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.

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 patatin-like protein with galactolipase activity is induced by drought stress in Vigna unguiculata leaves.

This paper reports the cloning of a cDNA (Vupat1) expressed in Vigna unguiculata leaves coding for a protein with 48% sequence homology to patatin, the major protein from potato tuber which has lipolytic acylhydrolase activity. Two cultivars differing in drought tolerance were examined in Northern-blot analyses. Expression of Vupat1 is stimulated by drought stress, especially in the drought-sensitive cultivar. Vupat1 was expressed in the baculovirus system as a fusion protein secreted in the culture medium. The recombinant protein displays lipolytic activity towards monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerols.

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.

Enzymatic activity and gene expression under water stress of phospholipase D in two cultivars of Vigna unguiculata L. Walp. differing in drought tolerance.

Phospholipase D, a major lipid-degrading enzyme in plants, was studied in two cultivars of Vigna unguiculata L.Walp, differing in their tolerance to drought (cv. EPACE-1, drought-tolerant, and cv. 1183, drought-susceptible). Enzymatic activities, measured with 14C-PC as substrate, increased when plants were submitted to water stress, the increase being much higher in the drought-sensitive cultivar. A 2911 bp cDNA encoding a putative phospholipase D (VuPLD1) was isolated from a cDNA library prepared from V. unguiculata leaves. The deduced amino acid sequence (809 residues) shows 85.5% identity and 91.3% similarity to that of PLD from Ricinus communis. The expression of the VuPLD1 gene in the leaves is differently modulated by water deficit, depending on the intensity of stress and the tolerance or sensitivity of the plants. In the drought-susceptible V. unguiculata cv. 1183, it readily increased under water stress, reaching maximum values at mild water deficit (-1.5 MPa). In the drought-tolerant cv. EPACE-1, VuPLD1 mRNA remained low throughout the whole drought treatment. Dehydration of leaves led to a dramatic increase in transcript level in both cultivars. Changes in protein amounts semi-quantified by immunoblotting correlated well with variations in transcript steady-state level. Taken together, these results showed that phospholipase D in cowpea plants is essentially regulated at the transcriptional level, and that gene expression is strongly stimulated even by moderate water deficit in the drought-sensitive plant. On the contrary, the drought-tolerant plant presents a remarkable stability of PLD gene expression in conditions of water stress.

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.

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.