Characterization of a cDNA clone encoding wheat CP

A cDNA library was constructed from 1-day germinating wheat seed mRNA. Use of oryzasin β and γ cDNAs as probes gave 38 positive clones from 500 000 plaques. These clones comprised four groups, all of which encoded CPs belonging to the papain family. All the CPs retained the consensus sequence for the catalytic triad composed of cysteine, histidine, and asparagine residues. One of these CPs, named gliadain, was subjected to further analyses. Gliadain was so named as it almost specifically digested gliadin, as discussed below. Gliadain shows 87.8% identity to barley EP-B [44] and 70.6% to rice CP [45]. The putative catalytic triad were found as 162Cys−301His−322Asn, and two potential N-glycosylation sites were detected in gliadain (Fig. 1).

Effects of exogenously added gibberellin on the expression of gliadain mRNA and protein

Northern blot analysis was performed to define the expression stage of gliadain. Although gliadain mRNA was somewhat detectable at the maturation stage, much clearer expression was observed during germination (Fig. 2A). Expression during germination was distinct in seeds but was not detected in shoots or roots. The enzyme concentration does, in fact, increase over time.

We then investigated the effects of the phytohormone, gibberellin, on the expression of gliadain at both the mRNA and protein levels, as its addition is known to induce the expression of hydrolytic enzymes, such as amylase [46] and CPs in germinating seeds [33,47]. Synthesis of gibberellin begins when a seed absorbs water. We examined the time course of changes in gliadain mRNA expression due to the action of gibberellin and its suppression by uniconazole, a specific gibberellin-synthesis inhibitor. Although no significant effect of the inhibitor was observed in the first 12 h after the start of imbibition, distinct suppression of mRNA occurred at 24 h and thereafter (Fig. 2B). The lack of a significant suppressive effect at this initial stage may be due to the presence of a certain amount of gibberellin initially. Inhibition of gliadain mRNA expression also occurred, probably with the same time course, during the stage from 1 to 5 days after the start of imbibition (Fig. 2B). Our observations indicate that the expression of gliadain mRNA is markedly affected by gibberellin.

To confirm these results, we examined the expression of gliadain protein by immunoblotting analysis using anti-gliadain serum. Our results showed that, in the absence of added uniconazole, a cross-reactive band of 30 kDa appeared approximately 1 day after the start of imbibition (Fig. 2C). This band may have been due to mature gliadain molecules with sugar chains, as the sizes of pro- and mature gliadain molecules estimated from their putative primary structures are 38.6 and 25.3 kDa, respectively. Two potential N-glycosylation sites exist in the probable maturation enzyme region. The level of this band of 30 kDa increased much more at 3 days than 1 day after the start of imbibition. No particular increase in band intensity was observed when uniconazole was added. Although gliadain was detected at low levels in the presence of the inhibitor, this may have been due to gliadain synthesis prior to its inhibition by uniconazole. These results are consistent with northern blotting data showing that a large quantity of gliadain mRNA was expressed 2 days after the start of imbibition (Fig. 2B). The use of uniconazole thus indicated that the levels of an antigen-positive protein decreased 1 day after the start of imbibition, with little change in quantity for the next 4 days. In summary, gibberellin-inducible gliadain is expressed distinctly during germination.

Histochemical detection of gliadain occurring in 1-day germinating seeds

Compartmentalization of gliadain expression was dissected by immunostaining with an anti-gliadain serum. Immunopositive staining was detected in the aleurone layer and also in the endosperm region in the vicinity of the embryo (Fig. 3A). Expression of gliadain in the endosperm was limited to the region near the embryo in which protein bodies had already been degraded (Fig. 3B). No significant staining was detected in the regions in which the protein bodies remained, which are somewhat distant from the embryo (Fig. 3A). Expression of gliadain was also detected in the aleurone, with an increase in expression intensity at sites near the embryo. These signals were not detected without anti-gliadain serum (Fig. 3C,D). Significant staining was observed in the embryo and epidermis, but it is not clear whether this signal is due to gliadain itself or some artificial high background induced by the secondary antibody used, because the same was also observed without anti-gliadain serum. The observed results strongly suggest that gliadain is involved in the proteolysis of storage proteins.

Biochemical properties of recombinantly produced gliadain

Immunostaining with anti-gliadain serum suggested that gliadain was involved in the degradation of storage proteins. To obtain a more detailed insight into the biochemical properties of gliadain in vitro, we prepared recombinant gliadain. For this, a GST–progliadain expression plasmid was constructed and introduced into Escherichia coli. The expected fusion protein was produced as an insoluble inclusion body. The product was then purified according to the method of Matsumoto et al. [48]. The molecular mass of GST–progliadain is 60 kDa (Fig. 4A).

To identify the endogenous substrate of gliadain, we reacted GST–progliadain with gliadin and glutenin, both of which are the prolamins according to the Osborne classification. Gliadin is comprised of five elements, high molecular mass (HMM)-gliadin species of 104 000–125 000 Da, α-, β-, and γ-gliadin that migrate together on SDS/PAGE, and ω-gliadin as a minor component. In this experiment, ω-gliadin and HMM-gliadin were not extracted, and α-, β-, and γ-gliadin of 25 000–30 000 Da, were examined. Reacting GST–progliadain, we found that α-, β-, and γ-gliadin were degraded into smaller fractions, while the enzyme had no appreciable effect on HMM-glutenin or low molecular mass (LMM)-glutenin (Fig. 4B). These results suggested that gliadain digests gliadin specifically, and so we named this proteinase gliadain. To confirm the substrate specificity of gliadain, we attempted to react GST–progliadain with rice protein composed of glutelin, globulin, and prolamin. We extracted rice protein bodies from seeds, and then incubated them with GST–progliadain; none of these proteins was digested by GST-progliadain (data not shown). Taken together, these results indicated that gliadain digests only wheat seed gliadin.

Next, we investigated the substrate specificity using synthetic peptides and found that GST–progliadain hydrolyzed benzyloxycarbonyl benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH₂-Mec) much better than Z-Arg-Arg-NH₂-Mec and Arg-NH₂-Mec. The K_m value for the hydrolysis of Z-Phe-Arg-NH₂-Mec was 9.5 µm, which was comparable with the case when the same substrate was treated with cathepsin L. In addition, GST–progliadain was most active at pH 4–6, as in cathepsin L. These observations, together with the amino acid sequence similarity (data not shown), suggest that gliadain belongs to the cathepsin L family.

Inhibitory activity of cystatins against gliadain

Information is available on the inhibition of CPs by cystatins. As expected, GST–progliadain was inhibited by E-64 and egg white cystatin at IC₅₀ concentrations in the order of 10⁻⁸ m, as well as by the two wheat cystatins, WC1 and WC2, at IC₅₀ concentrations in the order of 10⁻⁸ m(Table 1).

Wheat seeds

Seeds of T. aestivum L., cultivar Norin 61, were harvested on the Tama Experimental Farm at the University of Tokyo, Japan.

Construction of a cDNA library and isolation of wheat CP cDNA clones

Total RNA was extracted from 1-day germinating wheat seeds using the phenol–SDS method, and mRNA was purified using an oligo(dT) cellulose column. Double-stranded cDNA was constructed with a Time Saver cDNA synthesis kit (GE Healthcare, Chalfont St Giles, UK). The cDNA was ligated into a λgt10 (EcoRI/CIAP) phage vector (Stratagene, La Jolla, CA). After in vitro packaging, phages were grown on E. coli C600Hfl. Recombinant plaques were transferred onto nylon membranes (Hybond N, GE Healthcare) and hybridized at 55 °C for 24 h with a mixture of oryzain α and γ cDNA fragments labeled with [³²P]CTP[αP] as a probe for screening. Membranes were washed at 55 °C in 2× NaCl/Cit containing 0.1% SDS and exposed to X-ray film.

Germination of wheat seeds and treatment with plant hormones

Wheat seeds were soaked in 5% hypochlorous acid containing 0.02% Triton X-100 and then washed in water. Seeds were placed on a fully moistened vermiculite sheet and germinated at 26 °C in the dark. Germinating seeds were treated with the following plant hormones at various concentrations in the medium: gibberellin A₃ at 3.1 mm; and abscisic acid at 1 mm; and uniconazole as a gibberellin synthesis inhibitor at 30 mg·L⁻¹.

Northern blotting analysis

Total RNA was extracted from mature, immature, and germinated seeds using the phenol–SDS method. Shoots and roots were taken from germinated seeds and then total RNA fractions were also extracted. Aliquots of 10 µg of the RNA were electrophoresed on 1% agarose gels and blotted onto nylon membranes. Hybridization was performed at 42 °C with [³²P]CTP[αP]-labeled gliadain cDNA as a probe.

Preparation of anti-gliadain serum

Two primers, 5′-TCCGGATCCGGACAATGACCTGGAG-3′ (P1) and 5′-AAAGAATTCAGACACGCATA-3′ (P2), were synthesized. P1 corresponding to the N-terminal region of gliadain has a BamHI site at the 5′-end (underlined). P2, corresponding to the C-terminal region has an EcoRI site at the 5′-end (underlined). PCR was performed using these two primers and gliadain cDNA as a template. The amplified fragments were digested with both BamHI and EcoRI prior to insertion into pUC19. The resultant gliadain expression plasmid was introduced into E. coli strain YA21. The transformant was grown for 24 h at 37 °C, and cultured for a further 2 h after addition of 1 mm isopropyl thio-β-d-galactoside. Cells were collected by centrifugation, lyzed by ultrasonication, and subjected to SDS/PAGE. A band with the expected molecular mass of gliadain was cut from the gel and used to immunize a rabbit. An anti-gliadain serum was purified from the serum according to the method of Matsumoto et al. [48]. Animals were treated in accordance with criteria established by the Animal Care and Use Committee at the University of Tokyo.

Western blotting analysis

Germinated wheat seeds were harvested at 1, 3, and 5 days after imbibition, washed in water, and stored at −80 °C. Frozen samples were crushed in solid carbon dioxide and suspended in buffer A (50 mm phosphate buffer, pH 6.6, containing 150 mm NaCl, 5 mm EDTA, and 0.1 mm E-64). The suspension was centrifuged at 3000 g and the precipitate was discarded. Ammonium sulfate was dissolved at a concentration of 80% in the supernatant and the solution was centrifuged at 10 000 g for 10 min. The precipitate was collected and dialyzed against buffer A. The resulting diffusible fraction was concentrated and electrophoresed. Samples were electroblotted onto polyvinylidene fluoride membranes, immunoblotted with anti-gliadain serum diluted 1000-fold, and then reacted with horseradish peroxidase-conjugated anti-(rabbit IgG) for visualization with ECL Plus (GE Healthcare).

Immunohistochemical analysis

Wheat seeds germinating for 1 day were stored at −80 °C. Frozen seeds were each sliced at a thickness of 3 mm and fixed in 10% formaldehyde. Each slice was washed three times with 50 mm phosphate buffer (pH 7.3), embedded in O.C.T. compound, embedding medium for frozen tissue specimens reagent, and further sectioned at a thickness of 4 µm. The sections were soaked in 0.3% H₂O₂/methanol for 30 min, blocked in 5% normal goat serum, and reacted with 1 : 1000-fold diluted anti-gliadain serum at room temperature for 30 min. Visualization was performed with streptavidin and biotinylated horseradish peroxidase.

Expression of GST–progliadain

The expression plasmid for gliadain was digested with BamHI and EcoRI, and inserted into the pGEX-3X expression vector (GE Healthcare). The resultant plasmid, pGEX–progliadain, was introduced into the ad 202 E. coli strain. The transformed cells were cultured in LB medium containing 100 mg·mL⁻¹ ampicillin until the medium reached an absorbance of 0.6 at 600 nm. Then, isopropyl thio-β-d-galactoside was added at a final concentration of 1 mm and the resultant mixture was cultured for a further 2 h. Cells were collected by centrifugation, washed once with 20 mm Tris/HCl (pH 8.0) containing 5 mm EDTA, and resuspended in the same buffer containing 1 mm phenylmethanesulfonyl fluoride, 5 mmβ-mercaptoethanol, and 1%N-lauroylsarcosyl. After sonication, a 20% volume of 10% Triton X-100 was added, mixed with a vortex at 4 °C, and centrifuged at 18 000 g for 20 min. The supernatant was diluted with a mixture of 140 mm NaCl, 2.7 mm KCl, 10 mm Na₂HPO₄, and 1.8 mm KH₂PO₄ (NaCl/P_i), and then loaded onto a glutathione–Sepharose 4B column (GE Healthcare), and washed with NaCl/P_i. The GST fusion protein was eluted with 50 mm Tris/HCl (pH 8.0) containing 10 mm glutathione and confirmed for purity by SDS/PAGE.

Assay of proteinase activity and inhibition of GST–progliadain by cystatins

To detect the proteinase activity of affinity-purified GST–progliadain, 5 µL of 1 mm Z-Phe-Arg-NH₂-Mec and 250 µL of 200 mm Mes-NaOH (pH 6.2) were added to 500-µL aliquots of the reaction mixture. The hydrolytic activities toward Z-Arg-Arg-NH₂-Mec and Arg-NH₂-Mec were measured at various pH values. The reaction was performed for 10 min at 37 °C and stopped by adding 500 µL of ethanol/HCl (473 : 27, v/v). Fluorometry was conducted at excitation and emission wavelengths of 370 and 461 nm, respectively. Each reaction mixture contained 1.0 × 10⁻⁵ m Z-Phe-Arg-NH₂-Mec and 6.0 × 10⁻⁷ m GST–progliadain in 100 mm Mes-NaOH buffer (pH 6.2). Florescence intensity was measured in the presence of each inhibitor at 10 nm to 10 µm tο obtain the IC₅₀.

Expression and purification of wheat seed cystatins

Two wheat cystatins, WC1 and WC4, were expressed in E. coli and purified by DE52, MonoQ and Superose 12 column chromatography according to the methods of Kuroda et al.[40]. The homogeneity of these proteins was also checked by SDS/PAGE.

Extraction of wheat storage proteins and digestion of them by GST–progliadain

Wheat gliadin and glutenin were extracted primarily by the method of Osborne [52] with some modifications. Wheat flour was purchased from Nisshin Flour Milling Corporation (Tokyo, Japan) and proteins were extracted with 0.1 m Tris/HCl (pH 8.6) containing 4 m urea. The extract was dialyzed against water and then lyophilized. The lyophilized sample was dissolved in distilled water and the soluble fraction was discarded. The remaining fraction was washed with water, and dissolved in 0.15 m NaCl to extract the soluble fraction. The residue was washed twice with water and once with 70% ethanol. Gliadin was obtained by extracting the residue with 70% ethanol. The resulting residue was used as glutenin. Gliadin and glutenin were lyophilized and suspended in 100 mm sodium acetate buffer (pH 4.5) containing 1.0 mm EDTA, 2.5 mmβ-mercaptoethanol at a concentration of 1.0% (w/v). GST–progliadain (4.01 × 10⁻³ units) was added to 100 µL of each protein suspension prior to incubation for 24 h at 37 °C. One unit of proteolytic activity of gliadain is defined as that which liberated 1 µmol of NH₂-Mec from Z-Phe-Arg-NH₂-Mec in 1 min under the conditions described above.