Figure 1.
Developmental aberration of germline and nursery cells in the mel2 mutant.
(A) A schematic illustration of the germline cell development in the anther and ovule in rice. Red-colored cells indicate the archesporial cells; magenta, sporogenous cells; light green, primary parietal cells; yellow, endothecium; brown, secondary parietal cells; dark green, middle layer cells; blue, tapetal cells; mandarin, meiocytes at premeiotic interphase. (B–L) Plastic sections of wild-type (B–F) and mel2 (G–L) reproductive organs during sporogenesis. (B, G) Premeiotic interphase. The callose accumulation among PMCs (a small arrow) was absent in the mutant. A large arrow indicates asynchronous, aberrant mitosis of the PMC. (C, H) Meiotic I prophase (diakinesis). (D, I) Post-meiotic microspore stage. Tapetal cells were pseudo-colored in magenta. (E, J) TUNEL assay. White arrows indicate TUNEL signals representing apoptotic DNA fragmentation found in nuclei of disrupted PMCs. (F, K, L) Ovules at post-meiosis. Three of tetrad spores (open arrowheads) were degenerated, and a single megaspore (an open arrow) underwent megagametogenesis after the completion of wild-type meiosis, whereas meiosis progression was stagnant at various steps in the mutant (K, L). Closed arrowheads indicate an equal size of tetrad spores. Bars, 10 µm.
Figure 2.
Rice MEL2 is the RRM protein partially homologous to human DAZAP1.
The single RRM of MEL2 is followed by a proline-rich sequence (yellow) like human DAZAP1, whereas DAZAP1 carries a doublet of RRMs. MEL2 possesses three additional motifs; The N-myristoylation target, the ankyrin repeats (red), and the RING finger motif (green). The peptide sequence of each motif is aligned and compared with known consensus sequence and/or sequences of other organisms, in which identical amino-acid residues are highlighted. Os, rice (Oryza sativa); Sb, Sorghum bicolor; Zm, Zea mays; Bd, Brachypodium distachyon; DAZAP1, huma DAZAP1 (UniProt/Swiss-Prot: Q96EP5); xPrrp, Xenopus Proline-rice RNA binding Protein (Vg1) (Q98SJ2).
Figure 3.
MEL2 mRNA was expressed in male and female germline and parietal cells before meiosis.
(A) The initiation stage of archesporial cells. A faint MEL2 signal (blue) appeared at hypodermis of stamen (st) and ovule primordium (op). (B) Early premeiotic mitosis stage. (C) A magnified image of (B). In op, plural hypodermal cells expressed MEL2. (D) The late premeiotic mitosis stage. (E, F) Magnified images of (D). The faint MEL2 signal was observed in female sporogenous cell (fsp) (E). Microsporocytes (msp) strongly expressed MEL2, and parietal layer cells (pl) adhered to msp were also stained (F). (G) No MEL2 signal was detected in and after meiosis. (H) A sense probe as a negative control gave no signal in the same stage with (D). Bars, 100 µm.
Figure 4.
Synchronous initiation of premeiotic S was disrupted among mel2 germ cells.
The longitudinal sections of wild-type (A–E), and mel2 anthers (F–J). (A, F) During sporogenous cells undergoing premeiotic mitosis, CDKB2;1 mRNA, the marker of G2/M transition, was expressed in patches in both wild-type and mel2 anthers (arrowheads). (B, G) During premeiotic interphase, CDKB2;1 expression was synchronously suppressed in wild-type anthers (B), whereas it was still in patches in mel2 anthers (G). (C, H) During premeiotic S or the onset of meiosis I, histone H4 mRNA, the marker of S-phase, was expressed simultaneously (C), while only a few germ cells expressed the H4 (H). (D, I) During sporogenous cells undergoing premeiotic mitosis, MEL1 mRNA, the marker of archesporial and sporogenous cells, was strongly expressed in sporogenous cells. (E, J) In early meiosis I, MEL1 expression was drastically downregulated in wild-type anthers (E), whereas kept at a higher level in mel2 anthers (J). Bars, 20 µm.
Figure 5.
The mel2 mutation abolished synchronous incorporation of bromodeoxyuridine (BrdU) in an anther at premeiotic interphase.
(A) The number of flowers with pollen mother cells (PMCs) emanating the BrdU signal was counted. Flowers at premeiotic interphase (with about 0.4-mm anthers) were provided for the BrdU incorporation experiment. Flowers emanating the BrdU signal in 0%, less than 50%, and more than 50% of PMCs were classified into classes 1, 2 and 3, respectively. (B) A percentage of BrdU-incorporated PMCs in an anther was measured in three plants (#: the plant number) each of the wild type and mel2 mutant. (C) Longitudinal sections of BrdU-incorporated anthers at premeiotic interphase. The wild-type anthers frequently contained a lot of PMCs emanating the intense BrdU signal in an anther locule (top), while the mel2 anthers contained a few (bottom). Yellow arrows indicate the nuclei emanating the BrdU signal. Bars, 20 µm.
Figure 6.
The absence of homologous chromosome synapsis in mel2 mutant anthers.
Chromosomes were counter-stained with DAPI (blue). (A, B) Meiocyte nuclei at early zygotene in the wild type (A) and mel2 mutant (B). (C, D, E) Meiocyte nuclei at late zygotene in the wild type (C) and mel2 mutant (D, E). In 79.7% of mel2 meiocytes, PAIR2 and ZEP1 were aberrantly accumulated, and the arrangement of OsCenH3 foci was soma-like (D). The remaining 20.3% of the meiocyte nuclei exhibited filamentous PAIR2 signals, comparable to those in wild-type early zygotene, and the centromere arrangement became meiotic (E). Bars, 10 µm.
Figure 7.
Subcellular localization of the T7 peptide-tagged MEL2 in anthers of transgenic rice plants.
Plastic cross-sections of transgenic anthers were counter-stained with DAPI (blue). Pollen mother cells (PMCs) were outlined with the broken lines. (A–C) Transgenic anthers expressing the C-tagged MEL2 (magenta). (A) Premeiotic mitosis stage. (B) Premeiotic interphase. A yellow arrow indicates the anti-T7 signals observed in the cytoplasm of tapetal cells. (C) Early meiosis I. (D) A transgenic anther expressing the N-tagged MEL2 (magenta) in premeiotic interphase. (E) An anther in premeiotic interphase transformed with the empty vector as a negative control. Bars, 10 µm. (F) The summary of MEL2 subcellular localization observed in (A–C). Smaller circles within larger ones indicate nuclei of germ cells. The pink and red indicate the subcellular localization of MEL2 at the cytoplasm and the perinuclear region, respectively. Broken and solid arrows indicate the cell division cycle and the cell maturation without the division.