Analysis of putative laccase isoforms in a Miscanthus transcriptome
Using more than 70 laccase protein sequences from both monocot and dicot plants, we performed a local tBLASTn search using the published Miscanthus transcriptome (Barling et al., 2013). More specifically, 17 laccase sequences from Arabidopsis thaliana (The Arabidopsis Information Resource [TAIR]; http://www.arabidopsis.org), 25 laccase sequences from Sorghum bicolor (Phytozome; http://www.phytozome.net/sorghum) and 29 laccase sequences from Brachypodium distachyon (Plant Genome and Systems Biology; http://pgsb.helmholtz-muenchen.de/plant/brachypodium) were used as queries.
Contigs containing full-length protein-coding sequences were further analyzed. Alignment of laccase sequences was performed using ClustalW alignment in MegAlign (DNASTAR, Madison, WI). A phylogenetic tree was generated using the Neighbour-Joining method (http://www.phylogeny.fr/) with bootstrap tests for 1000 replicates. Based on phylogeny and co-expression analyses, MsLAC1 was selected as a putative AtLAC17 ortholog.
To identify the promoter sequences of identified laccases, genomic DNA from Miscanthus sinensis (identification number: Sin-13) was sequenced to generate a partial genome database. The generated contig library was used to search for promoter sequences via BLAST search using the 5′-terminal sequences of laccases as query, and promoter sequences of corresponding genes were retrieved from the database.
Plant material and growth conditions
Miscanthus sinensis seeds (identification number: Sin-13) were a gift of Iris Lewandowski and are descendant of plants originally collected in Honshu, Japan [45]. Plants were grown in the greenhouse as previously described [9]. For over-expression of MsLAC1 in Arabidopsis Columbia-0 (Col-0) plants and complemented lines of the Arabidopsis lac4–2 lac17 double mutant (gift from Dr. Richard Sibout, Institut Jean-Pierre Bourgin), were grown in soil under short-day conditions (21 °C, 8 h light/16 h dark, 110 μmolm2s) until rosette stage, and then transferred to long-day conditions (21 °C, 16 h light/8 h dark, 110 μmol/m2s) in the greenhouse. For phenotyping of transgenic Arabidopsis lines, plants were cultivated under continuous light in a growth chamber (21 °C, 60% relative humidity, 135 μmol/m2s).
Histochemical staining of lignin
Arabidopsis inflorescence stems samples were embedded in 6% agarose and cut by hand using a razor blade [46]. Lignin was stained with HCl-phloroglucinol staining solution (2% phloroglucinol in absolute ethanol, mixed with equal volume of HCl before use). O-4-Linked coniferyl and sinapyl aldehydes in lignified cell walls were stained red [47] and images were captured using a Leica DM IRB inverted microscope.
Tissue sampling, RNA extraction and quantitative RT-PCR
For 10-day to 3-month-old Miscanthus plants, different tissues (leaf, stem, and root) were separated and collected as a pool. 6-month-old Miscanthus stems and developing leaves were cut and dissected at their nodes, and seven internodes and five leaves were sampled. All samples were immediately frozen in liquid nitrogen.
After grinding, 30 mg or 50 mg of tissue from Miscanthus or Arabidopsis, respectively, were used for RNA extraction as previously described [9]. Subsequently, 0.5 μg of total RNA was reverse-transcribed by AMV reverse transcriptase (Roboklon) [48]. Transcript abundance was determined by quantitative RT-PCR using reference genes as described in [9]. Gene-specific primers used for Miscanthus are listed in Additional file 1: Table S2.
Cloning of transcription factors and MsLac1 promoter
Following the Gateway® cloning protocol (Fisher Scientific), the entry vector pDONR201 was used for initial cloning, and two different destination vectors, pART7 and pLuc, were used as effector and reporter plasmids, respectively, as previously reported [48].
Transcription factors MsSND1 and MsSCM2–4 were previously cloned [9], and used in this study. The promoter of MsLAC1 as well as two additional transcription factors, MsVND7 and MsMYB52, were cloned with gene-specific primers containing gateway overhangs (Additional file 1: Table S1). DNA fragments were cloned into pDONR201 via BP reaction according to the manufacturer’s instructions, subsequently sequenced and transferred into the corresponding destination vectors using the LR reaction.
Promoter activation via dual luciferase assay
To determine activation of the MsLAC1 promoter by transcription factors related to lignin biosynthesis, dual luciferase assays were employed using grapevine (Vitis vinifera) suspension cells, as previously described [48]. As indicated in Fig. 4, all transcription factor ORFs were expressed under control of the CaMV35S promoter in pART7, these plasmids being used as effectors. In the reporter plasmid, firefly luciferase was expressed under control of MsLAC1 promoter (pMsLAC1); thus, the intensity of fluorescence indicated promoter induction via the respective transcription factors. Reporter plasmid, effector plasmid (and internal control, see below) were then coated onto gold particles and bombarded into grapevine suspension cells. After a two-day culture in the dark, bombarded cells were ground and fluorescence intensity of firefly and Renilla luciferase (internal control for transformation efficiency) in the isolated supernatants were quantified. The ratio between firefly and Renilla luciferase for each transfection experiment was normalized against the Renilla luciferase plasmid pRluc to represent the relative fold-activation of pMsLAC1 via each transcription factors. All measurements were repeated three times (technical repeats), and all experiments were carried out independently at least twice.
Sub-cellular localization of MsLAC1 protein by transient expression in Nicotiana benthamiana leaves
To determine the sub-cellular localization of the MsLAC1 protein, Agrobacterium tumefaciens ASE (pSOUP+) was transformed using the Greengate expression vectors [49] and subsequently infiltrated into leaves of 4-week-old Nicotiana benthamiana plants for transient expression of fluorescent protein-labelled MsLAC1 protein. Since an N-terminal signal peptide was predicted for the full-length MsLAC1 coding sequence, mCherry was fused to its C-terminus. Lt16b-GFP was used as a plasma membrane marker [33]. Agrobacterium strains containing this marker, mCherry tagged MsLAC1, and the p14 silencing inhibitor plasmid were dispersed in transformation buffer (10 mM MgCl2, 10 mM MES, 150 μM acetosyringone, pH 5.6) to an OD600 of 0.4, 0.4, and 0.1, respectively, and then mixed at equal volumes. After incubation at room temperature (RT) for 2 h, bacterial mixtures were co-infiltrated into tobacco leaves with a 2 ml sterile needle-less syringe. Dummy gene [49] replacing MsLAC1 was used as a control construct that lacked the MSLAC1 coding sequence. Infiltrated leaves were collected after 3 days. To test whether MsLAC1 localizes to the cell wall, leaf discs were incubated in 30% sucrose solution for 2 h before microscopy to plasmolyze cell walls from the plasma membrane. Details regarding the Greengate constructs used in this study are presented in the legend of Fig. 5.
Images were captured using the Perkin-Elmer UltraView VoX spinning disk confocal mounted on a Leica DM16000 inverted microscope with a Hamamatsu 9100–02 CCD camera. The GFP filter (excitation 488 nm, emission 525 nm) was used to image GFP-tagged constructs. The RFP filter (excitation 561 nm, emission 595-625 nm) was used to image mCherry-tagged constructs. Samples were imaged using a Leica oil immersion 20× or 63× objective. All images were processed using Volocity image analysis software (Improvision).
Heterologous expression of MsLAC1 protein in Pichia pastoris
The Pichia pastoris expression vector pPICZαA containing both MYC and HIS tags was used for heterologous expression of MsLac1 and CiFEH. The purified PCR products of MsLAC1 and the vector pPICZαA were digested with EcoRI and XbaI, and then ligated at 4 °C overnight using T4 DNA ligase (Thermo Fisher Scientific, Catalog number: EL0014). Yeast extract Peptone Dextrose medium (YPD) containing 1% yeast extract, 2% peptone and 2% dextrose was used for general culture. Selected yeast strains were cultivated in buffered complex media containing glycerol (BMGY) and methanol (BMMY) for induction of protein expression. Competent yeast strain X33 cells were prepared fresh, and 2 μg of the ligated constructs was used for transformation. Positive colonies were selected on YPD plates containing 50 μg/mL Zeocin™, followed by colony PCR using the α-factor and the 3′ AOX1 Sequencing Primers (Additional file 1: Table S1). Colonies were then screened on minimal methanol histidine medium and minimal dextrose histidine medium plates supplemented with Zeocin™ for fast methanol utilization (Mut+) phenotype [34].
To check the expression and secretion of recombinant MsLAC1 protein, verified Mut + colonies were cultured in 5 mL BMGY medium in 50 mL tubes (30 °C, 180 rpm) and harvested by centrifugation (5000 rpm, 5 min), when cultures had reached an OD600 of 3.0. Pellets were washed and subsequently re-suspended in BMMY medium (supplemented with 0.3 mM CuSO4) and diluted to a final OD600 of 1.0 in 30 mL BMMY media in 300 mL flasks, cultured at the same conditions (30 °C, 180 rpm). Methanol (final concentration: 1%, v/v) was added every day to maintain inducing conditions. After the onset of induction, aliquots were collected daily by centrifugation (5000 rpm, 5 min). Supernatants were dialyzed against pH 5.0 20 mM sodium acetate buffer overnight at 4 °C (cold room) and stored for further analysis at − 80 °C. The pPICZαAfeh strain expressing recombinant fructan exohydrolase (FEH IIa) protein from Cichorium intybus was cultivated under the same conditions and used as a control.
Measurement of laccase activity and protein concentration
Laccase activity was determined with ABTS (2,2-azino-bis3-ethylbenzothiazoline-6-sulfonic acid) as substrate [50]. Sample was diluted in pH = 5.0 50 mM HAc/NaAc buffer to 180 μL and mixed with 20 μL ABTS. 180 μL buffer without enzyme was mixed with ABTS in the same way and used as control. After incubation in 30 °C for 3 mins, absorbance at 420 nm was measured and the increased absorbance comparing with control (ΔA) was used for the calculation of enzyme activity:
$$ \mathrm{Laccase}\ \mathrm{activity}\ \left(\mathrm{U}\bullet {\mathrm{L}}^{-1}\right)=\frac{V_t\times N\times \Delta \mathrm{A}\times {10}^6}{V_e\times \varepsilon \times \Delta \mathrm{T}} $$
Where, Vt, total volume; N, dilution fold; Ve, sample volume; ε, Molar extinction coefficient for ABTS (3.6 × 104 M-1 cm-1); ΔT, time of incubation. To select the appropriate wavelength for each substrate, we measured substrate spectra at different concentrations. After calculating the correlation coefficients, we selected the wavelength with the highest absorbance for each substrate: 288 nm for synalpyl alcohol, P-coumaroyl alcohol, and coniferyl alcohol, as well as 296 nm for cinnamyl alcohol. One unit of enzyme activity was defined as the amount of enzyme that oxidizes 1 μmol substrate per minute at 30 °C. Protein concentration was determined via the Bradford assay using a calibration curve based on bovine serum albumin (0.1–1 mg/mL) with Bradford Reagent from Sigma (Product number: B6916).
Enzymatic oxidation of monolignols by recombinant MsLAC1 protein
Cinnamyl alcohol (CAS No.: 104–54-1), p-coumaryl alcohol (CAS No.: 3690-05-9), coniferyl alcohol (CAS No.: 458–35-5), and sinapyl alcohol (CAS No.: 537–33-7) were used as substrates to evaluate the ability of recombinant MsLAC1 protein to oxidize the canonical monolignols. In the assay, 0.02 U enzyme was mixed with 0.5 mM of each substrates [51]. Reactions were performed in 1.5 mL reaction vials in 50 mM NaAc-HAc buffer, pH 5.0, at 30 °C. Absorbance of the reaction mixtures were monitored between 250 to 650 nm [52]. For sinapyl alcohol, the reaction mixture was scanned at 5, 10, 20, 30 and 60 min after reaction start, respectively, whereas for other monolignols reaction mixtures were scanned at 0.5, 1, 2, 6, 12 h, respectively. The reaction was terminated by adding two drops of 10 mM NaN3. Water replacing the recombinant MsLAC1 protein was used as the control in different mixtures and measured in the same time periods.
Native-PAGE, SDS-PAGE and Western blot analysis
Recombinant protein samples were mixed with Roti®-Load 1 or Roti®-Load 2 followed by incubation at 100 °C for 5 min, for SDS-PAGE and Native-PAGE, respectively. Both types of electrophoresis were performed on a 4.5% stacking gel and a 12% separating gel, using chambers from Bio-Rad®. Sample loading, running conditions and ABTS staining procedures were as previously described [51].
After SDS-PAGE, proteins were electroblotted onto an Immobilon-P PVDF membrane (Sigma-Aldrich). Recombinant MsLAC1 protein was detected by Western Blot analysis, using anti-MYC (Thermo Fisher Scientific, MA1–980) as a primary antibody, detected by using SuperSignal West Dura Extended Duration Substrate for HRP (Thermo Fisher Scientific, Catalog number: 34075) after incubation with anti-mouse IgG secondary antibody (Bio-Rad, Catalog number: 172–1011).
Generation of transgenic Arabidopsis lines
All transgenic plant lines were generated using the Agrobacterium tumefaciens strain ASE (pSOUP+) transformed with Greengate constructs using the floral dip method [53]. The Arabidopsis lac4–2 lac17 double mutant was complemented with the pAtLAC17::MsLAC1 construct (i.e., expressing MsLAC1 under control of the AtLAC17 promoter). In addition, MsLAC1 was ectopically expressed in Arabidopsis Col-0 plants, using a p35S::MsLAC1 construct. The Greengate constructs used for over-expression are depicted in Additional file 1: Figure S4. For analysis of complementation and ectopic expression homozygous F3 plants were used.
Lignin analysis
Mature stems of Arabidopsis were dried and all siliques and leaves removed. Approximately 12 cm long stem segments were ground and then extracted with hot acetone in a Soxhlet column overnight to remove soluble compounds. The contents of acid soluble lignin (ASL) and insoluble lignin (IL) were measured according to [54]. Neutral cell wall carbohydrate contents were determined via liquid chromatography [55], while lignin composition was determined by gas chromatography after thioacidolysis as previously described [56]. All measurements were done in technical triplicates, using two independent transgenic lines.
Accession numbers
All sequence data from this paper can be found in GenBank under the following accession numbers. Promoter sequences: pMsLAC1 (MK310212). Coding sequences: MsLAC1 (MK310209), MsSND1 (KY930620), MsVND7 (MK310211), MsSCM2 (MF996502), MsSCM3 (KY930622), MsSCM4 (MF996501), MsMYB52 (MK310210).
Statistical treatment of data
Three independent experiments were carried out for all measurements. The SD value indicates the standard deviation calculated from the mean. For statistical analysis, the Student’s T-test was performed, and asterisks were used to represent the significant differences (*, P < 0.05; **, P < 0.01;***, P < 0.001). One-way ANOVA followed by Tukey test was also used to determine significant differences for lignin composition.