A polyclonal antibody against a recombinantly expressed Triticum aestivum RHT-D1A protein

Background Reduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein. Results To generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A–agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid. Conclusion The purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.


Background
Dwarfing alleles (reduced height, Rht) are an important breeding tool for increasing wheat grain yields. Rht genes containing lesions are most extensively used in worldwide wheat breeding programs. Among them, mutations Rht-B1b and Rht-D1b were the major factors in the Green Revolution, and more than 70% of the wheat cultivars all over the world carry at least one of them [1]. Rht-B1b and Rht-D1b homeoalleles originate from a Japanese variety, Norin 10, and were successfully exploited in wheat breeding programs in the 1950s [2,3]. Alternative semidwarfing alleles Rht-B1d and Rht-B1e have found some limited commercial applications, while more strongly dwarfing alleles, such as Rht-B1c and Rht-D1c, have not so far been exploited because they reduce biomass and the crop yield [4]. Both Rht-B1b and Rht-D1b involve a point mutation giving rise to a TAG stop codon in the N-terminal coding region [5]. These mutations decrease the plant's capacity to respond to gibberellic acid (GA), and wild-type plant height is not restored by exogenous application of this hormone. Wild-type Rht genes are orthologous to Arabidopsis genes RGA and GAI, which are negative regulators of the GA response [6]. Genes RGA and GAI encode DELLA proteins, which are transcriptional regulators suppressing the GA signal transduction pathway. The mechanism of DELLA protein-mediated GA signaling in Arabidopsis has been elucidated via biochemical, genetic, and structural analyses [7]. It is assumed that SLR1 (Slender Rice-1) in rice and SLN1 (Slender 1) in barley perform a function similar to that of RGA and GAI [8]. In the nucleus, a wild-type Arabidopsis DELLA protein binds to GA receptor GID1, GA, and the SCF E3 ubiquitin ligase complex. Such a large complex is recognized by the 26S proteasome and is destroyed. The degradation of DELLA proteins induces GA-responsive plant processes such as seed germination, stem and root elongation, and fertility [7].
DELLA research in wheat has focused on phenotypic effects of Rht-B1b and Rht-D1b on yield-related traits, without studies on the underlying mechanisms [7]. In wheat, although an interaction between RHT-1 and GID1 has been observed in yeast two-hybrid experiments [9], the RHT-1 protein has yet to be detected in plant tissues, owing to a lack of appropriate molecular tools. To detect the RHT-1 protein in wheat tissues, an antibody specific for this protein is needed. There are currently no commercial high-specificity anti-RHT-1 antibodies available.
In this study, the wheat Rht-D1a cDNA gene was synthesized and expressed in E. coli, then a polyclonal antibody was generated using the recombinant protein as an antigen, and the suitability of this polyclonal antibody for immunoblotting, western blot hybridization, and immunoprecipitation (IP) was analyzed.

Plant material
Wheat varieties Saratovskaya 29 and Norin 10 were identified and kindly provided by Dr. M.A. Yessimbekova, Department of the Field Crops Gene Pool of Kazakh Research Institute of Agriculture and Plant Growing, Almaty, Kazakhstan (no voucher specimen of this material has been deposited in a publicly available herbarium).

DNA extraction and PCR analysis
Genomic DNA was isolated from fresh leaf tissues of 4day-old wheat seedlings using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Alleles Rht-B1b and Rht-D1b were detected as recommended by Ellis et al. [10] by means of a primer combination specific for tall and dwarfing alleles ( Table 1). The PCR products were separated on 2% agarose gels and visualized after ethidium bromide staining by standard procedures [11].

cDNA synthesis
Seeds were surface-sterilized by washing first with 70% ethanol for 2 min, then with 1% sodium hypochlorite for 30 min, and finally with sterile distilled water. The sterilized seeds were next grown at 20°C on moistened filter paper. Total RNA was extracted from 100 mg of fresh leaf tissues of wheat (Triticum aestivum) variety Saratovskaya 29 in liquid nitrogen using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Intactness and high quality of RNA were confirmed by the presence of two intense 28S and 18S ribosomal RNA bands in ethidium bromide-stained agarose gels visualized under UV light. Five micrograms of DNA-free total RNA was converted into singlestranded DNA by means of a mix of oligo-dT 18 primers

Expression and purification of RHT-D1A proteins
To generate an expression plasmid for 6xHis-tagged RHT-D1A, the full-length coding sequence of Rht-D1a was excised with restriction enzymes EcoRI and HindIII from plasmid pBluescript II SK (+)-Rht-D1a and was subcloned into the pET-28c vector at the same sites. The resulting expression plasmid, pET-28c-Rht-D1a, yields the respective protein with an N-terminal 6xHistag sequence. E. coli DH5α served as a cloning host for the propagation of the expression vector. Three expression strains of E. coli, BL21(DE3), Rosetta-gami 2(DE3), and ArcticExpress (DE3), were prepared and transformed by the standard protocols [12]. The RHT-D1A protein was purified from a cell extract of IPTG-induced E. coli strain ArcticExpress (DE3) (Agilent Technologies) carrying the plasmid with the Rht-D1a gene. Briefly, the transformed E. coli cells were grown to optical density at 600 nm (OD 600 ) of 0.6 at 37°C, then the temperature was reduced to 13°C, and the cells were incubated for 30 min. After the desired temperature was reached, 500 μM IPTG was added, and expression was continued for 16 h. Due to strong expression in ArcticExpress (DE3), it was possible to purify RHT-D1A to homogeneity by only single-step affinity purification.
All purification procedures were carried out at 4°C. For purification of recombinant RHT-D1A (rRHT-D1A) in the native nondenatured state, the bacteria were harvested by centrifugation, and the cell pellets were lysed with a French press at 124.1 MPa in a buffer consisting of 50 mM Tris-HCl (pH 9.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 5% of glycerol, 1 mM DTT, 10 mM βmercaptoethanol, and 2% of Triton X-100 and supplemented with the Complete Protease Inhibitor Cocktail (Roche Diagnostics, Switzerland). The EDTA was intended to inhibit proteases, and β-mercaptoethanol and DTT were necessary for the maintenance of a reducing environment. Milder nonionic detergent Triton X-100 is good at solubilizing membrane proteins and for isolation of cytoplasmic proteins. Proteins retain their native conformation in the presence of this detergent. The lysates were cleared by centrifugation at 40,000 × g for 60 min at 4°C, the buffer of the resulting supernatant was adjusted to 500 mM NaCl and 20 mM imidazole, and the sample was loaded onto a HisTrap HP column (GE Healthcare) charged with Ni 2+ . The bound proteins were eluted in a 20-500 mM imidazole gradient. The purified protein samples were stored at − 20°C in 50% glycerol. The homogeneity of the protein preparations was verified by SDS-PAGE.

Preparation of the polyclonal anti-RHT-D1A antibody
This antibody was raised against the full-length recombinant 6xHis-tagged RHT-D1A protein (rRHT-D1A). For primary immunization, we subcutaneously injected~1 mg of the purified rRHT-D1A protein mixed with an equal volume of Freund's complete adjuvant (F5881, Sigma-Aldrich, Canada) at five spots on the dorsal back of a rabbit. Then, reimmunization was done three times with 1 mL of a solution containing 0.5 mg of the purified rRHT-D1A protein in 0.5 mL of elution buffer and 0.5 mL of incomplete Freund's adjuvant (F5506, Sigma-Aldrich, Canada) with a 14-day interval. One week after the last injection, blood was collected, and ammonium sulfate was added to 3 mL of the obtained rabbit antiserum to achieve 50% saturation. The precipitate was collected by centrifugation, and the pellet was dissolved in purified water and dialyzed against 10 mM potassium phosphate buffer (pH 7.0). The obtained immunoglobulin fraction was applied to a column with protein A-agarose beads equilibrated with the above buffer. After a wash with the same buffer, antibodies were eluted with 100 mM glycine buffer (pH 3.0). The IgG-containing fractions were pooled, and pH was adjusted to 7.0 with 1.0 M Tris base. The resulting suspension was kept at 4°C.

Antiserum titer determination by an ELISA
The titers of antisera were determined by an indirect ELISA. Each well of a 96-well ELISA plate (Corning Inc., USA) was coated with 1 μg of rRHT-D1A dissolved in 100 μL of 50 mM carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. After three washes with phosphate-buffered saline (PBS) Tween buffer (PBST; 0.05% of Tween 20 in PBS, pH 7.4), the wells were blocked with 100 μL of 3% BSA in PBST for 1 h at 37°C and then washed again twice with PBST. After blocking, 100 μL of serially diluted anti-RHT-D1A serum (1:1000 to 1:128000) was added into the antigen-coated wells. The plate was covered with an adhesive plastic and incubated for 2 h at room temperature and then washed four times with PBST. At the next step, a 1:30,000-diluted alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Sigma, Canada) was added at 100 μL/well and incubated for 1 h at 37°C. After a wash, 100 μL of a freshly prepared p-Nitrophenyl phosphate substrate solution was added into each well, and the plate was incubated at room temperature in a dark place. Finally, an absorbance was measured at 405 nm (A 405 ) on a multiskan FC (Thermo Scientific, MA, USA). All samples were tested in triplicate, with each plate containing control wells with positive serum samples and control wells with negative reference serum.

A dot blot assay
Next, 0.5-1.0 μg of each protein preparation was spotted onto a dry polyvinyl difluoride membrane (Pierce PVDF Transfer Membrane) and dried. The membranes were blocked with 5% nonfat dry milk in 1× Tris-buffered saline containing Tween 20 (TBST; 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.005% of Tween 20) for 1 h at room temperature. After that, the membranes were incubated in blocking buffer containing the anti-RHT-D1A (1:10, 000 dilution in the blocking solution with 0.1% of Tween 20) or anti-Ape1L polyclonal antibody (1:5000 dilution in the blocking solution with 0.1% of Tween 20) on a rocker overnight at 4°C. The membranes were then washed thrice in 1× TBST and incubated with a goat anti-rabbit antibody (ab6702, Abcam, Cambridge, UK) at a 1:20,000 dilution on a rocker for 1 h at room temperature. Next, each membrane was washed five times in 10 mL of 1× TBST for 5 min each time. A working substrate solution was prepared by mixing equal volumes of a H 2 O 2 solution and luminal/enhancer solution. Each membrane was incubated in the working solution for 2 min in darkness, and Kodak X-Omat was exposed to the membrane. The 6xHis-tagged RHT-D1A protein was also detected with the anti-His antibody raised in rabbits (1:1000 dilution; sc-803; Santa Cruz Biotechnology) and an anti-rabbit Ig horseradish peroxidase-conjugated antibody (1:10,000 dilution).

Plant protein extraction
Wheat grains were sterilized in 2% (v/v) NaOCl for 20 min and washed twice with sterile water, once with 0.01 M HCl, and then thoroughly with sterile distilled water. The grains were allowed to germinate at room temperature on sterile filter paper soaked in water or 10 μM GA in the presence or absence of 100 μM paclobutrazol (PBZ). After 4 days, the seedlings were excised from the seeds and the de-embryonated seeds were ground up in liquid nitrogen and then resuspended in RIPA buffer consisting of 50 mM Tris-HCl (pH 7.6), 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ mL leupeptin, and 1 mg/mL pepstatin; the cell debris were pelleted, and the protein concentration was determined with the Bradford Protein Assay Kit (Bio-Rad, France).
Total protein samples (25 μg) from each extract were fractionated by SDS-PAGE in a 10% gel and analyzed by western blotting with a 20,000-fold dilution of the anti-RHT-D1A polyclonal antiserum and a 30,000-fold dilution of a peroxidase-conjugated goat anti-rabbit IgG antibody (ab6702, Abcam, Cambridge, UK). The protein extracts were also subjected to detection of α-amylase with a polyclonal antibody against wheat α-amylase, kindly provided by Dr. A. Khakimzhanov (Aitkhozhin Institute of Molecular Biology and Biochemistry, Kazakhstan).

Zymogram analysis of α-amylase
For this purpose, nondenaturing polyacrylamide gel electrophoresis (native PAGE) was performed according to the method presented by Laemmli [13]. Four-day-old endosperm of wheat seedlings were ground up in liquid nitrogen and then resuspended in 10 mM CaCl 2 . The samples were mixed with 50% saccharose and loaded onto a polyacrylamide gel (4% and 10% polyacrylamide for the stacking and resolving gels, respectively). Electrophoresis was conducted at 100 V and 4°C. After that, the gel was incubated in 10 mM CaCl 2 for 30 min at room temperature. Next, the gel was incubated in a 1% (w/v) starch solution at 30°C and shaken for 60 min. After washing of the gel with distilled water, it was stained with the Lugol solution (1.3% I 2 and 3% KI). The signals of α-amylase activity appeared as bright bands on a dark background and were photographed.

Western blotting
Next, 0.5-1.0 μg of purified proteins or 5-10 μg of cell lysates was separated by SDS 10% PAGE. The gel was then electroblotted onto a PVDF membrane in a Bio-Rad Mini-transblot Cell according to the manufacturer's instructions. After that, each membrane was gently shaken in the blocking solution consisting of 5% milk and 0.1% Tween 20 in 1× TBS (Tris-buffered saline: 50 mM Tris-HCl pH 7.5, 20 mM NaCl) for 1 h at room temperature. After the removal of the blocking solution, the membrane was incubated in 10 mL of a solution of the affinity-purified polyclonal anti-RHT-D1A antibody (1:10,000 dilution in the blocking solution with 0.1% of Tween 20) or the anti-Ape1L polyclonal antibody (1:10, 000 dilution in the blocking solution with 0.1% of Tween 20) overnight at 4°C. The membrane was washed five times in 10 mL of wash buffer (1× TBS with 0.1% of Tween 20), for 5 min each time. After that, the membrane was incubated in 10 mL of a secondary antibody solution (1:20,000 dilution in the blocking solution with 0.1% of Tween 20) for 1 h at room temperature. Then, the membrane was washed five times in 10 mL of wash buffer, for 5 min each time. The working substrate solution was prepared by mixing equal volumes of an H 2 O 2 solution and luminal/enhancer solution and was used at 0.1 mL per cm 2 of the blot area. The membrane was incubated in the working solution for 2 min in darkness, and Kodak X-Omat was exposed to the membrane. The 6xHis-RHT-D1A protein was also detected with an anti-His antibody raised in rabbits (1:1000 dilution) and an anti-rabbit Ig horseradish peroxidase-conjugated antibody (1:10,000 dilution).
Immunoprecipitation E. coli cells expressing the rRHT-D1A protein were grown to OD 600 of 0.6 at 37°C, then the temperature was reduced to 13°C, and the cells were incubated for 30 min. After the desired temperature was reached, 50 μM IPTG was added, and expression was continued for 4 h. The cells were pelleted in lysis buffer (50 mM NaH 2 PO 4 , pH 7.4, 150 mM NaCl, 1 mM EDTA, 5% of glycerol, 0.2% of NP-40, 10 mM MgCl 2 , 1 U of DNase I, 2 mg/mL RNase, 0.1 mg/mL lysozyme, and the protease inhibitor cocktail). The suspension was incubated at 37°C for 30 min and sonicated thrice for 20 s at 1-min intervals on ice. Cell debris were removed by centrifugation at 10,000×g for 5 min. The cell lysates were diluted 1:10 with lysis buffer. For IP, 4 μg of the anti-RHT-D1A polyclonal antibody was added to the cleared cellular lysates containing 1 mg of total protein followed by incubation with rotation at 4°C for 2 h. After 1-h incubation with 45 μL of a 25% protein G-agarose slurry (Thermo Scientific, MA, USA), immunoprecipitates were washed four times with lysis buffer and analyzed by western blotting.

Rht-D1a cDNA gene
In wheat, three DELLA genes, Rht-A1, Rht-B1, and Rht-D1 (on chromosomes 4A, 4B, and 4D, respectively), are known and share high sequence similarity with one another. Rht-B1 and Rht-D1 have many alleles that differ from the wild-type alleles by single-nucleotide substitutions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key role in the creation of high-yielding wheat varieties. They are now present in more than 70% of current commercial wheat cultivars [1]. Both alleles Rht-B1b and Rht-D1b involve a point mutation generating a TAG stop codon in the N-terminal coding region [5].
This point mutation may cause amino acid misincorporation and/or truncation of the polypeptide, thus affecting correct heterologous expression of the RHT-D1A protein. Furthermore, this point mutation lies within the DELLA domain and thereby may alter the epitopes recognized by the immune response. For these reasons, before synthesizing the cDNA gene of RHT-D1, we decided to test for the presence of these mutant dwarfing alleles in wheat variety Saratovskaya 29. Previously, Ellis et al. designed primers for detecting alleles Rht-B1b and Rht-D1b [10]. This PCR-based method can detect the point mutations responsible for the two major semidwarfing alleles Rht-B1b and Rht-D1b in wheat. To test for the presence of these dwarfing alleles, DNA samples from Saratovskaya 29 were analyzed by PCR with primer combinations specific for the tall and dwarfing alleles ( Table 1 and Fig. 1). As a positive control, we used double-dwarf wheat variety Norin 10 carrying alleles Rht-B1b and Rht-D1b. As expected, the double-dwarf wheat variety tested positive with the Rht-B1b-and Rht-D1b-specific primers (Fig. 1a). By contrast, wheat variety Saratovskaya 29 yielded an amplicon of the expected size (237 bp) with the primer combination specific for tall alleles Rht-D1a and Rht-B1a; no amplification was detectable with the Rht-B1b-and Rht-D1b-specific primers (Fig. 1b). Thus, these data indicated that the Saratovskaya 29 wheat variety does not carry mutant alleles Rht-B1b and Rht-D1b.
A cDNA containing a gene for the putative RHT-D1A protein was obtained as described in the "Methods" section. The cDNA was subcloned at the EcoRI and HindIII sites into pBluescript II SK (+) and subjected to DNA sequencing. The cDNA consists of 1872 bp and contains a single open reading frame predicted to code for a protein of 623 amino acid residues. The calculated molecular weight of RHT-D1A is 65.3 kDa. Alignment of the RHT-D1A protein sequence revealed 100% similarity with the translated sequences of previously published Rht-D1a mRNA (GenBank accession number HE585643.1).
Consequently, in addition to the results of PCR analysis with allele-specific primers, sequencing of synthesized cDNA of RHT-D1A with subsequent alignment of translated sequences confirmed that the chosen wheat variety does not carry mutant alleles Rht-B1b and Rht-D1b.

Plasmid construction and optimization of Rht-D1a gene expression
The most widely used recombinant systems for obtaining immunogenic antigens are fusion proteins tagged with glutathione S-transferase (GST) [14]. Nonetheless, some animals have antibodies reacting with the GST part of the fusion protein; hence, all obtained antisera have to be checked not only against the recombinant antigen but also against GST alone. The polyhistidine tag is widely and preferably used for recombinantimmunogen production because of its low immunogenicity and rather weak interference with the protein's function or activity [15]. Therefore, we decided to use a histidine-tagged expression system for rRHT-D1A production. The Rht-D1a gene expression vector encoding the 6xHis-tag at the protein's N terminus was constructed as described in the "Methods" section. The expression plasmid with the cloned Rht-D1a gene was designated as pET-28c-Rht-D1a. To achieve strong immunological responses against rRHT-D1A, it is desirable to express it as a highly soluble protein. With the aim to improve the recombinant-protein folding and solubility, at first, three E. coli expression strains were tested. E. coli BL21 (DE3) is the most common prokaryotic strain used for the expression of recombinant proteins [16]. Rosetta-gami 2(DE3) is a strain engineered to enhance the expression of genes containing rare codons. Besides, it can promote disulfide bond formation to stabilize the recombinant protein owing to mutations of genes trxB and gor [17,18]. ArcticExpress (DE3) competent cells have been engineered to increase the recovery of a soluble protein because of their adaptation to lowtemperature cultivation [19].
The pET-28c-Rht-D1a plasmid was transformed into E. coli cells followed by cultivation in a standard manner by the method described in the "Methods" section. Protein expression was monitored by SDS-PAGE analysis. The total amount of protein produced by E. coli BL21 (DE3) and Rosetta-gami 2(DE3) was lower, and the protein of interest remained mostly in the insoluble fraction. Therefore, these competent cells were not used in further experiments. The most soluble protein was achieved by expression in E. coli Arctic Express (DE3) competent cells as a host. From the protein solubility point of view, 16 h of postinduction incubation at 13°C was found to be optimal for producing the most soluble protein. In SDS-PAGE analysis, one major band with apparent molecular weight 70 kDa revealed the main difference between the bacterial cell lysates before and after the induction with IPTG. This band clearly emerged after the induction in all the chosen clones carrying the rRHT-D1A gene (Fig. 2, lane 3). The RHT-D1A protein fused with 6xHis at the N terminus was purified from the bacterial cell lysate by affinity chromatography on the HisTrap HP 1 ml column (GE Healthcare). The 6xHis-tagged RHT-D1A fusion protein appeared as a single band (after purification by affinity chromatography and SDS-PAGE) with an approximate  (Fig. 2, lane 4). The yield of the recombinant 6xHis-RHT-D1A protein was 4 mg/L of bacterial culture.

Production of the polyclonal antibody
The purified 6xHis-RHT-D1A protein was employed as an antigen to generate a polyclonal antibody against wheat RHT-D1A. After the rabbits were immunized four times with the purified 6xHis-RHT-D1A protein, a polyclonal antibody against rRHT-D1A was first precipitated with different concentrations of ammonium sulfate and was further purified on the protein A-agarose column, following the manufacturer's instructions. The purified antibody showed high purity and consisted of two bands according to electrophoresis: one was the heavy chain (5 0 kDa), and the other was the light chain (~25 kDa), as assessed by SDS-PAGE in a 10% gel (Fig. 3a). The titers of the obtained RHT-D1A antisera were tested by an indirect ELISA. The ELISA results indicated that the titer of the purified polyclonal anti-RHT-D1 antibody is 1:64, 000, suggesting that the purified polyclonal antibody has good sensitivity to rRHT-D1A (Fig. 4). The antibody titer is defined as the highest dilution of antiserum at which the ratio of A 405 (A 405 of postimmunization serum/A 405 of preimmunization serum) is > 2:1.

Evaluation of the anti-RHT-D1A polyclonal antibody by immunoblotting and IP
Western blot analysis was conducted to validate the reactivity and specificity of the purified polyclonal antibody by means of various amounts of purified rRHT-D1A protein (1 and 3 μg). The purified antisera reacted   (Fig. 3c).
It is important to verify whether the antibody is specific to a protein's native, nondenatured state or to the denatured protein because the denaturing treatment of protein samples prior to SDS-PAGE may alter the exposure and availability of the epitope, thus affecting antibody-binding affinity [20]. The capacity of the generated polyclonal anti-RHT-D1A antibody to identify nondenatured forms of RHT-D1A was tested by an immunodot assay involving recombinant 6xHis-RHT-D1A. The purified recombinant 6xHis-RHT-D1A protein, which was used for rabbit immunization, and an E. coli lysate containing the 6xHis-RHT-D1A protein were dotted on a PVDF membrane. A purified 6xHis-tagged recombinant APE1L protein [21], which was cloned and tagged in the same way as the RHT-D1A protein was, was dotted as a negative control. These control proteins were also utilized to test specificity and to rule out the possibility that the polyclonal antibody recognizes the 6xHis tag. The purified 6xHis-tagged rRHT-D1A and APE1L proteins were first analyzed by SDS-PAGE in gels stained with Coomassie brilliant blue (Fig. 5a) and then by dot and western blot hybridization (Fig. 5b, c).
Thus, the dot blot and western blot analyses indicated that the anti-RHT-D1A antibody recognizes both a nondenatured and denatured RHT-D1A protein. Hence, these results also rule out the possibility that the anti-RHT-D1A polyclonal antibody recognizes the 6xHis tag, which was present on the 6xHis-tagged RHT-D1A immunogen used to generate the polyclonal antibody.
The IP assay is an important tool for targeted protein purification, analysis of protein-protein interactions, and identification/analysis of protein complexes. A specific antibody that can selectively bind to the target epitope with high affinity is required for IP. For example, for some IP applications, the antibody should recognize the target protein in its native conformation; this property requires the accessibility of an epitope on the surface of the target protein [22]. The anti-RHT-D1A antibody was assessed in the IP of rRHT-D1A. The IP of RHT-D1A was performed in the cell lysate with protein G-agarose beads. As a control, the same IP procedure was performed with protein G-agarose beads lacking antibodies. The polyclonal anti-RHT-D1A antibody precipitated the 70-kDa rRHT-D1A protein. Proteins that were 25 and 50 kDa also stained in SDS-PAGE gels and represent antibody heavy and light chains (Fig. 6a). Western blot hybridization also revealed that the polyclonal anti-RHT-D1A antibody precipitated RHT-D1A (Fig. 6b). Control precipitate samples did not yield any significant bands either in SDS-PAGE with staining with Coomassie brilliant blue (Fig. 6a) or in western blots (Fig. 6b).
Thus, these data provided additional evidence that the obtained anti-RHT-D1A antibody can be used to detect the RHT-D1A protein in both undenatured and denatured states as validated by IP and dot and western blots.
Detection of RHT-D1A in wheat seedlings using the polyclonal anti-RHT-D1A antibody Our final goal was to obtain an antibody that could serve as a sensitive tool with which we can measure the endogenous level of an RHT-D1A protein in a tissue where this protein normally operates. Therefore, the final checkpoint for the newly raised polyclonal antibody was to determine whether this antibody specifically detects the endogenous RHT-D1A protein.
Because DELLA protein degradation upon GA engagement by receptors called GID is the main regulatory mechanism in GA signaling, we analyzed the effect of a GA biosynthesis inhibitor, PBZ, on RHT-D1A stability. For this purpose, wheat seeds were germinated with water or 10 μM GA in the presence or absence of 100 μM PBZ. As shown in Fig. 7, inhibition of GA synthesis completely blocked α-amylase production and  activity, indicating that the inhibitor successfully repressed the synthesis of gibberellins. Furthermore, the finding that PBZ blocked the α-amylase response in 4-day-old endosperm of wheat seedlings treated with water but did not inhibit the α-amylase production in the presence of GA meant that the observed effects of these inhibitors on GA responses are not attributable to nonspecific effects or the poisoning of cellular metabolism. The anti-RHT-D1A antibody recognized 70-and 55-kDa proteins in the PBZ-treated seedlings. The 70-kDa protein is close to the predicted molecular mass of the endogenous RHT-D1A protein (65.3 kDa). We found that the 70-kDa protein band was not detectable in GAtreated wheat seedlings, confirming the high affinity of the polyclonal anti-RHT-D1A antibody. Most likely, the 55-kDa protein is unrelated to RHT-D1A because this protein was present in the GA-treated seedlings too. Consequently, our data clearly demonstrate that RHT-D1A disappears in response to exogenous and endogenous GA, as previously described for barley SLN1 [23] and rice SLR1 [24] proteins.

Discussion
DELLA proteins play an essential part in the regulation of plant growth and adaptation to unfavorable environmental conditions [8]. In wheat, the majority of DELLAregulated processes affect the seed yield and quality [25,26]; accordingly, understanding the molecular basis of DELLA function holds great promise for improvement of the crop yield. To date, most studies in this field have dealt with a phenotypic effect of dwarf mutants of the Rht gene on yield-related traits, and researchers have not expressed the DELLA proteins of Triticum species. Furthermore, to further reveal the functions of the wheat RHT protein, it is necessary to have a highly sensitive and specific antibody against RHT. Accordingly, in this study, we prepared and characterized an anti-RHT-D1A polyclonal antibody.
In an initial experiment, with primer combinations specific for the tall and dwarfing alleles, we showed that the Saratovskaya 29 wheat variety does not carry mutant alleles Rht-B1b and Rht-D1b. Subsequently, we synthesized cDNA encoding the putative wheat RHT-D1 protein, and sequencing of the synthesized cDNA confirmed that the chosen wheat variety does not carry the mutant Rht-D1 allele.
Previously, N-terminal domains of DELLA proteins from both Arabidopsis and Malus domestica have been isolated and expressed in E. coli. To ensure strong immune responses to small antigens such as the Nterminal domain of DELLA proteins, it is essential that these antigens be expressed as soluble proteins [27]. Of note, in that study, all the N-terminal domains of DELLA proteins were expressed at adequate levels of soluble proteins when MBP was employed as the protein fusion partner [27]. In our study, the most soluble fulllength 6xHis-tagged rRHT-D1A was obtained by protein production in E. coli ArcticExpress (DE3) competent cells as a host. Soluble recombinant 6xHis-RHT-D1A was easily purified and was applied as a potent immunogen for the production of the anti-RHT-D1A polyclonal antibody.
The usual dose of a soluble protein administered with Freund's adjuvant to rabbits is in the range of 50 to 1000 μg, and for mice, it is 10-200 μg; for goats or sheep, the typical dose is 250-5000 μg [28]. Nevertheless, for primary injection, most investigators use doses 100-200 μg [29,30] or even less than 25-50 μg [31,32]. It should be stressed that in some studies concerning the polyclonal antibody production in rabbits, for primary immunization, researchers have used doses of 400 to 500 μg [33,34] even up to 1.0 mg [35,36]. These observations point to the dependence of the antigen concentration required for primary immunization on immunogenic potency of the antigen. In our study, for the primary immunization,~1 mg of the protein in Freund's complete adjuvant was inoculated subcutaneously. The resultant polyclonal antibody was precipitated with 50% ammonium sulfate and further purified by affinity chromatography on protein A-agarose. Our indirect ELISA showed that the purified polyclonal antibody has good sensitivity to the rRHT-D1A protein (Fig. 4).
It is known that the specificity of an antibody is in part dependent on the type of an immunogen: a peptide or purified protein. Nonetheless, an antibody raised against a peptide may not work well when the respective protein in an immunoblot is in its native conformation. Such antibodies may not be useful for IP experiments but may bind the protein of interest after it is fully denatured. The opposite may also be true, especially if the immunogen was a purified protein, namely, an antibody works well for the protein in its native conformation but not when denatured [37].
Our anti-RHT-D1A polyclonal antibody successfully recognized the nondenatured protein in dot blot and IP analyses. Furthermore, the anti-RHT-D1A polyclonal antibody yielded strong signals by recognizing denatured RHT-D1A in western blotting. Here, this antibody was found to be rather specific to RHT-D1A because in dot and western blotting, the antibody did not react with the control recombinant TaAPE1L protein that was expressed and tagged in the same way as the RHT-D1A protein was [21] (Fig. 2).
In 4-day-old endosperm of wheat seedling extracts, the endogenous RHT-D1A protein was detected as a single band of the expected molecular mass, but the anti-RHT-D1A polyclonal antibody also reacted with another protein with molecular mass 55 kDa (Fig. 7). The 70-kDa protein degraded after treatment of plants with GA, as previously reported for barley (SLN1) and rice (SLR1) DELLA proteins [23,24]. Our antibody recognized epitope(s) on the 55-kDa protein in all treatment groups, and the protein in question was not degraded in the presence of GA. Of note, in rice, two sequences have been identified that are homologous to SLR1: SLR1-like 1 and SLR1-like 2 (SLRL1 and SLRL-2). SLRL1 and SLRL2 contain regions with high similarity to the Cterminal conserved GRAS domains of SLR1 but lack the N-terminal conserved region of the DELLA proteins. DELLA proteins are members of the GRAS family of transcriptional regulators [38] containing two distinct domains: an N-terminal regulatory domain and a Cterminal functional GRAS domain. The N-terminal domain is required for binding the GID1-GA receptor complex, a process that ultimately triggers DELLA degradation and promotes GA-responsive growth. Furthermore, in contrast to DELLA proteins, SLRL1 is not degraded upon GA treatment, and SLRL1 overexpression is not responsive to GA [39]. Moreover, no sequences have been found in the Arabidopsis genome that are homologous to SLRL1 and SLRL2. Given that maize contains a sequence highly similar to SLRLs, it has been suggested that SLRL-type GRAS proteins exist in monocots but not dicots [39]. Although we do not know the identity of the 55-kDa protein, we can speculate that it may be a wheat protein homologous to SLRL1 and SLRL2. Further studies are needed to identify the 55-kDa protein. On the other hand, we cannot rule out the presence in RHT-D1A of a specific proteolytic cleavage site that can generate a truncated form of RHT-D1A. The biological activity of many proteins, including transcription factors, is regulated by posttranslational modifications involving a controlled proteolytic activity. Proteolysis can remove segments of a protein to enable or prevent its biological function and in addition may cause changes of subcellular localization by removing localization sequences [40].
Although bacterially expressed rRHT-D1A should not have post-translational modifications and an optimal folding environment present in plants, this situation did not decrease the immunogenicity of this recombinant protein in rabbits. Our results indicate that the use of the rRHT-D1A antigen generated in the bacterial expression system did not lead to substantial alterations in the antigen-antibody recognition reaction for RHT-D1A. The purified polyclonal antibody raised against the RHT-D1A protein is sensitive and useful for research on DELLA-regulatory mechanisms in wheat.