Identification Of New Phenotypes And Preliminary Molecular Genetic Analysis Of Two Novel Tmm Allelic Mutants In Arabidopsis Thaliana

In this study,M2 seeds from ethyl methanesulfonate-mutagenized seeds of Arabidopsis thaliana(Columbia ecotype) were used for screening.For screening,the stomatal phenotype was evaluated at light microscope.Two mutants which had clustered stomata were obtained.Complementation analysis of these mutants demonstrated that these two mutants were tmm alleles.To determine mutation sites of mutants,the TMM gene was amplified and sequenced.Considering the phenotypic and genetic characterization of two mutants,we certified that these two mutants were new alleles of tmm.Three tmm alleles have been reported so far,so we designated them as tmm-4 and tmm-5 respectively.New phenotypes and characteristics of these tmm allelic mutants were depthly studied in this research.As in tmm-1,the minimal one-celled spacing pattern of stomatal distribution was also disrupted in tmm-4 and tmm-5.The clustered phenotype was observed and stomatal density was increased obviously in tmm alleles.But there were some difference existed in stomatal distribution of tmm mutants.The stomatal density and density of clustered stomata were higher in tmm-4 than other mutants in the initial stage of leave development,which indicated that the mutation sites of TMM in tmm-4 might mainly regulate stomata patterning at initial stage of leave development.The percentage of stomata in clusters was higher in tmm-5 in comparison of other mutants,which suggested that there were more stomata disrupted normal pattern and formed stomatal clusters.The seedlings growth and seeds germination of tmm-5 were inhibited by exogenous ABA evidently.The seed dormancy is mainly controlled by ABA and tmm-4 displayed defective in seed dormancy.Mutations in tmm-4 and tmm-5 leaded to insensitivity response of ABA at high concentration in ABA-induced stomatal closing.The percentages of water loss in detached leaves were evident higher in tmm alleles compared with wild-type.All these results showed that tmm allelic mutants displayed altered sensibility for responding ABA at various aspects of growth events and suggested that TMM participated in some ABA-induced physiological process.TMM transcript was detected in all of tmm allelic mutants by RT-PCR analysis,and TMM was transcribed at roughly equivalent levels in wild-type and mutants plants.It suggested that the difference of mutation sites did not affect the transcription of TMM in mutants.Though the stomatal density and distribution of transgenic plants seemed to be similar with wild type,the number of trichomes and branching was visibly decreased in TMM-overexpressing plant leaves,which suggest that the overexpression of TMM gene affected the formation of trichomes and the decision of trichome branch.However,the number of trichomes and branching were normal levels in tmm allelic mutants which indicated that there were cooperator of TMM existed and both of them could regulate the generation of trichomes and the formation of branching redundantly.These new features and functions of TMM gene needed to be further verified.