Molecular Genetics of Arabidopsis Titan Mutants

Oklahoma State University, Stillwater OK

Investigators

Abstract

0091754 Meinke The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. The common feature among these mutants is that endosperm nuclei become enlarged and highly polyploid. Mutant embryos differ in cell size, morphology, and viability, depending on the locus involved. Three titan mutants with distinct phenotypes were first identified in the Meinke laboratory 4 years ago. During the previous grant period, 10 additional titan mutants were recovered and 5 TITAN genes cloned in collaboration with Novartis (Syngenta). Several of these genes encode condensin (SMC) proteins required for chromosome condensation and dosage compensation. Another gene product is related to the ARF class of GTP binding proteins associated with vesicle trafficking. The remaining genes are involved with other cellular processes. What remains to be determined is how these diverse proteins interact to influence nuclear division and cell viability during seed development. This project will make it possible to continue the analysis of titan mutants and determine the relationship between gene function, nuclear dynamics, and cell growth during seed development. Emphasis will be placed on the SMC class of titans because these proteins appear to be most directly linked to the mutant phenotype. Project objectives are to identify the remaining SMC gene knockouts through reverse genetics, characterize nuclear and cytoskeletal defects in more detail through fluorescence and electron microscopy, analyze phenotypes of multiple SMC knockouts, and determine how SMC functions in plants compare with those established in other organisms. The ARF class of titans will be examined first by attempting to complement ttn1 with an ARF-GAP that maps to the TTN1 region. This should complete a map-based cloning effort initiated during the last grant period. The other class of titans will be examined in part by analyzing more tagged endosperm mutants from the Novartis collection, including those that have intermediate titan phenotypes. These additional mutants will be compared with existing titans to establish the complex network of genes that influence endosperm development. The titan phenotype demonstrates that Arabidopsis endosperm tissue provides an ideal environment for proliferation of aberrant nuclei defective in mitosis and cell cycle control. Detailed analysis of these mutants should provide valuable insights into novel features of nuclear dynamics during endosperm development and evolution. The long-term goal is to understand the complex mechanisms that regulate endosperm formation in angiosperms.