1-Aminocyclopropane-1-carboxylic acid stimulates tomato pollen tube growth independently of ethylene receptors.

The plant hormone ethylene plays vital roles in plant development, including pollen tube (PT) growth. Many studies have used the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as a tool to trigger ethylene signaling. Several studies have suggested that ACC can act as a signal molecule independently of ethylene, inducing responses that are distinct from those induced by ethylene. In this study, we confirmed that ethylene receptor function is essential for promoting PT growth in tomato, but interestingly, we discovered that ACC itself can act as a signal that also promotes PT growth. Exogenous ACC stimulated PT growth even when ethylene perception was inhibited either chemically by treating with 1-methylcyclopropene (1-MCP) or genetically by using the ethylene-insensitive Never Ripe (NR) mutant. Treatment with aminoethoxyvinylglycine, which reduces endogenous ACC levels, led to a reduction of PT growth, even in the NR mutants. Furthermore, GUS activity driven by an EIN3 Binding Site promoter (EBS:GUS transgene) was triggered by ACC in the presence of 1-MCP. Taken together, these results suggest that ACC signaling can bypass the ethylene receptor step to stimulate PT growth and EBS driven gene expression.

Fine monitoring of major phenolic compounds in lettuce and escarole leaves during storage.

Two varieties of lettuce (Lactuca sativa L. var. crispa and capitata) and one variety of escarole (Cichorium endivia var. latifolium) were chosen due to their different browning sensitivity during storage as minimally processed products. The changes in the compositions and contents of the primary polyphenolic compounds were investigated during the first few hours of storage and then after 1, 2, 3, and 6 days of storage at 6°C and revealed sharp variations. Browning development, activity of phenylalanine ammonia lyase, and concentration of ascorbic acid were also examined during storage. The content of chicoric acid, which was the most abundant phenolic compound, varied substantially during the first 24 hr of storage and between the different varieties. Oak leaf lettuce, which was the most sensitive variety to browning, was characterized by a higher maintained chicoric acid level with a constant decreased chlorogenic acid level during the storage period. PRACTICAL APPLICATIONS: Browning development is responsible for the short shelf life of minimally processed vegetables, such as lettuce (Lactuca sativa). Polyphenolic compounds, as substrates of enzymatic reactions, are involved in the browning susceptibility of leaves. Comparisons of the profiles and contents of these compounds in different leaves showed dramatic variations during storage. This study provides additional information to better control, optimize minimally processed produce and select more suitable leaves for the fresh-cut industry.

Bioassay-guided isolation and UHPLC-DAD-ESI-MS/MS quantification of potential anti-inflammatory phenolic compounds from flowers of Inula montana L.

ETHNOPHARMACOLOGICAL RELEVANCE: Flowers of Inula montana L. (Asteraceae), commonly known as "Arnica de Provence", are used in the traditional medicine of Provence in France with the same indication as Arnica montana, for the relief of bruises, as an anti-inflammatory agent. AIMS OF THE STUDY: The aim of our study is to evaluate its anti-inflammatory properties and to justify its traditional uses. Its potential valorization is evaluated in order to propose Inula montana as an alternative to Arnica montana. MATERIALS AND METHODS: Bio-guided fractionation of ethanolic extract allowed the isolation of compounds responsible of the inhibition of NO production. The fractionation was realized using chromatographic techniques and structure elucidation was conducted by ESI-MS and NMR spectral data. Anti-inflammatory effect of ethanolic extract, different fractions and isolated pure compounds was studied in vitro on immortalized mouse macrophages RAW 264.7. An analytical UHPLC-DAD-ESI-MS/MS method was developed for the identification of these compounds in the herbal drug. This UHPLC-DAD method was validated and was used to compare the phenolic profile and content in plant material from the two collection sites: Bonnieux and Merindol. RESULTS: Eleven compounds were identified by UHPLC-MS. Chlorogenic acid (1), Luteolin (2), Nepetin (3), 3,5-O-Dicaffeoylquinic acid (4), 1,5-O-Dicaffeoylquinic acid (5), Nepitrin (6), Hispiduloside (7) and Jaceosid (8) were isolated and identified by NMR. Compounds 9, 10 and 11 were confirmed to be 6-Hydroxykaempferol 3,7-dimethyl ether, Hispidulin and Chrysosplenol C, respectively by comparing retention times and MS/MS data with those of the authentic substances. Six compounds: 1 and 4-8 are reported for the first time in Inula montana L. Compounds 2-8 showed promising anti-inflammatory activity with the release of NO with IC50 value < 7 µM. The UHPLC-DAD method of quantification of three major bioactive compounds (1, 3 and 5) was validated. CONCLUSION: Flowers extracts and isolated compounds present promising anti-inflammatory activity which provides a scientific basis for the traditional use of Inula montana and may be proposed in the same indications as Arnica montana. The developed and validated simple, accurate and rapid UHPLC method can be used for the quality control of the herbal drug.

New sesquiterpene acid and inositol derivatives from Inula montana L.

A phytochemical investigation of the ethanol extract of leaves and flowers of Inula montana L. led to the isolation of one new sesquiterpene acid called Eldarin (1) and four new inositol derivatives, Myoinositol,1,5-diangelate-4,6-diacetate (2), Myoinositol,1,6-diangelate-4,5-diacetate (3), Myoinositol-1-angelate-4,5-diacetate-6-(2-methylbutirate) (4), Myoinositol-1-angelate-4,5-diacetate-6-isovalerate (5) isolated for the first time, along with eleven known compounds described for the first time in Inula montana, 1ß-Hydroxyarbusculin A (6), Artemorin (7), Santamarin (8), Chrysosplenol C (9), 6-Hydroxykaempferol 3,7-dimethyl ether (10), Reynosin (11), Calenduladiol-3-palmitate (12), Costunolide (13), 4-Hydroxy-3,5-dimethoxybenzenemethanol (14), 9ß-Hydroxycostunolide (15) and Hispidulin (16). Structural elucidation has been carried out by spectral methods, such as 1D and 2D NMR, IR, UV and HR-ESI-MS. These compounds have been tested in vitro for anti-inflammatory and cytotoxic activity on macrophages RAW 264.7. As a result, compounds 2, 3, 7, 13, 14, 15 and 16 showed a release of NO with IC50 value <30µM on macrophages.

Alternation of light/dark period priming enhances clomazone tolerance by increasing the levels of ascorbate and phenolic compounds and ROS detoxification in tobacco (Nicotiana tabacum L.) plantlets.

The effect of the alternation of light/dark periods (AL) (16/8 min light/dark cycles and a photosynthetic photon flux density (PPFD) of 50 µmol photons m(-2) s(-1) for three days) to clarify the mechanisms involved in the clomazone tolerance of tobacco plantlets primed with AL was studied. Clomazone decreased PSII activity, the net photosynthetic rate (Pn), and the ascorbate and total polyphenol contents and increased H2O2 and starch grain accumulation and the number of the cells that underwent programmed cell death (PCD). The pretreatment with AL reduced the inhibitory effect of clomazone on the PSII activity and photosynthesis, as indicated by the decreases in the H2O2 and starch grain accumulation and the PCD levels, and increased the content of ascorbate and certain phenolic compounds, such as chlorogenic acid, neochlorogenic acid and rutin. The AL treatment could promote photorespiration via post-illumination burst (PIB) effects. This alternative photorespiratory electron pathway may reduce H2O2 generation via the consumption of photochemical energy, such as NADH+H(+). At 10 days (D10) of AL treatment, this process induced moderate stress which stimulates H2O2 detoxification systems by increasing the activity of antioxidant enzymes and the biosynthesis of antioxidant components. Therefore, the PCD levels provoked by clomazone were noticeably decreased.

Tolerance to clomazone herbicide is linked to the state of LHC, PQ-pool and ROS detoxification in tobacco (Nicotiana tabacum L.).

In this study, plantlets of two tobacco (Nicotiana tabacum L.) varieties that are clomazone-tolerant (cv. Xanthi) and clomazone-sensitive (cv. Virginie vk51) were subjected to low concentration of clomazone herbicide. The oxygen-evolving rate of isolated chloroplasts, chlorophyll a fluorescence transients, JIP-test responses, hydrogen peroxide contents, antioxidant enzyme activities, cytohistological results and photosynthetic pigment contents were recorded. The results indicated that the carotenoid content was 2-fold higher in Virginie, which had greater clomazone sensitivity than Xanthi. Virginie exhibited noticeable decreases in the LHC content (Chl a/b ratio), the maximum photochemical quantum efficiency of PSII (Fv/Fm), the performance index on the absorption basis (PIabs), and the electron flux beyond the first PSII QA evaluated as (1-VJ) with VJ=(FJ-F0)/(Fm-F0) as well as increases in the rate of photon absorption (ABS/RC) and the energy dissipation as heat (DI0/RC). These results suggest that PSII photoinhibition occurred as a consequence of more reduced PQ-pool and accumulated QA(-). The oxygen evolution measurements indicate that PSI electron transport activity was not affected by clomazone. The more significant accumulation of H2O2 in Virginie compared to Xanthi was due to the absence of ROS-scavenging enzymes, and presumably induced programmed cell death (PCD). The symptoms of PCD were observed by cytohistological analysis, which also indicated that the leaf tissues of clomazone-treated Virginie exhibited significant starch accumulation compared to Xanthi. Taken together, these results indicate that the variable tolerance to clomazone observed between Virginie and Xanthi is independent of the carotenoid content and could be related to the state of the LHC, the redox state of the PQ-pool, and the activity of detoxification enzymes.

Pretreatment with alternation of light/dark periods improves the tolerance of tobacco (Nicotiana tabacum) to clomazone herbicide.

This work analyses the effects of alternation of light/dark periods pretreatment (AL) in tobacco plantlets (Nicotiana tabacum L. cv.Virginie vk51) growing in solution with low concentration of the clomazone herbicide. The experimentation has been carried out by exposing the plantlets to successive and regulated periods of light (16min light/8min dark cycles, PAR 50µmolm(-2)s(-1)) for three days. The photosynthesis efficiency was determined by mean of the chlorophyll fluorescence and JIP-test. The AL pretreatment improved the clomazone tolerance; this has been observed by the increase in the leaf area of the plant, the maximal photochemical quantum efficiency of PSII (Fv/Fm), the actual PSII efficiency (ФPSII), the performance index (PIabs), the electron flux beyond Quinone A (1-VJ), and also by the diminution of the energy dissipating into heat (DI0/RC). Furthermore, AL pretreatment led to low accumulation of hydrogen peroxide (H2O2) which proves that the scavenging enzymatic system have been activated before clomazone treatment. In the plantlets pretreated with AL, with regard to the ascorbate content, some of antioxidant enzyme whose function is associated with it have continued to scavenge reactive oxygen species (ROS) induced by clomazone, such as ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). So, the observed photooxidative damages induced by clomazone herbicide were noticeably reduced.

Effect of salt stress on tomato fruit antioxidant systems depends on fruit development stage.

The effects of different levels of salt stress on the oxidative parameters (H2O2 and MDA), the total pool sizes of ascorbate, the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the activities and relative transcript levels of the enzymes of ascorbate-glutathione cycle; ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were studied in fruits of tomato. Plants were treated by three concentrations of NaCl (50, 100 and 150 mM) and fruits at different development stages were harvested after 3 and 6 days of stress. The concentrations of ascorbate (AsA) and dehydroascorbate (DHA) generally changed with salt stress treatments. Moreover, changes in SOD and CAT activities and DHAR, MDHAR, APX and GR activities and relative transcript levels were dependent on the fruit development stage and the intensity and duration of salt stress. These results suggest that the response of antioxidant systems of tomato fruits to oxidative stress induced by salt stress treatments was different depending on the fruit development stage.

Metabolism in orange fruits is driven by photooxidative stress in the leaves.

In plants, stress signals propagate to trigger distant responses and thus stress acclimation in non-exposed organs. We tested here the hypothesis that leaves submitted to photooxidative stress may influence the metabolism of nearby fruits and thus quality criteria. Leaves of orange trees (Citrus sinensis (L.) Osbeck cv. 'Navelate') were acclimated to shade for 1 week and then submitted to full (FL) and medium light (ML) conditions. As expected, photoinhibition was detected in leaves of both FL and ML treatments as revealed by stress indicators (Fv /Fm , Performance Index) for at least 99 h after treatments. In the fruits near the stressed leaves, we then determined the activities of enzymes related to oxidative stress, superoxide dismutase, catalase and the enzymes of the ascorbate (AA)/glutathione cycle, as well as the contents in sugars, organic acids and carotenoids. Ascorbate peroxidase and monodehydroascorbate reductase activities in the pulp of fruits were dramatically higher in both treatments when compared to the control. AA and total sugars were not affected by the photooxidative stress. However, the FL treatment resulted in a 16% increase in total organic acids, with succinic acid being the major contributor, a shift towards less glucose + fructose and more sucrose, and a 15% increase in total carotenoids, with cis-violaxanthin being the major contributor. Our observations strongly suggest the existence of a signal generated in leaves in consequence of photooxidative stress, transmitted to nearby fruits. Exploiting such a signal by agronomic means promises exciting perspectives in managing quality criteria in fruits accumulating carotenoids.

Response of the physiological parameters of mango fruit (transpiration, water relations and antioxidant system) to its light and temperature environment.

Depending on the position of the fruit in the tree, mango fruit may be exposed to high temperature and intense light conditions that may lead to metabolic and physiological disorders and affect yield and quality. The present study aimed to determine how mango fruit adapted its functioning in terms of fruit water relations, epicarp characteristics and the antioxidant defence system in peel, to environmental conditions. The effect of contrasted temperature and light conditions was evaluated under natural solar radiation and temperature by comparing well-exposed and shaded fruit at three stages of fruit development. The sun-exposed and shaded peels of the two sides of the well-exposed fruit were also compared. Depending on fruit position within the canopy and on the side of a well-exposed fruit, the temperature gradient over a day affected fruit characteristics such as transpiration, as revealed by the water potential gradient as a function of the treatments, and led to a significant decrease in water conductance for well-exposed fruits compared to fruits within the canopy. Changes in cuticle thickness according to fruit position were consistent with those of fruit water conductance. Osmotic potential was also affected by climatic environment and harvest stage. Environmental conditions that induced water stress and greater light exposure, like on the sunny side of well-exposed fruit, increased the hydrogen peroxide, malondialdehyde and total and reduced ascorbate contents, as well as SOD, APX and MDHAR activities, regardless of the maturity stage. The lowest values were measured in the peel of the shaded fruit, that of the shaded side of well-exposed fruit being intermediate. Mango fruits exposed to water-stress-induced conditions during growth adapt their functioning by reducing their transpiration. Moreover, oxidative stress was limited as a consequence of the increase in antioxidant content and enzyme activities. This adaptive response of mango fruit to its climatic environment during growth could affect postharvest behaviour and quality.

Effect of water stress on antioxidant systems and oxidative parameters in fruits of tomato (Solanum lycopersicon L, cv. Micro-tom).

The effects of different levels of water stress on oxidative parameters (H2O2 and MDA), the total pool sizes of ascorbate, the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the activities and relative transcript levels of the enzymes of ascorbate-glutathione cycle ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were studied in the fruit of tomato (Solanum lycopersicum L. cv. Micro-Tom). Plants were subjected to three levels of water stress (S50, S25 and S0) and fruits at different development stages were harvested after 3, 6 and 10 days of stress. Changes in H2O2 and MDA contents indicated that water stress induced oxidative stress in fruits. The concentrations of ascorbate (AsA) and dehydroascorbate (DHA) generally modified with water stress treatments. Moreover, changes in SOD and CAT activities and DHAR, MDHAR, APX and GR activities and relative transcript levels were dependent on the fruit development stage and the intensity and the duration of water stress. These results suggest that the response of antioxidant systems of tomato fruits to oxidative stress induced by water stress treatments was different depending on the fruit development stage.

Microplate quantification of enzymes of the plant ascorbate-glutathione cycle.

Here, we describe microplate assays for determining the specific activities of four enzymes that constitute the ascorbate-glutathione cycle: APX, MDHAR, DHAR, and GR. In plants, these enzymes play a major role in detoxifying reactive oxygen species produced in cells under environmental stress. This work presents the development of plate reader assays to allow rapid analysis of the ascorbate-glutathione cycle activity using tomato fruits subjected to salt stress as a model. With this method, it is possible to analyze easily in one day the activities of the four enzymes for 30 experimental samples, all in triplicate and with blanks.

Photosynthetic activity of Lolium perenne as a function of endophyte status and zinc nutrition.

Grass infection by endophyte fungi can confer host resistance to different types of stress, but limited evidence is available on the related effects on the photosynthetic mechanism of the grasses. Zinc has direct and indirect effects on this mechanism and is one of the more important environmental pollutants. To measure whether photosynthesis of the host plant is affected by fungal infection when endophyte-free and endophyte-infected grasses contain similar excess zinc concentrations in their leaves, two batches of Lolium perenne L. cv. Apollo plants were established, one batch was infected with Neotyphodium lolii, the other was not. Both batches were then treated for 8 d with a nutrient solution containing 0, 1, 5, 10 or 20 mm ZnSO4. The increase in zinc concentration induced some reduction in photosystem II (PSII) activity but not enough to account for the total drop in the net photosynthetic rate. Endophyte fungus favoured maintenance of the PSII activity, but did not significantly modify the net photosynthesis and similar zinc concentration levels were observed in leaves of both types of plant. Interactive effects of zinc and light induced less photodamage to the PSII of the host, which is able to react to an increase in photon flux density (PFD). In endophyte-free plants, the reaction centre contributed more than antenna complexes to energy dissipation. In endophyte-infected plants, the quenching of the reaction centre and antenna complexes rose simultaneously and at a constant rate, as zinc concentrations increased.

Use of commercial plant species in a hydroponic system to treat domestic wastewaters.

The objectives in this work were to investigate a conceptual layout for an inexpensive and simple system that would treat primary municipal wastewater to discharge standards. A commercial hydroponic system was adapted for this study and the wastewater was used to irrigate wooly digitalis (Digitalis lanata Ehrh.) and foxglove (Digitalis purpurea L.). These plants are medicinal and produce cardenolide compounds. Influent and effluent samples were collected once a month for six months and analyzed to determine the various parameters relating to water quality. The legal discharge levels for total suspended solids (SS), biochemical oxygen demand (BOD5), and chemical oxygen demand (COD) were reached for the two tested plants after 48 h of wastewater treatment; the removal was 82, 93, and 79%, respectively, for wooly digitalis and 92, 92, and 84%, respectively, for foxglove. Similar results were obtained during a 6-mo period although the sewage composition varied widely. The system tended to be unable to remove N and P to concentrations below regulated levels. Compared with the nutrient solution composition, the wastewater was more concentrated in Na+ and Cl- and less in N, K+, and Ca2+. These variations can lead to the decline of wooly digitalis plants. Foxglove developed a significant root system to increase mineral absorption wastewater being used as the unique nutritive source. After 10 wk all the wooly digitalis seedlings were dead. Despite this fact, however, the root system remained in place for a significant time (< 4 mo), thus continuing to filter wastewater and to be used as a bacterial support thus making it possible to have a security period to replace the dead plants.

Treatment of domestic wastewater by an hydroponic NFT system.

The objectives in this work were to investigate a conceptual layout for an inexpensive and simple system that would treat primary municipal wastewater to discharge standards. A commercial hydroponic system was adapted for this study and the wastewater was used to irrigate Datura innoxia plants. Influent and effluent samples were collected once a month for six months and analysed to determine the various parameters relating to the water quality. The legal discharge levels for total suspended, biochemical oxygen demand and chemical oxygen demand were reached with the plant system after 24 h of wastewater treatment. Total nitrogen and total phosphorus reduction were also obtained. NH4(+)-N was reduced by 93% with nitrification proving to be the predominant removal process. Significant nitrification occurred when the BOD5 level dropped 45 mg/l. Similar results were obtained for six months although the sewage composition varied widely. D. innoxia develops and uses the wastewater as the unique nutritive source.

Treatment of domestic wastewater using the nutrient film technique (NFT) to produce horticultural roses.

Removal of organic matter, nitrogen and phosphorus was investigated in a pilot based on the nutrient film technique system used for horticultural production. Rosebushes producing commercial roses were set on an inclined impermeable surface over which a thin film of domestic wastewater flowed directly through the root matrix. The roses produced with wastewater as nutrient solution were qualitatively and quantitatively similar to those produced traditionally and can be marketable. In the presence of the rosebushes, the lowering of chemical oxygen demand (COD), biological oxygen demand (BOD5) and suspended solids (SS) increased with their influent strengths in a linear fashion correlation. Whatever the pollutant load of the influent, within the range studied, the level of organic pollution required to allow the discharge of the effluent into water courses was reached after 24 h of treatment. At this time, the COD reached 39 +/- 13 mg L(-1), the BOD5 7 +/- 4 mg L(-1) and the SS 8 +/- 6 mg L(-1). The removal percentages were 89, 95 and 94, respectively, whereas without plants they were 55, 33 and 53, respectively. The rosebushes had a beneficial effect on the removal of phosphorus from 20% to 23% greater in their presence than without. In presence of plants, the nitrate was the principal form of nitrogen from 48 h of treatment whereas without rosebushes, nitrite represent more than 20% of initial nitrogen whatever the treatment duration. The root matrix served not only as a filter for the organic matter, but also provided a microhabitat suitable for nitrifying bacteria and heterotrophic micro-organisms, which responded rapidly to the pollutant load.

Urban wastewater treatment by a nutrient film technique system with a valuable commercial plant species (Chrysanthemum cinerariaefolium Trev.).

Urban wastewater causes rapid eutrophication of natural waters and requires treatment before discharge. This is expensive and produces huge quantities of sludge. In the European Community, it will no longer be lawful to dispose of this sludge as landfill after 2005 (European Directive 91/271/CEE of May 21, 1991). Wastewater treatment by the Chrysanthemum cinerariaefolium plants in horizontal flow was investigated using the nutrient film technique (NFT), a widely used hydroponic system in the commercial greenhouse industry. After a 48 h plant treatment, the purification efficiency was 95%, 91%, and 99% with respect to suspended solids (SS), biochemical oxygen demand (BOD5), and chemical oxygen demand (COD), and the elimination of nutrients (total nitrogen and total phosphorus) varied between 40% and 80%. SS and thus indirectly BOD5 and COD were removed by filtration and adsorption; the solids trapped in the root systems were then decomposed and mineralized. The system with 25 plants purified 30 L of wastewater in 48 h. One-hundred people communities wastewater could be treated with a 6 m2 area of production. Pyrethrin contents and chlorophyll a fluorescence of plants grown on raw urban waters were not significantly different from those grown on a standard nutrient solution.