Hyperaccumulators Long Nightshade (solanum Nigrum L.) Physiological And Molecular Mechanisms Of The Tolerance And Accumulation Of Cd

Solanum nigrum L. is a newly discovered Cd-hyperaccumulator occurring in natural or contaminated soils with cadmium. The ability to uptake and accumulate large amounts of cadmium in its leaves (124.6 mg/kg.DW) makes this plant an effective phytoremediator. To our knowledge, no research concerning the effect of in vitro cadmium tolerance and Cd-responsive genes in S. nigrum have been reported. In this study, we choose Cd-hyperaccumulator Solanum nigrum as material to (ⅰ) establish a high frequency in vitro shoot regeneration system; (ⅱ) investigate the protective effects of proline against cadmium toxicity of callus and regenerated shoots of S. nigrum; (ⅲ) examine the NO accumulation in roots under Cd stress and the role of NO in the physiological responses of plant to Cd stress; (ⅳ) identify the Cd-responsive genes in S. nigrum by DDRT.1. Optimum concentrations of plant growth regulators for shoot regeneration were determined using 11 combinations of NAA and BA concentrations. Hypocotyl segments and leaf explants in the presence of all plant growth regulators combinations resulted in callus formation. Of the two explants cultured, regenerated shoots from hypocotyl explants showed the better response in culture, producing shoots per explant in all combinations of NAA and BA. A regeneration frequency of 100% was recorded and the production up to 42 shoots per explant cultured on medium supplemented with 5 mg/L BA and 0.5 mg/L NAA by the end of the 3rd week. Leaf explants also showed a good response to in vitro culture, a regeneration frequency of 100% and 28 shoots per explant were obtained on MS medium supplemented with 3 mg/L BA and 0.5 mg/L NAA in three weeks. Almost all of the regenerated shoots were rooted ex vitro by transferring to half strength Hoagland solution after about 7 days.2. The protective effects of proline against cadmium toxicity of callus and regenerated shoots of S. nigrum are investigated based on a high frequency in vitro shoot regeneration system. Proline pretreatment reduces the Reactive Oxygen Species (ROS) levels and protects the plasma membrane integrity of callus under cadmium stress, and therefore improves the cadmium tolerance in S. nigrum. ICP-MS analysis shows that exogenous proline increases the cadmium accumulation in callus and regenerated shoots of S. nigrum. Further analysis indicates that the improvement of cadmium tolerance caused by proline pretreatment is correlated with an increase of superoxide dismutase (SOD) and catalase (CAT) activity and intracellular total glutathione content. The interaction between proline and enzymic or non-enzymic antioxidants is discussed.3. We found that Cd toxicity inhibited the seedling growth of S. nigrum while induced the occurrences of lateral roots and adventitious roots. Further study indicated that nitric oxide (NO) is involved in the processes. Exposure to Cd enhanced NO accumulation in roots. Supplementation with NO donor sodium nitroprusside (SNP) further improved the development of lateral root and adventitious root and Cd accumulation in shoots. Application of NO-scavenger 2-(4- carboxyphenyl)- 4,4,5,5- tetramethylimidazoline- 1-oxyl-3-oxide (cPTIO) reversed the effects of NO on modulating root system architecture and Cd accumulation in leaves. These results suggested that manipulation of NO level is an effective approach to improve Cd tolerance in plants by modulating root system architecture and promote Cd accumulation in leaves, and will provide insights into novel strategies for phytoremediation.4.To identify Cd-responsive genes in S. nigrum, a non-radioactive differential display reverse transcription polymerase chain reaction (DDRT-PCR) technique was applied to isolate of genes whose transcription was altered in seedlings under Cd stress. A total of 48 DD bands were identified; from these bands, fragments corresponding to 7 cDNAs were cloned. Reverse northern dot-blot analysis confirmed the different expression patterns of these genes under cadmium toxicity. The homology analysis revealed that five of these cDNAs had a clear identity to Solanum species, and putative functions were assigned, including calmodulin, ascorbate peroxidase, catalase, glutamylcysteine synthetase and iron-regulated transporter. Finally, the involvement of these genes in heavy metal tolerance is discussed.