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For. Snow Landsc. Res. 76, 3: 415–419 (2001) 415 Chestnut seed proteins involved in stress tolerance Luis Gomez and Cipriano Aragoncillo Departamento de Biotecnologia, Escuela Tecnica Superior de Ingenieros de Montes, Universidad Politecnica de Madrid, 28040 Madrid, Spain [email protected] Abstract A thorough understanding of the biochemical and physiological basis of stress responses in plants is needed to rationally manipulate tolerance traits. Most studies have focused so far on the identification of stress-responsive genes in herbaceous plants. Forest trees, by contrast, have been largely ignored. Here we summarize our recent findings on the functional characterization of two chestnut seed proteins, the molecular chaperone CsHSP17.5 and the endochitinase CsCh3, which are produced when plants are affected by thermal stress and microbial infection. Keywords: antifungal proteins, Castanea sativa, chestnut, chitinases, thermal stress 1 Introduction Our studies of stress adaptation have been mainly conducted with chestnut seeds. The rationale for this is that: 1) many stress-inducible proteins have abundant, developmentallyregulated seed homologues (e.g., KITAJIMA and SATO 1999), 2) chestnut seeds should contain high levels of certain defensive proteins because of their unusually high water content, and 3) the abundance of tannins and other phenolic compounds in woody plants makes it very difficult to isolate active proteins from vegetative tissues. The proteins identified by us include pathogenesis-related proteins, such as endochitinases (COLLADA et al. 1992; ALLONA et al. 1996) and a thaumatin-like protein (GARCIA-CASADO et al. 2000), as well as a low-molecular weight heat-shock protein (COLLADA et al. 1997, SOTO et al. 1999). All these polypeptides belong to structurally diverse families associated with plant defensive responses (WATERS et al. 1996; KITAJIMA and SATO 1999) and accumulate at high levels in chestnut seeds. All of them have stress-inducible homologues in chestnut stems, roots and/or leaves. The work described here was presented at different meetings of the EC COST Action “Multidisciplinary Chestnut Research” between 1998 and 2001. 2 Small heat-shock proteins (sHSPs) One of the most abundant low-molecular weight proteins of C. sativa cotyledons, termed CsHSP17.5 (Chestnut sHSP17.5), can form high-molecular weight complexes in vitro (Fig. 1 and COLLADA et al. 1997). Its complete primary structure was determined from the fulllength cDNA and showed homology with small heat-shock proteins (sHSPs) in plants (SOTO et al. 1999). Since molecular chaperone activity had already been shown for plant sHSPs using model enzymes (COLLADA et al. 1997; LEE et al. 1997), we analyzed the effects of CsHSP17.5 on the refolding of an endogenous substrate, the seed endochitinase CsCh1. As shown in Table 1, the refolding yields of denatured CsCh1 were about five times higher in the presence of the sHSP than in control reactions with lysozyme (used as a negative control because its size and isoelectric point are similar to those of CsHSP17.5). The activity of sHSP genes during seed maturation and germination was also analyzed. As shown in Figure 2, 416 Luis Gomez, Cipriano Aragoncillo when RNA from cotyledons was probed with the coding region for CsHSP17.5, a single hybridizing band was detected in all cases. During seed development the signal was maximal at mid-maturation stage and subsequently decreased. By contrast, there was a steady decrease in the amount of transcript during germination. sHSP expression was also analyzed in chestnut plantlets. In non-stressed controls, a weak band was observed in stems, but not in leaves or roots (Fig. 2). However, when plants were subjected to heat stress, increased transcript abundance was observed in all organs. D ND kD kD 450 240 67 160 45 67 25 Fig. 1. Electrophoretic behavior of the seed protein CsHSP17.5 (arrows) under dissociating (D) and non-dissociating (ND) conditions. The protein was purified by selective extraction and differential ammonium-sulfate precipitation as in COLLADA et al. (1997). P: purified chestnut sHSP; M: molecular weight markers. 17.8 12.3 P P M S 0 1 Leaves 3 5 M 7 0 1 Stems 3 5 7 0 1 Roots 3 5 7 A Seed maturation 1 2 3 1 Seed germination 2 3 4 B Fig. 2. Induction of sHSP transcripts in 20-week old chestnut plantlets (A) or in seeds (B). Plantlets were kept in growth chambers as described in SOTO et al. (1999) and then treated at 40 °C for 0, 1, 3, 5, and 7 h. Seed maturation and germination stages are numbered as in PERNAS et al. (2000). In all cases RNA was extracted and analyzed by Northern blot hybridization as previously described (SOTO et al. 1999), using as probe the first 662 bp of the Cs hsp17.5 cDNA (EMBL accession AJ009880). S: mature seeds. Table 1. Effect of CsHSP17.5 on the refolding of chitinase CsCh1. This enzyme was denatured in 6 M guanidine hydrochloride and then placed under refolding conditions in the presence of equimolar amounts of CsHSP17.5 or lysozyme (negative control). At the times (min) indicated, aliquots were taken and assayed for chitinase activity as described in ALLONA et al. (1996). Results are mean relative activities (%) of at least three independent assays. Duration (min) 0 15 30 45 60 120 sHSP Lysozyme 9.4 8.3 35.1 14.1 71.5 16.4 94.4 17.3 98.9 18.2 100 19.6 417 For. Snow Landsc. Res. 76, 3 (2001) 3 Endochitinases Basic chitinases are amongst the most abundant soluble proteins of chestnut seeds (COLLADA et al. 1992, ALLONA et al. 1996). We used chestnut chitinase CsCh3 to undertake a structure-activity analysis within this protein family. Based on the X-ray structure of barley Horv2 protein, a model was constructed for the catalytic domain of CsCh3. The overall fold corresponds to a globular all-α domain with ten helical segments (Fig. 3). Comparisons with structurally-related enzymes and theoretical considerations led us to identify potential catalytic residues (GARCIA-CASADO et al. 1998). To test our hypotheses, we performed single residue substitutions and expressed the mutant enzymes in bacteria. A comparison was then made of the specific activities shown by wild type (wt) and mutated enzymes (Fig. 4). Class I chitinases (like CsCh3) contain an N-terminal chitin-binding extension besides the catalytic domain. To better define the antifungal properties of each domain, wt CsCh3 and its mutated forms were assayed against the fungus Trichoderma viride (GARCIACASADO et al. 1998). While all enzymes tested were able to inhibit fungal growth, close examination of the mycelia revealed substantial differences. Thus, CsCh3 or any of its mutant forms caused increased branching of young hyphae (Fig. 5). By contrast, chitinase CsCh1, which has antifungal activity but lacks a chitin-binding domain, caused no visible alterations. Interestingly, the antifungal activity of chestnut chitinases is synergistically enhanced by a thaumatin-like protein recently purified from chestnut cotyledons (GARCIACASADO et al. 2000). C-term Fig. 3. Ribbon diagram of the catalytic domain of CsCh3 (residues 58 to 297). Atomic coordinates were predicted using SWISS-MODEL (http://www.expasy.ch/swissmod/ SWISS-MODEL.html) and crystallographic data for the Horv2 protein (PDB entry 2BAA). 100 Thr175Ala Asn254Ile Gln173Leu Glu146Gln Glu146Asp Glu124Asp wt/Ch3 0 Glu124Gln 50 Control Residual specific activity (%) Fig. 4. Hydrolytic activity of mutant chitinases. The activity of recombinant proteins was measured by a colorimetric method that uses CM-chitin-RBV (Loewe Biochemica GmbH) as a substrate. Assay conditions were as in GARCIA-CASADO et al. (1998). Residual specific activity relative to wt recombinant CsCh3 are presented (%). Results are means of at least six independent assays (SD was less than 5%). N-term 418 A Luis Gomez, Cipriano Aragoncillo B Fig. 5. Representative micrographs of the morphological changes induced in Trichoderma viride hyphae upon exposure to chitinases. Fungal spores were plated out on potato dextrose agar and incubated for 40 h at 25 ºC. Then different amounts of protein solutions were applied to sterile paper discs laid on the agar surface. Micrographs were taken 18 h later with an inverted light microscope (Prior Scientific Instruments, UK). (A) negative control; (B) mutant Thr175Ala. 4 Conclusions The seeds of C. sativa have proven to be a good source material to isolate proteins involved in stress tolerance. For example, they contain an abundant molecular chaperone, CsHSP17.5, that accumulates at levels comparable to those of major storage proteins (COLLADA et al. 1997). Like them, it forms oligomeric complexes in vitro. Its deduced amino acid sequence shows homology with cytosolic sHSPs (WATERS et al. 1996). In line with this finding, immuno-electron microscopy analyses of cotyledonary cells showed an overall cytoplasmic localization for CsHSP17.5 (SOTO et al. 1999). We have shown here and elsewhere (COLLADA et al. 1997) that this protein has molecular chaperone activity, as is the case for some other sHSPs (JINN et al. 1995, LEE et al. 1995). Our results support a role of CsHSP17.5 in protecting seed tissues against thermal stress, a notion reinforced by the finding that homologous transcripts are induced in vegetative tissues by heat treatments. Recently, CsHSP17.5 has been shown to protect bacterial cells against thermal stress in vivo (SOTO et al. 1999). Chitinases are highly abundant in chestnut seeds as well (COLLADA et al. 1992). We have used chitinase CsCh3 to analyze structure-activity relationships. Through sequence and structural comparisons potentially relevant residues were identified (GARCIA-CASADO et al. 1998). Our results point towards Glu124 as the general acid catalyst and Glu146 as the general base. The latter probably activates a water molecule for nucleophilic attack. The mutant chitinases generated in this study were also tested for their ability to inhibit fungal growth. It has been suggested that the chitin-binding domain present in class I chitinases is not essential for antifungal activity (ISELI et al. 1993). However, different peptides related to this domain have been shown to inhibit fungal growth (BROEKAERT et al. 1989). Analysis of the morphological changes caused in the hyphal tips suggests that both domains of CsCh3 alter apical growth, although through different mechanisms. Several lines of evidence have substantiated the potential of chitinases to counter fungal disease in plant (GRISON et al. 1996). The structure-function analysis of CsCh3 should contribute to optimizing their applicability to the genetic engineering of disease-resistant plants. Acknowledgments We thank all members of our research groups for their dedication to the work presented here. Financial support was obtained from Ministerio de Educación y Cultura of Spain (grants BIO960441 and BIO99-0931 to L.G.) and Comunidad Autónoma de Madrid (grants 07B-012-97 and 07M-0047-2000 to C.A.). For. Snow Landsc. Res. 76, 3 (2001) 5 419 References ALLONA, I.; COLLADA, C.; CASADO, R.; PAZ-ARES, J.; ARAGONCILLO, C., 1996: Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons. Plant Mol. Biol. 32: 1171–1176. BROEKAERT, W.F.; VAN PARIJS, J.; LEYNS, F.; JOOS, H.; PEUMANS, W.J., 1989: A chitin-binding lectin from stinging nettle rhizomes with antifungal properties. Science 245: 1100–1102. COLLADA, C.; CASADO, R.; FRAILE, A.; ARAGONCILLO, C., 1992: Basic endochitinases are major proteins in Castanea sativa cotyledons. Plant Physiol. 100: 778–783. COLLADA, C.; GOMEZ, L.; CASADO, R.; ARAGONCILLO, C., 1997: Purification and in vitro chaperone activity of a class I small heat-shock protein abundant in recalcitrant chestnut seeds. Plant Physiol. 115: 71–77. 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