Fig 1.
Aborted pollen development in smg7-6 mutants.
(A) Anthers of indicated mutants after Alexander staining. Viable pollen stain red. (B) Anther loculi in telophase II (Col-0, smg7-6 left panel) and assumed aberrant anaphase II (smg7-6, middle panel) stained with DAPI. Scale bar corresponds to 20 μm. (C) Alexander staining of pollen produced by the indicated mutants. The qrt1-2 mutation causes pollen from one tetrad to remain together. (D) Percentage of tetrads with one, two, three, four or no viable pollen. 245 tetrads from qrt1-2 smg7-6 plants were counted.
Fig 2.
Meiosis in smg7 and tdm1 mutants.
(A) Immunodetection of spindles using anti-α-tubulin antibody (red) in pollen mother cells. DNA is counterstained by DAPI (blue). AII—anaphase II, TII—telophase II, MIII—metaphase III, AIII—anaphase III. (B) Structure of chromosomes counterstained by DAPI in the third meiotic division.
Fig 3.
Live imaging of meiotic progression in smg7-6 PMCs.
(A) Time-lapse series of PMCs containing microtubules labeled with the pRPS5A::TagRFP:TUB4 construct. Timepoints relate to nuclear envelope breakdown at the end of prophase I. Scale bars correspond to 5 μm. (B) Duration of indicated meiotic stages inferred from spindle dynamics in time-lapse movies of pRPS5A::TagRFP:TUB4. Five individual PMCs from each wild type and smg7-6 plants are shown. (C) Duration of postmeiotic cycles of spindle assembly and disassembly in individual PMCs from two separate anther lobes of smg7-6 mutants.
Fig 4.
Characterization of the EMS30 and EMS155 suppressor lines.
(A) Five week-old plants of indicated genotypes. (B) Analysis of silique length along the main inflorescence bolt in wild type (n = 16), smg7-6 (n = 15), EMS30 (n = 16), EMS155 (n = 15) and cenh3-4 (n = 16) plants. Position 1 corresponds to the oldest and position 40 to the youngest silique scored on the main bolt. Error bars represent standard deviations. (C) Anthers of indicated lines after Alexander staining. (D) Number of viable pollen per anther from flowers at positions 5–15 and 25–35 along the main inflorescence bolt. Error bars represent standard deviations (n = 9).
Fig 5.
Meiotic progression in the EMS30 line.
(A) Immunocytological analysis of EMS30 PMCs with spindles visualized with anti-α-tubulin antibody (red). DNA is counterstained with DAPI (blue). MI, MII, MIII—metaphase I, II, III; AI, AII, AIII—anaphase I,II, III; TII—telophase II. Scale bar represents 5 μm. (B) Anther loculi stained with DAPI. Wild type and smg7-6 loculi contain tetrads and polyads, respectively. EMS30 plants contain tetrads (middle panel) and a mixture of polyads, tetrads, and AIII in their loculi (right panel). The inset shows a detail of a PMC in the MIII/AIII stage. Scale bar represents 20 μm. (C) Quantification of PMCs in late stages of meiosis. Number of PMCs analyzed for each genotype is indicated in parentheses.
Fig 6.
Molecular characterization of the cenh3-4 allele.
(A) Diagram of the CENH3 gene with exons marked as boxes. DNA sequence surrounding the intron 3 is shown; capital letters depict exons. Amino acids encoded by canonically spliced mRNA are indicated. The cenh3-4 mutation at the splice donor site of exon 3 is indicated in red, stop codon in the intron of unspliced mRNA in blue. (B) Relative abundance of spliced and unspliced CENH3 mRNA determined by quantitative RT-PCR. Two sets of primers were designed for fully spliced mRNA matching either wild type (CENH3-G) or cenh3-4 (CENH3-A) allele sequences. (C) Western blot detection of total CENH3 protein by anti-CENH3 antibody. Protein loading is shown on a Ponceau-S stained membrane (bottom panel). (D) Abundance of SMG7 mRNA relative to wild type determined by qRT-PCR. (E) NMD efficiency assessed by qRT-PCR analysis of alternatively spliced variants of the AT2G45670 gene with or without a premature termination codon (PTC) relative to wild type. Error bars in (B), (D) ad (E) represent standard deviations from 3 biological replicas.
Fig 7.
Effect of cenh3-4 mutation on the localization of CENH3 on Arabidopsis centromeres.
(A) Immunodetection of CENH3 in PMCs and tapetum cells using α-CENH3 antibody (green); DNA is counterstained with DAPI (blue). Longer exposure times for EMS30, EMS155, and cenh3-4 were applied to detect the signal. Scale bar corresponds to 5 μm. (B) Association of CENH3 with the CEN180 satellite repeat determined by chromatin immunoprecipitation with CENH3 antibody and dot blot hybridization. Antibody against histone H3 was used as a control. (C) Quantification of the CEN180 repeat in the ChIP experiments by qPCR. Error bars represent standard deviation of three technical replicates. Three independent experiments are presented.
Fig 8.
Immunodetection of CENP-C, KNL2 and MIS12 in root nuclei of cenh3-4 and wild type.
DNA is counter-stained with DAPI (blue). Longer exposure times for cenh3-4 were applied to detect a signal. Scale bar = 5 μm.
Fig 9.
Mitotic defects in cenh3-4 mutants.
(A) Seedlings grown on vertically oriented agar plates for 10 d after germination. (B) Dynamics of root growth on vertical agar plate. Error bars represent standard deviations (n = 17 to 39). (C) An example of mitotic progression in a root cell visualized by the HTA10:RFP marker. Classification of stages whose duration was quantified in Table 1 is indicated. (D) Time-lapse series of mitotic nuclei from wild type and cenh3-4 plants that were treated with 1.5 μM oryzalin (Ory) during the experiment.
Table 1.
Duration of mitotic phases in root cells.
NEB was defined as a time point when nuclei lost round shape (Fig 9C). None of the examined cells in cenh3-4 roots treated with oryzalin that entered the mitosis formed a metaphase plate (n = 12).