Physiological and structural modifications in snail medic (Medicago scutellata L.) plants exposed to salinity.

Seeds of snail medic (Medicago scutellata L.) were assessed for their response to salt at the germination and seedling stages. NaCl at concentrations 86 and 170 mM decreased the final germination percentage. Embryonic axis length, water content and dry weight of embryonic axis and cotyledons were also reduced by salt treatment. Furthermore, 28-d-old plants were grown hydroponically with different NaCl concentrations (0, 86 and 170 mM). After 7 days of treatment, growth, water content and development of the different organs of M. scutellata plant were affected especially at the highest NaCl concentration (170 mM). However, NaCl did not affect root length and the number of stem shoots but reduced stem length and total leaf area. Salt treatment increased markedly the concentration of Na+ in leaf and root tissues while reduced that of K+ only in root and stem tissues. Lipid peroxidation revealed the damage of the membranes of roots and leaves. Moreover, showed a more intense suberization and lignification at the cambial zone of roots of M. scutellata, were observed under the effect of NaCl.

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.

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.

Cadmium tolerance and accumulation characteristics of mature flax, cv. Hermes: contribution of the basal stem compared to the root.

The potential of mature flax plants (cv. Hermes) to tolerate and accumulate cadmium (Cd) was studied to determine which part of the plant would be the key organ for phytoremediation purposes. After 4 month-growth on sand substrate containing 0.1mM Cd in a greenhouse, the roots and stems were separated and the stems were divided into three parts. The effects of Cd were studied on growth parameters, histology and mineral nutrition. No visible toxic symptoms were observed. Tolerance-index values calculated from growth parameters and nutrients remained relatively high, allowing the development of the plant until maturity and formation of seeds. The roots and bottom stem accumulated the highest quantity of Cd (750 and 360 mg/kg dry matter), values which largely exceeded the threshold defined for hyperaccumulators. On the other hand, basal stem had a high bioconcentration factor (BCF=32) and translocation factor TF' (2.5) but a low TF (0.5), indicating that this basal part would play a major role in phytoremediation (phytostabilization rather than phytorextraction). Therefore, the high tolerance to Cd and accumulation capacity make possible to grow Hermes flax on Cd-polluted soils.

Up-regulation of leucine aminopeptidase-A in cadmium-treated tomato roots.

The effects of cadmium (Cd) on aminopeptidase (AP) activities and Leucine-AP (LAP) expression were investigated in the roots of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 10 days in the presence of 0.3-300 µM Cd and compared to control plants grown in the absence of Cd. AP activities were measured using six different p-nitroanilide (p-NA) substrates. Leu, Met, Arg, Pro and Lys hydrolyzing activities increased in roots of Cd-treated plants, while Phe-pNA cleavage was not enhanced after Cd treatments. The use of peptidase inhibitors showed that most of the Leu-pNA hydrolyzing activity was related to one or several metallo-APs. Changes in Lap transcripts, protein and activities were measured in the roots of 0 and 30-µM Cd-treated plants. LapA transcript levels increased in Cd-treated roots, whereas LapN RNAs levels were not modified. To assess amount of Leu-pNA hydrolyzing activity associated with the hexameric LAPs, LAP activity was measured following immunoprecipitation with a LAP polyclonal antiserum. LAP activity increased in Cd-treated roots. There was a corresponding increase in LAP-A protein levels detected in 2D-immunoblots. The role of LAP-A in the proteolytic response to Cd stress is discussed.

Toward characterizing seed vigor in alfalfa through proteomic analysis of germination and priming.

Alfalfa, the most widely grown leguminous crop in the world, is generally exposed to severe salinity stress in Tunisia, notably affecting its germination performance. Toward a better understanding of alfalfa seed vigor, we have used proteomics to characterize protein changes occurring during germination and osmopriming, a pretreatment that accelerates germination and improves seedling uniformity particularly under stress conditions. The data revealed that germination was accompanied by dynamic changes of 79 proteins, which are mainly involved in protein metabolism, cell structure, metabolism, and defense. Comparative proteomic analysis also revealed 63 proteins specific to osmopriming, 65 proteins preferentially varying during germination, and 14 proteins common to both conditions. Thus, the present study unveiled the unexpected finding that osmopriming cannot simply be considered as an advance of germination-related processes but involves other mechanisms improving germination such as the mounting of defense mechanisms enabling osmoprimed seeds to surmount environmental stresses potentially occurring during germination. The present results therefore provide novel avenues toward understanding the mechanisms of invigoration of low vigor seeds by priming treatments that are widely used both in commercial applications and in developing countries (on farm seed priming) to better control crop yields.

Characteristics of cadmium tolerance in 'Hermes' flax seedlings: contribution of cell walls.

Most flax (Linum usitatissimum) varieties are described as tolerant to high concentrations of Cd. The aim of the present paper was to better characterize this tolerance, by studying the responses of flax plantlets, cv Hermes, to 18d growth on 0.5mM Cd. In Cd-treated seedlings, the majority of Cd was compartmentalized in the roots. Analysis of other elements showed that only Fe concentration was reduced, while Mn increased. Growth parameters of Cd treated flax were only moderately altered, with similar mass tolerance-indices for roots and shoots. Tissue anatomy was unaffected by treatment. The effect on lipid peroxidation, protein carbonylation and antioxidative activities appeared low but slightly higher in roots. The most important impacts of Cd were, in all organs, cell expansion, cell-wall thickening, pectin cross-linking and increase of cell-wall enzymatic activities (pectin methylesterase and peroxidase). Thus, the role of the cell wall in Cd tolerance might be important at two levels: (i) in the reinforcement of the tissue cohesion and (ii) in the sequestration of Cd.

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.

Nitrogen and carbon relationships between the parasitic weed Orobanche foetida and susceptible and tolerant faba bean lines.

The parasitic weed Orobanche foetida (Poiret) is an emergent agronomical problem on faba bean in Tunisia. The Tunisian breeding programs for faba bean resistance to O. foetida have produced several tolerant lines including the line XBJ90.03-16-1-1-1, which limits both parasite attachments to the host roots and growth of the attached parasites. The present study aims to provide a better understanding of the nutritional relationships between the parasite and this tolerant line in comparison with the susceptible Bachaar genotype. Phloem saps of faba bean were harvested using phloem exudation experiments. The major organic compounds potentially transferred from both faba bean genotypes to the parasite were identified as sucrose, raffinose, stachyose, citrate, malate, asparagine (ASN), aspartate (ASP), glutamine, glutamate, serine, alanine and GABA. However, the phloem exudates of the tolerant line were highly deficient in nitrogen when compared to that of the susceptible line. When attached to roots of the tolerant line, the parasite displayed limited activities of soluble invertases in tubercles, and especially in shoots, suggesting that the low performance of the broomrapes attached to the tolerant line resulted from a reduced capacity to utilize the host-derived carbohydrates. On the other hand, the mechanisms involved in the osmotic adjustment and primary metabolism of the parasite did not differ significantly according to the host genotype: mineral cations, especially potassium and calcium, predominated as the major osmotically-active compounds in both tubercles and shoots; shoots accumulated preferentially hexoses as organic solutes although tubercles accumulated preferentially starch and soluble amino acids, especially ASP and ASN. This suggests an important role for a glutamine-dependent asparagine synthetase (EC 6.3.5.4) in the N metabolism of the parasite.

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.

Assessment of enzyme induction and aerenchyma formation as mechanisms for flooding tolerance in Trifolium subterraneum 'Park'.

The objective of this study was to evaluate the role of enzyme induction and aerenchyma formation in prolonged tolerance to soil flooding in a variety of underground clover (Trifolium subterraneum 'Park') previously selected for resistance. Seedlings were grown in hydroponic tanks, initially with aeration for 3 weeks and subsequently in the absence of aeration for up to 3 weeks. After 1 h in the absence of aeration, the oxygen concentration in the hydroponic medium had decreased to 1.5 %. During the 3 weeks of extreme oxygen deficiency, primary roots died and were replaced by considerable numbers of adventitious roots. Activities of many glycolytic and fermentative enzymes increased in adventitious roots. Excised adventitious roots were capable of immediate induction of ethanol in the absence of lactate production, in association with energy charge higher than that in excised roots of aerobically maintained controls. Energy charge was even higher when measured in adventitious roots in planta. Interestingly, haemoglobin protein could be correlated with energy charge. Aerenchyma was readily visualized in adventitious roots by optical microscopy of longitudinal and transverse sections. We conclude that avoidance of root anoxia via aerenchyma is the major mechanism for prolonged root tolerance in Trifolium subterraneum 'Park'.