ABSTRACT
The plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and regulates meiosis. Here, we reveal the molecular mechanism underlying this reprogramming. We demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by 24-nucleotide small interfering RNAs (siRNAs) transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) through activity of CLSY3, a chromatin remodeler absent in other anther cells. Tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Tapetum-derived siRNAs also silence germline transposons, safeguarding genome integrity. Our results reveal that tapetal siRNAs are sufficient to reconstitute germline methylation patterns and drive functional methylation reprogramming throughout the male germline.
Subject(s)
Arabidopsis/cytology , Arabidopsis/genetics , Epigenesis, Genetic , Paternal Inheritance , Pollen/genetics , RNA, Small Interfering/genetics , DNA Methylation , Meiosis/genetics , Mitosis/geneticsABSTRACT
B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.
Subject(s)
Chromosomes, Plant/genetics , Evolution, Molecular , Pollen/genetics , Pregnancy Proteins/genetics , Zea mays/genetics , Meiosis/genetics , Mitosis/geneticsABSTRACT
Because zinc sulfate (ZnSO4) is widely used in many fields such as biomedicine, electronics, and chemistry, it is important to evaluate its toxic effects. In this study, the cyto-genotoxic effects of ZnSO4 on meristematic cells in the root tip of Allium cepa L. were investigated. After calculating the effective concentration (EC50 = 70 ppm) of ZnSO4, A. cepa root tip cells were suspended for 24, 48, 72, and 96 h in solutions of 35 ppm (EC50/2), 70 ppm (EC50), and 140 ppm (EC50 × 2) concentrations. Using the counts of dividing cells, the mitotic index (MI) was calculated. Chromosome aberration index (CAI) was determined from percentages of abnormal cells. When the obtained data were statistically evaluated, it was determined that all application concentrations caused a significant decrease in MI and an increase in CAI compared to the control group (distilled water). It was concluded that increased ZnSO4 dose concentrations and exposure times caused cytotoxicity and genotoxicity in the root cells of A. cepa L.
Subject(s)
Chromosome Aberrations/chemically induced , Meristem/drug effects , Mitosis/drug effects , Onions/drug effects , Onions/growth & development , Onions/genetics , Plant Roots/drug effects , Zinc Sulfate/toxicity , Adult , Cytotoxins/toxicity , Female , Humans , Male , Maximum Tolerated Dose , Meristem/genetics , Meristem/growth & development , Middle Aged , Mitosis/genetics , Mutagens/toxicity , Occupational Diseases/chemically induced , Occupational Exposure , Plant Roots/genetics , Plant Roots/growth & development , Risk AssessmentABSTRACT
Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.
Subject(s)
DNA Replication/genetics , Nuclear Pore/pathology , Telomere-Binding Proteins/genetics , Telomere/genetics , Cell Line, Tumor , DNA Damage/genetics , Humans , Mitosis/genetics , Mutation , Neoplasms/genetics , Neoplasms/physiopathology , Shelterin Complex , Telomere/metabolism , Telomere-Binding Proteins/metabolismABSTRACT
Dimethyl fumarate (DMF) has emerged as a first-line treatment for the relapsing-remitting multiple sclerosis (RRMS) subtype. It is hypothesized that DMF has anti-inflammatory and antioxidant effects although mechanisms are not fully understood. This study used RNA-seq to profile gene expression responses to DMF in cultured astrocytes. Responses were compared with those of isosorbide di-(methyl fumarate) (IDMF), a newly designed fumarate that may partially replicate DMF activity with fewer adverse effects. Both compounds altered the expression of MS-associated genes, including those near MS susceptibility loci and genes dysregulated in MS patient astrocytes. The shared DMF/IDMF transcriptome response involved altered expression of antioxidant genes (e.g., HMOX1) and genes linked to extracellular matrix integrity (TIMP3, MMP9) and migration of pro-inflammatory cells into CNS (CCL2). IDMF-specific transcriptome responses included down-regulation of mitotic genes associated with a proliferative reactive astrocyte phenotype (ICAM1) and repression of genes encoding NF-kappaB subunits (NFKB2, RELA, RELB) and NF-kappaB targets (NCAPG, CXCL1, OAS3). Overall, these results identify astrocyte-centered mechanisms that may contribute to the established efficacy of DMF as an RRMS treatment. Furthermore, our findings support a rationale for further studies of IDMF as a novel fumarate, which may have unique suppressive effects on astrocyte reactivity and glial scar formation. [200 words].
Subject(s)
Astrocytes/drug effects , Dimethyl Fumarate/analogs & derivatives , Astrocytes/metabolism , Cells, Cultured , Dimethyl Fumarate/pharmacology , Down-Regulation/drug effects , Drug Evaluation, Preclinical , Genetic Predisposition to Disease , Humans , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase Inhibitors/pharmacology , Mitosis/drug effects , Mitosis/genetics , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Multiple Sclerosis, Relapsing-Remitting/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , Phenotype , Protein Biosynthesis/drug effects , Transcriptome/drug effectsABSTRACT
Circadian control of cell division is well established in diverse organisms. Recent single-cell studies on mouse fibroblasts have shown that the circadian clock and cell cycle systems are robustly phase-coupled in a bidirectional manner. In healthy cells, coupling of clock and cell cycle results in timed mitosis and rhythmic DNA replication. However, little is known about the interplay between these two oscillators in cancer cells, which often display de-regulated cell proliferation and circadian gene expression. Here we review the molecular organization of the circadian clock and the cell cycle, as well as the reciprocal interaction between the circadian clock and the cell cycle in normal and in cancer cells. Understanding how the circadian clock and cell cycle are coupled in cancer cells will be instrumental to optimally take advantage of chronotherapy in cancer treatment, as efficiency of therapy benefits from asynchrony in timed mitosis between the host and the malignant cells in order to predict the optimal time of treatment.
Subject(s)
Cell Cycle/genetics , Circadian Clocks/genetics , Circadian Rhythm/genetics , Mitosis/genetics , Animals , Cell Proliferation/genetics , DNA Replication/genetics , Humans , Mice , Single-Cell AnalysisABSTRACT
Signalling events through small peptides are essential in multiple aspects of plant reproduction. The ScRALF3 Solanum chacoense Rapid Alkalinization Factor (RALF) peptide was previously shown to regulate multiple aspects of cell-cell communication between the surrounding sporophytic tissue and the female gametophyte during ovule development. We analysed the global expression pattern of ScRALF3 with GUS reporter gene under control of the ScRALF3 promoter and validated it with in situ hybridisation. To better understand the role of ScRALF3 we used three different RNA interference (RNAi) lines that reduced the expression of ScRALF3 during pollen development. Both expression methods showed the presence of ScRALF3 in different tissues, including stigma, style, vascular tissues and during stamen development. Down-regulation of ScRALF3 expression through RNAi showed drastic defects in early stages of pollen development, mainly on the first mitosis. These results suggest that the ScRALF3 secreted peptide regulates the transition from sporogenesis to gametogenesis in both male and female gametophytes.
Subject(s)
Gene Expression Regulation, Plant , Germ Cells, Plant , Mitosis , Plant Proteins , Pollen , Signal Transduction , Solanum , Mitosis/genetics , Peptides/metabolism , Plant Proteins/metabolism , Pollen/cytology , Signal Transduction/genetics , Solanum/cytology , Solanum/genetics , Solanum/growth & developmentABSTRACT
Centrosomes and primary cilia are usually considered as distinct organelles, although both are assembled with the same evolutionary conserved, microtubule-based templates, the centrioles. Centrosomes serve as major microtubule- and actin cytoskeleton-organizing centers and are involved in a variety of intracellular processes, whereas primary cilia receive and transduce environmental signals to elicit cellular and organismal responses. Understanding the functional relationship between centrosomes and primary cilia is important because defects in both structures have been implicated in various diseases, including cancer. Here, we discuss evidence that the animal centrosome evolved, with the transition to complex multicellularity, as a hybrid organelle comprised of the two distinct, but intertwined, structural-functional modules: the centriole/primary cilium module and the pericentriolar material/centrosome module. The evolution of the former module may have been caused by the expanding cellular diversification and intercommunication, whereas that of the latter module may have been driven by the increasing complexity of mitosis and the requirement for maintaining cell polarity, individuation, and adhesion. Through its unique ability to serve both as a plasma membrane-associated primary cilium organizer and a juxtanuclear microtubule-organizing center, the animal centrosome has become an ideal integrator of extracellular and intracellular signals with the cytoskeleton and a switch between the non-cell autonomous and the cell-autonomous signaling modes. In light of this hypothesis, we discuss centrosome dynamics during cell proliferation, migration, and differentiation and propose a model of centrosome-driven microtubule assembly in mitotic and interphase cells. In addition, we outline the evolutionary benefits of the animal centrosome and highlight the hierarchy and modularity of the centrosome biogenesis networks.
Subject(s)
Centrosome/metabolism , Centrosome/physiology , Cilia/metabolism , Animals , Cell Cycle , Cell Differentiation , Centrioles/metabolism , Centrioles/physiology , Cilia/genetics , Humans , Microtubule-Organizing Center/physiology , Microtubules/physiology , Mitosis/genetics , Organelles/metabolism , Organelles/physiologyABSTRACT
B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A. speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to > 93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A. speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.
Subject(s)
Aegilops/genetics , Chromosomes, Plant/genetics , Nondisjunction, Genetic , Base Sequence , Cell Nucleus/genetics , Centromere/metabolism , Conserved Sequence/genetics , Mitosis/genetics , Pollen/genetics , Repetitive Sequences, Nucleic Acid/genetics , Secale/genetics , Spindle Apparatus/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation, Plant , Meiosis/genetics , Pollen/cytology , Pollen/growth & development , Zea mays/cytology , Zea mays/growth & development , Cell Differentiation , Mitosis/genetics , Mutation , Pollen/metabolism , Sequence Analysis, RNA , Single-Cell Analysis , Transcription, Genetic , Transcriptome , Zea mays/geneticsABSTRACT
BACKGROUND: Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology. METHODS: Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed. RESULTS: Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions. CONCLUSION: All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.
Subject(s)
Blood Platelets/metabolism , Cell Differentiation , Mitosis , Up-Regulation , Adipose Tissue, White/cytology , Bone Marrow Cells/cytology , Cell Differentiation/genetics , Cell Proliferation , Cytokines/metabolism , Humans , Immunophenotyping , Intercellular Signaling Peptides and Proteins/metabolism , Kruppel-Like Factor 4 , Mitosis/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stromal Cells/cytology , Stromal Cells/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Umbilical Cord/cytology , Up-Regulation/geneticsABSTRACT
Three-dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining or mass spectrometry, applied to 2D cell culture with limited physiological relevance or tissue sections. Here, we present 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening. This workflow comprises iterative antibody staining, high-content 3D imaging, and machine learning for detection of mitoses. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, a basis for model-based prediction of spatially resolved affinities of proteins. As a proof-of-concept, we mapped twelve epitopes in 3D-cultured spheroids and investigated the network effects of twelve mitotic cancer drugs. Our approach reveals novel insights into spindle fragility and chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways. 3D SPECS's ability to map potential drug targets by multiplexed immunofluorescence in 3D cell culture combined with our automated high-content assay will inspire future functional protein expression and drug assays.
Subject(s)
Drug Screening Assays, Antitumor , Epitopes/genetics , Mitosis/genetics , Proteins/genetics , Cell Line, Tumor , Drug Evaluation, Preclinical/methods , Epitopes/immunology , Gene Expression Regulation/drug effects , High-Throughput Screening Assays , Humans , Proteins/drug effectsABSTRACT
Currently, more than half of newly diagnosed cancer patients receive radiation treatment. However, the radioresistance of tumor cells as well as the early and late side effects limit the beneficial outcome of radiotherapy. Accordingly, the innovative approaches to maximize tumor killing and/or minimize radiation toxicity remain a major focus of interest. In the past decade, several pieces of evidence have shown the importance of different modes of regulated cell death (RCD) in the radioresponse of malignant and normal tissues. Furthermore, the biological modulation of radiation-induced RCDs has come to attention as a novel therapeutic means. Here, we review the major signaling pathways that orchestrate all types of RCD initiated by exposure to ionizing radiation. The latest advances in the development of small-molecule RCD modulators (both natural and synthetic) that are intended for widening the therapeutic window of radiotherapy are also discussed.
Subject(s)
Cell Death/radiation effects , Radiation, Ionizing , Animals , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/immunology , Apoptosis/radiation effects , Autophagy/drug effects , Autophagy/genetics , Autophagy/immunology , Autophagy/radiation effects , Cell Death/drug effects , Cell Death/genetics , Cell Death/immunology , Cell Line, Tumor , Cellular Senescence , Drug Evaluation, Preclinical , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Mitosis/drug effects , Mitosis/genetics , Mitosis/radiation effects , Necrosis/drug therapy , Necrosis/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/radiotherapy , Radiation-Sensitizing Agents/pharmacology , Radiation-Sensitizing Agents/therapeutic use , Reactive Oxygen Species , Signal Transduction/drug effectsABSTRACT
In angiosperms, the key step in sexual reproduction is successful acquisition of meiotic fate. However, the molecular mechanism determining meiotic fate remains largely unknown. Here, we report that OsSPOROCYTELESS (OsSPL) is critical for meiotic entry in rice (Oryza sativa). We performed a large-scale genetic screen of rice sterile mutants aimed to identify genes regulating meiotic entry and identified OsSPL using map-based cloning. We showed that meiosis-specific callose deposition, chromatin organization, and centromere-specific histone H3 loading were altered in the cells corresponding to pollen mother cells in Osspl anthers. Global transcriptome analysis showed that the enriched differentially expressed genes in Osspl were mainly related to redox status, meiotic process, and parietal cell development. OsSPL might form homodimers and interact with TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factor OsTCP5 via the SPL dimerization and TCP interaction domain. OsSPL also interacts with TPL (TOPLESS) corepressors, OsTPL2 and OsTPL3, via the EAR motif. Our results suggest that the OsSPL-mediated signaling pathway plays a crucial role in rice meiotic entry, which appears to be a conserved regulatory mechanism for meiotic fate acquisition in angiosperms.
Subject(s)
Meiosis , Oryza/cytology , Oryza/metabolism , Plant Proteins/metabolism , Arabidopsis Proteins/metabolism , Cell Differentiation/genetics , Gametogenesis, Plant/genetics , Gene Expression Regulation, Plant , Genes, Plant , Mitosis/genetics , Models, Biological , Mutation/genetics , Nuclear Proteins/metabolism , Oryza/genetics , Oxidation-Reduction , Phylogeny , Plant Proteins/genetics , Pollen/cytology , Pollen/metabolism , Protein Binding , Protein Multimerization , Transcription, GeneticABSTRACT
In flowering plants, germ lines are induced from somatic meristems within reproductive organs. Within anthers, germinal cell initials first undergo several rounds of mitotic proliferation before synchronously entering meiosis. Our understanding of the progression and the molecular basis of this mitosis to meiosis transition is still limited. Taking advantage of the correlation between anther length and premeiotic germinal cell development in maize (Zea mays), we studied the transcriptome dynamics of germinal cells at three sequential stages, mitotic archesporial cells, enlarging pollen mother cells at the premeiosis interphase, and pollen mother cells at the early prophase of meiosis, using laser microdissection-based expression profiling. Our analysis showed that cells undergoing the mitosis-meiosis switch exhibit robust transcriptional changes. The three stages are distinguished by the expression of genes encoding transcription factor subsets, meiotic chromosome recombination proteins, and distinct E3 ubiquitin ligases, respectively. The transcription level of genes encoding protein turnover machinery was significantly higher in these three stages of germinal cells than in mature pollen, parenchyma cells, or seedlings. Our experimental results further indicate that many meiotic genes are not only transcribed, but also translated prior to meiosis. We suggest that the enlarging pollen mother cells stage represents a crucial turning point from mitosis to meiosis for developing germinal cells.
Subject(s)
Gene Expression Regulation, Plant , Meiosis/genetics , Mitosis/genetics , Plant Proteins/genetics , Transcriptome , Zea mays/genetics , Flowers/genetics , Flowers/growth & development , Flowers/physiology , Gene Expression Regulation, Developmental , Germ Cells, Plant , Meristem/genetics , Meristem/growth & development , Meristem/physiology , Pollen/genetics , Pollen/growth & development , Pollen/physiology , Transcription Factors/genetics , Zea mays/growth & development , Zea mays/physiologyABSTRACT
Fifty years ago, Lynn Margulis proposed a comprehensive hypothesis on the origin of eukaryotic cells with an emphasis on the origin of mitosis. This hypothesis postulated that the eukaryotic cell is a composite of different parts as a result of the symbiosis of various different bacteria. In this hypothesis, she integrated previously proposed ideas that mitochondria and chloroplasts were descendants of endosymbionts that originated from aerobic bacteria and blue-green algae (now cyanobacteria), respectively. However, the major part of her hypothesis, which she believed to be original, was the origin of mitosis. The core of her postulate involved a chromosome partition mechanism dependent on DNA-microtubule binding, which originated from a hypothetical centriole-DNA complex, with an ability to replicate. Surprisingly, her complete lack of real experimental works in the cytoskeleton, cell motility, or paleontology did not prevent this 29-year-old junior scientist from assembling archival knowledge and constructing a narrative on the evolution of all organisms. Whether the centriole-DNA complex originated from a spirochete or not was a minor anecdote in this initial postulate. Unfortunately, this hypothesis on the origin of mitosis, which she believed to be a holistic unity, testable by experiments, was entirely refuted. Despite falsification of her original narrative as a whole, her success as a founder of endosymbiotic theory on the origin of mitochondria and chloroplasts is undoubted. We will discuss the reasons for her success in terms of the historical situation in the latter half of the 20th century.
Subject(s)
Mitosis/genetics , Models, Theoretical , Symbiosis , Chromosomes , DNA Replication , History, 20th Century , PlastidsABSTRACT
In order to study the function of kinesin-14 motor protein KIFC1 during spermatogenesis of Procambarus clarkii, the full length of kifc1 was cloned from testes cDNA using Rapid-Amplification of cDNA Ends (RACE). The deduced KIFC1 protein sequence showed the highest similarity between Procambarus clarkii and Eriocheir senensis (similarity rate as 64%). According to the results of in situ hybridization (ISH), the kifc1 mRNA was gathered in the acrosome location above nucleus in the mid- and late-stage spermatids. Immunofluorescence results were partly consistent with the ISH in middle spermatids, while in the late spermatids the KIFC1 was distributed around the nucleus which had large deformation and formed four to six nuclear arms. In the mature sperm, KIFC1 and microtubules were distributed around the sperm, playing a role in maintaining the sperm morphology and normal function. Overexpression of P. clarkii kifc1 in GC1 cells for 24 hours resulted in disorganization of microtubules which changed the cell morphology from circular and spherical into fusiform. In addition, the overexpression also resulted in triple centrosomes during mitosis which eventually led to cell apoptosis. RNAi experiments showed that decreased KIFC1 protein levels resulted in total inhibition of spermatogenesis, with only mature sperm found in the RNAi-testis, implying an indispensable role of KIFC1 during P. clarkii spermiogenesis.
Subject(s)
Acrosome/physiology , Arthropod Proteins/genetics , Cell Nucleus/metabolism , Kinesins/genetics , Nephropidae/physiology , Spermatogenesis , Animals , Apoptosis , Arthropod Proteins/metabolism , Cells, Cultured , Cloning, Molecular , Humans , Kinesins/metabolism , Male , Microtubules/metabolism , Mitosis/genetics , RNA, Small Interfering/genetics , Sequence Homology, Amino AcidABSTRACT
Epigenetic reprogramming occurring during reproduction is crucial for both animal and plant development. Histone H3 Lys 4 trimethylation (H3K4me3) is an evolutionarily conserved epigenetic mark of transcriptional active euchromatin. While much has been learned in somatic cells, H3K4me3 deposition and function in gametophyte is poorly studied. Here, we demonstrate that SET DOMAIN GROUP2 (SDG2)-mediated H3K4me3 deposition participates in epigenetic reprogramming during Arabidopsis male gametogenesis. We show that loss of SDG2 barely affects meiosis and cell fate establishment of haploid cells. However, we found that SDG2 is critical for postmeiotic microspore development. Mitotic cell division progression is partly impaired in the loss-of-function sdg2-1 mutant, particularly at the second mitosis setting up the two sperm cells. We demonstrate that SDG2 is involved in promoting chromatin decondensation in the pollen vegetative nucleus, likely through its role in H3K4me3 deposition, which prevents ectopic heterochromatic H3K9me2 speckle formation. Moreover, we found that derepression of the LTR retrotransposon ATLANTYS1 is compromised in the vegetative cell of the sdg2-1 mutant pollen. Consistent with chromatin condensation and compromised transcription activity, pollen germination and pollen tube elongation, representing the key function of the vegetative cell in transporting sperm cells during fertilization, are inhibited in the sdg2-1 mutant. Taken together, we conclude that SDG2-mediated H3K4me3 is an essential epigenetic mark of the gametophyte chromatin landscape, playing critical roles in gamete mitotic cell cycle progression and pollen vegetative cell function during male gametogenesis and beyond.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , Chromatin/metabolism , Gametogenesis, Plant , Histones/metabolism , Lysine/metabolism , Mitosis , Arabidopsis/genetics , Cell Nucleus/metabolism , Gametogenesis, Plant/genetics , Gene Expression Regulation, Plant , Germination/genetics , Heterochromatin/metabolism , Meiosis/genetics , Methylation , Mitosis/genetics , Mutation/genetics , Pollen/growth & development , Pollen/metabolism , Pollen Tube/genetics , Pollen Tube/growth & development , Retroelements/geneticsABSTRACT
KEY MESSAGE: Phenotype identification, expression examination, and function prediction declared that the anther-preferential expressing gene PMR may participate in regulation of male gametophyte development in rice. Male germline development in flowering plants produces the pair of sperm cells for double fertilization and the pollen mitosis is a key process of it. Although the structural features of male gametophyte have been defined, the molecular mechanisms regulating the mitotic cell cycle are not well elucidated in rice. Here, we reported an anther-preferential expressing gene in rice, PMR (Pollen Mitosis Relative), playing an essential role in male gametogenesis. When PMR gene was suppressed via RNAi, the mitosis of microspore was severely damaged, and the plants formed unmatured pollens containing only one or two nucleuses at the anthesis, ultimately leading to serious reduction of pollen fertility and seed-setting. The CRISPR mutants, pmr-1 and pmr-2, both showed the similar defects as the PMR-RNAi lines. Further analysis revealed that PMR together with its co-expressing genes were liable to participate in the regulation of DNA metabolism in the nucleus, and affected the activities of some enzymes related to the cell cycle. We finally discussed that unknown protein PMR contained the PHD, SWIB and Plus-3 domains and they might have coordinating functions in regulation pathway of the pollen mitosis in rice.
Subject(s)
Flowers/metabolism , Flowers/physiology , Mitosis/physiology , Oryza/metabolism , Oryza/physiology , Pollen/physiology , Cell Cycle/genetics , Cell Cycle/physiology , Flowers/growth & development , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Mitosis/genetics , Oryza/growth & development , Plant Proteins/genetics , Plant Proteins/physiology , Pollen/genetics , Pollen/growth & developmentABSTRACT
Wild Helianthus species are a valuable genetic resource for the improvement of cultivated sunflower. We report the discovery and characterization of a unique high frequency production of triploids when cultivated sunflower was pollinated by specific accessions of diploid Helianthus nuttallii T. & G. and H. maximiliani Schr. Genomic in situ hybridization (GISH) analyses indicated that the triploid F1s had two genomes from the wild pollen sources and one from the cultivated line. Mitotic chromosome analyses indicated that the frequency of triploid progenies from the crosses of cultivated lines × H. nuttallii accession 102 (N102) was significantly higher than those of unexpected polyploid progenies from the crosses of wild perennial species × N102, and no unexpected polyploids were obtained from the reverse crosses. Pollen stainability analysis suggested the existence of a low percentage of unreduced (2n) male gametes in some accessions, especially N102 and H. maximiliani accession 1113 (M1113), which were generated at the telophase II and tetrad stages of meiosis. The triploid F1s could be the results of preferred fertilization of the low frequency of 2n male gametes with the female gametes of the cultivated sunflower, due to the dosage factors related to recognition and rejection of foreign pollen during fertilization. The triploids have been used to produce amphiploids and aneuploids. Future studies of the male gametes' fate from pollination through fertilization will further uncover the mechanism of this whole genome transmission. Studies of the genetic control of this trait will facilitate research on sunflower polyploidy speciation and evolution, and the utilization of this trait in sunflower breeding.