State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
Plant J. 2018 Apr;94(2):288-303. doi: 10.1111/tpj.13855. Epub 2018 Mar 14.
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/- 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.
复制因子 C（RFC）是一种保守的真核复合物，由 RFC1/2/3/4/5 组成。它在酵母和果蝇的 DNA 复制和细胞周期中发挥着重要作用。然而，除了拟南芥（At）亚基 AtRFC1 和 AtRFC3 之外，RFC 亚基在高等植物中的功能尚不清楚。在这项研究中，我们研究了 AtRFC4 的功能，发现 AtRFC4 功能丧失会导致早期孢子体致死，最早发生在伸长的合子阶段，所有缺陷胚胎都在二至四细胞胚胎正常阶段停止发育，胚乳具有六到八个游离核。通过胚胎特异性 DD45pro 和 ABI3pro 或胚乳特异性 FIS2pro 驱动的 AtRFC4 表达来互补 rfc4-1/+，不能完全恢复缺陷胚胎或胚乳，而这三个启动子的组合在 rfc4-1/+ 中使退化的胚珠发育成有活力的种子。这表明 AtRFC4 同时在胚乳和胚胎中发挥作用，胚乳的增殖对胚胎成熟至关重要。rfc4-1/+ 中的 DNA 含量测定证实，胚乳和胚胎中的 DNA 复制被打乱，导致有丝分裂受阻。此外，我们观察到 rfc4-1/- 幼苗中晚期 S 期和 M 期细胞的比例降低，表明不完全的 DNA 复制导致根尖分生组织细胞的细胞周期停滞。因此，我们得出结论，AtRFC4 是 DNA 复制的关键基因。
