Journal of Experimental Botany, Vol. 52, No. 365, pp. 2283-2289,
December 1, 2001
© 2001 Oxford University Press
Original Papers |
A class I chitinase from soybean seed coat
1 Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, Canada N5V 4T3
2 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
Received 1 May 2001; Accepted 2 July 2001
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Abstract |
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Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS–PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.
Key words: Gene, glycosyl hydrolase, pathogenesis-related protein, Phytophthora sojae, testa.
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Introduction |
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Chitinases are enzymes that catalyse the hydrolysis of ß-1,4-N-acetylglucosamine linkages present in chitin. As chitin is a major component of fungal cell walls, and is absent in plants, chitinases play a role in plant defence against pathogens. Supportive evidence for the defensive role of chitinases includes chitinase inhibition of fungal growth in vitro (Schlumbaum et al., 1986
), enhanced resistance to pathogens in plants that constitutively express high levels of chitinase (Broglie et al., 1991), and visualization of in vivo chitin breakdown (Benhamou et al., 1993). Chitinase induction is often co-ordinated with the expression of specific ß-1,3-glucanases and other PR proteins in response to pathogen attack, as well as in response to treatment with elicitors and abiotic factors. Additional non-defensive roles of chitinases in nodule development (Goormachtig et al., 1998), and in host specificity of rhizobia (Staehelin et al., 1994) have been proposed. Chitinase genes are differentially regulated in response to development or by colonization of plant tissues by microorganisms (Salzer et al., 2000).
Plant chitinases are classified into six groups based on their primary structure (Neuhaus, 1999). Classes I and IV are characterized by the presence of an N-terminal, cysteine-rich, chitin-binding domain that is also found in proteins such as hevein and in non-leguminous plant lectins. Class II chitinases lack the chitin-binding domain but are otherwise similar to class I chitinases. Class III and class V are more distantly related. Class I plant chitinases and other proteins containing the hevein domain have been identified as major food and contact allergens and are implicated in latex-fruit syndrome (Breiteneder and Ebner, 2000). Allergenicity to banana, avocado, chestnut, and latex products commonly results from sensitization to class I chitinases and proteins bearing hevein-like domains.
In this study, it is shown that a class I chitinase is an abundant protein in soluble extracts from soybean seed coat tissues. The 32 kDa protein was catalytically active and could be purified in one step by affinity chromatography on chitin beads. Isolation and sequence analysis of the cDNA and genomic copies corresponding to this seed coat chitinase revealed a class I chitinase precursor protein of 320 amino acids encoded within three exons. Expression of this chitinase gene was associated with senescence, ripening, and response to pathogen infection.
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Materials and methods |
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Protein fractionation and analysis
Processed soybean (Glycine max [L.] Merr) seed hulls were obtained from ADM Agri-Industries, Windsor, ON. For extraction of proteins, 10 g of processed seed hulls were added to 0.2 l 10% (v/v) ethanol and gently agitated for 16 h at 4 °C. This solution was filtered (Miracloth, Calbiochem, La Jolla, CA) and the filtrate clarified by centrifugation (10000 g, 20 min). Carrier ampholytes (pH range 3.5–10, Amersham Pharmacia Biotec Inc, Baie d'Urfé, Quebec, Canada) were added to a final concentration of 2% (v/v) prior to IEF (Rotofor apparatus, BioRad, Hercules, CA). Fractions were analysed by SDS–PAGE using 12% acrylamide gels and proteins visualized by silver staining (Blum et al., 1987
). For N-terminal microsequencing, IEF fractions containing the target protein were concentrated by ultrafiltration (Centricon, Millipore, Bedford, MA) before electrophoresis and transfer to PVDF membrane (following the method of Moos et al., 1988).
Binding assays and chitinase activity measurements
Soybean seeds were from the collection at Agriculture and Agri-Food Canada. Seeds of cultivar Harosoy 63 were briefly soaked in water and seed coat tissues dissected and frozen in liquid N2. Samples of 1 g tissue were ground using a mortar and pestle in 3 ml extraction buffer (0.1 M NaHPO4, pH 6.0). Insoluble material was removed by centrifugation (10000 g, 10 min), and the supernatant was used directly for chitin-binding or catalytic assays.
For chitin-binding assays, the protein suspension was added to 100 mg chitin beads (New England Biolabs) in 15 ml plastic tubes. After gentle agitation for 1 h at 4 °C, the beads were pelleted (5000 g, 5 min), and washed extensively with extraction buffer. The chitin beads were resuspended in SDS–PAGE loading buffer (Sambrook et al., 1989) and boiled for 5 min to release any bound proteins. This eluate was directly analysed by SDS–PAGE, along with a sample of the crude protein extract. For chitinase activity assays, samples were analysed on 12% SDS-polyacrylamide gels containing 0.01% (w/v) glycol chitin (according to the method of Trudel and Asselin, 1989). After electrophoresis, gels were washed with 1% (v/v) Triton X-100 in 100 mM sodium acetate buffer, pH 5.0 at 37 °C. Gels were visualized under UV light following staining with Caclofluor White M2R. Lytic zones were identified as non-fluorescent dark bands.
Library screening and DNA sequencing
Methods for the construction and screening of soybean cv. Harosoy 63 cDNA and genomic 
libraries have been described (Gijzen, 1997). Positive clones were plaque purified and subcloned into a plasmid vector (pBluescript, Strategene, La Jolla, CA) for sequence analysis. Automated sequencing of DNA was accomplished using dye-labelled terminators and fragments separated in acrylamide gels (model 377, Applied Biosystems, Foster City, CA). Plasmid inserts containing cDNA clones were sequenced on both strands by primer walking. An 11.3 kb EcoRI genomic clone was shotgun sequenced by random transposon insertion (GPS-1, New England Biolabs, Beverly, MA), and gaps were closed by primer walking. The finished 11368 bp of sequence was assembled from 171 sequencing runs representing 119519 bp of raw data, resulting in an average 10-fold coverage (Seqman, Lasergene, DNAStar, Inc., Madison, WI).
RNA and DNA blot analysis
Total RNA was purified from soybean tissues using a modified phenol–chloroform method (Wang and Vodkin, 1994). Samples of 10 µg of RNA were electrophoretically separated on 1.0% formaldehyde gels and transferred to nylon membranes according to standard procedures (Sambrook et al., 1989). Soybean genomic DNA was isolated according to the method described previously (Dellaporta et al., 1983). Restriction digestion of 30 µg DNA and blotting to membranes followed standard protocols (Sambrook et al., 1989). Filters were preincubated at 65 °C for 4 h in 0.25 M Na2HPO4 (pH 7.2), 1% BSA, 7% SDS, and 1 mM EDTA. The hybridization solution was identical to that used for pre-incubation, except that 2.5 ng ml-1 of 32P-cDNA probe was added. Filters were hybridized for 16 h at 65 °C, then washed four times at 68 °C and twice at 22 °C in 0.02 M Na2HPO4 (pH 7.2), 1% SDS, and 1 mM EDTA, 20 min per wash.
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Results |
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Purification and analysis of seed hull proteins identifies new constituents
To determine the composition of proteins present in seed hulls, concentrated protein extracts were separated by IEF and SDS–PAGE, and the resulting gels were silver stained. A representative separation is shown in Fig. 1
. The results show that seed hulls contain a mixture of soluble proteins that vary in size, charge, and relative abundance. Of the five proteins indicated in Fig. 1A, proteins I, II, and III were identified based upon their size, isoelectric point, comparison with known standards, published reports, and previous microsequencing analyses that had been undertaken. These proteins correspond to the seed coat peroxidase, the Kunitz trypsin inhibitor, and the hydrophobic protein/Gly m 1 allergen, as shown in Table 1. Complete amino acid and cDNA sequences have been described for these three proteins. Proteins IV and V could not be identified and were subject to further purification and N-terminal microsequencing analysis. Peptide sequences were then compared to known and putative protein sequences in public databases. Sequence data for protein IV consisted of a stretch of 18 amino acids that did not match any known soybean protein, but had high similarity to the chitin-binding domain of hevein and class I chitinases, as shown in Fig. 1B. Sequence data for protein V was similar to an N-terminal sequence reported for a peptide released from soybean seeds following immersion in hot water (Hirano et al., 1992), however, the full length sequence and identity of this 24 kDa protein is unknown.
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Databases at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) were also searched for reading frames matching the peptide sequences from protein IV and protein V, to identify cDNA transcripts corresponding to these two proteins. From this analysis, the protein IV peptide sequence exactly matched an EST from a soybean/Phytophthora sojae (Kaufmann and Gerdemann) infection site cDNA library (Qutob et al., 2000
). An EST corresponding to protein V could not be identified, and remains unrepresented in public databases at the time of writing (dbEST release 121500, including 136,958 soybean ESTs).
Soybean seed coat chitinase is a modular protein with distinct domains
The soybean–P. sojae infection site EST matching protein IV was sequenced and used to probe a seed coat cDNA library to identify transcripts expressed in the seed coat. This resulted in the isolation of several seed coat cDNA transcripts identical in sequence to the original soybean/P. sojae infection site cDNA. The 1.2 kb cDNA transcript encoded a preprotein of 320 amino acids that shares features of class I chitinases isolated from other plant species, as shown in Fig. 2A. A signal peptide leader sequence of 23 amino acids followed by a chitin-binding domain, a proline hinge region, and a catalytic domain were all represented in the peptide sequence. The mature protein of 297 amino acids has a calculated molecular mass of 31.9 kDa and does not possess any obvious vacuolar targeting signals or N-glycosylation sites. Two crucial Glu residues required for catalytic activity for this class of glycosyl hydrolases are conserved in the soybean chitinase and correspond to Glu146 and Glu168 of the precursor protein. The soybean chitinase peptide sequence was most similar (74% identity) to a chitinase (GenBank Accession no. X63899) isolated from pea (Chang et al., 1995). The best Arabidopsis match (62% identity) corresponded to a basic chitinase encoded by the T2E22.18 gene on chromosome 3 (Samac et al., 1990; The Arabidopsis Information Resource, http://www.Arabidopsis.org/).
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The chitin-binding and catalytic domains of the protein are functionally active
Chitin-binding and catalytic assays were performed on protein extracts from seed coat tissues dissected from fully mature, desiccated soybeans to test the functional properties of the protein. The chitinase could be highly purified from crude extracts by adsorption to chitin affinity beads, as shown in Fig. 2B
. A single protein band of 32 kDa was detected after elution of chitin-binding proteins. Purified protein IV, from preparative IEF separations shown in Fig. 1, displayed identical chitin-binding properties (not shown). Fig. 2B also shows a gel-based enzyme assay, indicating that seed coat extracts possess a single band of chitinolytic activity corresponding to the 32 kDa chitin-binding protein. In contrast, protein extracts of whole seeds displayed several chitinolytic activity bands (not shown). These results indicate that seed coat tissues predominantly contain a single chitinase isozyme that retains chitin-binding and catalytic activity.
The chitinase gene contains two introns and is flanked by TA repeats
Genomic DNA blot analysis was carried out under high stringency conditions to estimate copy number and to determine size of restriction enzyme fragments containing the chitinase gene. These results are shown in Fig. 3. Using full length cDNA as a probe, a hybridization pattern consistent with that of a single or low copy gene was observed. Soybean genomic libraries were then screened by hybridization with the cDNA clone to isolate the corresponding gene. This resulted in the isolation of an 11.3 kb EcoRI fragment containing the chitinase gene, as shown in Fig. 4. Sequence analysis of this genomic clone showed that the chitinase gene is encoded within a 2.4 kb stretch that includes two introns. The gene is flanked by A+T rich-tracts consisting of (TA)n repeats of 11–41 bp in length.
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The entire 11368 bp of sequence was searched for matches to other genes, proteins, and ESTs within public databases, and for predicted genes by genscan (Burge and Karlin, 1997
). Many ESTs and proteins from a variety of plant species produced significant hits matching the soybean chitinase (the following GenBank accession numbers are shown in parentheses). In addition to the original EST from P. sojae-infected soybean tissues (BE584126), four other soybean ESTs matched the chitinase sequence. Sequences from these ESTs were 98–100% identical to the seed coat chitinase cDNA transcript and probably correspond to the same gene. These four soybean ESTs originated from cDNA libraries constructed from cotyledons (AW200675), root tips (AW620882), whole seedlings (BE611523), and root nodules (AI973935). A second gene predicted by genscan occurred downstream from the chitinase gene and in the opposite orientation. This predicted gene encoded a 35 kDa hypothetical protein similar (E value of 10-37) to that encoded by Arabidopsis gene F8D20.230 on chromosome 4.
The chitinase is highly expressed in maturing seed coat tissues
Tissue and developmental expression patterns of the chitinase gene were examined by RNA gel blot analysis. Figure 5 shows that the greatest level of expression was observed in the seed coat during the later stages of seed ripening and maturation. Strong hybridization signals were also observed for RNA samples isolated from senescing pod, root, and leaf tissues. Low levels of transcript were discernible in embryo and stem tissues from late stages of development. The gene is also induced in response to pathogen infection, and could be detected in hypocotyl tissues 48 h after inoculation with a virulent strain of P. sojae.
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Discussion |
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Soybean seed coats are comprised of several cell layers that arise from the inner and outer integument of the ovule. Many different cell types are represented in the mature seed coat, including the epidermal palisade cells (macrosclerids), the hourglass cells (osteosclerids), the tracheid cells of the vascular region, and various forms of intact and crushed parenchyma cells (Miller et al., 1999
). The seed may also bear an extraneous layer of material derived from cells of the inner epidermis of the pod endocarp (Gijzen et al., 1999b). Thus, the overall composition of the seed coat is influenced by a variety of highly differentiated cells that encapsulate and protect the embryo until suitable conditions for germination occur, whereupon the seed coat is discarded. In the present study, proteins derived from extracts of seed hulls have been separated and an abundant 32 kDa chitinase enzyme identified by N-terminal sequence analysis. Other abundant proteins that have been previously characterized were also identified, including seed coat peroxidase enzyme, soybean trypsin inhibitor, and hydrophobic protein/Gly m 1 allergen. Purification and microsequence analysis of a basic 24 kDa protein from the seed hull extracts showed that this protein was similar to a peptide released from seeds in hot water, but otherwise remains unknown.
From the peptide sequence data for the 32 kDa protein a corresponding EST originating from a mixed soybean/P. sojae cDNA library has been identified, and identical cDNA transcripts from a seed coat cDNA library have subsequently been isolated. The deduced full length amino acid sequence of the chitinase showed that this protein is translated with a secretory leader peptide that is cleaved during maturation. The soybean seed coat chitinase is somewhat unusual in that it is a basic isozyme that is apparently secreted to the apoplast, since it does not possesses a C-terminal vacuolar targeting signal that occurs in most other class I chitinases. It is not known whether the chitinase protein is associated with a particular cell layer, such as that observed for the seed coat peroxidase enzyme that accumulates in the hourglass cells (Gijzen et al., 1993, 1999a).
Chitinases may be abundant proteins occurring in monocot (Huynh et al., 1992; Swegle et al., 1992) and dicot seeds (Collada et al., 1993). Previous work has shown that soybean seeds contain at least five chitinase isozymes and a gene encoding a class III chitinase from soybean seeds has been described (Wadsworth and Zikakis, 1984; Yeboah et al., 1998). The symbiosis-stimulated induction of chitinase isozymes in soybean roots has also been studied (Xie et al., 1999). Results from an analysis of the seed coat shows that this tissue contains predominantly one isozyme that retains chitin-binding and catalytic activity. Thus, it may be possible to develop soybean seed hulls as a source of chitinase enzyme for medical, commercial or industrial applications.
The most similar protein in Arabidopsis is a basic chitinase that is 62% identical to the soybean seed coat chitinase and is encoded by a single gene on chromosome 3. Like the soybean chitinase gene, the Arabidopsis basic chitinase gene is flanked by (AT)n repeats. These extended (AT)n tracts are susceptible to local denaturation and unwinding, may occur adjacent to highly expressed or induced genes, and may function in association with matrix attachment regions to organize DNA superstructure (Holmes-Davis and Comai, 1998).
Aside from these repetitive (AT)n tracts, DNA regions flanking the soybean and Arabidopsis chitinase genes did not share any obvious similarities or microsynteny. An additional gene encoding a hypothetical protein was predicted to occur within the 11.3 kb fragment encompassing soybean chitinase gene. The equivalent region in Arabidopsis is predicted to encode at least four additional genes, reflecting the higher gene density in this species. The soybean hypothetical protein occurring adjacent to the chitinase shares sequence similarity to several hypothetical proteins predicted from the sequencing of the Arabidopsis genome. However, none of these Arabidopsis genes occur near the basic chitinase. Given the relatively low gene density of soybean, more sequence data are required to assess the regional synteny with Arabidopsis in the vicinity of this gene.
In addition to their role in plant defence, class I chitinases are emerging as a distinct group of panallergens causing cross-sensitization to different foods and materials in susceptible persons (Diaz-Perales et al., 1998; Sanchez-Monge et al., 1999). Sensitization is usually limited to raw or uncooked foods, since IgE-mediated recognition of the chitin-binding domain is lost upon heat denaturation. Several different allergenic proteins have been identified from soybeans including those that cause food and inhalant allergies, but chitinases have not been included among these to date. The finding that a class I chitinase is an abundant component of the soluble protein fraction from seed coats indicates that this protein should be considered as a potential determinant of allergenicity to raw or uncooked soybean products.
In summary, soybean seed coats are particularly rich in defence-related proteins and peptides, although there are abundant proteins that have yet to be identified. Besides the biological role of providing defence and protection of the seed until germination occurs, seed coat tissues affect the overall quality and value of soybean food and feed products. Thus, characterization of seed coat constituents and their corresponding genes is important from a biological, nutritional, and economic standpoint in a widely grown crop species such as soybean.
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Acknowledgments |
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We thank Richard I Buzzell and Vaino Poysa (Agriculture and Agri-Food Canada, Harrow, ON) for soybean seed; ADM Agri-Industries (Windsor, ON) for processed seed hulls; Aldona Gaidauskas-Scott for technical assistance; Jenn McMurray, Sandra Miller, and Ida van Grinsven for DNA sequencing; B Patrick Chapman for data processing; the Biotechnology Service Centre (University of Toronto, ON) for peptide microsequencing; and Krzysztof Szczyglowski for critical reading of the manuscript. This research was supported in part by a grant from the Ontario Soybean Growers (Chatham, ON).
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Notes |
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3 To whom correspondence should be addressed. Fax: +1 519 457 3997. E-mail:gijzenm{at}em.agr.ca
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Abbreviations |
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IEF, isoelectric focusing; PR, pathogenesis-related.
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