Regulatory Mechanisms of Microtubule Functions by MAP Kinase Cascade

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We isolated a semi-dominant Arabidopsis thaliana mutant propyzamide hypersensitive 1-1 (phs1-1) which showed hypersensitivity toward a microtubule-depolymerizing drug, propyzamide, and demonstrated that PHS1 codes for a novel protein belonging to a mitogen-activated. protein kinase (MAPK) phosphatase family. The phosphatase in phs1-1 had an amino acid exchange at the conserved MAPK-interaction domain at the N-terminus, retained a phosphatase activity, and acted dominant negatively.Several T-DNA insertion null alleles of PHS1 which lacked expression of the PHS1 protein grew normally and did not show any obvious phenotypic abnormalities. We isolated many genetic suppressors of phs1-1, and found 7 intragenic suppressor alleles of PHS1, further demonstrating that homozygous PHS1 null alleles do not show obvious phenotypes. Other phosphatases may have redundant functions. The suppressor screening also recovered several extragenic suppressor mutants. Some of these extragenic suppressor alleles were found to have mutations in tubulin genes. Responsible genes for the remaining suppressor mutants are being cloned by a map-based approach.Expression of the PHS gene was analyzed in transgenic Arabidopsis plants in which a GUS reporter gene was inserted in the C-terminus of the PHS1 protein in the genomic region. Basal GUS activities were detected in almost all cell types but the strong expression was observed in rapidly elongating cells. When GFP-PHS1 was expressed under its own promoter and terminator, wild-type PHS1 was found uniformly in the cytosol whereas the phs1-1 version of PHS1 was localized in a dot-like pattern in the cytoplasm.