OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels.

Singlet oxygen produced from triplet excited chlorophylls in photosynthesis is a signal molecule that can induce programmed cell death (PCD) through the action of the OXIDATIVE STRESS INDUCIBLE 1 (OXI1) kinase. Here, we identify two negative regulators of light-induced PCD that modulate OXI1 expression: DAD1 and DAD2, homologs of the human antiapoptotic protein DEFENDER AGAINST CELL DEATH. Overexpressing OXI1 in Arabidopsis (Arabidopsis thaliana) increased plant sensitivity to high light and induced early senescence of mature leaves. Both phenomena rely on a marked accumulation of jasmonate and salicylate. DAD1 or DAD2 overexpression decreased OXI1 expression, jasmonate levels, and sensitivity to photooxidative stress. Knock-out mutants of DAD1 or DAD2 exhibited the opposite responses. Exogenous applications of jasmonate upregulated salicylate biosynthesis genes and caused leaf damage in wild-type plants but not in the salicylate biosynthesis mutant Salicylic acid induction-deficient2, indicating that salicylate plays a crucial role in PCD downstream of jasmonate. Treating plants with salicylate upregulated the DAD genes and downregulated OXI1 We conclude that OXI1 and DAD are antagonistic regulators of cell death through modulating jasmonate and salicylate levels. High light-induced PCD thus results from a tight control of the relative activities of these regulating proteins, with DAD exerting a negative feedback control on OXI1 expression.

Actin filament reorganisation controlled by the SCAR/WAVE complex mediates stomatal response to darkness.

Stomata respond to darkness by closing to prevent excessive water loss during the night. Although the reorganisation of actin filaments during stomatal closure is documented, the underlying mechanisms responsible for dark-induced cytoskeletal arrangement remain largely unknown. We used genetic, physiological and cell biological approaches to show that reorganisation of the actin cytoskeleton is required for dark-induced stomatal closure. The opal5 mutant does not close in response to darkness but exhibits wild-type (WT) behaviour when exposed to abscisic acid (ABA) or CaCl2 . The mutation was mapped to At5g18410, encoding the PIR/SRA1/KLK subunit of the ArabidopsisSCAR/WAVE complex. Stomata of an independent allele of the PIR gene (Atpir-1) showed reduced sensitivity to darkness and F1 progenies of the cross between opal5 and Atpir-1 displayed distorted leaf trichomes, suggesting that the two mutants are allelic. Darkness induced changes in the extent of actin filament bundling in WT. These were abolished in opal5. Disruption of filamentous actin using latrunculin B or cytochalasin D restored wild-type stomatal sensitivity to darkness in opal5. Our findings suggest that the stomatal response to darkness is mediated by reorganisation of guard cell actin filaments, a process that is finely tuned by the conserved SCAR/WAVE-Arp2/3 actin regulatory module.

Singlet Oxygen-Induced Cell Death in Arabidopsis under High-Light Stress Is Controlled by OXI1 Kinase.

Studies of the singlet oxygen ((1)O2)-overproducing flu and chlorina1 (ch1) mutants of Arabidopsis (Arabidopsis thaliana) have shown that (1)O2-induced changes in gene expression can lead to either programmed cell death (PCD) or acclimation. A transcriptomic analysis of the ch1 mutant has allowed the identification of genes whose expression is specifically affected by each phenomenon. One such gene is OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) encoding an AGC kinase that was noticeably induced by excess light energy and (1)O2 stress conditions leading to cell death. Photo-induced oxidative damage and cell death were drastically reduced in the OXI1 null mutant (oxi1) and in the double mutant ch1*oxi1 compared with the wild type and the ch1 single mutant, respectively. This occurred without any changes in the production rate of (1)O2 but was cancelled by exogenous applications of the phytohormone jasmonate. OXI1-mediated (1)O2 signaling appeared to operate through a different pathway from the previously characterized OXI1-dependent response to pathogens and H2O2 and was found to be independent of the EXECUTER proteins. In high-light-stressed plants, the oxi1 mutation was associated with reduced jasmonate levels and with the up-regulation of genes encoding negative regulators of jasmonate signaling and PCD. Our results show that OXI1 is a new regulator of (1)O2-induced PCD, likely acting upstream of jasmonate.

Light-induced acclimation of the Arabidopsis chlorina1 mutant to singlet oxygen.

Singlet oxygen (¹O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, ¹O2-induced transcriptomic changes result in acclimation to ¹O2. Here, using a chlorophyll b-less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of ¹O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to ¹O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing ¹O2 formation, indicating acclimation to ¹O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in ¹O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2.

The dual effect of abscisic acid on stomata.

The classical view that the drought-related hormone ABA simply acts locally at the guard cell level to induce stomatal closure is questioned by differences between isolated epidermis and intact leaves in stomatal response to several stimuli. We tested the hypothesis that ABA mediates, in addition to a local effect, a remote effect in planta by changing hydraulic regulation in the leaf upstream of the stomata. By gravimetry, porometry to water vapour and argon, and psychrometry, we investigated the effect of exogenous ABA on transpiration, stomatal conductance and leaf hydraulic conductance of mutants described as ABA-insensitive at the guard cell level. We show that foliar transpiration of several ABA-insensitive mutants decreases in response to ABA. We demonstrate that ABA decreases stomatal conductance and down-regulates leaf hydraulic conductance in both the wildtype Col-0 and the ABA-insensitive mutant ost2-2. We propose that ABA promotes stomatal closure in a dual way via its already known biochemical effect on guard cells and a novel, indirect hydraulic effect through a decrease in water permeability within leaf vascular tissues. Variability in sensitivity of leaf hydraulic conductance to ABA among species could provide a physiological basis to the isohydric or anisohydric behaviour.

Toxicity of copper excess on the lichen Dermatocarpon luridum: antioxidant enzyme activities.

The aim of this study was to investigate the toxicity of copper on the aquatic lichen Dermatocarpon luridum focusing on the activities of some antioxidant enzymes. Investigations were conducted using increasing copper concentrations (0.00, 0.25, 0.50, 0.75 and 1.00 mM CuSO(4) x 5H(2)O) in synthetic freshwater that emulated the major ion compositions of its natural water biota; time course measurement was 0, 3, 6, 12, 24 and 48 h. The copper concentration in thalli increased with its increase in the medium and the duration of treatment. Copper induced lipid peroxidation, measured using the hydroperoxi-conjugated dienes (HPCD) concentration. The decrease in the protein concentrations was similar in thalli exposed to copper concentrations above 0.50 mM and the decrease was twice lower in thalli exposed to 0.25 mM copper. The activities of antioxidant enzymes measured were differently affected by copper excess. For 0.25 mM copper, the activities of SOD (superoxide dismutase) and APX (ascorbate peroxidase) were unchanged when compared with unstressed thalli whereas the CAT (catalase) activity increased and the GR (glutathione reductase) activity decreased. The activities of SOD and APX increased in thalli exposed to concentrations above 0.50mM copper. The CAT activity increased after the first 3h of experiments at these concentrations and then decreased with the duration of treatment at an activity lower than in the unstressed plant. Whereas the APX activity increased, the GR activity similarly decreased for the copper concentration tested whatever the duration of the experiment.

Use of the aquatic lichen Dermatocarpon luridum as bioindicator of copper pollution: accumulation and cellular distribution tests.

Laboratory experiments were conducted to investigate the potential use of the aquatic lichen Dermatocarpon luridum as bioindicator of copper pollution. Lichen thalli were exposed to 0.00, 0.25, 0.50, 0.75 and 1.00 mM copper in synthetic freshwater to solve the problems of metal bioavailability. The mineral composition of this media was prepared so that it corresponded to the ion composition of natural waters in D. luridum ecosystems. Sequential elution procedures using NiCl2 or Na2-EDTA (20 mM) were used to determine the distribution of metals at different cellular sites. The copper concentration extracted from thalli was correlated with pollution intensity, the greater correlation being with the Na2-EDTA extractant. The malondialdehyde concentration in thalli can be used as indicator of copper pollution; however, similar membrane degradation was observed for 0.25 and 0.50 mM copper and for 0.75 and 1.00 mM copper.

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.