Study Of A Rescued Arabidopsis Pipe Mutant LL-2 And PIPE Site-Directed Mutagenesis

We isolated an epidermal cell mutant pipe by screening the ethylmethanesulphonate mutagenized Arabidopsis pool. Through map-based cloning technique, the mutant gene was identified, encoding a protein phosphatase.In this study, the pipe mutants were mutagenized with EMS again to establish a new mutant pool. And we identified one recovery line LL-2 from this pool. The phenotype of pipe was partially rescued by the LL-2. The height of LL-2 was significantly higher than pipe. Compared to pipe mutant, the phyllotaxy and rosette leaves of LL-2 were recovered to normal phenotype. But the number of rosette leaves of LL-2 was less than that of wild-type. Unlike the brick-like shape of leaf epidermal cells from pipe mutant, the LL-2 shows jigsaw-puzzle appearance of leaf epidermal cells, similar to that of wild type. Genetic analysis showed that the mutant gene of LL-2 is tightly linked to PIPE. We next sequenced the PIPE gene of LL-2, and found a C to T single-nucleotide substitution that resulted in the conversion of histidine to tyrosine in amino acid 183. Subsequently, we verified that pipe can not interact with DELLA protein in yeast-two-hybrid assay. Transforming 35S--GFP into wild-type plants had no abnormal phenotypes and transforming 35S--GFP into pipe had no recovery effect on the mutant. To further investigate the function of PIPE, three active sites were mutated through site-directed mutagenesis. As a result, there was no obvious difference between overexpressing plants and wild-type or pipe background.We also investigated the function of CKI on GA signaling. The T-DNA insertion lines of three CKI kinase gene were identified. But they can not rescue the mutant phenotype of pipe. Therefore, these three CKIs may be not involved in GA signaling pathway.