Introduction

Lignans are phenolic compounds resulting from the stereo-selective dirigent protein-mediated coupling of the 8 and 8′ C atoms of the side chains of two coniferyl alcohol moieties (Davin et al. 1997). They constitute a widespread family of plant secondary metabolites and present a huge variety of structures due to additional modifications that occur after dimerization (Umezawa 2003). The role of lignans in planta is not yet completely clarified. The strong antioxidant activity of lignans suggests a role in preventing free-radical-induced damage, especially in the case of flaxseeds, rich in unsaturated fatty acids (Kitts et al. 1999; Wiesenborn et al. 2003). Lignans have been investigated for numerous biological activities, which could provide benefits to human health (Adlercreutz 2007; Peterson et al. 2010).

The main flax lignan is secoisolariciresinol (SECO) (Fig. 1), predominantly found in its glycosylated form (secoisolariciresinol diglucoside, SDG) and stored as a hydroxymethyl glutaryl ester-linked complex (SDG-HMG) in the flaxseed coat (Ford et al. 2001; Ghose et al. 2014; Dalisay et al. 2015). The biosynthesis of SECO proposed by Ford et al. (2001) requires a PLR enzyme. Isolation, cloning, and evaluation of the enantioselectivity of two PLRs showed that LuPLR1 is responsible for the formation of (+)-SECO in seeds and LuPLR2 is involved in the early steps of (−)-SECO biosynthesis and derived lignans (yatein, hinokinin, bursehernin, thujaplicatin, and matairesinol dimethyl ether) accumulated mainly in leaves (Fig. 1) (von Heimendahl et al. 2005; Hemmati et al. 2010). These major enzymes are, therefore, two NADPH-dependent pinoresinol–lariciresinol reductases with opposite enantioselectivity. The involvement of the PLR1 enzyme in SDG synthesis was confirmed in flaxseed by detailed investigation of the spatio-temporal regulation of LuPLR1 gene expression, which revealed that LuPLR1 was actively expressed in the flaxseed coat in good correspondence with the location of SDG accumulation (Hano et al. 2006). Furthermore, the LuPLR1 promoter contained putative regulatory boxes related to transcriptional activation by the plant hormone abscisic acid (ABA), regulating a number of physiological events during seed development and maturation (Hano et al. 2006). The results confirmed an enhanced transcriptional activity of LuPLR1 gene and an increased production of SDG in the seed coat after treatment with exogenous ABA (Renouard et al. 2012), which demonstrated the potential improvement in lignan accumulation.

Fig. 1
figure 1

Proposed biosynthetic pathway of lignans in flax (Linum usitatissimum L.) adapted from Ford et al. (2001), Ghose et al. (2014) and Dalisay et al. (2015). LuPLR1 (pinoresinol-lariciresinol reductase 1) enzyme is responsible for the biosynthesis of the major lignan (+)-secoisolariciresinol accumulated in the seedcoat in its diglucosylated form SDG, whereas LuPLR2 (pinoresinol-lariciresinol reductase 2), which is expressed in leaves, leads to the accumulation of another type of lignans derived from the (−)-secoisolariciresinol enantiomer, such as (−)-yatein. pino, pinoresinol; lari, lariciresinol; seco, secoisolariciresinol; SDG, secoisolariciresinol diglucoside; DIR, dirigent protein oxidase complex; UGT, UDP-glucosyl transferase

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Since a growing number of useful properties of lignans are being discovered, it has become of great interest to elucidate the gene expression regulation of these enzymes for a better understanding of lignan natural production. Regarding PLR expression in different flax organs, both LuPLR1 and LuPLR2 transcripts were present in seeds and roots, while only LuPLR2 expression could be detected in stems and leaves (Hemmati et al. 2010). To elucidate further the role of LuPLR2 in flax, we first investigated its spatio-temporal gene expression in relation to yatein biosynthesis in aerial parts using RT-qPCR analysis and observed its promoter-driven expression in transgenic promoter-reporter plants. For this purpose, a 5′ flanking region (putative promoter) of the LuPLR2 gene was isolated and cloned prior to this study. Second, since lignans are thought to be involved in plant defense mechanisms, treatments with methyl jasmonate (MeJA), salicylic acid (SA), and wounding were carried out to provide better insight into LuPLR2 gene expression regulation. The accumulation of yatein, the main flax lignan accumulated in aerial parts, was monitored concomitantly.

Materials and methods

Plant materials

All flax (Linum usitatissimum L.) plant material was generated from the linseed cultivar Barbara supplied by Coopérative Terre de Lin (St Pierre le Viger, France).

Chemicals

All reagents were of analytical grade or higher available purity and were purchased from Thermo-Fisher (Courtaboeuf, France). Deionized water was purified by a Milli-Q water-purification system from Millipore (Molsheim, France). All solutions prepared for HPLC were filtered through 0.45-µm nylon syringe membranes prior to use and o-coumaric acid from Sigma (St Quentin Fallavier, France) was used as the internal standard.

Genomic DNA preparation and LuPLR2 promoter isolation by PCR walking

The total genomic DNA of flax leaves was extracted according to Doyle and Doyle (1990) with some modifications described by Roger et al. (2001). Genomic DNA templates were prepared using the PCR walking approach described by Devic et al. (1997). Isolated genomic DNA was digested to completion with HpaI, EcoRV, ScaI, DraI, PvuII, or SspI restriction enzymes (Promega, Charbonnières-les-Bains, France), creating blunt-ended fragments that were then used independently. DNA fragments were ligated to an adaptor oligonucleotide duplex as described by Devic et al. (1997). The six adaptor-ligated DNA fragment libraries were used as DNA templates for further PCR.

The flax promoter nucleotide sequence was amplified using a nested PCR strategy. Specific primers were designed after reference to the nucleotide sequence of LuPLR2 cDNA (Hemmati et al. 2010; GenBank accession number EU029951). A first PCR reaction was carried out in a 50-µl reaction volume using 5′-CCTCCGATCACCAGGACCTTGCTCTTG-3′ (27-mer, WPLR2a external gene specific primer) as 3′ primer at 300 nM and 5′-GGATCCTAATACGACTCACTATAGGGC-3′ (27-mer, AP1, external adaptor oligonucleotide-specific primer) as 5′ primer at 300 nM, 300 nM of each dNTP, 5 ng of stock DNA, 2-mM MgCl2, and 0.8 µl of a high fidelity polymerase mix (Pfu, Promega). A three-step PCR was performed for 40 cycles, including 45 s of denaturation at 94 °C, and 45 s of annealing at 67 °C and 5-min elongation at 70 °C followed by 7 min of additional elongation. Then, 2 µl of the PCR product was used for a second round of amplification to re-amplify the products of the first PCR using 5′-CCGACGGTGGCGTAGGCCGGGAGGG-3′ (25-mer, WPLR2b, internal gene specific primer) as a 3′ primer at 400 nM and 5′-CTATAGGGCTCGAGCGGC-3′ (18-mer, AP2, internal adaptor oligonucleotide-specific primer) as 5′ primer at 350 nM, 350 nM of each dNTP, and 0.8 µl of Pfu polymerase. The secondary three-step PCR was performed for 35 cycles with the same protocol with an annealing temperature of 62 °C. Here, a 1207-bp-long promoter-containing sequence (excluding the 5′UTR from the numbering) using the DraI restriction fragment library from flax genomic DNA as template was amplified using those primers, purified using a gel extraction kit (Qiagen, Courtabeuf, France), tailed and cloned into the pGem-T Easy vector (Promega), and sequenced on both strands (MWG Biotech AG, Ebersberg, Germany).

Promoter-reporter plasmid constructions

The putative promoter fragment was cloned upstream of the GUSint reporter gene (containing an intron) of the pKGWFS7 plasmid (Karimi et al. 2002), to generate a promoter-reporter construct. The following set of specific primers was used to generate the deletion for directional cloning upstream of the GUSint reporter gene: pLuPLR2-R: 5′-TTTGTCGGATTCGTCGGAGATGG-3′ associated with the following forward primers pLuPLR2-F1: 5′-CACCCTATAGGGCTCGAGCGGCCGCC-3′, or pLuPLR2-F2: 5′-CACCAATATTGTTGTGCTACATGATCG-3′, or pLuPLR2-F3: 5′-CACCGACCAGTCACATTCAAAATTAATC-3′ or pLuPLR2-F4: 5′-CACCGTAGATCAACACAAGTAAGC-3′ yielding, respectively, the 1-1207:GUS, 1-826:GUS, 1-473:GUS, and 1-170:GUS LuPLR2 promoter-reporter constructs. Amplified fragments were purified using a gel extraction kit (Qiagen). The purified fragments were subcloned into the pENTR/D TOPO cloning vector (Invitrogen, Villebon sur Yvette, France) following the manufacturer’s recommendations. The resulting constructs were then transferred into the pKGWFS7 vector (Karimi et al. 2002) using Gateway technology (Invitrogen). The plasmid was then transferred into the disarmed A. tumefaciens strain GV3101 (pGV2260) by triparental mating with E. coli strain HB101 (pRK2013) as helper.

Flax transgenesis with promoter-reporter fusion construct

Transgenic flax (Barbara cv.) plants were obtained following the protocol described by Lacoux et al. (2003). Briefly, 5-day-old seedling hypocotyls were excised and co-cultivated for 2 days with A. tumefaciens. Timentin (500 mg/l; Duchefa, Haarlem, The Netherlands) was used to eliminate agrobacteria in the kanamycin-containing (300 mg/l) selective medium. Putatively transformed hypocotyl-derived calli, actively growing on selective medium, were subcultured every 2 weeks to allow bud production on MS-derived medium (Murashige and Skoog 1962), and were maintained at 25 °C under a 16-h photoperiod. After 6 months of subcultures on selective medium as described in Lacoux et al. (2003), shoots were weaned gradually and cultured in a growth room with a 16-h light period. A parallel transformation was carried out with a construct containing the CaMV35S promoter fused to the GUSint gene.

Generation of RNAi transgenic plants

The generation of the RNAi plants and the homozygous transgenic lines were obtained as previously described in Renouard et al. (2014). Flax transgenesis was performed as described above for promoter-reporter transgenic plants.

Subcellular location of LuPLR2 in tobacco leaves

For cellular location, N-terminal and C-terminal fusions of LuPLR2 with EGFP were generated. The amplified fragments obtained using the primers N-LuPLR2-F: 5′-CACCGCCGCGGGATTCCTATTCC-3′ and N-LuPLR2-R: 5′-TTAAACATATCGTTTCATATACTC-3′ for the N-GFP construct and C-LuPLR2-F: 5′-CACCATGGCCGCGGGATTCCTATTCC-3′ and C-LuPLR2-R: 5′-AACATATCGTTTCATATACTC-3′ for the c-GFP construct were purified and subcloned into the pENTR/D TOPO cloning vector (Invitrogen) following the manufacturer’s recommendations. The resulting constructs were then transferred into the pK7WGF2 and pP7FWG2 vectors (Karimi et al. 2002) using Gateway technology (Invitrogen) to obtain the EGFP fusion vectors named NEGFP::LuPLR2 and CEGFP::LuPLR2, respectively.

The plasmids were then transferred into the disarmed A. tumefaciens strain GV3101 (pGV2260) with E. coli strain HB101 (pRK2013) as helper.

Pieces of tobacco leaves were wounded and incubated in MS medium with agrobacteria for 3 days in a dark room. Then, tobacco leaves were washed four times with 10 ml of MS medium containing 1 g/l of timentin (15:1, w/w) mixture of ticarcillin disodium and potassium clavulanate (Duchefa) to eliminate bacteria and then plated onto agar MS medium containing 250 mg/l kanamycin and 500 mg/l timentin (Duchefa) and incubated at 25 °C in the dark.

Fluorescence microscopy

Microscopy was performed using transformed tobacco leaves placed in water on a glass slide and covered with a cover slip. An Olympus system TCS SP2 (Mannheim, Germany) was used for imaging the GFP signal with an argon laser. Cells were observed under bright field or fluorescence microscopy using a 488-nm excitation filter and a 515–565-nm wavelength pass filter. Pictures were taken with an 8 megapixel eye sight camera (MD239 F/A).

Hydroponic plant growth

Five-day-old seedlings were placed in a hydroponic system containing Hoagland solution (Hoagland and Arnon 1938; Tocquin et al. 2003) as described by Quéro et al. (2014). Plantlets were treated with MeJA at 100 µM or SA at 100 μM. Plantlets were harvested by cutting leaves above the cotyledons: 8 and 24 h after each treatment for RNA extraction and RT-qPCR analysis. For reporter gene activity and lignan extraction, plantlets were harvested after 48 h and 96 h, respectively. All the samples were immediately frozen in liquid nitrogen and stored at −80 °C prior to analysis.

Wounding of flax leaves

Leaves of L. usitatissimum plants grown in the greenhouse (maintained at 25 °C under a 16-h photoperiod (30 µmol m−2 s−1 photosynthetically active radiation) were wounded with serrated forceps at several places along each leaf. Wounded and control leaves were harvested after 8 and 24 h for qPCR analysis, 48 h for β-glucuronidase (MUG) assay and 96 h for lignan analysis. All the samples were immediately frozen in liquid nitrogen and stored at −80 °C prior to analysis.

RNA extraction and quantitative PCR

Total RNA was extracted from 100 mg of ground frozen tissues using the RNeasy Plant Kit (Qiagen) and the RNase free DNase set (Qiagen) to prevent DNA contamination, as described in Hano et al. (2006). RNA was quantified using a fluorometer and the Quant-iT RNA Assay Kit (Invitrogen) adapted for the Qubit fluorometer according to the manufacturer’s protocol. The reverse transcriptions were performed using the first-strand cDNA synthesis kit (Thermo Fisher).

Quantitative PCR was performed in 96-well plates with a PikoReal real-time PCR system (Thermo Fisher) using DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo Fisher). The reaction was carried out in a 20-µl volume (1-µl diluted cDNAs, 10 µL of 2× SYBR Green mix, and primer pairs at 1 µM). All PCR reactions were carried out with the same following protocol: 7 min at 95 °C, 40 cycles of 10 s at 95 °C, 10 s at 55 °C, and 30 s at 72 °C. The specificity of the amplified product was confirmed for each primer pair using a melting curve. Data were analyzed with the Pikoreal software (Thermo Fisher). Three biological replicates combined with two technical repetitions were used for each sample. Relative transcript levels of the LuPLR2 gene were obtained using the specific primers qLuPLR2–F: 5′-ACTTTGCTCGGAGACGGTAA-3′ and qLuPLR2–R: 5′-CACGGACAGGGTTGTCTTTT-3′ designed using the Primer3 software and normalized using the comparative ΔΔCq method with two validated housekeeping reference genes selected by Huis et al. (2010): LuCYC encoding cyclophilin (qLuCYC-F: 5′-TGATTGCGGTCAGCTGTAG-3′ and qLuCYC-R: 5′-AGGTGAAACGCTAGGCAGAA-3′) and LuETIF5A encoding a Eukaryotic Translation Initiation Factor 5A (qLuETIF5A-F: 5′-TGCCACATGTGAACCGTACT -3′ and qLuETIF5A-R: 5′-CTTTACCCTCAGCAAATCCG-3′).

β-Glucuronidase (GUS) activity

Quantitative assays for GUS (β-glucuronidase) activities were performed by measuring 4-methylumbelliferone (4-MU, Sigma) produced from the glucuronide precursor 4-methylumbelliferyl-β-d-glucuronide (4-MUG, Sigma). Cells (50 mg) were ground and homogenized in 500 μl of extraction buffer containing 50-mM sodium phosphate; pH 7.0, 10-mM EDTA, 0.1% (v/v) Triton X-100, 0.1% sarcosyl, and 10-mM β-mercaptoethanol at 4 °C. Following centrifugation for 15 min at 12,000g at 4 °C, GUS activity was determined in the supernatant using a fluorescence spectrophotometer (BioRad). Protein concentrations were quantified using a Qubit fluorimeter and the Quant-iT Protein Assay Kit (Invitrogen) according to the manufacturer’s protocol. β-glucuronidase activity was expressed as RFU (relative fluorescence units) per mg of soluble proteins.

Histochemical assays of β-glucuronidase (GUS)

Histochemical staining for GUS activity was performed as described by Jefferson et al. (1987) and modified by Kogushi et al. (1990) to avoid background (i.e., with 20% methanol in the 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) solution). Fragments of stems, roots, and leaves (plants grown in vitro) as well as bolls and seeds (greenhouse plants) were hand-cut and subsequently incubated with 1-mg/l X-Gluc (Duchefa). Staining was allowed to proceed at 37 °C until a blue stain had developed in the samples. Samples were cleared of chlorophyll by incubation in 95% ethanol.

Yatein extraction and quantification

Yatein was extracted by adapting the protocol described above and was quantified by HPLC using o-coumaric acid as the internal standard. It was extracted twice with ethanol under reflux conditions for 4 h, evaporated to dryness, resuspended in ethanol, and filtered (0.45 µm) before HPLC injection. The separation was performed, using the same system previously described, at 35 °C on a Purospher (Merck, Fontenay sous Bois, France) RP-18 column (250 × 4.0 mm i.d.; 5 µm). The mobile phase consisted of 0.2% acetic acid in water (solvent A) and methanol (solvent B). The composition of the mobile phase varied during the run according to a nonlinear gradient at a flow rate of 0.8 ml.min−1 as follows: from 0 to 40 min of A-B: 90:10 (v/v) to 30:70 (v/v), from 41 to 50 min of A-B: 30:70 (v/v) to 0:100 (v/v), and A–B: 0:100 (v/v) from 51 to 60 min. Detection was performed at 280 nm. Yatein was identified on the basis of its retention time and quantified using the calibration curve of yatein standard ranging from 0.0125 to 1 mg/ml with a correlation coefficient of 0.999. Yatein standard was purified as described in Doussot et al. (2017).

Statistical analysis of data

All experiments presented in this study are the means and the standard deviations of at least three independent replicates. Comparative statistical analyses of groups were performed using Student’s t test or one-way analysis of variance according to the data.

Results

In vivo functional characterization of the LuPLR2 gene and its role in yatein biosynthesis in flax leaves

Our first objective was to provide direct in vivo evidence for the involvement of the LuPLR2 gene in yatein biosynthesis. RNAi technology was previously applied to downregulate LuPLR1 gene expression in flaxseeds (Renouard et al. 2014). Given the high sequence similarity between LuPLR1 and LuPLR2, we analyzed the impact of RNAi-mediated PLR gene silencing on both LuPLR1 and LuPLR2 gene expression in flax leaves, which constitute the main accumulation site of yatein, thought to result from the LuPLR2-mediated formation of (−)-secoisolariciresinol given both its expression profile and stereospecific activity (Hemmati et al. 2010). For this purpose, homozygous cell lines of R2 progeny plants were analyzed for their LuPLR1 and LuPLR2 expressions in leaves by RT-qPCR. As expected, a significant decrease in LuPLR1 gene expression was observed in some lines (Fig. 2a), but it was less marked than that previously observed in seed (Renouard et al. 2014), certainly due to the lower steady-state level of LuPLR1 mRNA in leaf tissues. Not surprisingly given their high sequence identity, this RNAi approach also had a significant but contrasting impact on LuPLR2 gene expression, with an increase observed in the Pi1-AJ line, probably due to gene expression compensation, whereas significant decreases in the LuPLR2 mRNA steady-state level were observed in the four other analyzed cell lines, especially for the Pi1-Y line (Fig. 2a).

Fig. 2
figure 2

a Relative LuPLR1 and LuPLR2 gene expression estimated by RT-qPCR. b Yatein accumulation. c Correlation between gene expression level and yatein accumulation in photosynthetic leaves from wild-type and interfered LuPLR transgenic flax (Pi1-AJ, Pi1-AK, Pi1-AM, Pi1-Y, and Pi1-E independent R2 lines). Data are expressed as the mean (n = 3) ± standard deviation of the mean. Significant differences compared to the corresponding control at P < 0.05:*P < 0.01: **P < 0.001: ***according to Student’s t test

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To assess the impact of these PLR gene expression modifications on lignan biosynthesis, the leaf extracts of these plants were subjected to HPLC and LC–MS analysis. A strong decrease in yatein content was observed in the Pi1-AK, -AM, and -Y lines (Fig. 2b). Note that, in these plants, the yatein decrease did not result in a significant increase in pinoresinol as we previously observed in seed (Renouard et al. 2014). However, a significant increase in total flavonoids was noted, probably as a result of a greater availability of the earlier phenylpropanoid precursors in this pathway in these plants. On the contrary, the Pi1-AJ plants showed a slight increase in their yatein accumulation compared to untransformed plants, which was also consistent with their LuPLR2 gene expression. Taken together, these results revealed a strong and significant correlation (0.876) between LuPLR2 gene expression and yatein accumulation in these transgenic plants, evidencing for the first time the direct in vivo involvement of LuPLR2 in yatein biosynthesis from (−)-secoisolariciresinol (Fig. 2c). For comparison, the correlation coefficient between LuPLR1 gene expression and yatein accumulation was as low as 0.260 (Fig. 2c).

To identify the subcellular location of LuPLR2-derived lignan biosynthesis in flax, transgenic tobacco mesophyll cells were generated that express LuPLR2-eGFP (enhanced green fluorescent protein) fusion proteins. As shown in Fig. 3, high levels of the GFP fusion proteins were detected in the transgenic cells when LuPLR2 was fused to eGFP by either its N- or its C-terminal end, and the same location for the GFP fluorescence was observed on the cell surface (Fig. 3a). Further analysis of these mesophyll cells plasmolyzed in a 0.5-M NaCl solution gave poor additional information for the precise location of the fused proteins; therefore, the proteins were fractionated by centrifugation to analyze the fluorescence of the resulting fractions (Fig. 3b). This approach located the LuPLR2-GFP fusion proteins in the soluble fraction, indicating a preferential cytoplasmic location.

Fig. 3
figure 3

a Subcellular location of LuPLR2-GFP fusion proteins in tobacco mesophyll. Control cells were obtained following empty vector transformation. GFP was fused in either the N or the C-terminal end of PLR2. Pictures are representative of independent transformation events. b Relative GFP quantification following protein extraction and fractionation into soluble and insoluble fractions

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Isolation and in silico analysis of the LuPLR2 gene promoter

To clarify the transcriptional regulation of the LuPLR2 gene from flax, a PCR walking strategy was employed to isolate the 5′ flanking genomic region of this gene as described by Devic et al. (1997). The resulting 1,266 bp DNA fragment (GenBank accession number KY126318), obtained with the DraI restriction fragment library as template, was confirmed as the LuPLR2 gene upstream sequence by sequencing and, more recently, by in silico analysis on the Phytozome database (Goodstein et al. 2012) based on the lately released flax genome sequencing results (Wang et al. 2012) (Online Resource S1).

In silico scans of this 5′ flanking sequence upstream of the translation initiation codon of 1,266 bp using the public database PlantPAN (Chang et al. 2008) confirmed its promoter nature and revealed the presence of putative cis-acting elements (Online Resource S2). A TATA box (TATAAAT) matching the consensus for the plant was located 35-bp upstream of the putative transcription start determined as being adenine surrounded by another adenine on each side (+1). A CAAT box was found 65-bp upstream of the transcription start. These distances are regular features of eukaryotic promoters (Zhu et al. 1995). The ATG of the translation start was located 51-bp downstream of the transcription start (ATG) and this was confirmed by cloning the full-length cDNA. The 5′UTR was AT-rich (around 60%) and fitted well the consensus for dicot UTR (Joshi et al. 1997). The Kozak sequence CAAAATGGG corresponded well with the consensus for terrestrial plants (Lütcke et al. 1987). In this promoter sequence of 1,207 bp, several cis-acting elements have been identified as sequences involved in tissue-specific expression, hormonal regulation, and the response to biotic and/or abiotic stresses (Online Resource S2). Several cis-acting elements are potentially involved in the spatial expression in leaves, such as the RAV1, GATABOX or MYBCORE elements, and in seed, such as the AACACOREOSGLUB1, CAATBOX1, RYREPEATATGMGY1, SEF1MOTIF, SEF3MOTIFGM, and SEF4MOTIFGM7S (Online Resource S2). This putative promoter sequence also contains several regulatory motifs required for different hormone signaling pathways such as for auxins (ASF1MOTIFCAMV and D2GMAUX28), ABA (ATHB6COREAT, MYB2CONSENSUSAT, and MYC2CONSENSUSAT), gibberellins (CAREOSREP1, GAMYB, and GAREAT) or ethylene (ERF1) (Online Resource S2). Other interesting putative regulatory sequences involved in the response to biotic (elicitors and wounding) and/or abiotic (UV and dehydration) stresses were also detected in this promoter region (Online Resource S2).

Spatio-temporal LuPLR2 gene expression pattern in relation to yatein biosynthesis

Transgenic flax plants were grown in a greenhouse up to seed production. The LuPLR2 gene expression was measured by RT-qPCR and promoter transcriptional activities were studied in different organs [roots, stem, old (yellowish senescing) leaves, young (photosynthetically active) leaves, and maturing seeds] from wild-type and transgenic LuPLR2 promoter-driven reporter gene expression plants (Fig. 4). In our hands and contrary to the first isoform highly expressed in seed (i.e., LuPLR1), the LuPLR2 gene encoding this second PLR isoform appeared highly expressed in vegetative tissue (Fig. 4a), especially in photosynthetic leaves; it was expressed at the highest levels in young leaves and to a lesser extent in old leaves and stem (Fig. 4b, c). The expression of the LuPLR2 gene in young leaves was almost 20 times higher than that of the LuPLR1 gene (Online Resource S3).

Fig. 4
figure 4

Spatial LuPLR2 gene expression in flax leaves, stem, and root. a Histochemical location of β-glucuronidase expression driven by the LuPLR2 gene promoter. b Quantification of β-glucuronidase activity resulting from LuPLR2 gene promoter-driven expression. c Normalized RT-qPCR analysis of LuPLR2 gene expression in leaves, stem, and root using LuCYC (encoding cyclophilin) and LuETIF5A (encoding a eukaryotic translation initiation factor 5A). d Yatein accumulation in these tissues. Data are expressed as the mean of n = 4 independent experiments ± standard deviation of the mean and different letters indicate significant differences between conditions (P < 0.05). PS, young photosynthetically active leaves; SC, old yellowish senescing leaves

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These results are in agreement with the histochemical location and quantification of the transcriptional activity driven by the LuPLR2 gene promoter obtained from promoter-reporter transgenic plants. In these plants, the amount of blue staining in plant tissue after the X-Gluc assay indicated the location and intensity of LuPLR2 promoter transcriptional activity. The staining obtained in leaves was mainly located in the distal region (Fig. 4a). A faint staining of stem tissue appeared after a prolonged incubation time and no GUS staining was detected in untransformed plants (Fig. 4a). The LuPLR2 gene promoter activity was then quantified using the MUG assay. The highest expression was obtained in young leaves followed by old leaves and stem (Fig. 4b). The lowest promoter-driven activity was detected in roots (Fig. 4b) consistent with the X-Gluc staining and the RT-qPCR analysis. All these expression data were consistent with the measurements of yatein content (Fig. 4c) showing a preferential accumulation in foliar tissue and a poor content in roots.

The expression of LuPLR2 during germination started as soon as the imbibition was performed and reached a plateau at 6 h (Fig. 5a). On closer observation, it appeared that LuPLR2 gene expression and yatein synthesis were almost restricted to the cotyledons, confirming the preferential foliar location of the activity of this gene (Fig. 5b, c, d).

Fig. 5
figure 5

LuPLR2 gene expression during flaxseed germination. a Normalized RT-qPCR analysis of LuPLR2 gene expression during flaxseed germination (HAI: hour after imbibition). b Histochemical location of β-glucuronidase expression driven by the LuPLR2 gene promoter in both wild-type (CTL) and transgenic (LuPLR2:GUS) flax seedlings. c Quantification of β-glucuronidase activity resulting from LuPLR2 gene promoter-driven expression in whole seedlings (WS), radicule (Rd), hypocotyl (Hp), and cotyledon (Cot). d Yatein accumulation in these tissues. Data are expressed as the mean of n = 4 independent experiments ± standard deviation of the mean and different letters indicate significant differences between conditions (P < 0.05)

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LuPLR2 gene expression pattern and yatein biosynthesis in response to stress

As lignans are probably involved in plant defense mechanisms and given the presence of several cis-regulatory elements potentially involved in the plant defense response revealed by the in silico analysis of LuPLR2, LuPLR2 gene expression was investigated in response to different stresses (hormones, elicitors, and wounding). Hydroponically grown plantlets treated with methyljasmonate (MeJA) and salicylic acid (SA) were assayed for gene expression by both MUG assays and RT-qPCR and for their yatein content. The results are presented in Fig. 6: MeJA triggered the expression of LuPLR2 leading to an over-accumulation of yatein, while SA failed to act strongly on the expression of this gene and yatein accumulation was not stimulated.

Fig. 6
figure 6

a Representative in vitro flax culture used for the experiments. b Quantification of β-glucuronidase activity resulting from LuPLR2 gene promoter-driven expression in untreated (CTL), and SA- and MeJA-treated (100 µM for each treatment) leaves of hydroponic in vitro flax culture (measured 48 h after treatment). c Normalized RT-qPCR analysis of LuPLR2 gene expression quantified 8 h and 24 h after treatment. d Yatein accumulation determined 96 h after treatment. Data are expressed as the mean of n = 4 independent experiments ± standard deviation of the mean and stars indicate significant differences between conditions (*P < 0.05, **P < 0.01, ***P < 0.005)

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Since lignans have been related to plant protection against pathogen and herbivore attack (McRae and Towers 1984; Harmatha and Dinan 2003), it was assumed that injuries mimicking an insect attack would increase the transcriptional activity of genes involved in their biosynthesis, including LuPLR2. To verify this hypothesis, leaves of greenhouse-grown plants were wounded with pliers and the resulting LuPLR2 expression was monitored by RT-qPCR. The activity of the LuPLR2 promoter after wounding was tested by X-Gluc and MUG assays and lignan production was monitored by HPLC analysis. LuPLR2 promoter-driven reporter gene expression in plants revealed by the X-Gluc assay that the β-glucuronidase (and therefore LuPLR2 gene expression) was located at the wounded site (Fig. 7a), while the MUG assay results clearly showed an increase in LuPLR2 transcriptional activity in wounded plants (Fig. 7b). Not surprisingly, the LuPLR2 gene expression analyzed by RT-qPCR showed a clear induction in wounded leaves compared to the control, which was slightly higher 8 h after wounding than after 24 h (Fig. 7c). Nevertheless, this lower induction after 24 h could have been due to the decay of leaf tissue after wounding as well as the result of a transient response. Yatein production was higher in wounded leaves than in control samples (Fig. 7d), fitting well with the gene expression results, and thus, we can state that mechanical wounding induced LuPLR2 gene expression and consequently stimulated lignan production.

Fig. 7
figure 7

LuPLR2 gene expression and yatein accumulation in flax leaves subjected to mechanical wounding. Flax wounded leaves and histochemical location of β-glucuronidase expression driven by the LuPLR2 gene promoter in flax leaves subjected to mechanical wounding. a Quantification of β-glucuronidase activity resulting from LuPLR2 gene promoter-driven expression (48 h after wounding). b Normalized RT-qPCR analysis of LuPLR2 gene expression 8-h and 24-h post-wounding. c Yatein accumulation 96 h after wounding in flax control (CTL) or subjected to mechanical wounding (W) leaves. Data are expressed as the mean of n = 4 independent experiments ± standard deviation of the mean and stars indicate significant differences between conditions (*P < 0.05, **P < 0.01, ***P < 0.005)

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Identification of putative LuPLR2 promoter cis-acting elements responsive to environmental stimuli

To identify the putative cis-elements involved in both developmental regulation (leaf senescence) and the stress response (MeJA treatment and wounding), transgenic flax plants carrying promoter deletions of the LuPLR2 promoter region fused to the uidA reporter gene were used (Fig. 8a). The results indicated that the cis-elements, responsible for gene expression in response to stress, are located in separate portions of the LuPLR2 promoter (Fig. 8b).

Fig. 8
figure 8

a Schematic representation of deletions of LuPLR2 gene promoter and their corresponding β-glucuronidase activity in seeds of stably transformed flax plants. Bars correspond to the lengths of the truncated LuPLR2 gene promoter fragments fused to the GUS reporter gene. b Location of the most significant cis-acting elements present in each deletion (for details, see Online Resource S2; distance from TS: transcription start). c β-Glucuronidase activity analysis of LuPLR2 gene promoter deletion; photosynthetic or senescent leaves. PS, young photosynthetically active leaves; SC, old yellowish senescing leaves. d β-Glucuronidase activity analysis of LuPLR2 of MeJA-treated or wounded leaves relative to CTL (untreated control) of stably transformed flax plants. The relative β-glucuronidase activity of each deletion is reported as a percentage of 1-1207:GUS activity measured in photosynthetic (control) leaves. Data are expressed as the mean of n = 4 independent experiments ± standard deviation of the mean for leaves development: different letters indicate significant differences between conditions (P < 0.05), whereas for MeJA and wounding treatments: stars indicate significant differences between conditions (*P < 0.05, **P < 0.01, ***P < 0.005)

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These results also confirmed that the LuPLR2 promoter is less active in senescent leaves than in younger photosynthetic leaves (Fig. 8c). The relative β-glucuronidase activity measured for the 4 deletions in these tissues indicated a stronger impact of the deletions of the regions from −826 to −473 and −473 to −170 in senescent tissue (Fig. 8c). A search for homology within these regions among known cis-motifs from plants revealed the presence of two MYB-binding sites (MYBplant, Fig. 8b) which have been described as playing a major role in the control of lignan biosynthesis (Zhao et al. 2014). Moreover, a binding site for the RAV1 transcription factor, responsible for high gene expression levels in rosette leaves in Arabidopsis (Kagaya et al. 1999), was identified in this promoter region. We can also note two other putative MYB-binding sites (MYBplant) located in the more distal region from −1207 to −826 (Fig. 8b).

Concerning the LuPLR2 promoter stress responsiveness, MeJA treatment and wounding resulted in the same relative β-glucuronidase activity pattern for all the four deletions tested (Fig. 8d). A first significant decrease in the relative β-glucuronidase activity was observed with the deletion of the region from −826 to −473, which contains two putative two binding sites for the WRKY transcription factor, involved in the plant defense response (Yamamoto et al. 2004), and one for the MYC2 transcription factor involved in the MeJA response (Lorenzo et al. 2004) (Fig. 8b). Moreover, the deletion of the region from −473 to −170, containing four putative-binding sites for the WRKY transcription factor involved in the response to wounding or elicitors (Nishiuchi et al. 2004; Yamamoto et al. 2004), resulted in a complete loss of the response to both MeJA treatment and wounding.

Discussion

Lignan biosynthesis in flax is controlled by PLRs with distinct gene expression patterns

Understanding the spatio-temporal transcriptional regulation of the genes related to lignans biosynthesis could yield information about the role of these compounds in planta and about the specificity of the end-products of their enzymes; nevertheless, data are still scarce in this field. Moreover, whether the first steps in lignan biosynthesis are catalyzed by enzymes with weak or without enantioselectivity or by mixtures of enzymes with high but opposite enantioselectivity is still a subject of debate. Pinoresinol, lariciresinol, and secoisolariciresinol exist as mixtures of both enantiomers, whereas matairesinol is the first enantiomerically pure compound of lignan biosynthesis in most plant species investigated so far (Umezawa 2003). In Arabidopsis thaliana, two pinoresinol reductase (PrR) enzymes have been described in the formation of lariciresinol: one preferentially used (−)-pinoresinol (AtPrR2) as substrate, whereas the other (AtPrR1) was only regiospecific showing no enantioselectivity toward pinoresinol enantiomers (Nakatsubo et al. 2008). However, it is important to mention that these enzymes from A. thaliana are monofunctional and use only pinoresinol as a substrate; hence, they are named pinoresinol reductases (PrRs). In flax, two PLR enzymes, probably resulting from a gene duplication of a common ancestor in the progenitor of the L. usitatissimum species, have been described. We have previously studied the stereospecificity and regulation expression of the LuPLR1 gene responsible for the synthesis of (+)-SDG in flaxseed (von Heimendahl et al. 2005; Hano et al. 2006; Renouard et al. 2012; Corbin et al. 2013a, 2013b; Renouard et al. 2014) and a second PLR gene, LuPLR2, has been allocated to the formation of the opposite enantiomer, (−)-secoisolariciresinol, in vegetative tissues (Hemmati et al. 2010). In such a context, a comparative functional genomic work for the two PLRs from L. usitatissimum was of great interest. Here, we demonstrate that yatein biosynthesis control in response to wounding, MeJA, and elicitors in flax leaves is operated through the transcriptional regulation of this LuPLR2 gene. As already observed for LuPLR1 (Hano et al. 2006), LuPLR2 is expressed in the seedcoat of immature flaxseeds; however, this expression is almost five times lower than LuPLR1 gene expression in good agreement with the enantiomeric composition observed in flaxseed of 85–99% (+)-SDG versus 1–15% (−)-SDG (Ford et al. 2001).

In aerial parts, LuPLR2 is highly expressed in the distal part of young leaves with an expression level almost 20 times higher compared to LuPLR1 (Online Resource S3), which is coherent with the flax leaf content in lignans, such as yatein and its lignan derivatives, derived from (−)-SECO (Hemmati et al. 2010). Our results, therefore, indicate that lignan biosynthesis in flax is controlled by PLRs with opposite stereospecificity and that this control results from distinct gene expression regulation and not only from differences in the respective catalytic efficiencies of these isoforms.

LuPLR2 gene expression pattern is developmentally controlled

The availability of gene sequences corresponding to the lignan-specific biosynthetic enzymes PLRs provided a means to study the temporal and spatial regulation of lignan formation. As lignin and lignans share the same precursor, lignan accumulation in woody tissues of trees has been studied (Davin and Lewis 2003; Holmbom et al. 2003), so LuPLR2 activity is expected to be found in stem tissue. In flax stem lignifying tissue, both LuPLR1 (unigene c1251) and LuPLR2 (unigene c3993) gene activities were detected by Huis et al. (2012). The transcript detection of lignan biosynthetic genes in ligneous species such as Pinus taeda and Forsythia intermedia by in situ hybridization indicated that lignan biosynthetic genes, such as dirigent proteins and PLR genes, are expressed in xylem and other lignifying tissues (Burlat et al. 2001; Kwon et al. 2001). Interestingly, the pinoresinol reductase gene from Arabidopsis (AtPrPR1) was also found co-expressed with several genes involved in secondary cell wall biosynthesis and its promoter region was activated by MYB46, previously characterized as a master regulator of secondary cell wall biosynthesis (Zhao et al. 2014). Two other MYB transcription factors, PtMYB1 and PtMYB8, from loblolly pine (Pinus taeda) were able to trans-activate PLR gene expression in this gymnosperm species (Bomal et al. 2008). Here, the presence of several putative-binding sites for MYB transcription factors in the regions essential for LuPLR2 gene expression suggests an involvement of such regulators.

LuPLR2 gene expression was detected during germination and located in the cotyledons with an expression pattern similar to that evidenced by Kim et al. (2002) in Arabidopsis thaliana expressing promoter-reporter constructs from dirigent protein genes involved in lignan biosynthesis in western red cedar. It was assumed in A. thaliana that phenolics like flavonoids could alter physiological processes, such as seed dormancy and germination (Debeaujon and Koornneef 2000). LuPLR1 gene expression during seed maturation has been described previously (Hano et al. 2006; Renouard et al. 2012). The differential expression pattern observed for LuPLR1 and LuPLR2 genes could indicate distinct roles for their respective products during seed maturation and germination.

LuPLR2 gene expression was particularly high in the distal part of young leaves and cotyledons. This could indicate regulation by the auxin produced during the leaf-primordium development (Aloni et al. 2003) and this preferential location in young foliar tissue could be related to a role in plant defense against herbivores (see also below in the defense response). Barto and Cipollini (2005) evidenced that the relative levels between constitutive and inducible defense varied greatly with the ontogeny in A. thaliana leaves. Clearly, young tissues constitute a high potential part of the plant that needs to be better protected than the older leaves (Rhoades and Cates 1976). Similar behavior is observed during severe drought when old leaves are sacrificed for the benefit of the apical parts.

Evidences for a direct role of the LuPLR2 gene in the flax defense response

MeJA and SA are phytohormones both associated with stress response but involving different mechanisms and sometimes known to act antagonistically (Kunkel and Brooks 2002; Leon-Reyes et al. 2010; Schweiger et al. 2014). Here, consistent with these observations, MeJA applications had a stimulatory effect on LuPLR2 gene expression associated with an increase in yatein accumulation in the leaves of treated plants, while this was not the case with SA, evidencing that the LuPLR2 stress response is controlled by jasmonate signaling rather than by SA.

The ability of MeJA to stimulate the biosynthesis of plant secondary metabolites has been established. The production of lignans was enhanced by MeJA treatment in Forsythia x intermedia cell suspension (Schmitt and Petersen 2002). Such MeJA-dependent induction of lignan biosynthesis has been reported in Linum album and Linum nodiflorum using in vitro systems (Berim et al. 2005; van Fürden et al. 2005; Yousefzadi et al. 2010), which makes them interesting means for understanding the regulation of lignan biosynthesis in flax in the present study. Our results demonstrate that MeJA application could be an attractive way of enhancing lignan production, thus meeting the objective of biotechnological improvement of lignan accumulation using flax in vitro systems, as well as showing for the first time that this induction also works in a whole plant system.

Ralph et al. (2006) reported that the expression of dirigent protein genes, also related to lignan synthesis, was stimulated by a stem-boring insect in spruce. Since lignans can be assigned to plant protection against pathogen and herbivore injuries (MacRae and Towers 1984; Harmatha and Dinan 2003), it is assumed that such attacks could enhance the transcription activity of genes involved in their biosynthesis.

In the present work, LuPLR2 gene expression and yatein biosynthesis were induced in wounded leaves, indicating that mechanical wounding induced LuLR2 gene expression and consequently lignan production. Our results obtained with the truncated LuPLR2 promoters suggest that the same regulatory region is involved in the response to MeJA and wounding.

Consistent with this observation, MeJA is known as an important signal in the response against insects, herbivore, and wounding, while SA has an important role in plant defense against pathogens and viral infections. MeJA has been shown to induce the expression of a subset of the pathogenesis-related (PR) genes, which contribute to the pathogen-attack resistance in plants (Shah and Klessig 1996). Wounding of plant tissue leads to an increased production of defense compounds in plants. In flax, gene expression of allene oxide synthase (AOS), an enzyme involved in the biosynthetic pathway of jasmonate (JA), is induced after mechanical wounding of leaves leading to an increase in endogenous levels of JA (Harms et al. 1995). Similarly, endogenous JA biosynthesis was rapidly and transiently enhanced in mechanically wounded tobacco leaves (Han et al. 2012). Our study tends to confirm that the flax plant perceives wounding and responds to it using the same JA signaling pathway and lignan synthesis as when an elicitation is performed. Overall, the results shown here are in favor of a role for yatein in the defense against biotic stress.

To summarize, the present study provides new insights into the in planta regulation and function of lignans in flax. It is now clear that lignan biosynthesis in flax relies on different PLRs with opposite stereospecificity and with very distinct expression regulation. Our data constitute the first direct evidence for in vivo involvement of the LuPLR2 gene in the biosynthesis of (−)-yatein accumulated in flax leaves. The spatio-temporal expression of the LuPLR2 gene in the distal part of young foliar tissue leaves perfectly matches the location of (−)-yatein accumulation. This expression pattern was related to a possible role for this lignan in flax defense, particularly in response to wounding and MeJA, which is an important signal in this response. Some putative cis-acting elements located throughout the LuPLR2 promoter have been proposed. Further biochemical and genetic studies will focus on the LuPLR2 response to wounding and herbivore attacks to provide the complete characterization of the signaling cascade and the identification of transcription factor(s) involved in the regulation of LuPLR2 gene expression and yatein biosynthesis during these defense responses.

Author contribution statement

CC, SD, and IM performed the experiments and prepared the data. LM, CD, SR, TL, and FL contributed to the generation and analysis of the plant material. CH, EF, EL, and DA designed and supervised the study. JD supervised and contributed to the analytical chemistry experiments. CH and EF analyzed the data and wrote the manuscript. EL and DA edited the manuscript. All authors read and approved the final manuscript.