Visible Study Of Mercuric Ion Intracellular Partitioning In Living Cells Of Brassica Juncea

Exposure to mercury causes severe damage to plants, animals and even humans. Brassica juncea, as a plant species that has been reported as a new heavy metal hyper-accumulator recently, is used in this research. Brassica juncea can accumulate many divalent metals including Hg2+due to its particular transmembrane proteins. Calli of Brassica juncea were induced from cotyledons, stems and young leaves explants under the effect of hormonal supplementation (NAA,6-BA,2,4-D). The results showed that the stem is the best organ to obtain loose callus material. The MS medium containing1.0mg·L-1NAA+1.0mg·L-1-BA+2.0mg·L-12,4-D was found to be the most effective in callus induction. Suspension cells were established by transferring calli to a liquid MS medium in triangular flasks containing the same composition and same hormonal supplementation and were shaken for21days.By using EPNP as a highly selective and sensitive probe for Hg2+, the distribution of Hg2+in Brassica juncea can be detected. Our results show that EPNP can penetrate the cell wall and cell membrane, and is distributed inside the cell. With the increase of HgCl2concentrations from10μM to100μM in the cultured medium, a gradual enhanced fluorescence can be observed. A time-dependent experiment showed the fluorescence of the EPNP-Hg complex was still not quenched after3hours of exposure. It was also found that Hg2+mostly accumulated in lysosomes and not in the nucleus or mitochondria. These observations provide direct experimental evidence for the localization of Hg2+in Brassica juncea suspension cells in vivo. In vivo fluroseence imaging to visualize the transportation of Hg2+, the progress can be detected. Our results show that with the increase of HgCl2from10μM to100μM in the cultured medium, gradually enhanced fluorescence intensity can be observed. A time-dependent experiment showed the fluorescence of the EPNP-Hg complex in the plant was still accumulated. To the best of our knowledge, this is the first example of a probe-based methodology for imaging Hg2+in living plants and suspension cells. More applications of EPNP for fluorescence imaging of other plants grown in Hg2+polluted environment may be helpful to better understand plant poisoning.