Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two- to four-cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4-1/+ with AtRFC4 expression driven through the embryo-specific DD45pro and ABI3pro or the endosperm-specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4-1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4-1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S-phase and M-phase cells in the rfc4-1/-FIS2;DD45;ABI3pro::AtRFC4 seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.