Lebendzellbildgebung von Meiose in Antheren von Arabidopsis thaliana

Meiosis is a crucial event for sexual reproduction; during its course the chromosome number is halved, and recombination between homologs takes place. Understanding how meiosis is regulated in plants has a direct impact on breeding applications and, therefore, researchers invest constant effort in studying its fundamental aspects. Extensive knowledge about the meiotic progression results from the cytological analysis of fixed material. Although highly informative, this approach is not sufficient to understand the dynamics of meiosis; numerous works have demonstrated that key meiotic events as homologs paring and segregation are heavily dependent on chromosome movements and cytoskeleton rearrangements, underpinning the need of a spatiotemporal description of the cell division. This dissertation introduces a live cell imaging technique, based on confocal microscopy, which allows the observation of the entire meiotic division of pollen mother cells of Arabidopsis thaliana. In this study, the behavior of single meiocytes is monitored throughout the progression of meiosis by the simultaneous visualization of the meiotic subunit of cohesin, RECOMBINATION 8 (REC8), and microtubules. The double reporter line, named KINGBIRD (Kleisin IN Green, tuBulin In ReD), allows the description of five cellular features: cell shape, nucleus position, nucleolus position, chromosome conformation, and microtubule array. These features combine in a non-random manner to form cellular states; the analysis here performed led to the identification of 11 principal states, referred to as landmarks, which are convergent points of the meiotic progression. Using the landmark system as a reference, it was possible to describe a precise time course of meiosis, which included the duration of short and asynchronous phases, such as metaphases and anaphases. Taken together, the here established microscopy technique and landmark system constitute a novel approach, which opens new ways to the study of plant meiosis.Die Meiose ist ein essentieller Schritt der sexuellen Fortpflanzung; während ihres Verlaufs wird die Chromosomenzahl halbiert und eine Rekombination zwischen homologen Chromosomen ermöglicht. Unser Verständnis der Regulation der Meiose in Pflanzen ist für die Pflanzenzüchtung von direktem Interesse, weshalb große Anstrengungen unternommen werden, die grundlegenden Abläufe zu verstehen. Aus der zytologischen Analyse von fixiertem Material wurde bereits umfangreiches Wissen über den grundsätzlichen Ablauf der Meiose gewonnen. Obwohl sehr informativ, reicht dieser Ansatz aber nicht aus, um die Dynamik der Meiose im Detail zu verstehen. Zahlreiche Arbeiten haben gezeigt, dass meiotische Schlüsselereignisse wie Paarung der Homologen und deren Segregation stark von Chromosomenbewegungen und Zytoskelettumlagerungen abhängen, was die Notwendigkeit einer genauen räumlich-zeitlichen Beschreibung der meiotischen Zellteilung untermauert. Mit dieser Dissertation wird eine Technik zur Lebendzellbeobachtung während der Meiose eingeführt, die auf konfokaler Lasermikroskopie basiert und die Beobachtung des gesamten Ablaufs der meiotischen Teilung der Pollenmutterzellen von Arabidopsis thaliana ermöglicht Durch eine gleichzeitige Visualisierung der Mikrotubuli und der meiotischen Untereinheit von Kohäsin, RECOMBINATION 8 (REC8), kann die Entwicklung einzelner Meiozyten während des Verlaufs der Meiose genau mitverfolgt werden. Die hierfür konstruierte zweifache Reporterlinie namens KINGBIRD (Kleisin IN Green, tuBulin In ReD) ermöglicht die Beschreibung von fünf Zellmerkmalen: Zellform, Position des Zellkerns, Position des Nucleolus im Zellkern, Chromosomenkonformation und die Anordnung der Mikrotubuli. Die spezifische Kombination dieser Merkmale charakterisiert jeweils bestimmte meiotische Stadien. Die hier durchgeführte Analyse führte zur Identifizierung von 11 Hauptzuständen, sogenannten Referenzpunkten, die konvergente Punkte des meiotischen Ablaufs darstellen. Mit Hilfe des Referenzpunkt-Systems konnte ein genauer zeitlicher Verlauf der Meiose beschrieben werden, der es nun ermöglicht, auch die Dauer kurzer und asynchroner Phasen, wie Metaphase und Anaphase, präzise zu erfassen. Die hier etablierte mikroskopische Technik zur Lebendbeobachtung und das Referenzpunkt-System stellen einen innovativen Ansatz dar, der es ermöglicht, neue Wege in der Erforschung der Meiose in Pflanzen zu gehen

Caught in the Act: Live-Cell Imaging of Plant Meiosis

Live-cell imaging is a powerful method to obtain insights into cellular processes, particularly with respect to their dynamics. This is especially true for meiosis, where chromosomes and other cellular components such as the cytoskeleton follow an elaborate choreography over a relatively short period of time. Making these dynamics visible expands understanding of the regulation of meiosis and its underlying molecular forces. However, the analysis of meiosis by live-cell imaging is challenging; specifically in plants, a temporally resolved understanding of chromosome segregation and recombination events is lacking. Recent advances in live-cell imaging now allow the analysis of meiotic events in plants in real time. These new microscopy methods rely on the generation of reporter lines for meiotic regulators and on the establishment of ex vivo culture and imaging conditions, which stabilize the specimen and keep it alive for several hours or even days. In this review, we combine an overview of the technical aspects of live-cell imaging in plants with a summary of outstanding questions that can now be addressed to promote live-cell imaging in Arabidopsis and other plant species and stimulate ideas on the topics that can be addressed in the context of plant meiotic recombination.</jats:p

RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis

To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1-dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. One function of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase

International audienceChromosome 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

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase.

Accurate chromosome segregation at mitosis and meiosis is crucial to prevent genome instability, birth defect, and cancer. Accordingly, separase, the protease that triggers chromosome distribution, is tightly regulated by a direct inhibitor, the securin. However, securin has not been identified, neither functionnally nor by sequence similarity, in other clades that fungi and animals. This raised doubts about the conservation of this mechanism in other branches of eukaryotes. Here, we identify and characterize the securin in plants. Despite extreme sequence divergence, the securin kept the same core function and is likely a universal regulator of cell division in eukaryotes.journal articl

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase

Significance Accurate chromosome segregation at mitosis and meiosis is crucial to prevent genome instability, birth defect, and cancer. Accordingly, separase, the protease that triggers chromosome distribution, is tightly regulated by a direct inhibitor, the securin. However, securin has not been identified, neither functionnally nor by sequence similarity, in other clades that fungi and animals. This raised doubts about the conservation of this mechanism in other branches of eukaryotes. Here, we identify and characterize the securin in plants. Despite extreme sequence divergence, the securin kept the same core function and is likely a universal regulator of cell division in eukaryotes.</jats:p

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase

AbstractChromosome 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 homologues 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.</jats:p

RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis

Germ cells on demand Unlike animals, plants do not set aside a germline. Instead, germ cells are developed on demand from somatic lineages. Zhao et al. examined the regulatory pathways that manage the transition from somatic to germ cell development in the small plant Arabidopsis (see the Perspective by Vielle-Calzada). The transcription factor WUSCHEL (WUS) was needed early on for development of ovules. Soon after, a trio of inhibitors that work through a cyclin-dependent kinase allowed a transcriptional repressor to down-regulate WUS. This opened the door to meiosis, while restricting the number of reproductive units per seed to one. Science , this issue p. eaaf6532 ; see also p. 378 </jats:p