Anne Repellin

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.

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.

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).

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.

Analysis of arsenic and antimony distribution within plants growing at an old mine site in Ouche (Cantal, France) and identification of species suitable for site revegetation

One of the objectives of this study was to assess the contamination levels in the tailings of an old antimony mine site located in Ouche (Cantal, France). Throughout the 1.3 ha site, homogenous concentrations of antimony and arsenic, a by-product of the operation, were found along 0-0.5 m-deep profiles. Maximum concentrations for antimony and arsenic were 5780 mg kg(-1) dry tailings and 852 mg kg(-1) dry tailings, respectively. Despite the presence of the contaminants and the low pH and organic matter contents of the tailings, several patches of vegetation were found. Botanical identification determined 12 different genera/species. The largest and most abundant plants were adult pines (Pinus sylvestris), birches (Betula pendula) and the bulrush (Juncus effusus). The distribution of the metalloids within specimens of each genera/species was analysed in order to deduce their concentration and translocation capacities. This was the second goal of this work. All plant specimens were highly contaminated with both metalloids. Most were root accumulators with root to shoot translocation factors <1. Whereas contamination levels were high overall, species with both a low translocation factor and a low root accumulation coefficient were identified as suitable candidates for the complete revegetation of the site. Species combining those characteristics were the perennials P. sylvestris, B. pendula, Cytisus scoparius and the herbaceous Plantago major, and Deschampsia flexuosa.

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.

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.

Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome

Basal phosphoinositide-dependent phospholipase C (PI-PLC) activity controls gene expression in Arabidopsis suspension cells and seedlings. PI-PLC catalyzes the production of phosphorylated inositol and diacylglycerol (DAG) from phosphoinositides. It is not known how PI-PLC regulates the transcriptome although the action of DAG-kinase (DGK) on DAG immediately downstream from PI-PLC is responsible for some of the regulation. We previously established a list of genes whose expression is affected in the presence of PI-PLC inhibitors. Here this list of genes was used as a signature in similarity searches of curated plant hormone response transcriptome data. The strongest correlations obtained with the inhibited PI-PLC signature were with salicylic acid (SA) treatments. We confirm here that in Arabidopsis suspension cells SA treatment leads to an increase in phosphoinositides, then demonstrate that SA leads to a significant 20% decrease in phosphatidic acid, indicative of a decrease in PI-PLC products. Previous sets of microarray data were re-assessed. The SA response of one set of genes was dependent on phosphoinositides. Alterations in the levels of a second set of genes, mostly SA-repressed genes, could be related to decreases in PI-PLC products that occur in response to SA action. Together, the two groups of genes comprise at least 40% of all SA-responsive genes. Overall these two groups of genes are distinct in the functional categories of the proteins they encode, their promoter cis-elements and their regulation by DGK or phospholipase D. SA-regulated genes dependent on phosphoinositides are typical SA response genes while those with an SA response that is possibly dependent on PI-PLC products are less SA-specific. We propose a model in which SA inhibits PI-PLC activity and alters levels of PI-PLC products and substrates, thereby regulating gene expression divergently.

Protein carbonylation during natural leaf senescence in winter wheat, as probed by fluorescein-5-thiosemicarbazide.

Leaf senescence is characterised by a massive degradation of proteins in order to recycle nitrogen to other parts of the plant, such as younger leaves or developing grain/seed. Protein degradation during leaf senescence is a highly regulated process and it is suggested that proteins to be degraded are marked by an oxidative modification (carbonylation) that makes them more susceptible to proteolysis. However, there is as yet no evidence of an increase in protein carbonylation level during natural leaf senescence. The aim of our study was thus to monitor protein carbonylation level during the process of natural senescence in the flag leaf of field-grown winter wheat plants. For this purpose, we adapted a fluorescence-based method using fluorescein-5-thiosemicarbazide (FTC) as a probe for detecting protein carbonyl derivatives. As used for the first time on plant material, this method allowed the detection of both quantitative and qualitative modifications in protein carbonyl levels during the last stages of wheat flag leaf development. The method described herein represents a convenient, sensitive and reproducible alternative to the commonly used 2,4-dinitrophenylhydrazine (DNPH)-based method. In addition, our analysis revealed changes in protein carbonylation level during leaf development that were associated with qualitative changes in protein abundance and carbonylation profiles. In the senescing flag leaf, protein carbonylation increased concomitantly with a stimulation of endoproteolytic activity and a decrease in protein content, which supports the suggested relationship between protein oxidation and proteolysis during natural leaf senescence.