The Screening Of Acid-insensitive Mutants In Arabidopsis Thaliana

Acid rain will corrode buildings and cause great damage to plants and the entire ecosystem.The damage of acid rain to plants has been well-researched and the response of plants to acid stress has also been studied to some extent,but it is still unclear how plants sense acid stress.Ca²⁺is an important second messenger in plant cells.Calcium signals occur in response to environmental signals when sensed by plant cells to trigger downstream defense responses.Aequorin,a photoprotein produced by the jellyfish Aequorea victoria,can combined with Ca²⁺to fluoresce.Arabidopsis plants that stably express Aequorin were marked as AQ.A mutant library was established by inducing random mutation in AQ seeds using EMS,and the mutant seedlings were treated with acid stress to screen out those that are insensitive to acid signal.We treated AQ seedlings with acetic acid solutions of different concentrations and p H values and determined that the minimum condition to trigger calcium signal in Arabidopsis was p H 4.2 and 15 m M,which was used in the future screening for mutants.In the first round of screening,260 seeds were randomly selected and planted from each of 204 mutant pools,followed by acid treatment with a p H 4.2,15 m M acetic acid solution.Approximately 24 candidate mutant seedlings were selected from each pool and made up a total of 4752 seedlings from 53040 mutants,and 4053 bags of seeds were successfully collected.In the second round of screening,each mutant obtained from the first round was taken as a line,and 20-25 seeds were taken from each line along with 50 seeds from AQ line to conduct a comparative experiment.The lines with weaker calcium signal fluorescence compared to AQ line were selected.Such operation was repeated three times and candidate lines were selected for further verification of specificity using Sorbitol,Na Cl,Ca Cl₂,and H₂O₂ solutions as comparisons.89candidate mutant lines that were specifically insensitive to acid were screened out.The candidate mutant lines were crossed with AQ as female parent,and 27 lines of BF₁seedlings were selected and transplanted for having a fluorescence close to AQ seedlings and a weaker fluorescence in their M₄ seedlings.Seeds of their BF₂ generation was obtained afterwards,which would be further cultivated and sequenced to analyze the mutation sites in order to obtain the genes of acid signal receptors.Physiological experiments would be carried out on selected M₄ plants to analyze the physiological characteristics of acid-insensitive mutants.Our research on H⁺receptors in plant started from the perception of H⁺,by which the molecular mechanism of plants sensing acid rain can truly be explored.In addition,it is expected to obtain the genes of acid receptors in plants through this research and to fill the vacancy of acid receptors in plants,which would also contribute to the further understanding and improvement of the acid growth theory of plants.Furthermore,it could be conducive to cultivate crops or other plants that resist acid rain by changing and regulating relevant genes in crops,forages,forest tree species and other plants to manipulate their mechanism of H⁺sensation.Meanwhile,research on plants sensing acid rain can reveal the generation of plant adaptability and the mechanism of its dynamic changes under the background of global environmental changes.These studies will also help improve food security and plant ecosystem security in China.