A quantitative method for determination of PPDK concentration in miscanthus leaves

In this study, we used ELISA for quantification of PPDK in photosynthesizing leaves of miscanthus. We cloned a fragment of the gene encoding PPDK, purified the resulting protein by affinity chromatography, identified it using MALDI mass spectrometry, and obtained monoclonal antibodies by immunizing BALB/c mice. Selectivity of monoclonal antibodies was assessed by Western blot using the protein extracts of Soranovskii. The presence of PPDK was again verified by MALDI mass spectrometry. Therefore, we developed and tested the method for determining PPDK quantity in miscanthus using ELISA.


Introduction
The prospect of using plants with rapid growth as cellulose sources is currently widely investigated all over the world. However, there have been few attempts to grow them in cold climates. Plants that give high biomass yields in short vegetation period can allow one to use huge areas of marginal lands that are ineffective for traditional agriculture. Miscanthus (mostly M. 9 giganteus) is widely used as a plant biomass source. The area of its cultivation is constantly increasing (Lewandowski et al., 2000;Heaton et al., 2010;Meyer & Hong, 2011;Boersma & Heaton, 2012;Brancourt-Hulmel et al., 2014) and new processing methods are being developed (Slynko et al., 2013). However, most cultivars of miscanthus cannot be efficiently cultivated in cold climate (Farrell et al., 2006;Anderson et al., 2011). We developed the Soranovskii cultivar of miscanthus, which has high cold tolerance and high biomass yield. In 2013, it was added to the State register of selection achievements (certificate of authorship no 58540) and is currently regarded as new cellulose source for Russia.
Soranovskii cultivar has changes in its root system in comparison to the source species: there are long horizontal roots with growth buds that quickly colonize soil creating a solid plantation without tussocks (Shumny et al., 2010;Gismatulina, 2014). It has the following characteristics: • high biomass yields with minimal cultivation costs (up to 15 tons of dry mass per year for 15-20 years); • the ability to grow on marginal lands; • high holocellulose to lignin ratio (Shumny et al., 2010).
Miscanthus, as well as many other efficient agricultural cultures (corn, sugar cane), has the C4 photosynthesis pathway enabling rapid biomass growth rate (Ehleringer et al., 1997). Plant biomass yield in moderate climate depends on the ability to accumulate solar energy at low average temperatures during the vegetation period. Miscanthus is able to perform highly effective photosynthesis during short and cold vegetation periods, in contrast to corn and sugar cane (Mortaignie, 2014;Boersma et al., 2015), and is considered the most cold tolerant C4 crop.
ELISA is one of the most convenient methods for determining protein concentration. It allows one to assess the quantity of target protein in studied specimens using monoclonal antibodies. Monoclonal antibodies to a conservative hapten allow one to evade cross-reactivity and increase the accuracy of quantitative analysis (Lequin, 2005).
In this study, we used ELISA for quantification of PPDK in photosynthesizing leaves of miscanthus. We cloned a fragment of the gene encoding PPDK, purified the resulting protein by affinity chromatography, identified it using MALDI mass spectrometry, and obtained monoclonal antibodies by immunizing BALB/c mice. As the target sequence, we took the conservative PPDK_N domain of Miscanthus giganteus (GeneBank AAP34175.1) (Naidu et al., 2003). Selectivity of monoclonal antibodies was assessed by Western blot using the protein extracts of Soranovskii cultivar. The presence of PPDK was again verified by MALDI mass spectrometry. Therefore, we developed and tested the method for determining PPDK quantity in miscanthus using ELISA.

Cloning, extraction, and purification of recombinant PPDK
A known sequence of M. giganteus ppdk gene from GenBank (AAP34175) was used to obtain recombinant PPDK (Naidu et al., 2003). Since this gene is long, we selected the conserved PPDK_N domain (amino acid positions 106-425). This sequence was optimized for Escherichia coli expression and synthesized by ATG Service Gene (Saint-Petersburg, Russia). A histidine-containing sequence corresponding to the MRGSHHHHHH polypeptide was added to the 5 0 -terminus of the gene fragment. This polypeptide is used for protein purification by metal ion affinity chromatography (Hengen, 1995). The resulting gene fragment was cloned into the pQE30 plasmid under the T5 phage promoter with the Lac operator. E. coli XLblue strain was used for protein expression (Sambrook et al., 1989).
Initial clone selection was performed by PCR. Colonies containing the target fragment were cultivated in 0.8 ml of LB medium to 0.6 OD (k = 600 nm). Protein expression was initiated by adding IPTG to 1 mM with subsequent incubation at 37°C for 6 h with constraint stirring.
To select the clone with the maximum target protein yield, 50 ll of E. coli cells were sedimented by centrifugation at 16 100 g for 2 min, and 30 ll of loading SDS buffer was added (0.05 M Tris-HCl pH 6.8; 2% SDS; 0.002% bromophenol blue; 10% glycerin; 5% mercaptoethanol), resuspended and sonicated for 15 min. Lyzed cells were centrifuged for 2 min at 16 100 g; the supernatant was boiled for 5 min at 95°C and applied on a polyacrylamide SDS mini gel (8 9 10 9 0.75 cm) for electrophoresis according to the Laemmli method (Laemmli, 1970). Gels were stained by Coumassie G-250 (Bio-Rad, Hercules, CA, USA).
The selected clone was cultivated in 500 ml of LB medium containing ampicillin to 0.6 OD (k = 600 nm) with subsequent induction by adding IPTG to 1 mM. Cells were incubated at 32°C for 6 h with constant stirring. Cells were sedimented by ultracentrifugation (2500 g, 10 min) and the supernatant was discarded. Cells were dissolved in lysis buffer (10 mM imidazol, 300 mM KCl, 100 mM Tris-HCl, pH 8.0), sonicated for 5 min and centrifuged (12 000 g, 30 min) to remove cell debris. The supernatant was filtered through a 0.5 lm filter and transferred to a 10 ml Co +2 -Sepharose CL-6B (GE Healthcare, Pittsburgh, PA, USA) HIS-specific metal affinity column equilibrated by lysis buffer. The column was washed with 100 ml of lysis buffer. Immobilized proteins were extracted with 10 ml of elution buffer (250 mM imidazol, 300 mM KCl, 100 mM Tris-HCl, pH 8.0). Quantity of proteins was determined by spectrophotometry by Quick Start TM Bradford Protein Assay (Bio-Rad). Aliquots of the purified protein were electrophoresized in one-dimensional 12% SDS-PAGE with subsequent protein identification by mass spectrometry.
Obtaining monoclonal antibodies to purified recombinant PPDK Monoclonal antibodies were obtained by us in collaboration with RusBioLink (Moscow, Russia). To obtain monoclonal antibodies, BALB/c mice were immunized with purified recombinant PPDK. Hybridome strains producing monoclonal antibodies were then purified and injected into recipient mice. After the appearance of ascites tumor, mice were slaughtered and ascites fluid was isolated.
Ascites fluid was centrifuged at 4°C for 30 min at 1200 g. Supernatant was diluted 2-3 times using phosphate buffer saline (PBS). An equal volume of saturated sodium acetate was added, and the mixture was incubated on ice for 30 min at continuous stirring and centrifuged at 4°C for 15 min at 8000 g. The precipitate was dissolved in a small amount of PBS and dialyzed overnight against Buffer A (0.02 M NaH 2 PO 4 / Na 2 HPO 4 , pH 8.0). After dialysis, protein aggregates were removed by centrifugation at 4°C for 40 min at 12 000 g. Supernatant was filtered through a 0.22 lm filter and transferred to a Mono Q HR 10/10 column equilibrated with Buffer A for ion exchange chromatography. Protein sample was transferred to the column at 1 ml per min using the 'super loop' device. Chromatography was controlled by a 'UV control unit' flow spectrometer. Proteins were eluted from the column by linear NaCl gradient (0-0.3 M) at 3 ml min À1 and NaCl concentration increase rate of 10 mM min À1 . As the result, we obtained hybridome cell cultures and monoclonal antibodies that can be used for PPDK quantification in Miscanthus specimens.

Plant material and sample preparation
Five specimens of miscanthus of the Far Eastern collection of ICiG SB RAS, and an individual of Soranovskii were used for PPDK quantification.
Miscanthus plants were grown in the ICiG SB RAS hydroponic hothouse at the average temperature of 20°C. Eight circles (four from the 2nd leaf and four from the 3rd) were excised using a metal tube 8 mm in diameter, which yields 4 cm 2 of leaf surface. Samples were ground in liquid nitrogen, transferred into 1.7 plastic tubes with 0.4 ml of 0.01 M PBS with 1% v/v PMSF (Sigma-Aldrich, St. Louis, MO, USA), sonicated for 1 h on ice using a 4 l/1 Sapphire ultrasound bath, and centrifuged (30 min, 4°C, 16 100 g). Supernatants were stored at -70°C and used for Western blot and ELISA.

SDS-PAGE electrophoresis and Western blotting
Protein extracts were mixed with an equal volume of loading buffer (0.05 M Tris-HCl pH 6.8; 2% SDS; 0.002% bromophenol blue; 10% glycerin; 5% mercaptoethanol) and boiled for 5 min. Of each sample, 30 ll was loaded in lanes. Proteins were concentrated in 4% SDS-PAGE (37.5 : 1 acrylamide/bisacrylamide, 0.1% SDS, 0.125 mM Tris-HCl pH 6.8, 0.1% TEMED, 0.05% PSA) at 10 mA and separated in 12% SDS-PAGE (37.5 : 1 acrylamide/bisacrylamide, 0.1% SDS, 0.375 mM Tris-HCl pH 8.8, 0.05% TEMED, 0.05% PSA) at 15 mA in an electrophoresis Mini-PROTEAN â Tetra Cell (Bio-Rad). First gels were stained by Sypro Ruby Protein Stain and imaged using VersaDoc MP4000 (Bio-Rad) and used for mass spectrometry analysis. Second gels were placed to Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) for transfer of antigen to an Immun-Blot â PVDF membrane (0.2 lm, Bio-Rad). Transfer was run at 100 mA overnight, and subsequently at 500 mA for 30 min (running buffer 25 mM Tris, pH 8.3, 192 mM glycine, with 20% methanol). For a blocking of the remaining protein binding sites, a PVDF membrane with applied antigen was placed to 5% nonfat dry milk dissolved in TTBS (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 2 9 30 min at a room temperature on a shaker. After decanting of blocker, the membrane was incubated with primary antibody in TTBS for 90 min at a room temperature on a shaker with gentle agitation. Primary antibody was diluted to 1 lg ml À1 in TTBS. After decanting of primary antibody solution, the membrane was washed with five changes of a large volume of TTBS. Each wash was for 10 min at room temperature with strong agitation.
After decanting of washing solution, the membrane was incubated with secondary goat antimouse antibody conjugated to HRP, 1 : 10 000 dilution in TTBS, for 90 min at room temperature with gentle agitation. We used Immun-Star Goat Anti-Mouse (GAM)-HRP Conjugate (Bio-Rad).
After decanting of secondary antibody solution, the membrane was washed with six changes of a large volume of TTBS. Each wash was for 10 min at room temperature with strong agitation.
After decanting of washing solution, the membrane was incubated with mixture of luminol/enhancer and peroxide buffer solutions in 1 : 1 ratio for 5-10 min. After incubation, the membrane was placed to an Imager VersaDoc MP4000 (Bio-Rad) and imaged.

Mass spectrometry analysis
We performed a mass spectrometry analysis for the corresponding protein bands from a parallel gel. For mass spectrometry  analysis, selected protein bands were excised from the gel using an automated EXQuest Spot Cutter system (Bio-Rad). The dye was removed from excised gel fragments by washing in 200 ll of buffer containing 0.2 M ammonium bicarbonate and 50% acetonitrile at 37°C until gel pieces had no color. Then, the pieces were dried in 100% acetonitrile and 150 ll of buffer containing 0.2 M ammonium bicarbonate, 20 ll of 45 mM dithiothreitol and incubated at 37°C for 30 min. Afterward, the gels were dried again and incubated in 100 mM iodoacetamide in darkness for 30 min at room temperature. After incubation, gels were dried. Trypsinolysis was performed by rehydrating the gel in 15 lg ml À1 trypsin solution (Trypsin Gold, Mass Spectrometry Grade; Promega, Madison, WI, USA) containing 40 mM ammonium bicarbonate and 10% acetonitrile for 16 h at 37°C.

Database searches
Proteins were identified by their tryptic mass map using the Mascot search algorithm (http://matrixscience.com/ home.html). The following search parameters were used: mass error, AE0.05 Da; nonrestricted sites, 1; possible modifications, methionine oxidation and cysteine carbamylation; database, Plants-EST.

ELISA
For ELISA (Ngo, 1991), 0.1 ml aliquots of leaf protein extract were transferred to immunological plates (MICROLON â 200 96W Microplate, F-bottom, high binding, Greiner Bio-One GmbH). 0.1 9 dilutions (in 0.01 M PBS) were used. The plate was kept for 30 min at 25°C and constant shaking at 250 rpm on the thermo shaker PST-60 HL (Biosan, Riga, Latvia). After sorption, the aliquots were removed, the plate was washed twice with PBST (0.01 M PBS with 0.04% Tween 20), incubated at 25°C for 15 min and constant shaking (250 rpm) in 0.1 ml PBST for blocking of nonspecific sorption. Afterward, PBST was removed, and 0.1 ml of monoclonal antibody to PPDK (1 lg ml À1 ) in 0.01 M PBS was transferred to the plate. For quantitative binding, the antibody was incubated for 30 min at 25°C and constant shaking at 250 rpm. Afterward, the antibody solution was removed, the plate was washed four times with PBST, and 100 ll of goat antimouse antibody (0.05 lg ml À1 ) in 0.01 M PBS (Bio-Rad) conjugated with horseradish peroxidase was added to each cell. The plate was incubated for 30 min at 25°C with constant shaking at 250 rpm. Then, the antibody was removed and the plate was washed six times with PBST, 100 ll of 0.4 mg ml À1 o-phenylenediamine solution in 0.1 M Na-citrate buffer (pH 5.5) with 0.0004% of fresh 30% H 2 O 2 was added to each cell. Substrate oxidation was performed for 10 min at 25°C with constant shaking 250 rpm. The reaction was stopped by 50 ll of 0.5 M H 2 SO 4 . Color intensity was measured at 490 nm using an Epoch plate scanner (BioTek, Winooski, VT, USA).
Purified recombinant PPDK was used for calibration. A gel filtration column (Micro Bio-Spin TM 6 Columns, Bio-Rad) equilibrated with 0.01 M PBS was used to transfer the protein from denaturing buffer (300 mM KCl, 50 mM KH 2 PO 4 , 250 mM imidazole, 6 M urea) to 0.01 M PBS. Protein concentrations were measured according to Quick Start TM Bradford Protein Assay (Bio-Rad).

Results and discussion
Screening for recombinant clones Escherichia coli cells containing the target sequence were analyzed by PCR. Total protein fraction was analyzed by SDS-PAGE stained by Coumassie G-250 (Bio-Rad). The clone with the highest expression level of the target protein with the mass of~35 kDa (indicated by an arrow on Fig. 1) was selected. Sequence of the target insert was verified using Sanger dideoxy sequencing (Sanger et al., 1977).

Extraction, purification, and verification of the recombinant protein
The selected clone was grown in bacterial medium. Expression of the recombinant protein was performed as described in Materials and Methods. Recombinant protein was extracted from the total protein fraction by metal ion affinity chromatography, and electrophoresized in SDS-PAGE gel with subsequent staining by Coumassie G-250 (Bio-Rad) (Fig. 2). The target protein with the molecular mass of~35 kDa was excised from the gel, digested by trypsin, and analyzed by mass spectrometry.
We compared the mass spectrum of the digested protein ( Fig. 3) with the spectrum calculated based on the cloned sequence using the MASS program.
We demonstrated that experimentally detected peptides corresponded to calculated ones (Table 1).
The obtained purified protein was used for immunization of laboratory animals. Procedure for obtaining monoclonal antibodies is described in Materials and Methods. We obtained hybridome strains producing monoclonal antibodies. Binding of the obtained mouse antibodies with the target recombinant protein was verified by Western blot (Fig. 4).

PPDK identification in Miscanthus leaves
Total protein fraction was extracted from the leaves of Soranovskii cultivar according to the procedure described in Materials and Methods. SDS-PAGE electrophoresis and the corresponding western blot are given on Fig. 5. From Fig. 5, one can see that the protein fraction that binds to antibodies has much higher mass than (102 kDa). It is known that PPDK is active in homotetameric form (Wang et al., 2008), so it is conceivable to suggest that tetrameric PPDK complexes are sufficiently stable even in denaturing conditions. Protein bands from SDS-PAGE that bind to mouse monoclonal antibodies were excised, lyzed by trypsin, and analyzed by mass spectrometry. These protein bands were found to contain PPDK (C4-specific pyruvate orthophosphate dikinase [Miscanthus 9 giganteus]), (Table 2). Therefore, the obtained monoclonal antibodies are highly specific for miscanthus PPDK.   Applying the method of PPDK quantification on a sample of Miscanthus leaves PPDK quantification in photosynthesizing miscanthus leaves was performed using ELISA. Leaves were homogenized in PBS, and PPDK concentration was subsequently determined. Purified recombinant protein was used as a reference. Concentrations were expressed as lg cm À2 (Table 3). For mass spectrometry analysis, the studied protein extracts were transferred to one-dimensional SDS-PAGE and analyzed by Western blot. SDS-PAGE electrophoresis and the corresponding Western blot of protein from miscanthus leaves are shown on Fig. 6.
Protein bands from SDS-PAGE that bind to mouse monoclonal antibodies were excised, lyzed by trypsin, and analyzed by mass spectrometry. These protein bands were found to contain PPDK (C4-specific pyruvate orthophosphate dikinase [Miscanthus x giganteus]), the results are presented in Table 4.
We used specific monoclonal antibodies to recombinant PPDK to quantify PPDK content in photosynthesizing leaves of a collection of miscanthus specimens from the Far East and of the Soranovskii cultivar. PPDK content ranged from 0.318 (clones D36 from the Far East) to 0.534 lg cm À2 (Soranovskii cultivar), and two plants from the Far East (clones D153 and D187) had PPDK content close to zero. According to Western Blot analysis (Fig. 6b), D153 and D187 did not bind primary antibodies. One-dimensional electrophoresis demonstrated that these specimens differ from others (Fig. 6a) and did not have the high molecular weight fraction that corresponds to PPDK, but contain a thick band about 1/3 of the way down the gel. It is possible that it represents a PPDK isoform containing substitutions or deletions that impede the formation of the stable tetrameric form, which in turn may affect PPDK cold tolerance (Ohta et al., 1997). However, this issue requires additional studies, as we failed to identify this band.  In this study, we developed a method for determining PPDK content by ELISA. We transformed E. coli with a plasmid containing fragments of the gene encoding PPDK. The protein was purified using affinity chromatography. We obtained highly specific mouse monoclonal antibodies to purified recombinant PPDK. Gel electrophoresis, immunoblotting, and mass spectrometry were used to identify the protein and for subsequent screening of antibodies for specificity toward miscanthus PPDK. The developed method was used to quantify PPDK in photosynthesizing leaves of a collection of miscanthus specimens from the Far East, as well as of the Soranovskii cultivar of miscanthus.