Mechanisms Of TIR3 Gene Regulating Shoot Apical Meristem And Embryo Development In Arabidopsis

In both plants and animals,stem cells have the capacity for self-renewal,they generate daughter cells to produce new tissues.In plants,stem cells are located in shoot and root apical meristems.The cell division at these sites enables plants to continuously and repetitively generate new organs and structures throughout their lifetime.The shoot apical meristem(SAM)originates from the embryogenesis and gives rise to the aerial parts of plants.Therefore,it is very important to analyze the SAM development in plants.In this study,we identified a T-DNA insertion mutant which had a larger SAM compared to the wild type,and we termed it big shoot meristem(bom1).Then we analyzed the molecular functions of the mutant gene that mediated the SAM development.Our discoveries thus reveal a new framework for the regulation of stem cell specification during SAM development.The main results are as follows:(1)The aerial organ size of bom1 was decreased compared with the wild type,especially with the compact rosette leaves,shortened petioles and hypocotyls.However,the SAM and inflorescence meristem of the mutants were significantly larger than the wild type,with more rosette leaves and flowers.Through the identification of the T-DNA insertion site,it was found that a single T-DNA sequence was inserted into the TRANSPORT INHIBlTOR RESPONSE 3(TIR3)gene(AT3G02260),resulting in the loss-of-function of TIR3 gene and then enlarged SAM in bom1/tir3 mutants.(2)In order to analyze the mechanisms of TIR3-mediated regulation of SAM development,we examined the expression patterns of CLV3,WUS,and STM in bom1/tir3 mutants.We found that loss-of-function of TIR3 enhanced the expression of these genes in the mutants.Therefore,TIR3 regulates SAM development through inhibiting the expression of these genes.(3)Through genetic analysis,it was found that TIR3 inhibits the expression of WUS and STM genes.(4)In the wus and stm mutants,the expression of TIR3 gene was significantly enhanced.While in WUS and STM overexpressing plants,the expression of TIR3 gene was significantly inhibited.Therefore,WUS and STM inhibit the expression of TIR3.Through Chromatin Immunoprecipitation assays(Ch IP),it was found that STM directly bound to the promoter of TIR3 to inhibit TIR3 transcription.There is feedback regulation between TIR3 and WUS,STM in regulating the SAM development.(5)Through observating the embryogenesis of bom1/tir3 mutants and wild type,we found that there was no significant difference in the initiation of SAM.Therefore,TIR3 gene may mainly regulate the SAM maintenance during postembryonic development.However,the embryos of the bom1/tir3 mutants were significantly smaller than the wild type.The smaller embryos of the mutants were due to the suppression of cell division,and the expression of cell cycle-related genes in bom1/tir3 mutants was also inhibited.We further found that the mature seeds of the mutants were also significantly smaller than the wild type.(6)We further analyzed the transcriptomes of the bom1/tir3 and wild type ovules 3 days after fertilization.The results showed that the transcription level of the cytokinin synthesis gene IPT5 was significantly decreased in the ovules of the mutants.Furthermore,overexpression of the IPT5 gene restored the smaller embryo and seeds of the mutants.These results indicate that TIR3 gene regulates embryo and seed development through influencing the synthesis of cytokinin.