Evaluation of trace metal accumulation in six vegetable crops intercropped with phytostabilizing plant species, in a French urban wasteland.

The extensive development of agriculture in urban and peri-urban wastelands polluted with several trace elements (TE) poses risks to human health through contaminated food products. The objective was to explore the accumulation of TE in the various parts of vegetable crop plants (tomato, French bean, radish, potato, spinach, and leek) intercropped with phytostabilizing plant species (ryegrass and white clover, respectively). Field studies were conducted in a multicontaminated French urban wasteland with Cd, Cu, Pb and Zn, and an alkaline soil pH. Analyses of the respective non-edible parts of monocultured vegetable crops showed accumulation of all TE, mostly Zn, then Pb and Cu, and finally Cd. The corresponding TE accumulation factors (soil to plant) were all below 0.25. In the edible parts, average concentrations for TE were above the limit values, according to European and Chinese standards. TE contents in the phytostabilizing species chosen were in the same orders of magnitude and the same ranking as described for vegetable crops and most accumulation was in the roots. Unexpectedly, the presence of the phytostabilizing plants had a very strong positive impact on the soil to plant accumulation factor. Moreover, the edible plant parts were poorly impacted by the co-cropping with phytostabilizing plants.

Combined Effects of Ozone and Drought on the Physiology and Membrane Lipids of Two Cowpea (Vigna unguiculata (L.) Walp) Cultivars.

The interactive effects of drought and ozone on the physiology and leaf membrane lipid content, composition and metabolism of cowpea (Vigna unguiculata (L.) Walp.) were investigated in two cultivars (EPACE-1 and IT83-D) grown under controlled conditions. The drought treatment (three-week water deprivation) did not cause leaf injury but restricted growth through stomatal closure. In contrast, the short-term ozone treatment (130 ppb 12 h daily during 14 day) had a limited impact at the whole-plant level but caused leaf injury, hydrogen peroxide accumulation and galactolipid degradation. These effects were stronger in the IT83-D cultivar, which also showed specific ozone responses such as a higher digalactosyl-diacylglycerol (DGDG):monogalactosyldiacylglycerol (MGDG) ratio and the coordinated up-regulation of DGDG synthase (VuDGD2) and ω-3 fatty acid desaturase 8 (VuFAD8) genes, suggesting that membrane remodeling occurred under ozone stress in the sensitive cultivar. When stresses were combined, ozone did not modify the stomatal response to drought and the observed effects on whole-plant physiology were essentially the same as when drought was applied alone. Conversely, the drought-induced stomatal closure appeared to alleviate ozone effects through the reduction of ozone uptake.

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

[This corrects the article on p. 608 in vol. 5, PMID: 25426125.].

Molecular cloning, characterization and differential expression of novel phytocystatin gene during tropospheric ozone stress in maize (Zea mays) leaves.

In this study, a full-length cDNA encoding a novel phytocystatin gene, designated CC14, was identified in maize leaves. The CC14 gene sequence reported in this study has been deposited in the GenBank database (accession number JF290478). The CC14 gene was cloned into an expression vector pET30 EK/LIC and was then transformed into Escherichia coli strain BL21 (DE3) pLysS to produce a recombinant CC14 protein. The recombinant protein was purified by nickel nitrilotriacetic acid affinity chromatography after induction with 1 mM IPTG. The purified CC14 protein was electrophoresed on SDS-PAGE and a protein 25 kDa in size was observed. Antiprotease activities of the purified recombinant CC14 protein against cysteine proteases and commercially available papain were tested. The results showed that CC14 purified protein suppressed 100% activity of papain and 57-86% plant cysteine protease activity. Moreover, an upregulation of CC14 gene expression was observed after 20 days of ozone stress in maize leaves. Together, these observations concurred to conclude that CC14 gene could potentially be used as a basis for the development of transgenic crops and natural pesticides that resist biotic and abiotic stresses.

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.

Identification and characterization of MOR-CP, a cysteine protease induced by ozone and developmental senescence in maize (Zea mays L.) leaves.

Among the different classes of endoproteases, cysteine proteases are consistently associated with senescence, defense signaling pathways and cellular responses to abiotic stresses. The objectives of this work were to study the effects of various concentrations of ozone on gene expression and enzymatic activity for papain-like cysteine proteases (PLCPs), in the leaves of maize plants grown under field conditions. Leaves from ranks 12 and 10 (cob leaf) were harvested regularly over a long-term artificial ozone fumigation experiment (50 d). Tissues were tested for transcriptional and activity changes concerning cysteine proteases, using qRT-PCR for the newly identified ozone-responsive PLCP gene (Mor-CP) and synthetic oligopeptide Boc-Val-Leu-Lys-AMC as a PLCP-specific substrate, respectively. Results showed that developmental senescence induced a significant and progressive rise in CP activity, only in the older leaves 10 and had no effect on Mor-CP gene expression levels. On the other hand, ozone dramatically enhanced Mor-CP mRNA levels and global PLCP enzymatic activity in leaves 12 and 10, particularly toward the end of the treatment. Ozone impact was more pronounced in the older leaves 10. Together, these observations concurred to conclude that ozone stress enhances natural senescence processes, such as those related to proteolysis.

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.

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.

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.

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.

Selected carbohydrate metabolism genes show coincident expression peaks in grains of in vitro-cultured immature spikes of wheat (Triticum aestivum L.).

An in vitro culture system is useful to study grain development under defined conditions to minimize confounding effects associated with whole plant studies and metabolite movement into the developing grains. The objective of this study was to monitor the expression patterns of carbohydrate metabolism genes during grain development in an in vitro wheat spike culture system. Immature spikes were cultured prior to anthesis, and grains were collected at various days postanthesis (DPA). Grains from cultured spikes showed maximum expression of starch metabolic genes by 10 DPA, with a rapid decline thereafter. The rapid increase and decrease in expression rate in the in vitro system was thought to be due to fructan exohydrolase (1-FEH and 6-FEH) or sucrose transporter 1 (SUT1) and sucrose synthase (SuSy) genes being highly expressed. SUT1 reached peak expression at 8 DPA, two days earlier than the other genes, and may account for the rapid early stage trigger in expression of the other genes. However, expression of 1-FEH and 6-FEH genes in in vitro-cultured spikes peaked at 12 DPA, two days later than the other genes, and could indicate that fructan catabolism was not a factor in the rapid accumulation of starch in the in vitro-cultured spikes. Accumulation of GBSSI polypeptides generally showed similar patterns in both systems, with the maximum amount in the in vitro system observed four days later than in the in planta spikes, reflecting different turnover controls of GBSSI transcripts. The in vitro system offers opportunities for further refinement and detailed grain development studies.

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).

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.

Water deficit induces variation in expression of stress-responsive genes in two peanut (Arachis hypogaea L.) cultivars with different tolerance to drought.

Peanut (Arachis hypogaea L.) is an important subsistence and cash crop in the semi-arid tropics where it often suffers from drought stress. Although its ecophysiological responses are studied, little is known about the molecular events involved in its adaptive responses to drought. The aim of this study was to investigate the involvement of membrane phospholipid and protein degrading enzymes as well as protective proteins such as "late embryogenesis-abundant" (LEA) protein in peanut adaptive responses to drought. Partial cDNAs encoding putative phospholipase D alpha, cysteine protease, serine protease and a full-length cDNA encoding a LEA protein were cloned. Their expression in response to progressive water deficit and rehydration was compared between cultivars differing in their tolerance to drought. Differential gene expression pattern according to either water deficit intensity and cultivar's tolerance to drought were observed. A good correspondence between the molecular responses of the studied cultivars and their physiological responses previously defined in greenhouse and field experiments was found. Molecular characters, as they were detectable at an early stage, could therefore be efficiently integrated in groundnut breeding programmes for drought adaptation. Thus, the relevance of the target genes as drought tolerance indicators is discussed.

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.