Physiologia Plantarum 150: 493–504. 2014 © 2013 Scandinavian Plant Physiology Society, ISSN 0031-9317

Cloning and functional analysis of the promotersthat upregulate carotenogenic gene expression duringflower development in Gentiana luteaChangfu Zhua,b,†, Qingjie Yangc,d,†, Xiuzhen Nia, Chao Baib, Yanmin Shenga, Lianxuan Shid,Teresa Capellb, Gerhard Sandmanne and Paul Christoub,f,∗

aSchool of Life Sciences, Changchun Normal University, Changchun 130032, ChinabDepartament de Produccio Vegetal i Ciencia Forestal, Universitat de Lleida-Agrotecnio Center, Lleida 25198, SpaincCollege of Landscape Architecture, Northeast Forestry University, Harbin 150040, ChinadSchool of Life Sciences, Northeast Normal University, Changchun, 130024 ChinaeDepartment of Molecular Biosciences, J.W Goethe Universitaet, Frankfurt, D-60054 GermanyfInstitucio Catalana de Recerca i Estudis Avancats, Passeig Lluis Companys, Barcelona 08010, Spain

Correspondence*Corresponding author,e-mail: christou@pvcf.udl.cat

Received 17 June 2013;revised 24 October 2013

doi:10.1111/ppl.12129

Over the last two decades, many carotenogenic genes have been cloned andused to generate metabolically engineered plants producing higher levels ofcarotenoids. However, comparatively little is known about the regulation ofendogenous carotenogenic genes in higher plants, and this restricts our abilityto predict how engineered plants will perform in terms of carotenoid contentand composition. During petal development in the Great Yellow Gentian(Gentiana lutea), carotenoid accumulation, the formation of chromoplasts andthe upregulation of several carotenogenic genes are temporally coordinated.We investigated the regulatory mechanisms responsible for this coordinatedexpression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS,GlLYCB, GlBCH and GlLYCE) promoters by inverse polymerase chainreaction (PCR). Each promoter was sufficient for developmentally regulatedexpression of the gusA reporter gene following transient expression intomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCBand GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containingimmature green fruits, indicating a strict correlation between promoteractivity, tomato fruit development and chromoplast differentiation. As wellas core promoter elements such as TATA and CAAT boxes, all five promoterstogether with previously characterized GlZEP promoter contained threecommon cis-regulatory motifs involved in the response to methyl jasmonate(CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may representbinding sites for transcription factors responsible for co-regulation. Our dataprovide insight into the regulatory basis of the coordinated upregulation ofcarotenogenic gene expression during flower development in G. lutea.

Abbreviations – ABRE, abscisic acid responsive element; BCH, β-carotene hydroxylase; ERE, ethylene response element;GlBCH, Gentiana lutea β-carotene hydroxylase; GlLYCB, Gentiana lutea lycopene β-cyclase; GlPDS, Gentiana lutea phytoenedesaturase; GlLYCE, Gentiana lutea lycopene ε-cyclase gene; GlZDS, Gentiana lutea ζ -carotene desaturase gene; GlZEP,Gentiana lutea zeaxanthin epoxidase; IMG, immature green; LA-PCR, long accurate polymerase chain reaction; LYCB, lycopeneβ-cyclase; MeJA, methyl jasmonate; MG, mature green; PCR, polymerase chain reaction; PDS, phytoene desaturase; PSY ,phytoene synthase; UTR, untranslated region; ZDS, ζ -carotene desaturase; ZEP, zeaxanthin epoxidase.

†These authors contributed equally to this work.

Physiol. Plant. 150, 2014 493

Introduction

The carotenoids are a large class of yellow, orangeand red pigments derived from isoprenoid precursors.In higher plants, they accumulate in the chloroplastsof leaves and in the chromoplasts of many flowersand fruits. Carotenoids function as accessory pigmentsduring photosynthesis and help to prevent photo-oxidation (Frank and Cogdell 1996, Demmig-Adamsand Adams 2002). They are also precursors of theplant hormones abscisic acid (Creelman and Zeevart1984) and strigolactone (Gomez-Roldan et al. 2008,Umehara et al. 2008). In flowers and fruits, thebright colors imparted by carotenoids help to attractpollinating insects and seed-dispersing animals (Bartleyand Scolnik 1995), and the colors also provideagronomic value in fruit and vegetable crops as wellas ornamental plants (Bartley and Scolnik 1995, Zhuet al. 2010).

Carotenoids play a fundamental role in humannutrition as antioxidants and precursors of vitamin A,and a high dietary intake of carotenoids reduces the riskof several diseases (Fraser and Bramley 2004, Zhu et al.2009, Bai et al. 2011, Farre et al. 2011). Genes encodingcarotenogenic enzymes have therefore been cloned andused for metabolic engineering to increase the nutritionalvalue and health-promoting properties of plants byimproving the carotenoid content and composition(Sandmann et al. 2006, Zhu et al. 2007, 2013, Giulianoet al. 2008, Farre et al. 2010, 2011, Bai et al. 2011).However, the regulation of endogenous carotenoidformation in higher plants is poorly understood,restricting the extent to which the impact of metabolicengineering in crops can be predicted (Sandmann et al.2006, Giuliano et al. 2008, Farre et al. 2010).

The flowers in the Great Yellow Gentian (Gentianalutea) contain abundant amounts of β-carotene andxanthophylls, making it a useful model to investigate theregulation of carotenoid biosynthesis in flowers (Zhuet al. 2002, 2003). The petals possess chromoplaststhat originate from fully developed chloroplasts, andthere is a temporal correlation among the accumu-lation of carotenoids, the formation of chromoplastsand the induction of the carotenogenic genes PSY(phytoene synthase), PDS (phytoene desaturase), ZDS(ζ -carotene desaturase), LYCB (lycopene β-cyclase),BCH (β-carotene hydroxylase) and ZEP (zeaxanthinepoxidase) (Zhu et al. 2002, 2003) (Fig. 1). Transcriptionof carotenogenic genes in G. lutea controls the accu-mulation of carotenoids during flower development(Zhu et al. 2002, 2003). The G. lutea zeaxanthinepoxidase (GlZEP) promoter was recently cloned andcharacterized in transgenic tomato plants (Yang et al.

2012), where it was shown to drive the developmentallyregulated expression of gusA, encoding the reporterβ-glucuronidase (GUS). High levels of gusA expressionwere observed in chromoplast-containing fruits andpetals. In contrast, only low levels of expression wasseen in the immature green (IMG) chloroplast-containingfruits and leaves. GlZEP promoter activity was strictlyassociated with fruit development and chromoplastdifferentiation (Yang et al. 2012).

Here we describe the isolation and analysis of five G.lutea carotenogenic gene promoters (Gentiana lutea phy-toene desaturase gene (GlPDS), Gentiana lutea ζ -caro-tene desaturase gene (GlZDS), Gentiana lutea lycopeneβ-cyclase gene (GlLYCB), Gentiana lutea β-carotenehydroxylase (GlBCH) and Gentiana lutea lycopene ε-cyclase gene (GlLYCE) promoters, the latter controllingthe expression of lycopene ε-cyclase gene). We inves-tigated the regulatory basis of coordinated upregulationduring G. lutea flower development and searched forcommon cis-regulatory elements that could provideinsight into the hierarchal control of carotenoid biosyn-thesis in flower petals.

Materials and methods

Plant material

Gentiana lutea leaves were obtained from the HokkaidoExperimental Institute of Health Science, Japan. Thetissues were frozen in liquid nitrogen immediately afterharvesting and stored at −80◦C. Tomato (Solanumlycopersicum cv. Micro-Tom) plants were grown in thegreenhouse at 25◦C with a 16-h photoperiod.

Cloning the promoter sequences

Gentiana lutea Genomic DNA was extracted from 5 g ofleaf tissue according to Edwards et al. (1991). GenomicDNA (20 μg) was completely digested with a restrictionenzyme appropriate to the corresponding promotersequence (Table 1) and self-ligated using 10 Weiss unitsof T4 DNA Ligase (Invitrogen, Carlsbad, CA) to generatecircular molecules. These were used as templates foramplification by the long accurate polymerase chainreaction (LA-PCR), following the recommendationsprovided with the Takara LA-PCR Kit (Takara, Shuzo,Japan). The products were cloned into vector PCR® IITOPO® (TA Cloning Kit, Invitrogen, Carlsbad, CA) forsequencing using the Big Dye Terminator v3.1 CycleSequencing Kit on a 3130x1 Genetic Analyzer (AppliedBiosystems, Foster City, CA). The restriction enzymes andprimer sequences required for each of the five promotersare listed in Table 1.

494 Physiol. Plant. 150, 2014

Fig. 1. Carotenoid biosynthesis pathway in plants. Abbreviations: CRTISO, carotenoid isomerase; CYP97C, heme-containing cytochrome P450carotene ε-ring hydroxylase; GGPP, geranylgeranyl diphosphate; HYDB, β-carotene hydroxylase [non-heme di-iron β-carotene hydroxylase (BCH) andheme-containing cytochrome P450 β-ring hydroxylases (CYP97A and CYP97B)]; LYCB, lycopene β-cyclase; LYCE, lycopene ε-cyclase; PDS, phytoenedesaturase; PSY, phytoene synthase; VDE, violaxanthin de-epoxidase; ZDS, ζ -carotene desaturase; ZEP, zeaxanthin epoxidase; Z-ISO, ζ -caroteneisomerase. This figure was modified based on Zhu et al. (2010, 2013).

Physiol. Plant. 150, 2014 495

Table 1. PCR primers used to clone gene promoters, to construct promoter-gusA fusion plasmids and to clone genomic DNA to confirm the isolatedpromoter sequences.

Gene Purposes Primers

Positions (+1 is thefirst nucleotide

of cDNA)

Accession numbersfor cDNA or genomic

DNA (gDNA)

PDS Promoter cloning(digested with EcoRI)

F1: 5′-GGACACATATCTGCTGTTAACATAGGTAGGCAAGG-3′ +145 to +179 EF203257 (cDNA)R1: 5′-GGTAGGATAAAATTCACTAAGTTGAAGGTGAAAGGG-3′ +1 to +36

Promoter-GUS construct F2: 5′-GTCGACAATTCATGAGTTCAAACCCGTGATTCGTTC-3′ −1077 to −1048 DQ226992 (gDNA)R2: 5′-GGATCCATATCAAAGCTGGTACCAAACAGAGCAAAC-3′ −1 to −30

Genomic DNA cloning F3: 5′-AATTCATGAGTTCAAACCCGTGATTCGTTC-3′ −1077 to −1048 DQ226992 (gDNA)R3: 5′-GGTAGGATAAAATTCACTAAGTTGAAGGTG-3′ +7 to +36

ZDS Promoter cloning(digested with BamHI)

F1: 5′-AGGCTTGTTTCCACCGGAACCTGAACATTATCGG-3′ +415 to +448 EF203258 (cDNA)

R1: 5′-TCAATGCAATCCTGAAGAACTTAGACCATTGCTGT-3′ +123 to +157Promoter-GUS construct F2: 5′-GTCGACGATCCGTTTAACGCTTAGATCGTCGTCATC-3′ −766 to −737 JQ417647 (gDNA)

R2: 5′-GGATCCTTAGATTATGTTAAAACAAGCATCAAAGCT-3′ −1 to −30Genomic DNA cloning F3: 5′-GATCCGTTTAACGCTTAGATCGTCGTCATC-3′ −766 to −737 JQ417647 (gDNA)

R3: 5′-TCAACGCAATCCTGAAGAACTTAGACCATT-3′ +128 to +157LYCB Promoter cloning

(digested with EcoRI)F1: 5′-CACCCTTTATGTGGGTTTGTTGATAAAGTCTGCTCC-3′ +385 to +420 EF203253 (cDNA)

R1: 5′-CCAATTGTTGCCCAGAAAGCAAACTGAATTTCGAG-3′ +259 to +293Promoter-GUS construct F2: 5′-GTCGACAATTCAACATCAAATGGCTAGTTGGACTTT-3′ −1506 to −1477 JQ417648 (gDNA)

R2: 5′-GGATCCTCTGCGACACGTCTGACGGTGGAGTTAATT-3′ −1 to −30Genomic DNA cloning F3: 5′-AATTCAACATCAAATGGCTAGTTGGACTTT-3′ −1506 to −1477 JQ417648 (gDNA)

R3: 5′-CTTTTCTCTATTGGGTGTACTTTATTTCTT-3′ +313 to +342BCH Promoter cloning

(digested with PstI)F1: 5′-TTGGTCTCCGGTAGAAACAGCAACATTCATTGCCGT-3′ +135 to +170 EF203255 (cDNA)

R1: 5′-GTGACCGGAAACCGGAGAAACAGCTGAGAGGAGAG-3′ +73 to +107Promoter-GUS construct F2: 5′-GTCGACGCCTAGGCCTACAATGCACAACCTAATACC-3′ −1744 to −1715 EF203261 (gDNA)

R2: 5′-GGATCCACCACATGTTTTCTTTGGATTGTGAAACTC-3′ −1 to −30Genomic DNA cloning F3: 5′-GCCTAGGCCTACAATGCACAACCTAATACC-3′ −1744 to −1715 EF203261 (gDNA)

R3: 5′-TACCGGAAAATGGTGACCGGAAACCGGAGA-3′ +90 to +119LYCE Promoter cloning

(digested with BglII)F1: 5′-TATTCGAGACGATCAAGAAGAAGGAGGATTCTCAGTG-3′ +310 to +346 EF203256 (cDNA)

R1: 5′-GTTGTTTGAAGTGTGAGCGCAGGGCAGCCGCTAAAG-3′ +31 to +66Promoter-GUS construct F2: 5′-GTCGACGATCTTAAGTTATGTTCTAGTAAGTAAAGC-3′ −938 to −909 EU592045 (gDNA)

R2: 5′-GGATCCTCGTTTCAAGAGTTGCCACGTGGCTTGAAA-3′ −1 to −30Genomic DNA cloning F3: 5′-GATCTTAAGTTATGTTCTAGTAAGTAAAGC-3′ −938 to −909 EU592045 (gDNA)

R3: 5′-GTTTGGCCCTTTATTTGTGTAGTTTCCGTG-3′ +226 to +255

Promoter-gusA constructs

The promoter sequences were fused to the gusA genein vector pBI101 (Clontech Laboratories, MountainView, CA) (Jefferson et al. 1987) by amplifying eachpromoter using primers containing additional sequencesto provide appropriate restriction sites. For example, thefull-length GlPDS promoter region was amplified fromG. lutea genomic DNA using forward primer F2 (5′-GTCGAC AAT TCA TGA GTT CAA ACC CGT GAT TCGTTC-3′, introducing a SalI site in italics) and reverseprimer R2 (5′-GGA TCC ATA TCA AAG CTG GTA CCAAAC AGA GCA AAC-3′, introducing a BamHI site initalics). The 1077-bp amplified promoter fragment wasthen transferred to the PCR® II TOPO® vector usingthe Invitrogen TA Cloning® Kit, to yield intermediatevector pCR-GlPDSPro. The pCR-GlPDSPro and pBI101vectors were digested with SalI and BamHI, allowing

the GlPDSPro fragment to be inserted upstream of gusA,yielding the final construct pBI-GlPDSPro-GUS (PDS-gusA). The constructs pBI-GlZDSPro-GUS (ZDS-gusA),pBI-GlLYCBPro-GUS (LYCB-gusA), pBI-GlBCHPro-GUS(BCH-gusA) and pBI-GlLYCEPro-GUS (LYCE-gusA) wereconstructed in an analogous manner using the specificprimers listed in Table 1. The integrity of all intermediateand final constructs was confirmed by sequencing.

Transient expression of promoter-gusA constructsin tomato

Plasmids pBI101, pBI121 (35S-gusA), pBI-GlPDSPro-GUS (PDS-gusA), pBI-GlZDSPro-GUS (ZDS-gusA),pBI-GlLYCBPro-GUS (LYCB-gusA), pBI-GlBCHPro-GUS(BCH-gusA) and pBI-GlLYCEPro-GUS (LYCE-gusA)were introduced into Agrobacterium tumefaciens

496 Physiol. Plant. 150, 2014

strain LBA 4404 by electroporation (Mattanovich et al.1989). Individual colonies were seeded into 5-mlaliquots of YEM medium (0.5% beef extract, 0.1%yeast extract, 0.5% peptone, 0.5% sucrose, 2 mMMgSO4, pH 7.2) containing 50 μg ml−1 kanamycin and25 μg ml−1 rifampicin, and were shaken at 300 rpm,28◦C overnight. Each culture was then used toinoculate 50 ml of induction medium (YEM mediumsupplemented with 20 μM acetosyringone, 10 mMMES, pH 5.6) containing 50 μg ml−1 kanamycin and25 μg ml−1 rifampicin, followed by incubation as above.Bacteria were recovered by centrifugation (2700 g),resuspended in infiltration medium (10 mM MgCl2,10 mM MES, 200 μM acetosyringone, pH 5.6) to anOD600 of approximately 1.0, and then incubated atroom temperature with gentle agitation (20 rpm) for 3 h.Approximately 450 μl of the infiltration medium wasthen injected into fruits at the immature green stage (fruitdiameter 0.8–1.0 cm, about half full size, 15–20 daysafter anthesis. Fruits were not fully expanded and stillgreen, defined as ‘immature green’) (Akihiro et al. 2008),and 600 μl was injected into fruits at the mature green(MG) stage (fruit diameter 1.5–2.0 cm, 30–35 daysafter anthesis. Fruits were fully expanded and green,defined as ‘mature green’) (Akihiro et al. 2008), in bothcases through the stylar apex using a 1-ml syringe withneedle (Orzaez et al. 2006). Injected fruits were lefton the vine for 3 days before harvesting and sectioningfor histochemical staining. All the experiments wererepeated six times in six independent Micro-Tom plants.

Histochemical GUS assay

Histochemical GUS assays were carried out according toJefferson et al. (1987) with minor modifications. Tissueswere incubated at 37◦C for 12 h in the dark in 1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) in100 mM sodium phosphate (pH 7.0), 10 mM EDTA(ethylenediaminetetraacetic acid), 0.5 mM potassiumferricyanide, 0.5 mM potassium ferrocyanide, 0.3% (v/v)Triton X-100 and 20% (v/v) methanol to eliminateendogenous GUS expression (Kosugi et al. 1990). Afterstaining, the tissues were destained in an ethanol series(50, 70, 80 and 95%) to remove chlorophyll, and thenstored in 70% (v/v) ethanol, and photographed with adigital camera.

Results

Cloning the promoter sequences

The promoters for GlPDS, GlZDS, GlLYCB, GlBCH andGlLYCE were cloned by inverse PCR using cleaved and

circularized G. lutea genomic DNA as the templateand outward-facing primers based on the correspondingcDNA sequences (Zhu et al. 2002, 2003). The primers,GenBank accession numbers and restriction enzymesused to prepare the genomic templates are listed inTable 1. After sequencing the products, DNA fragmentsof 1113 bp (GlPDS), 923 bp (GlZDS), 1848 bp (GlLYCB),1863 bp (GlBCH) and 1193 bp (GlLYCE) were isolateddirectly from genomic DNA using gene-specific primers(Primers and GenBank accession numbers are listedin Table 1). All the fragments comprised the upstreampromoters and the 5′-untranslated region (UTR). Thefull-length promoter fragments were defined as 1077 bp(GlPDS), 766 bp (GlZDS), 1506 bp (GlLYCB), 1744 bp(GlBCH) and 938 bp (GlLYCE) with position +1 assignedto the first nucleotide of the cDNAs (Zhu et al. 2002,2003).

Construction of promoter-gusA fusion genesand transient expression in tomato fruits

The full-length promoter regions were amplified fromG. lutea genomic DNA using forward primers containinga SalI restriction site (5′-GTCGAC-3′) and reverse primerscontaining a BamHI restriction site (5′-GGATCC-3′). Thepromoter fragments were inserted upstream of the gusAgene in vector pBI101, which had been digested withSalI and BamHI.

Tomato fruits at the IMG and MG stages wereinjected with bacterial cultures carrying the vec-tors pBI101 (promoterless gusA), pBI121 (35S-gusA,constitutive Cauliflower mosaic virus 35S promoter),pBI-GlPDSPro-GUS (PDS-gusA), pBI-GlZDSPro-GUS(ZDS-gusA), pBI-GlLYCBPro-GUS (LYCB-gusA), pBI-GlBCHPro-GUS (BCH-gusA) and pBI-GlLYCEPro-GUS(LYCE-gusA). Fruits were harvested 3 days later andtransverse sections were stained for GUS expression.As expected, both IMG fruits (Fig. 2) and MG fruits(Fig. 3) showed significant GUS expression when trans-formed with the 35S-gusA construct but no GUS expres-sion when transformed with the promoterless controlvector pBI101. We evaluated the five carotenoid con-structs (PDS-gusA, ZDS-gusA, LYCB-gusA, BCH-gusAand LYCE-gusA) by transient expression in tomato fruitsas above, using the 35S-gusA vector as a control. Histo-chemical staining revealed no GUS expression in IMGfruits transformed with PDS-gusA, ZDS-gusA and LYCE-gusA, and with only low GUS expression levels in IMGfruits transformed with the LYCB-gusA and BCH-gusAconstructs (Fig. 2) but higher GUS expression for theseconstructs, as well as PDS-gusA and LYCE-gusA, in MGfruits (Fig. 3). The ZDS-gusA construct produced lowlevel GUS expression in the mesocarp of MG fruits

Physiol. Plant. 150, 2014 497

Fig. 2. Histochemical GUS staining of typical transgenic Micro-Tom IMG fruit expressing pBI101, 35S-gusA, PDS-gusA, ZDS-gusA,LYCB-gusA, BCH-gusA, LYCE-gusA, respectively. Abbreviations: pBI101,pBI101; 35S-gusA, pBI121; PDS-gusA, pBI-GlPDSPro-GUS; ZDS-gusA,pBI-GlZDSPro-GUS; LYCB-gusA, pBI-GlLYCBPro-GUS; BCH-gusA, pBI-GlBCHPro-GUS (BCH-gusA); LYCE-gusA, pBI-GlLYCEPro-GUS.

(Fig. 3). Overall, the GUS expression of the five con-structs during fruit development was similar, indicatingthat all cis-acting elements necessary to confer GUSexpression in tomato fruits are contained within theisolated promoter sequences.

Promoter analysis

A search of the PlantCARE database of plant cis-regu-latory elements (Lescot et al. 2002, http://bioinformatics.psb.ugent.be/webtools/plantcare/html) revealed poten-tial TATA and CAAT boxes in all five promoters (Table 2;Figs S1–S5, Supporting Information). In addition to thesecore promoter elements, we found that all five promoterscontained three common cis-regulatory motifs: CGTCA-motif which is linked to methyl jasmonate (MeJA)signaling (Rouster et al. 1997), and ATCTA-motif whichwas recently found in the AtPSY promoter and is knownto interact with the ethylene response transcriptionfactor RAP2.2 (Welsch et al. 2003, 2007). Another motifpresent was Skn-1_motif (GTCAT) that is required forendosperm expression (Takaiwa et al. 1991).

Carotenoid biosynthesis is known to be regulated bylight (Von Lintig et al. 1997, Simkin et al. 2003, Li et al.

Fig. 3. Histochemical GUS staining of typical transgenic Micro-Tom MG fruit expressing pBI101, 35S-gusA, PDS-gusA, ZDS-gusA,LYCB-gusA, BCH-gusA, LYCE-gusA, respectively. Abbreviations: pBI101,pBI101; 35S-gusA, pBI121; PDS-gusA, pBI-GlPDSPro-GUS; ZDS-gusA,pBI-GlZDSPro-GUS; LYCB-gusA, pBI-GlLYCBPro-GUS; BCH-gusA, pBI-GlBCHPro-GUS (BCH-gusA); LYCE-gusA, pBI-GlLYCEPro-GUS.

2008, Welsch et al. 2008), and we identified at leastone light-response motif in each promoter, e.g. BoxI, Box II, ATC, GT-1, TCT, Sp1, G-box, ACE and AE-box (Table 3; Figs S1–S5) providing the basis for theregulation of carotenogenic gene expression accordingto day length and other cues involved in the controlof flower development. At least one MYB binding site,involved in drought tolerance (Abe et al. 2003), was alsofound in each promoter (Table 3).

We also identified a number of additional cis-actingelements shared among some of the promoters, includingan ethylene response element (ERE) in the GlPDS andGlLYCE promoters, abscisic acid responsive element(ABRE) and auxin response element (TGA-element) inthe GlBCH and GlLYCE promoters, and a W1-box thatresponds to fungal elicitors in the GlLYCB and GlLYCEpromoters. The GlZDS, GlLYCB and GlLYCE promotersshared a GCN4 motif common to endosperm-specificpromoters, whereas the GlLYCE and GlBCH promoterscontained four TA-rich regions and an ATGCAAAT motifthat binds GCN4 in rice, respectively (Table 2).

Discussion

The carotenoid biosynthesis pathway (Fig. 1) hasbeen completely elucidated through a combinationof biochemical, genetic and transgenic approaches

498 Physiol. Plant. 150, 2014

Tab

le2.

Spec

ific

cis-

actin

gre

gula

tory

elem

ents

foun

din

diff

eren

tca

rote

noge

nic

gene

prom

oter

sfr

omG

entia

nalu

tea.

The

posi

tions

ofth

eci

s-ac

ting

regu

lato

ryel

emen

tsar

ede

note

dre

lativ

eto

the

posi

tions

ofcD

NA

(+1

isth

efir

stnu

cleo

tide

ofcD

NA

).G

enBa

nkac

cess

ion

num

bers

for

the

prom

oter

sequ

ence

san

alyz

edar

eas

follo

ws:

PDS,

DQ

2269

92;

ZDS,

JQ41

7647

;LY

CB,

JQ41

7648

;BC

H,

EF20

3261

;ZEP

,EF2

0326

2;LY

CE,

EU59

2045

.

Prom

oter

TATA

-box

CA

AT-

box

ERE

ABR

E

TGA

-box

(aux

inre

spon

se)

TGA

-ele

men

t(a

uxin

resp

onse

)Bo

x-W

1G

CN

4A

TGC

AA

AT

mot

ifTA

-ric

hre

gion

PDS (1

077

bp)

−76 (TA

TATA

TA)

−624

,−55

2(C

AA

T);−

287,

−39

(CA

AA

T);−

433,

−168

,−87

(CA

ATT

)

−103

6,−1

003

(ATT

TCA

AA

)

ZDS (7

66bp

)−4

7(T

AA

TA);

−16

(TTT

A)

−654

,−62

6,−2

85(C

AA

T);

−706

,−58

0,−5

60,−

501;

−380

,−60

(CA

AA

T);

−415

,−35

3,−1

57(C

AA

TT)

−302

(TG

TG

TCA

)

LYC

B(1

506

bp)

−44 (TA

TAA

A)

−926

,−73

5,−6

85,−

579,

−517

,−40

3,−1

45(C

AA

T);−

1496

,−13

61,

−725

,−30

8(C

AA

AT)

;−8

71,−

831,

−776

,−53

3,−3

81,−

158,

−151

,−49

(CA

ATT

)

−112

3(T

TGA

CC

)−6

26 (TG

AG

TCA

)

BCH (1

744

bp)

−142 (TA

ATA

)−1

733,

−155

0,−1

147,

−113

6,−3

53,−

22(C

AA

T);−

1390

,−13

10,

−116

3,−6

47,−

483

(CA

AA

T);−

1540

,−12

08,

−115

4,−9

35,−

928,

−264

,−19

5,−1

88,−

155,

−137

(CA

ATT

)

−167

0,−7

38(C

AC

GTG

);−7

0(T

AC

GTG

)

−114

4(T

GA

CG

-TA

A)

−139

9(A

AC

GA

C)

−116

6(A

TA-

CA

AA

T)

LYC

E(9

38bp

)−7

3 (TA

ATA

)−8

34(C

AA

T);−

683,

−599

,−9

3(C

AA

AT)

−157 (A

TTTC

AA

A)

−440

,−22

(CA

CG

TG);

−21

(AC

GTG

GC

)

−908 (A

AC

GA

C)

−696 (TTG

AC

C)

−27 (C

AA

GC

CA

)−3

91,−

389,

−387

,−38

5(T

ATA

TATA

TATA

TATA

TATA

TA)

Physiol. Plant. 150, 2014 499

Table 3. The consensus cis-acting regulatory elements in different promoters of carotenogenic genes from Gentiana lutea. The positions of thecis-acting regulatory elements are denoted relative to the positions of cDNA (+1 is the first nucleotide of cDNA). GenBank accession numbers for thepromoter sequences analyzed are as follows: PDS, DQ226992; ZDS, JQ417647; LYCB, JQ417648; BCH, EF203261; ZEP, EF203262; LYCE, EU592045.

Promoter

Cis-acting

regulatory element

involved in light

responsiveness

ATCTA-motif

(RAP2.2 motif

in AtPSY )

Cis-acting

regulatory element

involved in MeJA-

responsiveness

Skn-1_motif

required for

endosperm

expression

MYB binding

site involved

in drought-

inducibility

PDS (1077 bp) −1035, −1002 (Box I, TTTCAAA);−795, −607 (ATC-motif,

AGTAATCT)

−603 (ATCTA) −597 (CGTCA-motif,CGTCA);

−266 (TGACG-motif,TGACG)

−596 (GTCAT) −46 (MBS,TAACTG)

ZDS (766 bp) −420 (TCT-motif, TCTTAC);−397 (GT-1 motif, GGTTAAT);−49 (Box 4, ATTAAT)

−6 (ATCTA) −743 (CGTCA-motif,CGTCA)

−742, −299(GTCAT)

−546 (MBS,TAACTG)

LYCB (1506 bp) −1407 (TCT-motif, TCTTAC) −1352, −954(ATCTA)

−18 (CGTCA-motif,CGTCA)

−623, −190(GTCAT)

−1372 (MBS,CAACTG)

BCH (1744 bp) −1670, −738 (G-box, CACGTG);−1427 (G-box, CACGTT);−114 (G-box, TGACGTGG);−70 (G-box, TACGTG);−1516, −1254 (Box 4, ATTAAT);−1453 (AE-box, AGAAACTT);−1410 (AE-box, AGAAACAT);−112 (ACE, ACGTGGA);−1725, −1420 (MRE,

AACCTAA);−33 (GAG-motif, AGAGAGT)

−1637, −1617,−1529, −415(ATCTA)

−1144, −114(TGACG-motif,TGACG); −817(CGTCA-motif,CGTCA)

−1287, −967,−816, −227,−207 (GTCAT)

−1602 (MBS,TAACTG)

ZEP (2225 bp) −2217 (GT-1-motif,GCGGTAATT);

−2160, −275 (Box I, TTTCAAA);−1613 (G-box, CACATGG);−1585, −1355 (G-box,

CACGTC);−1567 (chs-CMA2a,

TCACTTGA);−1068 (GAG-motif, AGAGAGT);−599, −262, −237, −215 (Box

4, ATTAAT)

−2191, −1512(ATCTA)

−2064, −1527(CGTCA-motif,CGTCA); −950(TGACG-motif,TGACG)

−658, −557(GTCAT)

−1191, −997(MBS,CAACTG)

LYCE (938 bp) −614 (Sp1, GGGCGG);

−531 (ATC-motif, AGCTATCCA);−464 (G-box, CACATGG);−440, −22 (G-Box, CACGTG);−156 (Box I, TTTCAAA);−23 (Box II, CCACGTGGC)

−765, −83(ATCTA)

−588 (TGACG-motif,TGACG);

−485 (CGTCA-motif,CGTCA)

−539, −435(GTCAT)

−524 (MBS,CAACTG)

(Zhu et al. 2007, 2010, 2013, Giuliano et al. 2008, Farreet al. 2010). During flower development in G. lutea,carotenoids accumulate in the petals, accompanied bya shift in profile from lutein derived from α-caroteneto xanthophylls derived from β-carotene (Zhu et al.2003). PSY and ZDS mRNA levels increase six- tosevenfold during this process, whereas PDS and othergenes required for β-carotene formation and metabolism(LYCB, BCH and ZEP) are induced approximatelytwofold (Zhu et al. 2002, 2003). In contrast, theabundance of the LYCE transcript is reduced to lessthan half of its normal value (Zhu et al. 2003). These

antagonistic changes shift the relative abundance ofcarotenoids from the α to β branches. These data,combined with the developmental regulation of theGlZEP promoter during tomato fruit development andchromoplast differentiation (Yang et al. 2012), suggestthat carotenogenesis in the flower petal is regulated bymultiple developmental and environmental cues.

It is clear that carotenogenesis in G. lutea petalsduring flower development is under transcriptionalcontrol (Zhu et al. 2002, 2003), but the mechanisms arelargely unknown. The coordinated expression of severalgenes may reflect similarities between their promoters,

500 Physiol. Plant. 150, 2014

or potentially could reflect clustering within particularchromosomal domain (Caron et al. 2001, Winzer et al.2012). Similar expression profiles may also be causedby the coordinated action of more than one set oftranscription factors, in which case the promoter regionsof co-regulated genes would be heterogeneous, e.g.the well-known C1 and R transcription factors whichcontrol the flavonoid biosynthesis pathway (Grotewoldet al. 2000, and other references therein).

To investigate the basis of coordinated carotenogenicgene expression during G. lutea flower development,we cloned promoter fragments upstream of GlPDS,GlZDS, GlLYCB, GlBCH and GlLYCE directly fromG. lutea genomic DNA by inverse PCR. Sequencingidentified putative TATA and CAAT boxes in all thepromoters (Table 2). We investigated GUS expressiondriven by these promoters in ripening tomato fruits whichprovide as a useful model for studying the regulationof carotenogenic gene expression in chromoplast-containing petals (Yang et al. 2012). We evaluatedthe constructs PDS-gusA, ZDS-gusA, LYCB-gusA, BCH-gusA and LYCE-gusA by transient expression in tomatoand found that all the promoters were sufficient fordevelopmentally regulated reporter gene expression inthe tomato fruits.

The GlLYCB and GlBCH promoters sustained strongGUS expression in chromoplast-containing MG fruits(Fig. 3) but only low levels in chloroplast-containingIMG fruits (Fig. 2). This is similar to the endogenousgene expression profiles of the genes in G. lutea (Zhuet al. 2002, 2003) and the expression of the GlZEPpromoter in transgenic tomatoes (Yang et al. 2012),confirming that carotenogenic gene promoter activityis strictly associated with tomato fruit development andchromoplast differentiation.

The integration of expression profiles and promotersequences can help to identify common and putativefunctionally relevant cis-acting elements (Werner 2001,Kim and Kim 2006, Yamamoto et al. 2011). Comparativebioinformatic studies on promoter regions of carotenoidgenes may elucidate common binding motifs involvedin carotenoid formation (Fraser and Bramley 2004). Wetherefore compared the isolated promoter sequenceswith the GlZEP promoter (Yang et al. 2012) using thePlant-CARE and PLACE databases (Higo et al. 1999,Lescot et al. 2002). All the promoters contained at leastone CGTCA-motif involved in the response to MeJA, oneATCTA-motif that interacts with the ethylene responsefactor RAP2.2 in Arabidopsis thaliana (Welsch et al.2003, 2007), one Skn-1_motif (GTCAT) that is requiredfor endosperm expression, and consensus motifsinvolved in responses to light and drought (Table 3).These shared cis-acting elements could represent

binding sites for transcription factors responsible forco-regulation (Kreiman 2004, Haberer et al. 2006,Obayashi et al. 2007, Lenka et al. 2009). The existenceof CGTCA and ATCTA motifs representing two hormoneresponse pathways may give an example of functionalcooperation or independent activities.

The ATCTA motif in the AtPSY promoter bindsRAP2.2, a member of the APETALA2/ERE-bindingprotein family. Overexpression of RAP2.2 induced onlyminor changes in the carotenoid profile of non-greenArabidopsis tissues, suggesting that additional factorsmay contribute to the regulation of PSY (Welsch et al.2007). Single copy of the ATCTA motif is found in othercarotenogenic gene promoters such as Arabidopsis DXS(encoding deoxyxylulose phosphate synthase) and PDS(Welsch et al. 2007), tomato and maize PDS (Welschet al. 2007), and tomato CYC-B encoding lycopene β-cyclase (Dalal et al. 2010). This motif is also present inseveral promoters involved in tocopherol biosynthesis(Welsch et al. 2007).

MeJA response elements are often found in genesinvolved in plant pathogen interactions (Rouster et al.1997). Lycopene synthesis in fruits treated with MeJAshowed an inverted U-shaped dose response whichsignificantly enhanced the lycopene content of the fruitsand restored lycopene accumulation in mutants deficientin jasmonic acid (spr2 and def1) at a low concentrationof 0.5 μM (Liu et al. 2012). The tomato DXS, GGPS(geranylgeranyl diphosphate synthase), PSY1 and PDSgenes were up-regulated by MeJA treatment (Liu et al.2012). Several other hormone response elements wereidentified in the promoters, including those responsiblefor the transduction of auxin and ethylene responses(Tables 2 and 3; Figs S1–S5).

The GlLYCE promoter contained all the cis-regulatoryelements found in other carotenogenic gene promotersexcept the ATGCAAAT motif present in the GlBCHpromoter (Tables 2 and 3). The TA-rich region wasunique to the GlLYCE promoter (Table 2). The diversecis-regulatory elements of the GlLYCE gene promoterimply that diverse cis-acting elements may play crucialroles in determining the complex endogenous expressionpattern in G. lutea when GlLYCE expression is firstsuppressed in petals from stage 1 (S1, <1.5 cm longwith hard bud) to S2 (1.5–2.5 cm long), then rapidlyup-regulated from S2 to S3 (2.5–3.5 cm long with thefirst signs of pigmentation) and suppressed again fromS3 to S5 (fully mature open and pigmented flowers) (Zhuet al. 2002, 2003). The expression of GlLYCB, GlBCHand GlZEP genes was induced by twofold from S1 toS5, reflecting the abundance of carotenoid compoundsfrom the α- to β-branches of the pathway (Zhuet al. 2003).

Physiol. Plant. 150, 2014 501

Compelling experimental evidence from severalplant species supports the transcriptional control ofcarotenogenic gene expression and carotenogenesis (Luand Li 2008, Cazzonelli and Pogson 2010, Farre et al.2011). However, only two types of transcription factors(RAP2.2 and PIFs) have been identified that directlyinteract with the Arabidopsis PSY promoter (Welschet al. 2007, Toledo-Ortiz et al. 2010). Comparativeanalysis of the cis-acting elements in the G. luteacarotenogenic gene promoters not only supportsprevious data suggesting that the genes are regulatedby light and MeJA, but also indicates the involvementof other hormones, in particular auxin (GlLYCE andGlBCH promoters). The well-established interactionbetween auxins and the carotenoid-derived hormonestrigolactone was recently reported (Hayward et al.2009). The shared three common cis-acting elements(CGTCA-motif, ATCTA-motif and Skn-1_motif) mayrepresent binding sites for transcription factors responsi-ble for co-regulation of carotenogenic gene expressionduring G. lutea flower development. Thus, our data offernew insights into the regulatory mechanisms controllingthe coordinated upregulation of carotenogenic geneexpression during flower development in G. lutea.

Acknowledgements – This work was supported by theNational Natural Science Foundation of China (31270344);Fundamental Research Funds for the Central Universitiesto Q. Y. (DL13BA06); MICINN, Spain (BIO2011-23324;BIO2011-22525; PIM2010PKB-00746); European UnionFramework 7 European Research Council IDEAS AdvancedGrant (to P. C.) Program-BIOFORCE.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Fig. S1. Sequence and putative cis-acting regulatoryelements identified in the Gentiana lutea phytoenedesaturase gene (GlPDS) promoter.

Fig. S2. Sequence and putative cis-acting regulatoryelements identified in the Gentiana lutea ζ -carotenedesaturase gene (GlZDS) promoter.

Fig. S3. Sequence and putative cis-acting regulatoryelements identified in the Gentiana lutea lycopene β-cyclase gene (GlLYCB) promoter.

Fig. S4. Sequence and putative cis-acting regulatoryelements identified in the Gentiana lutea lycopene ε-cyclase gene (GlLYCE) promoter.

Fig. S5. Sequence and putative cis-acting regulatoryelements identified in the Gentiana lutea β-carotenehydroxylase (GlBCH) promoter.

Fig. S6. Sequence and putative cis-acting regulatoryelements identified in the Gentiana lutea zeaxanthinepoxidase (GlZEP) gene promoter.

Edited by V. Shulaev

504 Physiol. Plant. 150, 2014