Plant Cell Reports (1999) 18: 601–606
© Springer-Verlag 1999
R. Rompf · G. Kahl
An elicitor-induced cDNA from aerial yam (Dioscorea bulbifera L.) encodes a pathogenesis-related type 4 protein
Received: December 1997 / Revision received: June 1998 / Accepted: November 1998
Abstract We established Dioscorea bulbifera (aerial yam) cell suspension cultures to study the expression of defense-related genes upon elicitation with the yam pathogenic ascomycete Colletotrichum gloeosporioides. The induction of phenylalanine-ammonia lyase (PAL) mRNA, coding for a key enzyme involved in phytoalexin biosynthesis, was observed upon elicitation. Using RT-PCR, we isolated an elicited cDNA clone with an open reading frame of 453 nucleotides which showed high homology to cDNA sequences of pathogenesis-related proteins belonging to the PRP-4 group. Yam PRP-4 expression is increased in elicited cell cultures as well as in elicited leaves, and is encoded by a small multigene family. This is the first example for the cloning of a cDNA that might be involved in defense reactions of yam plants. Key words Dioscorea bulbifera · Cell suspension cultures · Elicitor-responsive genes · PRP-4 Abbreviations BAP 6-Benzylaminopurine · LS medium Linsmaier and Skoog medium · NAA 1-Naphthaleneacetic acid · PAL Phenylalanine ammonia-lyase · PRP Pathogenesis-related protein · SA salycylic acid
Introduction
Yams (vegetatively propagated tuber and bulbil crops belonging to the genus Dioscorea) are an economically imCommunicated by H. Lörz R. Rompf · G. Kahl (½) Plant Molecular Biology, Biocentre, Johann Wolfgang Goethe-University, Marie-Curie-Strasse 9, D-60439 Frankfurt/Main, Germany Fax: +49-69-79829268 e-mail:
[email protected] The nucleotide sequence data reported will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession number AF013995
portant starchy staple food in tropical and subtropical regions of the world, especially for smallholders. However, yield losses caused by various pathogens are a serious problem in yam cultivation. Fungal diseases alone account for annual losses of 30–40% in Nigeria (Ikotun 1989) or even 100% in other regions (Kahl et al. 1991). The ascomycete Colletotrichum gloeosporioides is one of the major fungal pathogens causing necrotic symptoms (“anthracnosis”) on yam leaves that severely reduce photosynthesis and finally lead to the collapse of the leaves. Our objective is the characterization of yam genes induced during and after infection with C. gloeosporioides. Following pathogen attack, many plants activate defense-related genes coding for enzymes involved in phenylpropanoid metabolism, e.g., phenylalanine-ammonia lyase (PAL) (Dixon and Paiva 1995), or hydrolytic enzymes like chitinases and β-1,3 glucanases (Kombrink et al. 1988). Hydroxyproline-rich glycoproteins (Corbin et al. 1987), protease inhibitors (Ryan 1990) and various other proteins are induced as well (Bowles 1990). Polypeptides which are not present in healthy plants, but are synthesized only in response to pathological or related stress situations are coined “pathogenesis-related” proteins (PRPs). However, occasionally their tissue-specific or developmentally regulated expression can be observed (Cutt and Klessig 1992). Apart from pathogen infection, plant defense responses can also be triggered by elicitors, e.g., fungal cell wall compounds, plant-cell-wall-derived molecules, and a number of compounds including peptides, jasmonic acid, and salicylic acid (SA) (Benhamou 1996). The defense reactions in fungus-infected leaves resemble those in elicitor-treated cell cultures, as extensively shown in parsley (Hahlbrock et al. 1995). Despite the economic importance of yam and the detrimental effect of fungal infections on yam yield, almost nothing is known about the molecular interactions of yam and its major fungal pathogen, C. gloeosporioides. Therefore, we established Dioscorea bulbifera cell suspension cultures, and used them as well as leaves to study the induction of defense-related genes upon treatment with a cell wall preparation of C. gloeosporioides.
602
Materials and methods Initiation and maintenance of cell suspension cultures Bulbils from in-vitro-grown D. bulbifera plantlets were harvested under sterile conditions, cut into halves, and used as explants on LS medium (Linsmaier and Skoog 1965) with 2 mg/l 1-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzylaminopurine (BAP), solidified with 0.15% (wt/vol) Gelrite. Rapidly growing callus was subcultured on medium of the same composition every 3 weeks and kept at 25 °C in the dark. Cell suspension cultures were obtained by transferring callus into liquid LS medium with the above phytohormone composition. Cultures were shaken at 100 rpm and cultured at 25 °C in the dark. Subcultivation was performed every 10 days. The cells were sedimented, the old culture medium removed and the cells washed twice with liquid medium. Finally, the cells were resuspended in 2 vol of liquid medium and a pipette with a wide tip was used to transfer the cells into fresh sterile Erlenmeyer flasks.
Elicitation of leaves and cell cultures A crude preparation of C. gloeosporioides cell wall fragments was used for elicitation. C. gloeosporioides was grown in liquid medium (potato dextrose broth) for 3 weeks, mycelium harvested by centrifugation (5000 g, 20 min) and homogenized using an Ultra-Turraxtype homogenizer. The fungal material was washed twice with distilled water, autoclaved at 121 °C for 15 min, sedimented by centrifugation (10 000 g, 60 min) and used for elicitation without further purification. Leaves were spread with this cell wall preparation and subsequently wounded using sterile needles. Addition of 0.2 mg/ml elicitor to liquid cell cultures was performed 5 days after subcultivation. RNA isolation and Northern blot hybridization Total leaf RNA was isolated as described in Rompf and Kahl (1997). For RNA isolation from cell cultures, the material was harvested by vacuum-filtration and immediately frozen in liquid nitrogen. The cells (1.5 g) were ground to a fine powder in liquid nitrogen, then 12 ml isolation buffer [0.1 M NaCl, 10 mM Tris-HCl, pH 7.2, 1 mM EDTA, 1.0% (wt/vol) SDS] and 1 vol phenol/chloroform/isoamylalcohol (25/24/1) were added and vortex mixed for 5 min. Phases were separated by centrifugation (10 000 g, 15 min), and 0.1 vol NaAc (3 M, pH 5,2) and 2 vol EtOH added to the aqueous phase to precipitate nucleic acids for 30 min at –80 °C. After centrifugation (10 000 g, 20 min), the supernatant was removed and the pellet dissolved in double-distilled H2O. One volume of 6 M LiCl was added, and RNA precipitated for 1 h on ice. The RNA was pelleted by centrifugation (10 000 g, 20 min), washed with 70% EtOH and dissolved in 200 µl double-distilled H2O. For the preparation of Northern blots, total RNA was fractionated in a formaldehyde denaturing gel and blotted onto nylon membranes using alkaline transfer (Löw and Rausch 1994). The blots were hybridized to randomly primed (α32 P)-dCTP labelled PAL, yam PRP-4, or rDNA probes. Hybridization was performed overnight at 42 °C in 50% formamide, 6% SDS, 1 mM EDTA, 250 mM NaCl, and 250 mM sodium phosphate buffer (pH 7.2). Blots were washed 3 × 20 min in 1 ×SSC at room temperature, 1 ×30 min in 0.2 × SSC at 60 °C, and then exposed at –80 °C to Hyperfilm MP (Amersham) in the presence of an intensifying screen. Southern blot analysis of genomic DNA DNA was isolated from leaves according to Ramser et al. (1996). The genomic DNA was digested with several restriction enzymes. Samples of 7 µg DNA were electrophoresed in a 1% agarose gel, blotted onto nylon membrane (Sambrook et al. 1989) which was hybridized with a radioactively labelled yam PRP-4 cDNA probe, and washed as described for Northern blots.
Amplification of a cDNA fragment by RT-PCR Highly degenerate PAL consensus primers PAL-1 (5′-GCI GCI GCI ATH ATG GAR-3′) and PAL-2 (5′-AC RTC YTG RTT RTG YTG YTC-3′) were synthesized (Howles et al. 1994). cDNA was prepared from DNAse-treated total RNA using oligo(dT)15-primers and Superscript reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. In a total volume of 30 µl, the RT-PCR contained 100 ng cDNA as template, 1.5 µM of each primer, 200 µM of each dNTP, 1.5 mM MgCl2, 20 mM Tris-HCl (pH 8,4), 50 mM KCl and 1 U Taq DNA-Polymerase (Eurogentec). After an initial denaturation step for 30 s at 94 °C, amplification was performed for 30 cycles (94 °C for 30 s, 55 °C for 60 s, 72 °C for 2 min), and an extension period at 72 °C for 3 min. Cloning and sequence analysis of the RT-PCR product The RT-PCR product was cloned into the pGEM-T Vector System II (Promega), and sequenced on a Model 373 sequencer using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) with AmpliTaq DNA Polymerase (Roche Molecular Systems). A sequence homology search was performed using GenBank (release 98.0) and SwissProt (release 32.0) entries and the BLASTN and BLASTX similarity search programs (Altschul et al. 1990; Gish and States 1993).
Results and discussion
Callus initiation was observed 2 weeks after explanting bulbils from in-vitro-grown D. bulbifera plantlets on culture medium. The comparably rapid callus proliferation on bulbil explants is probably due to a layer of meristematic cells beneath the bulbil cortex (Asokan et al. 1983). After several rounds of subculturing soft and friable callus, cell suspension cultures could be established by suspending callus in liquid culture medium. Subculturing the suspensions using a pipet with a wide opening ensured that the cultures mainly consisted of small cell clumps below 1 mm in diameter. In the closely related yam species D. alata, D. opposita and D. rotundata, phenylpropanoid phytoalexins and PAL enzyme activity are induced in fungus- or Pseudomonasinfected tubers (Cline et al. 1989). Therefore, we first aimed at detecting PAL mRNA induction in elicited D. bulbifera cell cultures. Total RNA was isolated at different time points after elicitation (Rompf and Kahl 1997) and Northern blots were prepared. The heterologous PAL cDNA fragment Eli-4 (Somssich et al. 1989) was hybridized to the blotted Dioscorea RNA and a massive induction of PAL mRNA in cell cultures 1–24 h after elicitor addition was observed (Fig. 1). For the isolation of the corresponding gene, we tried to clone a yam PAL cDNA. RT-PCR using highly degenerate PAL consensus primers amplified a product with a mRNA template from elicited cell cultures, but not with mRNA from untreated cell cultures (not shown). The resulting 557-bp fragment was cloned and sequenced. Unexpectedly, a sequence derived from the PAL-1 primer was detected on both ends of the PCR fragment. A sequence homology analysis revealed high homology to PR-4 protein homologues, but no homology to PAL sequences. There-
603
Fig. 1 Northern blot analysis of PAL expression in elicited cell cultures. Total RNA (12 µg) isolated from cell cultures before, or 1, 4, and 24 h after addition of Colletotrichum gloeosporioides cell wall fragments was fractionated by gel electrophoresis, blotted onto nylon membranes and hybridized with a radioactively labelled PAL probe (upper panel). Blotting of equal amounts of RNA onto membranes was confirmed by hybridization to a genomic probe consisting of 5.8 S, 18 S and 26 S rDNA sequences (lower panel)
Fig. 2 Alignment of the amino acid sequences of the predicted yam PR-4 protein and related proteins. Amino acid sequences of PR-4b proteins from tobacco (Friedrich et al. 1991), a hevein-like protein from Arabidopsis (Potter et al. 1993), win1-protein from potato (Stanford et al. 1989), and barwin from barley (Svenson et al. 1993) are shown. Amino acid residues that are identical between yam PR-4-protein and related proteins are shaded. Colons represent gaps introduced to optimize the sequence alignment. Putative signal peptide cleavage sites are underlined
fore the clone was named yam PRP-4. The cloned PCR product contained an open reading frame of 453 bp, and an alignment of the predicted yam PRP-4 amino acid sequence with other PR-4 proteins could be performed (Fig. 2). The amino acid sequence of the yam PR-4 protein shows 79 and 78% homology to the PR-4B and PR4 A precursor proteins, respectively, from tobacco (Friedrich et al. 1991), 75% to a hevein-like protein precursor from Arabidopsis (Potter et al. 1993), on average 74% homology to barwin from barley and wheatwin proteins (Caruso et al. 1996; Svenson et al. 1993), 74% to potato win1 and win2 proteins (Stanford et al. 1989), 73% to pre-hevein from rubber tree (Broekaert et al. 1990), and 72% homology to the PRP-2 protein from tomato (Linthorst et al. 1991). Analysis of the hydrophobicity profile of the cDNA-encoded protein revealed a strong hydrophobic region at the N-terminal domain of the polypeptide. This putative signal peptide has 29 amino acids, a cleavage site between amino acids 29 (Ala) and 30 (Gln) and is in compliance with von Heijne’s rules (von Heijne 1986). A similar, highly hydrophobic sequence is found in the N-terminal domain of PRPs and functions as a signal peptide for translocation through the endoplasmic reticulum to the apoplastic space (Linthorst et al. 1991). The predicted yam PRP-4 lacks the hevein and hinge region found in win1, win2, and pre-hevein, and shows high homology to the C-terminal PRP-4 domain. Therefore the yam PR-4 protein would be grouped into
604
Fig. 3 Northern blot analysis of yam PRP-4 expression in elicited cell cultures, young and adult leaves. Total RNA (12 µg) isolated before, or 1, 4, and 24 h after elicitation was fractionated by gel electrophoresis, blotted onto nylon membranes and hybridized with a radioactively labelled yam PRP-4 probe. From left to right: total RNA isolated from cell cultures elicited by addition of C. gloeosporioides cell wall fragments; total RNA isolated from elicited young leaves; total RNA isolated from elicited adult leaves (upper panel). Leaves were sprayed with fungal cell wall fragments and subsequently wounded using sterile needles. Blotting of equal amounts of RNA onto membranes was confirmed by hybridization to a genomic probe consisting of 5.8 S, 18 S and 26 S rDNA sequences (lower panel)
class II of PR-4 proteins, which is represented by proteins consisting of the common PR-4 C-terminal domain only (Ponstein et al. 1994). PRP-4 class II proteins are supposed to function actively in plant defense against pathogens. For example, wheatwin1 and wheatwin2 possess antifungal activity against Botrytis cinerea and different wheat pathogenic Fusarium isolates (Caruso et al. 1996), and a barley PR-4 protein shows antifungal activity towards Trichoderma harzianum (Hejgaard et al. 1992). The specific mechanism of action against hyphal growth remains unclear, although a weak binding activity towards chitin was observed for the barley protein (Hejgaard et al. 1992). Several proteins belonging to the PRP-4 group are known to be pathogen or wound inducible. Tobacco PRP-4 expression increases during TMV infection (Friedrich et al. 1991) and SA treatment (Linthorst et al. 1991). Tomato plants accumulate PRP-2 mRNA during Cladosporium fulvum infection (Linthorst et al. 1991), and induction of hevein-like mRNA is observed following turnip crincle virus infection, ethylene, SA or 2,6-dichloroisonicotinic acid treatment in Arabidopsis leaves (Potter et al. 1993). Tobacco CBP20 is induced upon TMV infection and wounding (Ponstein et al. 1994). In contrast, barwin and wheatwin proteins have been isolated even from untreated seeds. Therefore, a function of the proteins as preformed defense compounds is assumed (Caruso et al. 1996; Svenson et al. 1993). The expression pattern of yam PRP-4 mRNA was analyzed in Northern blot experiments. A radioactively labelled yam PRP-4 cDNA probe was hybridized to blots
Fig. 4 Southern blot analysis of the yam PRP-4 gene. Dioscorea bulbifera DNA was digested with five different restriction enzymes. Samples of 7 µg were electrophoresed, blotted onto nylon membrane and hybridized with a radioactively labelled yam PRP-4 probe
containing total RNA from elicited cell cultures and RNA isolated from leaves that had been spread with the fungal cell wall elicitor and subsequently wounded. Young (less than 5 cm in diameter) and adult (5–20 cm in diameter) leaves were treated separately. RNA isolation and Northern blot hybridization were as described in Rompf and Kahl (1997). The results are shown in Fig. 3. The PRP-4 probe hybridizes specifically to a single mRNA species of approximately 600 bases. In elicited cell cultures (left panel), we observed a faint induction after 1 h, which became stronger after 4 and 24 h. A comparably rapid and strong induction of yam PRP-4 mRNA is also observed in elicited adult leaves (right
605
panel), whereas a basal level of yam PRP-4 mRNA is already present in untreated young leaves, which increases after elicitation (center). The observed expression patterns are similar to those of other proteins of the PRP-4 family and suggest a function of the yam PR-4 protein in defense against the anthracnose pathogen. The expression of yam PRP-4 seems also to be regulated developmentally (e.g., presence in young leaves, absence in older leaves). Similar to wheatwin and barwin proteins in seeds, yam PRP-4 might function as a preformed defense compound in young leaves. A low level of expression in intact tissue and an increase after wounding was also shown for win2 in potato leaves (Stanford et al. 1989) and rubber tree hevein (Broekaert et al. 1990). To estimate the copy number of yam PRP-4 genes in D. bulbifera, we performed Southern blot analyses. Hybridization of yam PRP-4 cDNA to genomic yam DNA digested with DraI and HindIII, which do not have internal recognition sites in the cDNA, revealed two strong and five weak hybridization signals (Fig. 4). Therefore yam PRP-4 appears to be encoded by a small multigene family of two to seven members. Our study is the first example of a defense-related gene from yam, which is similarly regulated in elicited cell cultures and leaves. Therefore, D. bulbifera cell cultures might serve as a model system to isolate defense-related yam genes, an approach shown to be successful in many other plant species (Hahlbrock et al. 1995). The rapid induction of PRP-4 mRNA upon elicitation in D. bulbifera leaves could explain the lower sensitivity of D. bulbifera towards anthracnosis as compared to more susceptible yam species. In the commercially more important species D. alata, cultivars both resistant and susceptible to C. gloeosporioides infection are found (Hahn et al. 1987). Since a delayed response of PR genes is often observed in susceptible host-pathogen interactions (Benhamou 1996), the timing of PRP-4 expression might serve to characterize resistant D. alata cultivars early in the infection process. Meanwhile, we have isolated the promotor of the yam PRP-4-1 gene, which will be used for the overexpression of other defense-related proteins like chitinases, β-1,3-glucanases and antifungal proteins in transgenic yam plants to improve their defense towards anthracnosis. Acknowledgements This research was supported by Deutsche Forschungsgemeinschaft (grant Ka 332/14-3), the GTZ (grant 95.2072.7-001.00), and the Herrmann Willkomm-Stiftung (Frankfurt am Main, Germany). R. R. appreciates a fellowship from Ev. Studienstiftung (Schwerte, Germany), and we thank Ryohei Terauchi for his critical comments on the manuscript.
References Altschul SF, Gish W, Miller W, Myers EW, Lipman D (1990) Basic local alignment search tool. J Mol Biol 215: 403–410 Asokan MP, O’Hair SK, Litz RE (1983) In vitro plant development from bulbil explants of two Dioscorea species. Hort Sci 18: 702–703
Benhamou N (1996) Elicitor-induced plant defense pathways. Trends Plant Sci 1: 233–239 Bowles D (1990) Defense related proteins in higher plants. Annu Rev Biochem 59: 873–907 Broekaert W, Lee HL, Kush A, Chua NH, Raikhel N (1990) Woundinduced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis). Proc Natl Acad Sci USA 87: 7633–7637 Caruso C, Caporale C, Chilosi G, Vacca F, Bertini L, Magro P, Poerio E, Buonocore V (1996) Structural and antifungal properties of a pathogenesis-related protein from wheat kernel. J Prot Chem 15: 25–44 Cline EI, Adesanya SA, Ogundana SK, Roberts MF (1989) Induction of PAL activity and dihydrostilbene phytoalexins in Dioscorea alata and their plant growth inhibitory properties. Phytochemistry 28: 2621–2625 Corbin DR, Sauer N, Lamb CJ (1987) Differential regulation of a hydroxyproline-rich glycoprotein gene family in wounded and infected plants. Mol Cell Biol 8: 1113–1122 Cutt JR, Klessig DF (1992) Pathogenesis related proteins. In: Boller T, Meins F Jr. (eds) Genes involved in plant defense. Springer, Berlin Heidelberg New York, pp 210–243 Dixon R, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085–1097 Friedrich L, Moyer M, Ward E, Ryals J (1991) Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein,Win-1 and Win-2. Mol Gen Genet 230: 113–119 Gish W, States DJ (1993) Identification of protein coding regions by database similarity search. Nat Genet 3: 266–272 Hahlbrock K, Scheel D, Logemann E, Nürnberger T, Parniske M, Reinold S, Sacks WR, Schmelzer E (1995) Oligopeptide elicitormediated defense gene activation in cultured parsley cells. Proc Natl Acad Sci USA 92: 4150–4157 Hahn SK, Osiru DSO, Akoroda MO, Otoo JA (1987) Yam production and its future prospects. Outlooks Agric 16: 105–110 Heijne G von (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14: 4683–4690 Hejgaard J, Jacobsen S, Bjorn SE, Kragh KM (1992) Antifungal activity of chitin-binding PR-4 type proteins from barley grain and stressed leaf. FEBS Lett 307: 389–392 Howles PA, Arioli T, Weinman JJ (1994) Characterization of a phenylalanine ammonia-lyase multigene family in Trifolium subterraneum. Gene 138: 87–92 Ikotun B (1989) Diseases of yam tubers. Int J Trop Dis 7: 1–21 Kahl G, Weising K, Hofman D, Knobloch I, Hergenröder W, Rompf R, Geistlinger J, Hüttel B, Ramser J, Kämmer D, Kost S, Bahl A (1991) Engineering fungus tolerance into Dioscorea (yam) plants. Proceedings of the 4th Conference of the International Plant Biotechnology Network (IPBNet), San Jose, Costa Rica, pp 15–22 Kombrink E, Schröder M, Hahlbrock K (1988) Several “pathogenesis-related” proteins in potato are β-1,3-glucanases and chitinases. Proc Natl Acad Sci USA 85: 782–786 Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100–112 Linthorst HJM, Danhash N, Brederode FT, Kan JAL von, Wit PJGM de, Bol JF (1991) Tobacco and tomato PR proteins homologous to win and pro-hevein lack the “hevein” domain. Mol Plant Microb Interact 4: 586–592 Löw R, Rausch T (1994) Sensitive, nonradioactive Northern blots using alkaline transfer of total RNA and PCR-amplified biotinylated probes. Biotechniques 17: 1027–1030 Ponstein A, Bres-Vloemans SA, Sela-Buurlage MB, Elzen PJM van der, Melchers LS, Cornelissen BJC (1994) A novel pathogen- and wound inducible tobacco (Nicotiana tabacum) protein with antifungal activity. Plant Physiol 104: 109–118 Potter S, Ukness S, Lawton K, Winter AM, Chandler D, diMaio J, Novitzky R, Ward E, Ryals J (1993) Regulation of a hevein-like gene in Arabidopsis. Mol Plant Microb Interact 6: 680–685 Ramser J, Lopez-Peralta C, Wetzel R, Weising K, Kahl G (1996) Genomic variation and relationships in aerial yam (Dioscorea bulbifera L.) detected by random amplified polymorphic DNA. Genome 39: 17–25
606 Rompf R, Kahl G (1997) mRNA differential display in agarose gels. Biotechniques 23: 28–32 Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28: 425–429 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Somssich IE, Bollmann J, Hahlbrock K, Kombrink E, Schulz W (1989) Differential early activation of defense-related genes in elicitor-treated parsley cells. Plant Mol Biol 12: 227–234
Stanford A, Bevan M, Northcote D (1989) Differential expression within a family of novel wound-induced genes in potato. Mol Gen Genet 215: 200–208 Svenson B, Svendsen I, Horup P, Roepstorff P, Ludvigsen S, Poulsen FM (1993) Primary structure of barwin: a barley seed protein closely related to the C-terminal domain of proteins encoded by wound-induced plant genes. Biochemistry 31: 8767–8770
