JXB Advance Access originally published online on November 10, 2006
Journal of Experimental Botany 2006 57(15):4245-4255; doi:10.1093/jxb/erl200
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RESEARCH PAPER |
Structural and functional diversity within the cystatin gene family of Hordeum vulgare
Laboratorio de Bioquímica y Biología Molecular, Dpto. de Biotecnología-Centro de Biotecnología y Genómica de Plantas-UPM, ETS Ingenieros Agrónomos. Ciudad Universitaria s/n, E-28040 Madrid, Spain
To whom correspondence should be addressed. E-mail: i.diaz{at}upm.es
Received 31 May 2006; Accepted 13 September 2006
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Abstract |
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Phytocystatins are inhibitors of cysteine proteinases from plants putatively involved in defence and as endogenous regulators of protein turnover. Seven genes encoding cystatins (HvCPI-1 to HvCPI-7), identified from EST collections and from an endosperm cDNA library, have been characterized. The intron–exon structure of their corresponding ORFs has been determined and the predicted three-dimensional models for the seven barley cystatins have been established, based on the known crystal structure of oryzacystatin I from rice. Only one out of the seven deduced proteins, HvCPI-7, had sequence variations affecting the three conserved motifs implicated in the enzyme–inhibitor interaction. In three cases, HvCPI-5, HvCPI-6, and HvCPI-7, amino acid differences lead to the prediction of important structural changes in their three-dimensional structures. Northern blot analysis indicated that the seven genes have different expression patterns in barley tissues. The recombinant proteins expressed in Escherichia coli showed distinct inhibitory properties in vitro, with different Ki values, against the three cysteine proteinases tested: papain, cathepsin B, and cathepsin H. Moreover, these recombinant proteins presented differential fungicidal characteristics inhibiting the growth of phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum in vitro. The resulting implications for the structural and functional diversity of the seven barley cystatins studied are discussed.
Key words: Antifungal activity, barley cystatin genes, cysteine proteinase inhibitor, intron–exon structure, three-dimensional structure prediction
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Proteolysis is a complex metabolic process required for protein processing and turnover in which exo- and endo-proteinases play an essential role. Plants contain a high number of proteinases putatively involved in different physiological processes, most of them belonging to the cysteine-, serine-, aspartic-, and metallo-proteinase classes (Rawlings and Barret, 1993). Specific inhibitors, which are involved with the intra- or extracellular control of proteolytic activity, have been described and classified according to the class of proteinases that they inhibit (De Leo et al., 2002).
Phytocystatins (PhyCys) are proteinaceous inhibitors of cysteine proteinases of the papain family C1A specifically from plants (MEROPS peptidase database, http:merops.sanger.ac.uk). They share with animal cystatins three motifs involved in the interaction with their target enzymes: the reactive site QxVxG, one or two glycine residues in the N-terminal part of the protein, and a tryptophan located downstream of the reactive site. In addition, PhyCys have a consensus sequence ([LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N) that conforms to a predicted secondary 
-helix structure and they are devoid of both disulphide bonds and putative glycosylation sites (Margis et al., 1998). Most of the PhyCys are small proteins with a molecular mass ranging from 11 to 16 kDa, although some of them contain a carboxy-terminal extension and have molecular masses of 
23 kDa (Misaka et al., 1996; Martinez et al., 2005b). Moreover, several 87 kDa multicystatins have been also described (Siqueira-Junior et al., 2002).
It has implicated that PhyCys play a role in defence due to their capability to inhibit proteinases from heterologous predators and pathogens, and they have been described as regulators of endogenous proteolytic activities during seed maturation and germination, and in programmed cell death (Solomon et al., 1999; Arai et al., 2002; Corre-Menguy et al., 2002; Belenghi et al., 2003). Their function in defence has been investigated widely, and it is supported by in vitro data on inhibition of digestive proteinases from insect pests and nematodes (Pernas et al., 1998; Koiwa et al., 2000; Siqueira-Junior et al., 2002; Haq et al., 2004), and by the protection that PhyCys genes overexpressed in transgenic plants confer against nematodes, insects, slugs, and viruses (Gutierrez-Campos et al., 1999; Walker et al., 1999; Atkinson et al., 2004; Alvarez-Alfageme et al., 2006). Antifungal and antimite activities have also been described for several PhyCys (Pernas et al., 1999, 2000; Soares-Costa et al., 2002; Martinez et al., 2003a, 2005b; Yang and Yeh, 2005; Christova et al., 2006).
Since the isolation of oryzacystatin I, the first cystatin to be purified, and the cloning of its cDNA from rice seeds (Abe et al., 1987a, b), >80 members from different plant species have been characterized. However, since the genome sequences of Arabidopsis thaliana and Oryza sativa were completed and the EST collections from these and other plants were available, the identification of complete cystatin gene families is increasing. Using bioinformatic resources, Martinez et al. (2005a) have annotated 12 different cystatin genes in rice, seven in Arabidopsis and seven ESTs in barley, and have proposed phylogenetic relationships among the cystatins from these plant species. Similarly, Massonneau et al. (2005) have published the sequence analysis and expression profiles of 10 maize cystatins in response to abiotic stresses.
In barley, the cDNA and genomic clones encoding the cystatin HvCPI-1 have already been characterized. The corresponding gene (Icy1) is ubiquitously expressed and its mRNA in leaves and roots is induced in response to anaerobiosis and cold shock (Gaddour et al., 2001). In the germinating seed, the Icy1 transcript accumulation decreases and its expression in aleurone cells is repressed by gibberellin (Martinez et al., 2003b, 2005c). The recombinant HvCPI-1 protein expressed in Escherichia coli is an efficient inhibitor of papain, chymopapain, ficin, and cathepsin B, as well as of the proteinases present in midgut extracts from the Colorado potato beetle (Gaddour et al., 2001; Martinez et al., 2003a; Alvarez-Alfageme et al., 2006). HvCPI-1 inhibits the growth of phytopathogenic fungi such as Botritys cinerea, Plectosphaerella cucumerina, and Colletotrichum graminicola in vitro (Martinez et al., 2003a).
The present study describes the molecular characterization of six new barley cystatin genes and makes comparisons with HvCPI-1, which has already been reported by Gaddour et al. (2001). Besides establishing the intron–exon organization of these genes and predicting the three-dimensional structure of their deduced proteins by bioinformatics analysis, their expression patterns in barley are explored. The inhibitory capacities of the corresponding recombinant proteins against several commercial cysteine proteinases and their antifungal properties towards the phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum have been determined. Possible structure–function relationships are discussed.
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Isolation and Southern blot analysis of barley cystatin clones
cDNAs encoding HvCPI-2 to HvCPI-7 were completely sequenced from Hordeum vulgare L. EST collection (The Clemson University Genomics Institute). The cDNA encoding HvCPI-1 had been purified previously from a developing barley endosperm cDNA library (Gaddour et al., 2001).
For Southern blot analysis, total DNA was isolated from etiolated leaves of barley cv. Bomi after 7 d of germination, following the procedure described by Taylor and Powell (1982). DNA samples were digested with HindIII, SacI, and XbaI restriction enzymes, electrophoretically separated on 0.7% agarose gels, and transferred onto Hybond N+ membranes (Amersham). Hybridization was performed under stringent conditions following standard protocols (Sambrook and Russell, 2001). The specific probes corresponding to the seven Icy genes were obtained by PCR using specific oligonucleotides derived from the 3' ends of the corresponding cDNAs to avoid cross-hybridization (Table 1).
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To determine the presence of introns, within the ORFs PCR amplification was carried out using total DNA from barley cv. Bomi leaves as template and the oligonucleotides indicated in Table 2. The PCR products were cloned in pGEM-Teasy vector (Promega) and then sequenced. Comparisons between the sequences of these fragments and those amplified from cDNAs were carried out. All the sequences were determined on both strands using vector-specific primers and an automated DNA sequencer (ABI PRISM TM 3100; Applied Biosystems).
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Cystatin sequence comparisons and analyses
Analysis of DNA and comparisons of deduced protein sequences were carried out with the current bioinformatics tools. Alignments of protein sequences within the Poaceae were performed at the DNA Data Bank of Japan (http://www.ddbj.nlg.ac.jp), and binary sequence comparisons (identity/similarity) between barley cystatins were made at the Network Protein Sequence Analysis, Pole Bioinformatique Lyonnais (http://npsa-pbil.ibcp.fr). In both cases, the CLUSTAL W program (Higgins et al., 1994), with default parameters and alignments manually corrected, was used. Signal peptide analysis was performed using the SignalP version 3.0 (http://www.cbs.dtu.dk/services/SignalP) program (Bendtsen et al., 2004). Phylogenetic analysis was conducted by the Molecular Evolutionary Genetic Analysis (MEGA) software, version 3.0 (Kumar et al., 2004), obtaining the phylogenetic tree from Neighbor–Joining analysis using the complete deletion and Poisson correction settings.
The three-dimensional structures of the barley cystatins were modelled by the automated SWISS-MODEL program (Peitsch, 1995, 1996). The known crystal structure of the rice oryzacystatin I (OC-I) (PDB identifier 1EQK [PDB] ) was used to construct the homology-based models. Structure analysis was performed using the RasMol 2.7 program (Sayle and Milner-White, 1995).
Northern blot analysis
For northern blots, seeds of barley (Hordeum vulgare) cv. Bomi were germinated at 22 °C in the dark for 7 d, and samples of leaves and roots were collected from the resulting plants. Developing endosperm, 10–22 d after flowering (daf), and immature embryos (18 daf) were prepared from kernels of plants grown in a greenhouse at 18 °C under a 18/6 day/night photoperiod. Mature embryos were isolated from dry barley Bomi seeds. Germinating embryos were obtained from imbibed seeds incubated for 8–48 h at 22 °C in the dark. All samples were frozen in liquid N2 and stored at –70 °C until used for RNA extraction.
Total RNA was extracted by the phenol/chloroform method, followed by precipitation with 3 M LiCl (Lagrimini et al., 1987). Denatured total RNA samples (8 µg each) were electrophoresed in 1.2% agarose gels containing 7% formaldehyde and blotted onto Hybond N+ membranes (Amersham). Hybridization and washings were carried out under stringent conditions, following standard procedures (Sambrook and Russell, 2001). The same 32P-labelled probes used in Southern blot analysis were used in northern blot assays.
Expression and purification of recombinant cystatins from E. coli
The cDNA fragments spanning the whole cystatin ORFs, devoid of their signal peptide sequences (HvCPI-1 to HvCPI-7 proteins), were amplified by PCR with appropriate oligonucleotide primers (Table 2). The PCR products were inserted in frame into the fusion expression vector pRSETB (Invitrogen). Escherichia coli BL21 CodonPlus (Stratagene) containing the recombinant plasmids were grown at 37 °C to an OD550 of c. 0.5 and induced with 1 mM IPTG (isopropyl ß-D-thiogalactopyranoside) for 2 h. Bacterial cells overexpressing the seven barley cystatins were harvested and processed. The fusion proteins with a histidine tail were purified using a His-Bind Resin (Novagen) following the manufacturer's instructions, and purification checked by SDS-PAGE.
Cysteine proteinase inhibitory activity
Inhibitory activity of the seven recombinant HvCPI proteins purified from E. coli was tested against commercial proteinases from Sigma: papain (EC 3.4.22.2
[EC]
), cathepsin B (EC 3.4.22.1
[EC]
), and cathepsin H (EC 3.4.22.16
[EC]
), using BANA (N-benzoyl-DL-arginine-ß-naphthylamide) as substrate, essentially as described by Gaddour et al. (2001). Protein concentrations were quantified by the Bio-Rad kit, with bovine serum albumin as standard, and the Ki values were determined from Dixon plots (1/V versus [I]).
Fungal growth inhibitory assays
Fungal strains (Fusarium oxysporum and Botrytis cinerea) taken from the laboratory collection, were maintained on potato dextrose agar medium. The in vitro inhibition assays were performed as described by Martinez et al. (2003a, 2005b). Approximately 103 spores of each strain were incubated in 100 µl of one-third potato dextrose broth at 28 °C for 48 h and agitated in the absence or in the presence of different concentrations (1.5 µM, 3 µM, 4.5 µM, 6 µM) of the recombinant barley cystatins (HvCPI-1 to HvCPI-7). The incubation was carried out in sterile microtitre plates and fungal growth was monitored by measuring absorbance at 492 nm and by observing them with a microscope. Results were expressed as a percentage of growth relative to that in the absence of the inhibitory agent, and the effective cystatin concentration for 50% growth inhibition (EC50) for each cystatin was calculated.
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Results |
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Gene copy number and intron–exon organization of barley cystatin genes
Total DNA from young etiolated leaves of barley cv. Bomi was digested with several restriction endonucleases and analysed by Southern blot under stringent conditions. Probes used were cDNA fragments derived from 3' ends of the corresponding cDNAs obtained by PCR (Table 1). As shown in Fig. 1a, the pattern of a single hybridization band, obtained in all cases, indicated that only one copy of each cystatin gene was present in the barley genome.
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The comparisons between the cDNA sequences and the PCR fragments amplified from genomic DNA have made it possible to establish that genes encoding HvCPI-1, HvCPI-2, and HvCPI-3 have one intron each within their ORFs. Although these introns are variable in length they are located at the same position between sequences encoding the LARFAV and the reactive site QxVxG. Genes encoding HvCPI-5, HvCPI-6, and HvCPI-7 had no introns within their ORFs, and the gene encoding HvCPI-4, the largest cystatin protein of this group, has three introns of 131, 1488, and 90 nt, respectively. The first intron is located in a similar position to those introns found in cystatin genes containing only a single intron, and introns 2 and 3 are positioned after the conserved PW residues located downstream of the reactive site (Fig. 1b).
The molecular mass calculated for six of the seven barley cystatins was in the 11–16.6 kDa range; only the HvCPI-4 protein, which has a long C-terminal extension, has a molecular mass (23.5 kDa) outside this range. The amino acid number varied from 107 to 243, and all cystatins, with the exception of HvCPI-1, contained a predicted signal peptide of 16–29 amino acids (Fig. 1b).
Comparison and phylogenetic analysis of the PhyCys proteins within the Poaceae
In order to evaluate the phylogenetic relationships of the barley cystatins with other PhyCys within the Poaceae, the deduced sequence, including the signal peptide, of the seven barley proteins HvCPI-1 to HvCPI-7 (genes Icy1 to Icy7; Martinez et al., 2005a) were compared with the amino acid sequences of the PhyCys proteins from the Poaceae family found in the databanks. An unrooted phylogenetic tree was constructed using the Neighbor–Joining method (Fig. 2). Bootstrap values higher than 50% allowed the tree to be organized into three major clusters, A, B, and C. These groups correlate with the major clusters of homologous genes obtained previously in a phylogenetic tree using the Arabidopsis, rice, and barley cystatins (Martinez et al., 2005a). A survey of the Poaceae phylogenetic tree indicates that the seven barley cystatins studied, although probably not representing the total number of cystatins of the barley genome, are representative of the three different clusters of homologous cystatin genes (Fig. 2).
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The pairwise percentage identity, determined for the mature deduced protein sequence of the seven barley cystatins, indicated that HvCPI-1 and HvCPI-2 shared the highest identity (62.5 identical; 76.7 similar residues), while the percentage of identity decreased to 20.5% (40.1 similar residues) between HvCPI-4 and HvCPI-5 (Table 3).
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Structural analysis of the barley cystatins
To search for amino acid variants that could lead to differences in the inhibitory capability of the barley cystatins, an alignment of all the barley cystatin sequences was performed by the CLUSTAL W program and appears in Fig. 3a, which includes OC-I from rice, the only phytocystatin whose three-dimensional structure has been determined (Nagata et al., 2000). OC-I is likely to be the orthologue of HvCPI-1 from barley (57.8% identical; 76.7% similar residues). All the protein signatures responsible for the cysteine proteinase inhibitory properties were conserved along the sequence of the seven barley cystatins compared, with the exception of HvCPI-7, which lacks the tryptophan present in the second loop (W104
F) between ß-sheets, ß4, and ß5, and the glycine in the fifth position of the conserved QxVxG reactive site (GE). In addition to the C-terminal extensions of variable length found in HvCPI-3 and HvCPI-4 cystatins, differences in the extent of the amino acid sequences corresponding to the loops connecting the ß-sheets and the -helix were also found for several cystatins (Fig. 3a).
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The predicted three-dimensional structures of the barley cystatins were established using as a molecular model the known crystal structure of the rice OC-I cystatin (Fig. 3b). Although these structures were predicted with variable degrees of accuracy, those for HvCPI-1 to HvCPI-4 share so many identical residues with rice OC-I that their protein structures must be similar, conserving the
-helix spanning the LARFAV motif and the four main ß-sheets (ß2, ß3, ß4, ß5). However, the other three barley cystatins (HvCPI-5 to HvCPI-7) show significant variations in their predicted three-dimensional structures. The N-terminal region of the HvCPI-5 protein, containing the -helix, could not be modelled by the program probably due to the longer sequence located between the putative -helix and the second ß-sheet. Besides, an extensive loop was predicted between its ß3 and ß4 sheets. Analysis of the HvCPI-6 model revealed a shorter loop than the rest of the cystatins. This loop contains the tryptophan involved in the interaction with the cysteine proteinases. The loss of an amino acid residue between the ß4 and ß5 sheets could be responsible for this variation. Finally, the predicted structure of the HvCPI-7 protein shows some distortions in the region of the ß3 sheet, probably due to the insertion of a glutamic acid instead of the G residue of the conserved motif QxVxG. Moreover, in the loop between the ß4 and ß5 sheets there are two more residues (ES), preceding positions of the conserved tryptophan that is changed (WF) in this cystatin.
Expression analysis of the barley cystatin mRNAs
The pattern of transcript accumulation for the barley Icy1 to Icy7 genes was investigated by northern blot analysis in the major barley tissues. Total RNA was isolated from developing endosperm (10–22 daf) and immature embryos (18 daf), from dry and germinating embryos (8–48 h after imbibition), and from 7-d-old leaves and roots. After electrophoresis and blotting, each filter was hybridized with the same specific probes as in the Southern blots described above. As shown in Fig. 4, the barley cystatin genes present different expression patterns. Icy1, Icy3, and Icy4 transcripts were expressed ubiquitously in all organs of the barley plant, although with different relative intensities. The expression of the Icy1 gene was abundant in developing endosperm, mature embryos, and roots, whereas Icy3 and Icy4 genes showed their highest expression levels in roots. The Icy2 mRNA accumulated mainly in germinating embryos and it was also detected in developing endosperm, leaves, and roots. Genes Icy5, Icy6, and Icy7 were preferentially expressed in the seeds: Icy6 and Icy7 transcripts were abundantly expressed in developing endosperm, while Icy5 mRNAs did accumulate at high levels in germinating embryos, although they were also detected in immature and mature embryos.
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Differential cysteine proteinase inhibition and fungicidal properties of the recombinant barley cystatins
The recombinant barley cystatins HvCPI-1 to HvCPI-7 were expressed in E. coli as fusion proteins, purified, and assayed against papain, cathepsin B, and cathepsin H, using BANA as substrate. Their Ki values against those enzymes were determined as previously described by Gaddour et al. (2001) and differences were shown against the different cysteine proteinases tested (Table 4). For four of the cystatins (HvCPI-1, HvCPI-2, HvCPI-3, and HvCPI-5) the estimated Ki value against papain was of the same order of magnitude (10–8 M), while the Ki values for HvCPI-4 and HvCPI-6 were 2.2x10–7 and 1.7x10–9 M, respectively. Concerning the inhibitory capacity towards cathepsins B and H, the cystatins HvCPI-1 and HvCPI-6 were good inhibitors of both cathepsins, as shown by the 10–6 M estimated anticathepsin B Ki data and the 3.7x10 –8 M and 6.2x10 –7 M Ki values for cathepsin H, respectively. The cystatin HvCPI-5 was the best inhibitor of cathepsin B (Ki=4.3x10–7 M), among the seven cystatins tested. No inhibitory activity against the enzymes mentioned was detected when HvCPI-7 cystatin was used.
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To study the effect of the seven barley cystatins on the growth of the phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum, in vitro bioassays were performed with the seven recombinant HvCPI-1 to HvCPI-7 cystatins. The assays were carried out by adding increasing concentrations of each cystatin to the culture medium of both fungi, and antifungal activity was measured by the inhibition of mycelium development. Six of the seven barley cystatins assayed were able to inhibit the fungal growth, HvCPI-2 and HvCPI-6 being the two best fungicidal proteins with effective concentration for 50% growth inhibition (EC50) <1.5 µM for both fungi (Table 4). The strongest inhibitory effect on B. cinerea mycelium growth was by HvCPI-2 (EC50: 0.91 µM), whereas the F. oxysporum growth was efficiently inhibited by the HvCPI-2, HvCPI-3, and HvCPI-6 proteins with EC50 values of c. 1 µM (Table 4; Fig. 5b). HvCPI-7 did not inhibit the fungal growth of any of the phytopathogenic fungi tested, even at 6.0 µM, the highest concentration assayed (Fig. 5b). The effects that the seven barley cystatins exerted on the growth of B. cinerea and F. oxysporum were corroborated by observations made with a microscope (Fig. 5a). As expected, no inhibition of growth was observed with the protein purified from E. coli, which had been transformed with the expression vector without insert, used as a negative control (data not shown).
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Discussion |
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Analyses of the structural diversity within several proteinase inhibitor gene families have recently been reported, including comparative phylogenetic analyses of cystatins from arabidopsis, rice, and barley (Martinez et al., 2005a). However, functional analyses of plant enzyme inhibitor families have been described in only three groups of Kunitz-type inhibitor genes from potato tubers and in a novel type of Bowman–Birk inhibitor gene family from rice (Heibges et al., 2003; Qu et al., 2003). The molecular and functional characterization of several members of a cystatin family from plants is presented here for the first time. Seven full-length cDNA sequences from barley encoding cystatins have been analysed and their functional properties correlated with their structural and molecular features.
The seven barley cystatin genes described are single copy genes that belong to three major classes, A, B, and C, of a phylogenetic tree, based on the deduced amino acid sequence comparison among cystatins within the Poaceae. This phylogenetic classification, together with their expression patterns and the inhibitory properties of the recombinant proteins against proteinases and phytopathogenic fungi, has made it possible to group cystatins into two functional groups: one group to include cluster A phytocystatins and the other group the phytocystatins allocated to clusters B and C.
The first evidence that supports the suggested dichotomy was the expression pattern of barley cystatin genes carried out by northern blot analysis. While the transcripts of Icy1, Icy3, and Icy4 (cluster A) were present in all tissues analysed and the transcripts of Icy2 (cluster A) accumulated in germinating embryos and vegetative tissues, the Icy5, Icy6, and Icy7 genes (clusters B and C) were only expressed in seeds, being the Icy6 and Icy7 mRNAs preferentially detected in the developing endosperms. The other cystatin genes clustered in group A, whose expression profile has already been reported, such as CCI, CCII, CC3, CC4, and CC5 from maize and WC1 and WC4 from wheat (Kuroda et al., 2001; Massonneau et al., 2005), are also ubiquitously expressed. It is possible that a broad expression pattern is related to less specialized functions, being simultaneously involved in the regulation of endogenous or heterologous cysteine proteinases, whereas a tissue-specific expression would support a more specialized role against specific cysteine proteinases. Moreover, the barley cystatin genes included in group A contained at least one intron in their structure, while the gene organization of Icy5, Icy6, and Icy7 had no introns within their ORFs.
The analysis of the predicted three-dimensional structures of the cystatin proteins, belonging to the first group (cluster A), revealed that HvCPI-1, HvCPI-2, HvCPI-3, and HvCPI-4 all showed a structure which is similar to rice OC-I and contain an -helix and at least four antiparallel ß-sheets (ß1, ß2, ß3, ß4). The inhibitory capacity of these four barley cystatins against papain, cathepsin B, and cathepsin H showed the existence of different specificities. HvCPI-1, HvCPI-2, and HvCPI-3 recombinant cystatins had Ki values towards papain of the same order of magnitude (10–8 M), whereas the Ki for HvCPI-4 was one order of magnitude higher (10–7 M), which could be related to the existence of a longer C-terminal tail.
Other cereal cystatins in cluster A, such as those from rice (OC-I, OC-II), corn (CCI), wheat (WC1, WC4), and sugarcane, are good inhibitors of cathepsin B and cathepsin H (Kondo et al., 1990; Abe et al., 1994; Kuroda et al., 2001; Oliva et al., 2004). These data suggest that minor modifications in the sequence and structure of the inhibitor affect its inhibitory properties. According to high amino acid identity (similarity), HvCPI-1 and HvCPI-2 are most probably the orthologues of rice cystatins OC-II and OC-I, respectively (Martinez et al., 2005a). Experimental data suggest that these cystatins could be implicated both in the regulation of endogenous protein turnover and as defence proteins against pests (Arai et al., 2002), and support the fact that the cystatins included in group A have a wider range of target enzymes.
The three cystatins (HvCPI-5, HvCPI-6, and HvCPI-7) belonging to groups B and C present significant variations in their sequence, when compared with OC-I and, consequently, their three-dimensional structures are less accurate to predict using bioinformatics tools. The HvCPI-5 modelling predicts an extensive loop between its ß3 and ß4 sheets and a longer sequence located between the putative -helix and the ß2 sheet. At the same time, this cystatin has the highest activity against cathepsin B (Ki=4.3x10–7 M) among the seven cystatins analysed. Likewise, the cystatin HvCPI-6 was a strong inhibitor of papain with a Ki=1.7x10–9 M, and it also inhibits cathepsin B and H, although to a lower degree. The structure of HvCPI-6 reveals the presence of a shorter loop containing the conserved tryptophan, involved in the interaction with its target proteinases. HvCPI-5 and HvCPI-6 proteins were clustered in groups B and C, together with eight proteins from rice and five from corn of unknown function. As cereal seeds are quite susceptible to insect and fungal attack, the fact that Icy5 and Icy6 genes were abundantly and specifically expressed in seeds supports the idea of the evolution of the genes of this cluster as a major implication in the defence mechanisms against pests and pathogens. HvCPI-7 cystatin is included within group C. This recombinant HvCPI-7 protein was unable to inhibit the three cysteine proteinases assayed. Several sequence variations could be responsible for this effect: the reactive site motif of HvCPI-7, QIVAE, had an amino acid substitution that changes a glycine into a negatively charged glutamic acid residue, which probably disrupts the interaction with the cysteine proteinases, and it lacks the tryptophan residue in the second half of the molecule, whose interaction with papain has been well documented (Koiwa et al., 2001). These changes did affect the predicted structure of HvCPI-7, with a clear distortion, both in the region of the ß3 sheet and in the loop between the ß4 and ß5 sheets. However, as two cystatin variants with reactive sites, DVVSA and NTSSA, with a low affinity against papain but a high inhibitory effect against cysteine proteinases from the coleopteran insect Acanthoscelides obtectus have been found (Melo et al., 2003), the possibility that HvCPI-7 could have inhibitory effects against specific cysteine proteinases cannot be dismissed.
The antifungal activity of PhyCys against phytopathogenic fungi has already been reported in cystatins from barley, chestnut, strawberry, sugarcane, taro, tomato, and wheat (Pernas et al., 1999; Siqueira-Junior et al., 2002; Soares-Costa et al., 2002; Martinez et al., 2003a, 2005b; Yang and Yeh, 2005; Christova et al., 2006). However, the mechanism by which PhyCys inhibits mycelium growth is not yet understood. Although Yang and Yeh (2005) have suggested that the inhibition of growth caused by the tarocystatin on the phytopathogenic fungus Sclerotium rolfsii was related to the direct inhibition of a fungal cysteine proteinase, Martinez et al. (2003a) have demonstrated, through site-directed mutagenesis of HvCPI-1, that the inhibition of the plant-pathogenic fungus Botrytis cinerea was not associated with its cysteine proteinase inhibitory properties. With the exception of HvCPI-7, all the barley cystatins studied were able to inhibit the fungal growth of B. cinerea and F. oxysporum, although with different EC50 values. The best antifungal barley cystatins were HvCPI-2 against B. cinerea and HvCPI-3 against F. oxysporum, while HvCPI-5 was least effective as a fungal inhibitor. It was not possible to detect any activity for HvCPI-7.
In spite of features in common and the conservation of the critical amino acid residues involved in the interaction with their target proteinases, the different barley cystatins described here have different functions, which could be related to their different structural features. These different inhibitory properties and a differential expression pattern could indicate that some of these proteins had evolved rapidly to inhibit specific cysteine proteinases and probably act as defence proteins against specific pests. A second group would comprise more ancient proteins which are probably involved in the endogenous regulation of plant enzymes, and with a new specific role in plant defence. Futhermore, the antifungal properties exhibited by certain barley cystatins combined with their capacity to inhibit different cysteine proteinases makes them good candidates for use as transgenes against agronomically important insect pests and fungal pathogens.
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Acknowledgements |
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We thank Mar Gonzalez for technical assistance. The financial support from the Ministerio de Educación y Ciencia of Spain (BFU2005–00603) is gratefully acknowledged.
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Footnotes |
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* Present address: Dpto. Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Carretera de La Coruña, Km 7.5, E-28040 Madrid, Spain.
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References |
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Abe M, Abe K, Iwabuchi K, Domoto C, Arai S. (1994) Corn cystatin I expressed in Escherichia coli: investigation of its inhibitory profile and occurrence in corn kernels. Journal of Biochemistry 116:488–492.
Alvarez-Alfageme F, Martinez M, Pascual-Ruiz S, Castañera P, Diaz I, Ortego F. (2006) Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant. Transgenic Research (in press).
Abe K, Emori Y, Kondo H, Suzuki K, Arai S. (1987a) Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin): homology with animal cystatins and transient expression in the ripening process of rice seeds. Journal of Biological Chemistry 262:16793–16797.
Abe K, Kondo H, Arai S. (1987b) Purification and characterization of a rice cysteine proteinase inhibitor. Agricultural and Biological Chemistry 51:2763–2768.[Web of Science]
Arai S, Matsumoto I, Emori Y, Abe K. (2002) Plant seed cystatins and their target enzymes of endogenous and exogenous origin. Journal of Agricultural and Food Chemistry 50:6612–6617.[CrossRef][Web of Science][Medline]
Atkinson HJ, Grimwood S, Johnston K, Green J. (2004) Prototype demonstration of transgenic resistance to the nematode Radopholus similes conferred on banana by a cystatin. Transgenic Research 13:135–142.[CrossRef][Web of Science][Medline]
Belenghi B, Acconcia F, Trovato M, Perazzolli M, Bocedi A, Polticelli F, Ascenzi P, Delledonne M. (2003) AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death. European Journal of Biochemistry 270:2593–2604.[Web of Science][Medline]
Bendtsen JD, Nielsen H, von Heijne G, Brunak S. (2004) Improved prediction of signal peptide: signal P 3.0. Journal of Molecular Biology 34:783–795.
Christova PK, Christov NK, Imai R. (2006) A cold inducible multidomain cystatin from winter wheat inhibits growth of the mold fungus, Microdochium nivale. Planta 223:1207–1218.[CrossRef][Web of Science][Medline]
Corre-Menguy F, Cejudo FJ, Mazubert C, Vidal J, Lelandais-Biere C, Torres G, Rode A, Hartmann C. (2002) Characterization of the expression of a wheat cystatin gene during caryopsis development. Plant Molecular Biology 50:687–698.[CrossRef][Web of Science][Medline]
De Leo F, Volpicella M, Licciulli F, Liuni S, Gallerani R, Ceci LR. (2002) PLANT-PIs: a database for plant protease inhibitors and their genes. Nucleic Acids Research 30:347–348.
Gaddour K, Vicente-Carbajosa J, Lara P, Isabel-LaMoneda I, Diaz I, Carbonero P. (2001) A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli. Plant Molecular Biology 45:599–608.[CrossRef][Web of Science][Medline]
Gutierrez-Campos R, Torres-Acosta J, Saucedo-Arias LJ, Gomez-Lim MA. (1999) The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nature Biotechnology 17:1223–1226.[CrossRef][Web of Science][Medline]
Haq SK, Atif SM, Khan RH. (2004) Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection. Archives of Biochemistry and Biophysics 431:145–159.[CrossRef][Web of Science][Medline]
Heibges A, Salamini F, Gebhardt C. (2003) Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.). Molecular Genetics and Genomics 269:535–541.[CrossRef][Web of Science][Medline]
Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673–4680.
Koiwa H, D'Urzo MP, Assfalg-Machleidt I, Zhu-Salzman K, Shade RE, An H, Murdock LL, Machleidt W, Bressan RA, Hasegawa PM. (2001) Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of cysteine proteinases. The Plant Journal 27:383–391.[CrossRef][Web of Science][Medline]
Koiwa H, Shade RE, Zhu-Salzman K, D'Urzo MP, Murdock LL, Bressan RA, Hasegawa PM. (2000) A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera). FEBS Letters 471:67–70.[CrossRef][Web of Science][Medline]
Kondo H, Abe K, Nishimura I, Watanabe H, Emori Y, Arai S. (1990) Two distinct cystatin species in rice seeds with different specificities against cysteine poteinases. Journal of Biological Chemistry 265:5832–5837.
Kumar S, Tamura K, Nei M. (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinformatics 5:150–163.
Kuroda M, Kiyosaki T, Matsumoto I, Misaka T, Arai S, Keiko A. (2001) Molecular cloning, characterization, and expression of wheat cystatins. Bioscience, Biotechnology and Biochemistry 65:22–28.[CrossRef][Medline]
Lagrimini LM, Burkhart W, Moyer M, Rosthein S. (1987) Molecular cloning of complementary DNA encoding the lignin forming peroxidase from tobacco: molecular analysis and tissue-specific expression. Proceedings of the National Academy of Sciences, USA 84:7542–7546.
Margis R, Reis EM, Villeret V. (1998) Structural and phylogenetic relationships among plant and animal cystatins. Archives of Biochemistry and Biophysics 359:24–30.[CrossRef][Web of Science][Medline]
Martinez M, Abraham Z, Carbonero P, Diaz I. (2005a) Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley. Molecular Genetics and Genomics 273:423–432.[CrossRef][Web of Science][Medline]
Martinez M, Abraham Z, Gambardella M, Echaide M, Carbonero P, Diaz I. (2005b) The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties. Journal of Experimental Botany 56:1821–1829.
Martinez M, Lopez-Solanilla E, Rodriguez-Palenzuela P, Carbonero P, Diaz I. (2003a) Inhibition of plant-pathogenic fungi by the barley cystatin HvCPI (gene Icy) is not associated with its cysteine proteinase inhibitory properties. Molecular Plant–Microbe Interactions 16:876–883.
Martinez M, Rubio-Somoza I, Carbonero P, Diaz I. (2003b) A cathepsin B-like cysteine protease gene from Hordeum vulgare (gene CatB) induced by GA in aleurone cells is under circadian control in leaves. Journal of Experimental Botany 54:951–959.
Martinez M, Rubio-Somoza I, Fuentes R, Lara P, Carbonero P, Diaz I. (2005c) The barley cystatin gene (Icy) is regulated by DOF transcription factors in aleurone cells upon germination. Journal of Experimental Botany 56:547–556.
Massonneau A, Condamine P, Wisniewski JP, Zivy M, Rogowsky PM. (2005) Maize cystatins respond to developmental cues, cold stress and drought. Biochemica et Biophysica Acta 1729:186–199.[Medline]
Melo FR, Mello MO, Franco OL, Rigden DJ, Mello LV, Genú AM, Silva-Filho MC, Gleddie S, Grossi-de-Sá MF. (2003) Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases. Biochimica et Biophysica Acta 1651:146–152.[Medline]
Misaka T, Kuroda M, Abuchi K, Abe K, Arai S. (1996) Soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin. European Journal of Biochemistry 240:609–614.[Web of Science][Medline]
Nagata K, Kudo N, Abe K, Arai S, Tanokura M. (2000) Three-dimensional solution of oryzacystatin-I, a cysteine proteinase inhibitor of rice, Oryza sativa L. japonica. Biochemistry 39:14753–14760.[CrossRef][Medline]
Oliva MLV, Carmona AK, Andrade SS, Cotrin SS, Soares-Costa A, Henrique-Silva F. (2004) Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane. Biochemical and Biophysical Research Communications 320:1082–1086.[CrossRef][Web of Science][Medline]
Peitsch M. (1995) Protein modeling by e-mail. Biotechnology 13:658–660.[CrossRef]
Peitsch M. (1996) ProMod and Swiss-Model: internet-based tools for automated comparative protein modeling. Biochemical Society Transactions 224:274–279.
Pernas M, Lopez-Solanilla E, Sanchez-Monge R, Salcedo G, Rodriguez-Palenzuela P. (1999) Antifungal activity of a plant cystatin. Molecular Plant–Microbe Interactions 12:624–627.
Pernas M, Sanchez-Monge R, Gomez L, Salcedo G. (1998) A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests. Plant Molecular Biology 38:1235–1242.[CrossRef][Web of Science][Medline]
Pernas M, Sanchez-Monge R, Lombardero M, Ateaga C, Castañera P, Salcedo G. (2000) Der p1 and Der f1, the highly related and major allergens from house mites, are differentially affected by a plant cystatin. Clinical Experimental Allergy 30:972–978.[CrossRef]
Qu L-J, Chen J, Liu M, et al. (2003) Molecular cloning and functional analysis of a novel type of Bowman-Birk inhibitor gene family in rice. Plant Physiology 133:560–570.
Rawlings ND and Barret AJ. (1993) Evolutionary families of peptidases. Biochemical Journal 290:205–218.
Sambrook J and Russell DW. (2001) Molecular cloning: a laboratory manual 3rd edn (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Sayle R and Milner-White EJ. (1995) RasMol: biomolecular graphics for all. Trends in Biochemical Sciences 20:374.[CrossRef][Web of Science][Medline]
Siqueira-Junior CL, Fernandes KVS, Machado OLT, Cunha M, Gomes VM, Moura D, Jacinto T. (2002) 87 kDa tomato cystatin exhibits properties of a defence protein and forms crystals in prosystemin over-expressing transgenic plants. Plant Physiology and Biochemistry 40:247–254.[CrossRef]
Soares-Costa A, Beltramini LM, Thiemann OH, Enrique-Silva F. (2002) A sugarcane cystatin: recombinant expression, purification and antifungal activity. Biochemical and Biophysical Research Communications 296:1194–1199.[CrossRef][Web of Science][Medline]
Solomon M, Belenghi B, Delledonne M, Levine A. (1999) The involvement of cysteine proteases and protease inhibitor genes in programmed cell death in plants. The Plant Cell 11:431–444.
Taylor B and Powell A. (1982) Isolation of plant DNA and RNA. Focus 4:4–6.
Walker AJ, Urwin PE, Atkinson HJ, Brain P, Glen DM, Shewry PR. (1999) Transgenic Arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug Deroceras reticulatum (Muller). Transgenic Research 8:95–103.[CrossRef][Web of Science][Medline]
Yang AH and Yeh KW. (2005) Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1). Planta 221:493–501.[CrossRef][Web of Science][Medline]
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