To follow the dynamics of meiosis in the model plant Arabidopsis, we have established a live cell imaging setup to observe male meiocytes. Our method is based on the concomitant visualization of microtubules (MTs) and a meiotic cohesin subunit that allows following five cellular parameters: cell shape, MT array, nucleus position, nucleolus position, and chromatin condensation. We find that the states of these parameters are not randomly associated and identify 11 cellular states, referred to as landmarks, which occur much more frequently than closely related ones, indicating that they are convergence points during meiotic progression. As a first application of our system, we revisited a previously identified mutant in the meiotic A-type cyclin TARDY ASYNCHRONOUS MEIOSIS (TAM). Our imaging system enabled us to reveal both qualitatively and quantitatively altered landmarks in tam, foremost the formation of previously not recognized ectopic spindle- or phragmoplast-like structures that arise without attachment to chromosomes.

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 Retinoblastoma homolog RBR1. In and 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 (), which ectopically accumulates in meiocytes of triple and mutants. Depleting in mutants restored the formation of only a single meiocyte.

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.

2 0 2 1 Chromosome distribution at anaphase of mitosis and meiosis is triggered by 2 2 separase, an evolutionarily conserved protease. Separase must be tightly regulated 2 3 to prevent the untimely release of chromatid cohesion and disastrous chromosome 2 4 distribution defects. Securin is the key inhibitor of separase in animals and fungi, but 2 5has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 2 6 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologues of securin. 7Disruption of PANS1 is known to lead to the premature separation of chromosomes 2 8 at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we 2 9show that PANS1 targeting by the anaphase-promoting-complex is required to trigger 3 0 chromosome separation, mirroring the regulation of securin. We showed that PANS1 3 1 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we 3 2 identified SEPARASE mutants, showing that PANS1 and SEPARASE have 3 3 antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 3 4 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 3 5 and PANS2 act as a plant securin. Remote sequence similarity was identified 3 6 between the plant patronus family and animal securins, suggesting that they indeed 3 7 derive from a common ancestor. Identification of patronus as the elusive plant 3 8 securin illustrates the extreme sequence divergence of this central regulator of 3 9 mitosis and meiosis.4 0 4 1 3

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