Planta (2006) 224:1291–1301

DOI 10.1007/s00425-006-0308-y

ORIGINAL ARTICLE

Pinoresinol–lariciresinol reductase gene expression and secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds

C. Hano · I. Martin · O. Fliniaux · B. Legrand · L. Gutierrez · R. R. J. Arroo · F. Mesnard · F. Lamblin · E. Lainé

Received: 28 February 2006 / Accepted: 25 April 2006 / Published online: 31 May 2006© Springer-Verlag 2006

Abstract The transcription activity of the pinoresi-nol–lariciresinol reductase (PLR) gene of Linum usita-tissimum (so-called LuPLR), a key gene in lignansynthesis, was studied by RT-PCR and promoter–reporter transgenesis. The promoter was found todrive transcription of a GUSint reporter gene in theseed coats during the Xax seed development. This Wttedwell with the tissue localization monitored by semi-quantitative RT-PCR of LuPLR expression. Accumu-lation of the main Xax lignan secoisolariciresinol dig-lucoside was coherent with LuPLR expression duringseed development. This three-way approach demon-strated that the LuPLR gene is expressed in the seedcoat of Xax seeds, and that the synthesis of PLRenzyme occurs where Xax main lignan is found storedin mature seeds, conWrming its involvement in SDGsynthesis.

Keywords Flaxseed · Lignans · Linum · Pinoresinol–lariciresinol reductase · Promoter · Secoisolariciresinol diglucoside

AbbreviationsGUS �-GlucuronidaseHMG Hydroxyl methylglutarylPLR Pinoresinol–lariciresinol reductaseRT-PCR Reverse transcription-polymerase

chain reactionSDG Secoisolariciresinol diglucosideX-Gluc 5-Bromo-4-chloro-3-indolyl-�-D-glucuronic

acid

Introduction

For the last decades, there has been an increasinginterest to use Xaxseed (Linum usitatissimum L., Lina-ceae) in diet in order to improve nutritional and healthstatus (Oomah 2001). The Xaxseed hull is rich in lign-ans and the embryo is rich in oil, with a high omega 3fatty acid content (Westcott and Muir 2003; Wiesen-born et al. 2003).

Lignans are phenolic compounds that result fromthe stereo-selective dirigent protein-mediated couplingof the 8 and 8� C-atoms of the side chains of two conife-ryl alcohol moieties (Davin et al. 1997). The role ofthese compounds in planta remains unknown. Thestrong antioxidant activity of lignans (Kitts et al. 1999)may suggest a role in protection against peroxidationof the highly unsaturated fatty acids that are abundantin Xaxseed. But, the phenylpropanoid nature of lignanscan also lead to the hypothesis that they are involved inplant defense against biotic stresses as it is the case for

C. Hano · I. Martin · B. Legrand · F. Lamblin · E. Lainé (&)Laboratoire de Biologie des Ligneux et des GrandesCultures, UPRES EA 1207, Antenne ScientiWque Universitaire de Chartres, 21 rue de Loigny la Bataille,28000 Chartres, Francee-mail: eric.laine@univ-orleans.fr

O. Fliniaux · L. Gutierrez · F. MesnardLaboratoire de Phytotechnologie et Génomique Fonctionnelle des Plantes, UPRES EA 3900,Université de Picardie Jules Verne, 1 rue des Louvels, 80037 Amiens cedex 01, France

R. R. J. ArrooLeicester School of Pharmacy—Natural Products Research, De Montfort University, The Gateway, Leicester,LE1 9BH, UK

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a number of other phenolic compounds (Harmathaand Nawrot 2002).

The beneWcial eVects of lignans on human health arewell documented (Westcott and Muir 2003; McCannet al. 2005) and in vitro studies conWrm the observed invivo eVect (Bylund et al. 2005). Lignans were shown toreduce the incidence of breast and prostate cancers bymodulating steroidal hormone synthesis (Adlercreutzand Mazur 1997). The important pharmacologicalproperties and physiological roles of lignans in plantahave led to numerous studies dealing with the biosyn-thesis and the accumulation of these metabolites (Fordet al. 2001; Seidel et al. 2002; Sicilia et al. 2003).

The main Xax lignan is lignan secoisolariciresinol,found mainly under its glycosylated form (secoisolaric-iresinol diglucoside, SDG) is stored as a hydroxym-ethyl glutaryl ester-linked complex (SDG-HMG; Fordet al. 2001). The biological activities of SDG, and itsmammalian lignan derivative enterolactone, have beenextensively studied. SDG mainly accumulates in theouter part of the seed (seed coats). This Wnding hasstimulated use of seed coats (“Flaxhull”) to provideenhanced levels of lignans for use as a functional food.Within this context several processes were developedto provide hull-enriched fractions (Madhusudhan et al.2000; Wiesenborn et al. 2003).

The following biosynthetic pathway of secoisolaric-iresinol in Xax has been proposed by Ford et al. (2001):it is speculated that a dirigent protein-assisted couplingof E-coniferyl alcohol leads to (¡)-pinoresinol which isconverted into (¡)-lariciresinol and (+)-secoisolaric-iresinol via the action of pinoresinol–lariciresinolreductase (PLR; Fig. 1), and Wnally into (+)-matairesi-nol. Thus the PLR, that catalyses two steps of the for-mation of Xax lignans (Fig. 1), would be a key enzymefor SDG synthesis.

The biosynthesis of lignans and isoXavonoidsinvolves phylogenetically related NADPH-dependentreductases: pinoresinol–lariciresinol reductase (PLR),phenylcoumaran benzylic ether reductase (PCBER)

and isoXavone reductase (IFR; Gang et al. 1999; Shojiet al. 2002), which products are presumed to sharecomparable physiological roles in plant defense (Ganget al. 1999). In the last few years, a large number ofthese reductases (so-called IFR “homologues”) havebeen identiWed in various plant species, woody andnon-woody angiosperms and gymnosperms (Davin andLewis 2003). Lewis et al. (2001) and von Heimendahlet al. (2005) have cloned and published Xax PLRencoding cDNAs. Previous experiments have led us toclone a number of Xax cDNAs involved in the regula-tion of phenylpropanoid pathway, amongst which theLuPLR (Hano et al. 2006) and LuPCBER (Attoumbreet al. 2006; AY837829) genes involved in lignan andneolignan metabolism. These two gene sequences werevery similar to the sequence published by von Heimen-dahl et al. (2005).

Expression-localization work has been performedby Kwon et al. (2001) on ligneous species (Forsythiaintermedia and Pinus taeda): in situ hybridizationexperiments showed that PLR and PCBER gene wereexpressed mainly in vascular tissue. Nevertheless, theprecise localization (tissue) and timing of synthesis ofthe Xax lignans are still hypothetical and deserve inves-tigation. There are no published data dealing with spa-tio-temporal regulation of expression of the key genesrelated to lignan synthesis in Xax. Such data could yieldinformation to support hypothesis on the role of thesecompounds in planta and also conWrm the involvementof PLR in Xax lignans synthesis.

To further elucidate the role of LuPLR in Xax, weinvestigated whether spatio-temporal regulation didexist at the transcriptional level for the LuPLR gene.Expression of the Xax PLR gene (LuPLR) was analy-sed by two means: semi-quantitative RT-PCR andobservation of the LuPLR promoter driven expressionin transgenic promoter–reporter plants. For this pur-pose, a 5� Xanking region (putative promoter) of theLuPLR gene was isolated and cloned. Then, a con-struct with a GUSint reporter gene driven by this

Fig. 1 Biosynthetic pathway leading to secoisolariciresinol,the major lignan present in Xax (adapted from Ford et al. 2001).

DP/oxidase dirigent-protein/oxidase complex, PLR pinoresinol–lariciresinol reductase

OH

OMe

OH

coniferyl alcohol x2

+

MeO

HO

O

O

OMe

OH

HH

(-) pinoresinol

8

(-) lariciresinol

OH

OH

OH

OMe

MeO

HO

(+) secoisolariciresinol

PLR

OH

OMe

HO

8’

MeO

HO

O

HO

OMe

OH

HHPLRDP oxidase

NADPH NADPH

8

8’

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potential regulatory upstream sequence was intro-duced into Xax via Agrobacterium tumefaciens. Accu-mulation of the main Xax lignan was monitoredconcomitantly.

Materials and methods

All L. usitatissimum plant material was linseed belong-ing to the cultivars Barbara (supplied by CoopérativeTerre de Lin, St Pierre le Viger, France) or Oliver(supplied by Institut Technique du Lin, Gamaches enVexin, France). Seed development stages were deWnedas described in Table 1.

The LuPLR sequence accession number AX191955(Lewis et al. 2001) was used to design the primers inuse in PCR walking and RT-PCR experiments. AllDNA quantiWcations were performed by Xuorometryusing Hoechst 33258 (Ex: 360 nm; Em: 460 nm; Sigma)following Ausubel et al. (1992) protocol. RNA wasquantiWed using the Ribogreen RNA Quantitation kit(Ex: 490 nm; Em: 520 nm; Molecular Probes).

RNA extraction

RNA was isolated from various linseed organs. Forimmature seeds, embryos were excised out of the seedcoats and were submitted separately to RNA isolationusing the following method. Total RNA was extractedfrom 200 mg of frozen tissues ground in liquid nitrogenand then transferred in 2 ml TLES buVer (100 mMTris–HCl pH 8.0, 100 mM LiCl, 10 mM EDTA, 5%SDS) and 2% PEG 8000 containing tube. After 10 minof incubation at room temperature (in order to get ridof the mucilaginous stuV and the phenolic compoundsthat hinder correct RNA isolation) and centrifugation(10 min, 12,000g), the supernatant was transferred to anew tube with 2 ml of 80°C heated phenol. After cen-trifugation (10 min, 12,000g), one volume of a 4 M LiClsolution was added to the supernatant and incubated

one night at room temperature. After centrifugation(10 min, 12,000g), the pellet was dissolved in 125 �l ofDEPC-treated water. Total RNA were then precipi-tated by sodium acetate (0.3 M, pH 5.2) and ethanol.After 1-h incubation at ¡80°C, RNA were washed with70% ethanol and dissolved in 20 �l DEPC-treatedwater. Additional puriWcation was performed by usingNucleoSpin RNA clean up (Macherey-Nagel) andRNA were then submitted to digestion with RNasefree DNase (Promega).

Semi-quantitative RT-PCR

First-strand cDNA was synthesized at 37°C for 1 h.BrieXy, 0.5 �g of total RNA was incubated in reversetranscription buVer with 4 units of Omniscript ReverseTranscriptase (Qiagen), 0.5 mM of each dNTP, 1 �M ofoligo-dT primer and 10 units of RNAse inhibitor (RNa-sin, Promega). A 939 bp fragment of the LuPLR cDNAwas ampliWed using PLRF1 forward primer (5�-ATGGGGCGGTGCAGAGTTCT-3�) and PLR-R1reverse primer (5�-TCAAAGGTAGATCATCAGA-3�)designed from a Xax PLR cDNA sequence (accessionnumber AX191955). To normalize the amount ofmRNA in each PCR reaction, a PCR product (632 bp)corresponding to the exon 2 of the ACTIN gene wasampliWed with ACT-F2 forward primer (5�-TCTGGAGATGGTGTGAGCCACAC-3�) and ACT-R2reverse primer (5�-GGAAGGTACTGAGGGAGGCCAAG-3�) designed from the tobacco sequence.cDNA fragments were ampliWed during 25, 27 and30 cycles.

Isolation of genomic DNA

Total genomic DNA of L. usitatissimum (Xax) leaveswas extracted according to Doyle and Doyle (1990)with some modiWcations earlier described by Rogeret al. (2001). Genomic DNA templates were preparedby using the PCR walking approach described by Devic

Table 1 Characterization of Xaxseed developmental stages. Fresh weights were determined as the mean of 100 seeds, seed-coats or em-bryos fresh weight measurements

ND not determined

Stages Day afterXowering

Whole seed FWper seed (mg)

Seed coat FWper seed (mg)

Embryo FW per seed (mg)

Morphological features

S0 4 3.6 3.6 – No visible embryoS1 10 9.56 8.28 0.28 0.5 mm embryoS2 16 8.65 7.50 1.15 From 2 to 3 mm embryoS3 20 8.22 6.26 1.96 From 4 to 5 mm embryoS4 24 9.57 5.29 4.28 5 mm embryoS5 35 10.2 ND ND Nearly mature seed

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et al. (1997). Isolated genomic DNA was digested tocompletion with several restriction enzymes, eitherHpaI, EcoRV, ScaI, DraI, PvuII or SspI, creating bluntended fragments that were then used independently.Adaptator oligonucleotide duplex were prepared andligated to the DNA fragments resulting from enzymaticdigestion as described by Devic et al. (1997). The sixadaptator-ligated DNA fragment libraries were used asDNA templates for further PCR.

PLR promoter cloning by PCR walking

The Xax promoter nucleotide sequence was ampliWedusing a nested PCR strategy. Two primers, namedPLR1 and PLR2, were designed after reference to thespeciWc nucleotide sequence of PLR cDNA (Lewiset al. 2001; GenBank accession number AX191955).

A Wrst PCR reaction was carried out using 5�-TCCGCTTGCCTATGTACCCGGTACC-3� (25-mer,external gene speciWc primer) as 3� primer (primerPLR1) and 5�-GGATCCTAATACGACTCACTATAGGGC-3� (27-mer, external adaptator oligonu-cleotide speciWc primer) as 5� primer (primer AP1).The primary PCR was done in a 50 �l reaction volumecontaining 300 nM of primer AP1, 300 nM of primerPLR1, 300 nM of each dNTPs, 5 ng of stock DNA and2 mM MgCl2. In order to decrease the probability ofobtaining mutations, 0.8 �l of a high Wdelity polymer-ase mix (pfu, Promega) was used. A three step PCRwas performed for 40 cycles, including 45 s of denatur-ation at 94°C, and 45 s of annealing at 67°C and 5 minelongation at 70°C followed by 7 min of additionalelongation. Then 2 �l of the PCR product was used fora second round of ampliWcation to re-amplify the prod-ucts of the Wrst PCR using 5�-CCACCAGAACTCTGCACCGCCCCAT-3� (25-mer, internal gene spe-ciWc primer) as a 3� primer (primer PLR2) and 5�-CTATAGGGCTCGAGCGGC-3� (18-mer, internaladaptator oligonucleotide speciWc primer) as 5� primer(primer AP2). The secondary three step PCR was per-formed for 35 cycles including 45 s of denaturation at94°C, 45 s of annealing at 62°C, 5 min of elongation at70°C, followed by a Wnal cycle of extension at 72°C for5 min with 350 nM of primer AP2, 400 nM of primerPLR2, and 350 nM of each dNTPs and 0.8 �l of pfupolymerase. PCR products were puriWed using gelextraction kit (Qiagen), tailed and cloned into pGem Teasy vector (Promega) and sequenced on both strands(MWG Biotech AG). VeriWcation of the position ofthe putative promoter sequence was achieved by PCRusing a 5� primer designed in the middle of thesequence obtained by PCR walking (5�-GGACCCAAGAGATTTTGAGC-3�) and a 3� primer

matching a region ca. 100 nucleotides downstream thestart codon (5�-GCTTGAGGACGTAAGTGTCG-3�).

Plasmid construction

To test expression speciWcity of the promoter in trans-genic plants a putative promoter fragment was clonedupstream the GUSint reporter gene (containing anintron) into the HindIII–XbaI sites of pGIBin19 plas-mid, in order to create a transcriptional fusion with theGUSint reporter gene. A promoter sequence 633 bplong was synthesized by PCR using the “DraI” geno-mic fragment cloned into pGem T easy as template.The following set of linker: primers was used to gener-ate the deletion for directional cloning upstream of theGUSint reporter gene: PDPPLR: 5�-CGATCTAGATGGTTGGATTTCTCTTGTCG-3� and PMPPLR:5�-CTTAAGCTTGGACCCAAGAGATTTTGAGC-3� yielding a 633 bp fragment.

Three nucleotides were added upstream of therestriction sites in order to facilitate enzymatic diges-tion of the PCR-generated fragments. AmpliWed frag-ments were puriWed using a gel extraction kit (Qiagen).

The resulting chimeric construct was designatedp638PLR-GUS, and its construction conWrmed by PCRanalysis using a 5� primer chosen within the nptIIselectable marker gene and a 3� primer within the GUSreporter gene. The plasmid was then transferred intothe disarmed A. tumefaciens strain GV3101 (pGV2260)by triparental mating with E. coli strain HB101(pRK2013) as helper.

Flax transgenesis with promoter–reporter fusion construct

Flax (cv. Barbara) transgenic plants were obtained fol-lowing the protocol described by Lacoux et al. (2003).BrieXy, 5-day-old seedling hypocotyls were excised andcocultivated for 2 days with A. tumefaciens. Timentin(500 mg/l; Kalys, France) was used to remove agrobac-teria in the kanamycin-containing (300 mg/l) selectivemedium. Putatively transformed hypocotyl-derivedcalli, actively growing on selective medium, were sub-cultured every 2 weeks and allowed for bud productionon MS derived medium (Murashige and Skoog 1962)containing both auxin and cytokinin, and were main-tained at 25°C under a 16-h photoperiod. After6 months of subcultures on selective medium, theabsence of remaining bacteria was checked, and thepresence of the transgene in the regenerated in vitrogrown plantlets was veriWed by isolating DNA andusing PCR to detect the presence of the LuPLRpromoter–GUSint fusions. Shoots were weaned

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progressively and cultured in growth room with 16-hlight period.

A parallel transformation was carried out with aconstruct containing the CaMV35S promoter fused tothe GUSint gene.

Histochemical assays of �-glucuronidase

Histochemical staining for �-glucuronidase (GUS)activity was performed as described by JeVerson et al.(1987) and modiWed by Kogushi et al. (1990) to avoidbackground (i.e. with 20% methanol in the 5-bromo-4-chloro-3-indolyl-�-D-glucuronic acid (X-Gluc) solu-tion). Fragments of stems, roots and leaves (in vitrogrown plants) as well as bolls and seeds (greenhouseplants) were hand-cut and subsequently incubated with1 mg/l X-Gluc (Kalys). Staining was allowed to pro-ceed at 37°C until blue stain had developed in the sam-ples. Samples were cleared of chlorophyll byincubation in 95% ethanol.

Lignan content evaluation

The lignan content was evaluated by an HPLC pro-cedure based on the method developed by Ford et al.(2001). BrieXy, each sample was ground after lyophil-ization and then extracted with methanol 70%. OnceWltered, the extract was either directly analysed, orsubmitted to an alkaline treatment (NaOH) prior toanalysis, in order to observe, respectively, the solubleor the ester-linked phenolic compounds. Forinstance, an alkali treatment will break up the SDG-HMG oligomers. HPLC analysis was performed witha KROMASIL® C18 column (5 �m, 250 mm£ 4.6 mm; Macherey Nagel) and with a mobile phaseconstituted of a mixture of acidiWed water andMeCN. Lignans and hydroxycinnamic acids wereidentiWed by comparison of their retention timeswith authentic standards and by mass measurements,which were obtained by HPLC electrospray ioniza-tion mass spectrometric analyses. LC–ESI–MS wererecorded on a Waters 2695 Alliance/ZQ 4000micromass system. The ESI–LC–MS operating

parameters were as follows: HPLC system (seebefore); electrospray capillary temperature at 250°C,capillary voltage at 3.5 V, gas Xow (N2) set at 350 l/hfor drying and at 50 l/h for nebulizing, spray voltageat 3.5 kV. All spectra were obtained in the positiveand negative-ion mode, over an m/z range of 50–1,950, at one scan every 2 s, and collected in the formof continuum data.

Stages 0 and 1, and stages 2 and 3 were pooled forlignan content analysis.

Statistical treatment of data

Statistical analysis was performed with SigmaStat 2.03.Means were compared by one-way analysis of vari-ance. All statistical tests were considered signiWcant atP < 0.05.

Results

Semi-quantitative RT-PCR monitoring of the endogenous LuPLR gene expression

The expression of lignan reductase gene in developingXax seed was monitored by semi-quantitative RT-PCR(Fig. 2). A number of 27 PCR-ampliWcation cycleswere chosen because it allowed the best results,enabling in the same time the visualization of ACTINgene and the LuPLR gene transcription. The PCRproducts were of the expected size for LuPLR, andACTIN control transcripts were detected in everysample.

The LuPLR expression was found in seed coats orwhole seeds at any developmental stage analysed. Incontrast, expression was absent or very low in isolatedembryos (Fig. 2). For example at stage 3 (described inFig. 4e–g) a strong LuPLR expression was detected inseed coats whereas it was nearly absent in embryos(Fig. 2). Expression in nearly mature seed (i.e. S5;Table 1) was lower than in previous stages.

Under the PCR-ampliWcation conditions used, noampliWed product could be detected in other testedorgans, i.e. leaves, stems and roots were devoid of visi-ble LuPLR transcription activity.

Cloning and analysis of the promoter sequence

Devic et al. (1997) used two-step PCR (annealing andpolymerization at the same temperature), but we per-formed three-step PCR to yield ampliWcation products.Optimal polymerization temperature for the enzyme

Fig. 2 RT-PCR analysis of LuPLR gene expression during Xax-seed development. Total RNA isolated from whole seed (WS),seed coat (SC) or embryo (EM) were subjected to RT-PCR semiquantitative analysis using an ACTIN gene as internal control.The diVerent stages (0–5) are described in Table 1. Ten microli-tres of 27-cycle PCR products were loaded on 1% (w:v) agarosegel

WS0 SC1 EM1 SC2 EM2 SC3 EM3 WS4 WS5

LuPLR

ACTIN

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we used was as high as 72°C, which was probably toohigh to allow annealing. A 1,188 bp fragment wasobtained after the second (nested) PCR using AP2 andPLR2 primers with the DraI restriction fragmentlibrary as template. Sequencing results conWrmed thatthis sequence was upstream the LuPLR gene (Fig. 3).

A TATA box (TCTCTATATATCG) matching theconsensus TCACTATATATAG, is located 33 basesupstream the putative transcription start determined asbeing the A (with a C two nucleotides downstream),this distance being a regular feature of eukaryotic pro-moters (Zhu et al. 1995). The transcription start posi-tion located 50 bp upstream the translation start (A ofATG) was conWrmed by rapid ampliWcation of cDNAend (RACE)-PCR. This 50 bp-long 5� UTR is AT richand Wts well the consensus for dicots UTR (Joshi et al.1997).

Putative cis-acting regulatory sequences (boxes)were detected using the data available on the followingdatabases: Plant CARE (http://www.sphinx.rug.ac.be:8080/PlantCARE:index.htm; Lescot et al. 2002) andPLACE (http://www.dna.aVrc.go.jp/PLACE/; Higoet al. 1999). The LuPLR promoter sequence isolatedusing PCR walking contained in the last 650 bp a num-ber of boxes that are thought to play a role in various

regulatory functions, including environmental controlof expression [elicitor (WRKY box), wounding (Wbox) or abscisic acid (ABRE, E-box) responsive ele-ments; Fig. 3]. Myb binding boxes (MYB1 and MYB2;Fig. 3) were also found in this sequence.

Promoter-driven expression in transgenic Xax cultures

As analysis of the 5� Xanking region of the LuPLRgene revealed that a number of putative cis-acting reg-ulatory elements were located in the last (proximal)650 bp of the putative LuPLR promoter (Fig. 3), weused this region for promoter–reporter experiments.

Transgenic Linum usitatisssimum plants were grownin greenhouse up to seed production. As shown inFig. 4, no expression could be detected in vegetativeorgans (stems, leaves, roots) of LuPLR-GUSint trans-genic plants (Fig. 4c, d). Control experiments per-formed with the CaMV35S-GUSint construct showedstrong staining in these organs (Fig. 4a, b).

On the other hand, a strong tissue speciWcity ofexpression in LuPLR-GUSint transgenic plants wasnoticed in developing seeds (Fig. 4n–q). This promoterdriven expression was mainly localized in seed coats(see arrows, Fig. 4n–q) while embryos did not exhibit

Fig. 3 Nucleotide sequence of the 5� non-coding region of theLuPLR gene (Genbank accession number AY654626). The nu-cleotides are numbered starting from the A(1) of the ATG startcodon (translation initiation, double boxed). The transcriptionstart (TS) and the putative TATA box are boxed with a single line.The oligonucleotides (PDPPLR and PMPPLR) used for PCRampliWcation of the fragments required for translational gene fu-sions are in bold and italic characters. The putative cis-regulatoryacting elements are boxed: ABRE and E box are putative bindingsites important for abscisic acid (ABA) gene expression regula-

tion (Hattori et al. 1995; Stalberg et al. 1996); MYB1 is a putativeMYB binding site present in promoters of phenylpropanoid bio-synthetic genes (Sablowski et al. 1994; Tamagnone et al. 1998);MYB2 is a putative site for a MYB factor involved in dehydrationand ABA induction gene expression (Abe et al. 1997; Busk andPages 1998); W box is a putative binding site involved in activa-tion of gene expression by wounding (Nishiuchi et al. 2004);WRKY is a putative binding site required for elicitor responsive-ness (Eulgem et al. 1999 )

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any detectable GUS staining (Fig. 4o, p). It is worth tonote that activity of the LuPLR promoter was quitelow as the blue staining was obtained only after anovernight incubation (Fig. 4p, q). While most of theseed coats exhibited blue staining, we noticed that theseed coats with aborted seeds did not exhibit any stain-ing (Fig. 4n), indicating that the seed presence seemsrequired for a correct expression regulation.

Control experiments with the CaMV35S-GUS con-struct showed strong staining mainly in vascularizedareas of the bolls (Fig. 4j, k) as well as in seed coats andembryos (Fig. 4l, m). No GUS staining was detected inseed bolls of untransformed plants (Fig. 4h, i).

Lignan content in seeds

Lignans and related metabolites contents wereexpressed per seed, in order to take into account themuch higher rate of weight increase of the whole seedin comparison with that of the seed coat (see Table 1).HPLC analyses performed before alkaline treatmentof methanol extracts conWrmed that SDG was storedcomplexed to HMG in the seeds, as previouslydescribed (Ford et al. 2001). Indeed, no SDG wasdetected in methanol extracts whereas a peak corre-sponding to SDG complex was observed (mainly SDG-di-HMG), increasing with the development stage of

Fig. 4 Histochemical “X-Gluc” assay of the GUS activity in Li-num usitatissimum wild type plants (h, i), CaMV35S (a, b, j–m)and LuPLR promoters-GUSint reporter (c, d, n–q) L. usitatissi-mum transgenic plants. The blue staining indicates the localiza-tion of the GUS activity. Aspect of wild type Xax boll, seed andembryo at developmental stage 3 (e–g). a Roots of CaMV35S-GUSint transgenic plant; b stem and leaves of CaMV35S pro-moter-GUSint transgenic plant; c roots of LuPLR-GUSint trans-genic plant; d stem and leaves of LuPLR-GUSint transgenicplant; e wild type boll at stage 3; f wild type seed at stage 3; g wild

type embryo at stage 3; h cross section of wild type boll; i crosssection of wild type seed; j boll cross section of CaMV35S-GUSinttransgenic plant; k detail of boll cross section of a Xax CaMV35S-GUSint transgenic plant; l seed cross section of CaMV35S-GU-Sint transgenic plant; m embryo cross section of CaMV35S-GU-Sint transgenic plant; n boll cross section of LuPLR-GUSinttransgenic plant; o detail of boll cross section of LuPLR-GUSinttransgenic plant; p seed cross section of LuPLR-GUSint trans-genic plant; q seed coat cross section of LuPLR-GUSint trans-genic plant. Bars represent 1 mm

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the seeds (Table 2). Moreover, HPLC analyses demon-strated that the contents of free glucosides of hydroxy-cinnamic acids rapidly decreased between the Wrststages S0–S1 (1,347 A.U. per seed) and stage S2–S3(222.3 A.U. per seed) of seed development (Table 2),probably as a consequence of their use as substrate forSDG synthesis. LC–MS experiments revealed that thispeak is composed of 3 hydroxycinnamic acid gluco-sides: ferulic (m/z 379.1 [M + Na]+), coumaric (349.1[M + Na]+) and caVeic acids (365.1 [M + Na]+).

After alkali treatment, SDG was released from itscomplexed form and was quantiWed in the seeds at fourdevelopment stages. SDG quantity per seed increasedas a function of time (Table 2). It increased by morethan 12 times from the Wrst stages S0–S1 (9.8 �g perseed) to nearly mature seed stage S5 (123.9 �g perseed; Table 2).

At the nearly mature stage S5, the SDG content is14.0 mg/gDW. Neither pinoresinol, nor lariciresinolwas detected in any extracts, suggesting a rapid conver-sion of these precursors (pinoresinol and lariciresinol)to secoisolariciresinol and then to SDG.

Discussion

Temporal and spatial expression of the LuPLR gene

In a previous study we have isolated several cDNAsencoding Xax monolignol- and lignan-speciWc biosyn-thetic enzymes, including a PLR gene (LuPLR), inorder to monitor their expression in response to bioticstresses in Xax cell suspension (Attoumbre et al. 2006;Hano et al. 2006). This gene and the heterologouslyexpressed enzyme have been characterized recently byvon Heimendahl et al. (2005).

The availability of gene sequences corresponding tothe lignan-speciWc biosynthetic enzyme PLR provideda mean to study the temporal and spatial regulation oflignan formation. This regulation of lignan biosynthesishas been studied in ligneous species such as P. taeda

and F. intermedia by in situ hybridization (Kwon et al.2001). These authors demonstrated PLR gene expres-sion in secondary xylem. Nevertheless there are nodata on this regulation in non-ligneous species.

Herein we attempted to deWne the expression pat-tern of LuPLR gene in relation with the accumulationof SDG in order to check if the gene expression takesplace where the Wnal accumulation is found, i.e. theseed coat. The second aim was to get an insight into thetiming of both expression of LuPLR and accumulationof SDG along the seed development.

For these purposes, we Wrst monitored LuPLR genetranscription activity by RT-PCR thus demonstratingthat the expression occurs mainly in seed coats what-ever the development stage considered. Expression innearly mature seed (i.e. S5, when the Wnal DW isalready reached) was lower than in previous stages.This could be attributed to the onset of desiccation andthe related decrease of metabolic activity characteriz-ing this developmental stage (Haughn and Chaudhury2005).

The upstream region of LuPLR gene was then iso-lated by PCR walking thus allowing to perform pro-moter–reporter transgenesis experiments in Xax.Except Kim et al. (2002) who expressed promoter-GUS constructs with promoters from dirigent proteingenes from western red cedar in Arabidopsis thaliana,there is no data promoter driven regulation in the Weldof lignan. The present promoter-GUS study performedwith the LuPLR gene promoter in L. usitatissimum isthe Wrst experiment on promoter–reporter experimentsin the Weld of lignan biosynthesis with a promoter fromone plant used to direct GUS expression in the sameplant in comparison to the lignan accumulation in thisplant. This putative LuPLR promoter region containsseveral putative cis-acting regulatory elements such asMyb factors that have been related to play a role inphenylpropanoid pathway control in seed coat(Sharma and Dixon 2005). Construct with the putativepromoter was able to drive expression in Xax cells,indicating that the minimal promoter was included in

Table 2 Lignans and related metabolites content in Xaxseed as a function of the developmental stage. The diVerences in the mean val-ues among the treatment groups were greater than would be expected by change indicating that there is a statistically signiWcant diVer-ence (n = 3 independent determinations)

Seed development stages S0–S1 S2–S3 S4 S5

Means of dry weight per seed (mg § SE) 1.9 § 0.9 6.0 § 1.6 8.5 § 1.7 8.8 § 0.9SDG concentration (mg/gDW § SE) 5.0 § 0.3 8.8 § 0.7 11.2 § 0.2 14.0 § 1.3SDG content (�g per seed § SE) 9.8 § 0.6 53.1 § 4.5 94.9 § 1.7 123.9 § 11.2SDG-complex area (A.U. per seed § SE) 910 § 10.8 8,556.0 § 80.7 17,130 § 95.5 19,446 § 219.7Free hydroxycinnamic acid glycosides area (A.U. per seed § SE)

1,347.1 § 88.9 222.3 § 12.6 147.9 § 33.2 210 § 7.1

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Planta (2006) 224:1291–1301 1299

this sequence. Localization of LuPLR promoter drivenexpression mainly in the seed coats Wts well with thetissue localization found using RT-PCR LuPLR geneexpression monitoring. The same localization ofexpression has been observed for genes involved inother phenolic compounds synthesis such as proantho-cyanidin Xavonoids in A. thaliana seed coat. For exam-ple RT-PCR, in situ hybridization and promoter–reporter experiments demonstrated the localization ofthe BAN gene encoding a dihydroXavonol reductase inA. thaliana seed coats (Devic et al. 1999; Debeaujonet al. 2003).

While most of the seed coats exhibited blue stain-ing, we noticed that empty seed coats (aborted seeds)did not exhibit any staining, indicating that theembryo development may be required for a correctexpression regulation. Indeed, seed coat growth anddiVerentiation are initiated by fertilization and pro-ceed co-ordinately with those of the embryo and theendosperm (Haughn and Chaudhury 2005). It can behypothesized that the ABA synthesized in the matur-ing seed plays a role, especially since an ABRE box(abscisic acid responsive element) is present in theLuPLR promoter, this box being also putatively aplace of interaction with a MYB transcription factor.Nevertheless, ABA is probably present only later inthe seed development, when desiccation begins tooccur.

This expression of LuPLR in the seed coat parttogether with the insect growth regulation activity ofsecoisolariciresinol (Torres et al. 2005) could suggest arole of this compound in seed protection againstinsects. Ralph et al. (2006) found that another lignansynthesis related gene expression (dirigent protein)was stimulated by a stem-boring insect in spruce.

Lignans and precursors contents during Xaxseed development

The progressive accumulation of SDG during seeddevelopment suggests that LuPLR gene expressionresults in a PLR activity allowing lignan synthesis.

Several glycosylated forms of precursors were foundabundantly in seed coats during early seed develop-ment. Then their concentration decreased, probably asa consequence of their use as substrate for SDG syn-thesis as has been proposed by Ford et al. (2001). Nev-ertheless, caution must be exercised when interpretingthe results of metabolism of various phenylpropanoidintermediates since they could be incorporated as wellinto the lignin found in the seed coat (Ford et al. 2001).

The SDG content at the nearly mature stage S5 is inagreement with the values reported in the literature for

mature seeds (Madhusudhan et al. 2000; Ford et al.2001; Eliasson et al. 2003).

In our L. usitatissimum seeds, neither pinoresinol,lariciresinol, matairesinol nor free secoisolariciresinolwere detected whatever the development stage is con-sidered. This could be explained by very low pool sizesof these compounds due to their rapid conversion intoSDG as it as been proposed by Ford et al. (2001).

Conclusion

The chemopreventive lignan SDG is accumulatedmainly in the seed coat of mature dry Xaxseeds (West-cott and Muir 2003; Wiesenborn et al. 2003).

The three-way approach performed in the presentwork allowed us to state that the Xax PLR gene isactively expressed in the seed coats of developing Xaxseeds, all along the seed development. This pattern ofLuPLR gene expression match perfectly with the local-ization of SDG accumulation in the seed coat, thusconWrming the involvement of PLR enzyme in SDGsynthesis in Xax. It also suggests that SDG synthesismost probably occurs at its storage site.

Acknowledgments We wish to thank J.P. Trouvé (CoopérativeTerre de Lin) for the gift of seeds, Institut Technique du Lin foraccess to the experimental Welds, and B. van Droogenbroeck(Ghent University) for seed pictures. Conseil Général d’Eure etLoir, Adittes and Région Centre funded this work. Part of thework was supported by the British Council’s and Alliance Pro-gramme (PN 04.078).

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