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
lgomez@montes.upm.es
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 identifi - cation 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 chest- nut 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, developmentally- regulated seed homologues (e.g., KITAJIMAand SATO1999), 2) chestnut seeds should con- tain 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;
ALLONAet al. 1996) and a thaumatin-like protein (GARCIA-CASADOet al. 2000), as well as a low-molecular weight heat-shock protein (COLLADAet al. 1997, SOTOet al. 1999). All these polypeptides belong to structurally diverse families associated with plant defensive responses (WATERSet al. 1996; KITAJIMAand SATO1999) 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. sativacotyledons, termed CsHSP17.5 (Chestnut sHSP17.5), can form high-molecular weight complexes in vitro(Fig. 1 and COLLADAet al. 1997). Its complete primary structure was determined from the full- length cDNA and showed homology with small heat-shock proteins (sHSPs) in plants (SOTOet al. 1999). Since molecular chaperone activity had already been shown for plant sHSPs using model enzymes (COLLADAet al. 1997; LEEet 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 con- trol 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,
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 tran- script abundance was observed in all organs.
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 COLLADAet al.(1997). P: purified chestnut sHSP; M: molecular weight markers.
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 SOTOet 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 PERNASet al.(2000). In all cases RNA was extracted and analyzed by Northern blot hybridization as previously described (SOTOet 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 ALLONAet al.(1996). Results are mean relative activities (%) of at least three independent assays.
Duration (min) 0 15 30 45 60 120
sHSP 9.4 35.1 71.5 94.4 98.9 100
Lysozyme 8.3 14.1 16.4 17.3 18.2 19.6
D kD
67 45
25
17.8
12.3
kD
450 240 160
67 ND
P M P M
Seed maturation 1 2 3
Seed germination 1 2 3 4 Leaves
0 1 3 5 7
Stems 0 1 3 5 7
Roots 0 1 3 5 7 S
A
B
3 Endochitinases
Basic chitinases are amongst the most abundant soluble proteins of chestnut seeds (COLLADAet al. 1992, ALLONAet 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-αdomain with ten helical segments (Fig. 3). Comparisons with structurally-related enzymes and theoretical considerations led us to identify potential cata - lytic residues (GARCIA-CASADOet 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 (GARCIA- CASADOet 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 (GARCIA- CASADOet al.2000).
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).
Fig. 4. Hydrolytic activity of mutant chitinases. The activity of recombinant proteins was measured by a col- orimetric 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 inde- pendent assays (SD was less than 5%).
C-term
N-term
Residual specific activity (%)
100
50
0
Asn254Ile
Thr175Ala
Gln173Leu
Glu146Asp
Glu146Gln
Glu124Asp
Glu124Gln
wt/Ch3
Control
4 Conclusions
The seeds of C. sativahave 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 (COLLADAet 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 (COLLADAet al.1997) that this protein has molecular chaperone activity, as is the case for some other sHSPs (JINNet al.1995, LEEet 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 (COLLADAet 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 (ISELIet al.1993). However, different peptides related to this domain have been shown to inhibit fungal growth (BROEKAERTet 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 (GRISONet al.
1996). The structure-function analysis of CsCh3 should contribute to optimizing their appli- cability 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 BIO96- 0441 and BIO99-0931 to L.G.) and Comunidad Autónoma de Madrid (grants 07B-012-97 and 07M-0047-2000 to C.A.).
Fig. 5. Representative micrographs of the morphological changes induced in Trichoderma viridehyphae upon ex - posure 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.
A B
5 References
ALLONA, I.; COLLADA, C.; CASADO, R.; PAZ-ARES, J.; ARAGONCILLO, C., 1996: Bacterial expression of an active class Ib chitinase from Castanea sativacotyledons. 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 vitrochaper- one activity of a class I small heat-shock protein abundant in recalcitrant chestnut seeds. Plant Physiol. 115: 71–77.
GARCIA-CASADO, G.; COLLADA, C.; ALLONA, I.; CASADO, R.; PACIOS, L.F.; ARAGONCILLO, C.;
GOMEZ, L., 1998: Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds. Glycobiology 8: 1021–1028.
GARCIA-CASADO, G.; COLLADA, C.; ALLONA, I.; SOTO, A.; CASADO, R.; RODRIGUEZ-CEREZO, E.; GOMEZ, L.; ARAGONCILLO, C., 2000: Characterization of an apoplastic basic thaumatin- like protein from recalcitrant chestnut seeds. Physiol. Plant. 110: 172–180.
GRISON, R.; GREZES-BESSET, B.; SCHNEIDER, M.; LUCANTE, N.; OLSEN, L.; LEGUAY, J.-J.;
TOPPAN, A., 1996: Field tolerance to fungal pathogens of Brassica napus constitutively expressing chimeric chitinase gene. Nat. Biotechnol. 14: 643–646.
ISELI, B.; BOLLER, T.; NEUHAUS, J.-M., 1993: The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol. 103: 221–226.
JINN, T.L.; CHEN, Y.M.; LIN, C.Y., 1995: Characterization and physiological function of class I low- molecular-mass, heat-shock protein complex in soybean. Plant Physiol.108: 693–701.
KITAJIMA, S.; SATO, F., 1999: Plant pathogenesis-related proteins: molecular mechanisms of gene expression and protein function. J. Biochem.125: 1–8.
LEE, G.J.; POKALA, N.; VIERLING, E., 1995: Structure and in vitromolecular chaperone activity of cytosolic small heat shock proteins from pea. J. Biol. Chem.270: 10432–10438.
LEE, G.J.; ROSEMAN, A.M.; SAIBIL, H.R.; VIERLING, E., 1997: A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state. Eur. Mol. Biol. Organ. J. 16: 659–671.
PERNAS, M.; SANCHEZ-MONGE, R.; SALCEDO, G., 2000: Biotic and abiotic stress can induce cystatin expression in chestnut. Fed. Eur. Biochem. Soc. Lett. 467: 206–210.
SOTO, A.; ALLONA, I.; COLLADA, C.; GUEVARA, M.A.; CASADO, R.; RODRIGUEZ-CEREZO, E.;
ARAGONCILLO, C.; GOMEZ, L., 1999: Heterologous expression of a plant small heat-shock protein enhances Escherichia coliviability under heat and cold stress. Plant Physiol. 120:
521–528.
WATERS, E.R.; LEE, G.J.; VIERLING, E., 1996: Evolution, structure and function of the small heat shock proteins in plants. J. Exp. Bot. 47: 325–338.
Accepted 29.1.02
