SCC3 is required for sister chromatid cohesin during mitosis

(A) Comparison between seedlings and root tips in wildtype and scc3.

(B) Comparison between wildtype and scc3 mutant plants.

(C) Comparison between wildtype and scc3 mutant panicles.

(D) Pollen grains stained in 1% I2-KI solution in wildtype and scc3. Bars, 50 μm.

(E) The chromosome behaviors of root tip cells in wildtype and scc3, stained with DAPI. Individual chromosomes are marked by red boxes. The distance between the sister chromatids increased significantly at prometaphase, and the sister chromatids separated in advance at metaphase in scc3. Bars, 5 μm.

(F) FISH analysis of mitotic cells with centromere-specific fluorescently labeled probes in the wildtype and scc3. The distance between centromeres increased significantly in scc3. CentO (red) signals indicate centromeres. Chromosomes were stained with DAPI (blue). Bars, 5μm.

(G) The distance between sister chromatids in different mitotic chromosomes. In normal condition, two sister chromatids form tight stick. In scc3, the distance between chromosome arms and telomeres were increased significantly. Curve diagrams show the distance between the arms and the telomeres as measured by IMAGEJ. Bars, 1 μm.

(H) Graphical representation of the frequency of each type of chromosome morphology. The classification was assigned when >50% chromosomes in a spread displayed the indicated morphology.

SCC3 affects the replication process of sister chromatids during mitosis

(A) The dynamic process of chromosome replication in early mitosis as revealed by pooled oligos specific to chromosome 12 (red). In the wildtype, S1 and S2 indicate the sister chromatids that are replicating. However, in scc3 sister chromatids exhibit many chromosome fragmentation during replication at interphase. Mitotic chromosomes in wildtype and scc3 were stained with DAPI (blue). Bars, 5 μm.

(B) The loading pattern of SCC3 (green, from mouse) and CENH3 (red, from rabbit) in wildtype root tip cells. Chromosomes were stained with DAPI (blue). Bars, 5 μm.

SCC3 is an axial element during meiosis

(A) In meiosis, SCC3 (green, from mouse) colocalizes with REC8 (red, from rabbit) from the leptotene to diplotene. Bars, 5 μm.

(B) During diakinesis and metaphase I, SCC3 (green, from mouse) gradually dispersed and finally retained in the centromeres indicated by CENH3 (red, from rabbit). Chromosomes were stained with DAPI (blue). Bars, 5 μm.

(C) Immunostaining of SCC3 (cyan, from mouse) and REC8 (red, from rabbit) in wildtype meiocytes at pachytene. Two parallel linear SCC3 signals colocalize with the REC8 linear signals, indicating chromosomal axial elements. Magnified images of the blocked regions are shown on the right. Bars, 5 μm.

(D) Immunostaining of SCC3 (cyan, from mouse) and ZEP1 (red, from rabbit) in wildtype meiocytes at pachytene. Two linear SCC3 signals wrap the ZEP1 signals, indicating chromosomal central elements are wrapped by axial elements. Magnified images of the blocked regions are shown on the right. Bars, 5 μm.

SCC3 is required for sister chromatid cohesin in early meiosis

(A) Meiocyte chromosome morphology at the preleptotene and leptotene in wildtype, scc3, rec8 and rec8 scc3. Chromosomes at preleptotene exhibited a hairy rodlike appearance and formed thin threads at leptotene in wildtype. However, chromosomes in scc3 were dispersed with blurred outlines and failed to from thin threads at leptotene. Bars, 5 μm.

(B) The process of chromosome replication in preleptotene is revealed by pooled oligos specific to chromosome 12 (red). Preleptotene chromosomes in wildtype, scc3, rec8 and rec8 scc3. Chromosomes were stained with DAPI (blue). Bars, 5 μm.

Homologous pairing and synapsis are disturbed in scc3

(A) Meiotic chromosome behavior in wildtype and scc3. Bars, 5 μm.

(B) Immunolocalization of ZEP1 (green, from mouse) and REC8 (red, from rabbit) in wildtype and scc3 meiocytes. ZEP1 was severely suppressed from zygotene to late pachytene in scc3. Bars, 5 μm.

SCC3 affects recombination progress and CO formation

(A) Immunolocalization of histone γH2AX phosphorylation (red), COM1 (green, from mouse), DMC1 (green, from mouse), RAD51 (green, from rabbit), ZIP4 (green, from mouse) and HEI10 (green, from mouse) in wildtype and scc3. REC8 (red, from mouse and rabbit) signals were used to visualize chromosome axes. Bars, 5 μm.

(B) Box scatter plot of histone γH2AX phosphorylation, COM1, DMC1, RAD51 and ZIP4 in wildtype and scc3. No difference of histone γH2AX phosphorylation and COM1 were shown between the wildtype and scc3. DMC1, RAD51 and ZIP4 foci were significantly decreased in scc3 compared with wildtype. Values are means ± SD. *** represents P < 0.001, two-tailed Student’s t-tests was performed.

(C) Chromosome behavior in scc3, rec8, rec8 scc3, dmc1 and dmc1 scc3 from diakinesis to anaphase I. Bars, 5 μm.

SCC3 loading onto meiotic chromosomes depends on REC8

(A) Immunolocalization of γH2AX (red, from rabbit), COM1 (green, from mouse), RAD51 (green, from rabbit), SCC3 (green, from mouse), PAIR2 (green, from rabbit) and PAIR3 (green, from rabbit) in rec8 meiocytes at zygotene. CENH3 (red and green, from rabbit and mouse) was used to indicate the centromeres. Bars, 5 μm.

(B) Immunolocalization of REC8 (red, from rabbit) and SCC3 (green, from mouse) in spo11, com1, dmc1, zep1, pair2 and pair3 meiocytes at zygotene. Bars, 5 μm.

(C) SCC3 interacts with RAD21.1 in yeast-two-hybrid assays. Interactions between bait and prey were examined on SD/DDO (SD-Leu-Trp) control media and SD/QDO (SD-Ade-His-Leu-Trp) selective media. AD, prey vector; BD, bait vector.

(D) Bimolecular fluorescence complementation assays show the interactions between SCC3 and RAD21.1 in rice protoplasts. Bars, 5 μm.

SCC3 acts as the cohesin and inhibits inter-sister chromatids repair during rice meiosis

(A) Structure model of the cohesin complex. The SCC3 subunit is associated with the middle region of SCC1.

(B) During meiosis, SCC3’s localization pattern probably changes to the root of DNA loop with lateral elements (LE), which wraps synaptonemal complex (SC). This indicates SCC3 is a meiotic LE essential for homologous chromosome pairing and synapsis, also affects recombination progress and CO formation.

(C) Meiotic recombination exhibits an intrinsic and mechanistical default towards selection of homolog bias in wildtype. Cohesin is responsible for the connection of sister chromatids intervening in this mechanism to inhibit recombination into use of sisters. However, in scc3, sister bias is required, with the defects of homologous pairing and synapsis. This outcome may be achieved by the residue function of DMC1 and RAD51 promoting DSBs end association with sister. While this situation in rec8 is quite different with most meiotic proteins fail to localize, thus forced to NHEJ repair pathway causing the sticky chromosomes and fragmentation.