Molecular Genetics Analysis Of CEO2 Gene Function In Arabidopsis Thaliana

Reactive oxygen species (ROS) was induced by several abiotic stresses including heavy metal pollution. It is well known that ROS play a dual role in plant cells. On the one hand heavy metals were damage to cells. On the other hand as important signalling molecules, they mediated diverse physiological prosesses such as growth and development and stress responses. Many signalling components acting downstream of ROS signals have been identified, but the molecular mechanism by which plants to perceive ROS and regulate genes expression is still not completely understood. This research analysed the function of CEO2, a potential transcription regulator factor, and the molecular mechanism of heavy metal injury.By bioinformation analysis of the amino acid sequences between CEO2 and CEO1, we found both protein were 65.0% identical and 72.3% similar, and there are three regions enriched in Arg and Lys which may act as nuclear localization signals (NLS). We identified CEO2 T-DNA insertion mutants, and detected the expression of CEO2 by RT-PCR, the results has shown that the CEO2 transcript reduced significantly in homozygous ceo2-1 mutant when compared with WT. By histochemical detection of GUS activity, high levels of GUS expression were found in metabolism tissues such as seedling, stigma and anther, and CEO2 maybe localized in nuclear.Under normal growth condition, there is no distinguishable difference in seed germination and development between ceo2-1 and WT. While treated with HgCl₂ or H₂O₂, the growth of ceo2-1 was repressed dramatically, the true leaves shown an etiolated phenotype and the number of leaves reduced. When detected further by electrolyte leakage, membrane integrity of ceo2-1 has been damaged more severely than WT. DAB staining and confocal microscope experiments have shown that heavy metal stress induced more H₂O₂ accumulated in ceo2-1 than WT, suggesting that the mutation of CEO2 might affect H₂O₂ signalling transduction and scavenging. CEO2 did not response to MV which may differ with CEO1.By yeast two-hybrid interaction and GST pull-down assays, we confirmed that CEO2 was able to interact with GPX3 which might serve as an oxidative stress sensor and transducer in stress signalling. Real-time PCR results have shown that the expression of some abiotic stress-induced genes such as APX1 and RD28 were reduced under oxidative or heavy metal stress.All the results mentioned above suggeste that CEO2, acting downstream of H₂O₂ sensor, is involved in regulating H₂O₂ metabolism and gene expression, and play an important role in response to heavy metal stress in plants.