RESUMEN
Meiotic rapid prophase chromosome movements (RPMs) require connections between the chromosomes and the cytoskeleton, involving SUN (Sad1/UNC-84)-domain-containing proteins at the inner nuclear envelope (NE). RPMs remain significantly understudied in plants, with respect to their importance in the regulation of meiosis. Here, we demonstrate that Arabidopsis thaliana meiotic centromeres undergo rapid (up to 500 nm/s) and uncoordinated movements during the zygotene and pachytene stages. These centromere movements are not affected by altered chromosome organization and recombination but are abolished in the double mutant sun1 sun2. We also document the changes in chromosome dynamics and nucleus organization during the transition from leptotene to zygotene, including telomere attachment to SUN-enriched NE domains, bouquet formation, and nucleolus displacement, all of which were defective in sun1 sun2. These results establish A. thaliana as a model species for studying the functional implications of meiotic RPMs and demonstrate the mechanistic conservation of telomere-led RPMs in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Cromosomas de las Plantas , Meiosis , Membrana Nuclear , Telómero , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cromosomas de las Plantas/genética , Telómero/metabolismo , Centrómero/metabolismo , Profase , Profase Meiótica I , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMEN
The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.
Asunto(s)
Arabidopsis/citología , Arabidopsis/genética , Genes Esenciales , Genes de Plantas/genética , Células Germinativas/metabolismo , Alelos , Proteínas de Arabidopsis/metabolismo , División Celular , Supervivencia Celular/genética , Evolución Molecular , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Células Germinativas/citología , Meiosis , Familia de Multigenes , Especificidad de Órganos , Polen/crecimiento & desarrollo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Semillas , Transgenes , Proteína de Unión al GTP ran/metabolismoRESUMEN
[This corrects the article DOI: 10.3389/fpls.2018.00368.].
RESUMEN
Chromosome distribution at anaphase of mitosis and meiosis is triggered by separase, an evolutionarily conserved protease. Separase must be tightly regulated to prevent the untimely release of chromatid cohesion and disastrous chromosome distribution defects. Securin is the key inhibitor of separase in animals and fungi, but has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologs of securin. Disruption of PANS1 is known to lead to the premature separation of chromosomes at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we show that PANS1 targeting by the anaphase-promoting complex is required to trigger chromosome separation, mirroring the regulation of securin. We showed that PANS1 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we identified SEPARASE mutants, showing that PANS1 and SEPARASE have antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 and PANS2 act as a plant securin. Remote sequence similarity was identified between the plant patronus family and animal securins, suggesting that they indeed derive from a common ancestor. Identification of patronus as the elusive plant securin illustrates the extreme sequence divergence of this central regulator of mitosis and meiosis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Cromosomas de las Plantas/metabolismo , Securina/metabolismo , Separasa/metabolismo , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Cromosomas de las Plantas/genética , Secuencia Conservada , Regulación de la Expresión Génica de las Plantas , Meiosis , Mutación/genética , Unión Proteica , Factores de TiempoRESUMEN
Genetic screens have been crucial for deciphering many important biological processes, including meiosis. In Arabidopsis thaliana, previous forward screens have likely identified almost all the meiotic genes that when mutated lead to a pronounced decrease in fertility. However, the increasing number of genes identified in reverse genetics studies that play crucial roles in meiosis, but do not exhibit strong phenotypes when mutated, suggests that there are still many genes with meiotic function waiting to be discovered. In this study, we produced 897 A. thaliana homozygous mutant lines using Ethyl Methyl Sulfonate (EMS) mutagenesis followed by either single seed descent or haploid doubling. Whole genome sequencing of a subset of lines showed an average of 696 homozygous mutations per line, 195 of which (28%) modify a protein sequence. To test the power of this library, we carried out a forward screen looking for meiotic defects by observing chromosomes at metaphase I of male meiosis. Among the 649 lines analyzed, we identified 43 lines with meiotic defects. Of these, 21 lines had an obvious candidate causal mutation, namely a STOP or splicing site mutation in a gene previously shown to play a role in meiosis (ATM, MLH3, MLH1, MER3, HEI10, FLIP, ASY4, FLIP, PRD2, REC8, FANCL, and PSS1). Interestingly, this was the first time that six of these genes were identified in a forward screen in Arabidopsis (MLH3, MLH1, SGO1, PSS1, FANCL, and ASY4). These results illustrate the potential of this mutant population for screening for any qualitative or quantitative phenotype. Thus, this new mutant library is a powerful tool for functional genomics in A. thaliana. The HEM (Homozygote EMS Mutants) lines are available at the Versailles Arabidopsis stock center.
RESUMEN
Meiotic crossovers (COs) are essential for proper chromosome segregation and the reshuffling of alleles during meiosis. In WT plants, the number of COs is usually small, which limits the genetic variation that can be captured by plant breeding programs. Part of this limitation is imposed by proteins like FANCM, the inactivation of which results in a 3-fold increase in COs in Arabidopsis thaliana. Whether the same holds true in crops needed to be established. In this study, we identified EMS induced mutations in FANCM in two species of economic relevance within the genus Brassica. We showed that CO frequencies were increased in fancm mutants in both diploid and tetraploid Brassicas, Brassica rapa and Brassica napus respectively. In B. rapa, we observed a 3-fold increase in the number of COs, equal to the increase observed previously in Arabidopsis. In B. napus we observed a lesser but consistent increase (1.3-fold) in both euploid (AACC) and allohaploid (AC) plants. Complementation tests in A. thaliana suggest that the smaller increase in crossover frequency observed in B. napus reflects residual activity of the mutant C copy of FANCM. Altogether our results indicate that the anti-CO activity of FANCM is conserved across the Brassica, opening new avenues to make a wider range of genetic diversity accessible to crop improvement.
RESUMEN
Introduction of clonal reproduction through seeds (apomixis) in crops has the potential to revolutionize agriculture by allowing self-propagation of any elite variety, in particular F1 hybrids. In the sexual model plant Arabidopsis thaliana synthetic clonal reproduction through seeds can be artificially implemented by (i) combining three mutations to turn meiosis into mitosis (MiMe) and (ii) crossing the obtained clonal gametes with a line expressing modified CENH3 and whose genome is eliminated in the zygote. Here we show that additional combinations of mutations can turn Arabidopsis meiosis into mitosis and that a combination of three mutations in rice (Oryza sativa) efficiently turns meiosis into mitosis, leading to the production of male and female clonal diploid gametes in this major crop. Successful implementation of the MiMe technology in the phylogenetically distant eudicot Arabidopsis and monocot rice opens doors for its application to any flowering plant and paves the way for introducing apomixis in crop species.
Asunto(s)
Meiosis/fisiología , Mitosis/fisiología , Oryza/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/clasificación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromosomas de las Plantas/genética , Cromosomas de las Plantas/metabolismo , Diploidia , Genotipo , Mutación , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Fenotipo , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Ciclinas/metabolismo , Meiosis , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromosomas de las Plantas/genética , Ciclinas/genética , Epistasis Genética , Genes Dominantes , Pruebas Genéticas , Modelos Biológicos , Mutación/genética , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismo , Tetraploidía , Tubulina (Proteína)/metabolismoRESUMEN
Meiotic crossovers (COs) shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs) appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Emparejamiento Cromosómico , Cinesinas/metabolismo , Meiosis , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , MutaciónRESUMEN
BACKGROUND: At meiosis, two successive rounds of chromosome segregation lead to ploidy halving. This is achieved through a stepwise release of sister chromatid cohesion, along chromosome arms to allow homolog segregation at anaphase I and at centromeres to allow sister chromatid segregation at anaphase II. Cohesins, the protein complex that ensures cohesion, must then be protected at centromeres throughout meiosis, until the onset of anaphase II. Members of the Shugoshin protein family have been shown to protect centromeric cohesins at anaphase I, but much less is known about the protection of cohesion during interkinesis, the stage between meiosis I and meiosis II. RESULTS: Here, we (1) show that both Arabidopsis SHUGOSHINs paralogs are required for complete protection of centromeric cohesins during meiosis I, without apparent somatic function, and (2) identified PATRONUS (PANS1), a novel protein required for protection of meiotic centromeric cohesion. Although AtSGO1 and AtSGO2 protect centromeric cohesion during anaphase I, PANS1 is required at a later stage, during interkinesis. Additionally, we identified PANS2, a paralog of PANS1, whose mutation is synthetically lethal with pans1 suggesting that PANS genes are also essential for mitosis. PANS1 interacts directly with the CDC27b and the CDC20.1 subunit of the Anaphase Promoting Complex (APC/C), in a manner suggesting that PANS1 could be both a regulator and a target of the APC/C. CONCLUSIONS: This study reveals that centromeric cohesion is actively protected at two successive stages of meiosis, by SHUGOSHINs at anaphase I and by PATRONUS at interkinesis.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Meiosis , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/clasificación , Proteínas de Ciclo Celular/genética , Segregación Cromosómica , Mutación , Reacción en Cadena de la Polimerasa , Alineación de SecuenciaRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is a large multiprotein E3 ubiquitin ligase involved in ubiquitin-dependent proteolysis of key cell cycle regulatory proteins, including the destruction of mitotic cyclins at the metaphase-to-anaphase transition. Despite its importance, the role of the APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, we describe the identification of a plant-specific negative regulator of the APC/C complex, designated SAMBA. In Arabidopsis thaliana, SAMBA is expressed during embryogenesis and early plant development and plays a key role in organ size control. Samba mutants produced larger seeds, leaves, and roots, which resulted from enlarged root and shoot apical meristems, and, additionally, they had a reduced fertility attributable to a hampered male gametogenesis. Inactivation of SAMBA stabilized A2-type cyclins during early development. Our data suggest that SAMBA regulates cell proliferation during early development by targeting CYCLIN A2 for APC/C-mediated proteolysis.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Ciclina A/química , Mutación , Complejos de Ubiquitina-Proteína Ligasa/fisiología , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Ciclo Celular , Regulación de la Expresión Génica de las Plantas , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Fenotipo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Polen/metabolismo , Homología de Secuencia de Aminoácido , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis , Ciclina A1/genética , Ciclinas/genética , Meiosis/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Ciclosoma-Complejo Promotor de la Anafase , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Ciclina A1/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Epistasis Genética , Gametogénesis/genética , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Ratones , Mitosis/genética , Mutación , Oocitos/metabolismo , Fosforilación , Plantas Modificadas Genéticamente , Transducción de Señal , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidoresRESUMEN
Cloning through seeds has potential revolutionary applications in agriculture, because it would allow vigorous hybrids to be propagated indefinitely. However, asexual seed formation or apomixis, avoiding meiosis and fertilization, is not found in the major food crops. To develop de novo synthesis of apomixis, we crossed Arabidopsis MiMe and dyad mutants that produce diploid clonal gametes to a strain whose chromosomes are engineered to be eliminated after fertilization. Up to 34% of the progeny were clones of their parent, demonstrating the conversion of clonal female or male gametes into seeds. We also show that first-generation cloned plants can be cloned again. Clonal reproduction through seeds can therefore be achieved in a sexual plant by manipulating two to four conserved genes.
Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Ingeniería Genética , Semillas/genética , Semillas/fisiología , Segregación Cromosómica/genética , Cromosomas de las Plantas , Cruzamientos Genéticos , Diploidia , Genes de Plantas , Heterocigoto , Histonas/genética , Meiosis/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Reproducción AsexuadaRESUMEN
Arbuscular mycorrhiza (AM) is a root endosymbiosis between plants and glomeromycete fungi. It is the most widespread terrestrial plant symbiosis, improving plant uptake of water and mineral nutrients. Yet, despite its crucial role in land ecosystems, molecular mechanisms leading to its formation are just beginning to be unravelled. Recent evidence suggests that AM fungi produce diffusible symbiotic signals. Here we show that Glomus intraradices secretes symbiotic signals that are a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs), which stimulate formation of AM in plant species of diverse families (Fabaceae, Asteraceae and Umbelliferae). In the legume Medicago truncatula these signals stimulate root growth and branching by the symbiotic DMI signalling pathway. These findings provide a better understanding of the evolution of signalling mechanisms involved in plant root endosymbioses and will greatly facilitate their molecular dissection. They also open the way to using these natural and very active molecules in agriculture.
Asunto(s)
Lipopolisacáridos/metabolismo , Micorrizas/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Simbiosis , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Daucus carota/química , Daucus carota/metabolismo , Daucus carota/microbiología , Glomeromycota/metabolismo , Lipopolisacáridos/química , Medicago truncatula/química , Medicago truncatula/crecimiento & desarrollo , Medicago truncatula/metabolismo , Medicago truncatula/microbiología , Datos de Secuencia Molecular , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Raíces de Plantas/química , Raíces de Plantas/crecimiento & desarrollo , Transducción de Señal , Esporas Fúngicas/química , Esporas Fúngicas/metabolismoRESUMEN
Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions.