The mop1 mutation affects the recombination landscape in maize.

Meiotic recombination is a fundamental process that generates genetic diversity and ensures the accurate segregation of homologous chromosomes. While a great deal is known about genetic factors that regulate recombination, relatively little is known about epigenetic factors, such as DNA methylation. In maize, we examined the effects on meiotic recombination of a mutation in a component of the RNA-directed DNA methylation pathway, Mop1 (Mediator of paramutation1), as well as a mutation in a component of the trans-acting small interference RNA biogenesis pathway, Lbl1 (Leafbladeless1). MOP1 is of particular interest with respect to recombination because it is responsible for methylation of transposable elements that are immediately adjacent to transcriptionally active genes. In the mop1 mutant, we found that meiotic recombination is uniformly decreased in pericentromeric regions but is generally increased in gene rich chromosomal arms. This observation was further confirmed by cytogenetic analysis showing that although overall crossover numbers are unchanged, they occur more frequently in chromosomal arms in mop1 mutants. Using whole genome bisulfite sequencing, our data show that crossover redistribution is driven by loss of CHH (where H = A, T, or C) methylation within regions near genes. In contrast to what we observed in mop1 mutants, no significant changes were observed in the frequency of meiotic recombination in lbl1 mutants. Our data demonstrate that CHH methylation has a significant impact on the overall recombination landscape in maize despite its low frequency relative to CG and CHG methylation.

Dynamic localization of SPO11-1 and conformational changes of meiotic axial elements during recombination initiation of maize meiosis.

Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation.

Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships.

Whole-chromosome painting probes were developed for each of the 10 chromosomes of maize by producing amplifiable libraries of unique sequences of oligonucleotides that can generate labeled probes through transcription reactions. These paints allow identification of individual homologous chromosomes for many applications as demonstrated in somatic root tip metaphase cells, in the pachytene stage of meiosis, and in interphase nuclei. Several chromosomal aberrations were examined as proof of concept for study of various rearrangements using probes that cover the entire chromosome and that label diverse varieties. The relationship of the supernumerary B chromosome and the normal chromosomes was examined with the finding that there is no detectable homology between any of the normal A chromosomes and the B chromosome. Combined with other chromosome-labeling techniques, a complete set of whole-chromosome oligonucleotide paints lays the foundation for future studies of the structure, organization, and evolution of genomes.

ZmMTOPVIB Enables DNA Double-Strand Break Formation and Bipolar Spindle Assembly during Maize Meiosis.

The meiotic TopoVI B subunit (MTopVIB) plays an essential role in double-strand break formation in mouse (Mus musculus), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), and recent work reveals that rice MTopVIB also plays an unexpected role in meiotic bipolar spindle assembly, highlighting multiple functions of MTopVIB during rice meiosis. In this work, we characterized the meiotic TopVIB in maize (Zea mays; ZmMTOPVIB). The ZmmtopVIB mutant plants exhibited normal vegetative growth but male and female sterility. Meiotic double-strand break formation was abolished in mutant meiocytes. Despite normal assembly of axial elements, mutants showed severely affected synapsis and disrupted homologous pairing. Importantly, we showed that bipolar spindle assembly was also affected in ZmmtopVIB, resulting in triad and polyad formation. Overall, our results demonstrate that ZmMTOPVIB plays critical roles in double-strand break formation and homologous recombination. In addition, our results suggest that the function of MTOPVIB in bipolar spindle assembly is likely conserved across different monocots.

Understanding and Manipulating Meiotic Recombination in Plants.

Meiosis is a specialized cell division, essential in most reproducing organisms to halve the number of chromosomes, thereby enabling the restoration of ploidy levels during fertilization. A key step of meiosis is homologous recombination, which promotes homologous pairing and generates crossovers (COs) to connect homologous chromosomes until their separation at anaphase I. These CO sites, seen cytologically as chiasmata, represent a reciprocal exchange of genetic information between two homologous nonsister chromatids. This gene reshuffling during meiosis has a significant influence on evolution and also plays an essential role in plant breeding, because a successful breeding program depends on the ability to bring the desired combinations of alleles on chromosomes. However, the number and distribution of COs during meiosis is highly constrained. There is at least one CO per chromosome pair to ensure accurate segregation of homologs, but in most organisms, the CO number rarely exceeds three regardless of chromosome size. Moreover, their positions are not random on chromosomes but exhibit regional preference. Thus, genes in recombination-poor regions tend to be inherited together, hindering the generation of novel allelic combinations that could be exploited by breeding programs. Recently, much progress has been made in understanding meiotic recombination. In particular, many genes involved in the process in Arabidopsis (Arabidopsis thaliana) have been identified and analyzed. With the coming challenges of food security and climate change, and our enhanced knowledge of how COs are formed, the interest and needs in manipulating CO formation are greater than ever before. In this review, we focus on advances in understanding meiotic recombination and then summarize the attempts to manipulate CO formation. Last, we pay special attention to the meiotic recombination in polyploidy, which is a common genomic feature for many crop plants.

The Axial Element Protein DESYNAPTIC2 Mediates Meiotic Double-Strand Break Formation and Synaptonemal Complex Assembly in Maize.

During meiosis, homologous chromosomes pair and recombine via repair of programmed DNA double-strand breaks (DSBs). DSBs are formed in the context of chromatin loops, which are anchored to the proteinaceous axial element (AE). The AE later serves as a framework to assemble the synaptonemal complex (SC) that provides a transient but tight connection between homologous chromosomes. Here, we showed that DESYNAPTIC2 (DSY2), a coiled-coil protein, mediates DSB formation and is directly involved in SC assembly in maize (Zea mays). The dsy2 mutant exhibits homologous pairing defects, leading to sterility. Analyses revealed that DSB formation and the number of RADIATION SENSITIVE51 (RAD51) foci are largely reduced, and synapsis is completely abolished in dsy2 meiocytes. Super-resolution structured illumination microscopy showed that DSY2 is located on the AE and forms a distinct alternating pattern with the HORMA-domain protein ASYNAPTIC1 (ASY1). In the dsy2 mutant, localization of ASY1 is affected, and loading of the central element ZIPPER1 (ZYP1) is disrupted. Yeast two-hybrid and bimolecular fluorescence complementation experiments further demonstrated that ZYP1 interacts with DSY2 but does not interact with ASY1. Therefore, DSY2, an AE protein, not only mediates DSB formation but also bridges the AE and central element of SC during meiosis.

Maize multiple archesporial cells 1 (mac1), an ortholog of rice TDL1A, modulates cell proliferation and identity in early anther development.

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.

Regulation of cell divisions and differentiation by MALE STERILITY32 is required for anther development in maize.

Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 (ms32) encodes a basic helix-loop-helix (bHLH) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells. These cells become vacuolated and expand, and lead to failure in pollen mother cell development. ms32 expression is specific to the pre-meiotic anthers and is distributed initially broadly in the four lobes, but as the anther develops, its expression becomes restricted to the innermost somatic layer, the tapetum. The ms32-ref mac1-1 double mutant is unable to form tapetal precursors and also exhibits excessive somatic proliferation leading to numerous, disorganized cell layers, suggesting a synergistic interaction between ms32 and mac1. Altogether, our results show that MS32 is a major regulator in maize anther development that promotes tapetum differentiation and inhibits periclinal division once a tapetal cell is specified.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

Establishing an optimized ATAC-seq protocol for the maize.

The advent of next-generation sequencing in crop improvement offers unprecedented insights into the chromatin landscape closely linked to gene activity governing key traits in plant development and adaptation. Particularly in maize, its dynamic chromatin structure is found to collaborate with massive transcriptional variations across tissues and developmental stages, implying intricate regulatory mechanisms, which highlights the importance of integrating chromatin information into breeding strategies for precise gene controls. The depiction of maize chromatin architecture using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) provides great opportunities to investigate cis-regulatory elements, which is crucial for crop improvement. In this context, we developed an easy-to-implement ATAC-seq protocol for maize with fewer nuclei and simple equipment. We demonstrate a streamlined ATAC-seq protocol with four key steps for maize in which nuclei purification can be achieved without cell sorting and using only a standard bench-top centrifuge. Our protocol, coupled with the bioinformatic analysis, including validation by read length periodicity, key metrics, and correlation with transcript abundance, provides a precise and efficient assessment of the maize chromatin landscape. Beyond its application to maize, our testing design holds the potential to be applied to other crops or other tissues, especially for those with limited size and amount, establishing a robust foundation for chromatin structure studies in diverse crop species.

Isolation of Meiocytes and Cytological Analyses of Male Meiotic Chromosomes in Soybean, Lettuce, and Maize.

Meiosis is a specialized cell division that halves the number of chromosomes following a single round of DNA replication, thus leading to the generation of haploid gametes. It is essential for sexual reproduction in eukaryotes. Over the past several decades, with the well-developed molecular and cytogenetic methods, there have been great advances in understanding meiosis in plants such as Arabidopsis thaliana and maize, providing excellent references to study meiosis in other plants. A chapter in the previous edition described molecular cytological methods for studying Arabidopsis meiosis in detail. In this chapter, we focus on methods for studying meiosis in soybean (Glycine max), lettuce (Lactuca sativa), and maize (Zea mays). Moreover, we include the method that was recently developed for examination of epigenetic modifications, such as DNA methylation and histone modifications on meiotic chromosomes in plants.

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

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.

Reduced Representation Bisulfite Sequencing in Maize.

DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution ( Cokus et al., 2008 ). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies ( Smith et al., 2009 ). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore the original RRBS protocol is not suitable for plants. Accordingly, we developed an in silico pipeline to select specific enzymes to generate a region of interest (ROI)-enriched, e.g., promoter-enriched, reduced representation genome in plants ( Hsu et al., 2017 ). By digesting the maize genome with MseI and selecting 40-300 bp segments, we sequenced about one-fourth of the maize genome while preserving 84.3% of the promoter information. The protocol has been successfully established in maize and can be broadly used in any genome. Our in silico pipeline is combined with the RRBS library preparation protocol, allowing for the computational analysis and experimental validation.

Using Flow Cytometry to Isolate Maize Meiocytes for Next Generation Sequencing: A Time and Labor Efficient Method.

Meiosis is essential during sexual reproduction to generate haploid gametes. Genomic or epigenomic studies of meiosis in multicellular organisms using next-generation sequencing (NGS) methods have been limited because of the difficulty of collecting thousands to millions of meiocytes. Here, we describe a simple protocol to efficiently isolate maize male meiocytes from formaldehyde-fixed samples for NGS techniques that require chemical crosslinking to preserve complex interactions or chromatin architecture. Anthers at desired meiotic stages are selected, fixed with paraformaldehyde, and disrupted using a homogenizer. Cell walls are digested to produce a cell suspension containing small somatic cells and large individual meiocytes. The meiocyte fraction is enriched by size separation with cell strainers and further purified by flow cytometry. From 400 anthers, we can isolate 20,000 meiocytes at 98% purity in 6 to 8 hours. © 2018 by John Wiley & Sons, Inc.

Optimized reduced representation bisulfite sequencing reveals tissue-specific mCHH islands in maize.

BACKGROUND: DNA methylation plays important roles in many regulatory processes in plants. It is economically infeasible to profile genome-wide DNA methylation at a single-base resolution in maize, given its genome size of ~2.5 Gb. As an alternative, we adapted region of interest (ROI)-directed reduced representation bisulfite sequencing (RRBS) to survey genome-wide methylation in maize. RESULTS: We developed a pipeline for selecting restriction enzymes in silico and experimentally showed that, in the maize genome, MseI- and CviQI-digested fragments are precisely enriched in promoters and gene bodies, respectively. We proceeded with comparisons of epigenomes and transcriptomes between shoots and tassels and found that the occurrences of highly methylated, tissue-specific, mCHH islands upstream of transcription start sites (TSSs) were positively correlated with differential gene expression. Furthermore, 5' regulatory regions between TSS and mCHH islands often contain putative binding sites of known transcription factors (TFs) that regulate the flowering process and the timing of the transition from the vegetative to the reproductive phase. By integrating MNase-seq and siRNA-seq data, we found that regions of mCHH islands accumulate 21nt-siRNAs in a tissue-specific manner, marking the transition to open chromatin, thereby ensuring the accessibility of TFs for tissue-specific gene regulation. CONCLUSIONS: Our ROI-directed RRBS pipeline is eminently applicable to DNA methylation profiling of large genomes. Our results provide novel insights into the tissue-specific epigenomic landscapes in maize, demonstrating that DNA methylation and siRNA and chromatin accessibility constitute a critical, interdependent component that orchestrates the transition from the vegetative to the reproductive phase.

A termite symbiotic mushroom maximizing sexual activity at growing tips of vegetative hyphae.

BACKGROUND: Termitomyces mushrooms are mutualistically associated with fungus-growing termites, which are widely considered to cultivate a monogenotypic Termitomyces symbiont within a colony. Termitomyces cultures isolated directly from termite colonies are heterokaryotic, likely through mating between compatible homokaryons. RESULTS: After pairing homokaryons carrying different haplotypes at marker gene loci MIP and RCB from a Termitomyces fruiting body associated with Odontotermes formosanus, we observed nuclear fusion and division, which greatly resembled meiosis, during each hyphal cell division and conidial formation in the resulting heterokaryons. Surprisingly, nuclei in homokaryons also behaved similarly. To confirm if meiotic-like recombination occurred within mycelia, we constructed whole-genome sequencing libraries from mycelia of two homokaryons and a heterokaryon resulting from mating of the two homokaryons. Obtained reads were aligned to the reference genome of Termitomyces sp. J132 for haplotype reconstruction. After removal of the recombinant haplotypes shared between the heterokaryon and either homokaryons, we inferred that 5.04% of the haplotypes from the heterokaryon were the recombinants resulting from homologous recombination distributed genome-wide. With RNA transcripts of four meiosis-specific genes, including SPO11, DMC1, MSH4, and MLH1, detected from a mycelial sample by real-time quantitative PCR, the nuclear behavior in mycelia was reconfirmed meiotic-like. CONCLUSION: Unlike other basidiomycetes where sex is largely restricted to basidia, Termitomyces maximizes sexuality at somatic stage, resulting in an ever-changing genotype composed of a myriad of coexisting heterogeneous nuclei in a heterokaryon. Somatic meiotic-like recombination may endow Termitomyces with agility to cope with termite consumption by maximized genetic variability.

Analyzing maize meiotic chromosomes with super-resolution structured illumination microscopy.

The success of meiosis depends on intricate coordination of a series of unique cellular processes to ensure proper chromosome segregation. Many proteins involved in these cellular events are directly or indirectly associated with chromosomes, especially those required for homologous recombination. These meiotic processes have been explored extensively by conventional light microscopy. However, many features of interest, such as chromatin organization, recombination nodules, or the synaptonemal complex are beyond the resolution of conventional wide-field microscopy. Moreover, in most sample preparation techniques for light microscopy, meiotic cells are squashed, which destroys the spatial organization of the nucleus. Here, I describe a protocol to analyze maize meiotic chromosomes by three-dimensional structured illumination microscopy (3D-SIM), a recently developed high-resolution microscopy technique. This protocol can be used to examine protein localizations at a high resolution level by immunofluorescence.

Interlock formation and coiling of meiotic chromosome axes during synapsis.

The meiotic prophase chromosome has a unique architecture. At the onset of leptotene, the replicated sister chromatids are organized along an axial element. During zygotene, as homologous chromosomes pair and synapse, a synaptonemal complex forms via the assembly of a transverse element between the two axial elements. However, due to the limitations of light and electron microscopy, little is known about chromatin organization with respect to the chromosome axes and about the spatial progression of synapsis in three dimensions. Three-dimensional structured illumination microscopy (3D-SIM) is a new method of superresolution optical microscopy that overcomes the 200-nm diffraction limit of conventional light microscopy and reaches a lateral resolution of at least 100 nm. Using 3D-SIM and antibodies against a cohesin protein (AFD1/REC8), we resolved clearly the two axes that form the lateral elements of the synaptonemal complex. The axes are coiled around each other as a left-handed helix, and AFD1 showed a bilaterally symmetrical pattern on the paired axes. Using the immunostaining of the axial element component (ASY1/HOP1) to find unsynapsed regions, entangled chromosomes can be easily detected. At the late zygotene/early pachytene transition, about one-third of the nuclei retained unsynapsed regions and 78% of these unsynapsed axes were associated with interlocks. By late pachytene, no interlocks remain, suggesting that interlock resolution may be an important and rate-limiting step to complete synapsis. Since interlocks are potentially deleterious if left unresolved, possible mechanisms for their resolution are discussed in this article.

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9.

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.