PHOTOTROPIN-mediated blue light signaling orients the asymmetry of Marchantia polymorpha spores

18 Multicellular organisms produced by sexual reproduction develop from single cells 19 and the asymmetry of these cells can define the orientation of the earliest 20 developmental axes. The haploid multicellular stage of the plant, Marchantia 21 polymorpha, develops from a single cell – the spore – that divides asymmetrically, 22 producing an apical germ cell that generates the plant body and a smaller basal cell 23 that differentiates as an anchoring germ rhizoid cell. We show that the orientation of 24 this asymmetric cell division is controlled by an external, environmental cue – blue 25 light – that is perceived by the photoreceptor PHOTOTROPIN and signals in an 26 NCH1-dependent manner. This defines core elements of the mechanism by which a 27 directional environmental signal orients cell division, which in turn orients the first 28 axis of symmetry. 29 30

31 Marchantia polymorpha; Spore; Asymmetric Cell Division; Cell Division Orientation; 32 Blue Light Signaling; Phototropin; NCH1. 33 34

35 Land plants develop separate, multicellular haploid and diploid phases in their life 36 cycle. The diploid phase develops from a zygote produced by the fertilization of a 37 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 2 polarized egg (1). The polarity established in the egg is inherited by the zygote (2), 38 which orients the first cell division and aligns the apical-basal axis of the embryo 39 through a mechanism in which auxin modulates the activity of transcription factors in 40 different regions of the embryo (3). The haploid phase develops from the cells 41 produced by meiosis (spores). Spores are dispersed in an anhydrous state and 42 become activated upon contact with water, and swell, forming spherical cells. Spore 43 cells then divide asymmetrically, and this asymmetry in turn aligns the first body axis 44 that defines the position of the first meristem from which the mature plant body 45 develops (4-6). The cues that orient the spore cell asymmetry are unknown. 46 A dry M. polymorpha spore is approximately 7-10 µm in diameter and expands to 47 about 20 µm within the first 24 hours after imbibition. After approximately 28 hours, 48 the spore nucleus moves from the cell centroid to the cell cortex at the future basal 49 pole. There, the nuclear membrane breaks down, the mitotic spindle forms, 50 chromosomes are separated at mitosis, and a new cell wall forms near the basal 51 pole during cytokinesis, producing a small cell near the basal pole and a larger cell 52 which includes the entire apical hemisphere and most of the basal hemisphere. The 53 large apical cell divides to form a population of cells from which a meristem forms, 54 while the smaller basal cell differentiates into a germ rhizoid, which anchors the 55 developing plant to the substrate (7-9). The movement of the nucleus to the basal 56 pole requires reorganization of the microtubule and actin microfilament 57 cytoskeletons, which provide both the direction and force for nuclear movement (10-58 13). However, neither the cue nor the molecular mechanism that orients spore cell 59 asymmetry in M. polymorpha is known. Here, we report the discovery that a blue 60 light cue perceived by the PHOTOTROPIN photoreceptor and its interaction partner 61 NCH1 mediate the perception of this environmental signal. 62 63

64 Light positively regulates the division of the M. polymorpha spore 65 Dry M. polymorpha spores (Figure 1A) become spherical with a diameter of 66 approximately 7-10 µm, within 12 hours of imbibition (Figure 1D). By 24 hours after 67 initiation of imbibition, spores were approximately 20 µm in diameter, with dense 68 cytoplasm and a nucleus located near the centroid (Figure 1B, D). Spores divided 69 asymmetrically after approximately 30 hours, forming a large apical germ cell and a 70 smaller basal cell, separated by a new cell wall that was perpendicular to the apical-71 basal axis of the two-cell stage plant (Figure 1C-E) (10, 14). The larger daughter cell 72 is generative and proliferates into a mass of morphologically similar cells that form 73 the main body of a sporeling (Figure 1C), on which a meristem develops de novo 8-74 10 days after plating in white light (15). The basal cell differentiates as a tip-growing 75 rhizoid cell. 76 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 3 To test the role of light in sporeling development, we grew spores in darkness, white 77 light, red light, and blue light in nutrient media without a sucrose supplement. Spore 78 cells did not divide if grown in darkness for up to 7 days (2-day data, Figure 1F-G, 79 Figure S1A; 5-day data, Figure S1B; 7-day data, Figure S1C). By contrast, 76% of 80 spore cells divided after two days in white light, 83% in red light, and 75% in blue 81 light (Figure 1F-G, Figure S1A). These data indicate that light is required for cell 82 division (16). To test if the lack of cell division in darkness was due to carbon 83 starvation resulting from a lack of photosynthesis, we grew spores in nutrient media 84 supplemented with 1% (w/v) sucrose (Figure 1F, Figure S1A). Dark-grown spores did 85 not divide (0%) after two days, while spores grown in white (88%), red (80%), and 86 blue (81%) light divided (Figure 1G). These data indicate that light is required to 87 stimulate spore division in the first days after imbibition, and that monochromatic red 88 or blue light alone is sufficient to initiate division. Dark-grown spores divided after five 89 (73%) and seven days (86%) in darkness on sucrose-supplemented medium, 90 indicating that spores can eventually divide in the absence of light (Figures S1B-C). 91 From these data, we conclude that the first cell division of the spore is dependent on 92 light at approximately 30 h. 93 94 Blue light orients the first cell division 95 We reasoned that either light, gravity, or both would control the cell division 96 orientation. To determine if light directs nuclear migration that positions and orients 97 cell division, we developed a chamber in which spores were cultivated under 98 unidirectional light. The larger apical cell developed on the side nearest the incident 99 light, while the smaller basal cell developed on the relatively shaded side (opposite 100 the incident light direction) (Figure 2A). The perpendicular orientation of the new cell 101 wall relative to the incident light results in the apical cell being located on the 102 illuminated side and the basal cell on the relatively shaded side. We measured the 103 angles between the direction of incident light and the apical–basal axis (Figure S2A). 104 The angles were close to 0°, indicating that the light vector aligns with the apical–105 basal axis in spores grown under unidirectional white light (Figure 2A). These data 106 suggest that light orients the asymmetric cell division in the spore cell. However, in 107 these experiments, the light and gravity vectors were aligned, making it impossible to 108 distinguish between the two. 109 To test if gravity contributes to the orientation of spore cell asymmetry (17-19), we 110 aligned the light and gravity vectors at 180° to each other by shining light from below. 111 If gravity defined the direction of asymmetry, we would expect the smaller basal cell 112 to develop on the lower, illuminated side and the larger apical cell to develop on the 113 upper, shaded side. On the other hand, if light oriented asymmetry, we would expect 114 the smaller basal cell to develop at the upper shaded side and the large apical cell to 115 develop at the lower illuminated side. When illuminated from below, the smaller basal 116 cell formed at the upper shaded side, and the new cell wall was oriented 117 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 4 perpendicular to the incident light (Figure 2B). These data do not support the 118 hypothesis that gravity directs asymmetry. Instead, they indicate that light directs the 119 development of cellular asymmetry that orients asymmetric division of the spore. 120 To understand when the light signal acts during the orientation of spore asymmetry, 121 we tested the stability of light-induced asymmetry. We grew spores for varying 122 durations in white incident light from one direction before changing the direction of 123 the incident light by 180°. Measurements were taken after a total of 48 hours of light 124 exposure for all treatments. In controls where light was applied from one direction for 125 the duration of the experiment, cell division was asymmetric, and the smaller basal 126 cell formed on the relatively shaded side. When the direction of light was reversed by 127 180° at different time points, up to 29 hours, the position of the smaller basal cell 128 formed by asymmetric cell division was also reversed (Figure 2C, Figure S2B). This 129 indicates that the light-directed asymmetry is labile and changes if the direction of 130 incident light changes until approximately 29 hours. Reversal of the light direction at 131 24 and 29 hours resulted in a mixed population where the smaller basal cells 132 developed at either pole, or in some cases, division was symmetrical, forming two 133 equal-sized cells (Figure 2C, Figure S2B). Spore germination and cell division are 134 asynchronous, and nuclear migration occurs between 24 and 29 hours (10). The 135 mixed orientations observed at these late time points are consistent with 136 developmental asynchrony within the spore population, such that individual spores 137 were at different stages when the light direction was altered. Reversal of the light 138 direction at 35 hours did not affect the position of the smaller basal cell – the smaller 139 cell always developed on the shaded side relative to the direction of first illumination 140 (Figure 2C, Figure S2B). Since asymmetric cell division occurs at around 30 hours, 141 we conclude that the cell asymmetry remains labile until shortly before cell division. 142 To determine which light wavelengths direct cell wall orientation during the 143 development of cellular asymmetry, we grew spores in white, red, and blue light and 144 determined the position of the smaller basal cell and new cell wall orientation. For 145 spore orientation assays, we built a custom illumination system (Figure S2C; Data 146 S1, “Lightbox”) in which spores cultured in 0.4% agar in chambered microscopy 147 slides were illuminated by directional light. This system allowed us to image and 148 measure cell division orientation of spores in three-dimensional space. In white light, 149 the smaller basal cell developed on the relatively shaded side, and the new cell wall 150 was oriented perpendicular to the incident light (Figure 2D). In blue light, the smaller 151 basal cell also developed on the relatively shaded side, and the new cell wall was 152 oriented perpendicular to the incident light (Figure 2D). However, in red light, the 153 smaller basal cell was randomly positioned, and the new cell wall was randomly 154 oriented relative to the incident light (Figure 2D). When we grew spores in red light 155 supplemented with blue light, they oriented similarly to those grown in 156 monochromatic blue light, with the smaller basal cell forming on the shaded side and 157 the new cell wall oriented perpendicular to the incident light, further underpinning the 158 role of blue light in orienting spore cells (Figure 2D). We measured the angles 159 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 5 between the direction of incident light and the apical-basal axis (Figure S2A) under 160 the four tested light conditions: white, blue, red, and combined red and blue light. 161 Significant directional clustering occurred in both white and blue conditions (Rayleigh 162 test, p < 0.001, mean resultant length r = 0.99 and 0.98, respectively), as did the 163 combined red and blue light treatment (p < 0.001, r = 0.94). In contrast, angles of the 164 spores grown in red light were not clustered (p = 0.998, r = 0.009). Watson-Wheeler 165 tests revealed significant differences in angular distributions among all groups (multi-166 group test p < 0.001), with all pairwise comparisons also showing statistically 167 significant differences (p < 0.005). 168 To further test the role of light in orienting cell division, we determined the orientation 169 of cell division in those spores that divided after 7 days in the dark on sucrose-170 supplemented media (Figures S1B-C). The position of the smaller basal cell was not 171 aligned and was random as observed in spores grown for 48 hours in red light 172 (Figure S2D). These data indicate that blue light provides the directional cue that 173 orients the asymmetry of cell division. While red light stimulates cell division, it does 174 not orient cell asymmetry. 175 176 MpPHOT-mediated blue light sensing orients the first cell division 177 Because blue light was required to orient the asymmetric cell division in the spore 178 (Figure 2), we hypothesized that a blue light receptor mediated this response (20-179 22). To identify the blue light photoreceptor responsible for orienting spore cell 180 division, we generated transcriptomes from sporelings at 0, 15, and 30 hours after 181 imbibition and performed RNA sequencing. mRNA levels of several blue light 182 photoreceptors – PHOTOTROPIN (MpPHOT), CRYPTOCHROME (MpCRY), and 183 FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN (MpFKF) – were abundant 184 (Figure 3A). We hypothesized that MpPHOTOTROPIN would regulate the direction 185 of nuclear movement (23) because of its described role in directing chloroplast 186 movement (24-27). 187 To test if Mpphot mutants are defective in blue light signaling, we characterized 188 phototropic responses in independent mutant lines (28). Positive phototropism was 189 defective in Mpphot mutants: wild type gametangiophore stalks grew straight and 190 erect towards the light source. By contrast, Mpphot mutant gametangiophore stalks 191 grew in different directions, resulting in curled stalk morphology (Figure 3B). Stalk 192 curling was quantified using a stalk curling index defined as the ratio between the 193 shortest and within-tissue distance from the stalk base to the base of the antheridial 194 head. The index of straight stalks approaches 1, but is lower in curved stalks. The 195 average index of wild type was ~0.98, whereas the index of Mpphot m utants was 196 ~0.74 (Figure 3B). The defective positive phototropism in Mpphot mutants is 197 consistent with the hypothesis that blue light-mediated phototropism is defective in 198 these mutants. 199 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 6 Negative phototropism was also defective in Mpphot mutants. In wild type, rhizoids 200 of 10-day-old sporelings grew away from the blue light source, while Mpphot mutant 201 rhizoids were insensitive to light direction and grew in all directions (Figure 3C). 202 Similarly, under standard white-light conditions on horizontal plates, rhizoids were 203 visible in 11-day-old Mpphot sporelings but not in wild type when densely plated 204 spores were imaged from above (Figure 3D). The defective gametangiophore growth 205 and defective rhizoid negative phototropism in Mpphot mutants demonstrate that 206 MpPHOT is required for both positive and negative phototropic responses and that 207 blue light signaling is defective in the Mpphot loss-of-function mutants. 208 To determine if MpPHOT is required to orient cell division asymmetry relative to the 209 direction of incident blue light, we compared the position of the smaller basal cell and 210 the orientation of the new cell wall that forms in unilateral light in wild type and 211 Mpphot mutants. The smaller basal cell developed on the shaded side of the two-cell 212 sporeling, and the new cell wall was oriented perpendicular to the direction of 213 incident light in the wild type (Figure 2A, Figure 3E). By contrast, in Mpphot mutants, 214 the smaller basal cell developed in random positions, and the orientation of the new 215 cell wall was random with respect to the direction of light (Figure 3F). We measured 216 the angles between the direction of incident light and the apical-basal axis. Wild-type 217 clustered at consistently low angles, indicating that they all were oriented in the 218 same direction (Rayleigh test, p 0.5, r = 0.12 and 0.14, 220 respectively). Watson-Wheeler tests revealed significant differences in angular 221 distributions between wild type and both mutants (p < 0.001), but not between 222 Mpphot-3 and Mpphot-4 (p = 0.99). These data indicate that the position of the 223 smaller basal cell and the orientation of the new cell wall were defective in Mpphot 224 mutants. We conclude that MpPHOT is required for the development of blue light-225 directed cell asymmetry that orients asymmetric cell division of the spore. 226 227 MpNCH1 mediates MpPHOT-dependent orientation of asymmetric cell division 228 To identify proteins involved in MpPHOT-mediated blue light signaling, we performed 229 in vivo proximity labeling using a MpPHOT–miniTurbo(ID)-YFP fusion protein. 230 Biotinylated proteins from two independent proMpPHOT:MpPH OT -miniTurbo-YFP 231 lines were compared with those from a compartment control (proMpPHOT:MpPIP-232 miniTurbo-YFP) and the wild-type control (Tak-1). All constructs were introduced into 233 the same genetic background, Tak-1, by thallus transformation. Proteins biotinylated 234 in the vicinity of MpPHOT were identified by mass spectrometry. This identified 547 235 to 550 proteins that were significantly enriched (log2FoldChange >2, p-value < 0.05) 236 for two independently tested PHOT-miniTurbo-YFP lines against Tak-1 (Figure 4 A, 237 Table S1). From the enriched candidates, we prioritized eleven proteins for further 238 analysis based on their expression in the one-cell spore stage (≤ 30) transcriptomes 239 (Figure 3A) and their predicted roles in signal transduction (candidates highlighted in 240 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 7 Figure 4A). To test if any of the eleven proteins were required for blue light-oriented 241 division of the spore cell, we generated a total of 137 mutant lines carrying loss-of-242 function mutations in the respective genes (Figure S4A). 243 Next, we characterized light responses in the thallus and gametangiophores. While 244 some developed defective thalli, only mutants in one gene – MpNRL PROTEIN FOR 245 CHLOROPLAST MOVEMENT1 (MpNCH1) – developed the curled gametangiophore 246 phenotype characteristic of defective blue light signaling, as observed in the Mpphot 247 mutants (Figure 4B). The stalk curling index of Mpnch1 mutants was approximately 248 0.77 (Figure 4C) and similar to Mpphot mutants (Figure 3C), whereas the wild type 249 developed an index close to 1 (~0.97; Figure 4C). These data indicate that blue light 250 signaling is defective in Mpnch1 mutants. 251 We then compared the position of the smaller basal cell and new cell wall orientation 252 in Mpnch1 mutants and wild type. In wild type, the new basal cell formed on the 253 shaded side, and the new cell wall was oriented perpendicular to the incident light 254 (Figure 4D). However, in Mpnch1 mutants, the smaller basal cell developed in 255 random positions relative to the direction of illumination, and the new cell wall was 256 randomly oriented (Figure 4D). We measured the angles between the direction of 257 incident light and the apical-basal axis. The orientation angles of wild type are 258 clustered (Rayleigh test, p 0.9), whereas the orientation of the axis is 259 largely random with only weak residual directionality (r = 0.34) in Mpnch1 mutants. 260 Consistent with this, the angular distributions of wild type and Mpnch1 differ 261 significantly (Watson–Wheeler test, p < 0.001). These data indicate that MpNCH1 is 262 required for blue light-directed asymmetry that orients asymmetric cell division in the 263 spore. 264 MpNCH1 is a member of a family of NRL (NPH3/RPT2-like) proteins that have been 265 implicated in MpPHOT-mediated blue light signaling (29). MpNONPHOTOTROPIC 266 HYPOCOTYL3 (MpNPH3) is also an NRL protein family member that functions in 267 blue light signaling (30). To test if MpNPH3 is required for blue light-oriented spore 268 cell division, we generated lines carrying mutations in MpNPH3 and compared the 269 positioning of the smaller basal cell and new cell wall orientation in Mpnph3 loss-of-270 function mutants with wild type. Both the position of the small basal cell and the 271 orientation of the new cell wall in the Mpnph 3 spores were wild type-like (Figure 4E). 272 The orientation angles are clustered in Mpnph3, similar to wild type (Rayleigh test, p 273 0.9). These data indicate that MpNPH3 is not required for the orientation 274 of cell asymmetry in the M. polymorpha spore. 275 If MpPHOT and MpNCH1 interact during the establishment of cell asymmetry in the 276 spore, we predicted that the proteins should co-localize during this process. To test 277 this hypothesis, we generated marker lines expressing proMpNCH1:MpNCH1-278 VENUS and proMpPHOT:MpPHOT-mCherry in the same spore. Spores were grown 279 for 24 h and 36 h under standard white light, then fixed and cleared to remove 280 chloroplast autofluorescence. MpPHOT-mCherry and MpNCH1-VENUS co-localized 281 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 8 at the cell surface (Figure 4F, Figure S4B). These data are consistent with MpPHOT 282 and MpNCH1 acting in the same pathway to regulate blue light-oriented cell 283 asymmetry in the M. polymorpha spore. 284 285

286 We discovered that MpPHOT-mediated blue light signaling aligns the cellular 287 asymmetry that orients the first division of the spore and sets up the first 288 developmental axis of the M. polymorpha body plan. MpPHOT-mediated blue light 289 signaling that directs the orientation of cellular asymmetry requires MpNCH1 but not 290 the related MpNPH3 NRL protein (29). These data demonstrate that the orientation 291 of cell asymmetry and subsequent body axis orientation depend on an external blue 292 light cue from the environment. Such externally directed cellular asymmetry positions 293 the germ rhizoid on the shaded side of the spore. This rhizoid cell anchors the plant 294 to the substrate during its early development (4). The apical cell is on the illuminated 295 side, divides and forms a photosynthetically active sporeling that develops a 296 meristem from which the plant body develops (15). 297 In land plants, the diploid zygote develops within the maternal tissues in which the 298 egg cell formed: within the archegonium in bryophytes, lycophytes, monilophytes, 299 and gymnosperms, and within the embryo sac in angiosperms (1). Signals from the 300 parental tissues polarize the egg and zygote (3, 31). By contrast, haploid spores that 301 form in bryophytes and monilophytes develop in the environment and independent of 302 the plant on which they are produced. The cues that orient development do not come 303 from surrounding tissues (11). In free-sporing plants, including several fern species, 304 blue light has been shown to inhibit spore germination, as reviewed in Suo et al (32). 305 Instead, we show here that blue light orients the symmetry-breaking event that 306 occurs in spores in the first 30 hours of growth before cell division. This cell 307 asymmetry orients the first cell division, the first body axis, and subsequently the 308 negative phototropic growth of the germ rhizoid. In this way, MpPHOT-mediated blue 309 light signaling orients the development of the M. polymorpha body, tethering the 310 plant to its substrate. 311 The demonstration that both MpPHOT and MpNCH1 are required to direct cell 312 asymmetry in the spore cell suggests that a canonical MpPHOT signaling pathway 313 regulates this process. In addition to MpNCH1, proximity labeling revealed several 314 proteins previously linked to phototropin signaling, including a BLUS-like protein (33) 315 and multiple kinases and phosphatases (Figure 4A, Fig S4A). These associations 316 are consistent with engagement of a broader phosphorylation-dependent signaling 317 framework. Nevertheless, our functional analyses identify MpNCH1 as a key 318 downstream component required for orienting spore cell asymmetry. This discovery 319 re veals how plants exploit a directional environmental cue to align activities in the 320 single totipotent cell from which the multicellular haploid plant develops. 321 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 9 322 323

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We thank Katharina Jandrasits and Magdalena Mosiolek for 463 general laboratory support. We are grateful to the ProTech Facility of the Vienna 464 BioCenter for cloning the MpNCH1 CRISPR constructs, and to the Vienna BioCenter 465 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 12 Core Facilities for continuous support. This includes the BioOptics Facility, with 466 special thanks to Thomas Lendl for assistance with co-localization image analysis, 467 as well as the Plant Sciences Facility, Mechanical Engineering Center, Electron 468 Microscopy Facility, Proteomics Facility, Molecular Biology Service, Media Lab, Lab 469 Support, and the administrative staff at the VBC. 470 Transmission electron microscopy (TEM) of spores was performed with Marlene 471 Brandstetter at the Electron Microscopy Facility, Vienna BioCenter Core Facilities 472 (VBCF), member of the Vienna BioCenter (VBC), Vienna, Austria. Scanning electron 473 microscopy (SEM) was carried out with Daniela Gruber at the Core Facility Cell 474 Imaging and Ultrastructure Research (CIUS), University of Vienna, a member of 475 Vienna Life-Science Instruments (VLSI). RNA sequencing was performed at the 476 Wellcome Trust Centre for Human Genetics, University of Oxford. Proteomic 477 analyses were carried out by the Proteomics Facility at IMP/IMBA/GMI using the 478 Vienna BioCenter Core Facilities (VBCF) instrument pool. 479 We thank Haonan Bao, Zohar Meir, and Victoria Spencer for critically reading the 480 manuscript, and Zohar Meir for assistance with uploading the transcriptome data to 481 the repository. 482 483 Funding: This work was supported by a grant from the Austrian Academy of 484 Sciences (OEAW) to the Gregor Mendel Institute and by a European Research 485 Council Advanced Grant (DENOVO-P, project number 787613) to Liam Dolan. 486 Radka Slovak was supported by a European Molecular Biology Organization 487 (EMBO) Long-Term fellowship (2017). 488 489 Author contributions: 490 Conceptualization: JR, RS, LD 491 Methodology: JR, RS, ESW, SS, MC, LD 492 Investigation: JR, RS, ESW, NE, BA, SD 493 Visualization: JR, ESW 494 Funding acquisition: LD 495 Project administration: LD 496 Supervision: JR, LD 497 Writing – original draft: JR, LD 498 Writing – review & editing: JR, ESW, LD 499 500 Competing interests: 501 Liam Dolan is a co-founder, shareholder, and board member of MoA Technology. 502 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 13 503 Data, code, and materials availability: 504 Requests for resources and further information should be directed to the lead 505 contact, Liam Dolan ([email protected]). 506 All data reported in this paper will be made available by the lead contact upon 507 request following publication of the peer-reviewed paper. The raw and processed 508 data for the spore transcriptomes will be available in the NCBI Gene Expression 509 Omnibus (GEO) repository upon publication of the peer-reviewed paper. The mass 510 spectrometry proteomics data have been deposited in the ProteomeXchange 511 Consortium via the PRIDE partner repository (34, 35) with the dataset identifier 512 PXD072764 and will be available upon publication of the peer-reviewed paper. 513 Requests for plasmids and transgenic lines generated in this study should be 514 directed to the lead contact. Recipients are required to provide appropriate 515 documentation, such as import permits, for the transfer of transgenic material. 516 517

518 Plant material and growth conditions 519 Wild-type accessions of Marchantia polymorpha, Takaragaike-1 (Tak-1, male) and 520 Takaragaike-2 (Tak-2, female) (36), together with the respective mutant lines, were 521 cultivated on sterile solid medium. The growth medium consisted of half-strength 522 Gamborg’s B5 (37) supplemented with 0.5 g/L MES hydrate, 1% (w/v) sucrose, and 523 1% (w/v) plant agar, adjusted to pH 5.5 before autoclaving. The plants were 524 cultivated at 23 °C under continuous white light illumination in standard LED growth 525 chambers at the institute or in growth chambers manufactured by Poly Klima 526 Climatic Grow Systems (Hofmann Kühlung Company; firmware version 527 03.10.10(22), controller: WAGO 750-8212 PFC200 G2 2ETH R). For dark treatment 528 controls, plates were wrapped twice with aluminium foil, transferred into a light-529 impermeable container, and maintained in the same growth chamber. 530 Unless otherwise stated in the respective figures, the following light regimes were 531 applied: standard continuous white light at a photon flux density (PFD) of 50-60 μ mol 532 m⁻ ² s⁻ ¹; blue light at approximately 30-40 μ mol m⁻ ² s ⁻ ¹ ( λ max = 450 nm); and red 533 light at approximately 80-90 μ mol m ⁻ ² s ⁻ ¹ ( λ max = 660 nm). Dark controls were 534 prepared as described above and kept at 23 °C in the growth chamber. Light 535 intensities were quantified using a Spectral PAR Meter PG100N (UPRtek). 536 For gametangiophore induction, plants were transferred to soil in SacO /i2 Microbox 537 containers containing an autoclaved 3:1 mixture of Neuhaus N3 compost and 538 vermiculite, and grown under 50 μ mol m⁻ ² s⁻ ¹ white light supplemented with 45 μ mol 539 m⁻ ² s⁻ ¹ far-red light in a long-day regime (16 h light / 8 h dark) at 23 °C. 540 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 14 541 Scanning electron microscopy of dry Marchantia polymorpha spores 542 Sporangia from a cross between Tak-1 and Tak-2 were collected and dried over silica 543 gel at room temperature for 3-4 weeks. Dry M. polymorpha spores were mounted on 544 SEM stubs and sputter-coated with gold for 30 seconds using a JEOL JFC-2300HR 545 sputter coater to generate a conductive surface layer. 546 Samples were imaged using a JEOL IT300 scanning electron microscope equipped 547 with a LaB6 filament. Secondary electron and backscattered electron detectors were 548 used for image acquisition. SEM imaging was performed at the Core Facility Cell 549 Imaging and Ultrastructure Research (CIUS), University of Vienna, member of the 550 Vienna Life-Science Instruments (VLSI). 551 552 Transmission electron microscopy of Marchantia polymorpha spores 553 Marchantia polymorpha spores were cultured on cellophane-covered medium plates 554 under continuous white light conditions for 24 hours after plating. The spores were 555 then washed off the cellophane and immediately pre-fixed in 0.5% (w/v) 556 paraformaldehyde and 0.05% (v/v) glutaraldehyde in 0.1 M PHEM buffer for 10 557 minutes. Samples were washed twice in PHEM buffer, transferred to Gamborg 558 medium supplemented with 10% (w/v) BSA (fraction V), and cryo-immobilized using 559 a Wohlwend HPF Compact 01 high-pressure freezer. 560 Freeze-substitution was performed in a Leica EM AFS automated system over 3 561 days at -90 °C in acetone containing 0.1% (w/v) tannic acid and 0.5% (v/v) 562 glutaraldehyde. After substitution, samples were warmed to -20 °C over 12 h, 563 washed in pre-cooled acetone, and incubated in 2% (w/v) osmium tetroxide in 564 acetone for 24 h for infiltration and post-fixation. Samples were then warmed at ~5 565 °C/h to 4 °C, rinsed, incubated for 2 h at 4 °C, brought to room temperature, and 566 washed further in acetone. 567 Samples were infiltrated with Agar 100 epoxy resin through a graded resin:acetone 568 series and polymerized in pure resin at 60 °C for 48 hours. Ultrathin sections (70 nm 569 nominal thickness) were cut using a Leica UCT ultramicrotome and post-stained with 570 2% (w/v) uranyl acetate and Reynolds’ lead citrate. 571 TEM imaging was performed on an FEI Morgagni 268D transmission electron 572 microscope equipped with a tungsten filament emitter and a MegaView III CCD 573 camera (Olympus-SIS). The instrument was operated at 80 kV (nominal maximum 574 100 kV). Images were acquired using Morgagni software (v3.0) and iTEM (v5.0). 575 Digital images were recorded at 7100x magnification with an image size of 576 1376x1032 pixels (16-bit), corresponding to a pixel size of 9.151 nm. 577 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 15 TEM imaging of Marchantia spores was carried out at the Electron Microscopy 578 Facility, Vienna BioCenter Core Facilities (VBCF), member of the Vienna BioCenter 579 (VBC), Vienna, Austria. 580 581 Bright-field imaging of plants and spores 582 Plants and spores were imaged using a Keyence VHX-7000 digital bright-field 583 microscope equipped with VH-Z00R/Z00T and VH-ZST lenses and a VHX-7020 584 camera. Where necessary, non-specific background was removed, and images were 585 cropped to appropriate dimensions for figure preparation. 586 587 Spore generation, culture, and imaging 588 Crosses between male and female plants were performed by collecting sperm from 589 antheridiophores in sterile water for 15 min and transferring the suspension onto 590 archegoniophores. Fully developed sporangia were collected 4-5 weeks after 591 fertilization, placed in sealed polypropylene microboxes (OV80+OVD80) with 592 micropore tape, and dried over silica gel ( 36, 38). After a 4-week desiccation period, 593 sporangia were stored at -70 °C until use. 594 For germination, frozen sporangia were thawed and gently crushed in 0.1% (w/v) 595 sodium dichloroisocyanurate (NaDCC; Sigma-Aldrich, Cat. No. 218928) for 1-2 min. 596 Spores were pelleted (13,000 x g, 3 min), washed twice with sterile water, and 597 resuspended in sterile water. 598 Spores were plated on sterile medium consisting of half-strength Gamborg B5 599 supplemented with 0.5 g/L MES hydrate (pH 5.5), containing 1% sucrose or no 600 sucrose, and solidified with 1% plant agar or 1% phytoagar. Cultures were grown at 601 23 °C under continuous illumination. For dark controls, plates were wrapped twice in 602 aluminium foil, placed in a light-tight box, and maintained in the same growth 603 chamber. 604 For spore orientation assays, wild type and mutant spores expressing 605 proMpSYP13A:mCitrine:MpSYP13A were embedded in half-strength B5 medium 606 (1.5 g/L B5, 0.5 g/L MES, 1% sucrose, pH 5.5) solidified in 0.4% plant agar. The agar 607 medium was cooled to near-solidification before mixing with spores to generate a 608 homogeneous 3D culture, which was solidified in chambered microscopy slides 609 (Ibidi, Cat. No. 80286). Slides were placed in a custom-built illumination system 610 (“lightbox”). 611 All procedures involving spores for light assays were performed under dim green 612 light in a sterile laminar flow hood. 613 614 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 16 Custom-built illumination system (Lightbox) for spore orientation experiments 615 Spore orientation assays were performed using the custom-built Lightbox system 616 described in this section. This setup enables controlled directional illumination of 617 spores in 0.4% 3D agar culture in chambered microscopy slides. 618 Software and control 619 The Lightbox control software was developed mainly by Sebastian Seitner (PlantS 620 Facility, GMI Vienna). The system comprises four independent chambers, each 621 equipped with a dedicated sample holder and LED module (Figure S2C, Data S1). 622 All modules can be controlled independently. 623 Illumination regimes are defined as schedules and uploaded to the device via USB-C 624 in JSON (JavaScript Object Notation) format. Each schedule specifies the sequence, 625 duration, and intensity of light conditions for each chamber. The uploaded schedules 626 are selected via an integrated user interface consisting of a rotary control and LCD 627 display. 628 Each module contains 16 high-power LEDs (OSRAM), covering four spectral ranges: 629 white (3500 K), blue (455 nm), red (657 nm), and far-red (727 nm). 630 Light intensity is regulated using pulse width modulation (PWM) at ~30 kHz. PWM 631 enables stable low-intensity output without shifting emission spectra. LEDs of each 632 color are arranged in two electrically independent groups. These groups are driven 633 with a 180° phase offset. At intensities above 50%, at least one group remains active 634 at any time. This reduces temporal fluctuations in light delivery. 635 Hardware 636 The Lightbox hardware was developed by Martin Colombini and the Mechanical 637 Engineering Center (Vienna BioCenter). 638 The system consists of a base plate, a control unit, and a light-tight enclosure 639 containing four independent chambers. Each chamber fits one two-well chambered 640 coverslip from IBIDI (Cat. No. 80286). 641 The enclosure includes a rear panel supporting the LED boards, a front panel with 642 an access door, structural side panels, internal partitions, and top covers. Samples 643 are inserted through the front door and positioned precisely within each chamber. 644 Illumination parameters are set via the external control unit. 645 Thermal control is facilitated through active airflow: a top-mounted fan draws air from 646 the base and exhausts it upwards, cooling the LED assemblies. The rear LED 647 compartment is covered by a removable panel to maintain airflow while allowing 648 access for maintenance. Each chamber is additionally ventilated by side-mounted 649 fans. Air enters through a filtered inlet, passes through a light-blocking labyrinth, and 650 exits through a corresponding outlet. These structures prevent external light 651 contamination. 652 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 17 Optical isolation between chambers is ensured by internal partitions, diaphragm 653 elements, and sealing interfaces between the front panel and door. These features 654 prevent light leakage and cross-illumination. 655 The structural components (base, front, rear, and side panels) are constructed from 656 rigid PVC. Covers, partitions, and the door are made from anodized aluminium. 657 Internal rails are aluminium with a sandblasted finish to reduce reflections. Interior 658 panels consist of black acrylic. Components are assembled primarily using stainless 659 steel screws. 660 661 Previous setup for spore orientation experiments 662 Initial (re)orientation and gravity experiments were performed using a simplified 663 setup. Unidirectional illumination was provided by single LEDs mounted on a 664 breadboard. To expose spores to unidirectional light, chambered microscopy slides 665 (Ibidi, Cat. No. 80286) containing spores were placed inside a 3D-printed cover box, 666 in which all sides except one were enclosed by a light-proof black cover. This 667 configuration allowed light to enter from only one side of the chamber, creating 668 unidirectional illumination. 669 The entire setup was placed in an opaque enclosure (a black cardboard box) for the 670 duration of the experiment. Light intensity was measured using a Spectral PAR 671 Meter PG100N (UPRtek). 672 673 Spore orientation quantification – angle calculation 674 Angles between the light vector and the apical-basal axis were measured using FIJI 675 and visualized in RStudio. Two-dimensional or z-stacked confocal images were 676 acquired from wild-type and mutant spores expressing the fluorescent marker 677 proMpSYP13A:mCitrine:MpSYP13A to visualize cell division. Images were oriented 678 such that the direction of incident light corresponded to the negative y-axis. 679 In FIJI, the transverse cell wall of asymmetrically dividing sporelings was identified 680 manually. Using the multi-point tool, the two junctions where the transverse wall 681 meets the spore outline were marked sequentially, and XY coordinates were 682 exported for each image. Angles were calculated in RStudio using the exported 683 coordinate files. The apical-basal axis was defined as the vector orthogonal to the 684 transverse cell wall. The angle between this axis and the light vector (negative y-685 axis) was then computed. 686 All calculated angles from individual measurements for each genotype and 687 experiment were used to generate binned radial histograms. Visualization was 688 performed using the R packages circular, plotrix, and ggplot2. 689 690 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 18 Fixation, clearing, and cell wall staining of spores 691 Spores expressing nuclear and plasma membrane markers ( 10) 692 (proMpROP:mScarletI-N7 and pro MpUBE2:mScarletI-AtLTI6b; Figure 2D) and 693 spores expressing fluorescent reporters for co-localization ( proMpNCH1:MpNCH1-694 VENUS and proMpPHOT:MpPHOT-mCherry; Figure 4F) were cultivated on 695 cellophane-covered standard medium with sucrose for the indicated time periods. 696 Cellophane with spores was removed and submerged in 10% formalin containing 697 0.1% Brij L23 for 30 min. Fixed sporelings were pelleted (7,000 x g, 3 min), washed 698 in 1x PBS, and cleared in ClearSee α solution (10% w/v xylitol, 15% w/v sodium 699 deoxycholate, 25% w/v urea, supplemented with 6.3 mg/mL sodium sulfite 700 anhydrous) for approximately one week in the dark with gentle agitation. 701 Cleared sporelings were mounted in ClearSee α and imaged by confocal microscopy 702 (Zeiss LSM 880) using a 40x/1.2 LD LCI Plan-Apochromat water/glycerol DIC 703

with silicone immersion oil matched to the refractive index of ClearSee α . 704 For co-localization analysis (Figure 4F), single-channel z-stacks ( VENUS and 705 mCherry) were processed in ImageJ/Fiji (39 ), and co-localization was quantified 706 using JACoP (40). 707 For cell division analysis under different light conditions (Figure 1G; Figures S1A-C), 708 the same fixation and clearing protocol was used with the addition of 0.2% 709 Renaissance SR2200 applied overnight prior to imaging. The dye for cell wall 710 staining was excited at 405 nm, and emission was collected between 420-500 nm 711 using an upright widefield microscope. Images were acquired with an sCMOS 712 camera, and cell divisions were manually scored in FIJI (39). 713 714 RNA-seq and differential expression analysis of early spore development 715 To assess gene expression during early spore development, spores were harvested 716 in two biological replicates at three timepoints: 0, 15, and 30 hours after plating on 717 standard medium plates covered with cellophane. For each sample, six wild type 718 sporangia (derived from a cross of Tak-1 and Tak-2) were collected, and surface-719 sterilized with 1% NaDCC, followed by washing three times with sterile water. The 720 spore suspension was plated and maintained under continuous white light (PFD 50-721 60 µmol m⁻ ² s⁻ ¹) for the indicated durations. 722 For collection, the cellophane with adhered spores was peeled from the medium, 723 flash-frozen in liquid nitrogen, and ground to a fine powder using a mixer mill 724 (RETSCH MM 400) at 30 Hz for 4 minutes. Total RNA was extracted using the 725 RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions, 726 followed by Turbo DNase treatment to remove residual genomic DNA. 727 RNA sequencing was performed at the Wellcome Trust Centre for Human Genetics, 728 University of Oxford, using an Illumina HiSeq 4000 platform generating 75 bp paired-729 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 19 end reads. Each sample was sequenced in five technical replicates, with replicates 730 distributed across five independent sequencing lanes. Raw reads were returned as 731 fastq.gz files. Reads passing quality control (Phred +33 quality score >30 assessed 732 with FastQC) were trimmed by removing the first and last 10 bases, and adapters 733 were removed using Trimmomatic (v0.32) (41). Paired-end reads were interleaved. 734 Residual bacterial rRNA sequences were filtered with SortMeRNA (v2.1b) (42). 735 Reads were error-corrected using BayesHammer within SPAdes (v3.10.1) (43) and 736 aligned to the Marchantia polymorpha reference transcriptome (v3.1) (44) using 737 Salmon (45). 738 739 Differential gene expression analysis was performed with DESeq2 (46). Transcript-740 level quantifications were summarized to gene-level counts using the tximport 741 algorithm, and technical replicates were collapsed using the collapseReplicates 742 function in DESeq2 package (46) in R. Gene annotations were obtained from the 743 Marchantia polymorpha reference genome (v3.1) (functional annotation downloaded 744 from https://marchantia.info/download/v31/). Pairwise comparisons were conducted, 745 with an adjusted p-value (padj) <0.05 considered significant. Genes with 746 log2FoldChange 0 as upregulated, and NA 747 indicated no significant change. 748 Between 0 and 15 hours, 2,953 genes were upregulated, 2,947 downregulated, and 749 13,317 unchanged. Of the 2,953 upregulated genes, 360 were further upregulated, 750 108 downregulated, and 2,485 unchanged at 30 hours. Of the 2,947 downregulated 751 genes, 128 were upregulated, 97 further downregulated, and 2,722 unchanged at 30 752 hours. Among the 13,317 unchanged genes between 0 and 15 hours, 290 were 753 upregulated, 85 downregulated, and 12,942 remained unchanged at 30 hours. 754 755 Mutant generation using CRISPR/Cas9 mutagenesis 756 CRISPR/Cas9 mutagenesis was performed as described in detail in Roetzer et al. 757 (28), including sgRNA design and target site selection, plasmid generation, bacterial 758 transformation, generation of transgenic lines, Agrobacterium -mediated 759 transformation of Marchantia polymorpha sporelings, and mutant screening and 760 genotyping. 761 In brief, CRISPR/Cas9-mediated mutagenesis was used to generate mutant lines for 762 MpNCH1 (Mp5g07060) and Mp NPH3 (Mp5g17270). Single guide RNAs (sgRNAs) 763 were designed using CHOPCHOP (47) or manually for regions not listed among the 764 top hits, based on gene sequences from marchantia.info (MpTak v6.1r2) (48). Each 765 sgRNA consisted of a 20-nt target sequence followed by a canonical NGG PAM, and 766 candidate sgRNAs were checked for uniqueness against the M. polymorpha 767 genome. 768 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 20 sgRNA duplexes were cloned into OP-076 vectors (L2_lacZgRNA-Cas9-CsA) from 769 the OpenPlant kit ( 49), carrying dual antibiotic resistance markers. Recombinant 770 plasmids were introduced into E. coli DH5α cells, verified by Sanger sequencing, and 771 purified for transformation. Mutant lines were generated using spores derived from a 772 cross between Tak-1 and Tak-2 and Agrobacterium-mediated transformation with 773 strain GV3101 (36 ). Transformants were selected on appropriate antibiotic-774 containing media. 775 Genomic DNA from transformants was extracted and PCR-amplified across gRNA 776 target sites. The PCR products were sequenced using Sanger sequencing to identify 777 mutations, and lines harboring mutations predicted to generate premature stop 778 codons were selected. Mutant lines were propagated, and genotypes were 779 repeatedly confirmed. 780 781 Stalk curling index 782 Stalk curling was quantified by calculating a stalk curling index (SCI). For this 783 analysis, individual stalks bearing an antheridial head were imaged under 784 standardized conditions. For each genotype, four independent plants were used. 785 To determine the SCI, two distances between the stalk base and the base of the 786 antheridial head were measured. First, the ‘real length’ was determined by tracing 787 the within-tissue path along the curvature of the stalk from its base to the base of the 788 antheridial head. Second, the ‘shortest distance’ was measured as the linear 789 distance between these same two reference points by drawing a straight line. 790 The stalk curling index was calculated as the ratio of ‘shortest distance’ to ‘real 791 length’. An SCI value approaching 1 indicates a straight stalk, whereas lower values 792 reflect increasing degrees of curvature. Length measurements were performed using 793 the measurement software of the Keyence microscope VHX-7000. 794 795 Sporeling orientation assay 796 Spores were surface-sterilized as described above and resuspended in sterile water 797 (one sporangium per 500 µL). For vertical blue-light assays (Figure 3C, Figure S3A), 798 25 µL of the suspension was evenly distributed onto square plates containing 799 standard growth medium (½-strength B5 Gamborg with 0.5 g/L MES hydrate, 1% 800 (w/v) sucrose, 1% (w/v) plant agar, pH 5.5). All handling prior to illumination was 801 performed under dim green light. After spreading spores with a sterile cell spreader, 802 plates were air-dried for 15 min in a laminar flow hood. 803 To restrict directional illumination, plates were wrapped twice in black, light-804 absorptive aluminium foil, leaving only the upper edge of the plate exposed. Plates 805 were positioned vertically in a climate chamber (Poly Klima Climatic Grow Systems; 806 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 21 Hofmann Kühlung; firmware 03.10.10(22), controller WAGO 750-8212 PFC200 G2 807 2ETH R) at 23 °C under continuous blue light (~30–40 µmol m⁻ ² s⁻ ¹; λ max = 450 nm) 808 for 10 days. 809 For white-light sporeling orientation assays (Figure 3D), spores were sterilized and 810 handled as described above, except that 100 µL of suspension was plated per 811 square plate. Plates were incubated horizontally under continuous white light at 23 812 °C. Images were acquired after 11 days using a Keyence microscope VHX-7000 as 813 detailed above. 814 815 miniTurbo-based proximity labeling (50) and mass spectrometry 816 Transgenic Tak-1 Marchantia polymorpha lines expressing MpPHOT-miniTurbo-YFP 817 (proMpPHOT:MpPHOT-miniTurbo-YFP_line-1 and line-2) and a plasma membrane 818 control ( proMpPHOT:MpPIP-miniTurbo-YFP), as well as the Tak-1 wild-type 819 background, were grown from gemmae on half-strength B5 Gamborg medium 820 supplemented with 1% sucrose, 1% plant agar, and 0.5 g/L MES hydrate (pH 5.5). 821 Plants were grown for 10 days under standard white light on plates covered with 822 cellophane to facilitate rapid thallus harvesting. 823 Proximity labeling under standard continuous white light 824 Ten-day-old thalli were incubated in 100 μ M biotin dissolved in liquid half-strength B5 825 Gamborg medium for 16 h. To stop the biotinylation, plant material was strained and 826 rinsed three times with 200 ml ice cold water, patted dry with paper towels and flash-827 frozen in liquid nitrogen. The frozen plant material was ground into a fine powder by 828 a mortar and aliquoted into technical triplicates of 500 μ L plant powder per sample. 829 Proteins were extracted by adding 1 ml of extraction buffer (50 mM Tris pH 7.5, 150 830 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA, 1 mM EGTA, 831 1 mM DTT, 20 µM MG-132, 1x cOmplete, 1 mM PMSF), incubating for 15 min on a 832 rotor wheel at 4° C. To digest cell walls, 1 µl Lysonase (Millipore) was added per 833 sample, followed by another 15 min incubation at 4° C before sonication in a water 834 bath at high settings for 4x 30 sec with 90 sec breaks. After sonication, samples 835 were centrifuged for 15 min at 4° C, 15000 x g and the supernatants were desalted 836 using PD-10 columns (GE Healthcare) to remove free biotin. The eluate was 837 collected in 5 ml LoBind tubes (Eppendorf) and mixed with 200 μ l Dynabeads 838 MyOne Streptavidin C1 (Invitrogen) slurry that was activated according to the 839 manual and pre-washed with extraction buffer. Streptavidin beads were incubated 840 with the extracts for 16 h at 4 °C on a rotating wheel. Dynabeads were separated on 841 a magnetic rack in 1.5 ml LoBind tubes (Eppendorf) and washed 4x with 1.5 ml cold 842 equilibration buffer, followed by washes with 1x with cold wash buffer I (equilibration 843 buffer with 250 mM NaCl + 1% SDS), 1x with cold equilibration buffer, 2x with cold 844 wash buffer II (equilibration buffer with 250 mM NaCl), 2x with cold wash buffer III 845 (equilibration buffer with 500 mM NaCl), 1x with cold equilibration buffer and 2x with 846 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 22 Urea wash buffer (2 M Urea, 50 mM Tris pH 7.5, room temperature) as also 847 described in Mair et al (51) and Wallner et al (52). Beads were resuspended in 1 ml 848 cold 50 mM Tris pH 7.5 and transferred into a new 1.5 ml LoBind tube and all buffer 849 was removed using a magnetic rack. Beads were frozen at -70°C for further 850 processing. 851 Proximity labeling under dark and blue-light conditions 852 The same MpPHOT transgenic lines were grown and adapted in complete darkness 853 for 24 h. Plants were divided into three conditions: 1) labeling in darkness, 2) blue-854 light activation immediately upon transfer, and 3) continuous blue-light exposure for 4 855 h before labeling. Biotin treatment was performed with 200 μ M dissolved in liquid 856 half-strength B5 Gamborg medium for 4 h. Further processing of plant material was 857 done as described for white light conditions. 858 On-bead digestion 859 Beads were resuspended in 60 μ L 100 mM ammonium bicarbonate with 600 ng Lys-860 C (Fujifilm Wako) and incubated for 4 h at 37 °C on a thermoshaker (1200 rpm). The 861 supernatant was reduced with 10 mM TCEP for 30 min at 60 °C and alkylated with 862 40 mM MMTS for 30 min at room temperature. Digestion was completed with 600 ng 863 trypsin (Trypsin Gold, Promega) overnight at 37 °C. Digests were acidified with 10 μ L 864 10% TFA and submitted to LC-MS/MS. 865 nanoLC-MS/MS analysis 866 Peptides were separated on a Vanquish Neo UHPLC system coupled to an Orbitrap 867 Exploris 480 mass spectrometer with FAIMS pro interface and Nanospray Flex 868 source. Trap and analytical columns were PepMap Acclaim C18 (5 μ m, 100 Å) with 869 dimensions 5 mm × 300 μ m ID (trap) and 500 mm × 75 μ m ID (analytical). Peptides 870 were eluted at 230 nL/min using a 120-min linear gradient from 2% to 35% 871 acetonitrile in 0.1% formic acid. Data were acquired in data-dependent acquisition 872 mode (DDA) with full scans at 60,000 resolution, m/z 350–1200, and MS/MS scans 873 of the most abundant ions with HCD at 30% collision energy. Compensation voltages 874 of -45, -60, and -75 V were applied with cycle times of 0.9 s (CV -45 and -60) and 0.7 875 s (CV -75). Precursor ions of charge 2-6 were selected for fragmentation, with 876 dynamic exclusion of 45 s. 877 Data processing and analysis 878 RAW files were processed in Proteome Discoverer 2.5 with MSAmanda 2.0. 879 Peptides were searched against the UniProt Marchantia polymorpha database 880 (34,956 sequences) and common contaminants. Fixed modification: beta-881 methylthiolation of cysteine; variable modifications: methionine oxidation, 882 phosphorylation (S/T/Y), deamidation (N/Q), N-terminal pyro-glutamate, and N-883 terminal acetylation. FDR was set to 1% at PSM and protein levels using Percolator, 884 and a minimum Amanda score of 150 was applied. Proteins were quantified with 885 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 23 IMP-apQuant (MaxLFQ algorithm) and normalized using iBAQ with sum 886 normalization across samples. Match-between-runs was applied, and proteins were 887 filtered to require at least three quantified peptides per protein. 888 Volcano plot generation 889 Differential enrichment analysis was performed comparing each transgenic line to 890 wild type and the control line. Volcano plots were generated in RStudio using 891 packages ggplot2 and ggrepel. Significantly enriched proteins were determined by a 892 p-value 2. 893 Proximity labelling - data availability 894 Mass spectrometry proteomics data from the proximity labelling experiments have 895 been deposited to the ProteomeXchange Consortium via the PRIDE partner 896 repository with dataset identifier PXD072764 and will be available upon publication 897 of the peer-reviewed paper. 898 899 Quantification and statistical analysis 900 Sequence assemblies and plasmid maps were generated using CLC Genomics 901 Workbench v9.5.1 (Qiagen). Gene sequences were obtained from MarpolBase 902 (https://marchantia.info/) (48). All experiments were repeated with 3-5 independent 903 biological replicates. Sample sizes are indicated in the respective figure legends. 904 Data analysis and visualization were carried out using RStudio (v2022.07.2, Build 905 576) and GraphPad Prism (v8.1.1). Statistical analyses were performed in RStudio 906 and GraphPad Prism. 907 908 Figure Legends 909 Figure 1. Light positively regulates the division of the M. polymorpha spore. 910 (A) SEM image of dry M. polymorpha spores. Scale bar = 2 µm. (B) TEM image of a 911 wild type spore 24 h after initiation of imbibition, showing dense cytoplasm and a 912 nucleus (n) located near the cell centroid. Scale bar = 3 µm. (C) Representative 913 bright-field images of spores at the indicated times after plating. (D) Visualization of 914 nucleus position in spores expressing nuclear and plasma membrane markers 915 (proMpROP:mScarletI-N7 and proMpUBE2:mScarletI-AtLTI6b). Spores were imaged 916 after 12, 24, 29, and 32 h. Maximum intensity projections (xy) of 3D z-stacks from 917 five representative spores are shown. Scale bar = 10 µm. (E) Schematic of the two-918 cell spore stage following the first cell division, showing a larger apical cell and a 919 smaller basal cell. The newly formed cell wall is oriented perpendicular to the apical-920 basal axis. (F) Spores grown for 2 days in darkness, white light, blue light, or red 921 light on nutrient media with or without sucrose. Scale bars = 25 µm. (G) 922 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 24 Quantification of spore cell division after 48 h. Proportions of dividing and non-923 dividing spores (total n = 566) are shown for each condition. Chi-square tests of 924 independence revealed significant effects of light treatment both with sucrose ( χ ²(3) 925 = 161.7, p < 0.0001) and without sucrose ( χ ²(3) = 72.1, p < 0.0001). Different letters 926 indicate statistically significant differences (p < 0.05); identical letters indicate no 927 significant difference. 928 Figure S1. Light positively regulates spore division in M. polymorpha . Related 929 to Figure 1F-G. 930 (A) Overview images of spores grown for 2 days in darkness, white, blue, or red light 931 on nutrient media with or without sucrose. Each overview image shows several 932 spores used for quantification. Cell division occurred under all light conditions but not 933 in darkness. Scale bars = 50 µm. (B, C) Quantification of spore cell division under 934 white light and darkness after 5 days (B; n = 148) and 7 days (C; n = 123), with and 935 without sucrose. For both time points, light treatment had a significant effect on cell 936 division with sucrose (5 days: χ ²(1) = 4.56, p = 0.033; 7 days: χ ²(1) = 3.98, p = 0.046; 937 chi-square test) and without sucrose (5 and 7 days: Fisher’s exact test, p < 0.0001). 938 Different letters indicate statistically significant differences (p < 0.05); identical letters 939 indicate no significant difference. 940 941 Figure 2. Blue light orients the first cell division. 942 (A) Radial histogram of wild type spores (n = 51) grown for 48 h in unidirectional 943 white light in 3D agar culture in chambered microscopy slides. Illumination from 944 above aligned light and gravity vectors. The new cell wall formed perpendicular to 945 the incident light, with the apical cell on the illuminated side and the basal cell on the 946 relatively shaded side. (B) Radial histogram of wild type spores (n = 49) grown for 48 947 h in unidirectional white light with light and gravity vectors opposed (illumination from 948 below). The new cell wall formed perpendicular to the incident light, and the basal 949 cell developed on the relatively shaded side. These data indicate that light, not 950 gravity, orients asymmetric division. (C) Reorientation experiments show that division 951 orientation remains labile until shortly before division. Spores (n = 318) were grown 952 in unidirectional white light, and the direction of light was reversed by 180° at 953 different time points; all samples were analyzed after 48 h. In controls (never 954 reoriented), the basal cell formed on the relatively shaded side. (D) Orientation 955 assays under different light conditions show that blue light orients the first cell 956 division. Spores were grown for 48 h in white, red, blue, or red+blue light in 3D agar 957 culture. All orientation experiments were performed using wild type spores 958 expressing the fluorescent marker proMp SYP13A:mCitrine:MpSYP13A to visualize 959 cell division. 960 Figure S2. Quantification and experimental setup for spore division orientation 961 assays. Related to Figure 2. 962 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 25 (A) Schematic of cell division orientation measurement. When the new cell wall 963 (black) is oriented perpendicular to the light vector (yellow), the apical–basal axis 964 (green) aligns with the light vector (left). When the cell wall is not perpendicular to 965 the light vector, the axes are misaligned (right). The angle (blue) between the light 966 vector and the apical–basal axis was measured for all spores used in orientation 967 assays. (B) Radial histograms of spores used for reorientation experiments (Figure 968 2C). Only asymmetrically dividing spores were included. Light direction was reversed 969 at 4.5 h (n = 40), 7.5 h (n = 40), 17 h (n = 40), 24 h (n = 35), 29 h (n = 41), and 35 h 970 (n = 40), with a non-reoriented control (n = 38). All spores were imaged and 971 quantified after 48 h total illumination. (C) Image of the custom-built illumination 972 system (“lightbox”) used for spore orientation assays. The open-front view shows 973 four units for chambered slides enabling unidirectional illumination. Details are 974 described in Materials and Methods. (D) Radial histogram of spores dividing after 7 975 days in darkness on sucrose-supplemented medium (n = 35). Cell wall orientation 976 was random. 977 978 Figure 3. MpPHOT-mediated blue light sensing orients the first cell division. 979 (A) Blue light receptor genes are expressed early during spore development. Shown 980 are normalized mRNA read counts at 0 h (blue), 15 h (orange), and 30 h (green); two 981 biological replicates per time point; mean with SD. (B) Positive phototropism of 982 gametangiophore stalks is defective in Mp phot mutants. Stalk curling was quantified 983 in 8-week-old plants grown under inductive conditions (n = 46 across three 984 genotypes). Scale bar = 5 mm. Data were tested for normality (Shapiro-Wilk); as 985 assumptions were not met, a Kruskal–Wallis test with Dunn’s multiple comparisons 986 was performed. Different letters indicate significant differences (p < 0.05); identical 987 letters indicate no significant difference. (C) Rhizoids of Mp phot mutant sporelings 988 are defective in negative phototropism in blue light. In wild type, rhizoids of 10-day-989 old sporelings grew away from the blue light source, whereas mutant rhizoids grew 990 in many directions. Scale bar = 1 mm. (D) Rhizoids of 11-day-old Mp phot mutant 991 sporelings grew in many directions in white light. Under standard white-light 992 conditions on horizontal plates, rhizoids were visible in mutants but not in wild type. 993 Spores were densely plated and imaged from above. Scale bar = 2 mm. (E) 994 MpPHOT is required to orient asymmetric cell division. Wild type and Mpphot mutant 995 spores expressing pro MpSYP13A:mCitrine:MpSYP13A were grown for 48 h in 996 c ontinuous blue light. In wild type, the basal cell formed on the shaded side, and the 997 new cell wall was oriented perpendicular to the incident light. In Mp phot-3 and 998 Mpphot-4, the position of the basal cell and cell wall orientation were random. 999 Figure S3. Rhizoids of Mpphot-4 mutant sporelings are defective in negative 1000 phototropism in blue light. Related to Figure 3C. 1001 (A) Rhizoids of wild-type sporelings grew away from the blue light source, while 1002 Mpphot-4 mutant rhizoids grew in multiple directions. Sporelings were grown for 10 1003 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 26 days in continuous blue light on vertical plates with illumination from above. Scale 1004 bar = 1 mm. 1005 1006 Figure 4. MpNCH1 mediates MpPHOT-dependent orientation of asymmetric cell 1007 division. 1008 (A) Proximity-labeling-based proteomics identifies proteins in close proximity to 1009 MpPHOT. MpPHOT–miniTurbo(ID)–YFP fusion proteins were used to biotinylate 1010 proteins in continuous white light, followed by mass spectrometry. The volcano plot 1011 shows differential enrichment relative to wild type (Tak-1) from triplicate samples (p 2). MpPHOT and miniTurbo–YFP are enriched as expected (self-1013 labeling), together with MpNCH1, MpBLUS, MpNPH3, and additional proteins 1014 highlighted in the graph. Eleven proteins enriched in two independent lines and 1015 expressed during early spore development (0–30 h; spore transcriptomes, Figure 3) 1016 were selected for further analysis. Additional comparisons of independent 1017 experimental lines with compartment and wild-type controls are provided in Table S1. 1018 (B) Positive phototropism of gametangiophore stalks is defective in Mpnch1 mutants. 1019 Scale bar = 5 mm. (C) Quantification of stalk curling in 8-week-old plants grown 1020 under inductive conditions (n = 37 across three genotypes). Data were tested for 1021 normality (Shapiro-Wilk); non-normal data were analyzed using a Kruskal-Wallis test 1022 with Dunn’s multiple comparisons. Different letters indicate significant differences (p 1023 < 0.05); identical letters indicate no significant difference. (D) MpNCH1 is required to 1024 orient asymmetric cell division. Wild-type and Mp nch1 spores expressing 1025 proMpSYP13A:mCitrine:MpSYP13A were grown for 48 h in continuous blue light. In 1026 wild type, the basal cell formed on the shaded side and the new cell wall was 1027 oriented perpendicular to the incident light, whereas both were random in Mp nch1. 1028 (E) MpNPH3 is not required for orientation of asymmetric cell division. Mp nph3 1029 spores grown under the same conditions showed wild type-like orientation. (F) 1030 MpNCH1–VENUS and MpPHOT–mCherry co-localize in spores. The cytofluorogram 1031 shows pixel intensity correlation from a single plane of an individual cell 1032 (thresholded; Gaussian blur, radius = 2). Signals are strongly correlated (Pearson’s r 1033 = 0.878), indicating overlapping localization. Spores were grown for 36 h on 1034 cellophane-covered medium, fixed, cleared, and imaged by confocal microscopy. 1035 Scale bar = 10 µm. 1036 Figure S4. Selection of candidate genes and co-localization of MpNCH1–1037 VENUS and MpPHOT–mCherry in the undivided spore. Related to Figure 4. 1038 (A) A total of 137 loss-of-function mutant lines were generated for selected genes 1039 using CRISPR–Cas9. 1040 (B) MpNCH1–VENUS and MpPHOT–mCherry co-localize in spores before the first 1041 cell division. Representative single-plane confocal images of an undivided spore are 1042 shown, including individual channels and the merged image. Co-localization analysis 1043 revealed a strong correlation between the signals (Pearson’s r = 0.909), indicating 1044 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint 27 overlapping subcellular localization. Spores were grown for 24 h on cellophane-1045 covered medium, then fixed, cleared, and imaged by confocal microscopy. Scale bar 1046 = 10 µm. 1047 1048 Supplementary Material 1049 Figures: S1 to S4 1050 Table S1: MS_table 1051 Data S1: Lightbox 1052 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted March 24, 2026. ; https://doi.org/10.64898/2026.03.22.713455doi: bioRxiv preprint