Meiosis In Maize Research Articles

ZmASY1 interacts with ZmPRD3 and is crucial for meiotic double-strand break formation in maize.

During meiosis, recombination-mediated pairing and synapsis of homologous chromosomes begin with programmed DNA double-strand breaks (DSBs). In yeast and mice, DSBs form in a tethered loop-axis complex, in which DSB sites are located within chromatin loops and tethered to the proteinaceous axial element (AE) by DSB-forming factors. In plants, the molecular connection between DSB sites and chromosome axes is poorly understood. By integrating genetic analysis, immunostaining technology, and protein-protein interaction studies, the putative factors linking DSB formation to chromosome axis were explored in maize meiosis. Here, we report that the AE protein ZmASY1 directly interacts with the DSB-forming protein ZmPRD3 in maize (Zea mays) and mediates DSB formation, synaptonemal complex assembly, and homologous recombination. ZmPRD3 also interacts with ZmPRD1, which plays a central role in organizing the DSB-forming complex. These results suggest that ZmASY1 and ZmPRD3 may work as a key module linking DSB sites to chromosome axes during DSB formation in maize. This mechanism is similar to that described in yeast and recently Arabidopsis involving the homologs Mer2/ZmPRD3 and HOP1/ZmASY1, thus indicating that the process of tethering DSBs in chromatin loops to the chromosome axes may be evolutionarily conserved in diverse taxa.

Low cell number ChIP-seq reveals chromatin state-based regulation of gene transcription in the rice male meiocytes.

Low cell number ChIP-seq reveals chromatin state-based regulation of gene transcription in the rice male meiocytes.

Heat shock protein 101 contributes to the thermotolerance of male meiosis in maize.

High temperatures interfere with meiotic recombination and the subsequent progression of meiosis in plants, but few genes involved in meiotic thermotolerance have been characterized. Here, we characterize a maize (Zea mays) classic dominant male-sterile mutant Ms42, which has defects in pairing and synapsis of homologous chromosomes and DNA double-strand break (DSB) repair. Ms42 encodes a member of the heat shock protein family, HSP101, which accumulates in pollen mother cells. Analysis of the dominant Ms42 mutant and hsp101 null mutants reveals that HSP101 functions in RADIATION SENSITIVE 51 loading, DSB repair, and subsequent meiosis. Consistent with these functions, overexpression of Hsp101 in anthers results in robust microspores with enhanced heat tolerance. These results demonstrate that HSP101 mediates thermotolerance during microsporogenesis, shedding light on the genetic basis underlying the adaptation of male meiocytes to high temperatures.

ZmSPO11-2 is critical for meiotic recombination in maize.

Most plant species have three or more SPO11/TOPOVIA homologs and two TOPOVIB homologs, which associate to trigger meiotic double-strand break (DSB) formation and subsequent meiotic recombination. In Zea mays L. (maize), ZmSPO11-1 and ZmMTOPVIB have been reported to be indispensable for the initiation of meiotic recombination, yet the function of ZmSPO11-2 remains unclear. In this study, we characterized meiotic functions of ZmSPO11-2 during male meiosis in maize. Two independent Zmspo11-1 knock-out mutants exhibited normal vegetative growth but both male and female sterility. The formation of meiotic DSBs of DNA moleculeswas fully abolished in the Zmspo11-2 plants, leading to the defective homologous chromosome paring, synapsis, recombination, and segregation. However, the bipolar spindle assembly was not noticeably affected in Zmspo11-2 meiocytes. Overall, our results demonstrate that as its partner ZmSPO11-1 and ZmMTOPVIB, ZmSPO11-2 plays essential roles in DSB formation and homologous recombination in maize meiosis.

Frequent Spindle Assembly Errors Require Structural Rearrangement to Complete Meiosis in Zea mays.

The success of an organism is contingent upon its ability to faithfully pass on its genetic material. In the meiosis of many species, the process of chromosome segregation requires that bipolar spindles be formed without the aid of dedicated microtubule organizing centers, such as centrosomes. Here, we describe detailed analyses of acentrosomal spindle assembly and disassembly in time-lapse images, from live meiotic cells of Zea mays. Microtubules organized on the nuclear envelope with a perinuclear ring structure until nuclear envelope breakdown, at which point microtubules began bundling into a bipolar form. However, the process and timing of spindle assembly was highly variable, with frequent assembly errors in both meiosis I and II. Approximately 61% of cells formed incorrect spindle morphologies, with the most prevalent being tripolar spindles. The erroneous spindles were actively rearranged to bipolar through a coalescence of poles before proceeding to anaphase. Spindle disassembly occurred as a two-state process with a slow depolymerization, followed by a quick collapse. The results demonstrate that maize meiosis I and II spindle assembly is remarkably fluid in the early assembly stages, but otherwise proceeds through a predictable series of events.

An F‐box protein ACOZ1 functions in crossover formation by ensuring proper chromosome compaction during maize meiosis

Meiosis is an essential reproductive process to create new genetic variation. During early meiosis, higher order chromosome organization creates a platform for meiotic processes to ensure the accuracy of recombination and chromosome segregation. However, little is known about the regulatory mechanisms underlying dynamic chromosome organization in plant meiosis. Here, we describe abnormal chromosome organization in zygotene1 (ACOZ1), which encodes a canonical F-box protein in maize. In acoz1 mutant meiocytes, chromosomes maintain a leptotene-like state and never compact to a zygotene-like configuration. Telomere bouquet formation and homologous pairing are also distorted and installation of synaptonemal complex ZYP1 protein is slightly defective. Loading of early recombination proteins RAD51 and DMC1 is unaffected, indicating that ACOZ1 is not required for double strand break formation or repair. However, crossover formation is severely disturbed. The ACOZ1 protein localizes on the boundary of chromatin, rather directly to chromosomes. Furthermore, we identified that ACOZ1 interacts with SKP1 through its C-terminus, revealing that it acts as a subunit of the SCF E3 ubiquitin/SUMO ligase complex. Overall, our results suggest that ACOZ1 functions independently from the core meiotic recombination pathway to influence crossover formation by controlling chromosome compaction during maize meiosis.

ZmPRD1 is essential for double-strand break formation, but is not required for bipolar spindle assembly during maize meiosis.

Homologs of PUTATIVE RECOMBINATION INITIATION DEFECT 1 (PRD1) are known to be essential for meiotic double-strand break (DSB) formation in mouse (Mus musculus), Arabidopsis, and rice (Oryza sativa). Recent research has shown that rice PRD1 also plays an unanticipated role in meiotic bipolar spindle assembly, revealing that PRD1 has multiple functions in plant meiosis. In this study, we characterize the meiotic function of PRD1 in maize (Zea mays; ZmPRD1). Our results show that Zmprd1 mutant plants display normal vegetative growth but have complete male and female sterility. Meiotic DSB formation is fully abolished in mutant meiocytes, leading to failure in homologous pairing, synapsis, and recombination. ZmPRD1 exhibits a different pattern of chromosome localization compared to its rice homologs. The ZmPRD1 protein interacts with several DSB-forming proteins, but does not directly interact with the kinetochore proteins REC8 and SGO1. Possibly as a result of this, there are no significant abnormalities of bipolar spindle assembly in Zmprd1 meiocytes. Overall, our results demonstrate that ZmPRD1 is essential for DSB formation and homologous recombination in maize meiosis. However, the recently-identified function of PRD1 in bipolar spindle assembly during rice meiosis is not conserved in maize.

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.

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.

ZmRAD51C is Essential for Double-Strand Break Repair and Homologous Recombination in Maize Meiosis.

Radiation sensitive 51 (RAD51) recombinases play crucial roles in meiotic double-strand break (DSB) repair mediated by homologous recombination (HR) to ensure the correct segregation of homologous chromosomes. In this study, we identified the meiotic functions of ZmRAD51C, the maize homolog of Arabidopsis and rice RAD51C. The Zmrad51c mutants exhibited regular vegetative growth but complete sterility for both male and female inflorescence. However, the mutants showed hypersensitivity to DNA damage by mitomycin C. Cytological analysis indicated that homologous chromosome pairing and synapsis were rigorously inhibited, and meiotic chromosomes were often entangled from diplotene to metaphase I, leading to chromosome fragmentation at anaphase I. Immunofluorescence analysis showed that although the signals of the axial element absence of first division (AFD1) and asynaptic1 (ASY1) were normal, the assembly of the central element zipper1 (ZYP1) was severely disrupted. The DSB formation was normal in Zmrad51c meiocytes, symbolized by the regular occurrence of γH2AX signals. However, RAD51 and disrupted meiotic cDNA 1 (DMC1) signals were never detected at the early stage of prophase I in the mutant. Taken together, our results indicate that ZmRAD51C functions crucially for both meiotic DSB repair and homologous recombination in maize.

Defining the developmental program leading to meiosis in maize.

In multicellular organisms, the entry into meiosis is a complex process characterized by increasing meiotic specialization. Using single-cell RNA sequencing, we reconstructed the developmental program into maize male meiosis. A smooth continuum of expression stages before meiosis was followed by a two-step transcriptome reorganization in leptotene, during which 26.7% of transcripts changed in abundance by twofold or more. Analysis of cell-cycle gene expression indicated that nearly all pregerminal cells proliferate, eliminating a stem-cell model to generate meiotic cells. Mutants defective in somatic differentiation or meiotic commitment expressed transcripts normally present in early meiosis after a delay; thus, the germinal transcriptional program is cell autonomous and can proceed despite meiotic failure.

Following meiosis in maize

Plant Science Plants do not set aside a germ-cell lineage from early development as animals do, but instead generate germ cells on demand. Nelms and Walbot, working in maize, took advantage of a size differential between somatic and developing germ cells in the anthers at the top of the maize plant to isolate individual germ cells during the meiotic progression to pollen development. They used single-cell RNA sequencing to study changes in the transcriptome through meiosis. These studies revealed increasing specialization as meiosis progressed, with a reorganization of the transcriptome in a transition during the leptotene stage of meiosis. Science , this issue p. [52][1] [1]: /lookup/volpage/364/52?iss=6435

Anaphase asymmetry and dynamic repositioning of the division plane during maize meiosis.

The success of an organism is contingent upon its ability to transmit genetic material through meiotic cell division. In plant meiosis I, the process begins in a large spherical cell without physical cues to guide the process. Yet, two microtubule-based structures, the spindle and phragmoplast, divide the chromosomes and the cell with extraordinary accuracy. Using a live-cell system and fluorescently labeled spindles and chromosomes, we found that the process self- corrects as meiosis proceeds. Metaphase spindles frequently initiate division off-center, and in these cases anaphase progression is asymmetric with the two masses of chromosomes traveling unequal distances on the spindle. The asymmetry is compensatory, such that the chromosomes on the side of the spindle that is farthest from the cell cortex travel a longer distance at a faster rate. The phragmoplast forms at an equidistant point between the telophase nuclei rather than at the original spindle mid-zone. This asymmetry in chromosome movement implies a structural difference between the two halves of a bipolar spindle and could allow meiotic cells to dynamically adapt to errors in metaphase and accurately divide the cell volume.

И.Н.ГОЛУБОВСКАЯ –СОЗДАТЕЛЬ УНИКАЛЬНОЙ КОЛЛЕКЦИИ МУТАЦИЙ ГЕНОВ МЕЙОЗА У КУКУРУЗЫ И ТАЛАНТЛИВЫЙ ИССЛЕДОВАТЕЛЬ ПРОБЛЕМЫ ГЕНЕТИКИ МЕЙОЗА

Over 40 years, Inna Nikitichna Golubovskaya studied the fundamental problem of genetic control of meiosis, using meiosis in Zea mays as the model. She discovered more than half of 50 genes and gene alleles controlling meiosis in maize anthers and ovules, and studied even more of them. Having started with her researches at the Institute of Cytology and Genetics, Siberian Branch of the USSR Academy of Sciences (Novosibirsk) in 1972, she moved in 1986 to the N. I. Vavilov Institute of Plant Industry (Leningrad/St. Petersburg) to become the leader of a research group. In 1993, she started to collaborate with the Department of Biology, University of North Dakota (USA). There she worked during the field seasons until 1998. In 1999, Dr. Golubovskaya was invited to work with the Department of Cell and Molecular Biology, California State University at Berkeley. She developed and maintained a seed collection of genetic maize lines bearing mutations of meiotic genes, and in 2012 brought it to the Vavilov Institute. She was actively involved in cloning and studying major genes regulating meiosis in maize at the molecular level, and their expression in meiotic process. I. N. Golubovskaya’s contribution to the studies on maize genetics and genetic control of meiosis was expressly extolled by American geneticists.

Meiotic Chromosome Structure and Function in Plants

Chromosome structure is important for many meiotic processes. Here, we outline 3 main determinants of chromosome structure and their effects on meiotic processes in plants. Cohesins are necessary to hold sister chromatids together until the first meiotic division, ensuring that homologous chromosomes and not sister chromatids separate during anaphase I. During meiosis in maize, Arabidopsis, and rice, cohesins are needed for establishing early prophase chromosome structure and recombination and for aligning bivalents at the metaphase plate. Condensin complexes play pivotal roles in controlling the packaging of chromatin into chromosomes through chromatin compaction and chromosome individualization. In animals and fungi, these complexes establish a meiotic chromosome structure that allows for proper recombination, pairing, and synapsis of homologous chromosomes. In plants, information on the role of condensins in meiosis is limited, but they are known to be required for successful completion of reproductive development. Therefore, we speculate that they play roles similar to animal and fungal condensins during meiosis. Plants generally have large and complex genomes due to frequent polyploidy events, and likely, condensins and cohesins organize chromosomes in such a way as to ensure genome stability. Hexaploid wheat has evolved a unique mechanism using a Ph1 locus-controlled chromosome organization to ensure proper chromosome pairing in meiosis. Altogether, studies on meiotic chromosome structure indicate that chromosome organization is not only important for chromatin packaging but also fulfills specific functions in facilitating chromosome interactions during meiosis, including pairing and recombination.

The transcriptome landscape of early maize meiosis.

BackgroundA major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq.ResultsWe detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis.ConclusionsTaken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.

Telomeres act autonomously in maize to organize the meiotic bouquet from a semipolarized chromosome orientation.

During meiosis, chromosomes undergo large-scale reorganization to allow pairing between homologues, which is necessary for recombination and segregation. In many organisms, pairing of homologous chromosomes is accompanied, and possibly facilitated, by the bouquet, the clustering of telomeres in a small region of the nuclear periphery. Taking advantage of the cytological accessibility of meiosis in maize, we have characterized the organization of centromeres and telomeres throughout meiotic prophase. Our results demonstrate that meiotic centromeres are polarized prior to the bouquet stage, but that this polarization does not contribute to bouquet formation. By examining telocentric and ring chromosomes, we have tested the cis-acting requirements for participation in the bouquet. We find that: (a) the healed ends of broken chromosomes, which contain telomere repeats, can enter the bouquet; (b) ring chromosomes enter the bouquet, indicating that terminal position on a chromosome is not necessary for telomere sequences to localize to the bouquet; and (c) beginning at zygotene, the behavior of telomeres is dominant over any centromere-mediated chromosome behavior. The results of this study indicate that specific chromosome regions are acted upon to determine the organization of meiotic chromosomes, enabling the bouquet to form despite large-scale changes in chromosome architecture.

Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin.

Meiotic chromosome condensation is a unique process, characterized by dramatic changes in chromosome morphology that are required for the correct progression of pairing, synapsis, recombination and segregation of sister chromatids. We used an antibody that recognizes a ser 10 phosphoepitope on histone H3 to monitor H3 phosphorylation during meiosis in maize meiocytes. H3 phosphorylation has been reported to be an excellent marker for chromosome condensation during mitotic prophase in animal cells. In this study, we find that on maize mitotic chromosomes only pericentromeric regions are stained; there is little staining on the arms. During meiosis, chromosome condensation from leptotene through diplotene occurs in the absence of H3 phosphorylation. Instead, the changes in H3 phosphorylation at different stages of meiosis correlate with the differences in requirements for sister chromatid cohesion at different stages. Just before nuclear envelope breakdown, histone H3 phosphorylation is seen first in the pericentromeric regions and then extends through the arms at metaphase I; at metaphase II only the pericentromeric regions are stained. In afd1 (absence of first division), a mutant that is defective in many aspects of meiosis including sister chromatid cohesion and has equational separation at metaphase I, staining is restricted to the pericentromeric regions during metaphase I and anaphase I; there is no staining at metaphase II or anaphase II. We conclude that changes in the level of phosphorylation of ser10 in H3 correspond to changes in the cohesion of sister chromatids rather than the extent of chromosome condensation at different stages of meiosis.

Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase.

An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to approximately 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination.

The role of the ameiotic1 gene in the initiation of meiosis and in subsequent meiotic events in maize.

Understanding the initiation of meiosis and the relationship of this event with other key cytogenetic processes are major goals in studying the genetic control of meiosis in higher plants. Our genetic and structural analysis of two mutant alleles of the ameiotic1 gene (am1 and am1-praI) suggest that this locus plays an essential role in the initiation of meiosis in maize. The product of the ameiotic1 gene affects an earlier stage in the meiotic sequence than any other known gene in maize and is important for the irreversible commitment of cells to meiosis and for crucial events marking the passage from premeiotic interphase into prophase I including chromosome synapsis. It appears that the period of ameiotic1 gene function in meiosis at a minimum covers the interval from some point during premeiotic interphase until the early zygotene stage of meiosis. To study the interaction of genes in the progression of meiosis, several double meiotic mutants were constructed. In these double mutants (i) the ameiotic1 mutant allele was brought together with the meiotic mutation (afd1) responsible for the fixation of centromeres in meiosis; and with the mutant alleles of the three meiotic genes that control homologous chromosome segregation (dv1, ms43 and ms28), which impair microtubule organizing center organization, the orientation of the spindle fiber apparatus, and the depolymerization of spindle filaments after the first meiotic division, respectively; (ii) the afd1 mutation was combined with two mutations (dsy1 and as1) affecting homologous pairing; (iii) the ms43 mutation was combined with the as1, the ms28 and the dv1 mutations; and (iv) the ms28 mutation was combined with the dv1 mutation and the ms4 (polymitotic1) mutations. An analysis of gene interaction in the double mutants led us to conclude that the ameiotic1 gene is epistatic over the afd1, the dv1, the ms43 and the ms28 genes but the significance of this relationship requires further analysis. The afd gene appears to function from premeiotic interphase throughout the first meiotic division, but it is likely that its function begins after the start of the ameiotic1 gene expression. The afd1 gene is epistatic over the two synaptic mutations dsy1 and as1 and also over the dv1 mutation. The new ameiotic*-485 and leptotene arrest*-487 mutations isolated from an active Robertson's Mutator stocks take part in the control of the initiation of meiosis.