HighlightAtNSE1 and AtNSE3 are crucial factors for early embryo and seedling development, and mutations of AtNSE1 and AtNSE3 can affect cell division and the DNA repair process.

In the life cycle of flowering plants, the sporophytic generation takes up most of the time and plays a dominant role in influencing plant growth and development. The embryo cell and endosperm free nucleus divisions establish the critical initiation phase of early sporophyte development, which forms mature seeds through a series of cell growth and differentiation events. Here, we report on the biological functions of two Arabidopsis (Arabidopsis thaliana) mitochondrial proteins, TRANSLOCASE OF THE INNER MEMBRANE9 (TIM9) and TIM10. We found that dysfunction of either AtTIM9 or AtTIM10 led to an early sporophyte-lethal phenotype; the embryo and endosperm both arrest division when the embryo proper developed to 16 to 32 cells. The abortion of tim9-1 and tim10 embryos at the 16/32-cell stage was caused by the loss of cell viability and the cessation of division in the embryo proper region, and this inactivation was due to the collapse of the mitochondrial structure and activity. Our characterization of tim9-1 and tim10 showed that mitochondrial membrane permeability increased and that cytochrome c was released from mitochondria into the cytoplasm in the 16/32-cell embryo proper, indicating that mitochondrial dysfunction occurred in the early sporophytic cells, and thus caused the initiation of a necrosis-like programmed cell death, which was further proved by the evidence of reactive oxygen species and DNA fragmentation tests. Consequently, we verified that AtTIM9 and AtTIM10 are nonredundantly essential for maintaining the mitochondrial function of early embryo proper cells and endosperm-free nuclei; these proteins play critically important roles during sporophyte initiation and development in Arabidopsis.

Highlight Arabidopsis RNase J plays an important role in maintaining chloroplast function, and the impairment of chloroplast development in rnj mutants may lead to aberrant embryos with disrupted pattern formation.

Chaperonins are a class of molecular chaperones that assist in the folding and assembly of a wide range of substrates. In plants, chloroplast chaperonins are composed of two different types of subunits, Cpn60α and Cpn60β, and duplication of Cpn60α and Cpn60β genes occurs in a high proportion of plants. However, the importance of multiple Cpn60α and Cpn60β genes in plants is poorly understood. In this study, we found that loss-of-function of CPNA2 (AtCpn60α2), a gene encoding the minor Cpn60α subunit in Arabidopsis thaliana, resulted in arrested embryo development at the globular stage, whereas the other AtCpn60α gene encoding the dominant Cpn60α subunit, CPNA1 (AtCpn60α1), mainly affected embryonic cotyledon development at the torpedo stage and thereafter. Further studies demonstrated that CPNA2 can form a functional chaperonin with CPNB2 (AtCpn60β2) and CPNB3 (AtCpn60β3), while the functional partners of CPNA1 are CPNB1 (AtCpn60β1) and CPNB2. We also revealed that the functional chaperonin containing CPNA2 could assist the folding of a specific substrate, KASI (β-ketoacyl-[acyl carrier protein] synthase I), and that the KASI protein level was remarkably reduced due to loss-of-function of CPNA2. Furthermore, the reduction in the KASI protein level was shown to be the possible cause for the arrest of cpna2 embryos. Our findings indicate that the two Cpn60α subunits in Arabidopsis play different roles during embryo development through forming distinct chaperonins with specific AtCpn60β to assist the folding of particular substrates, thus providing novel insights into functional divergence of Cpn60α subunits in plants.

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.