The ultimate purpose of this study was to investigate AHP2 protein function in Arabidopsis; AHP2 protein is known to form a heterodimer with MND1. But to do this an existing chromosome spreading protocol had to be modified to reproducibly provide large numbers of well preserved chromosomes and a multi-criteria meiotic staging method was developed to accurately identify chromosome spreads at specific prophase I substages. As well a technique for combined immuno-cytochemistry and fluorescent in situ hybridization (FISH) had to be developed. Coimmuno-localization of AHP2 and MND1 proteins, in wild type meiocytes, revealed synchronous temporal organization (signal initially peaks, for both, during zygotene) but spatially the AHP2 signal appeared exclusively as chromosome axis-associated foci whereas the MND1 signal was diffuse with some foci suggesting that MND1 may also be localizing to loop regions. This finding strongly suggests that MND1 also functions independently of AHP2 during meiosis. We coupled transmission electron microscopy (TEM) analysis of ultrathin sections of meiotic nuclei with light microscope (LM) analysis of chromosome spreads and demonstrated that ahp2 meiocytes fail to stabilize chromosome close alignment that normally occurs during zygotene. My method of combining 2-Bromo-5-deoxyUridine (BrdU) - determination of meiosis duration and the relative durations of each substage allowed us to calculate the absolute duration of each prophase I substage in wild type and revealed that early to mid-zygotene was prolonged in the ahp2 mutant. This finding was consistent with the ahp2 mutant’s overall lack of stabilized chromosome alignment. Scanning electron microscopy (SEM) showed that the Arabidopsis ahp2 short stamen filament was due to reduced cell elongation in filament parenchyma (epidermal) cells. Detection of illegitimate connections between chromosomes at anaphase I may trigger cell-to-cell signals that result in reduced epidermal cell elongation in the stamen filament. Finally, I report that the short arms of NOR-bearing chromosomes 2 and 4 homologously pair and synapse in the ahp2 mutant despite an overall lack of stabilized pairing. This NOR phenomenon has been observed in Drosophila but ours is the first report of the ability of NORs to locally induce pairing and synapsis in plants.

The ultimate purpose of this study was to investigate AHP2 protein function in Arabidopsis; AHP2 protein is known to form a heterodimer with MND1. But to do this an existing chromosome spreading protocol had to be modified to reproducibly provide large numbers of well preserved chromosomes and a multi-criteria meiotic staging method was developed to accurately identify chromosome spreads at specific prophase I substages. As well a technique for combined immuno-cytochemistry and fluorescent in situ hybridization (FISH) had to be developed. Coimmuno-localization of AHP2 and MND1 proteins, in wild type meiocytes, revealed synchronous temporal organization (signal initially peaks, for both, during zygotene) but spatially the AHP2 signal appeared exclusively as chromosome axis-associated foci whereas the MND1 signal was diffuse with some foci suggesting that MND1 may also be localizing to loop regions. This finding strongly suggests that MND1 also functions independently of AHP2 during meiosis. We coupled transmission electron microscopy (TEM) analysis of ultrathin sections of meiotic nuclei with light microscope (LM) analysis of chromosome spreads and demonstrated that ahp2 meiocytes fail to stabilize chromosome close alignment that normally occurs during zygotene. My method of combining 2-Bromo-5-deoxyUridine (BrdU) - determination of meiosis duration and the relative durations of each substage allowed us to calculate the absolute duration of each prophase I substage in wild type and revealed that early to mid-zygotene was prolonged in the ahp2 mutant. This finding was consistent with the ahp2 mutant’s overall lack of stabilized chromosome alignment. Scanning electron microscopy (SEM) showed that the Arabidopsis ahp2 short stamen filament was due to reduced cell elongation in filament parenchyma (epidermal) cells. Detection of illegitimate connections between chromosomes at anaphase I may trigger cell-to-cell signals that result in reduced epidermal cell elongation in the stamen filament. Finally, I report that the short arms of NOR-bearing chromosomes 2 and 4 homologously pair and synapse in the ahp2 mutant despite an overall lack of stabilized pairing. This NOR phenomenon has been observed in Drosophila but ours is the first report of the ability of NORs to locally induce pairing and synapsis in plants.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.