Model experimental systems for plant redox regulation and analysis of the stress-tolerance mechanism

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To establish a model experimental system for plant redox regulation of gene expression, we made transgenic tobacco BY-2 cells and Arabidopsis plants harboring overexpession and silencing system for ascorbate peroxidase (APX), a key enzyme of H_2O_2-scavenging in plants, inducing by estrogen supplementation. We hired pMDC7 and developed pXR as the vector for the aim. The resultant tobacco BY-2 line transformed with antisense construct for cytosolic APX, named AtAS3, showed 40 % less APX activity compared with untransformed BY-2 cells after addition of 25 μW estrogen. The cellular H_2O_2 level in AtAS3 was significantly increased by the silencing of cytosolic APX. Interestingly, AtAS3 showed increasing tolerance against salt and heat share after estrogen induction. These results indicated that this line is useful fir the model system of redox gene regulation. By the screening of suppressive subtraction hybridization (SSH) method, I have collected several possibilities for redox-responsible genes, including the known genes like HSPs.Furthermore, transgenic Arabidopsis plants silencing chloroplastic APX expression with RNAi by pXR introduction showed lower chloroplastic APX gene expression and its enzyme activity. The effect of the silencing on the APX expression was stable and lasting at least for six days after estrogen treatment. Microsaray analysis showed that approximately 400 and 250 genes were upregulated and downregulated, respectively. Among them, the genes related to signal transduction and metabolism were occupied, indicating there are many possibilities including chloroplast-to-nucleus signal transduction. Beside the APX-suppression systems describing above, we have also developed APX-overexpressors in tobacco BY-2 and Arabidopsis plants.Overall, we have proved that APX plays a central rule on cellular redox regulation, and have successfully developed the model experimental system fir elucidating redox gene regulation mechanism in plants.