Variability in response to salinity stress in natural Tunisian populations of Hordeum marinum subsp. marinum.

Soil salinity is one of the most serious environmental factors affecting crop productivity around the world. In this study, we analysed morpho-physiological variation in responses to salt stress in Tunisian populations of Hordeum marinum subsp. marinum. The plants were grown under two treatments (0 and 200 mm NaCl) until maturity. A total of 19 quantitative traits were measured before and during the harvest. It was observed that most studied traits are influenced by the increasing salinity. High to moderate broad-sense heritability (H2 ) were noted for most of parameters under control and salt treatment, implying that salt tolerance is moderately heritable and environmental variation plays an equally important role. The majority of correlations between measured traits under the two treatments are positive, where the strongest correlations were between spike number (SN) and weight (SW). Based on the salt response index (SRI) values, SN and SW are the most affected by salinity. The 150 studied lines formed three groups according to the SRI values of the 19 quantitative parameters, of which 101 were moderately sensitive, 27 tolerant and 22 highly tolerant. Overall genetic variation of H. marinum in response to salt stress may provide novel insight to identify genes responsible for salt tolerance.

Structural and functional integrity of Sulla carnosa photosynthetic apparatus under iron deficiency conditions.

The abundance of calcareous soils makes bicarbonate-induced iron (Fe) deficiency a major problem for plant growth and crop yield. Therefore, Fe-efficient plants may constitute a solution for use on calcareous soils. We investigated the ability of the forage legume Sulla carnosa (Desf.) to maintain integrity of its photosynthetic apparatus under Fe deficiency conditions. Three treatments were applied: control, direct Fe deficiency and bicarbonate-induced Fe deficiency. At harvest, all organs of deficient plants showed severe growth inhibition, the effect being less pronounced under indirect Fe deficiency. Pigment analysis of fully expanded leaves revealed a reduction in concentrations of chlorophyll a, chlorophyll b and carotenoids under Fe deficiency. Electron transport rate, maximum and effective quantum yield of photosystem II (PSII), photochemical quenching (qP), non-photochemical quenching (qN) as well as P700 activity also decreased significantly in plants exposed to direct Fe deficiency, while qN was not affected. The effects of indirect Fe deficiency on the same parameters were less pronounced in bicarbonate-treated plants. The relative abundances of thylakoid proteins related to PSI (PsaA, Lhca1, Lhca2) and PSII (PsbA, Lhcb1) were also more affected under direct than indirect Fe deficiency. We conclude that S. carnosa can maintain the integrity of its photosynthetic apparatus under bicarbonate-induced Fe deficiency, preventing harmful effects to both photosystems under direct Fe deficiency. This suggests a high capacity of this species not only to take up Fe in the presence of bicarbonate (HCO3- ) but also to preferentially translocate absorbed Fe towards leaves and prevent its inactivation.

Sulla carnosa modulates root invertase activity in response to the inhibition of long-distance sucrose transport under magnesium deficiency.

Being the principal product of photosynthesis, sucrose is involved in many metabolic processes in plants. As magnesium (Mg) is phloem mobile, an inverse relationship between Mg shortage and sugar accumulation in leaves is often observed. Mg deficiency effects on carbohydrate contents and invertase activities were determined in Sulla carnosa Desf. Plants were grown hydroponically at different Mg concentrations (0.00, 0.01, 0.05 and 1.50 mM Mg) for one month. Mineral analysis showed that Mg contents were drastically diminished in shoots and roots mainly at 0.01 and 0.00 mM Mg. This decline was adversely associated with a significant increase of sucrose, fructose and mainly glucose in shoots of plants exposed to severe deficiency. By contrast, sugar contents were severely reduced in roots of these plants indicating an alteration of carbohydrate partitioning between shoots and roots of Mg-deficient plants. Cell wall invertase activity was highly enhanced in roots of Mg-deficient plants, while the vacuolar invertase activity was reduced at 0.00 mM Mg. This decrease of vacuolar invertase activity may indicate the sensibility of roots to Mg starvation resulting from sucrose transport inhibition. 14 CO2 labeling experiments were in accordance with these findings showing an inhibition of sucrose transport from source leaves to sink tissues (roots) under Mg depletion. The obtained results confirm previous findings about Mg involvement in photosynthate loading into phloem and add new insights into mechanisms evolved by S. carnosa to cope with Mg shortage in particular the increase of the activity of cell wall invertase.

Impact of microbial inoculation on biomass accumulation by Sulla carnosa provenances, and in regulating nutrition, physiological and antioxidant activities of this species under non-saline and saline conditions.

Bacteria (Pseudomonas sp. and Bacillus sp.) and/or the arbuscular mycorrhizal (AM) fungus Rhizophagus intraradices were able to improve growth, physiological and biochemical characteristics of four Sulla carnosa Desf. provenances (Sidi khlif, Thelja, Kalbia and Kerker) from Tunisia under both saline and non-saline conditions. S. carnosa is a salt-tolerant legume plant, native from North Africa. The intrinsic bacterial characteristics evidenced the fitness of these bacteria to support salt stress and to stimulate plant growth. Bacillus sp. produced more indol acetic acid (IAA) than Pseudomonas sp. and showed a great surviving capacity under salt conditions supporting its capacity to improve plant growth under stress conditions. The microorganisms applied also have a different potential to increase the nutritional and related plant growth parameters. It is noticeable that some provenances reached the highest level of growth when inoculated with Bacillus sp. in Sidi khlif or by Bacillus plus AMF in Kalbia, which increased shoot by 318% and root by 774%. In contrast, in Thelja and Kerker the impact of the test microorganisms was mainly evidenced at increasing nutritional and physiological functions. Salinity reduced some growth and physiological variables as stomatal conductance, photosynthetic pigments and photosynthetic efficiency and increased electrolyte leakage. However, the microbial inoculants compensated these detrimental effects in a degree depending on the S. carnosa provenance. These microorganisms also orchestrate antioxidant activities involved in adaptative responses in S. carnosa provenances. The intrinsic ability of inoculants allow us to select the provenance/microorganism combination which maximizes S. carnosa growth, nutrition and physiological/biochemical responses under salt and non-salt conditions. The results obtained support that the target microbial inocula are beneficial for the ecological stability if this Mediterranean legume.

Root antioxidant responses of two Pisum sativum cultivars to direct and induced Fe deficiency.

The contribution of antioxidant defence systems in different tolerance to direct and bicarbonate-induced Fe deficiency was evaluated in two pea cultivars (Kelvedon, tolerant and Lincoln, susceptible). Fe deficiency enhanced lipid peroxidation and H2 O2 concentration in roots of both cultivars, particularly in the sensitive one grown under bicarbonate supply. The results obtained on antioxidant activities (SOD, CAT, POD) suggest that H2 O2 accumulation could be due to an overproduction of this ROS and, at the same time, to a poor capacity to detoxify it. Moreover, under bicarbonate supply the activity of POD isoforms was reduced only in the sensitive cultivar, while in the tolerant one a new isoform was detected, suggesting that POD activity might be an important contributor to pea tolerance to Fe deficiency. The presence of bicarbonate also resulted in stimulation of GR, MDHAR and DHAR activities, part of the ASC-GSH pathway, which was higher in the tolerant cultivar than in the sensitive one. Overall, while in the absence of Fe only slight differences were reported between the two cultivars, the adaptation of Kelvedon to the presence of bicarbonate seems to be related to its greater ability to enhance the antioxidant response at the root level.

Involvement of source-sink relationship and hormonal control in the response of Medicago ciliaris - Sinorhizobium medicae symbiosis to salt stress.

In order to explore the relationship between leaf hormonal status and source-sink relations in the response of symbiotic nitrogen fixation (SNF) to salt stress, three major phytohormones (cytokinins, abscisic acid and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid), sucrose phosphate synthase activity in source leaves and sucrolytic activities in sink organs were analysed in two lines of Medicago ciliaris (salt-tolerant TNC 1.8 and salt-sensitive TNC 11.9). SNF (measured as nitrogenase activity and amount of N-fixed) was more affected by salt treatment in the TNC 11.9 than in TNC 1.8, and this could be explained by a decrease in nodule sucrolytic activities. SNF capacity was reflected in leaf biomass production and in the sink activity under salinity, as suggested by the higher salt-induced decrease in the young leaf sucrolytic activities in the sensitive line TNC 11.9, while they were not affected in the tolerant line TNC 1.8. As a consequence of maintaining sink activities in the actively growing organs, the key enzymatic activity for synthesis of sucrose (sucrose phosphate synthase) was also less affected in the mature leaves of the more tolerant genotype. Ours results showed also that the major hormone factor associated with the relative tolerance of TNC 1.8 was the stimulation of abscisic acid concentration in young leaves under salt treatment. This stimulation may control photosynthetic organ growth and also may contribute to a certain degree in the maintenance of coordinated sink-source relationships. Therefore, ABA may be an important component which conserves sucrose synthesis in source leaves.

Interactive effects of salinity and phosphorus availability on growth, water relations, nutritional status and photosynthetic activity of barley (Hordeum vulgare L.).

The interactive effects of salinity and phosphorus availability on growth, water relations, nutritional status and photosynthetic activity were investigated in barley (Hordeum vulgare L. cv. Manel). Seedlings were grown hydroponically under low or sufficient phosphorus (P) supply (5 or 180 µmol KH(2) PO(4) plant(-1) week(-1) , respectively), with or without 100 mm NaCl. Phosphorus deficiency or salinity significantly decreased whole plant growth, leaf water content, leaf osmotic potential and gas exchange parameters, with a more marked impact of P stress. The effect of both stresses was not additive since the response of plants to combined salinity and P deficiency was similar to that of plants grown under P deficiency alone. In addition, salt-treated plants exposed to P deficiency showed higher salt tolerance compared to plants grown with sufficient P supply. This was related to plant ability to significantly increase root:shoot DW ratio, root length, K(+)/Na(+) ratio, leaf proline and soluble sugar concentrations and total non-enzymatic antioxidant capacity, together with restricting Na(+) accumulation in the upper leaves. As a whole, our results indicate that under concomitant exposure to both salt and P deficiency, the impact of the latter constraint is pre-dominant.

Stimulated growth rate by restriction of P availability at moderate salinity but insensitive to P availability at high salinity in Crithmum maritimum.

The halophyte Crithmum maritimum thrives in cracks of calcareous rocks or cliffs at seashores, a situation which associates limited phosphorus availability and high salinity. In order to understand the common patterns of colonization and zonation of this species, seedlings were cultivated for 34 d in inert sandy soil irrigated with a nutrient solution containing or not phosphorus at moderate (50 mM) or high (250 mM) NaCl level. Net assimilation rate and consequently relative growth rate increased in response to P deprivation at moderate saline level, but not at high salinity level. Parallelly, CO2 fixation rate, rubisco capacity, transpiration rate and stomatal conductance were diminished by P deprivation at moderate NaCl level. Intercellular CO2 concentration was therefore not affected. Chlororophyll fluorescence analysis revealed that photosynthetic systems were insensitive to change in P availability at moderate salinity level: neither pigment content, nor effective and maximum quantum yield, photochemical and non photochemical quenching, and electron transport rate were affected by P deprivation. On the contrary, at high salinity level when net photosynthesis, rubisco capacity and the quantum yields of PS2 were severely affected, P deprivation strongly augmented electron transport rate. Stomatal aperture and more modest increase in net photosynthesis, rubisco capacity, photosystem II effective quantum yield and photochemical quenching accompanied this response. This study shows the tolerance of C. maritimum to the phosphorus deprivation combined to moderate or to high saline level which may explain the common patterns of colonization and zonation of this species.

Physiological and anatomical adaptations induced by flooding in Cotula coronopifolia.

Cotula coronopifolia is a wild annual Asteraceae that grows in periodically-flooded prone environments and seems highly tolerant to periodic flooding. Seedlings of about 15 cm were collected directly from the edge of Soliman sabkha (N-E Tunisia, semi-arid stage) and grown under greenhouse conditions. Two treatments were considered: drainage and flooding. After 56 days of treatment, flooded plants showed a pronounced growth increase. This performance was essentially associated with significant increment in biomass production of both shoots and roots (about 220% of the control). The appropriate response to flooding was also characterized by the ability of the species to maintain its water status under such conditions. Neither water content nor water potential showed a significant variation as compared to those of non-flooded plants. However, transpiration rate decreased slightly but significantly in flooded plants (from 0.86 to 0.64 mmol H2O m-2 s-1). Na+ and K+ concentrations were practically maintained under waterlogging conditions, except a significant increase of Na+ content in roots of flooded plants (157% of the control). These responses were concomitant with maintenance of photosynthetic rate. However, the contents of chlorophylls a and b increased to 167% and 295%, respectively. It seems that the enhancement in these photosynthetic pigments together with a significant improvement in water use efficiency (from 4.66 to 6.07 mmol CO2 mol-1 H2O) allowed to the species to compensate the decrease in photosynthetic rate. At the anatomical level, this species responded to flooding by a significant development of its root aerenchyma (+63%) and an increase in the lignification of its stem xylem tissues (+37%). Based on the presented data, the plant fitness under flooding conditions was a result of dynamic readjustment of several morphological, physiological, and anatomical adaptive traits. Flood requirement together with salt tolerance are responsible for the predominance of C. coronopifolia in a large area in its natural biotope where most plants cannot tolerate interactive effects of flooding and salinity.

Differential response to salinity and water deficit stress in Polypogon monspeliensis (L.) Desf. provenances during germination.

The effects of provenance, salinity (0, 100, 200, 300 and 400 mm NaCl) and water deficit (0, -0.6, -1.1, -1.6 and -2.1 MPa mannitol solutions) on germination success of Polypogon monspeliensis were investigated. Eight Tunisian provenances from different bioclimatic origins were considered. Seed mass varied significantly between populations. Germination percentage was significantly affected by provenance, salinity and their interaction. Even at 300 mm NaCl, germination percentage of Tabarka, Kelbia and Kebili ranged from ca. 60% to ca. 85%, whereas Monastir, Gabes and El Haouaria succeeded in germinating in 200 mm NaCl. The 300 mm NaCl treatment highly reduced germination of Monastir and El Haouaria, and inhibited that of Gabes. Soliman and El Jem were the least salt-tolerant provenances. The severity of water deficit impact on seed germination was also provenance-dependent, especially at osmotic potentials of -1.1 to -1.6 MPa. At -1.6 MPa, germination percentage of Tabarka, Monastir and Kebili was close to 80%, while that of Gabes, El Jem and Kelbia was 0%, 5% and 20%, respectively. Regardless of provenance, germination was strongly impaired at -2.1 MPa. The variability of stress tolerance in P. monspeliensis could be of practical significance in programmes aimed at restoring arid and salt-affected lands since it allows use of provenances that germinate and establish successfully under unfavourable conditions prevailing in such zones.

Enhancement of acid phosphatase secretion and Pi acquisition in Suaeda fruticosa on calcareous soil by high saline level.

The aim of this study was to identify the relationship between the adaptive processes of Suaeda fruticosa for Pi acquisition and the physic-chemical and biological characteristics of two soil types under moderate and high saline conditions. Four treatments were established in pots: namely SS100, SS600, CS100 and CS600 where SS stood for sandy soil and CS for calcareous soil, and the indexes 100 and 600 were NaCl concentrations (mM) in irrigation distilled water. Assuming that Pi per g of plant biomass is an indicator of plant efficiency for P acquisition, the results showed that Pi acquisition was easiest on SS100 and was difficult on CS100. The differences in Pi acquisition between plants on SS100 and CS100 could be attributed to the low root surface area (-30%) and to the low alkaline phosphatases (Pases) activities (-50%) in calcareous rhizospheric soil. The high salinity level had no effect on the efficiency of P acquisition on SS but increased this parameter on CS (+50%). In the latter soil type, high acid phosphatase activities were observed in rhizospheric soil at high salinity level. Acid phosphatase seemed to be secreted from the roots. The higher secretion of acid phosphatase in this soil was related to the root lipid peroxidation in response to elevated salinity associated with the augmentation of unsaturated acids which might induce an oxidative damage of the root membrane. Thus we can conclude that in deficient soil such as calcareous, the efficiency of P acquisition in S. fruticosa which was difficult at moderate salinity level can be enhanced by high salinity level.

Nutrient uptake and management under saline conditions in the xerohalophyte: Tecticornia indica (Willd.) subsp. indica.

In the present investigation, we studied uptake and management of the major cations in the xerohalophyte, Tecticornia indica (Willd.) subsp. indica as subjected to salinity. Plants were grown under greenhouse conditions at various salinity levels (0, 100, 200 and 400 mM NaCl) over 110 days. At harvest, they were separated into shoots and roots then analyzed for water contents, dry weights (DW), and Na+, K+, Ca²+, and Mg²+ contents. Plants showed a growth optimum at 200 mM NaCl and much better tissue hydration under saline than non-saline conditions. At this salt concentration (200 mM NaCl), shoot Na+ content reached its highest value (7.9 mmol · g-⁻¹ DW). In spite of such stressful conditions, salt-treated plants maintained adequate K+, Ca²+, and Mg²+ status even under severe saline conditions. This was mainly due to their aptitude to selectively acquire these essential cations and efficiently use them for biomass production.

Salt excretion in Suaeda fruticosa.

Suaeda fruticosa is a perennial "includer" halophyte devoid of glands or trichomes with a strong ability of accumulating and sequestrating Na(+) and Cl(-). We were interested in determining whether leaf cuticle salt excretion could be involved as a further mechanism in salt response of this species after long-term treatment with high salinity levels. Seedlings had been treated for three months with seawater (SW) diluted with tap water (0, 25, 50 and 75% SW). Leaf scanning electron microscopy revealed a convex adaxial side sculpture and a higher accumulation of saline crystals at the lamina margin, with a large variability on repartition and size between treatments. No salt gland or salt bladder was found. Threedimensional wax decorations were the only structures found on leaf surface. Washing the leaf surface with water indicated that sodium and chloride predominated in excreted salts, and that potassium was poorly represented. Optimal growth of whole plant was recorded at 25% SW, correlating with maximum Na(+) and Cl(-) absolute secretion rate. The leaves of plants treated with SW retained more water than those of plants treated with tap water due to lower solute potential, especially at 25% SW. Analysis of compatible solute, such as proline, total soluble carbohydrates and glycinebetaine disclosed strong relationship between glycinebetaine and osmotic potential (r = 0.92) suggesting that tissue hydration was partly maintained by glycinebetaine accumulation. Thus in S. fruticosa , increased solute accumulation associated with water retention, and steady intracellular ion homeostasis confirms the "includer" strategy of salt tolerance previously demonstrated. However, salt excretion at leaf surface also participated in conferring to this species a capacity in high salinity tolerance.

Induced lead binding phytochelatins in Brassica juncea and Sesuvium portulacastrum investigated by orthogonal chromatography inductively coupled plasma-mass spectrometry and matrix assisted laser desorption ionization-time of flight-mass spectrometry.

The accumulation and transport of lead in Brassica juncea and Sesuvium portulacastrum plants and the possible formation of complexes of this element with bioligands such as phytochelatins was studied in roots and shoots of plants exposed to different amounts of Pb(NO(3))(2). Speciation studies on the plant extracts were conducted using size exclusion liquid chromatography and ion pair liquid chromatography coupled to UV and ICP-MS to monitor lead and sulphur. The identification of the species separated by chromatography was performed by MALDI-TOF-MS. In both types of exposed plants it was possible to identify the presence of the phytochelatin isoform PC(3). The results obtained suggest that both types of plants can be useful in studies of phytoremediation but the ability of S. portulacastrum to accumulate and redistribute Pb from root to shoot is more effective than B. juncea.

Interactive effects of salinity, nitrate, light, and seed weight on the germination of the halophyte Crithmum maritimum.

Interaction of salinity, nitrate, light, and seed weight on the germination of Crithmum maritimum was investigated. Seeds of three size categories were germinated at 0-200 mM NaCl with either 0, 5 or 20 mM KNO 3 . Experiments were done under darkness, white light, or red light. Regardless of seed weight, germination was maximal in distilled water. Under salinity, the smallest seeds showed the highest germination percentage. Salt impact was amplified by darkness, but was mitigated by nitrate supply, red light and their combination. At the same PPFD, germination of T2 seeds was higher, when exposed to red light than under white light, suggesting that germination was more influenced by the light type than by the PPFD. As a whole, not only salinity, nutrient availability, seed weight, and light, but also their interaction may control the germination of this halophyte.

Characterization of cosmetic cream with Mesembryanthemum crystallinum plant extract: influence of formulation composition on physical stability and anti-oxidant activity.

Cosmetic or pharmaceutical composition containing superoxide dismutase (SOD) was usually used in topical administration, particularly, in fighting against skin ageing and in the protection of the skin against radiation exposure. Mesembryanthemum crystallinum is a halophyte plant widely used in the traditional medicine, characterized by the presence of anti-oxidants enzymes in responses to abiotic stresses. In the present study, we prepared a formulation with M. crystallinum extract characterized by naturally occurring SOD and catalase in association with other anti-oxidants molecules. The SOD activity was measured by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide/riboflavin method, catalase by colorimetric method and the total anti-radical activity was measured by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) method. Formulations contain a significant SOD activity (8.33 U mg(-1)), a catalase activity (0.5 x 10(7) UC) and an anti-radical activity (30% of DPPH inhibition). The formulation storage (15 days at 4 degrees C) showed a marked loss of total anti-oxidant capacity. The addition of the M. crystallinum extract induced also a reduction in formulation viscosity and pH.

An interdisciplinary research strategy to improve symbiotic nitrogen fixation and yield of common bean (Phaseolus vulgaris) in salinised areas of the Mediterranean basin.

The main findings of a cooperative research group of agronomists, plant breeders, microbiologists, physiologists and molecularists to improve the symbiotic nitrogen fixation (SNF) and N2-dependent yield of common bean under moderate salinity in the Mediterranean basin are summarised. Agronomic surveys in reference production areas show large spatial and temporal variations in plant nodulation and growth, and in efficiency of utilisation of the rhizobial symbiosis. The latter was associated with a large rhizobial diversity, including new bean nodulating species. Macrosymbiont diversity in SNF and adaptation to NaCl was found. However, contrasts between plant genotypes could be altered by specific interactions with some native rhizobia. Therefore, variations in soil rhizobial population, in addition to agronomic practices and environmental constraints, may have contributed to erratic results observed in field inoculations. At the mechanistic level, nodule C and N metabolisms, and abcissic acid content, were related to SNF potential and tolerance to NaCl. Their relation with nodule conductance to O2 diffusion was addressed by in situ hybridisation of candidate carbonic anhydrase and aquaporin genes in nodule cortex. The limits and prospects of the cooperative strategy are discussed.