A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana

Abstract

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.

Figure 1.

Comparative Fertility of Wild-Type Arabidopsis and the mpa1 Mutant. (A) Wild-type siliques with a mean size of 12.78 mm (n = 500). (B) Alexander staining of a wild-type anther. (C) Alexander staining of wild-type pollen grains. Viable pollen grains are stained in red. (D) mpa1 siliques with a mean size of 3.99 mm (n = 500). (E) mpa1 anther. (F) mpa1 pollen grains. Nonviable pollen grains do not stain red and have different size and morphology compared with the viable grains. Bars in (A) and (D) = 5 mm; bars in (B) and (E) = 20 μm; and bars in (C) and (F) = 10 μm.

Figure 2.

Molecular Characterization of mpa1. (A) The MPA1 gene structure with exons (black boxes) and introns, the cDNA, and the corresponding protein. The triangle indicates the position of the T-DNA insertion (nucleotide 23,667,376) and a line indicates the Brd box in the 3′untranslated region (UTR) (white boxes). The protein domains are listed. (B) Expression analysis of MPA1 locus. RT-PCR products were amplified from RNA derived from flower buds at meiosis, stem, and leaf of both wild-type and mpa1 mutant plants. The GAPDH gene was used as a control.

Figure 3.

Cytogenetical Analysis of Wild-Type and mpa1 Meiosis. DAPI-stained meiotic stages. (A) to (E) Wild-type male meiosis. (A) Pachytene. Five fully synapsed bivalents. (B) Diakinesis. The bivalents are clearly distinguished. (C) Metaphase I. The five bivalents are coorientated on the spindle equator. Four ring bivalents (having chiasmata in both arms) and one rod bivalent (chiasmata in one arm only) can be distinguished. (D) Anaphase I. Segregation of five chromosomes to each pole. (E) Anaphase II. Separation of five chromatids toward each spindle pole. (F) to (J) mpa1 male meiosis. (F) Pachytene. The spacing between the homologous chromosomes appears greater than wild type, suggesting some abnormality in synapsis. (G) Diakinesis. Eight univalents and one bivalent. (H) Metaphase I. Only one rod bivalent and eight univalents. (I) Anaphase I. Chromosome missegregation, with seven chromosomes moving to one pole and three to the other one. (J) Anaphase II. Six groups with different numbers of chromosomes. (K) to (N) mpa1 female meiosis. (K) Pachytene. Abnormal synapsis. (L) Diakinesis. Ten univalents. (M) Metaphase I. One rod bivalent. (N) Anaphase II. Four groups containing 3, 4, 3, and 10 chromatids, respectively. (O) to (Q) Abnormal chromosome synapsis in mpa1. (O) Fully synapsed pachytene chromosomes, the BAC F24J10 on both homologs is visualized as a single FISH signal. (P) Abnormal synapsis with two discernable FISH signals, although still closely paired. (Q) Abnormal synapsis with two clearly separated FISH signals. The homologous chromosomes are broadly spaced although fully aligned. Bar = 10 μm.

Figure 4.

Inhibitors of PSA Phenocopy the mpa1 Mutation. (A) to (E) Puromycin (1 μM). (A) Inflorescences exhibit a semisterile phenotype, with short siliques and a reduced number of seeds per silique. (B) Pachytene. The homologous chromosomes exhibit slightly abnormal synapsis similar to mpa1. (C) Late diplotene. The condensation of the chromatin appears more diffuse than the wild type. (D) Metaphase I. Ten univalents. (E) Telophase II. Five groups of cells. (F) to (J) DAMPAQ-22 (10 μM). (F) Semisterile phenotype of the inflorescences. (G) Early diplotene. (H) Diakinesis. Two bivalents and six univalents. (I) Early anaphase I. (J) Metaphase II. Two groups of six and four chromosomes. (K) to (R) Fluorescent visualization of PSA with DAMPAQ-22 in the wild-type ([K] to [N]) and mpa1 ([O] to [R]) meiocytes. A fluorescent signal arising from binding of the bioprobe to MPA1 was detected during prophase I in the wild type. (K) Leptotene. Showing a high level of fluorescence. (L) Zygotene. (M) Pachytene. (N) Diakinesis. The fluorescent DAMPAQ-22 signal is absent in mpa1. (O) Leptotene. (P) Pachytene. (Q) Diakinesis (R) Telophase I. Four nuclei. Bar = 10 μm.

Figure 5.

Immunolocalization of Meiotic Proteins ASY1 and RAD51 in the Wild Type and mpa1. (A) and (B) Immunolocalization of ASY1 (green). (A) Wild-type pachytene. The axial/lateral elements are labeled. (B) mpa1 pachytene. Identical localization to that in wild type, but the fluorescence signal seems to be slightly more diffuse, consistent with the mutant phenotype observed at diplotene. (C) to (F) Immunolocalization of RAD51 (red). (C) Wild-type zygotene. RAD51 appears as numerous foci on the chromosomes. (D) mpa1 zygotene. In some cells RAD51 foci are detectable on the chromosomes. (E) and (F) mpa1 zygotene. In ∼90% of cells RAD51 accumulates within the cytoplasm. Bar = 10 μm.

Figure 6.

Immunolocalization of MSH4, MLH1, and α-Tubulin in the Wild Type and mpa1. (A) and (B) Immunolocalization of MSH4 (red). (A) Wild-type leptotene/zygotene. MSH4 is distributed as multiple foci on the chromatin. (B) mpa1 leptotene/zygotene. MSH4 is accumulated in the cytoplasm. (C) and (D) Immunolocalization of MLH1 (red) and ASY1 (green). (C) Wild-type zygotene. MLH1 foci are distributed on the chromosomes axes. (D) mpa1 zygotene. MLH1 is found in association with the nucleolus. (E) and (F) Localization of α-tubulin (green) in mpa1. Immunostaining of the mitotic spindle appears normal. (E) Mitotic metaphase. (F) Anaphase I. In all cases the slides were counterstained with DAPI (blue). Bar = 10 μm.