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Keywords:

  • arachidonic acid;
  • cystatin;
  • methyl jasmonate;
  • protease inhibitors;
  • tomato (Solanum lycopersicum)

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    We assessed the ability of the fungal elicitor arachidonic acid to induce cystatin genes in tomato (Solanum lycopersicum), using a cDNA expression library from arachidonate-treated leaves.
  • • 
    The cDNAs of two novel cystatins were isolated, coding for an approx. 11-kDa protein, SlCYS10; and for a 23.6-kDa protein, SlCYS9, bearing an N-terminal signal peptide and a long, 11.5-kDa extension at the C terminus. Both genes were induced by arachidonate but not by methyl jasmonate, an inducer of the 88-kDa eight-unit cystatin, multicystatin, accumulated in the cytosol of leaf cells upon herbivory.
  • • 
    A truncated form of SlCYS9, tSlCYS9, was produced by deletion of the C-terminal extension to assess the influence of this structural element on the cystatin moiety. As shown by kinetic and stability assays with recombinant variants expressed in Escherichia coli, deleting the extension influenced both the overall stability and inhibitory potency of SlCYS9 against cysteine proteases of herbivorous organisms.
  • • 
    These findings provide evidence for a multicomponent elicitor-inducible cystatin complex in tomato, including at least 10 cystatin units produced via two metabolic routes.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Protease inhibitors of the cystatin protein superfamily regulate proteolysis in various biological processes (Turk et al., 1997; Arai et al., 2002). Cystatins form a tight, reversible complex with cysteine proteases, acting as pseudosubstrates to enter the active site cleft of target enzymes and cause inhibition. Several roles have been attributed to cystatins in plants, including the control of endogenous cysteine proteases in physiological and developmental processes as diverse as organogenesis, seed development and maturation, storage protein turnover and programmed cell death (Kumar et al., 1999; Kuroda et al., 2001; Arai et al., 2002; Corre-Menguy et al., 2002; Belenghi et al., 2003; Rojo et al., 2004; Martinez et al., 2005a). Plant cystatins would also help plants to cope with abiotic stresses such as drought or cold temperatures, and inhibit the (exogenous) proteases of herbivorous organisms during herbivory or pathogenic infection (Pernas et al., 2000; Gaddour et al., 2001; Arai et al., 2002; Van der Vyver et al., 2003; Diop et al., 2004; Martinez et al., 2005b; Massonneau et al., 2005; Christova et al., 2006). Several lines of evidence suggest a significant role for cystatins in plant defense, including their inhibitory potency against the digestive cysteine proteases of herbivorous arthropods and parasitic nematodes (Zhao et al., 1996; Visal-Shah et al., 2001; Arai et al., 2002), their detrimental effects against pathogenic fungi (Pernas et al., 1999; Soares-Costa et al., 2002; Martinez et al., 2003, 2005b; Yang & Yeh, 2005; Christova et al., 2006), and the enhanced resistance of cystatin-expressing transgenic plants against herbivorous insects and pathogens (Guttierrez-Campos et al., 1999; Arai & Abe, 2000; Urwin et al., 2003; Outchkourov et al., 2004).

The induction of cystatin-encoding genes in leaves challenged with methyl jasmonate (MeJa), wounding or insect herbivory also support a protective role for plant cystatins (Bolter, 1993; Botella et al., 1996; Jacinto et al., 1998; Pernas et al., 2000; Wu & Haard, 2000; Belenghi et al., 2003; Bouchard et al., 2003). Current models for the stress-induced expression of protease inhibitors in plants point to the key role of α-linolenic acid, which is released from cell membranes upon wounding, then metabolized via the octadecanoid signaling pathway to give jasmonic acid, an inducer of defense-related genes (Farmer & Ryan, 1992; Koiwa et al., 1997; Gatehouse, 2002). In Solanaceae, several protease inhibitors, including the serine-type inhibitors, proteinase inhibitors I (Pin-I) and II (Pin-II); the Kunitz inhibitor cathepsin D inhibitor; the inhibitor of metalloproteases, metallo-carboxypeptidase inhibitor; and the eight-unit cysteine-type inhibitor, multicystatin are induced in leaves by wounding, insect herbivory, systemin, jasmonate, MeJa and/or jasmonate analogues or precursors including α-linolenate (Farmer & Ryan, 1992; Hansen & Hannapel, 1992; Hildmann et al., 1992; Bolter, 1993; Werner et al., 1993; Jacinto et al., 1998; Gleddie & Michaud, 2000; Wu & Haard, 2000; Moura & Ryan, 2001; Bouchard et al., 2003; Diez-Diaz et al., 2004). To document further the role of cystatins as an active player in the plant's defensive machinery, we assessed the ability of the fungal elicitor arachidonic acid to induce the expression of cystatin-encoding genes in tomato (Solanum lycopersicum).

Arachidonate, released from germinating spores of the late blight fungus Phytophthora infestans and related oomycetes during plant infection (Ricker & Bostock, 1992), is a potent inducer of systemic resistance to pathogens in plants (Bostock et al., 1981, 1986; Cohen et al., 1991; Coquoz et al., 1995; Fidantsef et al., 1999). In Solanaceae, this polyunsaturated fatty acid elicits programmed cell death and systemic defense responses via an α-linolenate/jasmonate-independent route presumably involving salicylic acid (Coquoz et al., 1995; Yu et al., 1997; Knight et al., 2001). Genes encoding a circadian rhythm-regulated protein of unknown function, DEA1, and specific forms of 3-hydroxy-3-methylglutaryl coenzyme A reductases and family 1 pathogenesis-related (PR) proteins were shown to be induced by arachidonate while remaining uninduced by jasmonate or wounding (Bostock et al., 1992; Choi et al., 1992, 1994; Fidantsef & Bostock, 1998; Fidantsef et al., 1999; Rivard et al., 2004; Weyman et al., 2006). Here we describe the differential inducing effects of arachidonate and jasmonate on cystatin-encoding genes, and provide evidence for the occurrence of a multicomponent, elicitor-inducible cystatin complex in tomato leaves.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Proteases and inhibitors

Trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane (E-64), papain (from papaya latex, EC 3.4.22.2), phenylmethylsulfonyl fluoride (PMSF), ethylenediamine tetraacetic acid (EDTA) and pepstatin A were purchased from Sigma (Oakville, ON, Canada). LdP30, a digestive cystatin-sensitive protease from the coleopteran insect Colorado potato beetle (Leptinotarsa decemlineata Say), was purified by affinity chromatography from third-instar larvae reared on potato plants, using oryzacystatin as an affinity ligand (Visal-Shah et al., 2001). The secreted cysteine proteases Mhp1 and Mip1, from the root-parasitic nematodes Meloidogyne hapla and Meloidogyne incognita, were prepared from preparasitic J2 larvae as described earlier (Michaud et al., 1996).

Plant material

Eight-wk-old glasshouse-grown tomato plants (Solanum lycopersicum) cv. Vendor were sprayed with 40 or 400 µm MeJa or arachidonate (Sigma) in 0.125% (v/v) Triton X-100. Control plants were treated with 0.125% (v/v) Triton X-100. After treatment, the plants were kept in different areas of the glasshouse to prevent cross-contamination between treatments. Leaves were harvested 0, 4, 8, 12, 16, 20 or 24 h after treatment, immediately frozen in liquid nitrogen, and stored at −80°C until use.

cDNA library construction and screening

A cDNA expression library was constructed with the ZAP Express cloning vector system (Stratagene, La Jolla, CA, USA) according to the supplier's instructions, using leaves harvested 16 h after treatment with 400 µm arachidonate as source of mRNA (see above). The library was screened with polyclonal antibodies raised in rabbits against purified potato multicystatin, according to Sambrook et al. (1989). After three rounds of purification, positive cDNAs were excised from the pBK-CMV phagemid vector. The plasmids were isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Mississauga, ON, Canada), and sequenced in both directions.

Phylogenetic reconstruction

Evolutionary relationships among tomato cystatins were assessed by reconstructing an unrooted phylogenetic tree with the DNA sequences of 26 plant cystatins (Table 1), including those isolated from the tomato leaf cDNA library. Cystatin gene sequences were first aligned using the multalin program (Corpet, 1988). An unrooted phylogenetic tree was then inferred from the alignments by the neighbor-joining distance method of Saitou & Nei (1987) using the Phylogenetic Inference Package (phylip) ver. 3.6, after generating a sequence similarity matrix based on Kimura's two-parameter model (Kimura, 1983).

Table 1.  Cystatin-encoding sequences used for the phylogenetic reconstruction
CystatinSpeciesAccession number*
  • *

    Data from the NCBI/GenBank database.

  • Tomato: formerly Lycopersicon esculentum.

  • Sequence retreived from the Solanaceae Genomics Network database (http://www.sgn.cornell.edu).

BcCYS1Brassica campestrisS65071
ClCYS1Coix lacryma-jobiAB037156
GmCYS1Glycine maxD31700
HvCYS1Hordeum vulgareY12068
IbCYS1Ipomoea batatasAF117334
MdCYS1Malus×domesticaAAO19652
OsCYS1Oryza sativaJ03469
OsCYS2O. sativaJ05595
RcCYS1Ricinus communisZ49697
SbCYS1Sorghum bicolorX87168
SlCYS5Solanum lycopersicumU153466
SlCYS8S. lycopersicumAF198390
SlCYS9S. lycopersicumAF198388
SlCYS10S. lycopersicumAF198389
SoCYS1Saccharum officinarumAY119689
StCYS1Solanum tuberosumL16450
StCYS2S. tuberosumL16450
StCYS3S. tuberosumL16450
StCYS4S. tuberosumL16450
StCYS5S. tuberosumL16450
StCYS6S. tuberosumL16450
StCYS7S. tuberosumL16450
StCYS8S. tuberosumL16450
TaCYS1Triticum aestivumAB038392
ZmCYS1Zea maysD10622
ZmCYS2Z. maysD38130

Northern blot analysis

Total RNA (10 µg), isolated from control and treated leaves according to Logemann et al. (1987) was resolved into 1.2% (w/v) formaldehyde–agarose gels and blotted onto nitrocellulose membranes. The membranes were hybridized for 20 h with appropriate 32P-labelle DNA probes and washed under stringent conditions. The filters were subject to autoradiography for 24 h at −80°C, using intensifying screens.

Heterologous expression in Escherichia coli

DNA sequences for the mature form of SlCYS9 (with no peptide signal), the mature form of SlCYS9 with no C-terminal extension (tSlCYS9, for truncated SlCYS9), and the eighth domain of tomato multicystatin (SlCYS8, formerly LeCYS8; Kiggundu et al., 2006) were amplified using the following primers including BamHI and EcoRI cleavage sequences: 5′-AAG GAT CCG CGA ACA GGG AAA ATC AGG AGG ATT CTG C-3′/5′-AGA ATT CTA GTT GTC AGG CTC CAT ACG ATT CAA GTG-3′ for SlCYS9; 5′-AAG GAT CCG CGA ACA GGG AAA ATC AGG AGG ATT CTG C-3′/5′AAG AAT TCT AGG TAG GAA CGT CTT CAA CAT GCT TGA A-3′ for tSlCYS9; and 5′-AAG GAT CCC AAA TCC TGG GGG CAT TAC CAA TGT TCC AT-3′/5′-AAG AAT TCA TTT CAC TTA GTG GCA TCA CCA ACA AGC TTG AAC TC-3′ for SlCYS8. After digestion with BamHI and EcoRI, the PCR amplicons were inserted into the protein expression vector pGEX-3X (Amersham Biosciences, Baie d’Urfé, QC, Canada), in frame with the glutathione S-transferase (GST)-encoding gene. This vector was introduced into E. coli strain Y1091 by electroporation, and used to produce the cystatins as described earlier for other plant cystatins (Michaud et al., 1994). The GST affinity partner was removed from cystatins by cleavage with human factor Xa (Novagen, San Diego, CA, USA) according to the supplier's instructions. Purity of the preparations was confirmed by 12% SDS–PAGE. Protein concentrations were determined according to Bradford (1976), with bovine serum albumin as a standard.

Estimation of Ki(app) values

The inhibitory activities of SlCYS8, SlCYS9 and tSlCYS9 were assayed by estimating apparent dissociation constants (Ki(app) values) for the complexes formed between these proteins and different cysteine proteases. Ki(app) values for papain and Ldp30 were determined by the monitoring of hydrolysis progress curves, according to Salvesen & Nagase (1989). Both enzymes were assayed in 50 mm Tris–HCl pH 6.0 with Z-Phe-Arg-para-nitroanilide (Bachem, Torrance, CA, USA) as a substrate. Proteolysis was allowed to proceed at 37°C in reduced conditions (5 mm l-cysteine), after adding a minimal volume of 50 mm Tris–HCl pH 8.0 (ctrl) or of either cystatin dissolved in the same buffer. Activity levels were monitored every 30 s over 10 min at 405 nm, using a Spectronic 1000 Plus spectrophotometer (Milton Roy, Rochester, NY, USA). Approximate Ki(app) values for Mhp1 and Mip1 were inferred by mildly denaturing gelatin/SDS–PAGE as described earlier (Michaud et al., 1996). Both enzymes were incubated with recombinant cystatins (5 pmole cystatin µl−1 nematode extract) for 10 min at 37°C before electrophoresis.

Cystatin stability assay

Cystatin stability in the presence of nontarget (insensitive) proteases was assessed by challenging SlCYS8, SlCYS9 and tSlCYS9 with a third-instar midgut extract from the herbivorous insect Colorado potato beetle (Michaud et al., 1995). The purified cystatins were incubated for various periods with the insect extract (1 µg insect protein pmole−1 cystatin). Proteolysis was stopped by adding SDS–PAGE sample buffer and incubating the whole mixture for 5 min at 100°C. Degradation of the cystatins was monitored on immunoblots after detection with antipotato multicystatin polyclonal antibodies. To identify proteases responsible for cystatin degradation, the insect extracts were preincubated for 30 min with either 100 µm E-64, 1 mm PMSF, 100 µm pepstatin A or 10 mm EDTA, before incubation with the cystatins (Michaud et al., 1995).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The tomato genome encodes (at least) three evolutionarily distinct cystatins

A cDNA expression library was prepared from tomato leaves treated with 400 µm arachidonate as source material. Two screens of 30000 plaque-forming units yielded several clones expressing proteins recognized by antipotato multicystatin polyclonal antibodies. Sequencing and homology searches showed these clones, also retrieved from a cDNA library prepared from γ-linolenic acid-treated leaves (not shown), to encode three different cystatin-like polypeptides. Some clones included an open reading frame for a cystatin of 235 residues referred to as SlCYS9 (GenBank accession no. AF198388), with a predicted signal peptide of 22 amino acids and a long, 103-aa extension at the C terminus (Fig. 1). Other clones encoded a 98-residue cystatin with no C-terminal extension, referred to as SlCYS10 (accession no. AF198389). The last clones encoded polypeptides showing high homology with each of the eight inhibitory domains of potato multicystatin. Sequence alignments, cross-reactions with antimulticystatin antibodies and Northern blot analysis (see below) strongly suggest that these clones, including the entire eighth inhibitory domain SlCYS8 (accession no. AF198390), encode parts of the MeJa-induced 88-kDa multidomain cystatin, multicystatin (Bolter, 1993).

image

Figure 1. Nucleotide and deduced amino acid sequences of the arachidonate-induced cystatin SlCYS9 (GenBank accession no. AF198388). Residue T132, in bold, corresponds to the C-terminal residue of the cystatin moiety (Fig. 2a). The amino acid sequence for the inhibitor's (predicted) signal peptide is in bold/italic. The stop codon is marked by an asterisk.

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Alignment of the three novel sequences with the model inhibitor oryzacystatin-I (OC-I, or OsCYS1 in this study; Abe et al., 1987), and with the eighth cystatin unit of potato multicystatin (PMC-8, or StCYS8; Waldron et al., 1993) revealed significant identity between all these cystatins, at least for the regions homologous to the 12-kDa cystatin, N-terminal moiety of SlCYS9 (Fig. 2a). The sequence of SlCYS9 corresponding to residues G39–T132 displayed 67, 56, 52 and 51% identity with the corresponding sequences of SlCYS10, OsCYS1, SlCYS8 and StCYS8, respectively. The new polypeptides included the typical inhibitory motifs of cystatins, namely a –GG– motif in the N-terminal trunk, the central signature inhibitory motif –QxVxG– (where x is any amino acid) of the first inhibitory loop, and a W residue charateristic of the second inhibitory loop in the C-terminal region, approx. 30 aa distal from the central inhibitory motif. SlCYS9 differed from the other cystatins by including a long, 11.5-kDa extension at the C terminus (Fig. 2a), similar to the extension of cystatins from other plants isolated in recent years (Fig. 2b).

image

Figure 2. Alignment of the deduced amino acid sequence of SlCYS9 (GenBank accession no. AF198388) with the sequences of SlCYS10 (AF198389), the eighth inhibitory domain of tomato multicystatin, SlCYS8 (AF198390), and other plant cystatins. (a) Alignment of SlCYS8, SlCYS9 and SlCYS10 with OsCYS1 (P09229) and the eighth inhibitory domain of potato multicystatin, StCYS8 (P37842). The predicted signal peptide of SlCYS9 is underlined. Key residues for cystatin activity are in bold. The arrow on the threonine residue (T132) marks the C-terminal end of the truncated version of SlCYS9. (b) Alignment of the C-terminal extension of cystatins from tomato (SlCYS9; this paper), sweet potato (IbCYS1; AF117334), soybean (GmCYS1; D31700), strawberry (FaCYS1; AJ845186), apple (MdCYS1; AY173139), castor bean (RcCYS1; Z49697) and field mustard (BcCYS1; S65071). Identity (id) and similarity (sim) percentages between each extension and the extension of SlCYS9 are given on the right.

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Evolutionary relationships among tomato cystatins and cystatins from other species were visualized by inferring an unrooted phylogenetic tree for the cDNA sequences of 26 plant cystatins (Table 1), using the neighbor-joining distance method of Saitou & Nei, 1987 (Fig. 3). As expected, SlCYS8 formed a clade with the fifth cystatin unit of tomato multicystatin (SlCYS5) and the eight units of potato multicystatin (StCYS1–StCYS8), while SlCYS9 formed a clade with cystatins from different plant families bearing the 11.5-kDa C-terminal extension. SlCYS10, with no C-terminal extension, grouped with Solanaceae multicystatins, but also showed significant homology with cystatins of other clades, suggesting the occurrence of at least three evolutionary distinct cystatin-encoding genes in the tomato genome.

image

Figure 3. Neighbor-joined phylogenetic tree for 26 cystatins from different plant taxa (Table 1). The unrooted tree was reconstructed using a distance matrix generated by Kimura's two-parameter model (Kimura, 1983). The analysis grouped the different cystatins into three clades (boxes), correlated with the taxon of origin (Solanaceae and Poaceae (monocot) cystatins) or the presence of a C-terminal extension (23-kDa cystatins). The tomato cystatins SlCYS8, SlCYS9 and SlCYS10 are in bold.

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SlCYS8, SlCYS9 and SlCYS10 are differentially induced by MeJa and arachidonate

The inducing effects of MeJa and arachidonate on expression of the three cystatin genes in tomato leaves were investigated by Northern blotting. A probe prepared with the cDNA sequence of SlCYS8 hybridized with an mRNA species approx. 2.5 kb in size (not shown), strongly suggesting that this cDNA was indeed encoding the C-terminal part of tomato multicystatin, homologous to the eighth inhibitory domain of potato multicystatin, StCYS8 (Waldron et al., 1993). As shown in Fig. 4(b,c), SlCYS8 was strongly induced by MeJa, but weakly induced by arachidonate. By contrast, SlCYS9 and SlCYS10 transcripts were present at a basal level in nontreated leaves, not induced further by MeJa, but strongly induced by arachidonate (Fig. 4b,c) and other unsaturated fatty acids including linoleic acid and γ-linolenic acid (not shown). As a control, the blots were probed with labelled cDNAs encoding the wound-induced serine-type inhibitor, Pin-II, and the PR-1 protein, protein P4 (Fig. 4a). In agreement with previous reports (Fidantsef et al., 1999; Rivard et al., 2004), the gene for Pin-II was induced by MeJa but not by arachidonate, while the gene for protein P4 was induced by arachidonate but not by MeJa, suggesting that the genes coding for SlCYS9, SlCYS10 and protein P4 were responding in a similar way to the fungal elicitor, presumably via an α-linolenate/jasmonate-independent pathway (Fidantsef et al., 1999). Overall, these observations suggest that MeJa and arachidonate induce the accumulation of distinct cystatins in tomato leaves, via either jasmonate-dependent or -independent pathways.

image

Figure 4. Northern blot analysis for the induction of Pin-II-, protein P4- and cystatin-encoding genes by methyl jasmonate (MeJa) and arachidonic acid (AA) in tomato leaves. (a) mRNA transcripts of the model proteins Pin-II and protein P4 16 h after treatment with 40 or 400 µm MeJa or AA. (b) mRNA transcripts of SlCYS8, SlCYS9 and SlCYS10 16 h after treatment with 400 µm MeJa or AA. (c) Relative accumulation of SlCYS8, SlCYS9 and SlCYS10 mRNA transcripts 0–24 h after induction with 400 µm MeJa or AA. Data are presented as relative values, compared with transcript signals 20 h post-treatment for each gene considered (value 1.0). Membranes were hybridized with 32P-labelled cDNA probes encoding either Pin-II, protein P4, SlCYS8, SlCYS9 or SlCYS10. Equal loading of RNA in each well was controlled by ethidium bromide fluorescence of total RNA fixed onto the membrane. In (c), relative amounts of mRNA transcripts on nitrocellulose membranes were estimated by densitometry using a Microtek ScanMaker II scanner (Microtek Laboratory, Torrance, CA, USA) and the image analysis software nih image 1.6 (National Institutes of Health, Bethesda, MD, USA). CTL, control plants sprayed with 0.125% (v/v) Triton X-100.

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The protease inhibitory profile of SlCYS9 is influenced by its C-terminal extension

To determine whether structural elements such as the C-terminal extension of SlCYS9 could influence the overall inhibitory profile of the inducible complement of cystatins in tomato, the activity of SlCYS9 against cysteine proteases was compared with the activity of SlCYS8, and with the activity of a truncated form, tSlCYS9, generated by removing 103 amino acids at the C terminus of the native inhibitor (arrow, Fig. 2a). The recombinant cystatins were produced in E. coli (with no signal peptide) and assayed for their respective inhibitory potency against papain and herbivorous pest cysteine proteases. The three cystatins were expressed in and purified from E. coli using the GST gene fusion system (Michaud et al., 1994), cleaved from the GST moiety (Fig. 5a), then assayed against papain, the herbivorous insect digestive protease Ldp30 and the major extracellular cysteine proteases of two root parasitic nematodes, Mhp1 and Mip1 (Visal-Shah et al., 2001). As shown in Table 2, the native form of SlCYS9 showed weak activity against papain and Ldp30, giving Ki(app) values in the micromolar range. By contrast, the truncated inhibitor tSlCYS9, structurally related to the model rice cystatin OsCYS1 (Fig. 5b), showed Ki(app) values in the nanomolar range for the same two enzymes, similar to SlCYS8. The same inhibitory pattern was observed for the nematode protease Mhp1, with estimated Ki(app) values in the nanomolar range for SlCYS8 and tSlCYS9, compared with a Ki(app) value in the micromolar range for SlCYS9. Noteworthily, cysteine proteases of the closely related nematodes M. incognita (Mip1; Table 2) and M. javanica (Mjp1; Michaud et al., 1996, not shown), were efficiently inhibited by SlCYS8 but not by SlCYS9 or tSlCYS9, pointing out a differential impact of the C-terminal extension on cystatin inhibitory activity, depending on the target protease assessed.

image

Figure 5. Heterologous expression of SlCYS8, SlCYS9 and tSlCYS9 in Escherichia coli and structural model for tSlCYS9. (a) Recombinant cystatins expressed using the glutathione S-transferase (GST) gene fusion system. The inhibitors were expressed (with no signal peptide) attached to GST, purified with reduced glutathione-embedded agarose beads, and cleaved from the GST affinity partner with human factor Xa. Purity of the preparations was confirmed here by 12% (w/v) SDS–PAGE followed by Coomassie Brilliant Blue staining. Values on the left refer to commercial molecular weight markers. (b) Tertiary structure of tSlCYS9, as inferred from structural coordinates of the model cystatin from rice, OsCYS1 (OC-I: 1EQK in Protein Data Bank; Nagata et al., 2000). The structure was constructed using modeller ver. 6.2 (Sanchez & Sali, 2000) and tested for energy distribution and stereochemical quality using the in-built Energy command of modeller and the procheck program (Laskowski et al., 1993), respectively. tSICYS9 was visualized using swiss-pdb software (Guex & Peitsch, 1997). The resulting model shows the typical structural elements of plant cystatins, including four antiparallel β sheets linked to an α-helix in the N-terminal region, two hairpin inhibitory loops entering the active site of target enzymes, and a protruding N-terminal trunk with the conserved –GG– motif presumably interacting with amino acid side chains of the target enzyme during the inhibitory process (Turk et al., 1997).

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Table 2.  Inhibition of papain and herbivorous pest cysteine proteases by SlCYS8, SlCYS9 and tSlCYS9 (see Materials and Methods)
InhibitorKi(app) (nm)
PapainLdp30Mhp1Mip1
SlCYS8  2.4  7.1 <10 <10
SlCYS9635545>100>100
tSlCYS9 11.1 11.6 <10>100

The C-terminal extension of SlCYS9 also influences its overall structure

Stability assays were carried out with SlCYS8, SlCYS9 and tSlCYS9 to assess the impact of the C-terminal extension on the overall structure of SlCYS9. To this end, the inhibitors were challenged with a larval midgut extract of the Colorado potato beetle, which contains digestive proteases from several mechanistic classes either sensitive or insensitive to plant cystatins (Michaud et al., 1995; Novillo et al., 1997). As seen in Fig. 6, a significant fraction of SlCYS8 and tSlCYS9 was hydrolyzed by the insect-insensitive proteases after incubation for 30 min under the conditions of the assay, the hydrolytic process being almost complete after 60 min (middle and lower panels). By contrast, SlCYS9 showed a very rapid degradation rate, being completely digested within a few seconds after adding the insect extract (Fig. 6, upper panel), with no degradation intermediate detectable on gel. Pre-incubation of the insect extract with the cysteine protease inhibitor E-64 prevented degradation of all three cystatins. By contrast, preincubation with PMSF (a serine-type inhibitor) or pepstatin A (an aspartate-type inhibitor) had only a partial and transient stabilizing effect, indicating that cystatin-insensitive cysteine proteases in the extracts – presumably cathepsin B-like enzymes (Michaud et al., 1995) – were responsible for cleaving the recombinant cystatins.

image

Figure 6. Degradation of SlCYS8, SlCYS9 and tSlCYS9 by digestive proteases of the Colorado potato beetle. The insect extract was preincubated with water (CTL, control), the low-molecular-weight cysteine protease inhibitor E-64, the serine protease inhibitor PMSF, the aspartate protease inhibitor pepstatin A (PEP), or the metalloprotease inhibitor EDTA before incubation with cystatins. Proteolysis was stopped after various periods by the addition of SDS–PAGE sample buffer, and cystatins remaining in the mixtures were visualized on immunoblots using antipotato multicystatin polyclonal antibodies. Purified SlCYS9 was also loaded alone as a control, as it was readily degraded in the presence of the insect proteases.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The main goal of this study was to compare the ability of arachidonate and (methyl) jasmonate to induce the expression of cystatin genes in tomato leaves. Several studies described the differential induction of defense-related genes by these two elicitors in Solanaceae, using as models a number of proteins including the serine-type inhibitors Pin-I and Pin-II, the PR-1 protein P4, and different forms of the metabolic effectors lipoxygenases and 3-hydroxy-3-methylglutaryl coenzyme A reductases (Choi et al., 1992, 1994; Fidantsef & Bostock, 1998; Fidantsef et al., 1999; Rivard et al., 2004). As a complement, we observed here that protease inhibitor- (cystatin-)encoding genes in tomato may respond not only to jasmonate, but also to the fungal elicitor arachidonate, presumably via a jasmonate-independent pathway.

The biological significance for the arachidonate-induced expression of cystatin genes in tomato remains to be clarified, but a protective role against invading pathogens appears plausible. Arachidonate is a potent elicitor of systemic defense responses in Solanaceae (Coquoz et al., 1995; Fidantsef et al., 1999; Weyman et al., 2006), notably triggering the expression of antimicrobial PR proteins in leaves (Fidantsef & Bostock, 1998; Fidantsef et al., 1999). Little information is available about the structural and functional characteristics of fungal proteases (St Leger et al., 1997; ten Have et al., 2004), but the involvement of secreted proteases – including cysteine proteases – during plant tissue infection by P. infestans and other pathogenic fungi is well documented (Ball et al., 1991; Murphy & Walton, 1996; Paris & Lamattina, 1999; Poussereau et al., 2001; ten Have et al., 2004). Strong antifungal effects have also been observed recently in vitro for two 23-kDa, C-tailed cystatins structurally related to SlCYS9 (Martinez et al., 2005b; Christova et al., 2006), again suggesting an antimicrobial role for this arachidonate-induced protein.

Despite these unsolved questions about the roles of SlCYS9 (and SlCYS10) in planta, our data clearly suggest the existence of a dynamic, elicitor-inducible ‘cystatin complex’ in tomato, consisting of at least 10 cystatin inhibitory units, SlCYS1–SlCYS10. These cystatins are induced in leaves in response to various stress signals including wounding, systemin and jasmonate (Bolter, 1993; Jacinto et al., 1998; Wu & Haard, 2000; this study), the fungal elicitor arachidonate (this study), and the bacterial phytotoxin coronatine (Gleddie & Michaud, 2000), a structural analogue of MeJa (Palmer & Bender, 1995). The occurrence of cystatin genes with distinct specificities and modes of induction in the tomato genome suggests the ability of this plant to synthesize cystatin forms active against a variety of (exogenous) cysteine proteases. From a functional viewpoint, the inducible cystatin complex in tomato would thus show plasticity at both the expression and protease inhibitory levels, making it effective and readily functional under a range of biotic stress conditions.

Structural elements such as the C-terminal extension or the N-terminal signal peptide for cellular secretion on SlCYS9 might also contribute to this functional plasticity. In contrast to SlCYS8, which forms insoluble crystals in the cytosol of tomato leaf cells after synthesis (Gleddie & Michaud, 2000), SlCYS9 bears an N-terminal signal peptide that presumably directs its movement through the cell secretory pathway. No additional sorting signal for the vacuole or the endoplasmic reticulum could be detected in SlCYS9 submitted to the WoLF PSORT Prediction database for plant sorting signals (http://psort.hgc.jp), which suggests an extracellular destination for this protein. N-terminal signal peptides for cellular secretion have been described recently for a number of stress- and developmentally regulated cystatins (Womack et al., 2000; Corre-Menguy et al., 2002; Rassam & Laing, 2004; Martinez et al., 2005b, 2005c; Massonneau et al., 2005). In vivo, such signals would allow the plant to accumulate cystatins in vacuoles upon wounding or insect herbivory, or to direct their secretion into the extracellular milieu upon fungal or bacterial attack. Whereas the final destination of SlCYS9 in tomato leaf cells still needs to be confirmed empirically, the apparently distinct intracellular targeting of SlCYS8 and SlCYS9 in MeJa- and arachidonate-treated leaves clearly contributes to the overall picture of a dynamic, multifunctional inducible cystatin complex in tomato.

The C-terminal extension of SlCYS9 might also have a certain influence in vivo. In contrast to an earlier study reporting a negligible impact for the C-terminal extension of a related cystatin from soybean seeds (Misaka et al., 1996), the C-terminal extension of SlCYS9 was shown here to influence strongly both the protease inhibitory potency and the tertiary structure of the protein. The differential stability of SlCYS9 and tSlCYS9 challenged with insect nontarget proteases indicated the occurrence of distinct sites for proteolytic cleavage at the surface of the two cystatin variants, which suggests that the influence of the C-terminal extension on SlCYS9 inhibitory activity was due, at least in part, to a general effect on the overall structure of the inhibitor moiety. At this point, our inhibitory data suggest a repressive, anticystatin effect for this structural element, but the existence of target proteases strongly inhibited by SlCYS9 in vivo, or a proteolytic deletion of the extension following secretion of the protein in the apoplast, cannot be ruled out. Similar extensions at the C terminus of plant cystatins have been described from sources as diverse as soybean seeds, field mustard flower buds, taro corms, strawberry fruits, wheat crowns and senescent leaves of sweet potato (Lim et al., 1996; Misaka et al., 1996; To et al., 1999; Martinez et al., 2005b; Yang & Yeh, 2005; Christova et al., 2006), but no clear function could be attributed to this ubiquitous structural element based on sequence homology searches in gene databases. After detecting the presence of amino acid strings possibly related to sequence motifs conserved among functional (inhibitory) cystatins, Martinez et al. (2005b, 2005c) recently suggested this extension to be a degenerated cystatin sequence resulting from an ancestral gene-duplication event followed by subsequent diverging evolution. Work is under way to assess this idea, also taking into account the reported occurrence in plants of protease inhibitors bearing inhibitor-independent antifeedant or antimicrobial functions (Maskos et al., 1996; Joshi et al., 1999).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Binh Nguyen-Quoc (CRH, Université Laval) and Line Cantin (CRCHUL, Université Laval) for fruitful discussions, and Thierry C. Vrain (Agriculture and Agri-Food Canada, Summerland, BC, Canada) for providing the nematode proteases. This work was supported by the Natural Science and Engineering Research Council of Canada, and by the Fonds Québécois pour la Recherche sur la Nature et les Technologies (Québec). C.G. was the recipient of a postdoctoral fellowship from the Centre de Recherche en Horticulture of Laval University. A.K. was the recipient of a fellowship from the Rockefeller Foundation.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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