Molecular cloning, characterization and differential expression of novel phytocystatin gene during tropospheric ozone stress in maize (Zea mays) leaves.

In this study, a full-length cDNA encoding a novel phytocystatin gene, designated CC14, was identified in maize leaves. The CC14 gene sequence reported in this study has been deposited in the GenBank database (accession number JF290478). The CC14 gene was cloned into an expression vector pET30 EK/LIC and was then transformed into Escherichia coli strain BL21 (DE3) pLysS to produce a recombinant CC14 protein. The recombinant protein was purified by nickel nitrilotriacetic acid affinity chromatography after induction with 1 mM IPTG. The purified CC14 protein was electrophoresed on SDS-PAGE and a protein 25 kDa in size was observed. Antiprotease activities of the purified recombinant CC14 protein against cysteine proteases and commercially available papain were tested. The results showed that CC14 purified protein suppressed 100% activity of papain and 57-86% plant cysteine protease activity. Moreover, an upregulation of CC14 gene expression was observed after 20 days of ozone stress in maize leaves. Together, these observations concurred to conclude that CC14 gene could potentially be used as a basis for the development of transgenic crops and natural pesticides that resist biotic and abiotic stresses.

Identification and characterization of MOR-CP, a cysteine protease induced by ozone and developmental senescence in maize (Zea mays L.) leaves.

Among the different classes of endoproteases, cysteine proteases are consistently associated with senescence, defense signaling pathways and cellular responses to abiotic stresses. The objectives of this work were to study the effects of various concentrations of ozone on gene expression and enzymatic activity for papain-like cysteine proteases (PLCPs), in the leaves of maize plants grown under field conditions. Leaves from ranks 12 and 10 (cob leaf) were harvested regularly over a long-term artificial ozone fumigation experiment (50 d). Tissues were tested for transcriptional and activity changes concerning cysteine proteases, using qRT-PCR for the newly identified ozone-responsive PLCP gene (Mor-CP) and synthetic oligopeptide Boc-Val-Leu-Lys-AMC as a PLCP-specific substrate, respectively. Results showed that developmental senescence induced a significant and progressive rise in CP activity, only in the older leaves 10 and had no effect on Mor-CP gene expression levels. On the other hand, ozone dramatically enhanced Mor-CP mRNA levels and global PLCP enzymatic activity in leaves 12 and 10, particularly toward the end of the treatment. Ozone impact was more pronounced in the older leaves 10. Together, these observations concurred to conclude that ozone stress enhances natural senescence processes, such as those related to proteolysis.

A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): cloning, characterization and relation to postharvest chilling stress resistance.

A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits.

The expression patterns of bromelain and AcCYS1 correlate with blackheart resistance in pineapple fruits submitted to postharvest chilling stress.

Blackheart is a physiological disorder induced by postharvest chilling storage during pineapple fruit export shipping. The aim of this study was to check the involvement of bromelain, the cysteine protease protein family abundantly present in pineapple fruits, and AcCYS1, an endogenous inhibitor of bromelain, in the development of blackheart. For this we checked the response to postharvest chilling treatment of two pineapple varieties (MD2 and Smooth Cayenne) differing in their resistance to blackheart. Quantitative RT-PCR analyses showed that postharvest chilling treatment induced a down-regulation of bromelain transcript accumulation in both varieties with the most dramatic drop in the resistant variety. Regarding AcCYS1 transcript accumulation, the varieties showed opposite trends with an up-regulation in the case of the resistant variety and a down-regulation in the susceptible one. Taken together our results suggest that the control of bromelain and AcCYS1 expression levels directly correlates to the resistance to blackheart development in pineapple fruits.

Cloning, characterization and differential expression of a Bowman-Birk inhibitor during progressive water deficit and subsequent recovery in peanut (Arachis hypogaea) leaves.

Bowman-Birk inhibitor (BBI) genes encode serine protease inhibitors well known for their anticarcinogenic properties and roles in plant defense against insects and pathogens. Here we investigated the expression of a BBI gene in response to water deficit, recovery and phytohormones. A full length cDNA encoding a novel BBI (AhBBI) was isolated from peanut (Arachis hypogaea L.) leaves. The deduced protein is a polypeptide of 11.5kDa containing a signal peptide of 20 amino acids which is missing from peanut seed full-length BBI. Sequence analysis showed that AhBBI presents the characteristic features of BBIs but its first inhibitory loop is unique among the Fabaceae species. Real-time PCR analyses indicated that in peanut leaves, AhBBI is upregulated by water deficit and exogenous jasmonic acid (JA) but repressed by abscissic acid (ABA) after 24h of treatment. The transcripts accumulation patterns during water deficit differed between two cultivars studied in relation to their tolerance levels to drought. AhBBI transcripts accumulated earlier and stronger in the tolerant cultivar (cv. Fleur11) compared to the susceptible one (cv. 73-30) suggesting that BBI genes are involved in drought stress tolerance. Subsequent rehydration reversed the accumulation of AhBBI transcripts in both cultivars but at different levels. The overall role of BBI in abiotic stress tolerance and the possible mechanisms of action are discussed.

Aluminum stress response in rice: effects on membrane lipid composition and expression of lipid biosynthesis genes.

The presence of aluminum (Al) in acidic soils is a major abiotic stress limiting the production of cultivated plants. Cell membranes are the main targets of environmental stresses and there is growing evidence for the involvement of membrane lipids in plant adaptation. The aim of this study was to evaluate the mid-long effects of Al on membrane lipid content and composition in the roots and shoots of rice plants grown under hydroponic conditions. Four rice cultivars were compared: two acknowledged as Al-resistant (Koshihikari) and Al-sensitive (Kasalath), respectively, and two Vietnamese cultivars, OM6073 and OM1490. Al treatment inhibited root and shoot growth in the sensitive cultivars and the observed changes in root and shoot lipid and fatty acid composition revealed patterns associated with Al sensitivity: larger decreases in lipid content and decreases in fatty acid unsaturation. In the roots, phospholipids, and particularly phosphatidylcholine (PC), decreased dramatically in the susceptible cultivars whereas the amount of lipid classes remained unchanged in the tolerant ones. In the shoots, the glycolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol as well as PC were mostly affected by Al treatment in the susceptible varieties. mRNA accumulation corresponding to genes coding for galactolipid synthases, enzymes of the PC and phosphatidylethanolamine biosynthetic pathways and fatty acid desaturases correlated well with changes in lipid contents in roots and partly explained lipid changes in leaves. The results suggested that the capacity to maintain the proper functioning of some lipid biosynthetic activities and hence the stability of lipid composition may help the rice plant to withstand Al stress.

Ozone and aging up-regulate type II metacaspase gene expression and global metacaspase activity in the leaves of field-grown maize (Zea mays L.) plants.

Maize plants (Zea mays L. cv. NK Perform) were exposed to O(3)-enriched air, using a new field fumigation system. Transcriptional changes for three type II-metacaspase genes were studied in the leaves (ranks 10 and 12), using quantitative real-time PCR. Global metacaspase activity was measured using metacaspase-specific synthetic tripeptide Boc-GRR-AMC. Aging had little effect on mRNA accumulation whereas four to six-fold increases were observed for the most O(3)-responsive type II metacaspase genes, in the older leaves 10. Global metacaspase activity increased by 257% and 333% in leaves 12 and 10, respectively, in response to the highest cumulated concentration. In non-fumigated plants, metacaspase activity progressively increased over the course of the experiment and always was higher in the older leaves 10. Together, these results suggest that metacaspase-mediated proteolysis is a crucial step in leaf responses to both O(3) and age-mediated senescence.

An aspartic acid protease from common bean is expressed 'on call' during water stress and early recovery.

A cDNA encoding a putative aspartic acid protease precursor (PvAP1) was cloned from the leaves of common bean (Phaseolus vulgaris). Sequence analysis showed that PvAP1 presents all the characteristic features of phytepsins, the typical plant APs. PvAP1 gene expression was tightly regulated by water stress, being significantly up-regulated under mild water stress (Ψ(w)=-1.0 MPa) for the drought-susceptible cultivar (Carioca) and moderate water stress (Ψ(w)=-1.5 MPa) for the more drought-tolerant cultivar (IPA). Protein gel blotting analysis under water stress revealed the presence of two main bands of calculated MW of 46 and 38 kDa, suggesting proteolytic processing of the enzyme precursor form under drought in both cultivars. Taken together, our results suggest that water stress regulates PvAP1 activity both at the transcriptional and post-transcriptional levels, and that the response occurs earlier and is stronger in the drought-susceptible cultivar.

Homoglutathione synthetase and glutathione synthetase in drought-stressed cowpea leaves: expression patterns and accumulation of low-molecular-weight thiols.

Glutathione (GSH) is an abundant metabolite and a major antioxidant in plant cells. However, in the Leguminosae, homoglutathione (hGSH) may replace glutathione (GSH) partially or completely. To date, cowpea (Vigna unguiculata) has been considered a non-hGSH-producing species, and no hGSHS cDNA has been isolated. Here we report on the cloning of a full-length cDNA coding for a hGSHS (EC 6.3.2.23) and the cloning of a partial cDNA coding for a putative glutathione synthetase (GSHS; EC 6.3.2.3) in cowpea leaf extracts. These cDNAs possess, respectively, the leucine/proline hGSHS signature and the alanine/alanine GSHS signature at the 3' end. Expression analysis showed a significant up-regulation of hGSHS during progressive drought stress that could be directly related to the drought tolerance of the cowpea cultivar used, while GSHS was mainly constitutively expressed. Nevertheless, quantification of low-molecular-weight thiols confirmed the previous findings that cowpea is essentially a GSH producing plant, as no hGSH was detected in the leaves. These findings raise new questions regarding the function, activity and substrate specificity of the cloned hGSHS cDNA. These questions are discussed.

Accumulation of lead in the roots of grass pea (Lathyrus sativus L.) plants triggers systemic variation in gene expression in the shoots.

The impact of lead nitrate (Pb(NO(3))(2); 0.5mM) on steady-state accumulation of messengers corresponding to stress responsive genes was studied in two local lines of 11-d grass pea (Lathyrus sativus L.) plants exposed for 96 h in a hydroponic system. Real-time reverse transcription polymerase chain reaction technique was used with grass pea-specific primers designed from newly isolated partial cDNA. Increases in accumulation of glutathione reductase, ascorbate peroxidase and glutathione S-transferase transcripts suggested that roots enhanced detoxification mechanisms involving glutathione. In the leaves where no lead was translocated, the pollutant indirectly triggered increases in expression of several genes. This process probably resulted from systemic signals originating from the roots where lead accumulated in large amounts, approximately 150 mg Pbg(-1) dry weight. A preventive and/or adaptive role for the signal is assumed, since it concerned genes implicated in reactive oxygen species scavenging (ascorbate peroxidase), protein protection (heat shock protein 70) and proteolysis (cysteine and aspartic proteases).

Molecular cloning and characterization of novel cystatin gene in leaves Cakile maritima halophyte.

Cakile maritima (Brassicaceae) is a halophyte that thrives on dunes along Mediterranean seashores, with high tolerance to salty and dry environments. We have previously shown that there is great morphological and physiological diversity between ecotypes. We investigated the expression of cysteine protease inhibitor (cystatin) genes in the response to hydric and saline constraints, as cystatins are known to participate in the response to environmental constraints in plants. We isolated, from C. maritime, a new cystatin cDNA (CmC) that encodes a 221 amino acid protein with a calculated molecular mass of 25 kDa. It displays a moderate-to-high amino acid sequence similarity with previously reported phytocystatin genes. The predicted protein is hydrophilic, with only one hydrophobic region, just at its N-terminus, and a calculated isoelectric point of 6.7. Sequence analysis revealed a monocystatin structure with one cystatin-like domain. The predicted protein CmC contains the main conserved motifs characteristic of the plant cystatins, and a putative site of phosphorylation by casein kinase II (TPSD). As some cystatins, it contains a C-terminal extension of 106 amino acid residues, with several conserved cystatin motifs. The expression was constitutive in non-stressed plants, with different levels between the ecotypes, and without apparent relation to the climatic area of origin. Augmented expression was observed under severe salinity except in the ecotype from the arid region. Water deficit also increased CmC expression in two ecotypes, with the highest value observed in the ecotype from the humid region. These results indicate that C. maritima responds to high salinity and water deficit by expressing a cystatin gene that is a known component of defense against abiotic constraints or biotic aggression and survival machinery.

Lead accumulation in the roots of grass pea (Lathyrus sativus L.): a novel plant for phytoremediation systems?

Eleven day-old grass pea plants (Lathyrus sativus L.) were grown hydroponically for 96 h in the presence of 0.5 mM lead nitrate (Pb(NO(3))(2)). The survival rate was 100%. The mean lead content (measured by ICP-OES) in root tissues was 153 mg Pb g(-1) dry matter. Over three quarters of the lead was not labile. Compared with control plants, lead-exposed plants showed a six-fold, two-fold and three and a half-fold reduction in their root calcium, zinc and copper contents, respectively. Together, these results suggested that Lathyrus sativus L. was tolerant to a deficiency in essential nutrients and able to store large amounts of lead in its root tissues. Therefore, it could be used for the development of new rhizofiltration systems.

Cloning and characterization of drought-stimulated phosphatidic acid phosphatase genes from Vigna unguiculata.

Under environmental stresses, several lipolytic enzymes are known to be activated and to contribute to membrane lipid turnover and generation of second messengers. In animal cells, phosphatidic acid phosphatase (PAP, EC 3.1.3.4), which dephosphorylates phosphatidic acid generating diacylglycerol, is long known as an enzyme involved in lipid synthesis and cell signalling. However, knowledge on PAP in plants remains very limited. The aim of this work was to isolate and characterize PAP genes in the tropical legume Vigna unguiculata (cowpea), and to study their expression under different stress conditions. Two cDNAs designated as VuPAPalpha and VuPAPbeta were cloned from the leaves of cowpea. Both proteins share sequence homology to animal type 2 PAP, namely, the six transmembrane regions and the consensus sequences corresponding to the catalytic domain of the phosphatase family, like the recently described Arabidopsis LPP (Lipid Phosphate Phosphatase) proteins. The recombinant protein VuPAPalpha expressed in Escherichia coli cells was able to convert phosphatidic acid into diacylglycerol. Unlike VuPAPbeta, VuPAPalpha has an N-terminal transit peptide and was addressed to chloroplast in vitro. Both genes are expressed in several cowpea organs and their transcripts accumulate in leaves in response to water deficit, including progressive dehydration of whole plants and rapid desiccation of detached leaves. No changes in expression of both genes were observed after wounding or by treatment with jasmonic acid. Furthermore, the in silico analysis of VuPAPalpha promoter allowed the identification of several putative drought-related regulatory elements. The possible physiological role of the two cloned PAPs is discussed.

Effects of progressive drought stress on the expression of patatin-like lipid acyl hydrolase genes in Arabidopsis leaves.

Patatin-like genes have recently been cloned from several plant species and found to be involved in stress responses and development. In previous work, we have shown that a patatin-like gene encoding a galactolipid acyl hydrolase (EC 3.1.1.26) was stimulated by drought in the leaves of the tropical legume, Vigna unguiculata L. Walp. The aim of the present work was to study the expression of patatin-like genes in Arabidopsis thaliana under water deficit. Expression of six genes was studied by reverse transcriptase polymerase chain reaction in leaves of plants submitted to progressive drought stress induced by withholding water and also in different plant organs. Three genes, designated AtPAT IIA, AtPAT IVC and AtPAT IIIA, were shown to be upregulated by water deficit but with different kinetics, while the other patatin-like genes were either constitutive or not expressed in leaves. The accumulation of transcripts of AtPAT IIA in the early stages of the drought treatment was coordinated with the upregulation of lipoxygenase and allene oxide synthase genes. AtPAT IIA expression was also induced by wounding and methyl jasmonate treatments. The in vitro lipolytic activity toward monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylcholine and phosphatidylglycerol was confirmed by producing the recombinant protein ATPAT IIA in insect cells. The analysis of free fatty acid pools in drought-stressed leaves shows an increase in the relative amounts of trans-3-hexadecenoic acid at the beginning of the treatment followed by a progressive accumulation of linoleic and linolenic acids. The possible roles of AtPAT IIA in lipid signaling and membrane degradation under water deficit are discussed.

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress.

Two cDNAs of the enzyme glutathione reductase (GR; EC 1.6.4.2) encoding a dual-targeted isoform (dtGR) and a cytosolic isoform (cGR), were cloned from leaves of common bean (Phaseolus vulgaris L.). Moderate drought stress (Psi w=-1.5MPa) followed by re-watering was applied to common bean cultivars, one tolerant to drought (IPA), the other susceptible (Carioca) and to cowpea (Vigna unguiculata L. Walp) cultivars, one tolerant to drought (EPACE-1), and the other susceptible (1183). mRNA levels were much higher for PvcGR than for PvdtGR in all cases. Moderate drought stress induced an up-regulation of the expression of PvcGR in the susceptible cultivars. On the contrary, PvdtGR expression decreased. In the tolerant cowpea EPACE-1, GR gene expression remained stable under drought. During recovery from drought, an up-regulation of the two GR isoforms occurred, with a peak at 6-10h after re-hydration. This suggests that moderate drought stress may lead to a hardening process and acclimation tolerance. The role of GR isoforms in plant tolerance and capacity to recover from drought stress is discussed.

Water deficit induces variation in expression of stress-responsive genes in two peanut (Arachis hypogaea L.) cultivars with different tolerance to drought.

Peanut (Arachis hypogaea L.) is an important subsistence and cash crop in the semi-arid tropics where it often suffers from drought stress. Although its ecophysiological responses are studied, little is known about the molecular events involved in its adaptive responses to drought. The aim of this study was to investigate the involvement of membrane phospholipid and protein degrading enzymes as well as protective proteins such as "late embryogenesis-abundant" (LEA) protein in peanut adaptive responses to drought. Partial cDNAs encoding putative phospholipase D alpha, cysteine protease, serine protease and a full-length cDNA encoding a LEA protein were cloned. Their expression in response to progressive water deficit and rehydration was compared between cultivars differing in their tolerance to drought. Differential gene expression pattern according to either water deficit intensity and cultivar's tolerance to drought were observed. A good correspondence between the molecular responses of the studied cultivars and their physiological responses previously defined in greenhouse and field experiments was found. Molecular characters, as they were detectable at an early stage, could therefore be efficiently integrated in groundnut breeding programmes for drought adaptation. Thus, the relevance of the target genes as drought tolerance indicators is discussed.

Drought stress and rehydration affect the balance between MGDG and DGDG synthesis in cowpea leaves.

Membranes are main targets of drought, and there is growing evidence for the involvement of membrane lipid in plant adaptation to such an environmental stress. Biosynthesis of the galactosylglycerolipids, monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG), which are the main components of chloroplast envelope and thylakoid membranes, could be important for plant tolerance to water deficit and for recovery after rehydration. In this study, galactolipid (GL) biosynthesis in cowpea (Vigna unguiculata L. Walp) leaves was analysed during drought stress and subsequent rewatering. Comparison of two cowpea cutivars, one drought tolerant and the other drought susceptible submitted to moderate drought stress, revealed patterns associated with water-deficit tolerance: increase in DGDG leaf content, stimulation of DGDG biosynthesis in terms of (14)C-acetate incorporation and messenger accumulation corresponding to four genes coding for GL synthases (MDG1, MGD2, DGD1 and DGD2). Similar to phosphate starvation, lack of water enhanced DGDG biosynthesis and it was hypothesized that the drought-induced DGDG accumulated in extrachloroplastic membranes, and thus contributes to plant tolerance to arid environments.

Glutathione reductase in leaves of cowpea: cloning of two cDNAs, expression and enzymatic activity under progressive drought stress, desiccation and abscisic acid treatment.

BACKGROUND AND AIMS: Reactive oxygen species are frequently produced when plants are exposed to abiotic stresses. Among the detoxication systems, two enzymes, ascorbate peroxidase and glutathione reductase (GR) play key roles. GR has also a central role in keeping the reduced glutathione pool during stress thus allowing the adjustments on the cellular redox reactions. The aim of this work was to study the variations in cytosolic and dual-targeted GR gene expression in the leaves of cowpea plants submitted to progressive drought, rapid desiccation and application of exogenous abscisic acid (ABA). METHODS: Two cowpea (Vigna unguiculata) cultivars, one drought-resistant ('EPACE-1'), the other drought-sensitive ('1183') were submitted to progressive drought stress by withholding irrigation. Cut-off leaves were air-dried or treated with exogenous ABA. Two GR cDNAs, one cytosolic, the other dual-targeted to chloroplasts and mitochondria were isolated by PCR and cloned in plasmid vectors. Reverse-transcription PCR was used to study the variations in GR gene expression. KEY RESULTS: Two new cDNAs encoding a putative dual-targeted and a cytosolic GR were cloned and sequenced from leaves of V. unguiculata. Drought stress induced an up-regulation of the expression of the cytosolic GR gene directly related to the intensity of the stress in both cultivars. The expression of dual-targeted GR was up-regulated by the drought treatment in the susceptible cultivar only. Under a fast desiccation, the '1183' cultivar responded later than the 'EPACE-1', although in 'EPACE-1' it was the cytosolic isoform which responded and in '1183' the dual-targeted one. Exogenous ABA enhanced significantly the activity and expression levels of GR in both cultivars after treatment for 24 h. CONCLUSIONS: These results demonstrate a noticeable activation in both cultivars of the antioxidant metabolism under a progressive water stress, which involves both GR genes in the case of the susceptible cultivar. Under a fast desiccation, the susceptible cultivar responded later than the resistant one, suggesting a weaker capacity of response versus the resistant one. Exogenous ABA probably acts on GR gene expression via a mediated signal transduction pathway.

Isolation and characterization of four ascorbate peroxidase cDNAs responsive to water deficit in cowpea leaves.

BACKGROUND AND AIMS: Abiotic stresses stimulate formation of active oxygen species in plant tissues. Among antioxidant mechanisms, H2O2 detoxication by ascorbate peroxidases (APX) plays an important role. Several APX isoforms exist in plant cells, and they have rarely been studied separately. The aim of this work was to study changes in cytosolic, peroxisomal, stromatic and thylakoid APX gene expression in response to progressive drought, rapid desiccation and application of exogenous abscisic acid in the leaves of cowpea (Vigna unguiculata) plants. METHODS: Two cowpea (V. unguiculata) cultivars, 'EPACE-1' which is drought-tolerant and '1183'which is drought-sensitive, were submitted to drought stress by withholding irrigation. Detached leaves were air-dried or treated with exogenous abscisic acid. APX cDNAs were isolated by PCR and cloned in plasmid vectors. Changes in gene expression were studied using reverse-transcription PCR. KEY RESULTS: Four new V. unguiculata cDNAs encoding putative cytosolic, peroxisomal and chloroplastic (stromatic and thylakoidal) APX were isolated and characterized. In response to the different treatments, higher increases in steady-state transcript levels of the cytoplasmic and peroxisomal APX genes were observed in '1183' compared with 'EPACE-1'. On the other hand, the expression of the chloroplastic APX genes was stimulated earlier in the tolerant cultivar when submitted to progressive drought. CONCLUSIONS: Water deficit induced differences in transcript accumulation of APX genes between the two cultivars that were related to their respective tolerance to drought. Chloroplastic APX genes responded early to progressive water deficit in the tolerant plant, suggesting a capacity to efficiently detoxify active oxygen species at their production site. The more sensitive '1183' was also able to respond to drought by activating its whole set of APX genes.

A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata (L.) Walp.) leaves.

Cystatins are protein inhibitors of cystein proteinases belonging to the papain family. In cowpea, cystatin-like polypeptides and a cDNA have been identified from seeds and metabolic functions have been attributed to them. This paper describes VuC1, a new cystatin cDNA isolated from cowpea leaves (Vigna unguiculata (L.) Walp.). Sequence analysis revealed a multicystatin structure with two cystatin-like domains. The recombinant VUC1 protein (rVUC1) was expressed in an heterologous expression system and purified to apparent homogeneity. It appeared to be an efficient inhibitor of papain activity on a chromogenic substrate. Polyclonal antibodies against rVUC1 were obtained. Involvement of the VuC1 cDNA in the cellular response to various abiotic stresses (progressive drought-stress, dessication and application of exogenous abscissic acid) was studied, using Northern blot and Western blot analysis, in the leaf tissues of cowpea plants corresponding to two cultivars with different capacity to tolerate drought-stress. Surprisingly, these abiotic stresses induced accumulation of two VuC1-like messages both translated into VUC1-like polypeptides. Difference in the transcript accumulation patterns was observed between the two cultivars and related to their respective tolerance level. Presence of multiple cystatin-like polypeptides and their possible involvement in the control of leaf protein degradation by cysteine proteinases is discussed.