Download Chestnut seed proteins involved in stress tolerance

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,
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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
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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.).
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Accepted 29.1.02