Molecular Genetics of Arabidopsis Titan Mutants

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0091754MeinkeThe titan (ttn) mutants of Arabidopsis exhibit dramatic alterations inmitosis and cell cycle control during seed development. The commonfeature among these mutants is that endosperm nuclei become enlarged andhighly polyploid. Mutant embryos differ in cell size, morphology, andviability, depending on the locus involved. Three titan mutants withdistinct phenotypes were first identified in the Meinke laboratory 4 yearsago. During the previous grant period, 10 additional titan mutants wererecovered and 5 TITAN genes cloned in collaboration with Novartis(Syngenta). Several of these genes encode condensin (SMC) proteinsrequired for chromosome condensation and dosage compensation. Anothergene product is related to the ARF class of GTP binding proteinsassociated with vesicle trafficking. The remaining genes are involvedwith other cellular processes. What remains to be determined is how thesediverse proteins interact to influence nuclear division and cell viabilityduring seed development. This project will make it possible to continue the analysis of titanmutants and determine the relationship between gene function, nucleardynamics, and cell growth during seed development. Emphasis will beplaced on the SMC class of titans because these proteins appear to be mostdirectly linked to the mutant phenotype. Project objectives are toidentify the remaining SMC gene knockouts through reverse genetics,characterize nuclear and cytoskeletal defects in more detail throughfluorescence and electron microscopy, analyze phenotypes of multiple SMCknockouts, and determine how SMC functions in plants compare with thoseestablished in other organisms. The ARF class of titans will be examinedfirst by attempting to complement ttn1 with an ARF-GAP that maps to theTTN1 region. This should complete a map-based cloning effort initiatedduring the last grant period. The other class of titans will be examinedin part by analyzing more tagged endosperm mutants from the Novartiscollection, including those that have intermediate titan phenotypes.These additional mutants will be compared with existing titans toestablish the complex network of genes that influence endospermdevelopment. The titan phenotype demonstrates that Arabidopsis endosperm tissueprovides an ideal environment for proliferation of aberrant nucleidefective in mitosis and cell cycle control. Detailed analysis of thesemutants should provide valuable insights into novel features of nucleardynamics during endosperm development and evolution. The long-term goalis to understand the complex mechanisms that regulate endosperm formationin angiosperms.