF12 (Sibéril et al., 2001; Suiet al., 2018), WRKY TF CrWRKY1 (Suttipanta et al.,2011), and the jasmonate ZIM domain (JAZ) proteins(Patra et al., 2018). ORCA3, ORCA4, and ORCA5,which form a physical cluster, regulate a number ofgenes in the TIA pathway through overlapping yetdistinct mechanisms (van der Fits and Memelink,2000; Paul et al., 2017). CrMYC2 activates ORCA3 bybinding to a qualitative sequence in the promoter(Zhang et al., 2011), whereas it indirectly activatesORCA4 and ORCA5 (Paul et al., 2017). In addition,CrMYC2 interacts with CrGBF1 and CrGBF2 to mod-ulate TIA biosynthesis (Sui et al., 2018). The CrMYC2-ORCA cascade has limited effects on the expressionof genes in the iridoid branch of the TIA pathway.However, a recent study has demonstrated thattransient overexpression of a derepressed CrMYC2(CrMYC2D126N) in C. roseus flower petals signifi-cantly activates expression of iridoid pathwaygenes (Schweizer et al., 2018). BIS1 and BIS2 aremajor regulators of the iridoid pathway (VanMoerkercke et al., 2015, 2016). Transient over-expression of CrMYC2D126N, BIS1, and ORCA3 sig-nificantly induces the indole and iridoid pathwaygenes, resulting in increased accumulation of stric-tosidine, 16-hydroxytabersonine, and horhammer-icine (Schweizer et al., 2018). The bHLH TF RMT1and JAZ proteins mediate cross talk between iridoidand terpenoid pathways to balance TIA accumula-tion (Patra et al., 2018). However, our knowledge islimited on the regulation of genes involved in thesequential conversion of tabersonine to vindoline inC. roseus leaves. Combinatorial overexpression ofwild-type or derepressed CrMYC2 (CrMYC2D126N),along with BIS1 and/or ORCA3, does not induce theexpression of vindoline pathway genes (Schweizeret al., 2018), suggesting that other TFs are likely in-volved in regulation of the vindoline pathway.In C. roseus, while tabersonine is produced in

leaves and roots, vindoline production is leaf specificand light dependent (De Luca et al., 1986). In addi-tion, previous studies suggest that the conversion oftabersonine to vindoline is phytochrome dependent(Aerts and De Luca, 1992). Light regulates myriadphysiological and developmental processes in plants,including photoperiodism, photomorphogenesis, seedgermination, and shade avoidance (Jiao et al., 2007).Light also plays crucial roles in the biosynthesis ofspecialized metabolites. The TFs in the bHLH, basicLeu zipper, and GATA families are known to controllight-responsive gene expression in plants (Richteret al., 2010; Toledo-Ortiz et al., 2014; Klermundet al., 2016). GATA TFs, widely distributed in eu-karyotes, are characterized by the class IV zinc fingermotif (CX2CX17-20CX2C; Lowry and Atchley, 2000).In Arabidopsis (Arabidopsis thaliana) and rice (Oryzasativa), GATA TFs are divided into four conservedand distinct classes, A through D (Reyes et al., 2004).Class B GATAs (B-GATAs) are further subdividedinto two families based on the presence of a conserved

LLM (Leu-Leu-Met) domain or HAN (HANABATARANU) domain (Behringer and Schwechheimer,2015). In Arabidopsis, the expression of two homol-ogous LLM B-GATAs, GNC (GATA-nitrate-inducible-carbon metabolism-involved) and GNL (GNC-like/cytokinin-responsive GATA factor1), is induced bylight known to regulate chlorophyll biosynthesis,chloroplast development, nitrate metabolism, seedgermination, flowering time, hypocotyl elongation,and stomata development (Richter et al., 2010, 2013a,2013b; Hudson et al., 2013; Klermund et al., 2016;Ranftl et al., 2016; Xu et al., 2017; Bastakis et al., 2018).In addition to GATA TFs, a small group of bHLH TFs,the Phytochrome Interacting Factors (PIFs), playcrucial roles in light-responsive gene expression anddownstream biological processes through interactingwith phytochromes in plants (Leivar and Monte,2014). Phytochromes are the receptor of red andfar-red light signals and exist in either the inactive Prform or the active Pfr form (Pham et al., 2018). PIFsphysically interact with the active Pfr, leading todegradation by the 26S/ubiquitin proteasome sys-tem. The PIF degradation triggers massive tran-scriptional reprogramming that regulates variousbiological processes (Leivar and Monte, 2014; Paiket al., 2017). Light-mediated anthocyanin accumula-tion in Arabidopsis is regulated by PIFs (Shin et al.,2007; Liu et al., 2015). In Arabidopsis, GNC and GNLare direct targets of PIFs in the regulation of seedgermination, flowering time, hypocotyl elongation,and stomata development (Richter et al., 2010;Klermund et al., 2016; Ranftl et al., 2016). In fungi, theGATA-type TF Csm1 has been reported to regulatebiosynthesis of the red pigments bikaverin andfusarubins in Fusarium fujikuroi (Niehaus et al., 2017);it is unclear, however, whether GATA TFs are in-volved in the regulation of specialized metabolitebiosynthesis in plants.The light- and phytochrome-dependent nature of

vindoline biosynthesis led us to hypothesize that alight-associated transcriptional cascade is involvedin the conversion of tabersonine to vindoline inC. roseus seedlings. In this study, we characterizeda light-induced C. roseus LLM domain B-GATA,termed CrGATA1, which predominantly expressesin the leaf. CrGATA1 activates the promoters ofkey vindoline biosynthetic genes in plant cells. Inaddition, transient overexpression or virus-inducedgene silencing (VIGS) of CrGATA1 in C. roseus seed-lings significantly altered vindoline pathway geneexpression and vindoline accumulation. We alsodemonstrated that a C. roseus PIF (CrPIF1) acts as anupstream negative regulator of CrGATA1, resultingin repression of vindoline biosynthetic genes in thedark. Derepression of CrGATA1, presumably throughdegradation of CrPIF1 under light, leads to increasedaccumulation of vindoline. Our findings reveal a pre-viously uncharacterized regulatory module in C. roseusinvolving PIF-GATA that governs light-induced vindolinebiosynthesis in seedlings.

Plant Physiol. Vol. 180, 2019 1337

CrPIF1-CrGATA1 Regulate Vindoline Biosynthesis

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RESULTS

The Vindoline Pathway Is Induced by Light in C.roseus Seedlings

Conversion of tabersonine to vindoline is respon-sive to developmental as well as environmental cues,such as light, in C. roseus seedlings (De Luca et al.,1986). Of the seven genes involved in the conversion,expression of deacetylvindoline-4-O-acetyltransferase(DAT) and desacetoxyvindoline-4-hydroxylase (D4H)is induced by light (Vazquez-Flota et al., 1997; St-Pierre et al., 1998; Vazquez-Flota and De Luca,1998). To determine the light-responsive expressionof tabersonine-vindoline conversion genes and alka-loid accumulation, 7-d-old etiolated C. roseus seed-lings were exposed to continuous white light for 1, 4,10, 24, 48, and 96 h, and the aerial parts of the seed-lings were collected for gene expression and alkaloidanalysis. Expression of tabersonine-16-hydroxylase2(T16H2), tabersonine-3-oxygenase (T3O), D4H, andDAT showed gradual increase upon exposure to lightthat peaked at 24 h. Expression of tabersonine-3-reductase (T3R) was highest at 48 h of light treat-ment and declined thereafter (Fig. 1A). However,expression of 16-hydroxytabersonine-O-methyltransferase

(16OMT) and 3-hydroxy-16-methoxy-2,3-dihydrotabersonineN-methyltransferase (NMT) did not significantly change inresponse to light (Fig. 1A), which corroborates earlierstudies showing that the activities of 16OMT and NMTwere less sensitive to light (St-Pierre and De Luca, 1995;Levac et al., 2008). We also measured the expressionof two upstream TIA pathway genes (SupplementalFig. S1), tryptophan decarboxylase (TDC) and strictosi-dine synthase (STR), in dark- and light-treated seed-lings. In contrast to the rapid light induction for thevindoline pathway genes (within 4 h), expression ofTDC and STR did not significantly change by lighttreatment up to 24 h (Supplemental Fig. S2A). Inaddition, we compared catharanthine, tabersonine,and vindoline in etiolated and light-treated C. roseusseedlings. We did not observe significant differencesin catharanthine accumulation between dark- andlight-treated seedlings (Supplemental Fig. S2B). How-ever, we detected a significant increase of vindolineand a decrease of tabersonine in seedlings exposedto light for 24 and 96 h (Fig. 1B). We also comparedlevels of catharanthine, tabersonine, and vindolinein normal, 11-d-old C. roseus seedlings grown undercontinuous light with those of dark-grown seed-lings exposed to light. We found that vindoline levels

Figure 1. Light-induced expression of vindoline pathway genes and vindoline production in C. roseus seedlings. A, Relativeexpression levels of seven vindoline pathway genes in aerial parts of C. roseus seedlings. Seven-day-old, dark-grown, etiolatedseedlings were exposed to white light for different lengths of time (1, 4, 10, 24, 48, and 96 h). Transcript levels of T16H2, 16OMT,T3O, T3R,NMT,D4H, andDATweremeasured by reverse transcription quantitative PCR (RT-qPCR). The RPS9 gene was used asan internal reference gene. B, Accumulation of tabersonine and vindoline in aerial parts of C. roseus seedlings. Here, 0 h refers to7-d-old etiolated seedlings, which were growing in dark (control) and then exposed to white light for 24 and 96 h. Alkaloids wereextracted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the concentrations of the alka-loidswere estimated based on peak areas comparedwith standards. Values represent means6 SD from three biological replicates.For each replicate, eight to 10 seedlings were combined. Statistical significance was determined by Student’s t test (*, P , 0.05and **, P , 0.01). DW, Dry weight.

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were significantly higher in light-grown seedlings(Supplemental Fig. S2C) compared with the etiolatedones exposed to light for 24 and 96 h (Fig. 1B). Takentogether, the results suggest that the expression offive of the seven vindoline pathway genes and vin-doline accumulation in C. roseus seedlings are lightinducible.

Putative Light-Responsive cis-Elements Are Present in thePromoters of Light-Inducible Vindoline Pathway Genes

The light-inducible expression of the five vindolinepathway genes prompted us to analyze their pro-moters for the presence of putative light-responsivecis-elements. We therefore retrieved the promoter se-quences (2 kb upstream of the first ATG of the codingframe) of the five light-responsive genes, T16H2, T3O,T3R,D4H, andDAT, from theMedicinal Plant GenomicsResource (http://medicinalplantgenomics.msu.edu/)and scanned the promoter sequences using PlantCARE(Lescot et al., 2002). We identified multiple knownlight-responsive cis-elements, of which the GATA, Box4, and Box I motifs are present in the promoters of all fivelight-responsive vindoline pathway genes (SupplementalTable S1). GATAandGATCcis-elementswere also found

in the promoters of two upstream vindoline pathwaygenes, TDC and STR, although their expression wasnot significantly induced by light. The GATA motif,consisting of WGATAR sequence (in which W de-notes A or T and R denotes A or G), and the GATCmotif are binding sites for light-inducible GATA TFs(Lowry andAtchley, 2000;Newton et al., 2001; Sugimotoet al., 2003; Manfield et al., 2007). Although the Box 4and Box I elements have been identified to be presentin many promoters of light-inducible genes, the TFstargeting these elements are unclear. Therefore, wefocused on GATA family TFs of C. roseus for theirpotential roles in regulating light-inducible vindolinebiosynthesis.

CrGATA1 Is Light Inducible and Coexpressed withVindoline Pathway Genes

We identified 24 putative GATA TFs in the C. roseusgenome. We next used C. roseus transcriptomic re-sources (Góngora-Castillo et al., 2012; accession no.SRA030483) to perform coexpression analysis of GATATFs and genes involved in vindoline biosynthesis. Hi-erarchical cluster analysis of the putative GATA TFsand the five light-inducible vindoline pathway genes

Figure 2. Identification of CrGATA1. A, Expres-sion of CRO_134526 (CrGATA1), CRO_117711,and five light-inducible vindoline pathway genesin aerial parts and roots of 7-d-old light-grownC. roseus seedlings. B and C, Expression ofCRO_134526 (CrGATA1) is quickly induced (B),while CRO_117711 is repressed by light in aerialparts ofC. roseus seedlings (C). For light treatment,7-d-old etiolated seedlings were exposed to whitelight for different lengths if time. Transcript levelsof vindoline pathway genes, CrGATA1, andCRO_117711, were measured by RT-qPCR withRPS9 as an internal reference gene. D,D4H,DAT,and CrGATA1 promoters are light inducible in N.benthamiana leaf. CaMV35S promoter was usedas a negative control. D4H, DAT, and CrGATA1promoters were cloned in pKYLX71 vector todrive expression of the GUS gene. The promoter-GUS plasmids were infiltrated in N. benthamianaleaves. Plants were kept in dark or light for 3 d.GUS activities were normalized by luciferase ac-tivities. Values represent means 6 SD from threebiological replicates. Statistical significance wasdetermined by Student’s t test (**, P , 0.01). E,eGFP is accumulated throughout the cell (left),whereas eGFP-CrGATA1 is localized to the nu-cleus (right). The experiment was repeated twotimes, and a representative result is shown here.





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revealed that two GATA TFs (CRO_T134526 andCRO_T117711) are tightly coexpressed with the vin-doline pathway genes (Supplemental Fig. S3). RT-qPCRwas performed tomeasure the expression of twoGATATFs and vindoline pathway genes in aerial parts androots of C. roseus seedlings. As shown in Figure 2A, thevindoline pathway genes, as well as CRO_T134526 andCRO_T117711, are preferentially expressed in aerialparts of the seedlings. Expression of CRO_T134526 andCRO_T117711was also measured in C. roseus seedlingsexposed to light for 1, 4, 10, 24, 48, and 96 h. Under lighttreatment, only CRO_T134526 expression was induced(Fig. 2B) and peaked at 4 h, while CRO_T117711 wasrepressed (Fig. 2C). Next, we cloned the CRO_T134526promoter to drive the expression of the GUS reportergene. Two light-dependent (D4H andDAT) and a light-independent (Cauliflower mosaic virus [CaMV] 35S)promoters driving the expression of the GUS reporterwere used as controls. The promoter-GUS reporterplasmids were used for agroinfiltration of Nicotianabenthamiana leaves. GUS activities were measuredin discs of infiltrated leaves collected from dark- orlight-incubated plants. As shown in Figure 2D, the GUSactivity in light-treated leaves, infiltrated with CrGATA1-GUS, D4H-GUS, or DAT-GUS, was significantly (3- to4fold) higher than that of the dark-incubated plants;however, we did not observe a significant change of GUSactivity in CaMV35S-GUS-infiltrated leaves of light- ordark-incubated plants, suggesting that the CRO_T134526promoter is light inducible. We selected CRO_T134526,hereafter designated as CrGATA1, for further characteri-zation and to elucidate its regulatory role in light-regulated vindoline biosynthesis.

Phylogenetic analysis showed that CrGATA1 is inthe same clade with the light-inducible ArabidopsisGATA TFs GNC and GNL (Supplemental Fig. S4;Manfield et al., 2007; Behringer and Schwechheimer,2015). Like GNC and GNL, CrGATA1 belongs to theLLM domain-containing B-GATAs. Amino acid se-quence alignment revealed that CrGATA1 shares 39%to 41% sequence identity with the Arabidopsis GATATFs GNC and GNL (Supplemental Fig. S5). To deter-mine the subcellular localization, CrGATA1 was fusedin frame to eGFP (enhanced GFP), and the fusion genewas expressed in tobacco (Nicotiana tabacum) proto-plasts.While the control eGFP accumulated throughoutthe cell, CrGATA1-eGFP fusion proteinwas localized tothe nucleus (Fig. 2E).

Transient Overexpression of CrGATA1 EnhancesVindoline Production in C. roseus Seedlings

To determine the role of CrGATA1 in vindoline bio-synthesis, we transiently overexpressed CrGATA1 in C.roseus seedlings using the fast agro-mediated seed-ling transformation (FAST) method (Weaver et al.,2014). Expression of CrGATA1 and the five light-responsive vindoline pathway genes was measuredin the aerial parts of young seedlings infiltrated either

with the empty vector control pCAMBIA1300 (EV) orthe overexpression vector pCAMBIA1300-CrGATA1(CrGATA1-OX). The RT-qPCR results revealed that,compared with EV, CrGATA1 overexpression resul-ted in 2.5- to 5fold increase in the expression ofT16H2, D4H, and DAT; the expression of T3O andT3R remained unchanged (Fig. 3A). In addition,vindoline accumulation was significantly increased,while tabersonine was decreased, in CrGATA1-OXseedlings relative to EV seedlings (Fig. 3B). Thesefindings suggest that CrGATA1 is a positive regula-tor of vindoline biosynthesis in C. roseus.

VIGS of CrGATA1 Reduces Vindoline Biosynthesis in C.roseus Leaves

VIGS was used to repress CrGATA1 expression inC. roseus leaves, as previously described (Liscombeand O’Connor, 2011). Expression of CrGATA1 andthe five light-responsive vindoline pathway genes was

Figure 3. CrGATA1 positively regulates vindoline biosynthesis in C.roseus. A and B, Transient overexpression of CrGATA1 in C. roseusseedlings elevates expression of vindoline pathway genes (A) and vin-doline production (B). A, Relative expression ofCrGATA1 and five light-inducible vindoline pathway genes in EV controls and CrGATA1-OXseedlings measured by RT-qPCR. B, Measurement of tabersonine andvindoline in EV controls and CrGATA1-OX lines. C, Expression ofvindoline pathway genes in CrGATA1-VIGS leaves. Relative expressionof CrGATA1 and five light-inducible vindoline pathway genes in EVcontrols and CrGATA1-VIGS leaves was measured by RT-qPCR. D,Measurement of tabersonine and vindoline in EV controls andCrGATA1-VIGS lines. Alkaloids were extracted and analyzed by LC-MS/MS, and the concentrations of the alkaloids were estimated basedon peak areas compared with standards. For RT-qPCR, the RPS9 genewas used as an internal reference gene. In all cases, values representmeans6 SD from three biological replicates. Statistical significance wascalculated using Student’s t test (*, P, 0.05 and **, P, 0.01). DW, Dryweight.


Liu et al.






measured in young leaves of C. roseus plants inoculatedeither with the pTRV2 EV control or the VIGS vectorpTRV2-CrGATA1 (CrGATA1-VIGS). We observed thatCrGATA1 expression was repressed by ;63% in VIGSleaves compared with the EV control (Fig. 3C). In ad-dition, transcript levels of T3O, T3R, and DAT werereduced by 43% to 58% in VIGS leaves compared withEV (Fig. 3C). Expression of T16H2 and D4H was notsignificantly affected in CrGATA1 VIGS lines. More-over, the amount of tabersonine was significantly ele-vated, while vindoline was decreased in leaves of theCrGATA1-VIGS plants compared with the EV plants(Fig. 3D). These results further support that CrGATA1is a positive regulator of vindoline biosynthesis in C.roseus. To determine whether CrGATA1 affects ex-pression of the upstream vindoline pathway genes, wemeasured the expression of TDC and STR in CrGATA1overexpression and VIGS lines. We did not detect sig-nificant changes in transcript levels compared with EV(Supplemental Fig. S6).

CrGATA1 Transactivates the Promoters of VindolinePathway Genes

To determine whether CrGATA1 can directly acti-vate the promoters of vindoline pathway genes, weperformed N. benthamiana leaf-based transactivationassays. The promoters of five light-inducible vindo-line pathway genes were cloned in the binary vectorpKYLX71-GUS to drive the expression of the GUS

gene. The resulting plasmids were mobilized toAgrobacterium tumefaciens and individually infiltratedinto N. benthamiana leaves together with EV (pCAM-BIA1300) or pCAMBIA1300-CrGATA1. GUS activityassay showed that transactivation of the T16H2, T3R,T3O, D4H, and DAT promoters by CrGATA1 was 1.8-to 2.9-fold compared with the EV control (Fig. 4A),suggesting that the vindoline pathway genes are reg-ulated by CrGATA1 in C. roseus.

The GATCMotif Is Crucial for CrGATA1 Activation of theD4H Promoter

Previous studies have demonstrated that GATA TFsbind to both GATA and GATC motifs (Newton et al.,2001; Sugimoto et al., 2003; Xu et al., 2017). The D4Hpromoter, highly activated by CrGATA1 (Fig. 4A), wasthus chosen for identification of potential binding sitesof CrGATA1. In silico analysis revealed that the D4Hpromoter contains two GATA and two GATC motifs(Fig. 4, B and C). First, we mutated the core sequence ofthe twoGATAmotifs (2431 to2426 and2139 to2134,relative to the first ATG in the coding frame) to GGCAby PCR-based site-directed mutagenesis (Pattanaiket al., 2010). Mutation of single or both GATA motifshad no effect on CrGATA1-mediated activation of theD4H promoter (Fig. 4B). However, transactivation ofthe D4H promoter by CrGATA1 was abolished whenboth GATC motifs (2617 to 2614 and 2541 to 2538,relative to ATG) were mutated to GAAA (Fig. 4C),

Figure 4. CrGATA1 transactivates promoters offive light-inducible vindoline pathway genesthrough GATC motifs. A, Transactivation ofT16H2, T3O, T3R, D4H, and DAT promoters(-pro), fused to the GUS reporter, by CrGATA1.Effector (CrGATA1) and reporter (promoter-GUS)constructs were coinfiltrated intoN. benthamianaleaves. A plasmid containing a luciferase reporter,driven by CaMV35S promoter and rbcS termina-tor, was used as a normalization control. Lucifer-ase and GUS activities were measured 2 d afterinfiltration. GUS activity was normalized againstluciferase activity. Control represents the reporterwith EV. B, Schematic diagram showing the GATAmotifs in theD4H promoter. Point mutations in theGATAmotifs are indicated by red letters. Mutationin the GATA motif has no effects on the activationof the D4H promoter by CrGATA1. C, Schematicdiagram showing the GATC motifs in the D4Hpromoter. Mutations in the GATC motifs are indi-cated by red letters. Mutation in the GATC motifaffects the activation of the D4H promoter byCrGATA1. Data presented are means6 SD of threebiological replicates. Statistical significance wascalculated using Student’s t test (*, P , 0.05 and**, P , 0.01). WT, Wild type.






suggesting that GATC motifs are crucial for activationof the D4H promoter by CrGATA1.

Phytochrome Is Likely Involved in Light-ResponsiveExpression of Vindoline Pathway Genes

Plants sense red and far-red light signals through thephotoreceptor phytochrome (Franklin andQuail, 2010).Previous studies have shown that the red light-inducedenzymatic activities of D4H and DAT in C. roseusseedlings are reversed by far-red light (Aerts and DeLuca, 1992; Vazquez-Flota and De Luca, 1998), sug-gesting a phytochrome-dependent regulation of vin-doline biosynthesis. To test this hypothesis, theexpression of CrGATA1 and five light-inducible vin-doline pathway genes was monitored in C. roseusseedlings exposed to red and then far-red light for dif-ferent durations. We used C. roseus ChlH, PORC, andRCA as experimental controls, as their orthologs inArabidopsis and rice respond to red and far-red light ina phytochrome-dependent manner (Liu et al., 1996;Moon et al., 2008; Inagaki et al., 2015; Fig. 5). Gene ex-pression analysis showed that red light significantlyinduced the expression of CrGATA1 and vindolinepathway genes, except for D4H (Fig. 5). The red light-mediated induction of vindoline pathway genes wasreversed following a 2-h exposure to far-red light(Fig. 5), suggesting the involvement of phytochrome-dependent regulatory factors in the vindoline pathway.

CrPIF1 Represses Vindoline Pathway Gene Expression andAlkaloid Accumulation in C. roseus Seedlings

PIFs act as negative regulators in the light signalingpathway. PIF accumulates in dark and degrades upon

interaction with phytochromes in light (Pham et al.,2018). We thus asked whether C. roseus PIFs are in-volved in the light regulation of vindoline biosynthesis.We identified three putative PIFs in the C. roseus ge-nome, designated here as CrPIF1, CrPIF3, and CrPIF4/5. CrPIFs share 35% to 41% amino acid sequence iden-tity with Arabidopsis PIF1, PIF3, and PIF4, while theyshare 52% to 60% identity with tomato (Solanum lyco-persicum) PIFs (Supplemental Fig. S7). In addition, C.roseus PIFs are phylogenetically closer to tomato PIFsthan those of Arabidopsis (Supplemental Fig. S8).Amino acid sequence alignment of PIFs (SupplementalFig. S7) showed that all three CrPIFs contain the con-served bipartite nucleus localization signal and phyto-chrome B-binding motif, indicating their conservedregulatory roles in the phytochrome-PIF pathway inplants. Similar to the tomato PIFs (Rosado et al., 2016),CrPIFs appeared to be also regulated at the transcrip-tional level, as their expression was affected by light(Supplemental Fig. S9). To determine the regulatoryroles of CrPIFs in vindoline biosynthesis, CrPIFs wereindividually overexpressed in C. roseus seedlings usingthe FAST method. CrPIF1 overexpression resulted insignificant down-regulation of CrGATA1 and the light-responsive vindoline pathway genes in dark (Fig. 6A).However, CrPIF3 or CrPIF4/5 had no effects on theexpression of CrGATA1 and the vindoline pathwaygenes (Supplemental Fig. S10). In addition, over-expression of CrPIF1 resulted in increased tabersonineand decreased vindoline (Fig. 6B), indicating thatCrPIF1 acts as a negative regulator of vindoline bio-synthesis in C. roseus. To determine the subcellularlocalization, CrPIF1 was fused in-frame to eGFP, andthe resulting CrPIF1-eGPF was expressed in tobaccoprotoplasts. While the control eGFP accumulatedthroughout the cell, the CrPIF1-eGFP fusion proteinwas localized to the nucleus (Fig. 6E).

Figure 5. Expression of CrGATA1 and five light-inducible vindoline pathway genes in C. roseusseedlings exposed to red (R) and far-red (FR) light.Seven-day-old etiolated C. roseus seedlings weretreated with red light for 4 h (measured at 2 and 4h), followed by far-red light for 2 h. Gene ex-pression in aerial parts of the seedlings was mea-sured by RT-qPCR. CrChlH, CrPORC, and CrRCAwere used as positive controls. The RPS9 genewasused as an internal reference gene. Data representmeans 6 SD of three biological samples. Differentletters denote statistical differences as assessed byone-way ANOVA and Tukey’s honestly significantdifference test (P , 0.05).


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VIGS of CrPIF1 Increases Vindoline Biosynthesis in C.roseus Leaves

VIGS was used to repress CrPIF1 expression in youngC. roseus leaves. Expression of CrPIF1, CrGATA1, and the

five light-responsive vindoline pathway genes (T16H2,T3O, T3R,D4H, andDAT) was measured in leaves of thedark-incubated VIGS plants. Plants inoculated with thepTRV2 EV served as a control. CrPIF1 expression was

Figure 6. CrPIF1 negatively regulates CrGATA1 and vindoline pathway genes through the G-box or PBE-box. A, Relative ex-pression of CrGATA1 and five light-inducible vindoline pathway genes in EV controls and CrPIF1-OX seedlings was measured by RT-qPCR. Seven-day-old seedlings were infiltrated and kept in dark for 3 d before sample collection. B, Measurement of tabersonine andvindoline in EV controls and CrPIF1-OX lines. C, Expression of CrGATA1 and vindoline pathway genes in CrPIF1-VIGS leaves. Relativeexpression of CrPIF1, CrGATA1, and five vindoline pathway genes in EV and CrGATA1-VIGS leaves was measured by RT-qPCR. D,Measurement of tabersonine and vindoline in EV controls and CrPIF1-VIGS lines. For RT-qPCR, the RPS9 gene was used as an internalreference gene. For tabersonine and vindoline contents, alkaloidswere extracted and analyzed by LC-MS/MS, and the concentrations ofthe alkaloidswere estimated based on peak areas comparedwith standards. E, Subcellular localization of CrPIF1 in tobacco protoplasts.eGFP is accumulated throughout the cell (left), whereas eGFP-CrPIF1 is localized to the nucleus (right). F, Transactivation of CrGATA1,T16H2, T3O, T3R, D4H, and DAT promoters (-pro), fused to the GUS reporter, by CrPIF1. Transactivation assays were carried out bycoinfiltrationof theCrGATA1expressionvectorwith apro-GUSconstruct intoN.benthamiana leaves. Theplantswere incubated indarkafter infiltration. A plasmid containing luciferase reporter, driven by CaMV35S promoter and rbcS terminator, was used as a normali-zation control. Luciferase andGUSactivitiesweremeasured2dafter infiltration.GUSactivitywas normalized against luciferase activity.Control represents the reporter with EV.G,Mutation in theG-box sequence (top) affects the transactivation of theCrGATA1 promoter byCrPIF1. H, Mutation in the PBE-box sequence (top) affects the transactivation of the DAT promoter by CrPIF1. All data presented aremeans6 SD of three biological replicates. Statistical significancewas calculated using Student’s t test (*, P, 0.05 and **, P, 0.01). DW,Dry weight; WT, wild type.





repressed by approximately 70% in VIGS leavescompared with the EV control (Fig. 6C). CrPIF1 si-lencing resulted in up-regulation of CrGATA1 and thefive vindoline pathway genes by two- to eightfold inVIGS lines compared with EV (Fig. 6C). Moreover,the amount of vindoline was elevated in leaves of theCrPIF1-VIGS plants compared with the EV plants(Fig. 6D). These results further suggest that CrPIF1 isa negative regulator of vindoline biosynthesis in C.roseus seedlings.

CrPIF1 Represses CrGATA1 and DAT through Binding tothe G/E/PBE-Box

To determine whether CrGATA1 and the vindolinepathway genes are regulated by CrPIF1, we per-formed transactivation assays in N. benthamianaleaves. The CrGATA1 promoter was cloned into theplasmid pKYLX71-GUS to drive expression of theGUS gene. The GUS reporter plasmids driven by thepromoters of CrGATA1 and five vindoline pathwaygenes were individually infiltrated into N. ben-thamiana leaves together with either EV (pCAM-BIA1300) or pCAMBIA1300-CrPIF1. We observed thatpromoter activities of CrGATA1, T16H2, and DATwere significantly repressed (40%–50%) by CrPIF1in dark (Fig. 6F), suggesting that CrPIF1 is a repres-sor of the vindoline pathway. It is well documentedthat PIFs bind to G-box (CACGTG) or PBE-box(CACATG/CATGTG) in the target promoters (Zhanget al., 2013). We identified a G-box (2142 to 2137 rel-ative to the first ATG) in the CrGATA1 promoter(Fig. 6G) and a PBE-box (21,106 to 21,101 relative tothe first ATG) in DAT promoter (Fig. 6H). The G-box inthe CrGATA1 promoter and the PBE-box (CACATG) inthe DAT promoter were mutated to CAAAAG andCACAAA, respectively. Transactivation assays usingN. benthamiana leaves were performed to evaluatethe effects of CrPIF1 on the mutated CrGATA1 andDAT promoters. We found that the repressive effectof CrPIF1 on the CrGATA1 and DAT promoters wasabolished by mutations in G-box and PBE-box, respec-tively (Fig. 6, G and H). These findings suggest thatCrPIF1 represses the activities of CrGATA1 and DATlikely by binding to the G-box or PBE-box motifs in thepromoters.

DISCUSSION

TIA biosynthesis in C. roseus is a highly complex andelaborated process that involves more than 30 differentenzymes, multiple cell types, and subcellular com-partments. A number of TFs regulating the biosynthesisof TIAs, such as strictosidine that serves as a precursorof tabersonine and catharanthine, have been charac-terized (Menke et al., 1999; van der Fits and Memelink,2000; Suttipanta et al., 2011; Zhang et al., 2011; Panet al., 2012; Li et al., 2013; Van Moerkercke et al., 2015,

2016; Paul et al., 2017; Patra et al., 2018; Schweizer et al.,2018; Sui et al., 2018). By comparison, our knowledge oftranscriptional regulation of the vindoline pathway andlight-mediated TIA biosynthesis is extremely limited.Here, we identified and characterized CrGATA1 andCrPIF1 for their roles in regulating vindoline biosyn-thesis in C. roseus seedlings.

TIA biosynthesis is developmentally regulated and isinfluenced by environmental cues, such as light.Tabersonine is present in both leaves and roots,whereas vindoline is predominantly found in leaves(De Luca et al., 1986). Moreover, dark-grown, etio-lated C. roseus seedlings accumulate a trace amount ofvindoline, which increases upon exposure to light.Correspondingly, the dark-grown seedlings accu-mulate a significant amount of tabersonine, whichdeclines upon exposure to light (De Luca et al., 1986).In addition, light induces the expression of D4H andDAT and their enzymatic activities in C. roseus seed-lings (De Luca et al., 1986; De Carolis et al., 1990; St-Pierre et al., 1998; Vazquez-Flota and De Luca, 1998).Our gene expression analysis revealed that, in addi-tion to D4H and DAT, the expression of T16H2, T3O,and T3R is significantly induced by light in C. roseusseedlings (Fig. 1A). In addition, the increase of vin-doline accumulation is accompanied by the decreaseof tabersonine upon exposure to light (Fig. 1B), fur-ther confirming the role of light in vindoline biosyn-thesis in C. roseus seedlings. Light is also known toaffect the biosynthesis of other specialized metabo-lites, including SGA in tomato (Wang et al., 2018)and anthocyanins in a number of plant species(Liu et al., 2018). Consistent with leaf-specificvindoline accumulation, CrGATA1 and the fivelight-responsive vindoline pathway genes are pref-erentially expressed in aerial parts of the seedling(Fig. 2A). The leaf-specific expression of CrGATA1and the vindoline pathway genes relative to those inroots (Fig. 2A) is significantly higher than light-induced expression in the seedling (Figs. 1A and2B), because the basal expression of these genes inroots is extremely low.

The cis-regulatory elements present in the gene pro-moters of metabolic pathways often provide cluesabout the potential TFs involved in the regulatory net-work and have been used as a tool for identification ofregulators. For instance, CrMYC2 was initially isolatedusing the G-box element present in C. roseus STR pro-moter and later demonstrated as a regulator of TIAbiosynthesis (Zhang et al., 2011; Schweizer et al., 2018;Sui et al., 2018). The presence of AT-rich motifs in thejasmonate-responsive element of the ORCA3 promoterled to the identification of the AT-hook regulators in C.roseus (Vom Endt et al., 2007). In addition, the presenceof putative MYB-binding sites in a betalain pathwaygene promoter (Polturak and Aharoni, 2018) andG-box/G-box-like sequences in tomato SGA biosyn-thetic pathway genes (Cárdenas et al., 2016) indicatesthe possible involvement of MYBs and MYC2, respec-tively, in the pathway regulations. Two R2R3 MYBs


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have since been identified as regulators of betalain bi-osynthesis in beet (Beta vulgaris; Polturak and Aharoni,2018). Our analysis of the vindoline pathway genepromoters revealed the presence of a number of light-responsive elements, including the GATA/GATC andG/E-box motifs (Supplemental Table S1). The presenceof GATA/GATC motifs in the five light-responsivepromoters in the vindoline biosynthetic pathway ledus to speculate that GATA family TFs are involved inthe light regulation of vindoline biosynthesis. In addi-tion, we noticed the presence of G/E-boxes that arerecognized by bHLH TFs, including PIFs, that areknown to be involved in light-responsive gene expres-sion in plants (Yadav et al., 2005; Pham et al., 2018).Transcriptomic and genomic resources are invalu-

able for the identification of missing genes encodingkey enzymes, transporters, and regulatory proteins inC. roseus (Geu-Flores et al., 2012; Van Moerkerckeet al., 2015; Larsen et al., 2017; Paul et al., 2017;Payne et al., 2017; Caputi et al., 2018; Patra et al., 2018;Qu et al., 2019). Coexpression analysis of vindolinebiosynthetic pathway genes and GATA TFs identi-fied two putative candidates for further characteriza-tion (Supplemental Fig. S3). Similar to the vindolinepathway genes (Vazquez-Flota et al., 1997; St-Pierreet al., 1998; Besseau et al., 2013), CrGATA1 is prefer-entially expressed in leaves (Fig. 2A) and significantlyinduced by light (Fig. 2B). Transient overexpressionand VIGS of CrGATA1 significantly altered the ex-pression of most of the vindoline pathway genes andvindoline accumulation in C. roseus (Fig. 3). Expres-sion of T3O and T3R was not increased in CrGATA1overexpression, while expression of T16H2 and D4Hdid not significantly change in VIGS lines. These ob-servations suggest that other regulators are also in-volved in the gene regulation. Similar observationshave been made for other regulators in the TIA path-way. For instance, a previous study has shown thatCrMYC2 regulates the AP2/ERF ORCA3 by bindingto the T/G-boxmotif in the promoter. However,CrMYC2overexpression does not significantly affect ORCA3transcripts, whereas silencing CrMYC2 in C. roseuscells reduces ORCA3 expression (Zhang et al., 2011).Similarly, ORCA3 is known to regulate the expres-sion of TDC. However, overexpression of ORCA3in C. roseus hairy roots does not significantly induceTDC expression (Peebles et al., 2009). Expression ofTDC and STR was not affected by overexpressionor VIGS of CrGATA1, indicating that CrGATA1 doesnot regulate the upstream TIA pathway genes inC. roseus seedlings.A transactivation assay in N. benthamiana leaves

showed that CrGATA1 activates the promoters of keyvindoline pathway genes (Fig. 4), suggesting thatCrGATA1 is an activator in vindoline biosynthesis.GATA TFs are involved in a number of developmentaland physiological processes. However, little is knownabout the roles of GATA TFs in light regulation ofspecialized metabolism. Two well-characterized Ara-bidopsis GATA TFs, GNC and GNL, are light inducible

and involved in chloroplast biogenesis and nitrogenmetabolism (Richter et al., 2010; Hudson et al., 2011).GNC and GNL bind the GATA motif in the GLU1promoter (Hudson et al., 2011), whereas the tobaccoGATA TF, AGP1, activates NtMYC2 by binding to theGATC motif in the NtMYC2 promoter (Sugimoto et al.,2003). Moreover, genome-wide binding analysis ofGNC and GNL reveals that both GATA and GATC cis-elements are enriched in the targets (Xu et al., 2017). Weidentified multiple GATA and GATC cis-elements inthe promoters of DAT, D4H, T3R, T3O, and T16H2.Mutation to the GATC element, but not the GATA el-ement (Fig. 4B), in the D4H promoter had a significanteffect on the transactivation (Fig. 4C), suggesting thatCrGATA1 likely recognizes the GATC element in theactivation of D4H.Activities of D4H and DAT increase significantly

upon exposure of C. roseus seedlings to red light, andthe effect is reversed by far-red light (Aerts and DeLuca, 1992; Vazquez-Flota and De Luca, 1998). In ad-dition, transcript levels of D4H increase following ex-posure to red light (Vazquez-Flota and De Luca, 1998).Phytochromes serve as receptors of red and far-redlight in plants and exist in two different forms, the in-active Pr and active Pfr. In the absence of red light, theinactive Pr accumulates in cytosol; however, uponperception of red light, Pr converts to the active Pfr thatis subsequently translocated to the nucleus (Franklinand Quail, 2010). Here, we demonstrated that expres-sion of CrGATA1, T16H2, T3O, T3R, and DAT in C.roseus seedling increases upon exposure to red light anddecreases following exposure to far-red light (Fig. 5),suggesting a phytochrome-dependent regulation ofvindoline biosynthesis. We did not observe an apparentincrease in D4H expression upon exposure to red light.This is most likely due to the duration of treatmentthat affects its expression in seedlings. PIFs are knownto interact with phytochromes and regulate light-responsive gene expression in plants. PIFs regulatethe biosynthesis of specialized metabolites, includinganthocyanins (Shin et al., 2007; Liu et al., 2015) andSGAs (Wang et al., 2018). For anthocyanin biosynthesisin Arabidopsis, PIF3 and HY5 act as activators (Shinet al., 2007), whereas PIF4 and PIF5 function as re-pressors, as overexpression of PIF4 or PIF5 reducesanthocyanin accumulation (Liu et al., 2015). We iden-tified three putative PIFs in the C. roseus genome.Transient overexpression or VIGS of CrPIF1 alteredvindoline pathway gene expression and vindoline ac-cumulation in C. roseus seedlings (Fig. 6, A–D). Previ-ous studies demonstrated that PIFs bind to theG/E-box/PBE elements in the target promoters. Scan-ning of the promoters of CrGATA1 and vindolinepathway genes revealed the presence of putative PIF-binding sites (Fig. 6, G andH). Transient transactivationassays showed that CrPIF1 repressed the promoter ac-tivities of CrGATA1 and vindoline pathway genes(Fig. 6, G and H). Site-directed mutagenesis of the PIF-binding sites in the CrGATA1 and DAT promotersabolished PIF-mediated repression, suggesting that







CrPIF1 directly regulates CrGATA1 and DAT by bind-ing to the G/E-box/PBE elements in the promoters. TheT16H2 promoter does not contain canonical PBE(CACATG) motifs but contains three E-box motifs(CAAATG, CAATTG, and CAGCTG) that are highlysimilar to the PBE. CrPIF1 likely suppresses T16H2expression by binding to the E-box (Fig. 6A). As TIAaccumulation in C. roseus is developmentally regulated,the difference in the alkaloid contents observed in thisstudy between overexpression (Figs. 3B and 6B) andVIGS (Figs. 3D and 6D) lines of CrGATA1 or CrPIF1 islikely due to the age of the seedlings used in theexperiments.

In conclusion, our work elucidates a mechanism bywhich light influences vindoline biosynthesis throughthe newly characterized CrGATA1 and CrPIF1. CrPIF1represses the expression of CrGATA1 and vindolinepathway genes in the dark, resulting in reduced vin-doline accumulation. CrPIF1 is possibly degraded inlight by the 26S/ubiquitin proteasome system, leadingto derepression of CrGATA1. The activation ofCrGATA1 leads to up-regulation of the vindolinepathway genes and increased vindoline accumulationin C. roseus seedlings (Fig. 7). Other regulators are likelyalso involved in vindoline biosynthesis. It remains un-clear what transactivator regulates CrGATA1, which,despite repression by CrPIF1, expresses at a low (basal)

level in the dark. Consequently, trace amounts of vin-doline can be detected in dark-grown C. roseus seed-lings. Nevertheless, our findings revealed a previouslyuncharacterized molecular mechanism that controlslight-mediated TIA biosynthesis in C. roseus. The role ofPIF-mediated GATA TF regulation is perhaps generallyconserved in plant light-regulated biosynthesis of spe-cialized metabolites.

MATERIALS AND METHODS

Plant Materials, Growth Conditions, and Treatments

Catharanthus roseus ‘Little Bright Eye’ seeds obtained from NESeed weresurface sterilized using 30% (v/v) commercial bleach for 6 min, rinsed fivetimes with sterile water, and incubated in dark at 28°C for germination on one-half-strengthMurashige and Skoogmedium. Seven-day-old etiolated seedlingswere treated with white light (40 mmolm22 s21), red light (31 mmolm22 s21), orfar-red light (52 mmol m22 s21). For white light treatment, seedlings were in-cubated for 1, 4, 10, 24, 48, or 96 h. For red and red/far-red light treatments, theseedlings were exposed to red light for 2 and 4 h and then exposed to far-redlight for 2 h. Aerial parts of seedlings were collected for RNA isolation or al-kaloid extraction.

RNA Isolation, cDNA Synthesis, and RT-qPCR

RNA isolation, complementary DNA (cDNA) synthesis, and RT-qPCRwereperformed as previously described (Paul et al., 2017). Primers used in RT-qPCRare listed in Supplemental Table S2.

Figure 7. Models depicting the regulatory roles of the CrPIF1-CrGATA1 module in light-induced vindoline biosynthesis. Left,CrPIF1 is likely accumulated in the dark and represses the expression of CrGATA1, T16H2, and DAT by binding to their pro-moters. Repression of CrGATA1 results in down-regulation of vindoline pathway genes, T16H2, T3O, T3R, D4H, and DAT,leading to limited vindoline production and increased tabersonine accumulation. One or more unknown TFs (TF?) mediates thelow (basal) expression of CrGATA1 despite CrPIF1 repression. Right, CrPIF1 is possibly degraded upon exposure of C. roseusseedlings to light, which is mediated by the red light sensed by phytochrome.Derepression ofCrGATA1 results in the activation ofvindoline pathway genes and vindoline accumulation. The seven genes involved in the conversion of tabersonine to vindoline arelisted in the inset box. Genes in red are responsive to light induction.


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In Silico Analysis of Putative Regulators of Vindoline inC. roseus

To identify C. roseus GATA TFs, sequences of all protein-coding genes weredownloaded from the latest version of the C. roseus genome from the DryadDigital Repository (Kellner et al., 2015). BLAST search was performed toidentify the putative GATA and PIF TFs. To further validate the BLAST searchresults, phylogenetic trees were constructed and visualized using the neighbor-joiningmethod throughMEGA5.1 software (Tamura et al., 2011). The statisticalreliability of individual nodes of the newly constructed trees was assessed bybootstrap analyses with 1,000 replications (Altschul et al., 1997). Alignments ofamino acids were conducted by the MAFFT method (Katoh and Standley,2013).

To analyze the expression patterns of C. roseus GATAs and vindolinepathway genes in five different tissues (seedling, mature leaf, immature leaf,stem, and root; Góngora-Castillo et al., 2012), hierarchical clustering was per-formed as previously described (Paul et al., 2017).

Subcellular Localization

To determine the subcellular localization of CrGATA1 or CrPIF1, full-lengthcDNA was translationally fused with the N terminus of eGFP in a pBluescriptplasmid as described earlier (Suttipanta et al., 2011). Expression of eGFP wasdriven by the CaMV35S promoter and the rbcS terminator. Plasmids wereelectroporated into tobacco (Nicotiana tabacum) protoplasts and visualized witha fluorescencemicroscope (Nikon Eclipse TE200; Nikon) after 20 h of incubationin dark at room temperature. A pBluescript plasmid expressing only eGFPserved as a negative control.

Plasmid Construction and Transient Gene Overexpressionin C. roseus Seedling

For transient overexpression in C. roseus seedlings, CrGATA1, CrPIF1,CrPIF3, or CrPIF4/5 was cloned into a modified pCAMBIA1300 vector con-taining CaMV35S promoter and rbcS terminator. C. roseus seedlings weretransiently transformed with the plasmids using the FAST method with somemodifications (Weaver et al., 2014). Briefly, Agrobacterium tumefaciens GV3101harboring the plasmid was grown on Luria-Bertani (LB) plates containing100 mg mL21 kanamycin, 50 mg mL21 gentamicin, and 30 mg mL21 rifampicin.A single colony was transferred to 2 mL of liquid LB medium containing sameantibiotics and incubated at 250 rpm and 28°C. Overnight-grownA. tumefacienscells were subcultured in 20 mL of liquid LB medium for 16 h at 250 rpm and28°C. A. tumefaciens cultures were then centrifuged, and the pellet was resus-pended in infiltration buffer (10 mM MgCl2, 10 mM MES, and 100 mM aceto-syringone) at an OD600 of 1, followed by incubation at 28°C for at least 3 h.Seven-day-old C. roseus seedlings were immersed in the A. tumefaciens culturesfor 1 h. After infiltration, seedlings were washed with sterile distilled water fivetimes and laid on petri dishes with autoclavedwet filter papers. After 3 d, aerialparts of seedlings were collected for RNA extraction and alkaloid analyses.

VIGS in C. roseus Plants

For VIGS (Liu et al., 2002), the plasmids pTRV2-CrChlH, pTRV2-CrGATA1,and pTRV2-CrPIF1 were generated by cloning partial coding sequences ofCrChlH (400 bp), CrGATA1 (358 bp), or CrPIF1 (183 bp) in the multiple cloningsites of the pTRV2 vector. C. roseus seedlings with two pairs of true leaves wereused to perform the VIGS assay using the pinchmethod as previously described(Liscombe andO’Connor, 2011). Harvest timewas guided by the appearance ofphotobleaching of the corresponding leaves in whichCrChlHwas silenced. As acontrol, plants were infected with a pTRV2 EV. The newly emerging pair ofleaves following inoculation was harvested, frozen in liquid nitrogen, and thenstored at 280°C until RNA and alkaloid extraction.

Plasmid Construction and Nicotiana benthamiana LeafInfiltration Assays

The reporter plasmids for N. benthamiana leaf infiltration assays were gen-erated by replacing the CaMV35S promoter in a modified pKYLX71 vectorcontaining the GUS reporter and rbcS terminator (Schardl et al., 1987) withT16H2 (1,129 bp), T3O (703 bp), T3R (1,129 bp), D4H (704 bp), DAT (1,317 bp),

or CrGATA1 (1,189 bp) promoter. Mutants of the following cis-elements, GATAmotifs (2430 to 2427 and 2138 to 2135) and GATC motifs in D4H promoter(2617 to 2614 and 2541 to 2538), G-box in CrGATA1 promoter (2142 to2137), and PBE-box in DAT promoter (21,106 to 21,101), were generated bysite-directed mutagenesis (Pattanaik et al., 2010). pCAMBIA1300 vectors con-taining CrGATA1 or CrPIF1 were used as the effector plasmids. A firefly lu-ciferase reporter, driven by CaMV35S promoter and rbcS terminator, was usedas an internal control in the leaf infiltration assays. Infiltration solutions wereprepared as described in the FAST method. Before infiltration, effector, re-porter, and internal control solutions were combined at a 1:1:1 ratio and mixedwell. Infiltration of N. benthamiana leaves was performed as previously de-scribed (Kumar and Bhatia, 2016). Two days after infiltration, leaf discs werecollected, frozen in liquid nitrogen, and ground to powder for protein extrac-tion. Firefly luciferase and GUS activities were measured as previously de-scribed (Pattanaik et al., 2010).

Alkaloid Extraction and Analysis

To extract alkaloids, light-treated seedlings, agroinfiltrated seedlings, orleaves collected for VIGS assay were frozen in liquid nitrogen and ground topowder. Samples were extracted twice in methanol (1:100, w/v) for 24 h on ashaker. Pooled extracts were dried using a rotary evaporator and diluted inmethanol. Samples were analyzed with HPLC, followed by electrospray in-jection in tandemmass spectrometry, as described previously (Suttipanta et al.,2011). The concentrations of the alkaloids were calculated using astandard curve.

Accession Numbers

Accession numbers are as follows: CrGATA1 (MK801106), CrPIF1(ALI87040.1), CrPIF3 (ALI87041.1), and CrPIF4/5 (ALI87042.1).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Simplified TIA biosynthetic pathway in C. roseus.

Supplemental Figure S2. Gene expression analysis and measurement ofTIAs in C. roseus seedlings.

Supplemental Figure S3. Coexpression of CrGATA genes with five light-inducible vindoline pathway genes in different tissues of C. roseus.

Supplemental Figure S4. Phylogenetic analysis of AtGATAs andCrGATAs.

Supplemental Figure S5. Multiple sequence alignment of CrGATA1 andArabidopsis GNC and GNL.

Supplemental Figure S6. TDC and STR expression is not altered inCrGATA1 overexpression and VIGS lines.

Supplemental Figure S7. Amino acid sequence alignment of PIFs from C.roseus, Arabidopsis, and tomato.

Supplemental Figure S8. Phylogenetic analysis of PIFs from C. roseus,Arabidopsis, and tomato.

Supplemental Figure S9. Expression of CrPIFs in response to lightand dark.

Supplemental Figure S10. CrGATA1 and vindoline pathway genes are notaltered by CrPIF3 and CrPIF4/5 overexpression in C. roseus seedlings.

Supplemental Table S1. Light-responsive cis-elements within promotersof five light-induced vindoline pathway genes.

Supplemental Table S2. Primers used in this study.

ACKNOWLEDGMENTS

We thank Dr. Bruce Downie of the Department of Horticulture, Universityof Kentucky, for assistance and advice on red/far-red light treatment of C.

















roseus seedlings and John May and Megan Combs (Department of Civil Engi-neering and Environmental Research Training Laboratories, University of Ken-tucky) for assistance on LC-MS/MS.

Received April 22, 2019; accepted May 3, 2019; published May 13, 2019.

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