The silver lining of antibiotic resistance: Bacterial-mediated reduction of tetracycline plant stress via antibiotrophy.

The reuse of water using effluents containing antibiotics from anthropogenic activities has been mainly linked to the development of antibiotic resistance. However, we report that the development of bacterial tolerance promotes plant growth. In the present study, we aimed to evaluate the efficiency of inoculation of a new antibiotic-degrading bacterium, Erwinia strain S9, in augmenting the tolerance of pea (Pisum sativum L.) plants to tetracycline (TET) (10 and 20 mg/L). Physiological parameters such as tissue elongation and biomass, as well as relative water content, were remarkably lower in plants exposed to TET than in the control. The inhibitory effects of TET were associated with reduced CO2 assimilation, stomatal conductance, transpiration, dark respiration, and light saturation point (LSP). High concentrations of TET-induced oxidative stress are attested by the overproduction of superoxide radicals (O2•-), hydrogen peroxide (H2O2), and hydroxyl radicals (HO•), resulting in increased malondialdehyde content and cell death. The high activity of antioxidant enzymes such as catalase, ascorbate peroxidase, and guaiacol peroxidase validated the proposed mechanism. Under TET stress conditions, supplementation with Erwinia strain S9 was beneficial to pea plants through osmotic adjustment, increased nutrient uptake, gas exchange optimization, and increased antioxidant activities. Its presence not only ensures plant survival and growth during antibiotic stress but also degrades TET via significant antibiotrophy. This strategy is a cost-effective environmental chemical engineering tool that can be used to depollute wastewater or to improve crop resistance in rhizofiltration treatment when treated wastewater is reused for irrigation.

Insights on strain 115 plant growth-promoting bacteria traits and its contribution in lead stress alleviation in pea (Pisum sativum L.) plants.

The present study aims to characterize the plant growth-promoting bacterial traits of Bacillus simplex (strain 115). This bacterium was inoculated in hydroponically conditions to improve pea (Pisum sativum L.) growth submitted to lead (Pb) toxicity. Root nodulation system was developed enough in 23-day-old plants attesting the interaction between the two organisms. In addition to its phosphate solubilization and siderophore production traits that reached 303.8 µg P mL-1 and 49.6 psu respectively, the Bacillus strain 115 exhibited Pb bio-sorption ability. Inoculation of Pb-stressed pea with strain 115 showed roots and shoots biomass recovery (+ 70% and + 61%, respectively). Similarly, water and protein contents were increased in Pb-treated plants after bacterial inoculation. In the presence of strain 115, Pb relative toxicity level decreased (- 39.3% compared to Pb stress only). Moreover, catalase and superoxide dismutase activities were upregulated in Pb-exposed plants (+ 56% and + 51%, respectively). After inoculation with strain 115, catalase and superoxide dismutase activities were restored by - 38% and - 44% respectively. Simultaneously, oxidant stress indicator (H2O2 and 4-hydroxynonenal) and osmo-regulators (proline and glycine-betaine) contents as well as lipoxygenase activity decreased significantly in Pb-treated plants after Bacillus strain's inoculation. Taken together, the results give some evidences for the plant growth-promoting capacity of strain 115 in helping alleviation of Pb stress.

Nitric oxide donor, sodium nitroprusside modulates hydrogen sulfide metabolism and cysteine homeostasis to aid the alleviation of chromium toxicity in maize seedlings (Zea mays L.).

The current research aimed to assess the protective role of nitric oxide (NO) against chromium (Cr) toxicity in maize seedlings. Chromium (200 µM) lowered osmotic potential in epicotyls and mostly in radicles (by 38% and 63%, respectively) as compared to the control. Sodium nitroprusside (SNP, NO donor) restored seedling biomass (+90% for both organs) and water potential, whereas application of Nω-nitro-L-arginine methylester (L-NAME, a NOS inhibitor) increased sensitivity to Cr. SNP suppressed Cr-triggered proline accumulation by inhibiting Δ1-pyrroline-5-carboxylate synthetase activity and stimulating proline dehydrogenase activity, leading to glutamate over-accumulation (~30% for both organs). Cr stimulated cysteine metabolism and this was further enhanced by SNP which stimulated serine acetyl-transferase and O-acetylserine (thiol) lyase activities. This was followed by an increase in endogenous hydrogen sulfide (H2S) generation by up-regulating L-cysteine desulfhydrase (+205%), D-cysteine desulfhydrase (+150%) and cyanoalanine synthase (+65%) activities in radicles compared to Cr-treatments plants. These positive effects were reduced in L-NAME compared to control. Combined Cr+SNP affected the levels of compounds involved in glutathione metabolism (γ-glutamyl-cysteinyl, γ-glutamyl-cysteinyl-clycine, γ-cysteinyl-glycine, and glycine.). All together, our findings indicate that NO and elicited cellular H2S act synergistically to alleviate Cr stress in maize seedlings by influencing a metabolic interplay between cysteine, proline, and glutathione.

Exogenous nitric oxide alleviates manganese toxicity in bean plants by modulating photosynthesis in relation to leaf lipid composition.

Nitric oxide (NO) is a signaling molecule controlling several steps of plant development and defense process under stress conditions. NO-induced alleviation of manganese (Mn) toxicity was investigated on bean plants submitted for 28 days to 500 µM MnCl2. Manganese excess decreased plant dry weight and elongation and increased levels of reactive oxygen species and lipid peroxidation leading to up-regulation of superoxide dismutase, catalase, and ascorbate peroxidase activities. The inhibitory effects of Mn on plant growth were associated to reduction of light-saturated carbon assimilation (Amax), stomatal conductance (gs), and transpiration (E). By contrast, Mn induced significant increase in the apparent quantum yield (ɸ) and light compensation point (LCP). Interestingly, intracellular CO2 (Ci) remains stable under Mn stress. Concomitantly, leaf membrane lipids have drastically reduced under high Mn concentration. After Mn exposition, leaf fatty acids exhibited a significant loss of linolenic acid, accompanied by an accumulation of palmitoleic, stearic, and linoleic acids leading to alteration of lipid desaturation. NO supply reversed Mn toxicity as evidenced by enhancement of growth biomass and recovery of Amax, E, ɸ, and LCP. Similarly, NO addition has positive effects on leaf lipid content and composition leading to restoration of lipid unsaturation. The modulation of fatty acid composition can be a way to reduce leaf membrane damages and maintain optimal photosynthesis and plant growth. Despite the absence of enough evidences in how NO is involved in lipid and photosynthesis recovery under Mn stress conditions, it is assumed that NO beneficial effects are attributable to NO/Mn cross-talk.

Gallic acid improves the antioxidant ability against cadmium toxicity: Impact on leaf lipid composition of sunflower (Helianthus annuus) seedlings.

In the present work, the effect of seed pre-soaking with gallic acid (GA; 3,4,5-triphydroxyl-benzoic acid) in conferring subsequent tolerance to Cd stress in sunflower (Helianthus annuus) seedlings was investigated. Exposing sunflower seedlings to increasing Cd concentrations (5, 10 and 20 µM) caused a gradual decrease in root and shoot biomass and increased the metal accumulation in both organs. Seed pretreatment with 75 µM GA significantly restricted Cd uptake, markedly alleviated Cd-induced plant growth inhibition, and mitigated the oxidative damages caused by this metal, as compared to plants directly exposed to Cd. GA pre-soaking prior to Cd stress also enhanced catalase, ascorbate peroxidase and glutathione reductase activities, while inhibiting that of superoxide dismutase. This was associated with increased levels of total thiols and glutathione along with a decreased level of oxidized glutathione in leaves. Moreover, GA pre-soaking led to changes in leaf fatty acid composition of seedlings challenged with Cd, as evidenced by the higher total lipid content and lipid unsaturation degree. As a whole, this study provides strong arguments highlighting the potential role of GA as a growth promoter for sunflower seedlings submitted to Cd stress, notably by boosting the antioxidant defense system and improving leaf membrane stability.

Nitric oxide and hydrogen sulfide protect plasma membrane integrity and mitigate chromium-induced methylglyoxal toxicity in maize seedlings.

The present study aims to analyse the potential crosstalk between nitric oxide (NO) and hydrogen sulfide (H2S) in triggering resilience of maize (Zea mays L.) seedlings to hexavalent chromium (Cr VI). Exogenous application of 500 µM sodium nitroprusside (SNP, as a NO donor) or sodium hydrosulfide (NaHS, as a H2S donor) to 9-day-old maize seedlings, countered a Cr (200 µM) -elicited reduction in embryonic axis biomass. Cr caused cellular membrane injury by enhancing the levels of superoxide and hydroxyl radicals as well as methylglyoxal, and 4-hydroxy-2-nonenal. The application of SNP or NaHS considerably improved the endogenous NO and H2S pool, decreased oxidative stress and lipid peroxidation by suppressing lipoxygenase activity and improving some antioxidant enzymes activities in radicles and epicotyls. Radicles were more affected than epicotyls by Cr-stress with enhanced electrolyte leakage and decreased proton extrusion as indicated by lesser H+-ATPase activity. H2S appeared to mitigate Cr toxicity through up-regulated H+-ATPase and glyoxalase pathways and by maintaining optimal GSH levels as downstream effects of ROS and MG suppression. Hence, H2S-mediated the regeneration of GSH pool is associated with the attenuation of MG toxicity by enhancing S-lactoglutathione and D-lactate production. Taken together, our results indicate complementary roles for H2S and GSH to strengthen membrane integrity against Cr stress in maize seedlings.

Exogenous application of hydrogen sulfide reduces chromium toxicity in maize seedlings by suppressing NADPH oxidase activities and methylglyoxal accumulation.

Chromium (Cr) represents an important source of metallic stress in plants. Working with maize (Zea mays) seedlings, we characterize the suppressive effects of exogenously applied NaHS (a hydrogen sulfide; [H2S] donor) on the toxic effects of Cr (VI). Heavy metal treatment reduced radicle and epicotyl lengths and fresh weights in seedlings at 6 and 9 days following germination. The negative Cr (200 µM) effect was countered by application with NaHS (500 µM) but this countering was reduced with the co-application of the H2S generation inhibitor hydroxylamine (HA) or the H2S scavenger hypotaurine (HT). The Cr-elicited H2O2 production was suppressed by NaHS and also by an inhibitor of the reactive oxygen species (ROS) generating NADPH oxidase (NOX). These effects were correlated with relative changes in carbomyl (-CO) and thiol (-SH) groups. Nitric oxide (NO) production increased by NaHS application with associated increase in S-nitrosoglutathione (GSNO) level, but low S-nitrosoglutathione reductase (GSNOR) activities indicating an elevated S-nitrosylation. Assessment of the role of the ascorbate-glutathione antioxidant cycle indicated that whilst ascorbate played at a best minor role, glutathione was more prominent. Methylglyoxal (MG) production was increased by Cr but reduced by NaHS through a mechanism which could be based on glutathione-S-transferase (GST) detoxification. Taken together data suggest that H2S acts to counter Cr effect in maize by NOX suppression, mostly likely by the well-characterised S-nitrosylation mechanism, as well as a reduction of MG accumulation.

Exogenous salicylic acid protects phospholipids against cadmium stress in flax (Linum usitatissimum L.).

Salicylic acid (SA) promotes plant defense responses against toxic metal stresses. The present study addressed the hypothesis that 8-h SA pretreatment, would alter membrane lipids in a way that would protect against Cd toxicity. Flax seeds were pre-soaked for 8h in SA (0, 250 and 1000µM) and then subjected, at seedling stage, to cadmium (Cd) stress. At 100µM CdCl2, significant decreases in the percentages of phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol (MGDG) and changes in their relative fatty acid composition were observed in Cd-treated roots in comparison with controls. However, in roots of 8-h SA pretreated plantlets, results showed that the amounts of PC and PE were significantly higher as compared to non-pretreated plantlets. Additionally, in both lipid classes, the proportion of linolenic acid (18:3) increased upon the pretreatment with SA. This resulted in a significant increase in the fatty acid unsaturation ratio of the root PC and PE classes. As the exogenous application of SA was found to be protective of flax lipid metabolism, the possible mechanisms of protection against Cd stress in flax roots were discussed.

Positive effects of salicylic acid pretreatment on the composition of flax plastidial membrane lipids under cadmium stress.

Interest in use of flax (Linum usitatissimum L.) as cadmium (Cd)-accumulating plant for phytoextraction of contaminated soils opened up a new and promising avenue toward improving tolerance of its varieties and cultivars to Cd stress. The aim of this study is to get insights into the mechanisms of Cd detoxification in cell membranes, by exploring the effects of salicylic acid (SA)-induced priming on fatty acids and lipid composition of flax plantlets, grown for 10 days with 50 and 100 µM Cd. At leaf level, levels of monogalactosyldiacylglycerol (MGDG), phosphatidylcholine (PC), phosphatidylglycerol (PG), and neutral lipids (NL) have shifted significantly in flax plantlets exposed to toxic CdCl2 concentrations, as compared to that of the control. At 100 µM Cd, the linoleic acid (C18:2) decreases mainly in digalactosyldiacylglycerol (DGDG) and all phospholipid species, while linolenic acid (C18:3) declines mostly in MGDG and NL. Conversely, at the highest concentration of the metal, SA significantly enhances the levels of MGDG, PG and phosphatidic acid (PA), and polyunsaturated fatty acids mainly C18:2 and C18:3. Furthermore, SA pretreatment seems to reduce the Cd-induced alterations in both plastidial and extraplastidial lipid classes, but preferentially preserves the plastidial lipids by acquiring higher levels of polyunsaturated fatty acids. These results suggest that flax plantlets pretreated with SA exhibits more stability of their membranes under Cd-stress conditions.

Selenium alleviates cadmium toxicity by preventing oxidative stress in sunflower (Helianthus annuus) seedlings.

The present study investigated the possible mediatory role of selenium (Se) in protecting plants from cadmium (Cd) toxicity. The exposure of sunflower seedlings to 20µM Cd inhibited biomass production, decreased chlorophyll and carotenoid concentrations and strongly increased accumulation of Cd in both roots and shoots. Similarly, Cd enhanced hydrogen peroxides content and lipid peroxidation as indicated by malondialdehyde accumulation. Pre-soaking seeds with Se (5, 10 and 20µM) alleviated the negative effect of Cd on growth and led to a decrease in oxidative injuries caused by Cd. Furthermore, Se enhanced the activities of catalase, ascorbate peroxidase and glutathione reductase, but lowered that of superoxide dismutase and guaiacol peroxidase. As important antioxidants, ascorbate and glutathione contents in sunflower leaves exposed to Cd were significantly decreased by Se treatment. The data suggest that the beneficial effect of Se during an earlier growth period could be related to avoidance of cumulative damage upon exposure to Cd, thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity.

Salicylic acid improves root antioxidant defense system and total antioxidant capacities of flax subjected to cadmium.

Cadmium (Cd) disrupts the normal growth and development of plants, depending on their tolerance to this toxic element. The present study was focused on the impacts of exogenous salicylic acid (SA) on the response and regulation of the antioxidant defense system and membrane lipids to 16-day-old flax plantlets under Cd stress. Exposure of flax to high Cd concentrations led to strong inhibition of root growth and enhanced lipid peroxides, membrane permeability, protein oxidation, and hydrogen peroxide (H2O2) production to varying degrees. Concomitantly, activities of the antioxidant enzymes catalase (CAT, EC 1.11.1.6), guaïcol peroxydase (GPX, EC 1.11.1.7), ascorbate peroxydase (APX, EC 1.11.1.11), and superoxide dismutase (SOD, EC 1.15.1.1), and the total antioxidant capacities (2,2'-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and ferric reducing antioxidant power (FRAP)) were significantly altered by Cd. In contrast, exogenous SA greatly reduced the toxic effects of Cd on the root growth, antioxidant system, and membrane lipid content. The Cd-treated plantlets pre-soaked with SA exhibited less lipid and protein oxidation and membrane alteration, as well as a high level of total antioxidant capacities and increased activities of antioxidant enzymes except of CAT. These results may suggest that SA plays an important role in triggering the root antioxidant system, thereby preventing membrane damage as well as the denaturation of its components.

Effects of long-term cadmium exposure on growth and metabolomic profile of tomato plants.

The response of tomato plants to long-term cadmium exposure was evaluated after a 90-days long culture in hydroponic conditions (0, 20, and 100 µM CdCl(2)). Cadmium preferentially accumulated in roots, and to a lower extent in upper parts of plants. Absolute quantification of 28 metabolites was obtained through (1)H NMR, HPLC-PDA, and colorimetric methods. The principal component analysis showed a clear separation between control and Cd treated samples. Proline and total ascorbate amounts were reduced in Cd-treated leaves, whereas α-tocopherol, asparagine, and tyrosine accumulation increased, principally in 100 µM Cd treated leaves. Carotenoid and chlorophyll contents decreased only in 100 µM Cd-mature-leaves, which correlate with a reduced expression of genes essential for isoprenoid and carotenoid accumulations. Our results show that tomato plants acclimatize during long-term exposure to 20 µM Cd. On the contrary, 100µM Cd treatment results in drastic physiological and metabolic perturbations leading to plant growth limitation and fruit set abortion.

Ultrastructure of Aeluropus littoralis leaf salt glands under NaCl stress.

The effects of salt uptake on the morphology and ultrastructure of leaf salt glands were investigated in Aeluropus littoralis plants grown for two months in the presence of 400 mM NaCl. The salt gland is composed of two linked cells, as observed in some other studied Poaceae species. The cap cell, which protrudes from the leaf surface, is smaller than the basal cell, which is embedded in the leaf mesophyll tissues and bears the former. The cuticle over the cap cell is frequently separated from the cell wall to form a cavity where salts accumulate prior to excretion. The basal cell cytoplasm contains an extensive intricate or partitioning membrane system that is probably involved in the excretion process, which is absent from the cap cell. The intricate membrane system seems to be elongated and heavily loaded with salt. The presence of 400 mM NaCl induced the disappearance of the collecting chamber over the glands and an increase in the number of vacuoles and their size in both gland cells. In the basal cell, salt greatly increased both the density and size of the intricate membrane system. The electron density of both gland cells observed under salt treatment reflects a high activity. All these changes probably constitute special adaptations for dealing with salt accumulation in the leaves. Despite the high salt concentration used, no serious damage occurred in A. littoralis salt gland ultrastructure, which consolidates the assumption that they are naturally designated for this purpose.

Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants.

The effects of cadmium (Cd) on cellular proteolytic responses were investigated in the roots and leaves of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 3 and 10 days in the presence of 0.3-300 microM Cd and compared to control plants grown in the absence of Cd. Roots of Cd treated plants accumulated four to fivefold Cd as much as mature leaves. Although 10 days of culture at high Cd concentrations inhibited plant growth, tomato plants recovered and were still able to grow again after Cd removal. Tomato roots and leaves are not modified in their proteolytic response with low Cd concentrations (< or =3 microM) in the incubation medium. At higher Cd concentration, protein oxidation state and protease activities are modified in roots and leaves although in different ways. The soluble protein content of leaves decreased and protein carbonylation level increased indicative of an oxidative stress. Conversely, protein content of roots increased from 30 to 50%, but the amount of oxidized proteins decreased by two to threefold. Proteolysis responded earlier in leaves than in root to Cd stress. Additionally, whereas cysteine- and metallo-endopeptidase activities, as well as proteasome chymotrypsin activity and subunit expression level, increased in roots and leaves, serine-endopeptidase activities increased only in leaves. This contrasted response between roots and leaves may reflect differences in Cd compartmentation and/or complexation, antioxidant responses and metabolic sensitivity to Cd between plant tissues. The up-regulation of the 20S proteasome gene expression and proteolytic activity argues in favor of the involvement of the 20S proteasome in the degradation of oxidized proteins in plants.

Salt impact on photosynthesis and leaf ultrastructure of Aeluropus littoralis.

The effects of salinity (400 mM NaCl) on growth, biomass partitioning, photosynthesis, and leaf ultrastructure were studied in hydroponically grown plants of Aeluropus littoralis (Willd) Parl. NaCl produced a significant inhibition of the main growth parameters and a reduction in leaf gas exchange (e.g. decreased rates of photosynthesis and stomatal conductance). However, NaCl salinity affected neither the composition of photosynthesis pigments nor leaf water content. The reduction in leaf gas exchange seemed to correlate with a decrease in mesophyll thickness as well as a severe disorganisation of chloroplast structure, with misshapen chloroplasts and dilated thylakoid membranes. Conspicuously, mesophyll chloroplasts were more sensitive to salt treatment than those of bundle sheath cells. The effects of NaCl toxicity on leaf structure and ultrastructure and the associated physiological implications are discussed in relation to the degree of salt resistance of A. littoralis.

Contribution of NaCl excretion to salt resistance of Aeluropus littoralis (Willd) Parl.

Aeluropus littoralis is a perennial halophyte, native to coastal zones. Although it is usually exposed to high saline, this plant grows normally without toxicity symptoms. In order to assess leaf salt excretion, different growth parameters, Na(+), K(+), Ca(2+), Mg(2+) and Cl(-) concentrations, as well as excreted ions were examined in plants grown for 2 months in the presence of various salinity levels (0-800 mM NaCl). In addition, salt crystals, salt glands and other leaf epidermal structures were investigated. Results showed that total plant growth decreased linearly with increase to medium salinity. This reduction concerns mainly shoot growth. In addition, this species was able to maintain its shoot water content at nearly 50% of the control even when subjected to 800 mM NaCl. Root water content seemed to be unaffected by salt. Sodium and chloride ion contents in shoots and in roots increased with salinity concentrations, in contrast to our observation for potassium. However, calcium and magnesium contents were not greatly affected by salinity. Excreted salts in A. littoralis leaves were in favor of sodium and chloride, but against potassium, calcium and magnesium which were retained in plants. Sodium and chloride were excreted from special salt glands, which were scattered on the both leaf surfaces. In addition to salt glands, papillae were the most frequent epidermal structure found on A. littoralis leaves, and are likely involved in A. littoralis salt resistance.

[Physiological and structural modifications induced by cadmium-calcium interaction in tomato (Lycopersicon esculentum)]. / Modifications physiologiques et structurales induites par l'interaction cadmium-calcium chez la tomate (Lycopersicon esculentum).

Tomato seedlings (Lycopersicon esculentum), initially cultivated in a basic nutrient solution during 12 days, were treated with increasing CdCl(2) concentrations for 10 days. The results showed that cadmium inhibited the weight growth depending on the metal concentration and the plant organ. In the presence of 20 microM CdCl(2), the addition of calcium, 0.1 to 10 mM of CaCl(2) in the culture medium, improved especially the biomass production and the mineral composition of the plants in concomitance with an increase in the contents of photosynthetic pigments. Histological study at the hypocotyle level revealed that cadmium (20 microM) induced a restriction of the tissue territories as well as meristem formations differentiating in a root structure. At this concentration, the addition of CaCl(2) (5 microM) was characterized by an opposite effect with absence of meristem structures. The overall results suggest that the alteration of some plant growth process after exposure to cadmium can be attenuated by an adequate calcium contribution in culture medium.