Journal of Experimental Botany, Vol. 54, No. 384, pp. 951-959,
March 1, 2003
© 2003 Oxford University Press
A cathepsin B-like cysteine protease gene from Hordeum vulgare (gene CatB) induced by GA in aleurone cells is under circadian control in leaves
Received 8 August 2002; Accepted 4 November 2002
Laboratorio de Bioquímica y Biología Molecular, Dpto. de Biotecnología, ETSI Agrónomos-UPM, Ciudad Universitaria s/n, 28040 Madrid, Spain
1 The nucleotide sequence data reported will appear in the EMBL Nucleotide Sequence Database under accession number AJ310426.
2 To whom correspondence should be sent. Fax:+34 913365757. e-mail: isabel{at}bit.etsia.upm.es
Abbreviations: GA, gibberellin; ABA, abscisic acid.
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Abstract |
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A barley cDNA clone encoding a putative cysteine protease with sequence homology to cathepsin B from mammalian cells has been characterized. This barley gene (CatB) is ubiquitously expressed, its mRNA being detected in leaves and roots, immature, mature and germinating embryos, in developing endosperms, and in aleurones upon germination, as assessed by northern blot analysis. The CatB mRNA expression in leaves increased by cold shock (6 °C), was not affected by wounding, and was under circadian control. These transcripts increased in the aleurone upon germination, whereas those for a cystatin encoding gene (Icy), that inhibits commercial cathepsin B in vitro, decreased. Gibberellin (GA) treatment of isolated barley aleurones induced and abscisic acid (ABA) repressed the steady-state levels of CatB mRNA, while Icy expression had an opposite pattern of mRNA accumulation in aleurones treated with GA. No response to GA or ABA was detected in leaves.
Key words: Abiotic stress, barley cathepsin-B expression, cDNA cloning, circadian rhythm, germination hormonal regulation.
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Introduction |
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The cysteine proteases are a group of enzymes identified in bacteria, yeast, animals, and plants, which play an important role in intracellular protein degradation (Barrett, 1986). They are synthesized as preproteins that are processed either autocatalytically or with the aid of a processing enzyme, and are stored in the vacuole or the lysosome, or are externally secreted. Among the cysteine proteases, the H and L cathepsins have been widely studied in mammals and, more recently, in plants (Ueda et al., 2000, and references therein). By contrast, only a few cathepsin B-like proteases of plant origin have been described so far.
A cDNA encoding a thiol protease similar to cathepsin B from mammalian cells was isolated from aleurone layers of wheat (Cejudo et al., 1992a). The corresponding mRNA accumulated in the scutellum and the aleurone layers of germinating grains where it was under the regulation of gibberellin (GA) and abscisic acid (ABA). The analysis of the promoter from the corresponding gene showed that the regulation of its expression was at the level of transcription (Cejudo et al., 1992b; Gubler et al., 1999). In Nicotiana rustica, a cDNA from a root library encoding a cathepsin B-like protease was also characterized. Analysis of its mRNA revealed that it was ubiquitously expressed—the accumulation of transcripts increased in leaves in response to wounding (Lidgett et al., 1995). A plant cathepsin B-like mRNA was also identified as an expressed sequence in Rhizobium-induced root nodules of Pisum sativum (Vincent et al., 2000). In addition, the sequences of cathepsin B-like proteases from Ipomoea batata and Arabidopsis thaliana are available in the data banks.
In barley, several cysteine proteases, supposedly involved in proteolysis during germination, have been described. A cDNA encoding the thiol protease aleurain, closely related to mammalian cathepsin H, was isolated from GA-treated aleurone cells, and also showed a high mRNA expression level in leaves and roots (Rogers et al., 1985). Two other cysteine proteases, EPA and EPB, with homology to mammalian cathepsin L, were also induced by GA in aleurones, and seemed to be expressed only in the seed scutellar epithelium and in aleurone layers upon germination (Koehler and Ho, 1988, 1990a, b; Mikkonen et al., 1996). However, no cathepsin B-like proteases have so far been described in barley.
Cystatins are a group of proteins isolated from animals, plants and yeast (Margis et al., 1998), specifically inhibiting cysteine proteases, that could be involved in the regulation of endogenous and heterologous proteases. Among the cereals, several cystatin isoforms diferentially expressed in seeds have been described in rice, maize and wheat (Kondo et al., 1990; Abe et al., 1995; Kuroda et al., 2001), and a cystatin from sorghum was detected in vegetative tissues (Li et al., 1996). In barley, a cDNA clone (Icy gene) encoding a cystein protease inhibitor (Hv-CPI) has recently been described by this group (Gaddour et al., 2001). It was expressed in embryos, developing endosperms, leaves, and roots, and its expression in vegetative tissues increased in response to anaerobiosis, dark and cold shock (6 °C).
The characterization of a cDNA clone encoding a cathepsin B-like cysteine protease from barley (gene CatB), its temporal and spatial expression patterns, and its response to abiotic stimuli such are cold temperatures and light is reported upon here. Its expression in aleurone cells upon germination and in GA- or ABA-treated aleurone layers is also reported and the pattern of expression of the CatB gene with that of the cystatin encoding gene Icy is compared. Possible physiological implications are discussed.
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Materials and methods |
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Screening of the cDNA library
Isolated aleurones of barley (Hordeum vulgare cv. Himalaya) from seeds previously imbibed in water for 4 d were incubated in the presence of 1 µM GA3 in succinate sodium buffer pH 5.2 containing 20 mM CaCl2, for 24 h. Total RNA was isolated and 2 µg were incubated with 2 units of DNAse RNAse free (Ambion) for 30 min, heated for 10 min, and reverse-transcribed to cDNA with the first-strand synthesis kit of Amersham-Pharmacia Biotech, using as primers the hexanucleotide mix of the kit. A DNA fragment corresponding to the barley homologue of the cathepsin B-like gene (Al21 gene) from wheat (Cejudo et al., 1992a) was amplified by PCR using two oligonucleotides derived from the sequence of this wheat gene (sense primer 5'-TCGCGAATTACACTATTGAGC-3'; antisense primer 5'-CACCGGTGATGTGCTTGTA-3'). The PCR-amplified 640 bp fragment was sequenced (Fig. 1) and used as a homologous probe in the screening of a cDNA library from developing endosperms of barley cv. Bomi (Mena et al., 1998). Positive cDNA clones were recovered in the pBluescript SK vector after in vivo excision and their DNA sequences were determined with the ABI PRISM 377 dye DNA sequence analyser (Perkin Elmer-Applied Biosystems). The cDNA with the longest insert, designated hereafter CatB cDNA, was chosen for further characterization.
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Southern and northern blot analysis
Total DNA was prepared from leaves of barley after 7 d of germination and restricted with appropriate endonucleases, separated on 0.8% agarose electrophoretic gels and transferred onto Hybond N membranes (Amersham). Hybridization was performed under stringent conditions following standard procedures (Sambrook et al., 1989) using as specific probe a 255 bp fragment derived from the 3' end of the CatB cDNA clone (Fig. 1).
RNA was purified from frozen tissues (developing endosperms, embryos, aleurones, young roots and leaves) by phenol/chloroform extraction followed by precipitation with 3 M LiCl (Lagrimini et al., 1987). Total denatured RNA was electrophoresed in 0.8% agarose gels containing 7% formaldehyde and blotted onto Hybond N membranes (Amersham). Hybridization and washings were done under stringent conditions, following standard procedures (Sambrook et al., 1989). The same CatB 32P-labelled probe, previously used for Southern blot analysis, and a specific probe of 198 nt derived from the 3' end of the Icy gene (Gaddour et al., 2001) were used in northern blots.
Plant treatments
Plants were grown during 6 d after germination under a photoperiod of 16:8 h (light/dark). At the end of the last dark period, plants were incubated for another 24 h at 6 °C, or their leaves sprayed with a solution of 100 µM ABA or 25 µM GA3 and incubated for 24 h. For the wounding treatment, leaves were pressed with sandpaper and similarly incubated for 24 h. To study the effect of light, plants grown for 6 d under the 16:8 h photoperiod, were illuminated with continuous white light (Osram 18W719 Tages light; 1200 lx) for 48 h at 25 °C, or maintained in the dark for 36 h at 25 °C after an initial light period of 12 h, or grown under a 12:12 photoperiod under identical light and incubation temperature conditions, taking samples at 4 h intervals. Leaf samples were frozen in liquid nitrogen until RNA extraction, as previously indicated. Quantification of the radioactive signal on northern blots was carried out by densitometry of the filters with a Scanner UMAX (UC-840) Data System and the NIH computer program.
Himalaya barley seeds (1992 harvest, Washington State University, Pullman) were de-embryonated and sterilized in 1.7 % (w/v) NaOCl for 10 min, treated with 0.01 M HCl for 5 min and thoroughly washed with distilled water. Seeds were placed at 22 °C in the dark on filter paper soaked with a solution containing 20 mM CaCl2, and 20 mM Na succinate, pH 5.2, for 24 h. Aleurone layers were isolated from the imbibed half-seeds under a dissecting microscope and incubated in Petri dishes at 22 °C in the dark with the buffer described above with no hormone, 10 µM ABA, or 1 µM GA3, or both. Samples were collected at 8 h intervals and aleurones were inmediately frozen in liquid nitrogen until RNA extraction.
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Characterization of a cathepsin B cDNA clone
The presence in wheat kernels of a cathepsin B gene (Cejudo et al. 1992a, b) led the search for its barley counterpart. To find such a gene, a barley cathepsin probe was generated by RT-PCR using as template for the cDNA synthesis poly(A)+ RNA from GA3-induced barley aleurone layers. For the PCR amplification, two oligonucleotides were derived from conserved regions of the cathepsin B gene (Al21) of wheat. Sequencing of PCR products identified a 640 bp fragment with homology to wheat cathepsin B (indicated in Fig. 1), which was used to screen at high stringency a
ZAPII cDNA library from developing barley endosperm (Mena et al., 1998). A library sample representing 80 000 plaque-forming units was plated after infection of the E. coli strain XL1-Blue MRF. After transferring onto Hybond N membranes, hybridization was performed with the 640 bp 32P-labelled barley probe described above. Approximately 200 positive plaques were identified and three of them further purified. The three shared an identical nucleotide sequence. The DNA fragment of 1260 bp contained an ORF encoding a protein of 344 amino acids with a deduced molecular mass of 37.2 kDa (Fig. 1). This protein shared a 97% identity with the deduced sequence for the wheat cathepsin B (Cejudo et al., 1992a). Among them, those amino acid residues putatively involved in the reactive site (C124, H279, N300). Sequence homology around the pro-sequence cleavage site (Fig. 1), suggested that barley cathepsin B might be processed as its wheat and mammalian counterparts, by cleavage of a signal peptide and a pro-sequence (Cejudo et al., 1992a). Similar to wheat cathepsin B, the hydrophobic core, common to actinidin and papain, was nearly unchanged and it also maintained the number and positions of the ten cysteine residues implicated in disulphide bridge formation. The barley CatB lacks the ERFNIN motif present in the cathepsin L/H proteases, which further indicates that this gene belongs to the cathepsin B subfamily of papain-like enzymes (Karrer et al., 1993). The barley cathepsin B-like protein (Hv-CATB) shares 81% identical residues with that from Nicotiana rustica, and 78% with those of Ipomoea batata and Arabidopsis thaliana, and 60% identity with human cathepsin B, which supports the cathepsin B-like nature of this protein. A comparison of their sequences is shown in Fig. 2.
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The phylogenetic dendrogram (Fig. 3), based on comparisons of the cysteine proteases of plants whose whole sequences are in the data banks, clearly indicates that the Hv-CATB is closely related to the other cathepsin B-like proteins described so far in plants, all of them belonging to a well-defined branch of the parsimony unrooted tree.
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Gene copy number
Total DNA from leaves of barley cv. Bomi was digested with two different restriction endonucleases (BamHI and SacI) and analysed by Southern blot under stringent conditions, using as a specific probe the 255 bp fragment spanning from position 995 to 1250 in the cDNA clone (Fig. 1). Although the number of cathepsin-like encoding genes varies among species, a single hybridization band was observed in both cases (Fig. 4), indicating that only one copy of the CatB gene is present in the barley genome.
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Pattern of expression and response to abiotic stimuli of the barley CatB and Icy genes
Since the recombinant Icy (Gaddour et al., 2001) encoded cystatin (Hv-CPI) was a good inhibitor in vitro of commercial bovine cathepsin B (Sigma) with a Ki=2.5x10–6 M (data not shown), it was hypothesized that the in vivo function of the CatB gene will be co-ordinated with that of the Icy gene, and decided, hereafter, to hybridize the northern blots with the specific probes for CatB and Icy in order to explore both their patterns of expression.
The spatial distribution of the CatB and Icy genes expression was examined among the major barley organs and tissues: leaves, roots, aleurones, endosperms at different stages of development, and embryos. Total RNA was isolated from immature (20 d after flowering, daf), mature and germinating (24 h) embryos, developing endosperms (10, 15, 20, and 25 daf), aleurone layers after 2 d of germination, 7-d-old roots and 7-d-old seedlings (Fig. 5A). After electrophoresis and blotting, filters were hybridized with the same specific probe (255 bp DNA) used in the Southern blot experiments. The barley cathepsin B transcripts were detected in all samples analysed, although the highest expression level appeared in the aleurone of germinating seeds (Fig. 5A) and the lowest in mature embryos (mE). The mRNA accumulation in the endosperm started early in development (10 daf) and increased as maturation progressed up to 25 daf (Fig. 5A). In embryos, the expression lowered with dessication (iE, immature 20 daf embryos; mE, embryos in dry seeds) and increased at 24 h of germination (gE). A similar trend in expression pattern was detected for the barley cystatin transcripts (Icy in Fig. 5) in leaves, roots and developing endosperms, whereas in those tissues related to the germinating seed, such as aleurones (A) and embryos (gE), an opposite expression pattern for the mRNAs of CatB and Icy genes was observed (Fig. 5A).
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The transcript accumulation in leaves in response to various abiotic stresses was also investigated. As shown in Fig. 5B, the steady-state level of CatB and Icy mRNAs increased about 3-fold when seedlings were incubated at 6 °C for 24 h (cold shock). No differential response, compared to the controls, was detected when 7-d-old seedlings were sprayed with solutions of 25 µM GA or 100 µM ABA, or when leaves were mechanically wounded (Fig. 5B).
Light regulation of CatB and Icy genes in barley leaves
Circadian fluctuation has been previously described for some plant cysteine proteases (Ueda et al., 2000), although, no such an analysis has been done for their putative inhibitors, the cystatins.
Figure 6 shows the results of the light regulation of the CatB and Icy genes. Northern blot analysis was performed with RNA recovered from leaves over two days at different time intervals and light conditions from barley plants that were previously grown for 6 d under a standard photoperiod of 16/8 h (light/dark). When the photoperiod of the 2 d experiment was 12/12 h (light/dark), both CatB and Icy steady-state mRNAs increased during the light period of the first day, then decreased, reaching a node at the end of the dark period (24 h). The whole cycle was repeated during the second day for the Icy gene, although the node was not clearly observed after 48 h for the CatB gene (Fig. 6A). When plants were incubated for 12 h in the light and were subsequently maintained for 36 h in the dark, a similar circadian pattern was observed for both CatB and Icy mRNAs; both transcripts accumulating during the first 12 h (light period), decreasing to initial levels at 24 h, and peaking again at 36 h, and decreasing thereafter (Fig. 6B). When the plants were incubated for 48 h in continuous light, although visibly stressed at the end of the incubation period, the up and down pattern every 12 h was maintained for the CatB gene while it was less obvious for the Icy gene (Fig. 6C). The control plants, under the standard photoperiod of 16/8 h (light/dark), had a circadian response similar to that in Fig. 6A (data not shown).
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Hormonal response of barley CatB and Icy genes in aleurone layers
Since the barley CatB transcript was detected in the aleurone sample upon northern blot analysis of germinating grains (Fig. 5A), the kinetics of the CatB mRNA accumulation was examined at different times after seed rehydration. As shown in Fig. 7A, the barley CatB message increased up to 24 h of grain imbibition. The blot was subsequently hybridized with a probe for the Icy gene, which was abundantly expressed at 8 h of grain imbibition and progressively decreased.
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The observed accumulation pattern of the barley CatB and Icy transcripts upon seed imbibition suggested that their expression may be hormonally regulated. So, isolated aleurone layers were used as a system to study the hormonal response of both genes to GA and ABA. These aleurone layers that do not synthesize GA, but are able to respond to external hormone treatment, were isolated from de-embryonated grains of Himalaya barley after 2 d of imbibition and incubated in the absence and presence of 1 µM GA3 or 10 µM ABA over different periods of time (8, 16 and 24 h). Total RNA was prepared from these samples, and from the controls incubated in the same buffer without hormones, and were used in northern blot analyses. The filters were hybridized first with the specific probe for CatB, and subsequently, with a probe for the Icy gene (Gaddour et al., 2001). As shown in Fig. 7B, CatB mRNA expression was detected after 8 h of seed imbibition and progressively increased up to 24 h in the control aleurones, whereas the Icy mRNA steady-state levels decreased, as ocurred in aleurones from germinating grains (Fig. 7A). When the barley aleurone layers were incubated in the buffer containing 1 µM GA3 the accumulation of the CatB mRNA transcripts increased while the incubation in 10 µM ABA reduced the CatB mRNA amounts. The treatment of GA+ABA exerts a counteracting effect in the CatB inducibility by GA3. For the Icy mRNA levels, a clear decrease of Icy transcripts was mediated by GA, although no effect of ABA could be perceived, and when added with GA3 it only partially counteracted the GA3 repression effect.
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Discussion |
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A cDNA clone from barley encoding a protein with sequence homology to cathepsin B from mammals (CatB gene) has been characterized. The deduced amino acid sequence for this barley protein has 97% of residues identity with the Al21 gene from wheat (Cejudo et al., 1992a, b), including the key amino acids of the protease active site and the cysteines of the putative disulphide bridges, indicating that CatB from barley and Al21 from wheat are homeologous genes. In fact, these proteins contain 15 cysteine residues, while in other cathepsin B-like encoding genes from plants the cysteine located in position 132 has been changed to a serine (Fig. 2). Cathepsin B-like proteases are present as small multi-gene families in some plant species, as in the genus Nicotiana with several genes involved (Lidgett et al., 1995). In wheat, at least three (one per haploid genome) and probably six variants have been described (Cejudo et al., 1992a). Similar multi-gene families have been also found for cathepsin H and L-like proteins in plants (Pechan et al., 1999; Li et al., 2000; Ueda et al., 2000). By contrast, the barley CatB gene is a single copy gene.
Protein degradation is the most obvious role for cysteine proteases, and all cysteine proteases described in cereals are expressed in seeds, with the exception of a maize gene (Yamada et al., 2000). Seed expression has been reported for cathepsin L- and H-like genes from rice (Watanabe et al., 1991; Kato and Minamikawa, 1996), barley (Rogers et al., 1985; Koehler and Ho, 1988, 1990a, b; Mikkonen et al., 1996), wheat (Sutoh et al., 1999), and maize (Domoto et al., 1995; Griffiths et al., 1997). The only cathepsin B-like protein gene characterized of germinating seeds, Al21 gene from wheat, is expressed both in the aleurone layers and in the scutellar parenchyma as assessed by in situ RNA hybridization (Cejudo et al., 1992a). The CatB gene from barley has been detected both in seeds and in vegetative tissues. In barley aleurone, CatB mRNA expression increases upon germination, suggesting a participation of this enzyme in the degradation of storage proteins in protein bodies to favour the mobilization of amino acids for plantule development before the start of its full autotrophic life. In vegetative tissues, as in 7-d-old seedlings, CatB mRNA is expressed while the mRNA for the Al21 gene of wheat was undetectable in young leaves after 2 d of germination (Cejudo et al., 1992a). Some cathepsin L and H-like genes from cereals are also expressed in vegetative tissues (Rogers et al., 1985; Kato and Minamikawa, 1996; Griffiths et al., 1997).
Plant cysteine proteases have been also associated with other cellular events such as programmed cell death (Solomon et al., 1999; Fath et al., 2000) and the degradation and turnover of proteins in response to biotic and abiotic stimuli. mir1, 2 and 3, cathepsin L-like protease genes from maize callus, and NtCYP-7 and 8, cathepsin H-like protease genes from tobacco are induced in leaves by wounding (Linthorst et al., 1993; Pechan et al., 1999), whereas NtCP-23, other cathepsin H-like encoding gene from tobacco is repressed in wounded leaves (Ueda et al., 2000). No mRNA induction of the barley CatB has been observed in response to wounding, which could indicate that Hv-CATB does not play a significant role in response to mechanical damage or to pathogen or predator attack. No induction by ABA or by GA was observed in leaves. The only significant variation in the pattern of CatB gene expression in leaves was produced by cold treatment, which increased about 3-fold the mRNA accumulation. A cathepsin H-like encoding gene (See1) from maize also presents a cold induction when 13-d-old plants were treated at 14–16/6 °C day/night for 8 d, conditions that also produce the normal senescence symptoms in maize leaves (Griffiths et al., 1997). The cold treatment (24 h at 6 °C) used in this study, did not cause senescence symptoms in barley plants, so Hv-CATB induction may be related to other processes associated with cold stress.
Circadian fluctuation has been reported for the mRNA accumulation of different cysteine proteases of tobacco: NtCP-23, NtCYP-7, NtCYP-8, and NRCATHB, (Linthorst et al., 1993; Lidgett et al., 1995; Ueda et al., 2000). In humans, cathepsins H and L also present a circadian rhythm, which has not been detected in cathepsins of the B type (Cimerman et al., 1999). In this study, it was found that the barley cathepsin B-like transcript also showed a rhythmic expression independently of the photoperiod investigated: either when plants were treated with a 12/12 h (light/dark) photoperiod for 2 d, or when plants were incubated for 12 h in the light and were subsequently maintained for 36 h in the dark, or when plants were exposed to continuous light for 48 h.
An in vitro inhibitor of bovine cathepsin B and of other plant cysteine proteases, such as papain, ficin, or chymopapain, is the HV-CPI protein encoded by the Icy gene (Gaddour et al., 2001). Similar Icy and CatB expression patterns have been detected in vegetative tissues (leaves and roots) and in developing endosperms and both genes are induced by cold treatment in leaves. A similar circadian pattern was also found in leaves for both Icy and CatB genes, although when exposed to continuous light for 48 h, this rhythmical pattern was not so evident for the Icy gene in these plants, which showed senescence symptoms probably due to the production of oxygen radicals under the continuous light exposure. This is the first time that a circadian expression pattern is reported for a cystatin encoding gene.
However, the Icy and CatB expression patterns were complementary in immature and mature embryos, as well as in germinating embryos and aleurones. They also respond in opposite ways to hormone treatments in aleurone layers: CatB being induced by GA and repressed by ABA while Icy was repressed by GA. Several cereal cathepsin genes are also induced by GA and repressed by ABA treatments in aleurone layers (Rogers et al., 1985; Koehler and Ho 1990a; Watanabe et al., 1991; Cejudo et al., 1992a, b; Mikkonen et al., 1996; Shintani et al., 1997; Sutoh et al., 1999). By contrast, GA or ABA treatments did not induce or repress the expression of the barley CatB and Icy genes in leaves.
In conclusion, these data show the ubiquitous expression of the CatB gene in barley, where its pattern of expression is similar in the vegetative tissues to that of the protease inhibitor encoding gene Icy, previously described here. The CatB and Icy genes have a common cold induction and circadian expression pattern in leaves. However, they show complementary expression patterns in pre and post-germinating embryos and different hormonal responses in aleurone layers. The capacity of the Icy encoded protein as an inhibitor of several cysteine proteases, specially of bovine cathepsin-B, suggests a possible in vivo role as an inhibitor of the proteolytic activity encoded by the CatB gene. However, this point deserves further investigation.
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Acknowledgements |
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Financial support from Comunidad de Madrid (project 07M/0015/2001) and from Ministerio de Ciencia y Tecnología (project BMC 2000-1483) are gratefully acknowledged. IR-S was the recipient of a PhD scholarship from Ministerio de Educación, Cultura y Deportes (Spain).
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