Anh Thu Pham Thi

Differences in growth and water relations among Phaseolus vulgaris cultivars in response to induced drought stress

Relatively little ecophysiological research has been conducted to determine the responses to drought of Phaseolus vulgaris. Four bean cultivars (cvs.) from Brazil, A320, Carioca, Ouro Negro and Xodó were submitted to an imposed water deficit in order to evaluate the importance of some adaptive mechanisms of drought resistance through the analysis of growth parameters, water status, gas exchange and indicators of tolerance mechanisms at the cellular level. During the drought treatment, relative growth rates were more reduced for A320 and Xodó than Carioca and Ouro Negro. A320 closed its stomata very rapidly and complete stomatal closure was obtained at Psi(w)=-0.6 MPa, in contrast to the other cvs. where stomata were fully closed only at Psi(w)=-0.9 MPa. Net assimilation rates were closely related to stomatal conductances. Mechanisms at the cellular level appeared to be mostly important for higher tolerance. Carioca and Ouro Negro, when compared to A320 and Xodó, were characterized by having better drought tolerance mechanisms and higher tissue water retention capacity leading to a better growth under water deficits. The leaf dehydration rates of those cvs. were slow whereas those of the drought sensitive cvs. were rapid. The results were confirmed by the electrolyte leakage test and leaf osmotic potential measurements, which indicated higher membrane resistance and osmotic adjustment in the two tolerant cvs. Carioca and Ouro Negro. It appears from this study that despite being cultivated in the same geographical region, the four cvs. of P. vulgaris displayed somewhat different drought adaptive capacities for prolonged drought during the vegetative phase.

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.

Efficient whole plant regeneration of common bean (Phaseolus vulgaris L.) using thin-cell-layer culture and silver nitrate

A method was designed to optimize rapid and high frequency direct shoot regeneration (without intermediate callus) of the commercially important common bean, Phaseolus vulgaris L., using the transverse thin cell layer (tTCL) method. The pretreatment of seeds with 10 µM TDZ significantly increased bud regeneration frequency on tTCL. A 2-week culture of tTCLs on 10 µM TDZ followed by a reduction in the TDZ concentration (1 µM) was needed to achieve optimal bud induction and further development of the neo-formed buds. An incubation period greater than 2 weeks of tTCLs with 10 µM TDZ concentration resulted in inhibitory effects on the development of shoots and roots. Shoot development was enhanced by 10 µM BAP and 10 µM AgNO(3) leading to 100% well developed shoots. Regenerated plants developed into true-to-type fertile plants.

Direct whole plant regeneration of cowpea [Vigna unguiculata (L.) walp] from cotyledonary node thin cell layer explants

Summary A simple protocol was established for high frequency direct shoot regeneration of cowpea [ Vigna unguiculata (L.) cv. EPACE-1]. Bud proliferation occurred at the cotyledonary nodes of cowpea seedlings three weeks after culture on a medium containing Murashige and Skoog salts (1962) and B5 vitamins (Gamborg et al. 1968) supplemented with TDZ. A 10 μmol/L TDZ pre-treatment, shoot tip removal and excision of longitudinal thin cell layers (TCL) at the level of the cotyledonary nodes with subsequent culture on a MSB5 medium supplemented with 1 μmol/L IBA and 1 μmol/L TDZ were the optimal conditions for maximum bud proliferation. Up to 32.5 regenerated shoot buds were produced per TCL. The regenerated plants (R 0 ) were true-to-type and successfully transferred to soil.

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.

Effects of an abscisic acid pretreatment on membrane leakage and lipid composition of Vigna unguiculata leaf discs subjected to osmotic stress

Abstract In leaf discs of Vigna unguiculata cv. EPACE-1 subjected to osmotic stress (PEG MW 8000, −1.3 MPa) a significant decrease in membrane integrity occurred, as evaluated by electrolyte efflux. This effect was significantly reduced when discs were treated with ABA (0.1 mM in Tris buffer) prior to PEG treatment. In PEG-treated discs, membrane lipids were drastically degraded, particularly the galactolipids. The percentage of linolenic acid in MGDG also decreased significantly. An ABA pretreatment of discs before osmotic stress significantly decreased galactolipid degradation and preserved their linolenic acid content. We concluded that ABA had a protective effect on membranes against osmotic stress, as attested by electrolyte leakage test and polar lipid analysis. Some possible roles for exogenous ABA in the acquisition of such an enhanced capacity to maintain membrane integrity under dehydration conditions are suggested.

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.

The role of membrane lipids in drought resistance of plants.

SummaryIn greenhouse as well as field-grown plants of Vigna unguiculata and Gossypium hirsutum submitted to drought, leaf membrane lipids show a series of modifications: 1 - A decrease in polar lipid content under severe water stress in sensitive and resistant varieties, due essentially to a decrease in monogalactosyl-diacylglycerol (MGDG) content.2 - A decrease in the degree of unsaturation of fatty acid in sensitive varieties, due to a degradation of linolenic acid (18:3).These modifications result from a slowing down of lipid biosynthesis and from an acceleration of degradative enzymatic processes.Comparison between lipid and fatty acid compositions of drought-susceptible and drought-tolerant varieties from various plant species, suggest that the linolenic acid content of the leaf membranes is related to a lack of ability of cells to withstand low water potentials. Clearly, plants having a low linolenic acid content from MGDG are more resistant than the others. This may be related to the susceptibility...

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.

Sur la reprise au cours de la rehydratation, du point de compensation de CO2, de la photorespiration et de l'ultrastructure, chez deux especes de cotonnier

Abstract Recovery during rehydration of CO 2 compensation point, photorespiration and ultrastructure in two cotton species With two cotton species, one drought-resistant, the other not, the evolution of CO 2 compensation point, photorespiration and leaf ultrastructure were followed after rehydration. The amount and speed of recovery depend on the intensity and duration of the previous osmotic treatment. Recovery is much easier in the resistant species. After recovery from a small water stress, net photosynthetic activity can be higher than the control. With severe stress, however, metabolic and structural alterations increase on rehydration. In general metabolic recovery parallels repair of structural membranes.