Agnès d'Arcy-Lameta

A novel patatin-like gene stimulated by drought stress encodes a galactolipid acyl hydrolase.

A cDNA (Vupat1) encoding a predicted 43 kDa protein was isolated from drought‐stressed cowpea (Vigna unguiculata) leaves. It has homology with patatin, a potato tuber storage protein with lipolytic acyl hydrolase activity. The recombinant protein VUPAT1 expressed in the baculovirus system displays preferentially galactolipid acyl hydrolase activity. Phospholipids are very slowly hydrolyzed and apparently triacylglycerols are not deacylated. Vupat1 promoter contains putative drought‐inducible sequences. Northern blots showed that gene expression is stimulated by drought stress and is more pronounced in a drought‐sensitive cultivar than in a drought‐tolerant one. An involvement in drought‐induced galactolipid degradation is proposed for VUPAT1.

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.

Aspartic protease in leaves of common bean (Phaseolus vulgaris L.) and cowpea (Vigna unguiculata L. Walp): enzymatic activity, gene expression and relation to drought susceptibility.

Four cultivars of related species, common bean and cowpea, which exhibit different degrees of drought resistance, were submitted to water stress by withholding irrigation. Drought induced an increase in endoproteolytic activity, being higher in susceptible cultivars (bean) than in tolerant ones (cowpea). An aspartic protease activity was found to be strongly induced especially in bean. From a cowpea leaf cDNA library, a full length aspartic protease precursor cDNA was obtained. Transcript accumulation in response to water stress indicated that the expression of the gene was constitutive in cowpea and transcriptionally up‐regulated in bean. The results showed that drought‐tolerant and drought‐susceptible bean plants differ regarding aspartic protease precursor gene expression.

Purification and characterization of a soluble lipolytic acylhydrolase from Cowpea (Vigna unguiculata L.) leaves

With the use of [14C]monogalactosyl diacylglycerol as substrate for enzymatic test, a lipolytic acylhydrolase (EC 3.1.1.26) was purified 263-fold with a yield of 2.0% from soluble leaf extract of Vigna unguiculata L. cv. EPACE-1. The procedure involved ammonium sulfate precipitation, Q-Sepharose Fast Flow chromatography, gel filtration on Sephacryl 300 HR and chromatofocusing on Mono-P, followed by a semi-preparative electrophoresis on polyacrylamide gel. The purified enzyme had a molecular mass of about 80 kDa, as determined by gel filtration. On SDS-PAGE, it showed a single band corresponding to a molecular mass of 40 kDa. The isoelectric point of the enzyme was estimated to be 5.0-5.1 by isoelectric focusing and chromatofocusing. The Km value was 0.119 mM for monogalactosyl-diacylglycerol. The hydrolytic activity of the enzyme on different substrates was determined: the relative rates were digalactosyl-diacylglycerol > monogalactosyl-diacylglycerol > phosphatidylcholine > phosphatidylglycerol. For all substrates, the products of hydrolysis were free fatty acids. Triacylglycerols were not hydrolysed. The enzyme was activated by calcium but was not calcium-dependent. Experiments concerning the enzyme stability as affected by temperature and pH demonstrated that it was quite stable.

Effect of drought on photodynamic peroxidation of leaf total lipophilic extracts

Abstract In vitro lipid peroxidation, induced by photochemically generated reactive oxygen species, was carried out either on purified α-linolenic acid (18:3n — 3) (1.32 mM) or on total lipophilic extracts ofleaf(standardized at 1.32 mM for 18:3n — 3 content). Three cultivars of plants differing in their sensitivity to water deficit were used: Vigna unguiculata L. cv. EPACE (drought-tolerant), Vigna unguiculata L. cv. IT-83D (moderately drought-tolerant) and Phaseolus vulgaris L. cv. Carioquinha (drought-sensitive). The time-course of 18:3n — 3 photoperoxidation was comprised of three phases: (i) monohydroperoxidation, characterized by formation of conjugated diene patterns, (ii) secondary oxidation, characterized by formation of conjugated triene patterns, (iii) oxidative fragmentation of peroxidized compounds, characterized both by the disappearance of conjugated polyenic patterns and by the formation of thiobarbituric acid reacting substances. Only the first and second phases were observed on photoperoxidation of total lipophilic extracts from leaves of well-hydrated plants. When each cultivar was subjected to drought treatment, the conjugated diene pattern maximum was attained earlier than for well-hydrated plants, suggesting that drought affects the resistance capacity of total lipophilic extracts from leaves to photoperoxidation. The more severe the drought, the more susceptible the leaf extracts were to lipid peroxidation induced by reactive oxygen species. The more resistant to drought the plant was, the slower the rate of formation of conjugated polyenic patterns. These results are discussed in relation with the chlorophyll a/b and carotenoids/18:3n — 3 molar ratios in the photoperoxidation apparatus. This work is the first attempt to correlate plant drought tolerance and the sensitivity of their leaf total lipophilic extracts to lipid peroxidation.

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.

Two novel plant cDNAs homologous to animal type-2 phosphatidate phosphatase are expressed in cowpea leaves and are differently regulated by water deficits.

Two cDNAs encoding putative phosphatidate phosphatases (PAPs) designated VuPAP-alpha and VuPAP-beta were cloned in cowpea (Vigna unguiculata L.) leaves. The predicted proteins have six membrane-spanning regions in common with animal type-2 PAPs. Unlike VuPAP-beta, VuPAP-alpha has an N-terminal transit peptide and is targeted in vitro to the chloroplasts. Gene expression of VuPAP-beta is stimulated by rapid air-desiccation of leaves and VuPAP-alpha transcripts increase during rehydration of plants exposed to drought-like conditions.

In vitro photodynamic lipid peroxidation of total lipophilic extracts from leaves of bean plants

In vitro lipid peroxidation, induced by reactive oxygen species photochemically generated, was carried out on purified alpha-linolenic acid (18:3(n - 3)) and on bean leaf total lipophilic extracts. The photosensitizer used was meso-tetraphenylporphine. The time-course of the reaction was evaluated by ultraviolet (UV) spectra analysis. The 18:3(n - 3) photoperoxidation kinetics comprised three steps: monohydroperoxidation, characterized by the appearance of conjugated diene patterns; dihydroperoxidation characterized by the appearance of conjugated triene patterns, oxidative cleavage of the mono- and dihydroperoxides, characterized by the disappearance of conjugated patterns. In contrast, for hydrated plant total lipophilic extract photoperoxidation, conjugated pattern appearance was slow and the maximum plateau was not obtained. In order to explain plant extract behaviour, we tested the effects of beta-carotene and quercetin (important components of the chloroplast membrane) on the time-course of 18:3(n - 3) photoperoxidation. The first step was inhibited by beta-carotene implying that type II photoperoxidation involving singlet oxygen (1O2) was predominant. Whilst the two last steps were inhibited by quercetin implying that type I photoperoxidation involving free radicals, prevailed. Since 18:3(n - 3) foliar content decreased under water deficit, we tested the behaviour of total lipophilic extracts from droughted plant leaves in presence of reactive oxygen species. In the case of droughted plants, the maximum of conjugated diene patterns was attained later than for hydrated plants, suggesting that drought affects the resistance capability of total lipophilic extracts from bean leaves to lipid photoperoxidation.