The full-length *N* gene (1269-bp long) was amplified by PCR on the template plasmid pQE30-N with *Pfu* polymerase with the forward primer 5 - GGCCATCGAT*GAATTC*GGATCCATC**ATG**AGAGGATCGC ATCACATCC-3 (*ClaI* restriction site: underlined, *EcoRI* site: italic, initiation translation codon: bold) and the reverse primer 5 -GACTTGTCGAC*GCGGCCGC***TTA** TGCCTGAGTTGAATCAGCAG-3 (*SalI* site: underlined, *NotI* site: italic, stop codon: bold). For mammalian cells expression, the PCR product was cut out by digestion with *EcoRI/NotI* and inserted into the pVAX1 vector (Invitrogen). For plant expression, the PCR product was cut out with *ClaI/SalI* and cloned into the pPVX201 plant vector (Baulcombe et al., 1995). In this vector, the full-length viral cDNA of the Potato virus X (PVX) is inserted between the constitutive 35S promoter of the cauliflower mosaic virus (CaMV 35S) and the transcription terminator (Nos-term) of the nopaline synthase gene of *A. tumefaciens*, necessary for the regulation of the viral genome expression upon infection with plasmid DNA. Characteristic components of the viral expression vector are the following: viral replicase gene (RdRp); triple gene block encoding protein for cell-to-cell movement (M1-3); viral coat protein gene necessary for encapsidation of viral RNA (CP); coat protein sub-genomic promoter (SgP; **Figure 1A**).

The full-length *M* gene (666-bp long) from the pCR2.1-TOPO TA vector was amplified in two subsequent steps. First, the forward primer 5 - GGCCATCGAT*GAATTC*GGATCCATC**ATG**GCAGACAACGG TACTATTAC-3 (*ClaI* restriction site: underlined, *EcoRI* site: italic, initiation translation codon: bold) and the reverse primer 5 -*GTGATGGTGATGA TG*CTCGAGTGCCTG**TACTAGCAAAGCAATATT**-3 (end of the His6 tag: italic, end of the *M* gene: bold) were used to add the His6 tag at the C-terminus. Subsequently, the same forward primer and the reverse primer 5 - GACTTGTCGAC*GCGGCCGC***TCA**ATG*G*TGATGGTGATG ATGCTCG-3 (*SalI* site*:* underlined, *NotI* site: italic, stop translation codon: bold) were used in order to add restriction sites useful for cloning.

For simplicity, we report the name of the recombinant genes *N-His*6 and *M-His*6 as *N* and *M* genes. The purified PCR products were cloned into the *Eco RV*-linearized pBlueScript SK(+; pBS) cloning vector (Stratagene; pBS-N and pBS-M), sequenced for authenticity and sub-cloned by *ClaI-SalI* in the pPVX201 plant vector (pPVX-N and pPVX-M) or by *EcoRI/NotI* in the pVAX1 vector (pVAX-N and pVAX-M). After XL1 blue cells transformation, pPVX-N and pPVX-M plasmid DNAs were purified (maxiprep kit, Qiagen) for plant infection, while pVAX-N and pVAX-M plasmids were purified with endotoxin-free purification kits (Plasmid Maxi kit LPS-free, Qiagen) for mammalian HEK-293 cells transfection.

The *M* gene was also sub-cloned by *XbaI/SalI* from the pBS-M construct into the pBI-121 plant binary vector (Clontech Laboratories, Acc. No. AF485783), obtaining the pBI-M construct. In this construct, the *M* gene is inserted between the CaMV 35S promoter and the NOS terminator. Characteristic components of the vector are: right and left borders (RB, LB), gene for kanamycin resistance (NPT II) under the transcription promoter and terminator of the nopaline synthase gene of *A. tumefaciens* (**Figure 6A**).

The pBI-M construct, obtained by substitution of the *GUS* gene with the *M* gene, was used to transform the GV3101 and C58C1 strains of *A. tumefaciens.*

## Expression and Purification of N and M Proteins in Bacteria

The expression and purification of the N and M proteins were done according to standard protocols (The QIAexpressionist, Qiagen). Since it was previously described that the M protein is toxic in bacteria (Carattoli et al., 2005), culture growth was performed at suboptimal temperature of 30◦C. In these conditions, we isolated a clone able to express the M protein in *E. coli*. Characterization revealed that it corresponded to a spontaneously mutated *M* gene, coding for a protein carrying the three substitutions K13 *>* R, F36 *>* L, and I160 *>* V (MRLV).

The MRLV and the N proteins were purified by Ni-NTA affinity chromatography in denaturing conditions. For the N protein, when purified in native conditions, imidazole concentration in the wash buffer was 30 mM.

Quantification of the N protein purified in denaturing and native condition was performed by Coomassie stained SDS-PAGE comparing the intensity of the band at 50 kDa to known amount of BSA using a Chemidoc ImageLab system with ImageLab 4.0 software (Bio-Rad).

#### Expression of N and M Proteins in Mammalian Cells

HEK-293 cell line was transfected in six wells plates with the pVAX-N or pVAX-M constructs according to standard protocols (Effectene Transfection Reagent, Qiagen). 24 h post-transfection (pt) some wells were added with 12.5 µM of the proteasome inhibitor MG-132. 48 h pt both samples, treated or not with MG-132, were harvested and prepared for protein expression analysis (immunoblotting and immunofluorescence). After three washes with cold phosphate-buffered saline (PBS: 21 mM Na2HPO4, 2.1 mM NaH2PO4, 150 mM NaCl, pH 7.2) cells were centrifuged at 800 rpm for 5 , recovered and re-suspended in SDS-loading buffer (10% glycerol, 60 mM Tris-HCl pH 6.8, 0.025% bromophenol blue, 2% SDS, 3% 2-mercaptoethanol) and boiled for 10 min.

For immunofluorescence analysis, HEK 293 cells were grown on multi-chamber glass slides and transfected at 40% of confluency. 24 h post transfection cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS. Samples were blocked with 5% no-fat dry milk in PBS. For detection of the N and M proteins, cells were incubated with specific polyclonal antibodies (pAb) validated in immuno-cytochemical assay on SARS-CoV infected and previously described (Carattoli et al., 2005). For N protein we used a mouse anti-N pAb, (obtained after immunization of animals with the purified His6- N protein produced in *E. coli*) at 1:800 dilution. For the M protein we used a mouse anti-M pAb (obtained by immunizing mice with the recombinant cytoplasmic domain, amino acids 138–222, of the M protein produced in *E. coli*) at 1:400 dilution. Cells were then incubated with a 1:300 dilution of an anti-mouse biotinylated secondary antibody (GE Healthcare) and a 1:50 dilution of Streptavidin-FITC conjugated (Sigma– Aldrich, GmbH, Steinheim, Germany). Nuclei were counterstained with DAPI (2 µg/ml) in PBS. Slides were examined using a fluorescence "Axiolab Zeiss" microscope (Oberkochen, Germany) interfaced with a Coolsnap CCD camera (Roper Scient., Princeton, NJ, USA).

#### Expression of N and M Proteins in *Nicotiana benthamiana*

Two leaves of *N. benthamiana* plants (4 week-old) were dusted with carborundum powder and inoculated with 10 µg of the plasmids pPVX-N, pPVX-M, or pPVX201 (empty vector, negative control) diluted in 100 µl bi-distilled water. Plants were also treated with 100 µl bi-distilled water (mock-infected) for monitoring viral infection symptoms. Plants were grown under 16 h daylight at 22◦C into a containment greenhouse (bio-safety level 2) and observed daily for PVX infection signs. Inoculated and symptomatic leaves were harvested and stored at – 80◦C until use.

Four week-old *N. benthamiana* plants were infiltrated with *A. tumefaciens* C58C1 and GV3101 cultures harboring the pBI-M construct that had been grown and induced as described (Kapila et al., 1997). Plants were subsequently grown as described and the leaf disks collected 3, 4, 5, 6, 7, and 10 days post-infiltration (dpi) were homogenized with an ultraturrax in three volumes (w/v) of SDS-loading buffer and boiled for 3 min.

To analyze N and M protein accumulation in plant tissues, soluble proteins were extracted from plant material using different buffers, depending on their hydrophobic properties. Crude plant extracts were prepared by grinding the tissue to a fine powder in liquid nitrogen. The powder was re-suspended and homogenized with an ultraturrax in three volumes (w/v) of PBS or, alternatively for the M protein, in one volume (w/v) of GB buffer (100 mM Tris-HCl pH 8.1, 10% glycerol, 400 mM sucrose, 5 mM MgCl2, 10 mM KCl, 10 mM 2-mercaptoethanol) containing protease inhibitors ("complete, EDTA-free," Roche Diagnostics, GmbH, Mannheim, Germany). Tissue homogenates were centrifuged at 4◦C, 12,000 × *g*, for 10 min. The supernatant was transferred to a fresh tube and kept on ice (or at 4◦C) until use and total soluble protein (TSP) content was estimated by the Bradford assay (Bio-Rad Inc., Segrate, Italy). Pellets were resuspended in appropriates volumes of SDS loading buffer, and prepared as described in the paragraph below, constituting the insoluble fraction of leaf extracts.

Homogenized tissues of pPVX-N infected plants were also used to inoculate *N. benthamiana* plants to propagate the infectious recombinant PVX particles until the third round of infection.

A small-scale purification of plant-produced N protein was performed. In brief, lyophilized leaf material was ground in liquid nitrogen, re-suspended and homogenized with an ultraturrax in 8 M urea, 100 mM NaH2PO4, 1 mM Tris, pH 8.0, and incubated for 1 h at RT with gentle shaking. Leaf material was finally lysed on ice by sonication at 10 Hz output (10 s each) for three times. Cell debris collected by centrifugation was discarded. The recovered supernatant was filtered (0.45 µm) and incubated for 2 h with the Ni-NTA resin. Plant N protein purification was performed by decreasing the pH (The QIAexpressionist). Protein elution was obtained at pH 4.5.

#### Immunoblotting

For immunoblotting analysis of the N protein, plant extracts containing 20 µg TSP and purified protein were boiled for 3 min in SDS-loading buffer. Immunoblotting detection of the M protein was performed on plant extracts incubated at 40◦C for 20' in SDS-loading buffer. Samples were separated by 12% SDS-PAGE and transferred onto PVDF membranes (Immobilon-P, Millipore). After membrane blocking with 5% non-fat dry milk in PBS (MPBS) over night (O/N) at 4◦C, membranes were incubated for 2 h at room temperature (RT) either with an anti-His6 monoclonal antibody (mAb; H1029-clone HIS-1, Sigma) or with specific polyclonal antibodies previously described (Carattoli et al., 2005). For N protein detection, we used a rabbit anti-N pAb, (obtained after immunization of animals with the purified His6-N protein produced in *E. coli*) at 1:3000 dilution. Immune complexes were revealed by 1 h incubation at RT with an antirabbit biotinylated secondary antibody (B8895, Sigma), at 1:5000 dilution, followed by Horseradish Peroxidase (HRP)-conjugated streptavidin (RPN1231, GE Healthcare) at 1:2000 dilution.

For M protein detection, membranes were incubated with the mouse anti-M pAb at 1:5000 dilution, followed by incubation with an anti-mouse HRP-conjugated secondary antibody at 1:10000 dilution (NA931, GE Healthcare).

For both proteins, the bound antibody was detected using the ECL Plus system ("Enhanced Chemi-Luminescence", GE Healthcare).

## Quantitative Triple Antibody Sandwich (TAS) ELISA Assay of Leaf Extracts

The amount of N protein in the plant extracts was estimated by a quantitative triple antibody sandwich (TAS) ELISA. Symptomatic systemic leaves, deriving from 15 plants infected with pPVX-N, were collected and pooled. Three independent extractions were performed and analyzed. Microtiter plates were coated with 100 µl/well of rabbit anti-N pAb (diluted 1:3000 in PBS) for 3 h at 37◦C followed by coating with 100 µl plant extract with a normalized TSP content (50 µg) for 16 h at 4◦C. Wells were then blocked with 150 µl/well of 5% MPBS at 37◦C for 3 h. The captured N protein was detected by incubating at 37◦C for 2 h with 100 µl/well of a 1:1500 dilution in 2% MPBS of a mouse anti-N pAb (obtained after immunization of animals with the purified His6-N protein produced in *E. coli*, Carattoli et al., 2005) followed by incubation with 1:10000 dilution of HRP-conjugated goat anti-mouse IgG antibody. After each step, wells were washed three times with PBS + 0.1% Tween 20 and one time with PBS. Enzymatic activity was measured by adding 100 µl/well v/v H2O2/ABTS [2 , 2 -azino bis-(3-etilbenzotiazolin) sulphuric acid] (KPL Inc., Gaithersburg, MD, USA) at RT in darkness condition. The absorbance of the samples was read after 30 at 405 nm on an ELISA microtiter plate reader. Known amounts of *E. coli-*purified N protein (0.5, 2, 5, 20, 50, 100, and 150 ng) were used as a standard. In order to give a better estimation of N protein yields in plant, the standard was diluted in *N. benthamiana* extract.

## Antigenicity Assay of the N Protein Produced in Plant with Patient Sera

A 'multi-strip' western blot assay was chosen to evaluate the antigenicity of the plant produced N protein. It is an immunoblotting procedure modified from Kiyatkin and Aksamitiene (2009), in which the sample, loaded in a single-well polyacrylamide gel (a standard gel where a single preparative well is cast), is blotted onto a membrane that is then cut into strips of the same width, each containing same amount of protein. Each strip is then incubated with different antibodies or sera and developed by a colorimetric assay (see Ciufolini et al., 1999; Di Bonito et al., 2002).

In our experiments, single-well 12% SDS-PAGE gels were loaded with one of the following preparations: (i) *E. coli* purified N protein (7.5 µg); (ii) plant-purified N protein (3.7 µg); (iii) plant extract from pPVX-N symptomatic leaves containing about 3.7 µg of N protein and 2 mg TSP; (iv) plant extract from pPVX201 symptomatic leaves containing about 2 mg TSP.

Samples (iii) and (iv) were prepared as following: the powder ground from pPVX-N or pPVX201 symptomatic leaves was resuspended and homogenized by an ultraturrax in one volume (w/v) of TN buffer (25 mM Tris-HCl pH 7.2, 150 mM NaCl) containing protease inhibitors. Tissue homogenates were centrifuged at 4◦C, 15000 × *g*, for 15 . TSP was calculated by Bradford assay and precipitated by trichloroacetic acid (TCA) at 25% final concentration. After 30' incubation in ice, samples were centrifuged, pellets were washed with cold 80% acetone, dried, re-suspended in SDS loading buffer. Gels were blotted using a semi-dry system (BIORAD) onto PVDF membranes.

After blocking with 5% MPBS, membranes were dried at RT and cut in 15 strips, 0.5 cm width. Each strip of samples (ii) and (iii) contained approximately 250 ng of the N protein (as plantpurified protein or in plant extract, respectively). As controls, strips containing 500 ng of *E. coli*-produced N protein [sample (i)], were prepared, as well as strips of plant extracts without the N protein [extract from pPVX201 symptomatic leaves, sample (iv)]. Before incubation with human sera, one strip from either plant-derived or *E. coli-*expressed N protein was incubated with the mouse anti-N pAb, as previously described (Carattoli et al., 2005), to confirm protein presence and to check that the amount of protein in the strips was sufficient for colorimetric detection.

The strips were incubated with pools of SARS sera collected during the SARS outbreak in Hong Kong in 2003 (Chan et al., 2005; 86 SARS patients pooled in seven groups) at 1:100 dilution in 3% MPBS, O/N at 4◦C. Blood samples were collected with informed written consent. The study was approved by the institutional human research ethics committee (The Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee).

As non-SARS controls, strips were incubated with three pools of sera from 30 patients affected by unrelated respiratory diseases (Carattoli et al., 2005). Antigen-antibodies complexes were revealed by a rabbit anti-human IgG (H + L) HRPconjugate (Southern Biotechnology Associates, Inc, Cat.No.6145- 05) diluted 1:5000 in 3% MPBS. After 1 h of incubation at RT colorimetric reaction on the strips was induced by adding 3,3 - Diaminobenzidine tetrahydrochloride substrate, DAB (Sigma D-5637) and Hydrogen peroxide.

# RESULTS

## Expression and Purification of Plant-Derived SARS-CoV N Protein

To produce the recombinant SARS-CoV N protein, *N. benthamiana* plants (4 weeks old) were infected with the pPVX-N DNA plasmid harboring the N gene (**Figure 1A**). As a control, plants were either mock infected or infected with the wild type pPVX201 vector. While mock infected plants

FIGURE 1 | Potato Virus X-mediated expression of SARS-CoV N protein in *Nicotiana benthamiana* plants. (A) Schematic representation of the pPVX-N construct used for the expression of SARS-CoV N protein in *N. benthamiana* leaves. CaMV 35S: constitutive 35S promoter from the cauliflower mosaic virus; Nos-ter: transcription terminator of the nopaline synthase gene of *Agrobacterium tumefaciens*; RdRp: PVX replicase gene; M1-3: PVX triple gene block for cell-to-cell movement; CP: PVX coat protein gene; SgP: coat protein sub-genomic promoter; N: SARS-CoV N gene; His6-tag: histidine tag. (B) Immunoblotting of TSP extracted from *N. benthamiana* plants infected with pPVX-N. For each sample, 20 µg TSP were loaded on gel. Lane 1: N protein purified from *Escherichia coli* under native conditions (40 ng); lane 2: pPVX-N inoculated leaves; lane 3: pPVX201 inoculated leaves (negative control); lane 4: pPVX-N symptomatic systemic leaves; lane 5: pPVX201 symptomatic systemic leaves (negative control); lane 6: molecular weight marker (Magic Mark, Invitrogen); lane 7: pPVX-N symptomatic systemic leaves extract stored at −20◦ C for 2 months. Immunoblotting was performed with the rabbit anti-N pAb.

showed no symptoms, typical symptoms (mainly chlorotic spots) generally appeared on the inoculated leaves of plants infected with pPVX-N or pPVX201 vectors 4–5 days post inoculation (dpi). The infection spread systemically to apical leaves, where symptoms appeared 7–10 dpi.

To examine whether the N protein accumulated in infected plants, soluble protein extracts were analyzed by ELISA and immunoblotting.

Interestingly, after infection with the pPVX-N plasmid, protein expression corresponded to symptoms in systemic leaves in all plants analyzed (about 100). This result indicates that the construct is stable and that the recombinant virus PVX-N can spread systemically within the inoculated plant. Immunoblotting of soluble protein extracts from plants infected with pPVX-N reveals a single band of about 50 kDa in systemic leaves (**Figure 1B**), suggesting the absence of proteolysis. On the contrary, when the N protein is expressed in *E. coli*, additional bands of lower molecular mass are present (**Figure 1B**). These bands probably correspond to protein degradation, in accordance with previous work demonstrating the intrinsic instability and/or autolysis of this protein when expressed in bacteria (Mark et al., 2008). Furthermore, pPVX-N-derived extracts, stored at – 20 ◦C for 2 months and then thawed (**Figure 1B**), as well as extracts obtained from freeze-dried pPVX-N-infected leaves showed an intact N protein (data not shown). Immunoblotting of mammalian cells transfected with the pVAX-N plasmid also revealed the presence of a single band of approximately 50 kDa corresponding to the N protein (**Figure 2A**). Immunofluorescence revealed a cytoplasmic localization of the N protein (**Figure 2B**).

Plant extracts deriving from PVX-N-infected leaves were also used for subsequent inoculations. N protein expression was confirmed at least until the third cycle of re-infection (Supplementary Figure S1), further demonstrating the stability of the construct and of the recombinant virion.

The amount of recombinant N protein expressed in leaves, as measured by TAS-ELISA, was approximately 3–4 µg/g fresh leaf weight, corresponding to 0.2% TSP (**Figure 3**).

Although the N protein accumulated mainly in the soluble fraction in all the expression systems used (plant, bacteria and mammalian cells), for purification the best recovery of the N protein from *E. coli* was obtained in denaturing conditions (3 mg of protein/liter of culture under denaturing conditions versus 0.4 mg protein/liter of culture under native conditions, **Figure 4A**). Therefore, we decided to perform N protein purification in denaturing conditions also from plant tissue. We performed a small-scale purification by loading plant extracts derived from freeze-dried leaves (obtained from a pool including primary-infected and re-infected systemic leaves) on a Ni-NTA

FIGURE 2 | Expression of SARS-CoV N protein in mammalian cells. (A) Immunoblotting of total proteins extracted from HEK-293 cells transfected with the pVAX-N plasmid. For each sample, total proteins from 1 × 105 cells were loaded on gel. Lane 1: N protein purified from *E. coli* under native conditions (20 ng); lanes 2, 3: HEK-293 cells, 24 h post-transfection with the pVAX-N plasmid, with or without the addition of proteasome inhibitor MG-132, respectively; lane 4: negative control, HEK-293 cells transfected with the pVAX empty vector. Immunoblotting was performed with the rabbit anti-N pAb. (B) Immunofluorescence of HEK-293 cells transfected with the pVAX-N plasmid (N) or with the pVAX empty vector [(−) CTR]. Immunofluorescence was performed with the rabbit anti-N pAb and nuclei were counter-stained with DAPI (100x objective, ZEISS ACHROSTIGMAT 100x/1,25 oil). Scale Bar = 5 µm.

(recorded at 30') of the following samples: bar 1: pPVX201 infected systemic leaves (negative control); bar 2: pPVX-N systemic leaves; bars 3–9: purified N protein produced in *E. coli* (0.5, 2, 5, 20, 50, 100, and 150 ng, respectively) diluted in *N. benthamiana* extract. Error bars represent standard deviations of three technical replicates, i.e., three separate extractions from a pool of leaves from 15 plants (pPVX201 or pPVX-N).

(Prestained Marker, Invitrogen); lanes 2–3: first and second elution fractions (purification performed under denaturing conditions); lane 4, 5, 6: BSA 1, 2, and 5 µg, respectively; lanes 7–8: first and second elution fractions (purification performed under native conditions). (B) Silver-stained SDS-PAGE of the N protein purified from *N. bethamiana* by affinity chromatography. Lane 1: N protein purified from *E. coli* under denaturing conditions (50 ng); lanes 2–3: first and second elution fractions of N protein purification from *N. benthamiana* (purification performed under denaturing conditions).

affinity purification column. In this way, we obtained yields of about 1 µg of purified N protein/g of fresh leaf weight (**Figure 4B**).

## Antigenicity of Plant-Derived SARS-CoV N Protein

The ELISA and immunoblot analysis gave a first indication of antigenic features of the plant-derived N protein. In fact, in such analysis the N protein was specifically recognized by rabbit and mouse anti-His6-N hyper-immune sera that had previously been shown to recognize the N protein in SARS-CoV infected cells (Carattoli et al., 2005).

To further characterize the N protein expressed in plant, we analyzed its reactivity with sera from SARS patients, collected during the SARS outbreak in Hong Kong in 2003. These SARS sera were previously screened by an ELISA assay with an *E. coli*expressed N protein (Di Bonito and Chan, data not shown). Here, to evaluate SARS-positive sera reactivity with N plant expressed protein, we used a 'multi-strip' western blot assay. In a first experiment, we evaluated the reactivity of the purified N protein with a pool of 5 SARS sera. As shown in **Figure 5A**, both preparations of purified N protein, from *E. coli* and from plant, are recognized by SARS sera as well as by the mouse anti-N pAb (positive control). Then, we validated the results obtained for the plant-purified N protein analyzing its reactivity with other six groups of SARS sera deriving from 86 patients and with three groups of sera from 30 patients affected by non-SARS respiratory diseases (**Figure 5B**). In this experiment, we observed that the N protein purified from plant is specifically recognized by all the groups of SARS sera analyzed, while no reactivity was observed with sera from patients affected by other respiratory diseases.

We also tested the reactivity of SARS and non-SARS sera with the unpurified N protein (soluble extract of pPVX-N symptomatic leaves) and with the extract from plant infected with the pPVX201 empty vector. A light reactivity with SARS sera was observed only for the unpurified N protein, while no reactivity was observed for the same preparation when using non-SARS sera (Supplementary Figure S2). Importantly, the pPVX201 leaf extract did not react with any human sera (Supplementary Figure S2).

To our knowledge, this is the first report of the antigenic properties of a plant-derived N protein as revealed by direct serology using SARS patient sera, suggesting its possible use for the development of SARS diagnostic assays.

# Expression of Plant-Derived SARS-CoV M Protein

We investigated the ability of plant expression systems to cope with the synthesis of M protein. We started our studies on M protein expression in *N. benthamiana* plants (4-week old) by infection with the pPVX-M plasmid harboring the *M* gene as described for the N protein. Also in this case typical symptoms generally appeared 4–5 dpi on inoculated leaves and 7–10 dpi on systemic leaves. However, M protein production was reported in just 2 plants out of 100 analyzed at detectable levels (data not shown).

Hence, we investigated the possibility to obtain the M protein by agroinfiltration. *N. benthamiana* leaves were infiltrated with *A. tumefaciens* suspensions (strains C58 and GV3101) harboring the pBI binary vector containing the *M* gene (pBI-M vector, **Figure 6A**). With this simple technology, and without the use of any post-transcriptional gene silencing suppressors, we were able to detect SARS-CoV M protein production in plant. The time course experiment revealed that the M protein is expressed mainly at 4 and 5 dpi (data not shown). For immunoblotting analysis, the M protein was extracted with an appropriate buffer (GB buffer) and, since it was previously reported that boiling causes M protein aggregation and precipitation (Lee et al., 2005), samples boiling was avoided. Interestingly, the M protein

in *E. coli* (500 ng); strips 2, 4: purified N protein produced in plant (250 ng). Strips 1 and 2: probed with a pool of 5 SARS patient sera. Strips 3 and 4: probed with the mouse anti-N pAb (positive control). (B) Validation of the results shown in panel A for the N protein produced in plant. Each strip derives from the blotting of a single-well SDS-PAGE gel (showed in C) loaded with 3.7 µg of purified N protein produced in plant. Each strip contains approximately 250 ng of N protein. Strip 1 was probed with the mouse anti-N pAb (positive control). Strips 2–7: probed with SARS patient sera (different pools deriving from several SARS patients). Strips 6–8: probed with pools of sera deriving from patients affected by non-SARS respiratory diseases (negative control). (C) Replica gel of the experiment shown in panel B stained by Coomassie Brilliant Blue for loading control of the purified N protein from plant.

accumulated almost totally in the soluble fraction of the plant extract (**Figure 6B**). As described in the previous paragraph for the N protein, to better characterize the plant-derived M protein we worked, at the same time, on M protein expression in bacteria and mammalian cells. Due to toxic properties of M protein for *E. coli* (Carattoli et al., 2005), we performed colony selection and M protein expression under sub-optimal growth conditions (30◦C). In this way, the M protein, with a mass of about 25 kDa was purified by Ni-NTA affinity chromatography in denaturing conditions, obtaining yields of about 100 µg/l culture (data not shown). DNA sequence analysis revealed the presence in the *M* gene of three spontaneous point mutations: R13 *>* K in the N-terminal domain, L36 *>* F in the first transmembrane domain and V160 *>* I in the cytoplasmic domain (MRLV). As the plant-derived recombinant M protein, the MRLV was also specifically recognized by the mouse anti-M pAb (**Figure 6C**) that had previously validated by Immunofluorescence Antibody Assay (IFA) in SARS CoV infected Vero cells (Carattoli et al., 2005). Although we were not able to express the M protein with its original aa sequence in bacteria, the MRLV protein was useful as standard for the plant-produced M protein characterization. Immunoblotting revealed that, contrary to prokaryotic cells, the plant system allowed the expression of the full-length M protein, especially by the use of the *Agrobacterium* C58C1 strain (**Figure 6B**). The M protein yield, calculated by immunoblotting using the quantified MRLV protein purified from *E. coli* as standard, was estimated to be 0.1–0.15% TSP. The mobility of the plant-derived M protein in SDS-PAGE was reduced compared to the mutated bacterial form (**Figure 6C**), suggesting a higher molecular mass of plant-expressed compared to the *E. coli*expressed M protein.

No attempts to purify the M protein from plant were done due to the expression estimates.

The M protein was also expressed in mammalian cells. Immunofluorescence on M-transfected HEK-293 cells using the mouse anti-M pAb revealed that M protein is primarily localized in the plasma membrane (**Figure 7**). Similar results were previously reported by Tseng and collaborators (Tseng et al., 2010), who described a perinuclear and plasma membrane localization of the M protein expressed in various cell lines. However, we did not observe M protein expression by immunoblotting, either in transfected cells or in the culture medium (not shown), probably because of its low expression level, as we reported in *N. benthamiana* plants by PVX-mediated infection.

#### DISCUSSION

The importance to develop effective therapeutic and preventive strategies, to be readily applied to new emergent pathogens is established by the two novel coronaviruses that have emerged in humans in the twenty-first century: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Both viruses cause acute respiratory distress syndrome and are associated with high mortality rates. There are no clinically approved vaccines or antiviral drugs available for either of these infections, thus their development represents a research priority (Graham et al., 2013).

Severe acute respiratory syndrome coronavirus was the first massive infectious disease outbreak and it still has the potential to cause large-scale epidemics in the future. The key to preventing and controlling a future outbreak of SARS is to develop rapid and specific diagnostic methods so that suspected

patients can be correctly assessed. Moreover, effective and safe treatment/vaccination will be extremely important in minimizing the damage of a new pandemic. Enormous efforts have been undertaken to these purposes. The four major diagnostic methods available for SARS include viral RNA detection by RT-PCR, virus induced antibodies by immunofluorescence assay (IFA), or by enzyme linked immunosorbent assay (ELISA) of nucleocapsid protein (N) and inoculation of patient specimens in cell culture (World Health Organization [WHO], 2003 SARS: Laboratory diagnostic tests).

The SARS-CoV N protein, expressed at early stage of infection and triggering a strong antibody response by the host, is considered to be the best diagnostic target (Surjit and Lal, 2008). Plasmon resonance-based biosensors (Huang et al., 2009; Park et al., 2009) and nanowire/carbon nanotube transistors (Ishikawa et al., 2009) have been developed for the detection of SARS-CoV N protein in patient sera. Such sensors offer real-time detection of nanomolar concentrations of the protein. Nevertheless, SARS tests should also have other useful features such as cost-effectiveness and ease of operation. Moreover, combinations of antigens may be necessary to provide a definitive diagnosis of SARS in humans and susceptible animals (Roper and Rehm, 2009).

The production of recombinant N protein has been achieved in a variety of heterologous expression systems. A synthetic gene with optimized codons has been expressed in *E. coli* at high yield (Das and Suresh, 2006) but it was demonstrated that bacterially expressed N protein produces false seropositivity owing to interference of bacterially derived antigens (Leung et al., 2006; Yip et al., 2007; Surjit and Lal, 2008) or cross-reacts with antisera of human coronaviruses (HCoV-OC43 and HCoV-229E)- infected patients (Woo et al., 2004). An interesting study correlated the phosphorylation state of the N protein with its antigenicity and specificity of antibodies recognition (Shin et al., 2007). These data underline the importance of producing the recombinant protein in eukaryotic platforms such as insect cells, yeast, or plants to set up more efficient and specific diagnostic tests. To date, it was demonstrated that the N protein produced in insect cells may be useful for the development of highly sensitive and specific assays to determine SARS infection (Shin et al., 2007). Later, the N protein was transiently expressed in plant by agroinfiltration, and its antigenicity was demonstrated in mice (Zheng et al., 2009) giving a proof of concept of its use in vaccine formulations. Previously, the S1 domain of SARS-CoV S protein had been stably expressed in tomato and low-nicotine tobacco plants obtaining about 0.1% TSP (Pogrebnyak et al., 2005). The plant-derived antigen was able to induce systemic and mucosal immune responses in mice.

While previous works have primarily focused on the S and N proteins, there is growing evidence of the potential of the M antigen both as an effective vaccine and diagnostic candidate. Thus, the obtainment of the recombinant full-length M protein in an eukaryotic expression system also represents a good way to develop an effective and safe SARS-CoV vaccine. In addition to the knowledge that the M protein elicits a strong humoral responses, and that a specific humoral and cellular immune response can be obtained by co-expressing S, M and E (Lu et al., 2007), it has been demonstrated that the M protein also contains T cell epitopes (Liu et al., 2010). The availability of recombinant M protein, in combination with other viral proteins might overcome the concern about the sensitivity and the specificity of N nucleoprotein-based assay, as described when using the N and the S proteins together (Woo et al., 2004; Haynes et al., 2007; Gimenez et al., 2009). This would help the development of more efficient reagents to detect antibodies in the infected human host.

Here, we propose the use of plants as an alternative system to produce the N and M antigens that could be useful to formulate new vaccines and diagnostic assays against SARS.

Since plant transformation and regeneration of stable transformants require considerable time, we used transient expression systems (PVX and agroinfiltration) to evaluate the ability of the plant expression system to cope with the synthesis of the SARS-CoV M and N proteins.

The N and M full-length genes of the human SARS-CoV Frankfurt I isolate were cloned, without codon optimization, into different expression vectors. For the SARS-CoV N protein, we assessed the successful ectopic expression in *N. benthamiana* plants by pPVX-mediated infection (**Figure 1**). We were able to obtain the N protein in systemic leaves in most primaryinfected plants as well as in 100% re-infected plants. These results demonstrate the stability of the construct, a condition not easily obtained especially when large sequences are inserted in the pPVX-derived expression cassette (the *N* gene is about 1300 bp). In fact, several studies report that the use of 'first generation' plant viral vectors (like the pPVX series) for the expression of proteins in plants can lead to insert elimination by natural selection over replication cycles as early as the first infection passage with a positive correlation between insert length and elimination rate (Avesani et al., 2007).

Unlike the observed prokaryotic expression pattern, no proteolysis products were detected in immunoblotting by using polyclonal sera in pPVX-N-derived extracts, fresh or stored at – 20◦C for 2 months (**Figure 1B**). These data demonstrate the stability of the recombinant N protein when transiently expressed in *N. benthamiana*. The same stability was observed when the N protein was expressed in mammalian cells, even in the absence of proteasome inhibitor (**Figure 2A**).

The purified plant-produced N protein is specifically recognized by sera from Chinese SARS patients of the 2003 outbreak, and not from patients affected by unrelated respiratory diseases (**Figure 5**). This result suggests that the plant-expressed SARS-N protein is suitable in SARS diagnosis.

It is interesting to note that, when using crude plant extracts, SARS human sera reveal a weak band, corresponding to the molecular weight of the N protein, without any cross-reaction with other components of the plant extract (Supplementary Figure S2). Taken together, the results indicate that plant-derived N protein is specifically detected by antibodies of SARS patients, using an assay where the antigen-antibody complex is revealed by a colorimetric method, less sensitive but more specific and suitable for hospital clinical laboratories. It should be noted that the only information available to date about plant-derived SARS-CoV N protein antigenicity were from Zheng et al. (2009) who immunized mice and evaluated the impact on the regulation of cytokines and on the elicited IgG subclasses. Our study is the first demonstration by direct serology that plant-derived N protein is able to reveal human N-specific antibodies present in sera of SARS patients, thus providing an adequate instrument to develop a rapid, low-cost, immune-based diagnostic assay to be used as an alternative or in association to molecular diagnosis.

For the M protein, we obtained a spontaneously mutated MRLV protein in *E. coli.* This allowed to overcome toxicity of the wild type protein when expressed in bacteria (Carattoli et al., 2005) and to purify the MRLV protein that was then used as positive control in our experiments. As the SARS-CoV M protein is difficult to express in recombinant form, the exact structure and function of the protein are not fully elucidated (Neuman et al., 2011; Tseng et al., 2013; Siu et al., 2014).

Unlike prokaryotic cells, the plant system allowed the expression of the full-length M protein by using *A. tumefacien*s (in particular the strain C58C1), demonstrating for the first time the possibility to express the SARS-CoV M protein in plants, without the need of codon optimization or addition of any further modifications. On the contrary, by PVX-mediated infection we observed very low expression levels for the M protein, only in 2 plants out of 100 analyzed. We can speculate that membrane M protein, that already proved to be toxic in *E. coli*, may be difficult to express by using a 'living vector' like PVX due to interference with virus replication and/or expression/assembly of viral components, while it is tolerated by the plant when expressed by agro-infiltration.

Compared to the mutated MRLV protein produced in *E. coli*, the M protein produced in plant shows a reduced electrophoretic mobility, suggesting a higher molecular mass (**Figure 6C**). The reason for this difference remains to be elucidated, but it could be due to modified residues in the MRLV protein or by the presence of glycosylation in the M protein produced in the eukaryotic system (the native protein is N-glycosylated at the fourth residue).

The plant-produced M protein yields were not sufficient to perform the test with human sera. Thus, for plant-derived M protein characterization, efforts should be made in order to enhance expression yields. This includes the use of secondgeneration viral vectors (Gleba et al., 2007) or other implemented platforms that have been developed in the last years (Moustafa et al., 2015; Paul et al., 2015; Sack et al., 2015).

# CONCLUSION

Our results add further insights to the characterization of the N and M proteins and provide a proof of principle for using plants as a robust, rapid and flexible production system for protein reagents suitable to face potential recurring SARS-CoV outbreaks.

#### AUTHOR CONTRIBUTIONS

RF, PDB concept and design of the research, analysis and interpretation of data, writing and revising the article, final approval of the version to be published. OCD, SM design of the research, acquisition of data, analysis and interpretation of data, drafting and writing the article. EI acquisition of data, analysis and interpretation of data. DDM writing and revising the article critically for important intellectual content. PKSC design and revising the article critically for important intellectual content and final approval of the version to be published.

#### FUNDING

The work was partially supported by the Italian 'Ministero della Salute,' grant 'Progetto Speciale Lotta alla SARS.'

#### REFERENCES

#### ACKNOWLEDGMENTS

We thank Dr. Alessandra Carattoli from 'Istituto Superiore di Sanità' for coordinating the SARS project. We also thank Orsola Bitti for technical assistance.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00054

syndrome-associated coronavirus-specific antibody. *Clin. Vacc. Immunol.* 6, 241–245. doi: 10.1128/CVI.00252-08

phosphorylation on immunoreactivity and specificity. *Virus Res.* 127, 71–80. doi: 10.1016/j.virusres.2007.03.019

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Demurtas, Massa, Illiano, De Martinis, Chan, Di Bonito and Franconi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in *Salvia miltiorrhiza*

*Zhichao Xu1, Hongmei Luo1, Aijia Ji1, Xin Zhang1, Jingyuan Song1\* and Shilin Chen1,2\**

*1 Institute of Medicinal Plant Development – Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China, 2 Institute of Chinese Materia Medica – Chinese Academy of Chinese Medical Science, Beijing, China*

#### *Edited by:*

*Eugenio Benvenuto, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Italy*

#### *Reviewed by:*

*Jianbo Xiao, University of Macau, China Laura Bassolino, CREA, Italy Shujuan Zhao, Shanghai University of Traditional Chinese Medicine, China*

#### *\*Correspondence:*

*Jingyuan Song<EMAIL_ADDRESS>Shilin Chen<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science*

*Received: 21 September 2015 Accepted: 19 January 2016 Published: 05 February 2016*

#### *Citation:*

*Xu Z, Luo H, Ji A, Zhang X, Song J and Chen S (2016) Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza. Front. Plant Sci. 7:100. doi: 10.3389/fpls.2016.00100*

Salvianolic acids are among the main bioactive components in *Salvia miltiorrhiza*, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the *S. miltiorrhiza* root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in *S. miltiorrhiza*.

Keywords: *Salvia miltiorrhiza*, hybrid-seq, full-length transcripts, phenolic acid biosynthesis, alternative splicing, cytochrome P450s, laccases

# INTRODUCTION

The alternative splicing events of mutiexon genes in multicellular eukaryotes can enhance the functional diversity of the encoded proteins and regulate gene expression through complex posttranscriptional mechanisms (Reddy et al., 2013). Recent alternative splicing analysis originating from next-generation sequencing (NGS, Illumina) has revealed that over 60% of multiexon genes undergo alternative splicing events in plants, such as *Arabidopsis thaliana* (Marquez et al., 2012), *Glycine max* (Shen et al., 2014), *Brachypodium distachyon* (Walters et al., 2013), and *Oryza sativa* (Zhang et al., 2010). However, the short-read assembly strategy of NGS limits its capacity to precisely quantify and predict alternative splicing events. In contrast, the long reads from SMRT sequencing (single molecule, real-time DNA sequencing using Pacific Biosciences RS II, PacBio) have demonstrated their advantage in sequencing full-length transcripts to identify and predict alternative splicing isoforms in human embryonic stem cells (Au et al., 2013; Roberts et al., 2013). Research has also addressed the disadvantage of high sequencing errors by correction with high-quality NGS reads (Au et al., 2012; Koren et al., 2012). Our recent study successfully

demonstrated the localization of tanshinones to the root periderm and revealed the molecular mechanism of tanshinone biosynthesis using hybrid-seq (next-generation and single molecular real-time sequencing, NGS and TGS) of the root transcriptome of *Salvia miltiorrhiza* (Xu et al., 2015).

*Salvia miltiorrhiza* Bunge is one of the most commonly used medicinal plants in Traditional Chinese Medicine (TCM), as its dried root or rhizome is of great phytochemical value in the treatment of cardiovascular diseases and inflammation, and as an anti-oxidant, among other uses (Cheng, 2006; Wang et al., 2007; Dong et al., 2011). The main active components of *S. miltiorrhiza* are hydrophilic salvianolic acids (SAs), such as rosmarinic acid (RA) and lithospermic acid B (LAB; Wang et al., 2007), and lipophilic diterpenoid components, such as tanshinones I/IIA, dihydrotanshinone, and cryptotanshinone (Lei et al., 2014). The availability of the nuclear and chloroplast genomes (Qian et al., 2013) and transcriptome (Hua et al., 2011; Luo et al., 2014), along with research related to the molecular regulation (Zhang et al., 2013, 2015; Tan et al., 2014; Li et al., 2015) and biosynthesis of its bioactive components (Guo et al., 2013, 2015; Bloch and Schmidt-Dannert, 2014), strongly favors *S. miltiorrhiza* as a potential model medicinal plant for TCM research.

There are two pathways for RA synthesis, namely, the phenylpropanoid pathway and the tyrosine-derived pathway, and many of the key genes encoding enzymes in *S. miltiorrhiza* have been identified (Di et al., 2013; Hou et al., 2013; Bloch and Schmidt-Dannert, 2014). In the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) sequentially catalyze the conversion of L-phenylalanine into 4-coumaroyl-CoA. In the tyrosine-derived pathway, tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate reductase (HPPR) sequentially catalyze the conversion of L-tyrosine into 4-hydroxyphenyllactic acid, which is then catalyzed into 3,4-dihydroxyphenllactic acid by an unknown CYP450 in *S. miltiorrhiza* (Di et al., 2013). Rosmarinic acid synthase (RAS) catalyzes conversion of the products from the two pathways to form 4-coumaroyl-3- , 4- -dihydroxyphenllactic acid (4C-DHPL), and SmCYP98A78 (allelic variant of SmCYP98A14, Chen et al., 2014) has been indicated as the specific hydroxylase that catalyzes the conversion of 4C-DHPL to RA (Di et al., 2013). Finally, oxidative dimerization of hydroxystilbene occurs, and laccase has been proposed to catalyze the oxidative reaction from RA to LAB (Giardina et al., 2010; Di et al., 2013). Although the phenolic acid biosynthetic pathway has in essence been proposed and identified, many homologous genes encoding key enzymes were uncovered by genome-wide strategy. Indeed, a total of 28 homologous genes of *SmPAL*, *SmC4H*, *Sm4CL*, *SmTAT*, *SmHPPR*, *SmRAS,* and *SmCYP98A78* have been identified by genome annotation (Wang et al., 2015).

In this study, using the hybrid-seq transcriptome of *S. miltiorrhiza* roots, we systematically analyzed the full-length transcripts and alternative splicing events of these 28 gene loci predicted as being related to RA biosynthesis. We then analyzed co-expression patterns and predicted candidate CYP450s and laccases related to the SA pathway. Our experiments not only reveal full-length transcript and alternative splicing data but also provide a reference tool for future studies on the genes involved in the biosynthesis of phenolic acids.

# MATERIALS AND METHODS

#### Plant Resources

*Salvia miltiorrhiza* (line 99-3) plants were cultivated at the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences (CAMS) in an open experimental field. Roots, stems, and flowers were collected from 3-yearsold plants growing in the field on May 27th, 2014. The roots were separated into three parts (periderm, phloem, and xylem) according to morphology and microstructure. Leaves with and without MeJA treatment (12 h, 200 µM; Sigma-Aldrich, St. Louis, MO, USA) were collected from tissue culture *S. miltiorrhiza* (line 99-3) plantlets at 25◦C under long-day condition of 16-h light/8 h dark. All of the collected tissues originated from the same clone of *S. miltiorrhiza* (line 99-3).

# Transcriptomic Data

Single molecule real-time DNA sequencing data from pooled root tissues (periderm, phloem, and xylem) using the PacBio RS II platform (Pacific Biosciences of California, USA; Accession, SRX753381) and RNA-seq reads from different root tissues (periderm, phloem, and xylem) using the Illumina Hiseq 2500 platform (Illumina, USA) are reported in our recent study (Xu et al., 2015; Accession, SRR1640458). RNA-seq reads for different organs (root, stem, and flower) were generated using the Illumina HiSeq 2000 platform (Illumina, USA; Accession, SRP028388), and Illumina reads from leaves with and without 12 h MeJA treatment were obtained in a previous study (Luo et al., 2014; Accession, SRP051564).

# Bioinformatic Analysis

Single molecule real-time DNA sequencing data were corrected with Illumina short reads using LSC 1.alpha software (Au et al., 2012). Alternative splicing isoforms were analyzed using IDP 0.1.7 software, employing SMRT sequencing reads, Illumina short reads, and genome scaffolds (Au et al., 2013). Differential gene expression in various root tissues, organs and under MeJA treatment was analyzed using Tophat 2.0.12 and Cufflinks 2.2.1 (Trapnell et al., 2012) by mapping the Illumina short reads to *S. miltiorrhiza* genome sequences. Heat maps were constructed using R statistical project (Gentleman et al., 2004).

# Gene Structures and Phylogenetic Analysis

The alternative splicing isoforms found by IDP were viewed using the IGV 2.3.34 software (http://www.broadinstitute.org/ software/igv/). The annotated gene sequences were corrected with the SMRT sequencing reads using Apollo software (Lee et al., 2013). Gene structures (e.g., intron, exon, intron phase) were also analyzed with Apollo. The full-length amino acid sequences of candidate CYP450s and laccases from *S. miltiorrhiza* and

other species were aligned with MEGA 6 (Tamura et al., 2013). Neighbor-joining trees were then constructed using the bootstrap method with 1,000 replications.

#### UPLC Analysis of LAB Content

The detection methods followed the Pharmacopeia of the People's Republic of China. Periderm, phloem, and xylem samples were ground into powder (with three biological replicates for each sample), and each weighed sample of ground powder (0.2 g) was extracted with 50 mL of 75% methanol. After 1 h of heating reflux extraction, 75% methanol was added to complement and maintain a constant weight, and the sample was filtered through a 0.45-µm syringe filter. In addition, an LAB standard was dissolved with 75% methanol at a concentration of 140 mg/L. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) with a mobile phase of 30% methanol, 10% acetonitrile, 1% methanoic acid, and 59% H2O in a Waters UPLC system (Waters, USA). The detection wavelength was set to 286 nm.

#### Gene Expression Analysis by qRT-PCR

Nine RNA samples were isolated from different *S. miltiorrhiza* tissues (periderm, phloem, xylem, root, stem, leaf, and flower), which were collected from experimental field, and leaves from tissue culture plantlets were treated with MeJA (control or 12-h MeJA treatment). Total RNA (three biological replicates for each sample) was isolated using the RNeasy Plus Mini kit (Qiagen, Germany). Reverse transcription was performed with PrimeScriptTM Reverse Transcriptase (TaKaRa, Japan). The qRT-PCR primers were designed with Primer Premier 6 (Supplementary Table S1), and their specificity was verified by PCR. The qRT-PCR analysis was performed in triplicate using SYBRR Premix Ex TaqTM II (TaKaRa, Japan), with *SmActin* as a reference gene, and a 7500 real-time PCR system (ABI, USA). The Ct value was calculated for analyzing relative expression levels using the 2−--CT method (Livak and Schmittgen, 2001). To detect differences in the expression of candidate genes among various tissues, one-way ANOVA was performed using IBM SPSS 20 software (IBM Corporation, USA). *P* < 0.01 was considered highly significant. Gene co-expression analysis of candidate genes was performed using Pearson's correlation test.

# RESULTS

# Localization of SA Accumulation in *S. miltiorrhiza* Root

The rhizome or root of *S. miltiorrhiza* is the primary medicinal part of this well-known herb. The hydrophilic phenolic acids in the *S. miltiorrhiza* root are mainly distributed in the phloem and xylem. UPLC identification demonstrated a similar LAB content in the phloem and xylem, which were five times higher than that in the periderm (**Figures 1A,B**). These results provided a

potential basis for co-expression analysis of SA biosynthetic genes in the *S. miltiorrhiza* root.

# Isoform Detection and Prediction of RA Biosynthetic Genes

Using the next-generation sequencing platform (Illumina), RNAseq data (a total of 867,864,885 reads) from *S. miltiorrhiza* periderm, phloem, xylem, root, stem, flower, leaf, and leaf after 12 h of MeJA treatment were collected. Using SMRT sequencing (PacBio RS II platform), full-length cDNA libraries from pooled periderm, phloem, and xylem samples were performed for a longread survey, and 796,011 subreads were employed to identify fulllength transcripts and alternative splicing events by hybrid-seq using the IDP (isoforms detection and prediction) pipeline.

A total of 28 candidate genes from the phenylpropanoid pathway and tyrosine-derived pathway, related to RA biosynthesis, were selected based on a genome-wide approach; these gene included *SmPALs* (3), *SmC4Hs* (2), *Sm4CLs* (10), *SmTATs* (3), *SmHPPRs* (3), *SmRASs* (6), and *SmCYP98A78* (Supplementary Table S2 and **Figure 1C**). The same approach was previously used to detect tanshinone biosynthetic genes (Xu et al., 2015). Fifteen gene loci were detected as full-length transcripts (Supplementary Figure S1), and their gene structures and intron phases are described in **Figure 2A**. *SmC4H2* might be a duplicated pseudogene of *SmC4H1* with an N-terminal deletion, as *SmC4H2* exhibits 74% homology with *SmC4H1* and is located at a distance of 7.5 kb from *SmC4H1* in the genome. *Sm4CL2*, *Sm4CL-like5*, *Sm4CL-like7,* and *SmTAT1* were identified as expressing alternatively spliced isoforms (**Figure 2B**), and all of the alternatively spliced junctions were characterized as intron retention. *Sm4CL2* and *Sm4CL-like5* each expressed two isoforms, whereas *Sm4CL-like7* and *SmTAT1* each expressed three isoforms (Supplementary Table S5). Among their respective alternative splicing events, *Sm4CL2-iso2*, *Sm4CLlike5-iso2*, and *SmTAT1-iso3* were the dominantly expressed isoforms (Supplementary Table S5), though three isoforms of *Sm4CL-like7* all exhibited similar expression. We found that all of the intron retentions introduced premature termination codons (PTCs), and the PTC locations in *Sm4CL2-iso1*, *Sm4CLlike5-iso1*, *Sm4CL-like7-iso2*, *Sm4CL-like7-iso3*, *SmTAT1-iso1*, and *SmTAT1-iso2* were in intron 4, intron 5, intron 2, intron 3, intron 4, and intron 4, respectively, (Supplementary Figure S2).

# Expression Profiles of Candidate RA Biosynthetic Genes

In this study, analysis of differentially expressed genes in the three root tissues showed that *SmPAL1*, *SmPAL3*, *SmC4H1*, *Sm4CL3*, *Sm4CL-like1*, *Sm4CL-like4*, *SmTAT1*, *SmHPPR3*, *SmRAS,* and *SmCYP98A78* exhibited low expression in the periderm and high expression in the phloem and xylem, in accord with the distribution of LAB (**Figure 1D**). In addition, the transcript levels of *SmPAL1*, *SmC4H1*, *Sm4CL3*, *Sm4CL-like1*, *SmTAT1*, *SmHPPR3*, *SmRAS,* and *SmCYP98A78* were significantly up-regulated after 12 h of MeJA treatment (Supplementary Table S2); however, the expression of *Sm4CL2*, *Sm4CL-like4*, *Sm4CL-like6*, and *SmHPPR2* was down-regulated after MeJA treatment. *SmTAT3*, *SmHCT2*, *SmHCT3*, and *SmHCT4* were identified as silenced genes. *SmHCT1* exhibited remarkably specific expression in the root xylem, yet *SmHCT5* showed only slight expression in the stem. Phylogenetic trees for 18 hydroxycinnamoyltransferase (HCT family) amino acid sequences including hydroxycinnamoyl-CoA:shikimate/quinate

hydroxycinnamoyltransferases (HCS/QTs), RASs, and hydroxycinnamoyl/benzoyltransferases (HCBTs), in different species revealed clustering of five unidentified HCTs from *S. miltiorrhiza* with RASs, rather than with HCS/QTs (Supplementary Figure S3).

#### Co-expression Analysis and Isoform Identification of Candidate CYP450s

Our RNA-seq results showed opposite expression patterns for *SmCYP76AH1* and *SmCYP98A78*, in accord with the different distribution of tanshinones and phenolic acids in the periderm, phloem, and xylem. Moreover, six CYP450s were selected as candidate CYP450s related to RA biosynthesis based on the criteria of a phloem/periderm FPKM greater than 1.5 and a xylem/periderm FPKM greater than 1.5. The selected CYP450s included *SmCYP749A39*, *SmCYP714C2*, *SmCYP92A73*, *SmCYP98A75*, and *SmCYP98A76* (Supplementary Table S3). A comprehensive evaluation of eight RNA-seq and qRT-PCR analyses of these CYP450s, including *SmCYP98A78* and *SmC4H1* (*SmCYP73A120*), indicated that expression level of the candidate CYP450s was significantly up-regulated by MeJA, with the exception of *SmCYP714C2*, which was not expressed in leaves (**Figures 3C** and **4**). Furthermore, Pearson's correlation analysis of the qRT-PCR results showed highly significant co-expression of *SmCYP98A75*, *SmCYP98A76*, *SmCYP98A78*, and *SmC4H1* (*P* < 0.01).

All six CYP450s were identified as full-length transcripts in the PacBio transcriptome, and their gene structures and intron phases are described in **Figures 3A,B**. In addition, gene loci *SmCYP749A39*, *SmCYP98A75*, and *SmCYP98A76* were found to undergo alternative splicing events, with each expressing two gene isoforms. All alternative splicing events of these candidate CYP450s were classified as intron retention. For their respective alternative splicing events, *SmCYP749A39-iso1*,

*SmCYP98A75-iso1*, and *SmCYP98A76-iso1* were found to be the dominantly expressed isoforms (Supplementary Table S5), and intron retention for *SmCYP749A39-iso2*, *SmCYP98A75-iso2*, and *SmCYP98A76-iso2* introduced PTCs in exon 3, intron 2, and intron 2, respectively (Supplementary Figure S4).

To better understand the putative functions of these candidate CYP450s, we constructed a phylogenetic tree with 31 full-length amino acid sequences from various species, including some functionally identified CYP98As. CYP92A73 clustered into one branch with CYP76AH1, which was identified as catalyzing the miltiradiene to ferruginol step in tanshinone biosynthesis (**Figure 3D**). These two CYP450s were found to be neighbors of C4H from *S. miltiorrhiza* and *A. thaliana* (**Figure 3D**). The other predicted CYP450s, SmCYP707A102, SmCYP749A39, and SmCYP714C2, were distant from the CYP98A subfamily (**Figure 3D**).

# Co-expression Analysis and Isoform Identification of Candidate Laccases

Eighty laccases were identified through genome-wide analysis in *S. miltiorrhiza*, with five identified based on the criterion of higher expression in the phloem and xylem than in the periderm: *SmLAC1*, *SmLAC2*, *SmLAC3*, *SmLAC4*, and *SmLAC5* (**Figure 5C**, Supplementary Table S4). Furthermore, RNA-seq and qRT-PCR analyses indicated that MeJA up-regulated the expression of *SmLAC1*, *SmLAC2* and *SmLAC5* (**Figures 5C** and **6**). According to the qRT-PCR data, *SmLAC5* was significantly co-expressed (*P* < 0.05) with *SmCYP98A78* and *SmC4H1*.

All five laccases were identified as full-length transcripts in the PacBio transcriptome, and their gene structures and intron phases are described in **Figures 5A,B**. In addition, the *SmLAC1*, *SmLAC3*, *SmLAC4*, and *SmLAC5* gene loci were found to undergo alternative splicing events, with *SmLAC1*, *SmLAC3*, and *SmLAC5* each expressing two isoforms and *SmLAC4* three isoforms. All of the alternative splicing events in these selected laccases were classified as intron retention. *SmLAC1-iso1*, *SmLAC3-iso2*, *SmLAC4 iso3*, and *SmLAC5-iso1* were the dominantly expressed isoforms among their respective alternative splicing events (Supplementary Table S5), and the intron retention in *SmLAC1-iso2*, *SmLAC3-iso1*, *SmLAC4-iso1*, *SmLAC4-iso2*, and *SmLAC5-iso2* introduced PTCs in intron 2, intron 4, intron 1, intron 4, and intron 5, respectively (Supplementary Figure S5).

To predict the functions of the candidate laccases, a phylogenetic tree was constructed using 32 amino acid sequences from *Populus trichocarpa*, *Picea abies*, *Oryza sativa*, and *S. miltiorrhiza*. SmLAC3, SmLAC4, and SmLAC5 were classified into different branches with other laccases that have been described as closely correlated with lignin biosynthesis in other species (**Figure 5D**).

# DISCUSSION

In this study, we analyzed full-length transcripts and alternative splicing events related to phenolic acid biosynthesis in different root tissue of *S. miltiorrhiza* by combining NGS and TGS technologies. Previous studies have only cloned a small number of full-length genes, such as *SmPAL1*, *SmC4H1*, *SmTAT1*, *SmHPPR1*, *SmRAS*, and *SmCYP98A78*, and identified their functions (Huang et al., 2008; Song and Wang, 2009, 2011; Xiao et al., 2009a, 2011; Di et al., 2013). Despite predicted locations and functions based on genome annotation, other full-length homologous genes and their functions have not yet

been identified (Wang et al., 2015). Among the 28 homologous genes identified as being involved in RA biosynthesis, the ability to detect 68% of full-length transcripts (15 full-length transcripts/22 expressed genes) and 27% of alternative splicing events at gene loci (4/15) indicates a significant advantage of hybrid sequencing in such discovery (Supplementary Table S2). Indeed, the availability of full-length transcripts will allow for establishing a metabolic engineering strategy with the aim of modulating the phenolic acid content, and the identification of alternative splicing events is beneficial for understanding the molecular mechanisms of phenolic acid biosynthesis in *S. miltiorrhiza*.

In line with our interest in phenolic acid biosynthesis, we found that not only the distribution of LAB but also the major expression of phenolic acid biosynthetic genes in the root occurred in the phloem and xylem (**Figure 1**). This agreement between phytochemical assay and gene expression in the root provided a basis for co-expression analysis. In addition, MeJA was found to dramatically promote the accumulation of phenolic acids and the expression of key genes (Xiao et al., 2009b). Although many genes related to RA biosynthesis have been cloned and identified in other species, 28 homologous genes based on genome-wide identification generated more candidates to assist in fully elucidating the RA biosynthetic pathway in *S. miltiorrhiza*. *4CL1* and *4CL2* of phenylpropanoid pathway have been cloned and their functions identified *in vitro* (Zhao et al., 2006); however, the 4CL catalyzing 4-cinnamic acid to 4-coumaroyl-CoA *in vivo* remains unknown. *Sm4CL3*, *Sm4CL-like1,* and *Sm4CL-like4* are most likely to be involved in the synthesis of RA. The overexpression of *SmHPPR1* in tyrosine-derived pathway resulted in the accumulation of 4 hydroxyphenylpyruvic acid, the substrate of SmHPPR (Xiao et al., 2011). However, *SmHPPR3*, rather than *SmHPPR1*, might participate in RA biosynthesis. An additional step from 4 hydroxyohenyllactic acid to 3,4-dihydroxyphenyllactic acid was found in *S. miltiorrhiza* using a C13 tracer. As this step is likely to be catalyzed by an unknown CYP450 (Di et al., 2013), we then selected six CYP450s that were more significantly expressed

in the phloem and xylem than in the periderm (Supplementary Table S3). According to phylogenetic tree and qRT-PCR analyses, CYP98A75 and CYP98A76 likely participate in RA biosynthesis rather than as 4-coumaroylshikimate/quinate 3-hydroxylases in quinic acid and shikimic acid biosynthesis (Chen et al., 2014). A previous study proposed that a laccase was potentially involved in the oxidative dimerization of RA to synthesize LAB (Di et al., 2013). To explore the reactions that convert RA to LAB, five laccases were identified as exhibiting higher expression in the phloem and xylem than in the periderm (Supplementary Table S4). Furthermore, *SmLAC5* was considered to be the best candidate for LAB synthesis. Further studies of these candidate CYPs and laccases using transgenic methods and biochemical reactions may accurately elucidate the mechanism of phenolic acid biosynthesis.

The complexity of alternative splicing events plays a potentially important regulatory role in SA biosynthesis, and many studies focusing on alternative splicing events in *Arabidopsis* have been reported (Filichkin et al., 2010; Marquez et al., 2012). In contrast to humans, the most common type of alternative splicing event in plants appears to be intron retention (Au et al., 2013; Reddy et al., 2013). In fact, all of the identified alternative splicing events in *S. miltiorrhiza* SA biosynthesis showed intron retention. A recent study reported that most of the intron retention isoforms in *Arabidopsis* are predicted to be targets of nonsense-mediated decay (NMD) to regulate mRNA stability. Expect for *Sm4CL-like7*, the alternative splicing events related to SA biosynthesis in *S. miltiorrhiza* expressed one predominant isoform (Supplementary Table S5). Low-expression isoforms have been described as splicing errors, which commonly trigger NMD to maintain mRNA stability (Filichkin et al., 2010; Zhang et al., 2010; Marquez et al., 2012; Reddy et al., 2013; Walters et al., 2013; Shen et al., 2014). In addition, the highly expressed genes *Sm4CL-like7-iso2* and *Sm4CL-like7-iso3*, which contain PTCs downstream of the splice junctions, might be subjected to NMD to eliminate incomplete transcripts (Supplementary Figure S2). Another prediction about these two PTC isoforms of *Sm4CL-like7* is that small interfering peptides with absent functional domains could form nonfunctional dimers that compete with and negatively regulate functional proteins. Our results clearly detected and predicted alternative splicing events related to SA biosynthesis, though the actual functions of the alternative splicing isoforms remain unknown. Thus, the systematic identification of co-expression, full-length transcripts and alternative splicing events related to the biosynthesis of lipophilic diterpenoid pigments (Xu et al., 2015) and hydrophilic phenolic acids in various root tissues of *S. miltiorrhiza* could better resolve the biology of the synthesis of such natural products.

In summary, we localized SA metabolism in the medicinal plant *S. miltiorrhiza* to the root phloem and xylem. We then identified full-length transcripts, encoding isoforms as well as alternative splicing events in SA biosynthesis and systematically analyzed six candidate CYP450s and five candidate laccases related to SA biosynthesis in *S. miltiorrhiza* using hybrid sequencing. Furthermore, our study provides a model for analyzing the full-length transcriptome and the biosynthesis of active constituents in other medicinal plants.

#### AUTHOR CONTRIBUTIONS

ZX, JS, and SC designed and coordinated the study. ZX, HL, XZ, and AJ performed experiments and analyzed the data. ZX, JS, HL, and SC wrote the manuscript.

#### ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (Grant no. 81573398), the Major Scientific

#### REFERENCES

and Technological Special Project for 'Significant New Drugs Creation' (Grant no. 2014ZX09304307001), the Program for Innovative Research Team at the Institute of Medicinal Plant Development (Grant no. IT1304), and the National Sciencetechnology Support Plan of China (Grant no. 2012BAI29B01).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.00100

experiments with TopHat and Cufflinks. *Nat. Protoc.* 7, 562–578. doi: 10.1038/nprot.2012.016

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Xu, Luo, Ji, Zhang, Song and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Efficient Rutin and Quercetin Biosynthesis through Flavonoids-Related Gene Expression in *Fagopyrum tataricum* Gaertn. Hairy Root Cultures with UV-B Irradiation

*Xuan Huang†, Jingwen Yao†, Yangyang Zhao, Dengfeng Xie, Xue Jiang and Ziqin Xu\**

*Provincial Key Laboratory of Biotechnology of Shaanxi, Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, Xi'an, China*

#### *Edited by:*

*Eugenio Benvenuto, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Italy*

#### *Reviewed by:*

*Heiko Rischer, VTT Technical Research Centre of Finland, Finland Silvia Massa, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Italy*

*\*Correspondence:*

*Ziqin Xu<EMAIL_ADDRESS>†These authors are co-first authors.*

#### *Specialty section:*

*This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science*

*Received: 18 September 2015 Accepted: 14 January 2016 Published: 04 February 2016*

#### *Citation:*

*Huang X, Yao J, Zhao Y, Xie D, Jiang X and Xu Z (2016) Efficient Rutin and Quercetin Biosynthesis through Flavonoids-Related Gene Expression in Fagopyrum tataricum Gaertn. Hairy Root Cultures with UV-B Irradiation. Front. Plant Sci. 7:63. doi: 10.3389/fpls.2016.00063*

Transformed hairy roots had been efficiently induced from the seedlings of *Fagopyrum tataricum* Gaertn. due to the infection of *Agrobacterium rhizogenes*. Hairy roots were able to display active elongation with high root branching in 1/2 MS medium without growth regulators. The stable introduction of *rol*B and *aux*1 genes of *A. rhizogenes* WT strain 15834 into *F. tataricum* plants was confirmed by PCR analysis. Besides, the absence of *vir*D gene confirmed hairy root was bacteria-free. After six different media and different sources of concentration were tested, the culturing of TB7 hairy root line in 1/2 MS liquid medium supplemented with 30 g l−1 sucrose for 20 days resulted in a maximal biomass accumulation (13.5 g l−1 fresh weight, 1.78 g l−1 dry weight) and rutin content (0.85 mg g−1). The suspension culture of hairy roots led to a 45-fold biomass increase and a 4.11-fold rutin content increase in comparison with the suspension culture of non-transformed roots. The transformation frequency was enhanced through preculturing for 2 days followed by infection for 20 min. The UV-B stress treatment of hairy roots resulted in a striking increase of rutin and quercetin production. Furthermore, the hairy root lines of TB3, TB7, and TB28 were chosen to study the specific effects of UV-B on flavonoid accumulation and flavonoid biosynthetic gene expression by qRT-PCR. This study has demonstrated that the UV-B radiation was an effective elicitor that dramatically changed in the transcript abundance of *ftpAL*, *FtCHI*, *FtCHS*, *FtF3H,* and *FtFLS-1* in *F. tataricum* hairy roots.

Keywords: tartary buckwheat, genetic transformation, hairy root, rutin, UV-B, flavonoid biosynthetic genes

# INTRODUCTION

As a significant food and medicinal species, tartary buckwheat (*Fagopyrum tataricum* Gaertn.; family Polygonaceae) is grown and used in the mountainous regions of Southwest China (Sichuan province), Northern India, Bhutan, and Nepal (Fabjan et al., 2003). The plant contains numerous functional components, including flavonoids, phenolic compounds, phytosterols, fagopyrins, d-chiro-inositol, and thiamin-binding proteins, which play essential role in antioxidant, hypocholesterolemic, and antidiabetic effects (Krkošková and Mrázová, 2005; Jiang et al., 2007; Tomotake et al., 2007; Yang and Ren, 2008; Yao et al., 2008; Qin et al., 2013).

The major functional components of *F. tartaricum*, such as rutin, quercetin, orientin, vitexin and kaempferol, had been demonstrated to be flavonoids In comparison with *Fagopyrum esculentum* (common buckwheat), as a source of dietary rutin and quercetin, *F. tartaricum* had higher contents of flavonoids and other phenolic compounds. As a secondary plant metabolite, rutin blocked the increase of capillary fragility related to hemorrhagic disease, reduced high blood pressure (Abeywardena and Head, 2001), decreased blood vessel permeability (with consequent antiedemic effect), lowered the risk of arteriosclerosis (Wojcicki et al., 1995), and displayed antioxidant activity (Watanabe, 1998; Park et al., 2000; Holasova et al., 2001; Krkošková and Mrázová, 2005). In comparison with common buckwheat, rutin content in tartary buckwheat was ∼3.2-fold higher in flowers, ∼3.1-fold higher in stems and ∼65-fold higher in seeds (Park et al., 2004). There had been increasing researches focusing on tartary buckwheat in recent years due to its remarkable health benefits associated with health.

Flavonoids are a class of secondary metabolites in plants involved in a great number of significant functions. They constitute a relatively diverse group of aromatic compounds derived from phenylalanine and malonyl-coenzyme. Phenylalanine ammonia lyase (PAL) catalyzes the conversion of phenylalanine to cinnamate. Based on this, *trans*-cinnamate is hydroxylated by cinnamic-4-hydroxylases (C4H) and is finally activated by the 4-coumarate/cinnamate coenzyme and 4-coumaryl-CoA-ligase (4CL), for the condensation of malonyl-CoA. As the major intermediates of flavonoid biosynthetic pathways chalcones are produced by the condensation of three molecules of malonyl-CoA and a single molecule of 4-coumaryl-CoA. The condensation of 4-coumaroyl-CoA and malonyl-CoA is conducted by chalcone synthase (CHS) to form either tetrahydroxy chalcone or trihydroxy chalcone. Chalcones are converted to the (2S)-flavanone naringenin by chalcone isomerases (CHIs) in a ring-closing step that forms the heterocyclic C-ring. From these central intermediates, the pathway diverges into several side branches for the synthesis of various classes of flavonoid molecules that are produced through the combined actions of functionalizing enzymes that could hydroxylate, reduce, alkylate, oxidize, and glycosylate the phenylpropanoid core structure (Kaneko et al., 2003; Fowler and Koffas, 2009; Santos et al., 2011); Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific 3-hydroxylation of (2S) flavanones to dihydroflavonols. Furthermore, dihydroflavonols are converted to flavonols and their glycosides through the corresponding flavonoid 3 -hydroxylase (F3 H) and flavonol synthase (FLS). These intermediates are further modified by varieties of hydroxylases, methyltransferases, reductases, and glycosyltransferases to form diverse flavonoids (e.g., quercetin and rutin) and isoflavonoids. For the biosynthesis of anthocyanins, dihydroflavonol reductase (DFR) catalyzes the stereospecific conversion of dihydroflavonols into the respective flavan-3,4-diols (leucoanthocyanins) through NADPHdependent reduction at the 4-carbonyl. The leucoanthocyanins are further converted to the anthocyanidins by anthocyanidin synthase (ANS; Winkel-Shirley, 2001; Pandey and Sohng, 2013).

Hairy root cultures, established by the transformation of plants with *Agrobacterium rhizogenes*, typically showed an increased production of secondary metabolites. They were genetically and biochemically stable at rapid growth rate. Besides, they could synthesize useful natural compounds at the levels comparable to those of wild-type (WT) roots (Guillon et al., 2006a,b). Hairy root cultures of many plant species have already been widely studied regarding the production of secondary metabolites which could be used as pharmaceuticals, cosmetics, and food additives (Crane et al., 2006; Georgiev et al., 2007; Thiruvengadam et al., 2014). Biotechnological approaches which used hairy root culture have greatly enhanced the production of rutin by common buckwheat (Lee et al., 2007; Kim et al., 2010) and the production of phenolic compounds by tartary buckwheat (Kim et al., 2009; Thwe et al., 2013).

UV-B radiation has already been proved to be an efficient biotic stress to stimulate secondary metabolite accumulation in plant cell and tissue culture (Binder et al., 2009; Hao et al., 2009). According to previous work, the flavonoids of *F. esculentum* sprouts were produced as protective substances against the UV-B radiation (Ožbolt et al., 2008; Tsurunaga et al., 2013). To the best of our knowledge, there was no previous report about the effect of UV-B on functional metabolites accumulation in the hairy root culture of *F. tartaricum*.

To elucidate the role of UV-B as an abiotic elicitor in regulating synthesis and a yield of flavonoids and other secondary metabolites, an efficient protocol is needed for stable genetic transformation of hairy roots of *F. tartaricum*. Therefore, we established such a protocol and applied it into the study on induced biosynthesis and accumulation of flavonoids in hairy roots of *F. tartaricum*. Furthermore, we carried out a research program in order to investigate the effects of UV-B light and an addition of various concentrations of sucrose to liquid culture medium so as to enhance the flavonoids production in this study. Moreover, the expression of flavonoid biosynthetic genes was examined through quantitative real time PCR in combination with the change of flavonoids content, in order to analyze the relation between genes expression and metabolic biosynthesis of flavonoids.

#### MATERIALS AND METHODS

#### Plant Material and Cultivation

Dehulled seeds of *F. tataricum* G. were surface-sterilized with 70% (v/v) ethanol for 1 min and 0.1% (v/v) mercuric chloride for 10 min, and then rinsed for four times in sterilized water. The treated seeds were sowed onto 1/2 MS medium (Murashige and Skoog, 1962) and solidified with 0.8% (w/v) agar. Before agar addition, the medium was adjusted to pH 5.8 and then sterilized through autoclaving at 121◦C for 20 min.

Germinating seeds cultured the temperature of 25 ± 2◦C in a growth chamber under a 16-h photoperiod with the flux rate of 35 μmol s−1 m−2. After 7 days, the hypocotyls and cotyledons of seedlings were cut into 0.5 cm × 0.5 cm pieces on a clean bench, and then transferred into Petri dishes, each of which contained 20 ml MS medium. The cut explants were cultured under the same conditions for 1–3 days as preculture before the inoculation.

Wild tartary buckwheat was planted in a test field at Northwest University (Xi'an, China) during the summers of year 2012 and 2013.

#### Preparation of *Agrobacterium rhizogenes*

Cultured *A. rhizogenes* WT strain 15834 was utilized for hairy root induction. The bacteria were started from glycerol stock and grown at 28◦C on 1.5% (w/v) of agar solidified YEB medium (Van Larebeke et al., 1977) with 250 mg/l penicillin for one night. Single colonies were grown at 28◦C with shaking (180 rpm) in 20 ml YEB liquid medium with 250 mg/L penicillin for selection. *A. rhizogenes* suspension culture was kept overnight until reaching the density of OD600 = 0.5. Cells were collected by centrifugation (4000 rpm, 5 min) and were resuspended in 1/2 MS liquid medium with supplement 30 g/L sucrose and acetosyringone 200 mmol/L. Cell suspensions at density OD600 = 0.6 were used for inoculation.

#### *A. rhizogenes* -Mediated Transformation

Hypocotyls and cotyledons from 7-days-old plants were cut into ∼0.5 cm pieces. Excised explants were dipped into *A. rhizogenes* 15834 suspensions in liquid inoculation medium for 10, 12, 15, or 20 min, blotted dry on sterile filter paper, and incubated in the dark at 25◦C on the agar-solidified MS medium. As a control, a few explants were placed in 1/2 MS liquid medium and were cultured in the same way. After 1∼3 days of coculture, explants were transferred onto solidified MS medium supplemented with 500 mg l−1 cefotaxime sodium. Explants that produced hairy roots (usually within 2 weeks after infection) were selected for further study. Roots (length 1.5–2.0 cm) that developed on the explants were excised aseptically, transferred onto MS medium supplemented with 400 mg l−1 cefotaxime in 9-cm Petri dishes, and incubated under the conditions described in Section "Plant Material and Cultivation." Roots were grown for 14 days. Furthermore, 0.3 g fresh weight (FW) was transferred into 250-ml Erlenmeyer flasks containing 50 ml MS, 1/2 MS, N6, 1 */*2 N6, B5 and 1/2 B5 liquid medium without growth regulators. Cultures were incubated on a shaker (100 rpm) as above, and roots were subcultured in every 14 days. Cefotaxime concentration was gradually reduced to zero in the MS liquid medium. Roots were kept at 25 ± 2◦C under standard cool white fluorescent tubes with the flux rate of 35 μmol s−1 m−2 and a 16-h photoperiod. Experiments were conducted in duplicate with three flasks per culture condition. As hairy roots can grew very well on medium without growth regulators, normal roots could hardly grow on the same medium. Therefore, as a control, roots excised from *in vitro* germinated seedlings were cultured in MS liquid medium without growth regulators. Meanwhile, transformation efficiency was calculated with each treatment. The transformation efficiency equaled to the number of explants inducing hairy roots/total number of explants × 100%.

# Genomic DNA Extraction and PCR Analysis

Genomic DNA was extracted from hairy roots and WT (seedling grew in 1/2 MS medium as control) roots of *F. tataricum* by the CTAB procedure (Doyle and Doyle, 1990). The Ri-plasmid of *A. rhizogenes* was extracted from strain 15834 by the SDS/alkaline lysis method (Petit et al., 1983), being used as a positive control. Integration of T-DNA responsible for hairy root formation was confirmed by PCR analysis using *rol*B, *aux*1, and *Vir*D specific primers. The sequences of primers used in the experiment were for *rolB* (Forward: 5 -GAT ATA TGC CAA ATT TAC ACT AG-3 ; Reverse: 5 -GTT AAC AAA GTA GGA AAC AGG-3 , the expected PCR product was 564 bp), *aux*1 (Forward: 5 -TTC GAA GGA AGC TTG TCA GAA-3 ; Reverse: 5 -CTT AAA TCC GTG TGA CCA TAG-3 , the expected PCR product was 350 bp) and *Vir*D (Forward: 5 -ATG TCG CAA GGC AGT AAG CCC A-3 ; Reverse: 5 -GCA GTC TTT CAG CAG GAC GAG CAA-3 , the expected PCR product was 438 bp). The PCR mixture consisted of 5 μl 10× PCR buffer (Takara Biotech; Japan), 2.5 μl of 100 nM dNTPs (Takara), 1 μl primer, 1 μl Taq polymerase (Takara), and 1 μl plant genomic DNA (or 1 μl plasmid DNA), in a final volume of 50 μl. The amplification conditions were: predenature for 5 min at 94◦C; denature for 1 min at 94◦C; anneal primer for 55 s at 52◦C (*rol*B)/ anneal primer for 55 s at 55◦C (*aux*1)/ anneal primer for 55 s at 56◦C (*Vir*D); extension for 1 min at 72◦C; repeat for 30 cycles; and final extension at 72◦C for 10 min. PCR results were checked by agarose gel electrophoresis (100 v/h) with DL5000 ladder marker (Takara), detected by ethidium bromide staining, and photographed by a gel documentation system (Bio-Rad; Hercules, CA, USA).

#### Total RNA Extraction and Expression Analysis of Flavonoid Biosythetic Genes by qRT-PCR

Total RNA was isolated from *F. tataricum* wild and transgenic hairy roots by utilizing the RNeasy Plant Mini Kit (Qiagen; Valencia, CA, USA). The RNA integrity was checked by 1.2% ethidium bromide stained RNA gel through the absorbance spectrum at 260: 280 nm wavelength by NanoVue Plus Spectrophotometer (GE Healthcare Bio-Science Crop., USA). The cDNA was synthesized from 1 μg of DNA free total RNA and reverse transcribed utilizing PrimeScriptR 1st Strand cDNA Synthesis Kit (Takara). The resulting cDNA products were used as the template for real time-PCR analysis.

Quantitative real-time PCR was performed for the transcriptional level analysis of flavonoid biosynthesis genes in a BIO-RAD CFX96 Real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). The gene-specific primer sets were designed as previous information described by Li et al. (2010). Real-time PCR was carried out in a 20 μl reaction volume including 0.5 μl of each primer, 5 μl of template cDNA and 10 μl of SYBR Green (SYBRR *premix Ex Taq*TM, Takara). According to Thwe et al. (2013), the program was executed. The histone H3 gene was used as reference gene (Timotijevic et al., 2010). Fluorescent intensity data were acquired during the extension step. The transcript levels were checked through utilizing a standard curve. Identical PCR conditions were used for all targets. The significant differences between cultivars were evaluated from three replicates of each sample.

# Measurement of Flavonoid Content (Rutin and Quercetin) by High-Performance Liquid Chromatography (HPLC)

Harvested hairy roots (1 g) were frozen in liquid N2, ground to a fine powder with a mortar and pestle, and extracted twice in methanol (50 ml) for 24 h at 5◦C. Extracts were vacuum-dried at 80◦C and dissolved in 10 ml methanol. The solution was filtered through a poly filter (pore size 0.45 μm) and diluted twofold with methanol. Extracts were used to analyze the rutin and quercetin by HPLC (Waters 2695 Sespartions Module and Waters 2996 Photodiode Array Detector) on a C18-column (Hypersil ODS, 250 mm × 4.6 mm) at 30◦C. The mobile phase consisted of methanol (solvent A) and 1% (v/v) glacial acetic acid (solvent B) with the flow rate of 1 mL min−1. The solvent gradient was from 40% solvent A/60% solvent B to 65% solvent A/35% solvent B over 35 min. Sample (rutin and quercetin) detection wavelength were 257 and 370 nm, respectively; injection volume equaled to 20 μl. The rutin and quercetin were detected and quantified with the authentic standards obtained from the Institute for Identification of Pharmaceutical and Biological Products (Beijing, China), which identified by the comparison with retention times and spectral characteristics of authentic standards. The quantitative values were calculated from the calibration curves (Supplementary Material, S1 and S2). All samples were run in triplicate (A HPLC chromatogram of hairy root line is shown in Supplementary Material, S3).

## Growth Kinetics of Cultured Transformed Roots

Hairy roots (0.3 g FW) were inoculated in a 250-ml Erlenmeyer flasks containing 50 ml basal liquid medium supplemented with sucrose. Biomass accumulation and flavonoids production were optimized through evaluating growth kinetics at various time intervals (4, 8, 12, 16, 20, 24 days). The media (MS, 1/2 MS, N6, 1/2 N6, B5, and 1/2 B5) and various concentrations of sucrose (10, 20, 30, 40, 50 g l−1) were evaluated to maximize the root biomass growth. Flasks were cultured with shaking (100 rpm) at 25 ± 2◦C with the flux rate of 35 μmol s−1 m−2 and a 16-h photoperiod. Hairy root biomass (FW and DW) and flavonoids production were measured during the 24 days of culture.

#### UV-B Light Stress Treatment

After hairy roots had grown to 2 g (FW) in 1/2 MS liquid medium, they were exposed to UV light (UV-B 313 lamps; Q-Panel; Cleveland, OH, USA) for 30 min. The lamps were wrapped in cellulose diacetate filters to block the UV-C range (wavelengths *<* 280 nm). The maximal radiation peak was at wavelength 302 nm. The UV-B light intensity on the sample surface was 1.26 μW/cm2, and the total energy supply was 0.34 J/cm2. The treatment continued for 3 days and the experiments of UV-B treatment were repeated at least three times. Both of rutin and quercetin contents were analyzed as in Section "Measurement of Flavonoid Content (Rutin and Quercetin) by High-Performance Liquid Chromatography (HPLC)." Nontransformed roots and wild plants were subjected to UV-B light stress as controls. All parts of the wild plants were examined by HPLC. Meanwhile, the expression of flavoniod biosynthesis genes was detected after the hairy roots were treated by UV-B irradiation. The changes of transcription abundance were analyzed by qRT-PCR as shown in Section "Total RNA Extraction and Expression Analysis of Flavonoid Biosythetic Genes by qRT-PCR."

## Statistical Analysis

All data analyses were performed using the Origin software program, V. 8.0. Values were expressed as mean ± SE. One-way ANOVA was applied in statistical analysis. Differences between means were evaluated by Duncan's multiple range test, and were considered to be significant for *P <* 0.05.

# RESULTS

# Establishment of Hairy Root Induction

The establishment of an efficient and reliable transformation system was proved to be highly desirable for the whole culture process of hairy root. Base on the preliminary experiments, explants age and co-cultivation time were investigated systematically in this work. Finally, 168 explants of *F. tatarium* were induced to hairy roots in 847 explants, and the transformation frequency was 19.83%.

# Effects of Preculture Time on Transformation Frequency of *F. tatarium*

Transformation frequency varied based on different preculture time (**Figure 1A**). Explants became more sensitive to the integration of T-DNA as preculture time increased. Transformation frequencies of explants were improved by 1–2 days preculture, in comparison with controls, reaching the maximum degree after 2 days of preculture. Besides, the induce rate could reach to 10.6 ± 0.6%. At longer preculture times, transformation frequencies decreased rapidly. Five to twelve hairy roots per stem were induced from hypocotyl cut ends or petiole within 2 weeks (**Figure 1B**). Meanwhile, no hairy roots were observed on explant without infection. Two to five hairy roots per explant were induced from cotyledons of tartary buckwheat with *A. rhizogenes* strain 15834 (**Figure 1C**).

# Effect of *A. rhizogenes* Infection Time on

Transformation Frequency Subjected to various transformation times, excised explants were dipped into *A. rhizogenes* in liquid inoculation medium. The optimal transformation times were about 20 min for explants, and the induce rate was 14.3 ± 0.5% (**Figure 1D**); All these

resulted in greater transformation efficiency and less damages to the explants.

## Confirmation of Genetic Transformation of Hairy Roots by PCR

In order to evaluate the genetic status of the selected hairy root, the PCR-based analysis was performed on the targeted *rol*B, *aux*1, and *vir*D genes. The *rol*B gene (located at the pRi TL-DNA segment) and the *aux*1 gene (located at the pRi TR-DNA segment), were diagnostic for T-DNA integration into the host genome of hairy root. The *Vir*D gene (located outside the pRi T-DNA segment) was used to check the presence of any remaining *Agrobacteria* in hairy root of *F. tatarium.* Furthermore, the root of aseptic plantlets and *A. rhizogenes* 15834 Ri plasmid were used as negative and positive controls respectively. According to the part results shown in **Figure 2**, the coexistence of *rol*B and *aux*1 genes indicated that the established hairy root lines integrated the pRi T-DNA of *A. rhizogenes* 15834 successfully. Furthermore, the absence of *vir*D gene confirmed that all those hairy roots were bacteria-free (The rest of results are shown in Supplementary Material, S4).

From the entire obtained 356 hairy root lines, we chose 60 lines which grew quickly in medium without plant growth regulators to the processing of PCR analysis. The presence of both of *rol*B and *aux*1 genes and the absence of *vir*D gene were observed in 20 hairy root lines. The results showed that these cultures had no *Agrobacterium* contamination, and that both of T-DNA fragments were integrated into the genome of these root lines.

## Effects of Various Media and Sucrose Concentration on Biomass Accumulation

We used several media (MS, 1/2 MS, B5, 1/2 B5, N6, 1/2 N6) for hairy root culture (**Table 1**). Hairy roots (TB7 line) were cultured for 20 days in six media for observation and for accurate measurement of FW and DW. Amongst the six media, 1/2 MS was the best for biomass accumulation (**Table 1**). Hairy roots were cultured in 1/2 MS liquid medium and presented the most rapid proliferation (**Figure 3**). The highest values of biomass accumulation (13.5 g l−1 FW; 1.78 g l−1 DW) were recorded for 1/2 MS medium, in which a thick mass grew in the bottle within 20 days. The growth cycles were generally similar in 1/2 MS solid vs. liquid media. However, the hairy root growth was slower in the solid medium. The condition of biomass accumulation in B5 and N6 media (including 1/2 B5 and 1/2 N6) was similar, which FW and DW of biomass were ∼50% lower than in 1/2 MS liquid medium, and the rutin content was lower than in 1/2 MS liquid medium.

As hairy roots were grew very well in 1/2 MS and MS media, we established various concentrations of sucrose (10–50 g l−1) in 1/2 MS medium, and examined their effects on hairy root growth. As the concentration of sucrose increased, hairy roots changed from white to brown, and became slower-growing. The

growth of hairy root reached its maximum in 30 g l−1 sucrose, and decreased greatly at the concentrations higher or lower than this value.

#### Kinetics of *F. tataricum* Hairy Root Growth and Flavonoids Accumulation

We detected flavonoids yield of 20 lines of hairy root which had been confirmed by PCR analysis. The result indicated that line TB7 had the highest content of rutin and quercetin (Data

TABLE 1 | Effects of different media and sucrose concentrations on biomass production of hairy root cultures in 1/2 MS medium.

*Roots (0.3 g FW per flask) were cultured for 20 days in 250-ml Erlenmeyer flasks containing 50 ml medium for 20 days. Data shown are mean* ± *SE of three replicates. Each experiment was performed in triplicate. Means with common letters are not significantly different at P < 0.05 according to Duncan's multiple range test.*

isn't shown). Therefore, TB7 were chosen to investigate the kinetics studies on biomass growth and flavonids accumulation in hairy root cultures of *F. tataricum* as shown in **Figure 4**. Hairy roots (initial FW 0.3 g) were cultured for 30 days in 1/2 MS medium containing 30 g l−1 sucrose. Root growth occurred primarily during the first 4–5 days, and then leveled off during days 20–24. Hairy roots were initially white or pale yellow, yet became brown and slow-growing after day 24, and required so subculturing in every 24 days. The maximal rutin yield was obtained (0.85 mg g−1) when the hairy root cultures were cultured on days 20. At the same time, the maximal FW and DW were 13.5 and 1.78 g, respectively. Both of biomass and rutin contents declined rapidly after 20 days.

## Effect of UV-B Light Stress Treatment on Flavonoids Production

We evaluated the resistance of wild tartary buckwheat plants and hairy roots to the UV-B light stress, in regard to possible enhancement of rutin and quercetin production (**Table 2**). TB7 hairy root lines were chosen to study the specific effects of UV-B on flavonoid accumulation. The rutin and quercetin content of stems, leaves, flowers, and WT (non-transformed) roots were compared with that of hairy roots. Following the UV-B stress treatment, the rutin content of hairy roots was strikingly higher than that in non-transformed roots. The increase in rutin content of treated hairy roots (from 0.93 to 4.82 mg g−1) was 5.18-fold higher than in WT roots. Hairy roots were more sensitive to UV-B stress treatment than to non-transformed roots, flowers, or stems. The relative order of rutin content increase under UV-B stress was leaves (9.35-fold) *>* hairy roots (5.18-fold) *>* stems (3.57-fold) *>* non-transformed roots (2.95-fold) *>* flowers (2.66 fold). Quercetin yield could not be detected in no-transformed root before the UV-B treatment. However, quercetin content was found in hairy root TB7 line, which increased drastically in hairy root with exposure due to UV-B (from 0.02 to 0.04 mg g−1). Meanwhile, quercetin was detected in leaves, flowers and stems of *F. tataricum*, and the yield was increased substantially after the treated UV-B irradiation.

#### Expression of Flavonoid Biosynthetic Genes in Hairy Roots of *F. tataricum* with UV-B Irradiation

To investigate biosynthesis of flavonoid genes in *F. tataricum*, the expression levels of biosynthesis genes in the hairy roots of *F. tataricum* were examined by qRT-PCR (**Figure 5A**). The expression levels of *ftpAL*, *FtC4H*, *Ft4CL*, *FtCHS*, *FtCHI*, *FtF3H*, *FtF3 H-1*, *FtF3 H-2*, *FtFLS-1*, *FtFLS-2*, *FtDFR*, and *FtANS* were shown. Although the gene transcripts for all of these enzymes were expressed in hairy roots (TB3, TB7, and TB28 lines) of *F. tataricum,* the expression levels were upregulated in TB7 than TB3 and TB28 hairy root lines except in *FtF3 H-1*, *FtF3 H-2*, *FtFLS-2,* and *FtANS*. In particular expression levels of *ftpAL, FtC4H*, *FtCHI*, *FtF3H,* and *FtFLS-1* in TB7 hairy root were significantly higher than the expression levels of TB3 and TB28 lines.

In order to understand the role of UV-B in regulating flavonoid biosynthesis, the transcript abundance for genes were involved in the flavonoid biosynthetic pathway and were analyzed by qRT-PCR (**Figure 5B**). The hairy root lines of TB3, TB7, and TB28 were also chosen to study the specific effects of UV-B exposure on gene expression. Although the gene transcripts for all the genes had increased the expression in three hairy roots lines, the expression levels were upregulated in TB7 than in TB3 and TB28 hairy root lines except in *FtF3 H-1*, *FtF3 H-2*, *FtDFR,* and *FtANS*. The key regulate genes of flavonoid biosynthesis, including *FtpAL*, *FtCHS*, *FtCHI*, *FtF3H*, and *FtFtFLS-1*, were found to be responsive to UV-B exposure drastically. *FtFLS-1* showed the highest transcript abundance in the UV-B exposure treatment, which was 30–40 fold higher than of no UV-B treatment. A significant UV-B induced was also observed in the transcript abundance of *FtCHI and Ft CHS*, which was 20–30 fold higher than no UV-B treatment. In contrast, the transcript abundance of *FtF3 H-1*, *FtF3 H-2*, *FtFLS-2*, *FtDFR,* and *FtANS* were slightly enhanced to response to the UV-B exposure treatments.

As shown in **Figure 5**, genes of flavonoid biosynthesis in tartary buckwheat have been almost entirely elucidated.

# DISCUSSION

*Agrobacterium rhizogenes* as a useful tool for gene transfer has been widely applied in many plant species. *A. rhizogenes* strain 15834 was often used to induce hairy root formation. Kim et al. (2010) obtained the hairy root of *F. esculentum* by *A. rhizogenes* 15834 successfully. *Taraxacum platycarpum* (Lee et al., 2004) and *Panax ginseng* (Yang and Choi, 2000) also underwent successful transformation with *A. rhizogenes* 15834. Meanwhile, Kim et al. (2009) and Park et al. (2011) reported that they obtained hairy root after inoculating sterile young stems of *F. tataricum* with *A. rhizogenes* strain R1000.

Transformation efficiency was affected by bacterial growth stage, infection time, pre-treatment of explant, as well as light and temperature conditions. Preculture of explants in MS medium for 2 days prior to transformation enhanced transformation efficiency. The optimal time of transformation was ∼20 min for explant. Plant tissues might be injured if the infection time was too long, and the infection time varied according to the plant

*After exposed to UV-B light for 30 min for 3 days, rutin and quercetin content of hairy roots were analyzed by HPLC. Data shown are mean* ± *SE of three replicates. Each experiment was performed in triplicate. Means with different letters are significantly different at P < 0.05 according to Duncan's multiple range test.*

species and type of explant. Chen et al. (2008) showed that longer preculture time might reduce explant viability, which resulted in injury. On the other hand, explants might wither more easily in the absence of a preculture process.

We obtained the maximal transformation frequency of *F. tataricum* by *Agrobacterium* only after suitable preculture. However, this conclusion might not be applied to all the plant species. Kim et al. (2004) used *Agrobacterium* to infect the *Perilla frutescens* explants, finding that the transformation frequency was much higher for non-precultured than for precultured explants.

The growth rate of hairy roots was greatly affected by the culture medium and various culture conditions. In present study, culturing in 1/2 MS liquid medium led to the most rapid proliferation of hairy roots, and biomass (FW) had increased ∼45-fold in 20 days. The growth of hairy root was exponential from days 0 to 20, and then entered into a stationary phase during days 20–25. These findings indicated that hairy root liquid cultures of tartary buckwheat were potentially useful for largescale biomass production. Park et al. (2011) also reported that the dry weight of hairy root enhanced 25-fold in the MS liquid medium during 21 days. *Polygonum multiflorum* Thunb. hairy root showed that the biomass (initially 0.5 g FW) increased 9.5 fold after being cultured in hormone-free MS liquid medium for 20 days (Thiruvengadam et al., 2014).

As the major carbon source, sucrose was extremely essential for the *F. tataricum* hairy root growth. In the present study, hairy roots grew rapidly in high sucrose concentrations. The optimal sucrose concentration was found to be 30 g l−1, at which biomass accumulation and rutin content were maximal. The growth of hairy root was strikingly lower at sucrose concentrations above or below 30 g l−1. Higher concentrations also resulted in the alteration of root morphology (e.g., root calluses, inhibition of lateral branching) probably due to osmotic stress (Hamill et al., 1987). Yu et al. (1996) found that the sucrose level was affected hairy root production in *Solanum avidare*. The levels of secondary metabolites produced by *in vitro* cultures could vary dramatically. As a matter of fact, most previous studies were focusing on nutrient composition in medium to achieve the optimized accumulation of metabolites in cultured cells (Rao and Ravishankar, 2002).

Since the biosynthesis of many secondary metabolites in plant is usually considered as a common defense response of plants to biotic and abiotic stresses, their accumulation could be stimulated by biotic and abiotic elicitors (Zhao et al., 2014a). Therefore, elicitation, as treatment of plant tissue cultures with elicitors, is one of the most effective strategies to enhance secondary metabolites production in plant tissue cultures. The most common and effective elicitors used in previous studies mainly included heavy metal ions, UV radiation (abiotic), the component of microbial cells, especially polyand oligosaccharides (biotic), and the signaling molecules in plant defense responses, such as salicylic acid (SA) and methyl jasmonate (MJ; Chen and Chen, 2000; Broeckling et al., 2005; Prakash and Srivastava, 2008; Smetanska, 2008; Ionkova, 2009; Zhao et al., 2010).

Flavonoids are produced as protective substances against UV-B radiation in plant. As an effective abiotic elicitor, some studies have described the production of flavonoids by buckwheat sprouts in response to UV-B irradiation (Kreft et al., 2002; Eguchi and Sato, 2009). Rutin (sometimes called vitamin P) displays strong antioxidant activity which could alleviate the damage from UV-B stress. Tsurunaga et al. (2013) found that rutin content and radical scavenging activity of buckwheat sprouts were enhanced under various levels of UV-B radiation. In the present study, rutin and quercetin content of hairy roots and all parts of tartary buckwheat were increased under UV-B stress. The maximal increase of rutin content (from 3.19 to 29.79 mg g−1, 9.35-fold) was observed in leaves. Interestingly, the next-highest increase of rutin content (from 0.93 to 4.82 mg g−1, 5.18-fold) was observed in hairy roots. This phenomenon might result from the insertion site of T-DNA during transformation; the underlying mechanism requires further investigation. In a previous study of buckwheat, Kim et al. (2010) found that rutin content was ∼2.4 fold higher in hairy roots than in WT roots. These findings are consistent with those of transformation studies on other plants, which suggested that *Agrobacterium* transfection might greatly enhance rutin content (Fu et al., 2006). According previous work, some work indicated that biotic elicitors can also enhance rutin and quercetin production in *F. tataricum* hairy root, e.g., Yeast polysaccharide (Zhao et al., 2014a) and exogenous fungal mycelia (Zhao et al., 2014b).

To understand the role of UV-B in regulating flavonoid biosynthesis, the transcript abundance for key enzymes genes involved in the flavonoid biosynthetic pathway were analyzed by qRT-PCR. Previous studies have extensively described the UV-B induction of those gene expressions (Frohnmeyer et al., 1992; Kubasek et al., 1992; Christie and Jenkins, 1996; Kliebenstein et al., 2002; Stracke et al., 2010). Flavonoid synthesis in plants is induced by perceiving the UV-B light with photoreceptors and by the expression of phenylpropanoid biosynthesis genes (Rizzini et al., 2011). These works considered the useage of flavonoid biosynthetic key enzymes PAL.C4H, CHI, CHS, FLC, F3H, etc. as an excellent UV-B signaling response marker. Amongst these genes, activated by the UV light, PAL played an important role in the first step of the flavonoid biosynthesis pathway. According to Hahlbrock et al. (2003), the promoter on PAL was in Box-P and that box-P-binding factor 1 was induced by UV stimulation and up-regulated the gene expression of PAL and followed the binding to Box-P. In the UV irradiation of rice seedlings, it has been reported that PAL increased at 12 h to its maximum expression and increases the contents of flavonoids (Jin et al., 2000). Hao et al. (2009) also reported that activation of PAL in *Ginkgo biloba* callus could be induced by UV-B, and flavonid biosynthesis could be stimulated. In *Arabidopsis*, the UV-B-mediate induction of *CHS* expression was UVR8-, COP1-, and HY-5-dependent with proteins belonging to the UV-B signaling pathway (Kliebenstein et al., 2002; Stracke et al., 2010). It has been widely known that HY5 can directly bind to the *CHS* promoter. However, this was not sufficient for the *CHS* transcriptional activation. However, the overexpression of HY5 fused with an activation domain was sufficient for the *CHS* expression induction, indicating that a presently unknown UV-B activated transcription factor must be involved as well (Stracke et al., 2010). In the present study, an irradiation with UV-B stimulated the expression of *PAL* in hairy roots of tartary buckwheat, showing significant expression after the irradiation (**Figure 5B**). Box-P was also conserved in the promoter region of other phenylpropanoid-biosynthesis genes and a UV responsible gene. For example, AtMYB4 in *Arabidopsis* represses the expression of the *C4H* gene, whereas UV irradiation canceled this repression and induced the expression of *C4H* (Jin et al., 2000). UV-B irradiation could trigger flavonol and anthocyanin biosynthesis in grapevine berries, which proved by up-regulated of key biosynthetic genes (*FLS1* and *UFGT*) and an increased anthocyanin concentration (Martinez-Lüscher et al., 2014). Those studies have provided useful evidence to prove the effect of UV-B exposure on the flavonoid biosynthesis.

This study demonstrated that UV-B as a major component changed dramatically in the transcript abundance of *FtpAL*, *FtCHI*, *FtCHS*, *FtF3H,* and *FtFLS-1* in *F. tataricum* hairy roots. Furthermore, *FtF3 H-1*, *FtF3 H-2,* and *FtFLS-2* genes were enhanced slightly after the UV-B treatment. A single irradiation with UV-B increased the production of rutin and quercetin for a maximum production correlated to the flavonoid biosynthesis enzyme gene expression. Through comparing with previous work, Thwe et al. (2013) investigated the various genes in the phenylpropanoid biosynthetic pathway to analyzed *in vitro* production of anthocyanin and phenolic compounds from hairy root cultures derived from two cultivars of tartary buckwheat. The result showed that phenylpropanoid biosynthetic pathway genes had expression in hairy roots, and the rutin and anthocyanin in hairy root of tartary buckwheat were identified. Thwe's work provides useful information on the molecular and physiological dynamic process that was correlated with phenylpropanoid biosynthetic gene expression and phenolic compound content in *F. tatarium* species. Base on those works, *F. tataricum* could be achieved through a complex regulation of genes involved in the flavonoid biosynthetic pathway.

### CONCLUSION

We have established an efficient protocol for *A. rhizogenes*mediated genetic transformation of tartary buckwheat (*F. tataricum*), and applied PCR analysis in the detection of hairy roots. Hairy roots cultured in 1/2 MS liquid medium supplemented with 30 g l−1 sucrose grew faster than normal roots under standard liquid culture conditions, and had higher rutin content. We also evaluated the effects of UV-B radiation on hairy roots and all other plant organs. We found that the rutin and quercetin content was significantly higher in hairy roots than in WT (non-transformed) roots. The expression of flavonoid biosynthetic genes was examined through quantitative real time PCR with UV-B treatment. The results showed that the expressions of key regulated genes were increased sharply in flavonoid biosynthetic pathway. Further studies on the dose-dependent UV-B irradiation could be used to determine the possible effects on tartary buckwheat flavonoids and anthocyanins incolved in the signal transduction pathway, which resulted in regulation by UV-B irradiation.

#### AUTHOR CONTRIBUTIONS

XH completed the *A. rhizogenes*-mediated transformation experiment, determination of the hairy roots rutin content and qRT-PCR experiment, JY completed effect of UV-B light stress treatment on rutin production of hairy roots experiment, YZ assisted with the *A. rhizogenes*-mediated transformation experiment, DX assisted with the rutin content determination experiment, and QZ guided the whole research as the corresponding author.

#### ACKNOWLEDGMENTS

This study was supported by grants from the National Natural Science Foundation of China (31300223), National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (J1210063), Natural Science Foundation of Shaanxi Province (2012JQ3003), Specialized Research Fund for the Doctoral Program of Higher Education<PHONE_NUMBER>0019), Opening Foundation of Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education (ZS12013), Key Scientific Research Project of Provincial Education Department of Shaanxi (15JS110), Provincial Training Programs of Innovation and Entrepreneurship for Undergraduate (1030), and Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry. In addition to this,

#### REFERENCES

the authors are grateful to Dr. S. Anderson for English editing of the manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.00063

hairy root culture of tartary buckwheat cultivars. *PLoS ONE* 8:e65349. doi: 10.1371/journal.pone.0065349

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer (Silvia Massa) and Handling Editor declared their shared affiliation, and the Handling Editor states that the process nevertheless met the standards of a fair and objective review.

*Copyright © 2016 Huang, Yao, Zhao, Xie, Jiang and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# *Cannabis sativa*: The Plant of the Thousand and One Molecules

#### *Christelle M. Andre\*, Jean-Francois Hausman and Gea Guerriero*

*Environmental Research and Innovation, Luxembourg Institute of Science and Technology, Esch-sur-Alzette, Luxembourg*

*Cannabis sativa* L. is an important herbaceous species originating from Central Asia, which has been used in folk medicine and as a source of textile fiber since the dawn of times. This fast-growing plant has recently seen a resurgence of interest because of its multi-purpose applications: it is indeed a treasure trove of phytochemicals and a rich source of both cellulosic and woody fibers. Equally highly interested in this plant are the pharmaceutical and construction sectors, since its metabolites show potent bioactivities on human health and its outer and inner stem tissues can be used to make bioplastics and concrete-like material, respectively. In this review, the rich spectrum of hemp phytochemicals is discussed by putting a special emphasis on molecules of industrial interest, including cannabinoids, terpenes and phenolic compounds, and their biosynthetic routes. Cannabinoids represent the most studied group of compounds, mainly due to their wide range of pharmaceutical effects in humans, including psychotropic activities. The therapeutic and commercial interests of some terpenes and phenolic compounds, and in particular stilbenoids and lignans, are also highlighted in view of the most recent literature data. Biotechnological avenues to enhance the production and bioactivity of hemp secondary metabolites are proposed by discussing the power of plant genetic engineering and tissue culture. In particular two systems are reviewed, i.e., cell suspension and hairy root cultures. Additionally, an entire section is devoted to hemp trichomes, in the light of their importance as phytochemical factories. Ultimately, prospects on the benefits linked to the use of the *-omics* technologies, such as metabolomics and transcriptomics to speed up the identification and the large-scale production of lead agents from bioengineered *Cannabis* cell culture, are presented.

Keywords: fibers, hemp, *Cannabis*, cellulose, lignin, cannabinoids, terpenes, lignans

#### INTRODUCTION

The current climatic and economic scenario pushes toward the use of sustainable resources to reduce our dependence on petrochemicals and to minimize the impact on the environment. Plants are precious natural resources, because they can supply both phytochemicals and lignocellulosic biomass. In this review, we focus on hemp (*Cannabis sativa* L.), since it is a source of fibers, oil and molecules and as such it is an emblematic example of a multi-purpose crop. We treat the aspects related to the use of hemp biomass and, more extensively, those linked to its wide variety of phytochemicals.

#### *Edited by:*

*Eugenio Benvenuto, ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Italy*

#### *Reviewed by:*

*Biswapriya Biswavas Misra, University of Florida, USA Felix Stehle, Technical University of Dortmund, Germany*

> *\*Correspondence: Christelle M. Andre<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science*

*Received: 27 October 2015 Accepted: 08 January 2016 Published: 04 February 2016*

#### *Citation:*

*Andre CM, Hausman J-F and Guerriero G (2016) Cannabis sativa: The Plant of the Thousand and One Molecules. Front. Plant Sci. 7:19. doi: 10.3389/fpls.2016.00019*

Known since the ancient times for its medicinal and textile uses (Russo et al., 2008; Skoglund et al., 2013), hemp is currently witnessing a revival, because of its rich repertoire of phytochemicals, its fibers and its agricultural features, namely quite good resistance to drought and pests, well-developed root system preventing soil erosion, lower water requirement with respect to other crops, e.g., cotton. This shows the great versatility of this fiber crop and encourages future studies focused on both *Cannabis* (bio)chemistry and genetic engineering. Hemp varieties producing oil, biomass or even both are currently cultivated and the availability of the hemp genome sequence greatly helps molecular studies on this important crop (van Bakel et al., 2011). In addition, the scientific community is very much interested in harnessing *Cannabis* pharmacological power: for example microorganisms are being engineered to produce -9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) (Taura et al., 2007a; Zirpel et al., 2015).

The final scope of this review is to discuss the potential of hemp for industry and to highlight its importance for the bio-economy. More specifically, we: (i) describe the use of hemp biomass (i.e., the fibers), (ii) discuss hemp molecules of industrial interest (namely cannabinoids, terpenes and phenolic compounds), (iii) describe the potential of hemp trichomes as pharma-factories and (iv) discuss the potential of genetic engineering, by describing the use of plant cell suspension and hairy root cultures.

# HEMP STEM: A SOURCE OF FIBERS WITH ANTIBACTERIAL PROPERTIES

Plant lignocellulosic biomass is an abundant renewable resource, which can provide biopolymers, fibers, chemicals and energy (Guerriero et al., 2014, 2015, 2016). Trees are important for the provision of wood, however, also fast-growing herbaceous species, like textile hemp (which has a THC content <0.3%; Weiblen et al., 2015), can provide high biomass quantities in a short time. The stem of this fiber crop supplies both cellulosic and woody fibers: the core is indeed lignified, while the cortex harbors long cellulose-rich fibers, known as bast fibers (**Figure 1**) (Guerriero et al., 2013).

This heterogeneous cell wall composition makes hemp stem an interesting model to study secondary cell wall biosynthesis, in particular the molecular events underlying the deposition of cortical gelatinous bast fibers and core woody fibers.

*Cannabis* woody fibers (a.k.a "hurds" or "shivs") are used for animal bedding because of their high absorption capacity and for the creation of a concrete-like material.

Hemp bast fibers are used in the biocomposite sector as a substitute of glass fibers. The automotive industry is particularly keen on using hemp bast fibers to produce bioplastics: this material is stronger than polypropylene plastic and lighter in weight (Marsh, 2003).

Beyond the applications in the construction and automotive industries, hemp fibers are attractive also in the light of their natural antibacterial property. Hemp bast fibers have been indeed described as antibacterial (Hao et al., 2014; Khan et al., 2015) and their use for the manufacture of an antibacterial finishing agent (Bao et al., 2014), surgical devices (Gu, 2006) or functionalized textiles (Cassano et al., 2013) has been reported. This property is linked to the chemical composition of hemp bast fibers: both free and esterified sterols and triterpenes have been identified, among which β-sitosterol and β-amyrin (Gutiérrez and del Río, 2005). These compounds possess known antibacterial properties (Kiprono et al., 2000; Ibrahim, 2012). Hemp bast fibers were also found to contain cannabinoids (2% of the total metabolite extract) (Bouloc et al., 2013 and references therein). More recently hemp hurd powder showed antibacterial properties against *Escherichia coli* (Khan et al., 2015). Since the hurd has a higher lignin content than the bast fibers, its antibacterial property may be linked to lignin-related compounds such phenolic compounds, as well as alkaloids and cannabinoids (Appendino et al., 2008; Khan et al., 2015).

## HEMP PHYTOCHEMICALS: THEIR PRODUCTION PATHWAYS AND MYRIAD OF BIOLOGICAL ACTIVITIES

Numerous chemicals are produced in hemp through the secondary metabolism. They include cannabinoids, terpenes and phenolic compounds (Flores-Sanchez and Verpoorte, 2008) and will be further described in the next sections. Although the pharmacological properties of cannabinoids have extensively been studied and are the most recognized hemp bioactives, the other components have no reasons to envy them, as they have also been associated with potent health-promoting properties. Research on *Cannabis* phytochemicals, as well as the widespread therapeutic use of *Cannabis* products, has been limited due to various reasons, including illegality of cultivation (due to its psychoactivity and potential for inducing dependence), variability of active components, and low abundance of some of them *in planta*. Further attentions is now drawn toward non-THC *Cannabis* active components, which may act synergistically and contribute to the pharmacological power and entourage effects of medicinal-based *Cannabis* extract (Russo, 2011).

#### Phytocannabinoids

Phytocannabinoids represent a group of C21 or C22 (for the carboxylated forms) terpenophenolic compounds predominantly produced in *Cannabis.* They have also been reported in plants from the *Radula* and *Helichrysum* genus (Appendino et al., 2008) but our knowledge on non-*Cannabis* source of cannabinoids is still in its infancy (Gertsch et al., 2010). More than 90 different cannabinoids have been reported in the literature, although some of these are breakdown products (ElSohly and Slade, 2005; Brenneisen, 2007; Radwan et al., 2009; Fischedick et al., 2010) and they are generally classified into 10 subclasses (Brenneisen, 2007). In this review, we will focus on the most abundant compounds found in the drug- and fiber-type *Cannabis*. The predominant compounds are THCA, CBDA and cannabinolic acid (CBNA), followed by cannabigerolic acid (CBGA), cannabichromenic acid (CBCA) and cannabinodiolic acid (CBNDA) (ElSohly and