Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

During meiotic prophase, telomeres attach to the inner nuclear envelope and cluster to form the so-called meiotic bouquet. Although this has been observed in almost all organisms studied, its precise function remains elusive. The coincidence of telomere clustering and initiation of chromosome synapsis has led to the hypothesis that the bouquet facilitates homologous chromosome pairing and synapsis. However, recent mutant analysis suggests that the bouquet is not absolutely required for either homologous pairing or synapsis but that it makes both processes much faster and more efficient. The initiation of bouquet formation is independent of the initiation of recombination. However, the progression through recombination and synapsis may be required for exit from the bouquet stage. Little is known about the mechanism of telomere clustering but recent studies show that it is an active process.

REC8 is a master regulator of chromatin structure and function during meiosis. Here, we dissected the functions of absence of first division (afd1), a maize rec8/α-kleisin homolog, using a unique afd1 allelic series. The first observable defect in afd1 mutants is the inability to make a leptotene chromosome. AFD1 protein is required for elongation of axial elements but not for their initial recruitment, thus showing that AFD1 acts downstream of ASY1/HOP1. AFD1 is associated with the axial and later the lateral elements of the synaptonemal complex. Rescuing 50% of axial element elongation in the weakest afd1 allele restored bouquet formation demonstrating that extent of telomere clustering depends on axial element elongation. However, rescuing bouquet formation was not sufficient for either proper RAD51 distribution or homologous pairing. It provides the basis for a model in which AFD1/REC8 controls homologous pairing through its role in axial element elongation and the subsequent distribution of the recombination machinery independent of bouquet formation.

Pairing, synapsis, and recombination are prerequisites for accurate chromosome segregation in meiosis. The phs1 gene in maize is required for pairing to occur between homologous chromosomes. In the phs1 mutant, homologous chromosome synapsis is completely replaced by synapsis between nonhomologous partners. The phs1 gene is also required for installation of the meiotic recombination machinery on chromosomes, as the mutant almost completely lacks chromosomal foci of the recombination protein RAD51. Thus, in the phs1 mutant, synapsis is uncoupled from recombination and pairing. The protein encoded by the phs1 gene likely acts in a multistep process to coordinate pairing, recombination, and synapsis.

To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A. Contrary to prior studies in rice and Arabidopsis in which mac1-like genes were inferred to act late to suppress trans-differentiation of somatic tapetal cells into meiocytes, we find that mac1 anthers contain excess archesporial (AR) cells that proliferate at least twofold more rapidly than normal prior to tapetal specification, suggesting that MAC1 regulates cell proliferation. mac1 transcript is abundant in immature anthers and roots. By immunolocalization, MAC1 protein accumulates preferentially in AR cells with a declining radial gradient that could result from diffusion. By transient expression in onion epidermis, we demonstrate experimentally that MAC1 is secreted, confirming that the predicted signal peptide domain in MAC1 leads to secretion. Insights from cytology and double-mutant studies with ameiotic1 and absence of first division1 mutants confirm that MAC1 does not affect meiotic cell fate; it also operates independently of an epidermal, Ocl4-dependent pathway that regulates proliferation of subepidermal cells. MAC1 both suppresses excess AR proliferation and is responsible for triggering periclinal division of subepidermal cells. We discuss how MAC1 can coordinate the temporal and spatial pattern of cell proliferation in maize anthers.

The recombination protein RAD51 is a component of the meiotic recombination pathway and has been proposed to play a role in the homology search, a process by which homologous chromosomes find each other before they pair in the prophase of meiosis. To study the relationship between recombination and chromosome pairing, we examined the distribution of RAD51 foci on meiotic chromosomes in maize mutants with defects in chromosome pairing. The patterns of RAD51 distribution showed dramatic variation among the meiotic mutants. The mutants generally exhibited significant decreases in the number of RAD51 foci at zygotene, corresponding to the degree of their pairing defects. These results provide evidence for a key role of RAD51 structures in the homology search.

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.chromosomes ͉ plant development ͉ genetics ͉ recombination

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAP-SIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.maize ͉ meiosis ͉ recombination ͉ chromosome pairing M eiotic prophase I encompasses a large number of tightly regulated and coordinated processes (1). Although many structural proteins involved in these processes are now known, mechanisms regulating meiotic prophase progression remain largely unexplored. In this study, we describe the function of the POOR HOMOLOGOUS SYNAPSIS (PHS1) gene in higher plants that regulates prophase I by coordinating recombination and homologous chromosome pairing (2). Meiotic recombination is initiated by SPO11-mediated formation of double-strand breaks (DSBs) in chromosomal DNA (3). In plants, this occurs in or before early leptotene (2, 4). The DSBs are subsequently resected by the MRN protein complex in plants and animals, and a similar MRX complex in S. cerevisiae, to generate singlestranded DNA (ssDNA) overhangs. The MRN complex contains MRE11, RAD50, and NBS1 (5, 6). MRE11 is a DNA-binding protein with endonuclease, exonuclease, and helicase activities that directly facilitate ssDNA overhang formation (6, 7). RAD50 most likely plays a structural role in the complex (6,8). NBS1 regulates the activity of MRE11 and signals DSB presence (5, 6). ssDNA tails created by DSB resection are coated by two DNA strand-exchange proteins, RAD51 and DMC1 (9). The nucleoprotein filaments formed in this way find and invade doublestranded DNA regions on the homo...