Isolation And Characterization Of Reactive Oxygen Species-insensitive Mutants In Arabidopsis Thaliana

Many environmental stresses result in the enhanced production of reactive oxygen species (ROS), including H202, 02, 0H and 1O2, in higher plants. H202 is the most attractive candidate for signaling via ROS because of its relatively long life and high permeability across membranes. A growing body of evidence suggests that H202 may be a new singal in plants and it performs many functions in plant physiological process, so the research on the signal transduction pathway of H202 is important evidencely. Undoubtedly, the acquisition of reactive oxygen species-insensitive mutant not only offers perfect material for unraveling the molecular mechanisms of reactive oxygen species generation in plant cells under various environmental stresses, but also provides new insights into oxidative signal transduction in plant cells. Isolation of the putative reactive oxygen species-insensitive mutants (ros) and characterization of mutants were performed by using root-bending assay and genetic analysis in Arabidopszs thaliana. The results showed that 1O4mol/L H202 was established for screening concentration based on the effects of H202 on root growth and stomata aperture. We screened 50,000 ethylmethane sulfonate (EMS)-mutagenized Arabzdopsis M2 seeds for reactive oxygen insensitive mutants, using root-bending assay on 1O4mol/L H202 agar plates. Of twenty-six putative reactive oxygen species-insensitive mutants were obtained. The mutants were crossed with the wild-type by rubbing stamens from the wild-type onto the stigma of emasculated mutant flowers. The H202 insensitive of mutants F1 and F2 seedlings arising from the crosses was determined by using root-bending assay on 1O4mol/L H202 plates The analysis of selfed F2 seedlings from a cross between ros-2 and the wild-type revealed a 3:1 segregation ratio ot wild-type seedlings and mutant seedlings. Therefore, the ros mutants were caused by single recessive nuclear mutations. -4- In addition, under H202 stress, the accumulation of MDA indicating the damage of membrane lipid was also studied. For the mutants, the accumula. on of MDA were smaller than that in wild-type. This suggested that ros mutant might be resist nce to oxidative stress and reduced the damage of membrane system. Epidermis strip bioassay showed that ABA induced the stomata closure in Arabidopsis, and CAT could partly reverse this effect, this suggests that H202 may be involved in the ABA stimulated stomata closure, namely, H202 was an intermediate in this signal chain in Arabidopszs. We directly examined the production of H202 in guard cells in wild-type ancf mutant by using laser scanning confocal microscopy based on 2,7-dichlorofluorescin diacetate. Time-course experiments showed that 100 ~tmol/L ABA induced H202 production in guard cells, and the region of chloroplast located occurred significantly earlier than that in the other regions of the guard cells in wild-type and mutant Arabidopsis. Howover intracellular DCF fluorescence intensity more distinct in wild-type than that in mutant after ABA treatments. This indicated that the mutation of ros mutant was possibly related to the scavenging enzymes of ROS, which abolished the unwanted H202 to avoid the damage of cell.