Such a conversion has not been reported for other meiotic Penitrem A mutants and implies that separase plays a role in microtubule organization or cell polarity in A. thaliana. Also implicating separase in regulating microtubule function is the radially swollen 4 mutant. This conditional mutant, isolated on the basis of temperature-dependent root swelling, was recently shown to harbor a mutation in separase. At the restrictive temperature, cells in the rsw4 root meristem have anomalous disjunction and, in addition, have disorganized cortical microtubules and abnormally high levels of Olomoucine cyclin B1;1. The latter findings taken together with those on meiocytes suggest that plant separase somehow regulates microtubule function. The mutation in rsw4 is an alanine to valine substitution at position 603 of AtESP. This substitution might be sufficient to destabilize the enzyme against high temperature. However, given that the mutation represents a relatively conservative change, and is far from the protease domain, at least some of the phenotypes seen in rsw4 might reflect gain-of-function effects due to the presence of an aberrant protein. To gain insight into the nature of this separase mutation, we examined meiocytes of rsw4 exposed to the restrictive temperature and compared them to the previous results with RNAi. Additionally, we took advantage of rsw4 affecting separase in all cells and examined gametophyte development. Here we report that the rsw4 phenotypes in meiocytes are strikingly similar to those observed with RNAi, arguing for a role for separase beyond being the traditional cleaver of cohesin. The effect of the rsw4 mutation on megagametogenesis was characterized by comparing ovules in wild type and rsw4 plants exposed to 30uC. Female gametophyte development was staged according to Christensen et al.. In wild-type plants, the functional megaspore undergoes mitosis to give rise to a two-nucleate embryo sac. A central vacuole is formed between the two nuclei, separating them. The two nuclei then undergo a second division, giving rise to a fournucleate embryo sac. After another round of division, which forms an eight-nucleate embryo sac, the two polar nuclei migrate toward the micropylar half of the developing female gametophyte and fuse to form the larger central cell. Abnormalities were identified in embryo sacs of rsw4 plants as early as stage FG1. Figure 5E shows an embryo sac in which both the functional megaspore, as well as the nonfunctional meiotic products, have degenerated. Degenerated megaspores were also observed at FG2 and FG3. However the majority of the embryo sacs appeared to develop normally and reached the mature FG7 stage. Therefore, although the developmental abnormalities in ovules of rsw4 exposed to 30uC are severe, they were relatively uncommon.