Localization of AtSCC4–GFP protein in root cells of Arabidopsis. (A) Presence of AtSCC4 in root cells was assessed in plants expressing the AtSCC4–GFP fusion protein under the control of the AtSCC4 promoter (pAtSCC4::AtSCC4-GFP). 35 individual lines were analyzed; images represent typical expression and localization patterns of the AtSCC4. AtSCC4 was detected in the meristematic, and transition and elongation zones of the roots. Regardless of the root zone, most of the AtSCC4 localized in nuclei and was excluded from nucleoli.  A small fraction of the protein was present in the cytoplasm of the cells. 5 µM FM4-64 was added to the medium to visualize cell membranes. Scale bars: 20 µm. (B) Changes in AtSCC4 localization during cell division: AtSCC4 was excluded from chromatin at prometaphase and decorated it again in late telophase. AtSCC4 localization was assessed in root cells of fixed wild-type seedlings expressing pAtSCC4::AtSCC4-GFP stained with DAPI. Three individual lines were used in the experiment; images represent the typical localization of AtSCC4. Arrows indicate dividing cells. Scale bars: 5 µm. (C) Schematic representation of a split nuclei iFRAP experiment. In the scenario shown on the top, a fluorescent protein of interest is immobilized. Bleaching of the selected region in a nucleus (red rectangle) only slightly affects the fluorescence signal in the region of interest (ROI, gray rectangle). In the scenario shown in the bottom, the fluorescent protein of interest is mobile, thus bleaching of the selected region causes a significant reduction of the signal in the ROI. (D) Split nuclei iFRAP assay of AtSCC4 reveals that a significant portion of nuclear AtSCC4 is immobilized, most probably by being associated with chromatin. Regions denoted by red rectangles were photobleached; ROIs are denoted by white rectangles. Nuclei within yellow or blue rectangles were fully bleached or unbleached, respectively. Scale bars: 20 µm. (E) Quantification of iFRAP efficacy. Difference in retained fluorescence intensity between bleached and non-bleached area of a nucleus is proportional to the size of immobile fraction of the protein. Regions exposed to a high-intensity laser (red rectangles in D) were used to quantify the bleaching efficacy for each nucleus. Regions not exposed to a high-intensity laser (white rectangles in D) were used to assess retardation of fluorescent protein. Fully bleached nuclei (yellow rectangles in D) were used to estimate possible delivery of newly synthesized fluorescent protein from the cytoplasm. Unbleached nuclei (blue rectangles in D) were used to assess photobleaching caused by scanning. Data represent mean±s.e.m., n=15. *P<0.0001 (Dunnett's test versus GFP). Roots of Arabidopsis plants were mounted in 0.5× MS medium.

Depletion of AtSCC4 leads to seed abortion. (A) Open siliques of Col-0 wild-type plants (WT), Atscc4 heterozygous T-DNA insertion lines (Atscc4-1/AtSCC4 and Atscc4-2/AtSCC4) and corresponding complemented lines (Atscc4-1/AtSCC4 pAtSCC4::AtSCC4-GFP and Atscc4-2/AtSCC4 pAtSCC4::AtSCC4-GFP). Aborted seeds are marked with a red asterisk. (B) Decrease of seed abortion frequency in the T1 generation of complemented lines confirms that the seed abortion phenotype was caused by the AtSCC4 deficiency. Data represent mean±s.e.m., n=10. Pairwise comparison of the means was performed using Student's t-test. Mean values showing statistically significant difference are annotated with different letters.

Loss-of-function mutants of AtSCC4 show an abnormal embryo phenotype but normal endosperm development. (A) Propidium iodide staining of AtScc4-1 and wild-type (WT) embryos. The earliest aberrations in the AtScc4-1 embryos were visible at the octant stage. (a–h) WT; (i–p) AtScc4-1 embryos. Embryos were imaged at the following stages; first cell division (a,i); second division (b,j); four cells (c,k); octant (d,l); dermatogen (e,m); globular (f,n); transition (g,o); heart (h,p). Images represent typical phenotype observed at the corresponding stage. The experiment was repeated three times with similar results. The developmental stage of mutant homozygous embryos was extrapolated from developmental stage of wild-type and heterozygous embryos from the same silique. Red arrows, cells undergoing abnormal cell divisions. Scale bars: 10 µm. (B) Deficiency in AtSCC4 does not cause abnormalities in endosperm development, while lack of AtSCC2 causes a severe phenotype. DIC microscopy images of WT, Atscc2-1 and Atscc4-1 endosperm. Scale bars: 100 µm.

Lack of AtSCC4 or AtSCC2 causes a shift of auxin response maxima preceding proliferation of the suspensor. Auxin response maxima in the wild-type (WT), Atscc4 and Atscc2 backgrounds were visualized using the reporter construct DR5rev::3xVENUS-N7, which encodes nuclei-targeted Venus fluorescent protein under the control of the auxin-responsive DR5rev promoter. An aberrant pattern of auxin response maxima in the mutants was detected at the early stages of development, prior to the onset of ectopic cell proliferation in suspensor (see globular stage). At later stages of development, mutant embryos exhibited inverse pattern of auxin response maxima, as compared to WT, with the peak response in the basal cells of the suspensors (see heart stage). Scale bars: 10 µm.

AtSCC4 interacts with the N-terminal domain of AtSCC2. (A) Yeast two-hybrid assay shows strong interaction between full-length AtSCC4 and the N-terminus of AtSCC2. Transgenic yeast cells expressing AD- (GAL4 activation domain) and BD- (DNA-binding domain) fusion proteins were preselected on double drop-out medium (DDO) by plating 30 µl of culture with an optical denisty at 600 nm (OD₆₀₀) of 0.1, 0.01 or 0.001. Protein interaction was verified using quadruple dropout medium (QDO) and the same plating method. Lamin+T7, positive control; p53+T7, negative control; AtSCC4+T7 and AtSCC2+lamin, controls for autoactivation of the GAL promoter. (B) AtSCC4 interacts with N-terminal domain of AtSCC2 in plants. An co-IP assay was performed using protein extracts of N. benthamiana leaves transiently expressing Myc-tagged AtSCC4 together with CFP-tagged AtSCC2 or with free CFP. Nuclear protein fractions were purified using anti-YFP columns and blotted with anti-CFP and anti-Myc.*, predicted molecular mass of Myc–AtSCC4; **, predicted molecular mass of CFP-tagged N-terminal domain of AtSCC2; ***, predicted molecular mass of free CFP.

AtSCC2 is not required for immobilization of AtSCC4-GFP in nuclei. (A) A split nuclei iFRAP assay of AtSCC4 in wild-type (WT) and AtSCC2-depleted (Atscc2-2) backgrounds reveals that AtSCC2 is not required for immobilization of AtSCC4 in nuclei. Red rectangles, photobleached regions; white rectangles, ROI; yellow rectangles, fully bleached nuclei. Dotted line designates shape of the embryo. Scale bars: 10 µm. (B) Quantification of iFRAP efficacy. The difference in retained fluorescence intensity between the photobleached area of a nucleus and ROI is proportional to size of the immobile fraction of the protein. Photobleached area (red rectangles in A) were used to quantify bleaching efficacy for each nucleus. ROIs (white rectangles in A) were used to assess retardation of fluorescent protein. Fully bleached nuclei (yellow rectangles in A) were used to estimate possible delivery of newly synthesized fluorescent protein from the cytoplasm. Unbleached nuclei were used to assess photobleaching caused by scanning. Data represent mean±s.e.m., n=10. The experiment was repeated twice. Student's two-tailed t-test revealed no statistically significant difference between iFRAP results for WT and Atscc2-2 backgrounds.