Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2) in Medicago truncatula plants.

CONCLUSIONS

Our study highlights the use of the DNA repair gene MtTdp2α as a tool for improving the plant response to heavy metal stress. Tyrosyl-DNA phosphodiesterase 2 (Tdp2), involved in the removal of DNA topoisomerase II-mediated DNA damage and cell proliferation/differentiation signalling in animal cells, is still poorly characterised in plants. The Medicago truncatula lines Tdp2α-13c and Tdp2α-28 overexpressing the MtTdp2α gene and control (CTRL) line were exposed to 0.2 mM CuCl2. The DNA diffusion assay revealed a significant reduction in the percentage of necrosis caused by copper in the aerial parts of the Tdp2α-13c and Tdp2α-28 plants while neutral single cell gel electrophoresis highlighted a significant decrease in double strand breaks (DSBs), compared to CTRL. In the copper-treated Tdp2α-13c and Tdp2α-28 lines there was up-regulation (up to 4.0-fold) of genes encoding the α and β isoforms of Tyrosyl-DNA phosphodiesterase 1, indicating the requirement for Tdp1 function in the response to heavy metals. As for DSB sensing, the MtMRE11, MtRAD50 and MtNBS1 genes were also significantly up-regulated (up to 2.3-fold) in the MtTdp2α-overexpressing plants grown under physiological conditions, compared to CTRL line, and then further stimulated in response to copper. The basal antioxidant machinery was always activated in all the tested lines, as indicated by the concomitant up-regulation of MtcytSOD and MtcpSOD genes (cytosolic and chloroplastic Superoxide Dismutase), and MtMT2 (type 2 metallothionein) gene. The role of MtTdp2α gene in enhancing the plant response to genotoxic injury under heavy metal stress is discussed.