Effect Of Pretreatment With H₂O₂ On Stress Tolerance Of Arabidopsis And Screening Of Salt-tolerance Mutants Of Arabidopsis

Chapter 1:Effect of Pretreatment with H₂O₂ on Stress Tolerance of ArabidopsisStress, such as high salt,oxidative stress and drought has an influence on plant growth. It is harmful for the development of agricultural production and ecological environment.There were many reports demonstrating that the pretreatment with many chemicals, such as SA, BABA could enhance stress tolerance of plants. H₂O₂ pretreatment has a similar effect with these chemicals. High concentrations of H₂O₂ is harmful for plants but low concentrations of H₂O₂ can enhance stress tolerance of many kinds of plants.First we determined the concentration of H₂O₂ which could limit the seed germination or growth of root to 50% in the 1/2 MS medium. The result showed that when the concentration of H₂O₂ was 12.5 mmol/L-13 mmol/L, the germination rate of Arabidopsis seeds was about 50%; when the H₂O₂ concentration in the medium was 0.8 mmol / L, the average root length is about half of that without H₂O₂; when the H₂O₂ concentration in the medium was 1.8-2.0 mmol / L the root growth was completely inhibited;then we also detected the germination of Arabidopsis seeds primed with different concentrations of H₂O₂ for different periods on filter papers containing different salinity levels. The results demonstrated that the Arabidopsis seeds soaked with 20μmol/L H₂O₂ for 12h or with 5-10μmol / L H₂O₂ for 48h could obviously increase the germination rate. But the further experiments in the 1/2MS medium with different stress factors found that the pretreatment of seeds primed with 5-10μmol / L H₂O₂ for 48h could reduce the germination rate and limit the growth of root. So the pretreatment of seeds soaked with 20μmol/L H₂O₂ for 12h could effectively improve seed germination and promote root growth under stress conditions to a certain extent.30 days old Arabidopsis seedlings cultured in soil are sprayed with 20μmol/L H₂O₂ or water for 12 h, then treated with 150 mmol/L NaCl solution or water for the following 4 days. Seedlings pretreated with H₂O₂ have enhanced fresh weight, dry weight than seedlings pretreated with water .The results demonstrated that the pretreatment of H₂O₂ mitigated the inhibition of salt stress on the growth of seedlings. Seedlings pretreated with H₂O₂ had higher levels of K⁺ content and lower levels of Na⁺ content, and higher K⁺/ Na⁺ ratio than the seedlings pretreated with water. The SOD activity was significantly enhanced by the pretreatment with H2O2, the MDA content of seedlings pretreated with H2O2 is lower than that of the water-pretreated, showing that the levels of lipid peroxidation caused by salt stress decreased and the damaged membrane system was rescued in some extent. In non-salt stress environment, seedlings pretreated with H2O2 have enhanced fresh weight and dry weight, higher levels of K⁺ and Na⁺ lowered K⁺/ Na⁺ ratio, enhanced SOD activity and lowered MDA levels than seedlings pretreated with water. The results demonstrated that the pretreatment with suitable concentrations of H2O2 could promote plants to accumulate some metabolic substances and to be in"priming"status to overcome the environment stress .Chapter 2:Screening of Salt-tolerance Mutants of ArabidopsisBecause salt stress could limit the growth of plants, the research to the mechanism of salt-tolerance become very important for plants to adapt to stress environments. Screening salt tolerance mutants from mutant pools and studying the functions of salt tolerance genes are the common strategies for the understanding of salt tolerance mechanisms. In this paper, we screened salt tolerance mutants from two mutant libraries——activation tagging mutant library and estradiol induced activation tagging mutant library. The screening condition is 200 mmol/L NaCl. We found that the T1 seedlings of lines 2846, 2824, 2834, 2841, 2852, 2853, 2859, 2868, 2892, 2819, 2824, 937, 942, 943, 944, 945, 962, 963, 964, 965, 549, 5636, 5607, 5646, 5625, 2209, 2205, 2222, 2223, 2278, 2279, 2272, 245, 246 had longer crooked roots than that of WT. Then we tested the salt tolerance of these lines for second time, only lines 2279, 2272, 2223, 945, 2846, 2205, 942, 2852, 2824, 943, 937, 2209, 2272, 2248, 2834, 944, 2853 had longer crooked roots than that of WT. we performed the same screening to the T2 seedlings of the above lines, only the seedlings of line 2834 had much more longer roots than WT. The ratio of the salt-tolerance seedlings to normal seedlings is 14:46.