SCC3 is required for sister chromatid cohesion during mitosis

(A) Morphology of seedlings and root tips in wild type and scc3.

(B) Morphology of plants in wild type and scc3.

(C) Morphology of panicles in wild type and scc3.

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

(E) The chromosome behaviors of root tip cells in wild type 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 probes in the wild type 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, respectively, 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 showing the indicated morphology.

SCC3 alters the structure of sister chromatids during mitosis

(A) The dynamic process of chromosome structure in early mitosis as revealed by pooled oligos specific to chromosome 12 (red). In the wild type, S1 and S2 indicate the replicated sister chromatids. However, in scc3 sister chromatids exhibited the variation of structure from interphase to prophase. Mitotic chromosomes in wild type 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 wild-type 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 wild-type 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 wild-type meiocytes at pachytene. Two linear SCC3 signals wrap the ZEP1 signals, indicating central elements of SC 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 cohesion in early meiosis

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

(B) The dynamic process of chromosome structure in preleptotene was revealed by pooled oligos specific to chromosome 12 (red). Preleptotene chromosomes in wild type, 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 wild type and scc3. Bars, 5 μm.

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

SCC3 affects recombination progress and CO formation

(A) Immunolocalization of histone γH2AX (red), COM1 (green, from mouse), DMC1 (green, from mouse), RAD51 (green, from rabbit), ZIP4 (green, from mouse) and HEI10 (green, from mouse) in wild type 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 wild type and scc3. No difference of histone γH2AX and COM1 were shown between the wild type and scc3. DMC1, RAD51 and ZIP4 foci were significantly decreased in scc3 compared with wild type. Values are means ± SD. *** represents P < 0.001, two-tailed Student’s t-tests was performed.

(C) Chromosome behaviors 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.