Maize AMEIOTIC1 is essential for multiple early meiotic processes and likely required for the initiation of meiosis

Abstract

Molecular mechanisms that initiate meiosis have been studied in fungi and mammals, but little is known about the mechanisms directing the meiosis transition in other organisms. To elucidate meiosis initiation in plants, we characterized and cloned the ameiotic1 (am1) gene, which affects the transition to meiosis and progression through the early stages of meiotic prophase in maize. We demonstrate that all meiotic processes require am1, including expression of meiosis-specific genes, establishment of the meiotic chromosome structure, meiosis-specific telomere behavior, meiotic recombination, pairing, synapsis, and installation of the meiosis-specific cytoskeleton. As a result, in most am1 mutants premeiotic cells enter mitosis instead of meiosis. Unlike the genes involved in initiating meiosis in yeast and mouse, am1 also has a second downstream function, whereby it regulates the transition through a novel leptotene-zygotene checkpoint, a key step in early meiotic prophase. The am1 gene encodes a plant-specific protein with an unknown biochemical function. The AM1 protein is diffuse in the nucleus during the initiation of meiosis and then binds to chromatin in early meiotic prophase I when it regulates the leptotene-zygotene progression.

Fig. 1.

Chromosome behavior in male meiocytes in wild-type maize and in the am1-1 and am1-praI mutants. (A) Telomere bouquet analysis using FISH with the telomere probe (green). (B) Chromosome pairing analyzed using FISH with the 5S rRNA locus probe (cyan). (C) Installation of meiotic cohesin AFD1 (green), the maize Rec8 homologue, on chromosomes. The pictured wild-type meiocyte is in zygotene. (D) Installation of ASY1 (green), a meiosis-specific protein associated with chromosome axis. (E) Installation of the RAD51 recombination protein (green) on meiotic chromosomes. (F) Cytologic detection of meiotic DSB formation using a modified TUNEL assay. Green: TUNEL assay staining meiotic DSBs. (A–F) Red: DAPI-stained chromatin. (Scale bars, 10 μm.)

Fig. 2.

(A) Diagram of the AM1 protein with marked positions of lesions in the am1-praI, am1-485, and am1-489 mutant alleles. Also noted are positions of 2 predicted coiled-coil domains and the protein region used to raise the anti-AM1 antibody. (B) Bayesian reconstruction of phylogeny of AM1 homologues in plants. Numbers next to branches are clade credibility values. (C) Expression of the am1 gene detected by RT-PCR in various wild-type maize tissues and in the 5 am1 mutants. (D) Western blot analysis of AM1 protein accumulation in wild-type maize tissues and in the 5 am1 mutants.

Fig. 3.

Immunolocalization of the AM1 protein in male meiocytes in wild-type maize. Red: DAPI-stained chromatin; green: AM1; purple: CENPC, which is a constitutive component of the inner kinetochore and marks centromeres. (Inset) Magnification of a centromeric region of one of the pachytene chromosomes, with CENPC identifying the kinetochore and AM1 staining in the pericentromeric region. (Scale bars, 10 μm; small bar Inset, 1 μm.)