Rice Cell Division Cycle 20s are required for faithful chromosome segregation and cytokinesis during meiosis

Abstract

Chromosome segregation must be under strict regulation to maintain chromosome euploidy and stability. Cell Division Cycle 20 (CDC20) is an essential cell cycle regulator that promotes the metaphase-to-anaphase transition and functions in the spindle assembly checkpoint, a surveillance pathway that ensures the fidelity of chromosome segregation. Plant CDC20 genes are present in multiple copies, and whether CDC20s have the same functions in plants as in yeast and animals is unclear, given the potential for divergence or redundancy among the multiple copies. Here, we studied all three CDC20 genes in rice (Oryza sativa) and constructed two triple mutants by CRISPR/Cas9-mediated genome editing to explore their roles in development. Knocking out all three CDC20 genes led to total sterility but did not affect vegetative development. Loss of the three CDC20 proteins did not alter mitotic division but severely disrupted meiosis as a result of asynchronous and unequal chromosome segregation, chromosome lagging, and premature separation of chromatids. Immunofluorescence of tubulin revealed malformed meiotic spindles in microsporocytes of the triple mutants. Furthermore, cytokinesis of meiosis I was absent or abnormal, and cytokinesis II was completely prevented in all mutant microsporocytes; thus, no tetrads or pollen formed in either cdc20 triple mutant. Finally, the subcellular structures and functions of the tapetum were disturbed by the lack of CDC20 proteins. These findings demonstrate that the three rice CDC20s play redundant roles but are indispensable for faithful meiotic chromosome segregation and cytokinesis, which are required for the production of fertile microspores.

Biochemical, biophysical, and functional characterisation of the E3 ubiquitin ligase APC/C regulator CDC20 from Arabidopsis thaliana

“…Full length AtCDC20.1 localised to the nucleoplasm ( Figure 5A ) and did not co-localise with an ER marker ( Figure 5B ) but with cytoplasmic RFP ( Figure 5C ) unequivocally indicating that AtCDC20.1-GFP was localised to nucleoplasm and cytoplasm. The observed subcellular localisation of full length AtCDC20.1-GFP ( Figure 5 ) was similar to the recently reported localisation of CDC20 from rice, where transient expression in rice protoplasts of CDC20-GFP fusions were mainly localised to the nuclei and to a lower extent in the cytoplasm ( Lin et al, 2022 ).…”

“…Moreover, yeast and animals harbour a single copy of the Cdc20 gene, whereas plants generally have multiple copies ( Capron et al, 2003 ; Lima et al, 2010 ). Indeed, a thorough evolutionary study of Cdc20 gene expansion identified multiple Cdc20 genes in the Charophyceae, Klebsormidiophyceae, Zygnematophyceae , and land plant species, indicating a tendency of Cdc20 gene expansion during streptophyte evolution ( Lin et al, 2022 ) and suggesting that Cdc20 gene duplications within-species were common during land plant evolution ( Lin et al, 2022 ).…”