The Response Of Sinorhizobium Meliloti-medicago Lupulina Symbiosis To Copper Stress And Its Potential Use For The Remediation Of Copper Contaminated Soils

Legumes are well known for their ability to form root nodules with compatible rhizobial strains, within which atmospheric nitrogen is reduced to ammonia for the benefit of both bacteria and plants; therefore, the symbiosis is of great environmental and agricultural importance. The effects of the copper-resistant strain Sinorhizobium meliloti CCNWSX0020 on the growth and antioxidant responses of Medicago lupulina in the presence of 200 mg kg-1 Cu2+ throughout different stages of symbiosis development were studied. Inoculation with S. meliloti CCNWSX0020 induced an increase in plant growth and nitrogen content irrespective of the presence of Cu2+. In the presence of 200 mg kg-1 Cu2+, the total amount of Cu uptake of inoculated plants significantly increased by 34.0% and 120.4%, respectively, in above-ground plant tissues and roots. Excess Cu was accumulated mainly in roots, with low Cu translocation to the aerial organs. The rate of lipid peroxidation was significantly decreased in both above-ground plant tissues and roots of inoculated vs. non-inoculated plants when measured either 8 days post inoculation(dpi), 13 dpi or 18 dpi. In comparison with non-inoculated plants, the activities of superoxide dismutase and ascorbate peroxidase of above-ground plant tissues of inoculated plants exposed to excess Cu were significantly elevated at different stages of symbiosis development; similar increases occurred in the activities of superoxide dismutase, catalase and glutathione reductase of inoculated roots. The symbiosis with S. meliloti CCNWSX0020 also up-regulated the corresponding genes involved in antioxidant responses in the plants treated with excess Cu. The results indicated that plants inoculated with the Cu-resistant bacterial strain S. meliloti CCNWSX0020 enhanced plant growth, and improved the responses of plant antioxidant defense to excess Cu stress.Rhizobial bacteria also have positive effects on plant growth through other mechanisms, including the solubilization of phosphate, the secretion of siderophores to sequester iron, the production of the phytohormone indole-3-acetic acid(IAA) and the synthesis of the enzyme ACC deaminase to lower stress ethylene levels in plants. Rhizobial species producing ACC(1-aminocycloproprane-1-carboxylate) deaminase have been shown to promote nodulation in various legumes via reducing ethylene levels in their host legumes. However, rhizobia produce a much lower level of ACC deaminase compared with most free-living plant growth-promoting bacteria containing ACC deaminase. While the endogenous rhizobial ACC deaminase is important in legume nodulation, it is not sufficient to protect host plants against environmental stresses. In this study, a genetically engineered Sinorhizobium meliloti strain overproducing ACC deaminase was constructed and its symbiotic performance in Medicago lupulina plants under moderate and severe Cu stress was assessed. The engineered strain, transformed with an exogenous ACC deaminase gene, expressed an almost 13-fold higher level of ACC deaminase activity compared with the wild-type strain. Plants nodulated with the engineered strain showed a greater dry weight, a decreased ethylene level in roots, a higher total Cu uptake but a lower level of Cu translocation to aerial parts, as compared with the plants nodulated with wild-type strain under Cu stress conditions. To evaluate the effect of severe Cu stress on acd S gene expression in symbiotic conditions, a quantitative RT-PCR of the exogenous acd S gene in the plants inoculated with S. meliloti(p RKACC) was performed. The m RNA level of this gene was increased fourfold in roots in the presence of 400 mg kg-1 Cu2+(4.02±0.27, n=3), compared with the roots under control conditions, while the expression of the acd S gene in above-ground plant tissues was not detected. The inoculation of ACC deaminase-overproducing strain induced the expression levels of Cu Zn SODc, Fe SOD, CAT and APX to a slight extent in the aerial parts of plants exposed to severe Cu stress. The expression levels of Cu Zn SODc, Cu Zn SODp, Fe SOD, Mn SOD, CAT, APX and GR also increased in roots, and was more pronounced when plant were grown under severe Cu stress. The expressions of these seven antioxidant genes were all remarkably increased by 2.8-3.7 folds in roots of plants exposed to severe Cu stress. Cu accumulated mainly in roots, with a lower level of Cu translocation to aerial parts for the plants nodulated with the engineered strain. These results were positively correlated with higher expression of antioxidant genes in the roots of these plants exposed to severe Cu stress. These findings suggest that the ACC deaminase-overproducing S. meliloti strain could further improve the growth and tolerance of M. lupulina plants to excess Cu, and enhance the antioxidant defense system directly or indirectly.