The Radially Swollen 4 Separase Mutation of Arabidopsis thaliana Blocks Chromosome Disjunction and Disrupts the Radial Microtubule System in Meiocytes

Abstract

The caspase-family protease, separase, is required at the onset of anaphase to cleave the cohesin complex that joins replicated sister chromatids. However, in various eukaryotes, separase has acquired additional and distinct functions. A single amino-acid substitution in separase is responsible for phenotypes of the Arabidopsis thaliana mutant, radially swollen 4 (rsw4). This is a conditional mutant, resembling the wild type at the permissive temperature (∼20°C) and expressing mutant phenotypes at the restrictive temperature (∼30°C). Root cells in rsw4 at the restrictive temperature undergo non-disjunction and other indications of the loss of separase function. To determine to what extent separase activity remains at 30°C, we examined the effect of the mutation on meiosis, where the effects of loss of separase activity through RNA interference are known; and in addition, we examined female gametophyte development. Here, we report that, at the restrictive temperature, replicated chromosomes in rsw4 meiocytes typically fail to disjoin and the cohesin complex remains at centromeres after metaphase. Meiotic spindles appear normal in rsw4 male meiocytes; however the mutation disrupts the radial microtubule system, which is replaced by asymmetric arrays. Surprisingly, female gametophyte development was relatively insensitive to loss of separase activity, through either rsw4 or RNAi. These effects confirm that phenotypes in rsw4 result from loss of separase activity and establish a role for separase in regulating cell polarization following male meiosis.

The Caspase-Related Protease Separase (EXTRA SPINDLE POLES) Regulates Cell Polarity and Cytokinesis inArabidopsis

“…Knockout (or constant knockdown) of ESP is embryo-lethal due to the failure of chromosome disjunction (Liu and Makaroff, 2006), consistent with the evolutionarily conserved role of ESP in daughter chromatid separation (Ciosk et al, 1998;Hauf et al, 2001;Moschou and Bozhkov, 2012). In addition to this canonical function, the defects in anisotropic (i.e., directionally dependent) cell expansion observed in rsw4 implicate ESP in the regulation of cell polarity (Yang et al, 2011). This duality of plant ESP function provides an interesting paradigm to study the role of a protease in the spatiotemporal relationship between karyokinesis and establishment of cell polarity.…”

Histone Deacetylase AtHDA7 Is Required for Female Gametophyte and Embryo Development in Arabidopsis

“…Among these genes, the separase AtAESP that was down-regulated in Athda7-2 may provide some insight into the role of AtHDA7. AtAESP is required for ovule and embryo development in Arabidopsis (Liu and Makaroff, 2006;Yang et al, 2011). Loss of separase activity dramatically slows the progression of megagametogenesis and embryogenesis.…”

“…S6B; Supplemental Table S1). For experimental validation, ARABIDOPSIS HOMOLOG OF SEPARASE (AtAESP) was selected, given its role in ovule and embryo development (Liu and Makaroff, 2006;Yang et al, 2011). As shown in Figure 10B, AtAESP was significantly down-regulated in Athda7-2 flower buds, with a change of 0.64-fold (P , 0.002) compared with the wild type.…”

Cytokinesis in plant male meiosis

“…AESP is a caspase family protease that is required for the proteolytic cleavage of the cohesin complex at the metaphase-to-anaphase transition and the release of sister chromatid cohesion during meiosis and mitosis 87 . Interestingly, besides a persisted chromatid cohesion and aberrant chromosome segregation, aesp meiocytes (e.g., RNAi and the conditional rsw4 mutant) also show clear alterations in telophase II RMA formation, including reduced MT extension, partial phragmoplast formation and complete loss of RMA establishment 88 , 89 . In addition, aesp PMCs often generate microspores with multiple nuclei, clearly indicating for a defect in meiotic cytokinesis.…”