Plant defense stimulation by natural isolates of bacillus depends on efficient surfactin production.

Some plant-associated Bacillus strains produce induced systemic resistance (ISR) in the host, which contributes to their protective effect against phytopathogens. Little is known about the variety of elicitors responsible for ISR that are produced by Bacillus strains. Working with a particular strain, we have previously identified the surfactin lipopeptide as a main compound stimulating plant immune-related responses. However, with the perspective of developing Bacillus strains as biocontrol agents, it is important to establish whether a central role of surfactin is generally true for isolates belonging to the B. subtilis/amyloliquefaciens complex. To that end, we set up a comparative study involving a range of natural strains. Their secretomes were first tested for triggering early defense events in cultured tobacco cells. Six isolates with contrasting activities were further evaluated for ISR in plants, based both on macroscopic disease reduction and on stimulation of the oxylipin pathway as defense mechanism. A strong correlation was found between defense-inducing activity and the amount of surfactin produced by the isolates. These results support the idea of a widespread role for surfactin as a nonvolatile elicitor formed by B. subtilis/amyloliquefaciens, and screening for strong surfactin producers among strains naturally secreting multiple antibiotics could be an efficient approach to select good candidates as biopesticides.

Reprogramming of fatty acid and oxylipin synthesis in rhizobacteria-induced systemic resistance in tomato.

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme of the oxylipin pathway, the lipoxygenase (LOX), leading to a faster accumulation of its product, the free 13-hydroperoxy octadecatrienoic acid (13-HPOT), 2 days after Botrytis cinerea inoculation. In the present study, we further investigated the stimulation of the oxylipin pathway: metabolites and enzymes of the pathway were analyzed to understand the fate of the 13-HPOT in ISR. Actually the stimulation began upstream the LOX: free linolenic acid accumulated faster in P. putida BTP1-treated plants than in control. Downstream, the LOX products 13-fatty acid hydroperoxides esterified to galactolipids and phospholipids were more abundant in bacterized plants than in control before infection. These metabolites could constitute a pool that will be used after pathogen attack to produce free fungitoxic metabolites through the action of phospholipase A2, which is enhanced in bacterized plants upon infection. Enzymatic branches which can use as substrate the fatty acid hydroperoxides were differentially regulated in bacterized plants in comparison to control plants, so as to lead to the accumulation of the most fungitoxic compounds against B. cinerea. Our study, which is the first to demonstrate the accumulation of an esterified defense metabolite during rhizobacteria-mediated induced systemic resistance, showed that the oxylipin pathway is differentially regulated. It suggests that this allows the plant to prepare to a future infection, and to respond faster and in a more effective way to B. cinerea invasion.

The elicitation of a systemic resistance by Pseudomonas putida BTP1 in tomato involves the stimulation of two lipoxygenase isoforms.

BACKGROUND: Some non-pathogenic rhizobacteria called Plant Growth Promoting Rhizobacteria (PGPR) possess the capacity to induce in plant defense mechanisms effective against pathogens. Precedent studies showed the ability of Pseudomonas putida BTP1 to induce PGPR-mediated resistance, termed ISR (Induced Systemic Resistance), in different plant species. Despite extensive works, molecular defense mechanisms involved in ISR are less well understood that in the case of pathogen induced systemic acquired resistance. RESULTS: We analyzed the activities of phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX), key enzymes of the phenylpropanoid and oxylipin pathways respectively, in tomato treated or not with P. putida BTP1. The bacterial treatment did not stimulate PAL activity and linoleate-consuming LOX activities. Linolenate-consuming LOX activity, on the contrary, was significantly stimulated in P. putida BTP1-inoculated plants before and two days after infection by B. cinerea. This stimulation is due to the increase of transcription level of two isoforms of LOX: TomLoxD and TomLoxF, a newly identified LOX gene. We showed that recombinant TomLOXF preferentially consumes linolenic acid and produces 13-derivative of fatty acids. After challenging with B. cinerea, the increase of transcription of these two LOX genes and higher linolenic acid-consuming LOX activity were associated with a more rapid accumulation of free 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids, two antifungal oxylipins, in bacterized plants. CONCLUSION: In addition to the discovery of a new LOX gene in tomato, this work is the first to show differential induction of LOX isozymes and a more rapid accumulation of 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids in rhizobacteria mediated-induced systemic resistance.

Ex Vivo Antioxidant Capacities of Fruit and Vegetable Juices. Potential In Vivo Extrapolation.

BACKGROUND: In support of claims that their products have antioxidant properties, the food industry and dietary supplement manufacturers rely solely on the in vitro determination of the ORAC (oxygen radical antioxidant capacity) value, despite its acknowledged lack of any in vivo relevance. It thus appears necessary to use tests exploiting biological materials (blood, white blood cells) capable of producing physiological free radicals, in order to evaluate more adequately the antioxidant capacities of foods such as fruit and vegetable juices. MATERIALS: Two approaches to assessing the antioxidant capacities of 21 commercial fruit and vegetable juices were compared: the ORAC assay and the "PMA-whole blood assay," which uses whole blood stimulated by phorbol myristate acetate to produce the superoxide anion. We described in another paper the total polyphenol contents (TPCs) and individual phenolic compound contents of all the juices were investigated. RESULTS: Ranking of the juices from highest to lowest antioxidant capacity differed considerably according to the test used, so there was no correlation (r = 0.33, p = 0.13) between the two assays when considering all juices. Although the results of the ORAC assay correlated positively with TPC (r = 0.50, p = 0.02), a much stronger correlation (r = 0.70, p = 0.004) emerged between TPC and % superoxide anion inhibition. In the PMA-whole blood assay, peonidin-3-O-glucoside, epigallocatechin gallate, catechin, and quercetin present in juices were found to inhibit superoxide anion production at concentrations below 1 µM, with a strong positive correlation. CONCLUSIONS: Associated with the determination of total and individual phenolic compounds contained in fruit and vegetable juices, the PMA-whole blood assay appears better than the ORAC assay for evaluating juice antioxidant capacity.

Compared Phenolic Compound Contents of 22 Commercial Fruit and Vegetable Juices: Relationship to ex-vivo Vascular Reactivity and Potential in vivo Projection.

The real impact of polyphenol-rich vegetable and fruit juice intake on cardiovascular health remains a matter of controversy. In the present study, rat aorta segments immersed in an organ bath (OB) were used to explore whether the total polyphenol content and/or individual phenolic compound contents of 22 commercial vegetable (n = 3) and fruit juices [(citrus (n = 5), berries (n = 10), apple (n = 2), pineapple (n = 2)] might be associated with vascular tone. Red juices (particularly blackcurrant) and lemon juice caused the most marked vasorelaxation, its amplitude being endothelium dependent or not according to the volume ratio of juice to initial OB solution Vjuice/VOBS). At volume ratios 5% and 10%, both the juice and OB total polyphenol for all juices and total anthocyanin contents for berry juices significantly correlated with aorta vasorelaxation intensity. This was not the case for total or individual flavonols (except kaempferol) or for total or individual flavanols (except epigallocatechin gallate). If one relates our measured concentrations of individual phenolic compounds in OB to what is known about their physiological concentrations, and given our evidenced correlations between compound concentrations and vasorelaxation intensity, kaempferol, epigallocatechin gallate and peonidin-3-O-glucoside seem to emerge as the interesting phenolic compounds likely to be responsible for the potent vasorelaxation observed with fruit juices, and more particularly blackcurrant ones. Clinical investigation is required, however, to confirm our observations.

Quantitative changes in protein expression of cadmium-exposed poplar plants.

Cadmium (Cd) pollution is a worldwide major concern having, among others, deleterious effects on plants. In the present work, the effects of a 20 microM Cd exposure in hydroponics culture during 14 days were evaluated in young poplar leaves. Proteins were analysed by 2-D DIGE, followed by MALDI-TOF-TOF identification. Additionally, growth and other physiological parameters were monitored during the experiment. Treated plants exhibited an inhibition of growth and visual symptoms appeared after 7 days. A significant accumulation of Cd in all organs was recorded by ICP-MS analysis. A number of changes in the expression of proteins with various functions were identified; in particular a decreased abundance of oxidative stress regulating proteins, whereas pathogenesis-related proteins showed a drastic increase in abundance. Furthermore, a large number of proteins involved in carbon metabolism showed a decrease in abundance, while proteins involved in remobilizing carbon from other energy sources were upregulated. In conclusion, the negative effect of Cd could be explained by a deleterious effect on protein expression from the primary carbon metabolism and from the oxidative stress response mechanism. Accumulation of Cd in stems of poplar, coupled with a low impact of Cd on physiological parameters, promotes the use of poplar trees for phytoremediation purposes.

Systemic resistance and lipoxygenase-related defence response induced in tomato by Pseudomonas putida strain BTP1.

BACKGROUND: Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR) in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes. RESULTS: In this study, we showed the protective effect of this bacterium in tomato against Botrytis cinerea. Following treatment by P. putida BTP1, analyses of acid-hydrolyzed leaf extracts showed an accumulation of antifungal material after pathogen infection. The fungitoxic compounds thus mainly accumulate as conjugates from which active aglycones may be liberated through the activity of hydrolytic enzymes. These results suggest that strain BTP1 can elicit systemic phytoalexin accumulation in tomato as one defence mechanism. On another hand, we have shown that key enzymes of the lipoxygenase pathway are stimulated in plants treated with the bacteria as compared with control plants. Interestingly, this stimulation is observed only after pathogen challenge in agreement with the priming concept almost invariably associated with the ISR phenomenon. CONCLUSION: Through the demonstration of phytoalexin accumulation and LOX pathway stimulation in tomato, this work provides new insights into the diversity of defence mechanisms that are inducible by non-pathogenic bacteria in the context of ISR.

Characterization and cloning of chitin deacetylases from Rhizopus circinans.

Chitin deacetylase catalyzes hydrolysis of the acetamido groups of N-acetylglucosamine of chitin in fungal cell walls. Here a chitin deacetylase secreted by Rhizopus circinans was purified to homogeneity and partially characterized. The enzyme exhibits an apparent molecular weight of approximately 75kDa. At 37 degrees C it shows optimal activity at pH 5.5-6. Its pH stability and thermal stability are good. Mn(2+) and Mg(2+) slightly enhance the activity of the enzyme and Cu(2+) strongly inhibits it. An R. circinans cDNA library was constructed and screened with a homologous probe synthesized by RT-PCR or with synthetic primers derived from the N-terminal amino-acid sequence of the native purified chitin deacetylase. Three chitin deacetylase cDNAs (RC, D2, and I3/2) were isolated from the cDNA library and sequenced. These cDNAs exhibit features characteristic of chitin deacetylase sequences: the presence of a polysaccharide deacetylase domain, a metal-binding triad, the conserved catalytic residues, and high homology with various chitin deacetylase genes. The cDNAs were cloned in a Pichia pastoris expression system and produced as polyhistidine-tagged proteins. Only one recombinant enzyme (called RC) was active under the tested conditions. It was purified to homogeneity in a single step and further characterized. The protein showed an apparent molecular mass of approximately 75kDa and, like the native enzyme, showed optimal activity at pH 5.5-6 at 37 degrees C. It was strongly inhibited by Cu(2+). The isolation of several chitin deacetylase cDNAs from the same microorganism is discussed.

The potency of commercial blackcurrant juices to induce relaxation in porcine coronary artery rings is not correlated to their antioxidant capacity but to their anthocyanin content.

OBJECTIVE: Polyphenol-rich products such as fruit juices have been found to have strong antioxidant capacities and to induce potent endothelium-dependent relaxation. We evaluated whether the commercial blackcurrant juices induced endothelium-dependent relaxation of isolated coronary arteries can be related to their antioxidant capacity and/or phenolic content. METHODS: Six different commercial blackcurrant juices were selected. Their main phenolic compounds were measured by ultra-performance liquid chromatography and antioxidant capacity was evaluated by spectrometric methods. Vascular reactivity studies with these juices were done using isolated porcine coronary arteries. RESULTS: The six different commercial blackcurrant juices induced relaxation ranging from 21% to 100% at the concentration of 0.5% volume per volume (v/v). The relaxation induced at 0.5% v/v was not correlated to their antioxidant capacity measured by either oxygen radical antioxidant capacity or DPPH (2,2-diphenyl-1-picrylhydrazyl) assays and also not to the ascorbic acid, total polyphenols, total flavanols, and total phenolic acid contents. In contrast, the amplitude of the relaxation was correlated to the total anthocyanins content and the individual anthocyanin concentration. CONCLUSIONS: Correlations between relaxation amplitude and total anthocyanin or individual anthocyanin contents are of interest for the development of functional blackcurrant beverages with the potential to promote vascular protection.

Evolution of antioxidant capacity during storage of selected fruits and vegetables.

Interest in the consumption of fresh fruits and vegetables is, to a large extent, due to its content of bioactive nutrients and their importance as dietary antioxidants. Among all of the selected fruits and vegetables, strawberries and black grapes have relatively high antioxidant capacities associated with high contents of total phenolic compounds, ascorbic acid, and flavonols. More interesting, the results of this study indicated that in most fruits and vegetables storage did not affect negatively the antioxidant capacity. Better, in some cases, an increase of the antioxidant capacity was observed in the days following their purchase, accompanied by an increase in phenolic compounds. In general, fruits and vegetables visually spoil before any significant antioxidant capacity loss occurs except in banana and broccoli. When ascorbic acid or flavonoids (aglycons of flavonols and anthocyanins) were concerned, the conclusions were similar. Their content was generally stable during storage.

Antioxidant capacity of black currant varies with organ, season, and cultivar.

Small berries such as black currant constitute one of the important sources of potential health-promoting phytochemicals because these fruits are rich sources of compounds with high antioxidant properties. In this work, antioxidant capacities of different parts (buds, leaves, fruits) of various black currant cultivars were compared throughout the growing season with the aim to prepare extracts with high antioxidant capacity. Buds (opened, at the end of March) and leaves (in June) had a higher content in phenolics and antioxidants than fully ripened berries (in July) and the best yield (per branch) was obtained with the leaves collected in June due to their higher biomass. The differences observed among the eight cultivars tested were small. Concerning flavonols, quercetin was dominant in all organs and cultivars, myricetin varied widely among the cultivars, and kampferol was very low.

Stimulation of the lipoxygenase pathway is associated with systemic resistance induced in bean by a nonpathogenic Pseudomonas strain.

Systemic defense reactions induced in bean by the non-pathogenic Pseudomonas putida BTP1 strain reduced disease caused by Botrytis cinerea. Phenylalanine ammonialyase activity and the level of endogenous free salicylic acid were compared in plant growth-promoting rhizobacteria-treated versus control plants, but no significant differences were detected. Furthermore, no enhanced fungitoxicity was detected in methanolic leaf extracts, suggesting that accumulation of bean phytoalexins was not part of the stimulated defense mechanisms. However, BTP1-inoculated plants showed increased levels of both linoleic and linolenic acids. On this basis, we further investigated whether the lipoxygenase pathway, leading to antifungal phytooxylipins, could have been stimulated. Two key enzymatic activities of this metabolic route, namely lipoxygenase and hydroperoxide lyase, were significantly stimulated during the first four days after challenging BTP1-treated plants with the pathogen. This was observed in parallel with a more rapid consumption of the respective substrates of these enzymes, as revealed by measurements of endogenous concentrations of linolenic acid and their hydroperoxide derivatives. Moreover, headspace-gas chromatography analyses showed significantly higher concentrations of the fungitoxic final product Z-3-hexenal in leaves from BTP1-inoculated beans as compared with control plants. Taken together, these results strongly suggest that the oxylipin pathway can be associated with enhanced disease resistance induced in bean plants by nonpathogenic rhizobacteria.

Cloning and expression analysis of cDNAs corresponding to genes activated in cucumber showing systemic acquired resistance after BTH treatment.

BACKGROUND: Infection of plants by necrotizing pathogens can lead to the rapid and localized induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. Subsequently, an increase of plant resistance against a broad spectrum of pathogens is observed systemically. This plant immunity is known as Systemic Acquired Resistance. To identify components of the transduction pathway, we cloned and analysed the expression pattern of several mRNAs accumulating in cucumber plants after induction of Systemic Acquired Resistance. RESULTS: We tested on cucumber different compounds known to induce systemic acquired resistance. Among these, BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester) proved to be very effective. mRNA RT-PCR differential display was used to identify mRNA sequences induced 24 hours after the application of 10 microM BTH to cucumber plants. A cDNA library constructed from cucumber plants sprayed with 10 microM BTH was screened to get corresponding full length cDNAs. Among the identified cDNAs were those coding for a putative ras-related GTP-binding protein, a putative beta-1,4-N-Acetylglucosaminyltranferase III and a putative pathogenesis related protein. The time course of accumulation of the three corresponding mRNAs was analysed by northern blotting in plants treated by BTH or in plants infected by Colletotrichum lagenarium. CONCLUSIONS: The mRNA RT-PCR differential display technique allowed the identification of three genes possibly involved in Systemic Acquired Resistance in cucumber. Pathogenesis-related proteins are known to be involved in plant defence against pathogens. GTP-binding protein and N-acetylglucosaminyltranferase III have been reported to be components of signal transduction pathways in mammals and plants.

Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response.

Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Hyperhydric shoots are characterised by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and a high water content. Hyperhydricity of Prunus avium shoots was expressed in vitro in one multiplication cycle by replacing the gelling agent agar (normal shoots: NS) by gelrite (hyperhydric shoots: HS). P. avium shoots evolving towards the hyperhydric state produced higher amounts of ethylene, polyamines (PAs) and proline, which are substances considered as stress markers. A higher activity of glutathione peroxidase (GPX; EC 1.11.1.9), involved in organic hydroperoxide elimination, suggested an increased production of these compounds in HS. The unchanged free fatty acid composition indicated no HS membrane damages compared to NS. The ploidy level of HS nuclei was not affected, but the bigger size and the lower percentage of nuclei during the S phase suggested a slowing down of the cell cycle. The results argued for a stress response of the HS, but no signs of oxidative damages of lipid membrane and nucleus were observed. The discussion points out paradoxical results in a classical analysis of stress and suggests an alternative way of defense mechanisms in HS, involving homeostatic regulation and controlled degradation processes to maintain integrity and vital functions of the cell.

Differential gene expression in two potato lines differing in their resistance to Phytophthora infestans.

Horizontal resistance to late blight in the potato is a primary objective of many breeding programs. Knowledge of the physiological and biochemical mechanisms underlying it, however, is scarce. The purpose of the present study was the identification of these physiological and biochemical factors in plant material obtained by crossing a late blight resistant Solanum phureja clone with a susceptible dihaploid of S. tuberosum subsp. tuberosum. The mRNA RT-PCR differential display method was used to compare the gene expression patterns of a resistant hybrid with that of a susceptible one. By sequence homology, we identified several genes with diverse functions, including genes known to be involved in resistance or stress responses and genes known to be involved in primary or secondary metabolism.

Comparative titration of ginsenosides by different techniques in commercial ginseng products and callus cultures.

The ginsenoside content of different ginseng species (Panax ginseng, P. quinquefolium, and P. vietnamensis) from different sources (roots from field-grown plants or from in vitro cultures, cells from solid calluses or from liquid cultures, commercial powders, and suspensions) is evaluated by means of a new high-performance thin-layer chromatography (HPTLC) technique combining an automatic TLC sampler and scanner. The results are compared with those obtained through more classical gross spectrometric and high-performance liquid chromatography (HPLC) techniques. HPTLC and HPLC allow the separation and estimation of the different ginsenosides. For this, HPTLC is faster and simpler than HPLC. Both techniques determine less amounts of ginsenosides than spectrophotometry, which displays overestimated values caused by light absorption by contaminating osides. In vitro cultured cells and roots contain the same ginsenosides as those produced by their mother plants, although at quite lower levels. The culture media also accumulates ginsenosides.

Antioxidant potential of different plum cultivars during storage.

Plums, the most commonly consumed fruits from Romania, are fruits rich in bioactive compounds, such as antioxidants. This research work was carried out to investigate the antioxidant potential of twelve plum cultivars, fresh and stored during 10days at 4°C by using different methods (DPPH, ORAC and erythrocyte resistance to haemolysis). The contents of total phenolic compounds and total anthocyanins were also determined by specific spectrometric methods. Significant differences between fresh and stored plum cultivars (p<0.05) were found. Storage at 4°C resulted in an increase in antioxidant potential and anthocyanins content of the autumn plum varieties. Autumn plum varieties also showed a higher antioxidant capacity than summer varieties, as assessed by the ORAC and the haemolysis resistance assays. Our results suggest that, even after storage, plums could be a good source of antioxidants, which may provide health-promoting effects for humans.

Cultivars, culture conditions, and harvest time influence phenolic and ascorbic acid contents and antioxidant capacity of strawberry (Fragaria x ananassa).

UNLABELLED: Strawberries are a good source of micronutrients, especially antioxidant phenolics. More information is needed to better exploit the health-promoting effect of such fruits. Several studies focused on the effects of genotype, cultural practices, and seasonal variation on the antioxidant potential of strawberries, but often yielding contradictory results and/or focusing on only 1 source of variability. In the present study, we showed that total phenolic compounds, ascorbic acid, and antioxidant capacity strongly differed between genotypes of strawberry. The precise results revealed the importance of genetic background for the antioxidant capacity and for the content of total phenolics (with up to 3.3-fold variations). Other parameters may also influence the antioxidant capacity in strawberry such as harvest time, culture conditions, and environmental factors. Moreover, in this study, the harvesting time (at the same ripening stage) appeared to be very important, more important than genotype. Variations of the antioxidant capacity of up to 4.1-folds were observed following the harvesting time of "Elsanta" cultivar. PRACTICAL APPLICATION: This article compares the antioxidant capacity and the content in ascorbic acid and phenolic compounds of strawberries of different varieties and of fruits harvested from April to December at the same ripening stage. The importance of strawberry antioxidant capacity resides in its benefits for human health.

Purification and characterisation of a 31-kDa chitinase from the Myzus Persicae aphid: a target for hemiptera biocontrol.

Hydrolytic enzymes involved in chitin degradation are important to allow moulting during insect development. Chitinases are interesting targets to disturb growth and develop alternative strategies to control insect pests. In this work, a chitinase from the aphid Myzus persicae was purified with a 36-fold purification rate in a three step procedure by ammonium sulphate fractionation, anion-exchange chromatography on a DEAE column and on an affinity Concanavalin A column. The purified chitinase purity assessed by 1D and 2D SDS-PAGE revealed a single band and three spots at 31 kDa, respectively. Chitinases were found to have high homologies with Concanavalins A and B, two chitinase-related proteins, a fungal endochitinase and an aphid acetylhydrolase by peptide identification by Maldi-Tof-Tof. The efficiency of two potent chitinase inhibitors, namely allosamidin and psammaplin A, was tested and showed significant rate of enzymatic inhibition.

Ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from Ribes nigrum.

Small fruits such as berries have low energy contents, but high contents of vitamins, micronutrients, and dietary fibers and constitute a good source of natural antioxidant compounds that are important constituents of the human diet. This study identified a large number of compounds in an extract of black currant showing high antioxidant activity and compared their profile in various parts of the plants (leaves, buds, and berries). If it was known that berries contained very high levels of natural phenolic compounds, this study showed that leaves and buds could also be considered good sources of natural antioxidants. Indeed, they contained high amounts of phenolic acids, flavonoids, and carotenoids. An acetone mixture can extract several classes of phenolic compounds with a good yield of flavonols, flavan-3-ols, and anthocyanins.