Haythem Mhadhbi

Antioxidant gene-enzyme responses in Medicago truncatula genotypes with different degree of sensitivity to salinity.

Antioxidant responses and nodule function of Medicago truncatula genotypes differing in salt tolerance were studied. Salinity effects on nodules were analysed on key nitrogen fixation proteins such as nitrogenase and leghaemoglobin as well as estimating lipid peroxidation levels, and were found more dramatic in the salt-sensitive genotype. Antioxidant enzyme assays for catalase (CAT, EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) were analysed in nodules, roots and leaves treated with increasing concentrations of NaCl for 24 and 48 h. Symbiosis tolerance level, depending essentially on plant genotype, was closely correlated with differences of enzyme activities, which increased in response to salt stress in nodules (except CAT) and roots, whereas a complex pattern was observed in leaves. Gene expression responses were generally correlated with enzymatic activities in 24-h treated roots in all genotypes. This correlation was lost after 48 h of treatment for the sensitive and the reference genotypes, but it remained positively significant for the tolerant one that manifested a high induction for all tested genes after 48 h of treatment. Indeed, tolerance behaviour could be related to the induction of antioxidant genes in plant roots, leading to more efficient enzyme stimulation and protection. High induction of CAT gene was also distinct in roots of the tolerant genotype and merits further consideration. Thus, part of the salinity tolerance in M. truncatula is related to induction and sustained expression of highly regulated antioxidant mechanisms.

Hydrogen peroxide scavenging mechanisms are components of Medicago truncatula partial resistance to Aphanomyces euteiches

The biochemical processes underlying the expression of resistance in the roots of Medicago truncatula against Aphanomyces euteiches infection was investigated, with emphasis on oxidative stress. The levels of H2O2, superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, soluble phenolics and lignin were measured in the roots of two lines, A17 partially resistant and F83005.5 susceptible to A. euteiches at three infection stages; penetration of the epidermis (1 dpi), colonization of the cortex (3 dpi) and invasion of the root stele (6 dpi). A rapid and large decrease of the H2O2 levels in A17 roots occurred. However, in F83005.5 roots, the decrease in H2O2 levels was delayed until 3 dpi. In A17 roots, the activities of ascorbate peroxidase, peroxidase and catalase were induced as early as 1 dpi, whereas a general decrease in the activity of the four antioxidant enzymes was observed in F83005.5 roots. The levels of soluble phenolics and lignin were increased in A17 roots at 3 and 6 dpi, respectively. The H2O2 levels were negatively correlated to ascorbate peroxidase, catalase and lignin production at 1, 3 and 6 dpi, respectively in A17 roots. Physiological concentrations of H2O2 found in M. truncatula infected roots had no detrimental effect on the in vitro growth of this oomycete. Our data suggest that H2O2 does not have a direct antimicrobial effect on M. truncatula resistance to A. euteiches, but is involved in cell wall strengthening around the root stele, preventing pathogen invasion of the vascular tissues.

Alternative oxidase 1 (Aox1) gene expression in roots of Medicago truncatula is a genotype-specific component of salt stress tolerance

Alternative oxidase (AOX) is the central component of the non-phosphorylating alternative respiratory pathway in plants and may be important for mitochondrial function during environmental stresses. Recently it has been proposed that Aox can be used as a functional marker for breeding stress tolerant plant varieties. This requires characterization of Aox alleles in plants with different degree of tolerance in a certain stress, affecting plant phenotype in a recognizable way. In this study we examined Aox1 gene expression levels in Medicago truncatula genotypes differing in salt stress tolerance, in order to uncover any correlation between Aox expression and tolerance to salt stress. Results demonstrated a specific induction of Aox1 gene expression in roots of the tolerant genotype that presented the lowest modulation in phenotypic and biochemical stress indices such as morphologic changes, protein level, lipid peroxidation and ROS generation. Similarly, in a previous study we reported that induction of antioxidant gene expression in the tolerant genotype contributed to the support of the antioxidant cellular machinery and stress tolerance. Correlation between expression patterns of the two groups of genes was revealed mainly in 48 h treated roots. Taken together, results from both experiments suggest that M. truncatula tolerance to salt stress may in part due to an efficient control of oxidative balance thanks to (i) induction of antioxidant systems and (ii) involvement of the AOX pathway. This reinforces the conclusion that differences in antioxidant mechanisms can be essential for salt stress tolerance in M. truncatula and possibly the corresponding genes, especially Aox, could be utilized as functional marker.

Medicago sativa - Sinorhizobium meliloti Symbiosis Promotes the Bioaccumulation of Zinc in Nodulated Roots

In this study we investigated effects of Zn supply on germination, growth, inorganic solutes (Zn, Ca, Fe, and Mg) partitioning and nodulation of Medicago sativa This plant was cultivated with and without Zn (2 mM). Treatments were plants without (control) and with Zn tolerant strain (S532), Zn intolerant strain (S112) and 2 mM urea nitrogen fertilisation. Results showed that M. sativa germinates at rates of 50% at 2 mM Zn. For plants given nitrogen fertilisation, Zn increased plant biomass production. When grown with symbionts, Zn supply had no effect on nodulation. Moreover, plants with S112 showed a decrease of shoot and roots biomasses. However, in symbiosis with S532, an increase of roots biomass was observed. Plants in symbiosis with S. meliloti accumulated more Zn in their roots than nitrogen fertilised plants. Zn supply results in an increase of Ca concentration in roots of fertilised nitrogen plants. However, under Zn supply, Fe concentration decreased in roots and increased in nodules of plants with S112. Zn supply showed contrasting effects on Mg concentrations for plants with nitrogen fertilisation (increase) and plants with S112 (decrease). The capacity of M. sativa to accumulate Zn in their nodulated roots encouraged its use in phytostabilisation processes.

Nodule Senescence in Medicago truncatula – Sinorhizobium Symbiosis Under Abiotic Constraints: Biochemical and Structural Processes Involved in Maintaining Nitrogen-Fixing Capacity

Nitrogen-fixing capacity (NFC) in nodules of four Medicago truncatula lines inoculated with four strains of Sinorhizobium was assessed, during the plant life cycle, in relation to parameters identified as indices of plant growth, photosynthetic capacity, nodule integrity, and functioning. Differences in duration of the NFC period were observed among symbiotic associations and were correlated with variability on plant biomass production. Senescence appearance and vigor varied in parallel with structural, physiological, and biochemical stability of nodules. Maintenance of a longer high-NFC period was correlated to a higher stimulation of antioxidant enzymes, mainly superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (POX, EC 1.11.1.7), and a consequent longer maintenance of membrane integrity and nodule structure within the first stages of senescence. Salinity and drought stresses interfered with nodule functioning and triggered fast and global nodule senescence, albeit a superiority of nodules having a long high-NFC period. The protective role of POX activity on salt- and drought-stressed nodules was revealed. On the other hand, SOD stimulation was independent of stress application. Another strategy allowing the maintenance of longer NFC in salt-stressed nodules could be the accumulation of starch granules in the senescence-functioning interface of nodules. This finding is currently under investigation. Interestingly, the symbioses with different behaviors of nodule senescence identified in this work would be useful bases for biochemical, genomic, and proteomic studies dissecting nodule senescence.

The antibiosis of nodule-endophytic agrobacteria and its potential effect on nodule functioning of Phaseolus vulgaris

The effect of the nodule-endophytic Agrobacterium strain 10C2 on nodulation, plant growth and nodule functioning of Phaseolus vulgaris was investigated using two rhizobial strains differing in their sensitivity to the in vitro antibiosis of the Agrobacterium strain. In the case of the sensitive strain, Agrobacterium sp. 10C2 induced a significant decrease in the proportion of pink nodules, probably by an antibiosis effect leading to the reduction in the number of bacteroids and thereby a decrease in total soluble proteins, leghaemoglobin content, photosynthesis and nitrogen fixation. In this case, the Agrobacterium strain behaved like a plant pathogen and the nodule reacted by increasing guaiacol peroxidase (POX) activity, which assures some physiological processes linked to pathogen control. By contrast, in the case of the resistant strain, the proportion of pink nodules increased, and thereby total soluble proteins, leghaemoglobin content, biomass production and nitrogen fixation were enhanced. The Agrobacterium strain is regarded in this case as a plant growth–promoting rhizobacterium and the POX-pathogen reaction was not observed. There was even a decrease in superoxide dismutase activity. The results suggested also that the Agrobacterium strain may be also involved in retarding nodule senescence in the case of the resistant strain.

Growth capacity and biochemical mechanisms involved in rhizobia tolerance to salinity and water deficit.

The aim of the present study was to evaluate abiotic stress tolerance of rhizobial strains belonging to Mesorhizobium, Sinorhizobium, and Rhizobium genera, as well as to investigate specie specific stress response mechanisms. Effect of NaCl and PEG on growth capacity, protein, lipid peroxydation (MDA), membrane fatty acid composition and antioxidant enzymes were investigated. Growth capacity and viability of overall rhizobia strains decreased proportionally to the increase of NaCl and PEG levels in the medium. Sinorhizobium strains appeared the most tolerant, where 4H41strain was able to grow at 800 mM NaCl and 40% PEG. On the other hand, growth of R. gallicum and M. mediterraneum was inhibited by 200 mM NaCl. The content of MDA was unchanged in Sinorhizobium strains under both stresses. For Mesorhizobium, only PEG treatment increased the content of MDA. Amount of the C19:0 cyclo fatty‐acid was increased in both Sinorhizobium and Mesorhizobium tolerant strains. NaCl stress increased Superoxide dismutase (SOD) activity of overall species; especially the most tolerant strain 4H41. Both treatments increased catalase (CAT) activity in 4H41, TII7, and 835 strains. Obtained results suggest that major response of tolerant Sinorhizobium and Mesorhizobium strains to NaCl and PEG stresses is a preferential accumulation of the C19:0 cyclo fatty acid within bacterial membrane as mechanism to reduce fluidity and maintain integrity. Cell integrity and functioning is also assured by maintaining and/or increasing activity of SOD and CAT antioxidant enzymes for tolerant strains to omit structural and functional damages related to reactive oxygen species overproduced under stressful conditions.

Growth and nitrogen-fixing performances of medicago truncatula-Sinorhizobium meliloti symbioses under salt (NaCl) stress: Micro- and macro-symbiont contribution into symbiosis tolerance

The effect of salt (NaCl) stress on plant growth and nitrogen-fixing apparatus was studied for symbiotic associations established between three Medicago truncatula lines inoculated and two Sinorhizobium meliloti strains. Salinity modulated all parameters analyzed as indices of growth performance, nitrogen-fixing capacity (acetylene-reduction assay) and nodule antioxidant system. Under stressful conditions, symbioses showed variability of response to salt application. Contrasting symbiotic associations were identified for nodulation, nitrogen-fixing capacity and salt tolerance. The total variance of analyzed performance indices under stressful conditions depended essentially on plant genotype factor. In nodules, the NaCl application had a decreasing effect on the rates of protein content and some antioxidant enzymes mainly catalase. Other enzymes such as guaiacol peroxidase increased in stressed nodules.

Cu-tolerant Sinorhizobium meliloti strain is beneficial for growth, Cu accumulation, and mineral uptake of alfalfa plants grown in Cu excess

In this work, we have compared the physiological responses of alfalfa plants inoculated with either Sinorhizobium meliloti strain S412 (Cu-tolerant) or S112 (Cu-sensitive) in the presence or absence of 0.5 mM of CuSO4. The addition of copper (Cu) introduced a decrease of nodule number and their dry weight (DW) in both symbioses. The interaction established with the Cu-sensitive strain is more affected by Cu than that with the tolerant one. In fact, plants inoculated with the sensitive strain revealed a decrease of shoot and root DW, larger than that found in plants inoculated with the tolerant strain. However, under copper supply, Medicago sativa with the Cu-tolerant strain did not show any significant changes in both shoot and root biomass production. Under Cu excess, high levels of Cu were detected in different parts of the plant in the two symbioses and a high translocation of Cu to aerial parts was shown with the strain S412. Plants with S412 were able to accumulate large quantities of calcium (Ca) in their roots and nodules. While Ca content decreased drastically in shoot at 0.5 mM of Cu treatment. Moreover, nodulation with S412 allowed plants to maintain high levels of magnesium (Mg) in all tissues and high iron (Fe) levels in nodules. Results suggest that this symbiotic pair could be used in phytostabilization of Cu-contaminated soils.