Fig 1.
dcm1 is a mutant with low male fertility.
(A) Phenotypic comparison of plant (Left), panicle (Upper Right, a few seeds of dcm1 indicated by an arrow) and pollen grains (Lower Right) of wild type and dcm1. Bars = 20μm. (B) Semi-thin sections of anthers from wild type and dcm1 at different developmental stages. Bars = 20μm. (C) Transmission electron micrographs of the anthers from both wild type and dcm1. Bars = 1μm in the left column and 200nm in the right column. AP, aborted pollen; Ba, bacula; Ex, exine; ML, middle layer; MP, mature pollen; McP, monocellular pollen; Ne, nexine; PMC, pollen mother cell; Se, sexine; T, tapetum; Ub, Ubisch body.
Fig 2.
Pollen grains with non-uniform size in dcm1.
(A) Scanning electron microscope observation of wild-type and dcm1 pollen grains. Large pollen grains are indicated by arrows. Bars = 50μm. (B) DAPI stained pollen grains in wild-type and dcm1. Bars = 10μm. (C) The arrangement of meiotic products and orientation of spindles in wild type and dcm1. Meiotic products are stained with acetocarmine. Spindles are immunolocalized with α-tubulin antibody. Bars = 5μm.
Fig 3.
Meiotic cytokinesis is defective in dcm1.
PMCs of wild type and dcm1 at different developmental stages are shown. Cell plates are indicated by arrows. Bars = 10μm.
Fig 4.
Prematurely dissolution of callosic plates and peripheral callose occur in dcm1 PMCs.
(A) Combined DAPI and aniline blue staining on wild-type and dcm1 PMCs at different developmental stages. Callose signals are indicated by arrows. Bars = 10μm. (B) Aniline blue staining on semi-thin section of anther in wild type and dcm1. Peripheral callose is indicated by arrows and callosic plates are indicated by arrow heads. Callose signals at dyad are indicated in yellow and blue frames. Bars = 10μm.
Fig 5.
Electron micrographs showing immunogold labeling of callose in wild type and dcm1.
(A) Dyads of the wild type. (B) Dyad-like stage in dcm1. CP, cell plate, enlarged in blue frames. CW, cell wall, enlarged in red frames. Bars = 1μm. (C) Quantification of immunogold labeling particles per unit area in the cell wall. Error bars represent SD (n = 50). Asterisks indicate significant differences according to Student’s t-test (***P<0.001).
Fig 6.
DCM1 encodes a protein with five tandem CCCH motifs.
(A) Gene structure of DCM1. Coding regions are shown as black boxes. 5’ and 3’ untranslated regions are shown as white boxes. Introns are shown as black lines. The triangles indicate the mutated sites in dcm1. Details of the mutated site are listed below. (B) Schematic representation of the conserved coiled-coil and tandem CCCH zinc finger domains in DCM1 protein. (C) Phylogenetic tree derived from the full length amino acid sequences of proteins containing the five tandem CCCH zinc finger motifs. The tree was constructed using Mega7 based on the neighbor-joining method. (D) Multiple sequence alignment of the tandem CCCH zinc finger domains from proteins used in phylogenetic tree construction.
Fig 7.
Expression pattern of the DCM1 gene.
(A) Real-time PCR analysis of DCM1 in different tissues. Ubiquitin is used as endogenous control. Error bars represent SD (n = 3). R, root; IN, internode; L, leaf; YP, young panicle; dcm1, young panicle of dcm1. (B) GUS activity in the pDCM1::GUS line. The developmental stages (from left to right): leptotene, pachytene, microspore stage, pollen grain stage. Bars = 1mm. (C) RNA in situ analysis of DCM1 in wild-type anthers at different developmental stages. Anther at leptotene with sense probe is shown as the negative control. Bars = 10um. ISPC, inner secondary parietal cell; Ms, microspore; P, pollen; PMC, pollen mother cell; SC, sporogenous cell; T, tapetum.
Fig 8.
DCM1 interacts with nuclear poly (A) binding proteins in nuclear speckles.
(A) Schematic diagram of full-length and truncated proteins of DCM1 used in yeast two-hybrid assays, the coiled-coil domain and tandem CCCH domain are annotated. (B) Schematic representation of OsPABN1 and OsPABN2. The conserved domains are documented. (C) DCM1 interacts with OsPABN1 and OsPABN2 in yeast two-hybrid assays, independently of the conserved tandem CCCH domain. (D) OsPABNs interacts with themselves and each other in yeast two-hybrid assays. (E) The interactions between DCM1 and OsPABNs are verified by BiFC assay. The corresponding plasmid pairs were listed. CFP, cyan fluorescent protein. DIC, differential interference contrast micrographs.
Fig 9.
Expression analysis of genes involved in callose metabolism and exine formation in wild-type and dcm1 anthers.
(A) The expression comparison of OsGSL1-OsGSL10 between wild-type and dcm1 anthers during meiosis. (B) The expression comparison of Osg1, OsA6, OsMYB80 and OsUGP1 between wild-type and dcm1 anthers during meiosis. (C) The expression comparison of OsABCG15 and DPW between wild-type and dcm1 anthers during meiosis. Ubiquitin is used as endogenous control. Error bars represent SD (n = 3). Asterisks indicate significant differences according to Student’s t-test (*P<0.05, **P<0.01 and ***P<0.001).