# Source paper archive - Source title: The cell polarity landscape in Arabidopsis male and female gametophytes - Author(s): Samuel Koh Wee Han, Monique van Schie, Catherine Albrecht, Joao Ramalho, Bodhi Voss, Dolf Weijers - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.10.30.763139v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.10.30.763139v1.full.pdf - PDF SHA-256: 1a1a033c5142ae4c429370a664bf8fd146ee233532239161ed346913f7b74324 - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 **The cell polarity landscape in Arabidopsis male and female gametophytes** 2 3 Samuel Koh Wee Han [1,] *, Monique van Schie [1,] *, Catherine Albrecht [1], João Jacob Ramalho [1,2] 4 Bodhi Voss [1], and Dolf Weijers [1,#] 5 1, Laboratory of Biochemistry, Wageningen University, Stippeneng 4, 6708WE Wageningen, 6 the Netherlands 7 2, Present address: Institute of Biodynamics and Biocomplexity, Department of Biology, 8 Utrecht University, Padualaan 8, Utrecht, the Netherlands 9 - These authors contributed equally 10 # Corresponding author: dolf.weijers@wur.nl 11 **Abstract** 12 Plant cell polarity underlies the capacity to directionally transport auxin and other small 13 molecules, but also precedes asymmetric cell division in the stomatal lineage. Except for 14 specific contexts, mechanisms underlying cell polarization are poorly understood. Most insights 15 on plant cell polarity have come from studies on flowering plant sporophytes, while no polar 16 protein localization has yet been reported in flowering plant gametophytes, despite clear 17 anisotropic growth and asymmetric cell divisions in this generation. Inspired by the 18 identification of deeply conserved SOSEKI proteins and their associated partners that mark 19 polar domains in bryophyte gametophytes and Arabidopsis sporophytes, we here investigate 20 cell polarity in Arabidopsis gametophytes. We discover a rich landscape of polar domains 21 throughout male and female gametophyte development, marked by one or more polar proteins. 22 We find that successive steps in male gametophyte polarization can be mechanistically 23 uncoupled, and the first polarization step precedes asymmetric cell division. Using ectopic 24 expression, we find that there is a near-universal capacity for polar protein localization in 25 gametophytes through a single polarity system. Our work offers the first insight into 26 gametophyte cell polarity in Arabidopsis, identifies novel polar domains, and presents a starting 27 point for future dissection of mechanisms underlying plant cell polarity. 28 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 29 **Keywords** 30 Gametophyte; cell polarity; Arabidopsis; Marchantia 31 32 **Introduction** 33 34 Cell polarity helps spatially organize cells. While in single-celled organisms, this can aid in cell 35 division, migration or local differentiation (e.g. flagella) (Chiou et al., 2017; Shapiro et al., 36 2009; Treuner-Lange and Søgaard-Andersen, 2014), in multicellular organisms, cell polarity 37 aids coordinated cell behavior and morphogenesis. Polar domains can create cell adhesions, 38 organize directional transport and enable oriented and asymmetric cell division (Knoblich, 39 2010; Rodriguez-Boulan and Macara, 2014). Polarity at the cellular level is marked by the local 40 accumulation of proteins, RNA, organelles, extracellular matrix or membrane components 41 (Martin and Ephrussi, 2009; St Johnston and Ahringer, 2010). Mechanisms guiding such local 42 accumulation therefore organize the polarity system. Mechanisms underlying cell polarization 43 in animals and yeasts have been studied in some detail and have led to the identification of key 44 pathways (Chiou et al., 2017; Nelson, 2003; St Johnston and Ahringer, 2010). Several polarity 45 localized proteins have been identified in plants, and these include the PIN-FORMED auxin 46 transporters (Gälweiler et al., 1998; Wisniewska et al., 2006), BOR1 and NIP5 borate 47 transporters (Takano et al., 2006; Takano et al., 2010; Wang et al., 2017), Rho of Plants (ROP) 48 GTPases (Fu et al., 2005; Xu et al., 2010), as well as BASL, POLAR and BRX scaffold proteins 49 (Dong et al., 2009; Houbaert et al., 2018; Koh et al., 2021; Marhava et al., 2018). Essentially 50 all of these plant polarly localized proteins are plant-specific and do not share clear homology 51 with non-plant polar proteins (Dettmer and Friml, 2011; Ramalho et al., 2022). Likewise, the 52 regulators of animal and yeast cell polarity are missing from plant genomes (Dettmer and Friml, 53 2011; Yang, 2008). Thus, plants must have invented their own cell polarity system, and its 54 identification offers an interesting challenge. More recently, SOSEKI proteins were identified 55 as edge-polarized proteins, conserved across land plants, and sharing homology with animal 56 Dishevelled polarity proteins (van Dop et al., 2020; Yoshida et al., 2019). Whether this is a rare 57 case of convergent evolution, or if there are more unexplored homologies between animal and 58 plant polarity systems, is an open question. 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 59 Almost all studies on plant cell polarity were performed in the flowering plant Arabidopsis, or 60 more generally in vascular plants (Dettmer and Friml, 2011; Ramalho et al., 2022; Yang, 2008). 61 Land plants are remarkable in that they have a clear alternation between generations: a haploid 62 gametophyte generation and a diploid sporophyte generation. The earliest land plant gave rise 63 to two domains: non-vascular plants (Bryophytes) and vascular plants (Tracheophytes). The 64 former group includes mosses, liverworts and hornworts, the latter group includes lycophytes 65 ferns, seed plants and flowering plants (Harris et al., 2022; One Thousand Plant Transcriptomes 66 Initiative, 2019; Puttick et al., 2018). A defining distinction between the two groups is that in 67 bryophytes, the haploid gametophyte is dominant and indeterminately growing, and the 68 sporophyte is reduced to a diminutive structure. Inversely, with ferns being the only exception, 69 in tracheophytes, the sporophyte dominates and grows indeterminately, while the gametophyte 70 is reduced to a structure of but a few cells. As mentioned, cell polarity has mostly been studied 71 in flowering plants, and exclusively in sporophytes, with the exception of tip-growing pollen 72 tubes, which reflect a unique type of polarity that may not reflect interphase polarity in non tip 73 growing cells (Hepler et al., 2001; Qin and Yang, 2011). It is therefore an open question whether 74 the tracheophyte sporophyte, which has complex tissue arrangements, has unique need for cell 75 polarity systems. The recent finding that SOSEKI proteins mark polar domains in the Bryophyte 76 gametophyte (Pukhovaya et al., 2026; van Dop et al., 2020), suggests that sporophytic and 77 gametophytic cell polarity have a common origin. The proteomic identification of common, 78 polarized SOSEKI-associated proteins in the sporophyte of the flowering plant _Arabidopsis_ 79 _thaliana_ and the gametophyte of the liverwort _Marchantia polymorpha_ (Pukhovaya et al., 2026) 80 further supports the notion of a deeply conserved polarity mechanisms across the two 81 generations of all land plants. However, despite decades of research on flowering plant 82 gametophytes, there are no reports on cell polarity during their development. 83 We here explore polarity in the male and female gametophytes of the flowering plant 84 _Arabidopsis thaliana_ . Both are multicellular: male gametophytes form from microspores, 85 following male meiosis (Berger and Twell, 2011; Borg et al., 2009; Twell, 2011). They undergo 86 a first, asymmetric division to generate a vegetative cell and a generative cell. The latter divides 87 once more to generate two sperm (Berger and Twell, 2011; Twell et al., 1998). The female 88 gametophyte forms from a megaspore, the survivor of the 4 meiotic products (Drews and 89 Koltunow, 2011; Schmidt et al., 2015; Yadegari and Drews, 2004). Following anisotropic 90 growth, it divides 3 times to generate 8 nuclei that are patterned in an embryo sac which, upon 91 cellularization, contains two synergids, an egg cell, a central cell and three antipodal cells. The 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 92 structure is highly polarized, but has an unusual developmental progression that includes a 93 coenocytic stage before cellularization (Drews and Koltunow, 2011; Sprunck and Gross-Hardt, 94 2011). Thus, both male and female gametophyte feature polarized structure, as well as 95 asymmetric cell division and/or polar patterning. However, whether this brief phase of 96 flowering plant development is marked by cell polarity remains an entirely open question. 97 Microspores are isolated from their sporophytic maternal tissue, the tapetum (Scott et al., 2004), 98 while female gametophytes grow within sporophytic maternal tissue, the nucellus (Ceccato et 99 al., 2013; Pagnussat et al., 2009), in which polar PIN protein localization has been reported. As 100 mentioned, it is however, an open question whether the gametophytes themselves are polarized. 101 Starting from a large collection of SOSEKI proteins and SOSEKI-associated proteins, 102 we here report that both male and female gametophytes are profoundly polarized. Both contain 103 multiple polar domains, marked by different proteins. In the male, we show that polar SOSEKI 104 protein localization precedes the earliest known sign of symmetry breaking. We show that 105 successive polarization steps can be mechanistically uncoupled. Lastly, we show that a single 106 polarity system appears to guide the localization of all proteins studied. This work reports a rich 107 landscape of cell polarization in the haploid generation in Arabidopsis and will mark a starting 108 point for the dissection of mechanisms guiding cell polarity in plants. 109 110 **Results** 111 112 **SOSEKI proteins mark polar domains during female reproductive development** 113 We initially explored the expression domains and subcellular localization patterns of the 114 Arabidopsis SOK family protein during female gametophyte development, and in the 115 sporophytic tissues in which the gametophyte is enclosed. For both this analysis and the analysis 116 presented in the following sections, localization patterns are summarized in Figure 8. 117 During stage I of female gametogenesis, the base of the ovule is formed by the 118 formation of finger-like protrusions of the placenta (Robinson-Beers et al., 1992; Schneitz et 119 al., 1995). At this stage, SOK2, 3, and 5 are expressed, each polarized inwards to the center of 120 the developing ovule (Fig. 1F, K, U). During stage II, the centrally localized Megaspore Mother 121 Cell (MMC) will undergo meiosis to form the tetrad. At the same time, the inner and outer 122 integuments are initiated from sporophytic cells (Robinson-Beers et al., 1992; Schneitz et al., 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 123 1995). We found all five SOKs to be expressed around stage II. Since we did not explicitly 124 address division of the MMC, we can not distinguish pre- versus post-meiotic patterns. Only 125 SOK1 is expressed in the MMC (or megaspore), while the other 4 proteins mark sporophytic 126 cells (Fig. 1, S1). SOK1 is polarly localized towards the chalazal pole of the MMC/megaspore 127 (Fig. 1B). SOK4 and 5 are both expressed in the cells neighboring the MMC/megaspore and 128 polarized towards it, with SOK5 in a broader expression pattern (Fig. 1Q, V). SOK3 is 129 ubiquitously expressed but seems polarized towards the MMC/megaspore in the neighboring 130 cells (Fig. 1L). SOK2 marks the outer edge cell layers of the funiculus (Fig. 1G). 131 During early stage III, the tetrad degenerates and only one nucleus remains in the newly 132 formed embryo sac. The outer integument starts to envelop the the structures underneath it. The 133 nucleus undergoes three rounds of mitosis while the surrounding tissues grow and start to curve 134 the ovule by differential growth rates. In late stage III, the eight nuclei in the embryo sac 135 cellularize to form the the egg cell, antipodals, synergids and central cell. Lastly, two nuclei 136 within the central cell fuse and the antipodal cells degenerate (Robinson-Beers et al., 1992; 137 Schneitz et al., 1995). During early stage III, SOK1 is expressed in the embryo sac, polar 138 towards the chalazal end, while at the last part of stage III the signal fades (Fig. 1C-E). During 139 most of stage III, SOK4 and 5 are expressed in the nucellus, polarized towards the embryo sac 140 (Fig. 1R-T, W-X). SOK2 keeps marking the outer edge cells of the funiculus (Fig. 1I, J), while 141 SOK3 is still broadly expressed (Fig. 1M-O). Following cellularization of the embryo sac, we 142 find only SOK5 to be expressed, and polarized inward in the two synergids (Fig. 1Y). 143 Thus, Arabidopsis female gametophyte development is marked by a landscape of polar 144 domains, both within the developing coenocytic gametophyte and the cellularized stage, and in 145 the surrounding sporophytic tissue. 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 146 147 **Figure 1. All SOK proteins are expressed and polarly localized during ovule development, only SOK1 is** 148 **restricted to the female gametophyte.** (A-Y) Confocal sections of the developing ovule in stage I, stage II and 149 III expressing pSOK1::gSOK1-YFP (A-E), pSOK2::gSOK2-YFP (F-J), pSOK3::gSOK3-YFP (K-O), 150 pSOK4::gSOK4-YFP (P-T) and pSOK5::gSOK5-YFP (U-Y). YFP, green; SR-2200, white. Images are displayed 151 with brightness normalization and in some cases rotated or inverted to facilitate comparison of images. Tissues 152 were fixed, cleared using Clearsee alpha (Kurihara et al., 2021) and stained with SR-2200 dye (Musielak et al., 153 2015) before imaging. Scale bars, 10 µm . 154 155 **SOSEKI proteins mark symmetry breaking and polar domains during male gametophyte** 156 **development** 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 157 Microspores undergo symmetry breaking, followed by nuclear migration (Oh et al., 2016; Park 158 et al., 1998) and pollen mitosis I (PMI), which divides the polarized microspore asymmetrically 159 into a large vegetative cell and a small generative cell (Eady et al., 1995; Mizuta et al., 2026). 160 The generative cell divides again at pollen mitosis II (PMII) to form two sperm cells (Berger 161 and Twell, 2011; Twell et al., 1998). 162 To characterize the developmental distribution of SOK proteins during male gametophyte 163 development, we imaged YFP fusions of all five Arabidopsis SOK proteins expressed under 164 their native promoters. Pollen was counterstained with Hoechst 33342 to identify nuclei and 165 assign the developmental stage (Fig. 2). 166 Among the five SOK proteins, only SOK1 and SOK3 were detected during pollen 167 development (Fig. 2). SOK1 showed a stage-dependent pattern. In unicellular microspores, it 168 accumulated in a discrete crescent at one side of the cell periphery before PMI (Fig. 2A). After 169 PMI, it outlined the generative cell (Fig. 2B). After PMII, it surrounded the two sperm cells 170 (Fig. 2C). SOK3-YFP was undetectable in unicellular and bicellular pollen but gave a diffuse 171 cytosolic signal in the vegetative cell of tricellular pollen (Fig. 2G–I). After PMI, the generative 172 cell is internalized by the vegetative cell. The generative cell, and later the sperm cells, are 173 therefore enclosed by a membrane derived from the vegetative cell plasma membrane. This 174 membrane is called the peri-germ cell membrane (PGCM), or inner vegetative plasma 175 membrane (Sugi et al., 2023; Sugi et al., 2024). The SOK1 pattern suggests that it may localize 176 to this PGCM. To test this, we combined SOK1-YFP with the PGCM marker pACA3::Lyn24 177 mTurquoise2 (Motomura et al., 2021; Sugi et al., 2023). SOK1-YFP closely overlapped with 178 Lyn24-mTurquoise2 around the germline compartment (Fig. 2P), and fluorescence intensity 179 profiles across the labelled membrane showed peaks of both reporters at the same positions 180 (Fig. 2Q). These observations are consistent with SOK1 localization to the PGCM during post 181 PMI development. However, given the close proximity of the PGCM to the germ cell plasma 182 membrane (Sugi et al., 2023), we can not exclude that some of the SOK1 signal is derived from 183 the germ cell plasma membrane. 184 We conclude that SOK1 marks an early step in symmetry breaking in the microspore, 185 prior to nuclear migration, and that it marks all successive steps in engulfment of the germ cell 186 and sperm, thus reporting on several key events associated with male gametophyte polarization. 187 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 188 196 200 where it marks two very different steps: symmetry breaking in the unpolarized microspore and 201 engulfment of the generative cell. It is unclear whether these two steps are part of the same, 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 202 continuous process, and if SOK1 protein is unique, or whether it reflects a generic SOK 203 property. To test the latter, we expressed each of the other 4 SOK proteins as YFP fusions from 204 the SOK1 promoter, alongside SOK1 as a control. We introduced these into the nuclear FB037 205 reporter line, which contains markers for nuclei (pACT11::H2B-mRFP) and germline nuclei 206 (pMGH3::MGH3-eGFP). 207 At the polarization stage, the late unicellular stage before PMI, SOK1 accumulated in a 208 polar domain at the cell periphery (Fig. 3A–B). Strikingly, SOK2, SOK3, SOK4, and SOK5 209 could all localize to this same domain when expressed in the young microspore (Fig. 3F, J, N, 210 R). Thus, the paralogs can adopt an early polar distribution resembling that of SOK1. After 211 PMI, all paralogs diverged from SOK1. While SOK1 surrounded the generative cell in 212 bicellular pollen (Fig. 3C) and the sperm cells in tricellular pollen (Fig. 3D), SOK2 (Fig. 3G), 213 SOK3 (Fig. 3K), SOK4 (Fig. 3O) and SOK5 (Fig. 3S) were mainly cytosolic and lacked the 214 membrane enrichment that SOK1 shows at the PGCM. In tricellular pollen, we detected no 215 signal from any of the SOK1 paralogs in the germline. Thus, when expressed in the SOK1 216 domain, the paralogs can form an early polar pattern but not localize to the PGCM. This 217 identifies differences among SOK proteins but also shows that the two steps are discontinuous. 218 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 219 225 SOKs-YFP domains. Numbers indicate pollen with the displayed pattern out of pollen scored from 4 independent 226 lines. Scale bars, 10 µm. 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 227 **SOK1 polarity precedes, and is independent of asymmetric microspore division** 228 SOK1 polarizes in microspores prior to nuclear migration (Fig. 2A, 3B), which has so far been 229 the earliest sign of polarization. The nuclear migration forecasts the asymmetric PMI division 230 (Mizuta et al., 2026; Park et al., 1998). Therefore, SOK1 polarization occurs prior to 231 asymmetric pollen mitosis. We asked if indeed, SOK1 polarization acts upstream and 232 independently of this event. Very few mutants are known to disturb PMI, but mutations in the 233 microtubule-associated GEM1/MOR1 protein disturb asymmetric PMI (Park and Twell, 2001; 234 Twell et al., 2002). GEM1/MOR1 is required for phragmoplast function (Twell et al., 2002): 235 _gem1_ microspores show incomplete nuclear migration and defective division asymmetry and 236 cytokinesis, producing symmetric or partial divisions (Park and Twell, 2001; Park et al., 1998). 237 We introduced SOK1-YFP into this mutant. In pollen from _gem1_ /+ plants, SOK1-YFP still 238 accumulated in a polarized domain at the cell periphery in microspores in which division had 239 failed (Fig. 4A–B″). All transgenic lines showed at least 80% of the microspores observed 240 displayed SOK1-YFP polar localization before PMI (Fig. 4C). SOK1 polarization can therefore 241 be established in microspores that do not complete the normal asymmetric division. This places 242 SOK1 polarization upstream of asymmetric division in microspores. 243 249 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 250 **SOSEKI-associated proteins collectively polarize during reproductive development** 251 Recent, systematic proteomic analysis identified a polar protein network centered on SOSEKI 252 proteins (Pukhovaya et al., 2026). This network includes SOSEKI proteins, as well as SOSEKI 253 INTERACTING BTB (SIB) proteins (SIB1/NRL2, SIB2/NRL16 and SIB3/NRL29), the 254 cytosolic kinase SICK, the receptor-like kinases SITK1 and SITK2, and the polarized S 255 acyltransferases PAT19, PAT20 and PAT22 (Pukhovaya et al., 2026). An independent study 256 placed NPT-clade NRL proteins, PATs and SICK in a lateral protein cluster in Arabidopsis roots 257 (Yoshinari et al., 2026). Thus, SOK proteins are markers for the existence of a more general 258 polarity system, and we explored whether other proteins in this network could inform about 259 properties of the gametophyte cell polarity system. To this end, we analyzed the expression and 260 localization of SIB1, SIB2, SIB3, SIB4, SIB5, SIB6, SICK, SITK1 and PAT19 in both female 261 and male gametophytes. 262 In the female gametophyte at Stage I, we found a consistent pattern of inward 263 localization for SIB1, SIB2, SICK, SITK1, and PAT19 (Fig. 5A, F, P, U, Z), similar to what was 264 observed for SOK2, 3, and 5 (Fig. 1F, K, U). SIB3, 4, 5 and 6 are not expressed during this 265 stage (Fig. S3). During Stage II and III SIB1, SIB2, SITK and PAT19 all show strongly 266 overlapping expression patterns in the sporophytic tissues, generally polarized inwards to the 267 developing gametophyte. SICK displays a mostly cytosolic localization, but is also partially 268 polar in some cells (Fig. 5). 269 During Stage II and early Stage III, SIB1, SITK and PAT19 are polarly expressed in the forming 270 inner and outer integuments (Fig. 5B, V, AA). During the late Stage III, SIB1, 2 and PAT19 271 strongly overlap within the female gametophyte, being expressed both at the chalazal end like 272 SOK1, and in the region where the egg cell and synergids will be formed (Fig. 1B-D, 5C-E, H 273 J, AB-AD). SICK shows a similar pattern as SOK5 in the synergid cells (Fig. 1Y, 5T). Lastly, 274 SIB5 is weakly expressed from Stage II onwards in the MMC/megaspore and more strongly 275 expressed towards the end of Stage III, where it has a similar pattern as SIB1, 2, and PAT19 276 within the female gametophyte (Fig. 5C-E, H-J, M-O, AB-AD). Thus, SOSEKI-associated 277 proteins show diverse patterns of polaraization that each match one or more SOSEKI proteins. 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 278 279 **Figure 5. Many SOK interacting proteins are expressed and polarly localized during ovule development,** 280 **only SIB5 is restricted to the female gametophyte.** (A-AD) Confocal sections of the developing ovule in stage 281 I, stage II and III expressing pSIB1::gSIB1-YFP (A-E), pSIB2::gSIB2-YFP (F-J), pSIB5::gSIB5-YFP (K-O), 282 pSICK::gSICK-TdT (P-T), pSITK::gSITK-YFP (U-Y) and pPAT19::gPAT19-mSC (Z-AD). YFP, TdT, mSC, 283 green; SR-2200, white. Images are displayed with brightness normalization and in some cases rotated or inverted 284 to facilitate comparison of images. Tissues were fixed, cleared using Clearsee alpha (Kurihara et al., 2021) and 285 stained with SR-2200 dye (Musielak et al., 2015) before imaging. Scale bars, 10 µm. 286 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 287 **SOSEKI-associated proteins reveal hierarchy of polarization in the male gametophyte** 288 We next analyzed the localization of all SOSEKI-associated proteins in male gametophytes. 289 Among the six SIB reporters, SIB1 and SIB4 reproduced the SOK1 pattern (Fig. 6A–C, J–L). 290 Both accumulated asymmetrically at the periphery of unicellular microspores, outlined the 291 generative cell after PMI, and surrounded the two sperm cells after PMII. SIB3, SIB5 and SIB6 292 could not be detected (Fig. 6G-I, M-O, P-R). SIB2 was detectable at all three stages, with 293 cortical signal in bicellular and tricellular pollen. SICK behaved like SOK1, SIB1 and SIB4. It 294 accumulated asymmetrically at the periphery of unicellular microspores, then surrounded the 295 generative cell after PMI and the two sperm cells after PMII (Fig. 6S–U). PAT19 formed 296 peripheral patches in unicellular microspores and was subsequently detected around the 297 generative and sperm cells, although the signal was weaker in tricellular pollen (Fig. 6Y–AA). 298 By contrast, SITK1 gave no detectable signal at any stage examined (Fig. 6V–X), in contrast to 299 its polar localization in roots (Pukhovaya et al., 2026). SOK1, SIB1, SIB4, SICK and PAT19 300 therefore share a developmental sequence from an early polar cortical domain to a domain 301 surrounding the generative and then the sperm cells. 302 Having multiple proteins with similar polar patterns in the same, simple structure, we next asked 303 if there is an order in which proteins polarize. We therefore crossed the SOK1-YFP line with 304 SICK-tdTomato or SIB1-tdTomato lines and co-localized the two fluorescent proteins. In both 305 cases, we found that SOK1 protein polarized before either of the other two proteins (Fig. 6AB, 306 AE). As suggested by individual patterns (Figs. 2A-B, S-T), each of the two pairs ultimately 307 co-localized to the microspore polar crescent (Fig. 6AC, AF) and the PGCM (Fig. 6AD, AG). 308 This suggests that SOK1 likely acts prior to SIB1 and SICK in the polarity network. 309 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 310 321 µm 322 323 324 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 325 **A universal polarity system guides localization in Arabidopsis female gametophytes** 326 The analysis of 14 polar proteins (5 SOK proteins, 6 SIB proteins, SICK, SITK1 and PAT19) 327 reveals novel polar cellular domains in both male and female gametophytes. However, each 328 protein marks a subset of these polar domains. Misexpression of all SOK proteins in the SOK1 329 domain shows that other proteins either localize to the SOK1 domain, or remain cytosolic. This 330 suggests that perhaps there are no alternative polarity domains at this stage. To test this directly, 331 we expressed SOK1 ubiquitously from the _RPS5A_ promotor (Weijers et al., 2001) and analyzed 332 localization in both male and female gametophytes. In male gametophytes, this line mirrored 333 native SOK1 accumulation (Fig. 2A-C, 7A-C). In female gametophytes, SOK1 strongly 334 overlapped with SIB1, 2, SITK and PAT19 (Fig. 5C-E, H-J, M-O, AB-AD, 7D-G). Strikingly, 335 SOK clearly localized the inner membrane of synergid cells (Fig 7G). This is very similar to 336 native SOK5 localization, as well as patterns of SICK, SIB5, and PAT19 (Fig. 1Y, 5O, T, AD). 337 This data suggests that these proteins collectively report on a single polarity system. 338 We next asked if cells in which we did not find native expression of SOSEKI proteins can 339 polarize the proteins. We therefore expressed SOK1 and SOK5 from the egg cell-specific _EC1_ 340 promotor (Sprunck et al., 2012). In these lines, SOK1 polarized towards the antipodal end of 341 the egg cell in a similar direction as under the native promotor, confirming that the pathway 342 that polarizes SOK1 is present in the egg cell (Fig. 7H). Interestingly, SOK5 is mostly cytosolic 343 and does not show any polarized expression, even though it is polarly expressed in the synergids 344 when expressed from its native promotor (Fig. 7I). Therefore, this data suggests that while there 345 is a common polarity system, SOK1 and SOK5 differ in their capacity to be polarly targeted, 346 similar to the case in pollen development. 347 We previously found that endogenous SOK proteins are polarized in gametophyte tissue in the 348 liverwort Marchantia and the moss Physcomitrium (Pukhovaya et al., 2026; van Dop et al., 349 2020). We however did not explore polarization in gametes. The finding that the Arabidopsis 350 egg cell is capable of polarizing SOK1 urges the question of whether, likewise, bryophyte egg 351 cells polarize proteins in the SOK network. We studied the localization of Marchantia SOK 352 (MpSOK) and SICK (MpSICK) proteins in Marchantia egg cells. Unlike Arabidopsis, 353 Marchantia produces large, spherical egg cells that are only covered by a single layer of cells. 354 We expressed MpSOK and MpSICK from the ubiquitous _EF_ promotor and the endogenous 355 promotor, respectively. In these lines, MpSOK is completely cytosolic in egg cells and shows a 356 punctate pattern (Fig. 7J). Likewise, MpSICK is also cytosolic, and also shows punctate pattern 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 357 (Fig. 7K). This data suggest that, in contrast to the Arabidopsis egg cell, the Marchantia egg 358 cell does not polarize SOK and associated proteins, and may not be not polarized. 359 360 **Figure 7. Misexpression of SOK1 shows that the described polar complex is one system not multiple,** 361 **furthermore egg cell polarity is not conserved during evolution.** (A-K) Confocal sections of the developing 362 microspores, bicellular, and tricellular pollen and ovule in stage II and III expressing pRPS5A::gSOK1-YFP. (A363 G) Confocal sections of ovules in late stage III expressing pEC1::SOK1-YFP and pEC1::SOK5-YFP (H, I). 364 Confocal sections of Marchantia egg cells, expressing EF::MpSOK-YFP and MpSICK::MpSICK-YFP (J, K). YFP, 365 TdT, mSC, green; SR-2200, white; Hoechst 33342, magenta. Images are displayed with brightness normalization 366 and in some cases rotated or inverted to facilitate comparison of images. Tissues were fixed, cleared using Clearsee 367 alpha (Kurihara et al., 2021) and stained with SR-2200 dye (Musielak et al., 2015) before imaging. Scale bars, 10 368 µm. 369 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 370 371 **Figure 8.** **Visual summary of the main findings of the paper** 372 373 374 18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 375 **Discussion** 376 Land plants have remarkable life cycles that alternate multicellular haploid and diploid 377 generations (Hofmeister, 1851). From an ancestral early land plant, two lineages emerged with 378 nearly opposing life histories regarding the role of each of the generations: Bryophytes feature 379 a dominant, and indeterminately growing haploid (gametophyte) phase and a generally 380 diminutive diploid (sporophyte) phase that is transient and serves to form spores following 381 meiosis (Shaw et al., 2011). With the exception of ferns, Tracheophytes have a dominant and 382 indeterminate sporophyte and a diminutive gametophytes (Kenrick, 2000) that divides a few 383 times to generate gametes (Maheshwari, 1950). Bryophytes gametophytes and Tracheophyte 384 sporophytes share complex multicellular development, patterning and meristem activity, but 385 these are entirely different phases of life and an important question is whether the same 386 mechanisms of development underlie both, how these relate to the other generation within each 387 lineage, and how these derive from the ancestral land plant. Here we explored the homology of 388 cell polarity systems in the generations within the flowering plant Arabidopsis. There has been 389 a rich documentation of cell polarity in the sporophyte (Muroyama and Bergmann, 2019), but 390 so far, no markers for cell polarity have been reported in the development of their gametophytes. 391 Our work was inspired by the recent discovery of deeply conserved cell polarity proteins, 392 including SOSEKI (van Dop et al., 2020; Yoshida et al., 2019), SOSEKI-INTERACTING BTB 393 (SIB), SIB1-INTERACTING CYTOSOLIC KINASE (SICK) and PROTEIN 394 ACYLTRANSFERASE19 (PAT19) (Pukhovaya et al., 2026; Yoshinari et al., 2026), that are 395 polarized both in Arabidopsis sporophytes and Marchantia gametophytes. This suggests that a 396 common polarity system me be shared by the two generations across these species, and by 397 inference, perhaps also within each species. Given that both male and female gametophytes in 398 Arabidopsis undergo multiple divisions, including asymmetric ones (Borg and Twell, 2010), 399 and the female gametophyte grows anisotropically (Drews and Yadegari, 2002), it is likely that 400 cell polarity plays a role in gametophyte development. 401 We report that both male and female gametophytes are marked by early polar 402 localization of several proteins. In fact, the polarization of SOK1 marks symmetry breaking in 403 microspore well before any other sign of asymmetry emerges. In the female gametophyte SOK1 404 likewise decorates one pole of the surviving megaspore prior to expansion growth. Hence, 405 establishment of cell polarity is indeed an early event in the Arabidopsis haploid phase. 406 Following this initial polarization, there are addition polarization events in both gametophytes: 407 in the male, there is polar targeting of a range of proteins to the PGCM, the membrane that 19 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 408 surrounds the generative cell and sperm (Sugi et al., 2024). In the female, there is polarization 409 within the two synergid cells, and there is the capacity of the egg cell to polarly localize 410 ectopically expressed SOSEKI protein. Hence, in both gametophytes there are at least two polar 411 domains, and perhaps multiple steps in polarization. In the case of the male, we could dissect 412 the independence of the initial polarization to a polar crescent in the microspore and the later 413 targeting to the PGCM. Ectopically expressing all SOK proteins from the SOK1 promoter 414 revealed that all SOK proteins can mark the polar crescent, while only SOK1has the ability to 415 localize to the PGCM. This is interesting, because the PGCM is contiguous with the initial polar 416 crescent. Since the crescent marks the region where the asymmetric division forms, the crescent 417 essentially “engulfs” the generative cell. Yet, the properties of this membrane must change 418 during the process, given the loss of SOK2/3/4/5 from it. Detailed analysis of SOK1 localization 419 during the first asymmetric microspore division through live imaging should help resolve 420 whether the polar domain is indeed inherited to form the PGCM, or whether there is dynamic 421 rearrangement of membrane and proteins during the process. 422 In the female, while there are multiple polar domains, these occur in different cells (megaspore 423 versus synergids versus egg cell), and there is no evidence that within the same cell, there are 424 different capacities to polarize, as in the male. Indeed, when ectopically expressing SOK1, it 425 was able to mark all polar domains discovered through the native expression of the other 426 proteins studied. We interpret that there is a single polarity system in the female gametophyte 427 that guides the localization of all the proteins we tested. 428 It is remarkable that the full set of conserved polarity-localized proteins (SOK, SIB, 429 SICK, PAT) localize to the same polar domains in gametophytes. This suggests that this set of 430 proteins may indeed represent a module that is functionally interlinked. So far, it has been 431 difficult to define the interdependencies within this module, given that there is such strong co 432 localization in the growing sporophyte. The gametophytes offer an exquisite context for 433 studying causalities, given that there is a linear, directional ontogeny where one stage precedes 434 the next. We co-localized SOK1 and SIB1, as well as SOK1 and SICK, and this revealed that 435 SOK1 appears to accumulate at the polar crescent in microspores before the other two proteins. 436 While we cannot exclude that there are functionally relevant polarized pools of SIB1 and SICK 437 contemporaneously or preceding SOK1 that are below the detection limit, the simplest 438 interpretation is that SOK1 acts upstream of SIB1 and SICK in microspore polarization. 439 Detailed analysis of mutants in each of these components should resolve such functional 440 relationships. In addition, there are enzyme-substrate relationships between some components, 20 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 441 such as palmitoylation of SOK1 by PAT19 (Pukhovaya et al., 2026), that lead to testable 442 hypotheses, such as that a Cysteine mutation in SOK1 should interfere with those processes that 443 require PAT19. Analysis of mutants is also critical to resolve the function of these polarity 444 proteins, and cell polarity in general, in the haploid generation. It is intuitive that asymmetric 445 cell division, anisotropic growth and membrane differentiation (e.g. PGCM) would depend on 446 polar targeting, but so far, our work only reports on the existence of polarity, not its relevance. 447 It is encouraging that the _pat19;20;22_ triple mutant has strong fertility defects (Liu et al., 2024) 448 that may be connected to gametophyte polarity defects. Unfortunately, genetic analysis of the 449 SOK family has do far not led to obvious phenotypes (Yoshida et al., 2019), but this may be 450 confounded by redundancies, and perhaps even the very gametophytic localizations we report 451 here. It is possible that there are complex requirements for SOK proteins in the haploid phase, 452 which would complicate the generation of mutants. 453 A major inspiration for exploring Arabidopsis gametophyte polarity was the 454 identification of conserved polar proteins between the Marchantia gametophyte and the 455 Arabidopsis sporophyte. Strikingly though, we found that neither MpSOK nor MpSICK are 456 polarized in the Marchantia egg cell. This is in contrast to AtSOK1, which does polarize in the 457 Arabidopsis egg cell when expressed there. There are several potential explanations: first, it is 458 possible that MpSOK requires geometric cell edges, which is where nearly all SOK localization 459 is found. The capacity to break symmetry in a spherical cell may be an evolutionary innovation 460 in AtSOK1. This is perhaps unlikely, given that all AtSOK proteins can break symmetry when 461 expressed in microspores. Alternatively, the Marchantia egg cell may simply not polarize. The 462 essentially symmetric division of the zygote (Kohchi et al., 2021) is consistent with this notion. 463 It is clear that the availability of molecular markers of cell polarity now allows asking and 464 answering new questions related to the evolution of polarity and the way it is integrated in 465 multicellular development. 466 More generally, while the current study describes the existence of a cellular and 467 molecular polarity system in flowering plant gametophytes, we consider this the starting point 468 for a range of previously inaccessible roads. First, using these novel markers for more detailed 469 and dynamic descriptions of cell polarity will lead to a solid, quantitively and hopefully 470 harmonizing framework for sporophyte and gametophyte development across the Bryophyte 471 Tracheophyte divide. Second, this work postulates roles for polarity proteins in gametophyte 472 development, which can now be tested. Third, these proteins – as markers – will help to place 473 cell polarization in the known frameworks of gametophyte development, as we did with the 21 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 474 _gem1_ mutant. Finally, it should be clear that the rich polarity landscape in both gametophytes, 475 and their potential connection to formative developmental events, suggests that gametophyte 476 polarization may be a developmental bottleneck, and may explain why mutagenesis and 477 screening at post-embryonic stages have so far produced so few general cell polarity regulators 478 in plants. We propose that gametophyte cell polarization offers a potentially fertile ground for 479 genetic exploration of plant cell polarity mechanisms in the future. 480 481 22 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 482 **Materials and methods** 483 484 **Plant material and growth conditions** 485 The _Arabidopsis thaliana_ Columbia-0 (Col-0) accession was the wild-type background for all 486 reporter lines produced in this study. The vegetative and gamete lineage reporter system FB037 487 containing both pHTR10:HTR10:eGFP and pACT11:H2B:mRFP has been described 488 previously (Borges et al., 2012). Seeds were surface-sterilized and then stratified for 2 days in 489 the dark at 4°C. Seeds were then germinated and grown under continuous white light at ∼120 490 μmol⋅m [−2] ⋅s [−1] and 22°C on vertically placed Petri dishes containing 0.5× Murashige and Skoog 491 (MS) medium (Murashige and Skoog, 1962) supplemented with 1% agar and 0.3% sucrose. 492 The seedlings were then transferred to soil and grown at 22 °C in long-day conditions (16 h 493 light / 8 h dark) in controlled-environment rooms, unless otherwise specified. Phenotypic 494 analysis was always carried out with pollen from at least four different plants. 495 496 **Vector construction and generation of transgenic lines** 497 The p _SOK1::SOK-YFP, pSOK2::SOK2-YFP, pSOK3::SOK3-YFP, pSOK4::SOK4-YFP,_ 498 _pSOK5::SOK5-YFP and pRPS5A-SOK1-YFP_ transgenic lines have previously been described 499 (Yoshida et al., 2019). The _pSIB1::SIB1-YFP_, _pSIB2::SIB2-YFP_, _pSIB3::SIB3-TdTomato,_ 500 _pSICK::SICK-TdTomato_ and pSITK1::SITK1-YFP have been described (Pukhovaya et al., 501 2026). Constructs for YFP-tagged SOK1-paralogs driven by SOK1 promoter were created 502 using the Golden Gate modular cloning method (Engler et al., 2014). Briefly, The SOK1 503 promoter (2004bp upstream of the start codon of SOK1) was amplified and cloned into 504 pICH41295. The genomic coding sequence of all SOK paralogs were amplified and cloned into 505 pICSL01005. To generate the level 1 module, the SOK1 promoter, individual SOK paralog, a 506 YFP-tag (cloned into pAGM1301) and the NOS terminator (pICH41421) were combinatorially 507 assembled into the Level 1 acceptor backbone (pICH47742). For the final Level 2 assembly, 508 each SOK Level 1 module was assembled into the Level 2 acceptor backbone (pICSL4723) 509 with a FAST-Red selectable marker (pICSL11015) and linker Ele2 (pICH41744). To generate 510 SIB4, SIB5 and SIB6 reporter lines, sequences containing the relevant promoter and coding 511 sequence were amplified and assembled upstream of sYFP and NOS terminator sequences in 512 HpaI-digested pPLV17 (De Rybel et al., 2011) using HiFi assembly (primers contain 20 bp tails 513 to facilitate homology-mediated assembly to neighboring fragments). A GGGSGGGGS flexible 23 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 514 linker was introduced between each coding sequence and sYFP. To generate pEC1::SOK1-YFP 515 and pEC1::SOK5-YFP, coding sequence SOK1 and SOK5, excluding the stop codon, were 516 cloned into linearized pDONR221 using NEBuilder® HiFi DNA Assembly (New England 517 Biolabs). The pDONR221 vector was amplified with the primers 5′ 518 GAAGCCTGCTTTTTTGTACAAAGTTGGC-3′ and 5′ 519 GACCCAGCTTTCTTGTACAAAGTTGGC-3′. The resulting entry clones were subsequently 520 recombined into a modified pMpGWB destination vector via an LR reaction using Gateway® 521 LR Clonase® II Enzyme Mix (Thermo Fisher Scientific). In this destination vector, a YFP 522 cassette had been inserted into the SacI restriction site of pMpGWB101, enabling the generation 523 of C-terminal YFP fusions of the target proteins. To drive expression in egg cells, the EC1 524 promoter fragment was cloned into the HindIII restriction site, replacing the original EF1α 525 promoter present in pMpGWB101. Primers used for generation of these constructs are listed in 526 Supplemental Table S1. All constructs were introduced into _Agrobacterium tumefaciens_ strain 527 GV3101pMP90 and transformed using the floral dip method (Clough and Bent, 1998). The 528 MpSOK and MpSICK transgenic Marchantia lines were previously described (Pukhovaya et 529 al., 2026). 530 531 **Confocal Imaging and Analysis** 532 Leica TCS SP5 confocal microscope was used with the following fluorescence excitation 533 emission setting to visualize reporter genes and staining signals: SR2200 and Hoechst 33342 534 excitation at 405 nm, emission 420 nm, YFP excitation 515 nm, emission 527 nm; eGFP 535 excitation 488 nm, emission 507 nm; RFP excitation 584 nm, emission 607 nm. Images were 536 taken with 20x water immersion objectives. For analysis, Fiji and Las X image analysis 537 software were used (Schindelin et al., 2012). Brightness was adjusted uniformly across the 538 entire image, and identical adjustments were applied when signal intensity was directly 539 compared. 540 541 **Imaging the microspore** 542 To detect fluorescent signals, anthers containing microspores and mature pollen were dissected 543 from fresh inflorescences in 0.3 m Mannitol solution. For nuclear staining, 0.1% Triton X-100 544 with 0.1% Hoechst 33342 were added to the Mannitol solution. 24 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 545 546 **Imaging the ovules and Marchantia egg cells** 547 Flowers were harvested in 4% PFA in PBS-T and fixed for 30 minutes under vacuum and 548 incubated at 5 degrees overnight. The next day, flowers were washed 3 times in PBS-T before 549 incubation in Clearsee-alpha (Kurihara et al., 2021) for approximately 2 weeks. During this 550 time, Clearsee-alpha solution was refreshed regularly. Afterwards, samples were washed using 551 PBS-T and incubated for 30 minutes under vacuum with 0.1% SR2200 (Musielak et al., 2015) 552 in PBS-T. For imaging, samples were washed once more in PBS-T and mounted in Vectashield 553 anti fading solution (Vector Labs). This protocol was adapted from (Vijayan et al., 2021). 554 555 **Acknowledgements** 556 The authors thank Prof. David Twell for the _gem1_ seeds, Jorg Becker for the FB037 reporter 557 system and our team members for helpful discussions. 558 559 **Competing interests** 560 The authors declare no competing interests. 561 562 **Authorship statement** 563 Conceptualization: S.K.W.H., M.v.S., C.A., D.W..; Validation: S.W.; Formal analysis: 564 S.K.W.H., M.v.S.; Investigation: S.K.W.H., M.v.S., B.V.; Resources: C.A., J.R.R.; Writing 565 original draft: S.K.W.H., M.v.S.; Writing - review & editing: S.K.W.H., M.v.S., C.A., J.R.R., 566 D.W.; Visualization: S.K.W.H., M.v.S.; Supervision: D.W.; Project administration: D.W.; 567 Funding acquisition: S.K.W.H., M.v.S., D.W.. 568 569 **Funding** 570 This work was supported by an EMBO Long-Term Fellowship (ALTF 391-2023) to S.K.W.H., 571 by a grant from the Graduate School Experimental Plant Sciences to M.v.S., by the European 25 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.30.763139) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 572 Research Council (AdG DIRNDL; contract number 833867) to D.W., and by a Gravitation 573 grant (GreenTE) of the Dutch Ministry of Education to D.W.. 574 575 **Data and resource availability** 576 All primary data is available upon request from the corresponding author. 577 578 **AI Statement** 579 No AI was used in the preparation of this paper 26 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.30.763139; this version posted November 2, 2026. 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