During land colonization, plants acquired a range of body plan adaptations, of which the innovation of three-dimensional (3D) tissues increased organismal complexity and reproductivity. In the moss, Physcomitrella patens, a 3D leafy gametophore originates from filamentous cells that grow in a two-dimensional (2D) plane through a series of asymmetric cell divisions. Asymmetric cell divisions that coincide with different cell division planes and growth directions enable the developmental switch from 2D to 3D, but insights into the underlying mechanisms coordinating this switch are still incomplete.Using 2D and 3D imaging and image segmentation, we characterized two geometric cues, the width of the initial cell and the angle of the transition division plane, which sufficiently distinguished a gametophore initial cell from a branch initial cell. These identified cues were further confirmed in gametophore formation mutants.The identification of a fluorescent marker allowed us to successfully predict the gametophore initial cell with > 90% accuracy before morphological changes, supporting our hypothesis that, before the transition division, parental cells of the gametophore initials possess different properties from those of the branch initials.Our results suggest that the cell fate decision of the initial cell is determined in the parental cell, before the transition division.

During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens. Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis. Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labeled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labeled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.

The phytohormone auxin and its directional transport through tissues play a fundamental role in plant development. This polar auxin transport relies on the PIN auxin exporters, whose polarization at the plasma membrane determines the overall direction of auxin flow. Hence, PIN polarization is crucial for development, but its evolution during the rise of developmental complexity in land plants remains unclear. Here, we investigated the evolution of PIN trafficking and polarization by comparing two model bryophytes, Physcomitrella patens and Marchantia polymorpha, with the angiosperm model Arabidopsis thaliana. All examined PINs show a conserved auxin export function, which is reflected by comparable bryophytic growth defects in overexpression lines. In bryophytes, bryophytic PINs polarize to filamentous apices, while Arabidopsis PINs distribute symmetrically on the membrane with additional intracellular puncta. In the Arabidopsis root epidermis, bryophytic PINs show no defined polarity. Pharmacological interference revealed a strong cytoskeleton dependence of bryophytic but not Arabidopsis PIN polarization, which, in contrast, strongly relies on Brefeldin A-sensitive trafficking. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues in individual species. These results collectively reveal a divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and suggest co-evolution of PIN sequence-based and cell-based polarity mechanisms.

Mosses are a cosmopolitan group of land plants, sister to vascular plants, with a high potential for molecular and cell biological research. The species Physcomitrium patens has helped gaining better understanding of the biological processes of the plant cell, and it has become a central system to understand water-to-land plant transition through 2D-to-3D growth transition, regulation of asymmetric cell division, shoot apical cell establishment and maintenance, phyllotaxis and regeneration. P. patens was the first fully sequenced moss in 2008, with the latest annotated release in 2018. It has been shown that many gene functions and networks are conserved in mosses when compared to angiosperms. Importantly, this model organism has a simplified and accessible body structure that facilitates close tracking in time and space with the support of live cell imaging set-ups and multiple reporter lines. This has become possible thanks to its fully established molecular toolkit, with highly efficient PEG-assisted, CRISPR/Cas9 and RNAi transformation and silencing protocols, among others. Here we provide examples on how mosses exhibit advantages over vascular plants to study several processes and their future potential to answer some other outstanding questions in plant cell biology.

MAP65 24 25 Summary statement: We performed a time-resolved localization screen of multiple 26 subunits of the exocyst complex throughout moss cytokinesis and show that each subunit 27 has a unique spatiotemporal recruitment pattern. 28 29 2 Abstract 30 31 During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed 32 from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially 33 restricted to the site of cell plate formation is unknown. Aggregation of cell-plate 34 membrane starts near regions of microtubule overlap within the bipolar phragmoplast 35 apparatus of the moss Physcomitrella patens. Since vesicle fusion generally requires 36 coordination of vesicle tethering and subsequent fusion activity we analysed the 37 subcellular localization of several subunits of the exocyst, a tethering complex active 38 during plant cytokinesis. We found that Sec6, but neither Sec3 or Sec5 subunits localized 39 to microtubule overlap regions in advance of cell plate construction started in moss. 40 Moreover, Sec6 exhibited a conserved physical interaction with an orthologue of the 41 Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle 42 fusion in Arabidopsis. Recruitment of PpKEULE and vesicles to the early cell plate was 43 delayed upon Sec6 gene silencing. Our findings thus suggest that vesicle-vesicle fusion is 44 in part enabled by a pool of exocyst subunits at microtubule overlaps that is recruited 45 independent of the delivery of vesicles. 46 47 56 2014; Boruc and Van Damme, 2015). Adaptations of canonical trafficking mechanisms 57 are however required because there is no pre-existing target membrane at the site of cell 58 3 division to which vesicles can fuse. Instead, membrane deposition is thought to be 59 initiated by 'homotypic' fusion of vesicles (Smertenko et al., 2017). This raises the 60 question of how vesicle fusion is spatially restricted to the site of cell division and not 61 spuriously throughout the cell when vesicles meet. It is thought that vesicles are 62 transported along polarized microtubules to the centre of the phragmoplast, a 63 cytoskeletal apparatus that supports cell plate assembly. Although locally concentrating 64 vesicles may enhance the fusion rates of fusion-competent vesicles, it is unclear whether 65 a transport-based mechanism alone can provide the spatial accuracy required to build a 66 straight and flat cell plate. Recently we identified short stretches of antiparallel 67 microtubule overlap at the midzone of phragmoplasts in the moss Physcomitrella patens 68 as sites where membrane build up is initiated (de Keijzer et al., 2017). It remained 69 however unclear whether there are molecules at overlaps that trigger vesicle fusion 70 locally. 71 In eukaryotic cells the fusion of transport vesicles with endomembrane compartments 72 and the plasma membrane relies on the combined action of fusion and tethering 73 complexes. The force driving the fusion of a vesicle and its destination membrane is 74 almost...

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CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.