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.

Lipid deacylating enzymes in plants: old activities, new genes.

Because lipids are major components of cellular membranes, their degradation under stress conditions compromises compartmentalization. However, in addition to having structural roles, membrane lipids are also implicated in signalling processes involving the activity of lipolytic enzymes. Phospholipases D and C, acting on the polar heads of phospholipids, have been relatively well characterized in plants. In contrast, knowledge of lipid deacylating enzymes remains limited. Lipid acyl hydrolases (LAH) are able to hydrolyse both fatty acid moieties of polar lipids. They differ from phospholipases A(1) or A(2) (PLA) acting on sn-1 or sn-2 positions of phospholipids, respectively, as well as from lipases which de-esterify triacylglycerols. The free polyunsaturated fatty acids generated by deacylating enzymes can be used in the biosynthesis of oxylipins and the lysophospholipids, provided by PLAs, are also bioactive molecules. In the four decades that have passed since the first description of LAH activities in plants some enzymes have been purified. In recent years, the widespread use of molecular approaches together with the attention paid to lipid signalling has contributed to a renewed interest in LAH and has led to the identification of different gene families and the characterization of new enzymes. Additionally, several proteins with putative lipase/esterase signatures have been identified. In the present paper we review currently available data on LAHs, PLAs, triacylglycerol lipases and other putative deacylating enzymes. The roles of lipid deacylating enzymes in plant growth, development and stress responses are discussed in the context of their involvement in membrane deterioration, lipid turnover and cellular signalling.

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.

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.

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.

Plant parasite control and soil fauna diversity.

The use of pesticides to control plant parasites and diseases has generated serious problems of public health and environmental quality, leading to the promotion of alternative Integrated Pest Management strategies that tend to rely more on natural processes and the active participation of farmers as observers and experimenters in their own fields. We present three case studies that point at different options provided by locally available populations of soil organisms, the maintenance of diverse populations of pests or increased resistance of plants to pest attacks by their interactions with earthworms and other useful soil organisms. These examples demonstrate the diversity of options offered by the non-planned agro-ecosystem diversity in pest control and the need to identify management options that maintain this biodiversity.

Effect of drought stress on lipid metabolism in the leaves of Arabidopsis thaliana (ecotype Columbia).

BACKGROUND AND AIMS: Cell membranes are major targets of environmental stresses. Lipids are important membrane components, and changes in their composition may help to maintain membrane integrity and preserve cell compartmentation under water stress conditions. The aim of this work was to investigate the effects of water stress on membrane lipid composition and other aspects of lipid metabolism in the leaves of the model plant, Arabidopsis thaliana. METHODS: Arabidopsis thaliana (ecotype Columbia) plants were submitted to progressive drought stress by withholding irrigation. Studies were carried out in plants with hydration levels ranging from slight to very severe water deficit. Enzymatic activities hydrolysing MGDG, DGDG and PC were measured. Expression of several genes essential to lipid metabolism, such as genes coding for enzymes involved in lipid biosynthesis (MGDG synthase, DGDG synthase) and degradation (phospholipases D, lipoxygenase, patatin-like lipolytic-acylhydrolase), was studied. KEY RESULTS: In response to drought, total leaf lipid contents decreased progressively. However, for leaf relative water content as low as 47.5 %, total fatty acids still represented 61 % of control contents. Lipid content of extremely dehydrated leaves rapidly increased after rehydration. The time-course of the decrease in leaf lipid contents correlated well with the increase in lipolytic activities of leaf extracts and with the expression of genes involved in lipid degradation. Despite a decrease in total lipid content, lipid class distribution remained relatively stable until the stress became very severe. CONCLUSIONS: Arabidopsis leaf membranes appeared to be very resistant to water deficit, as shown by their capacity to maintain their polar lipid contents and the stability of their lipid composition under severe water loss conditions. Moreover, arabidopsis displayed several characteristics indicative of a so far unknown adaptation capacity to drought-stress at the cellular level, such as an increase in the DGDG : MGDG ratio and fatty acid unsaturation.