SWI3 regulates male sex determination in Marchantia polymorpha

SWI3 regulates male sex determination in Marchantia polymorpha | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results SWI3 regulates male sex determination in Marchantia polymorpha View ORCID Profile Maria Lozano-Quiles , View ORCID Profile Parth K. Raval , View ORCID Profile Stefan A Rensing , View ORCID Profile Sven B. Gould doi: https://doi.org/10.1101/2025.10.29.685095 Maria Lozano-Quiles 1 Institute for Molecular Evolution, Heinrich–Heine–Universität Düsseldorf , 40225 Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maria Lozano-Quiles Parth K. Raval 1 Institute for Molecular Evolution, Heinrich–Heine–Universität Düsseldorf , 40225 Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Parth K. Raval Stefan A Rensing 2 Faculty of Chemistry and Pharmacy, Albert-Ludwigs-Universität Freiburg , 79104 Freiburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefan A Rensing Sven B. Gould 1 Institute for Molecular Evolution, Heinrich–Heine–Universität Düsseldorf , 40225 Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sven B. Gould For correspondence: gould{at}hhu.de Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Land plants alternate between multicellular haploid and diploid phases, a life cycle requiring tight coordination between vegetative growth and sexual reproduction. Chromatin regulators such as SWI3, a core subunit of the SWI/SNF complex, are ancestral and highly diversified in angiosperms. As their functions outside of angiosperms remain unclear, we investigated SWI3 of the liverwort Marchantia polymorpha , a non-vascular dioecious land plant. A mutation in the promoter of Mp SWI3 affected gametangiophore development and spermiogenesis in males, revealing its male-specific role in reproductive development. The Mp SWI3 mutant line amplifies vegetative propagation in males under conditions that would normally induce reproductive growth. The phenotype was underpinned by transcriptomic changes, showing that MpSWI3 modulates key regulators of gametangiophore initiation (e.g. BONOBO, GLID), sperm development and motility (e.g. DUO1, PKAR), and asexual reproduction (e.g. KAI2). Together, this positions Mp SWI3 as a chromatin-level regulator balancing vegetative and reproductive phases, highlighting an ancient epigenetic function that coordinates developmental phase transitions in land plants. Introduction Land plants (Embryophyta) exhibit a haplodiplontic life cycle, in which both haploid and diploid phases are multicellular and proliferate through mitosis ( Niklas and Kutschera 2010 ; Bowman et al. 2017 ; de Vries and Archibald 2018 ). The haploid gametophyte produces gametes mitotically, whereas the diploid sporophyte produces spores meiotically that develop into new gametophytes. This ‘alternation of generations’ contrasts with the diplontic life cycle of animals and the haplontic cycle of many streptophyte algae, where only the diploid and haploid phase, respectively is multicellular ( Niklas and Kutschera 2010 ; Bowman et al. 2017 ). Nevertheless, all eukaryotic life cycles progress through an encounter and fusion of gametes that marks the onset of the diploid phase with the formation of a zygote. In most eukaryotes, including algae ( Kalshoven et al. 1990 ; McCourt et al. 2004 ), this encounter depends on flagellated male gametes (sperms or spermatozoids). Following the transition of the streptophyte algal ancestor of land plants onto land, early plants still depended on flagellated spermatozoids and liquid media or a moist substrate for sperm motility and fertilization. While this is still true for the majority of land plant lineages, flagellated spermatozoids were lost twice, within conifers and angiosperms( Meyberg et al. 2020 ). Extant gymnosperms such as cycads produce both flagellated sperm and pollen, whereas angiosperms (flowering plants) rely entirely on pollen for fertilization ( Renzaglia 2001 ; Niklas and Kutschera 2010 ). Bryophytes, which diverged from vascular plants soon after plant terrestrialization, retain flagellated spermatozoids and in contrast to spermatophytes are gametophyte (haploid) dominant( Pandey et al. 2022 ). As such, they provide a valuable system to study the regulation and molecular control of male reproductive development in plants( Renzaglia 2001 ). Reproductive development, which mediates the transition between haploid and diploid phases through gamete fusion, is orchestrated by genetic and epigenetic regulators that likely date back to the origin of eukaryotes ( Goodenough and Heitman 2014 ; She and Baroux 2014 ; Speijer et al. 2015 ). In plants, this regulation involves DNA-binding proteins, chromatin modifiers, and transcription factors that together form networks coordinating developmental transitions through epigenetic and protein–protein interactions ( She and Baroux 2014 ; Borg and Berger 2015 ). Among these, chromatin-remodeling complexes (CRCs) form a nexus between regulatory signaling and chromatin-based transcriptional control ( Clapier and Cairns 2009 ; Han et al. 2013 ). SWI3, a key subunit of the SWITCH/SUCROSE NONFERMENTING (SWI/SNF) CRC, modulates nucleosome architecture via H3 acetylation at lysine 27 (H3K27), thereby activating reproductive genes and counteracting PRC2-mediated H3K27 methylation ( Sarnowski et al. 2005 ; Zheng and Chen 2011 ; Wu et al. 2012 ). In Arabidopsis , SWI3-dependent acetylation of floral identity and reproductive genes (e.g., AP3 and SEP) ensures proper flowering timing and viable seed formation ( Molitor et al. 2014 ; Yan et al. 2019 ). This activity counteracts H3K27me3-mediated repression that silences key reproductive regulators such as FLOWERING LOCUS C (FLC) and maintains AGAMOUS, APETALA3 , and SEPALLATA genes inactive until floral induction ( Jiang et al. 2013 ; Zheng et al. 2019 ) . In flowering plants, the SWI3 family has diversified into four members forming two phylogenetic groups, SWI3A/B and SWI3C/D. Likely due to an ancestral genome reduction event( Zhang et al. 2020 ; Donoghue et al. 2021 ; Harris et al. 2022 ; Linde et al. 2023 ), however, bryophytes encode only a single SWI3A/B-type gene ( Genau et al. 2021 ). In the moss Physcomitrium patens , SWI3A/B mutants fail to degrade cell-wall layers during spermatogenesis, resulting in impaired sperm motility, incomplete cytoplasmic reduction, infertility, and defective male reproduction ( Genau et al. 2021 ). While SWI3A/B function has been investigated in Arabidopsis and Physcomitrium , both species are monoecious, producing male and female reproductive organs on the same individual ( Koornneef and Meinke 2010 ; Rensing et al. 2020 ). As a result, possible sex-specific roles of SWI3 in gametophyte differentiation, transcriptional regulation, and parental contribution remain unclear. Marchantia polymorpha , a dioecious bryophyte with sex chromosomes determining distinct male and female individuals ( Bowman 2016 ), hence provides a system to address this issue. Furthermore, genes controlling gametangial development are only expressed during gametangiogenesis in Physcomitrium ( Perroud et al. 2018 ), but they are already differentially expressed during the vegetative stage in Marchantia , hinting at their sex-specific regulation in the liverwort already before its reproductive organs form( Hisanaga et al. 2019 ). Here, we establish that Marchantia SWI3 is a critical regulator in coordinating the transition from vegetative to reproductive growth in male plants and that it governs key aspects of sexual development. Remarkably, Mp SWI3 overexpression mutants reinforce vegetative propagation instead of sexual reproduction, suggesting MpSWI3 acts as an epigenetic control point between these distinct strategies of survival and reproduction. Results SWI3 regulates reproductive development and spermiogenesis in Marchantia To investigate the role of SWI3 in the haploid-to-diploid transition and sexual development, we attempted to generate distinct male and female lines with mutations in Marchantia SWI3 , using CRISPR/Cas9( Sugano et al. 2018 ). sgRNAs targeting the MpSWI3 coding region never yielded any viable transformants, but sgRNA targeting the 5’ upstream region provided us with male and female mutants carrying insertions, deletions, or substitutions in their promoter (Fig. S1a). Quantitative real-time PCR showed all lines to overexpress MpSWI3 in comparison to the WT (Fig. S1b), which is why we refer to them as swi3 OE-♂ and swi3 OE- ♀ hereafter. The vegetative growth was unaffected in both the swi3 OE lines ( Fig. 1a ). After two weeks under far red light – which induces the transition to reproductive phase – swi3OE -♂, however, produced approximately half as many antheridiophores (male gametangiophores) as the wild type (WT). In contrast, archegoniophore (female gametangiophores) production in the mutants was comparable to the WT ( Fig. 1a-b ). These results established Mp SWI3 as a gametangiophore development regulator in males. Download figure Open in new tab Fig. 1: Mp SWI3 mutants show defective antheridiophore formation and altered spermiogenesis. (a) Representative 30 days-old male and female plants from wild-type and swi3 OE lines. Full view of the thalli with their gametangiophores highlighted by circles, which are shown enlarged in the squares. (b) Gametangiophore maturation over time (n=15). (c) Representative images of sperms (left) and sperm motility patterns (right) from WT-♂ and swi3 OE - ♂. Scale bar: 20μm. (d) Mean speed, linearity of forward progression, and mean straight line speed of sperms from WT and swi3 OE-♂ lines (n=10). Statistically significant differences (P value < 0.05) are indicated by asterisks. (e) LogFC values for a key subset of genes differentially expressed in swi3 OE-♂ as compared to the WT-♂ under far-red light conditions; genes with significant differences in their expression are marked by a blue outline. Given the role of Mp SWI3 in antheridiophore development, we next examined whether swi3 OE-♂ antheridiophores produced motile sperms. WT spermatozoids matured to stage-5 as expected, whereas those of swi3 OE-♂ frequently arrested at the earlier stages ( Fig. 1c ). In WT, 38.5% of sperm remained capsulated, whereas in swi3 OE-♂, this proportion increased to 55.6% ( Fig. 1c ). Swi3 OE-♂ sperms also exhibited a significantly higher straight-line speed, greater linearity of forward progression, and overall increased swimming velocity ( Fig. 1d ). These results indicate that Mp SWI3 influences both early spermiogenesis and motility dynamics of mature sperms. To uncover the molecular basis of SWI3’s role in regulating reproductive development and spermiogenesis in male Marchantia plants, we performed comparative RNA-seq analysis of plants grown under far-red light. Swi3 OE-♂ showed an upregulation of 584 genes and a downregulation of 409 (|log 2 FC| > 2) (Fig. S2a, Table S1). Functional annotation of differentially expressed genes revealed enrichment for categories related to plant defence, peroxidase activity, and cell-wall remodelling (Fig. S2b). We next examined genes specifically linked to sexual and asexual reproduction. Among those involved in gametangiophore and gametangia development, Mp BONOBO (Mp BNB ) and Mp GLID were strongly upregulated (log 2 FC > 3), whereas Mp PIF , Mp CKI1 , Mp CPS , and Mp KOL2/3 were downregulated ( Fig. 1e ). Genes associated with the haploid-to-diploid transition showed a mixed pattern: Mp BELL1 and Mp BELL5 were upregulated, while Mp KNOX1 and Mp BELL4 were downregulated. Transcripts linked to sperm differentiation and motility, including Mp DUO1 (a master regulator of spermiogenesis in land plants ( Higo et al. 2018 )), Mp PKAR (a regulator of sperm motility( Yamamoto et al. 2024 )) and a flagellar radial spoke protein homolog (Mp6g07660), were upregulated ( Fig. 1e , Table S2). These patterns are consistent with the morphology and motility changes observed in swi3 OE-♂ sperms in Marchantia ( Fig. 1c–d ) as well as Physcomitrium , where SWI3a/b loss of function led to impairment in late maturation( Genau et al. 2021 ). SWI3 overexpression reinforces vegetative growth in lieu of reproductive development Because Mp SWI3 influenced both gametangiophore development and sperm maturation under far red light, we examined whether it also affected the vegetative to reproductive transition under far red light. WT plants showed the expected morphological responses (e.g. thallus flattening, gametangiophore formation, and empty gemma cups; gemma are vegetative propagules). However, swi3 OE-♂ produced significantly more gemma cups (16.2 ± 1,5) than WT-♂ (10.5 ± 1,2), whereas swi3 OE -♀ and WT-♀ showed no difference (Fig. 3a–b). Interestingly, while WT and swi3 OE-♀ suppressed gemmae production, swi3 OE-♂ produced 13.5-fold more gemmae at day 10 ( Fig. 2 ). These results demonstrate that Mp SWI3 overexpression promotes a shift to asexual reproduction through excessive gemma production under far-red light, instead of sexual reproduction via gametangiophore development. This shift was facilitated by expression changes in a number of genes involved in vegetative growth ( Fig. 2d ). This included the substantial overexpression of Mp KAIB, a key gemmae-formation factor contributing to the excessive gemma formation in swi3 OE-♂( Komatsu et al. 2023 ) ( Fig. 2 ). Moreover, several bHLH transcription factors ( Mp SETA, Mp BHLH29, Mp BHLH30, Mp BHLH31, Mp BHLH21, and Mp PIF) were downregulated, whereas late embryogenesis-related genes such as Mp LEA-like44 and Mp LEA-like45 were strongly upregulated (Fig. S2c). Together, these transcriptional changes suggest that Mp SWI3 may regulate genes related to sexual differentiation as well as asexual propagation, positioning it as a chromatin-level switch in the liverwort, balancing reproductive and vegetative growth, rather than unidirectional control of reproductive onset. Download figure Open in new tab Fig. 2: swi3 OE-♂ overproduce gemmae under far-red light. (a) Representative plants of 14d-old wild-type and swi3 OE lines. Plants outlined with a grey dashed border were grown under continuous white light, whereas those outlined in red were supplemented with far-red light. Scale bar: 0.4cm. (b) Closer view of representative gemmae cups of plants grown with supplemented far-red light. Scale bar: 0.2cm. (c) Gemma cup formation over time and number of gemmae per cup counted; n=15 for thalli and cups analysed. (d) LogFC values for key genes involved in gemma formation in swi3 OE-♂ (as compared to the WT-♂) under far-red light conditions. Genes with significant differences in expression are marked by a blue outline. Discussion Sexual reproduction is a defining trait of eukaryotic life, ensuring genetic recombination through the fusion of haploid gametes to form a diploid zygote( Goodenough and Heitman 2014 ; Speijer et al. 2015 ; Garg and Martin 2016 ; Raval et al. 2022 ; Jeffries et al. 2025 ). Across eukaryotic lineages, this fundamental process is elaborated by mechanisms that regulate the differentiation, compatibility, and union of gametes – from mating-type systems in unicellular algae and fungi to complex sexual organs in multicellular plants and animals( Heitman 2015 ; Yadav et al. 2023 ; Rizos et al. 2024 ; Becker et al. 2025 ). This diversification accompanied the transition from aquatic to terrestrial habitats, where new reproductive structures and regulatory cues evolved to ensure gamete encounter and fertilization in plants( Niklas and Kutschera 2010 ; She and Baroux 2014 ; Mori et al. 2015 ; Becker et al. 2025 ). These reproductive structures range from specialized gametangia to the evolution of pollen and seeds, which together enable fertilization independent of a wet habitat. Soon after the algal transition to land, and prior to the extensive diversification of reproductive structures seen in vascular plants, the ancestor of all bryophytes emerged after genome reformatting( Zhang et al. 2020 ; Donoghue et al. 2021 ; Harris et al. 2022 ; Linde et al. 2023 ). Consequently, extant bryophytes allow to explore the emergence of developmental and regulatory complexity underlying sexual differentiation, and the transition between vegetative and reproductive phases from a different perspective. Much of our understanding of the regulatory networks governing the transition between vegetative and reproductive phases, however, comes from studies in angiosperms. Here, numerous transcription factors, signalling cascades, and chromatin-remodelling complexes have been characterized(Blazquez & Weigel 2000; Andrés and Coupland 2012 ; He 2012 ). A key role for SWI3 is evident in Arabidopsis , where it regulates multiple reproduction-related developmental traits, including flowering time and reproductive organ formation( Sarnowski et al. 2005 ). Loss of At SWI3A or At SWI3B results in embryo abortion and male sterility, respectively, while mutations in At SWI3C or At SWI3D alter sexual-organ development( Sarnowski et al. 2005 ). Our findings extend this role to the bryophyte Marchantia , establishing Mp SWI3 as a key regulator of the vegetative–reproductive balance in a sister lineage to seed plants. Sexual reproduction in Marchantia is induced through the coordinated action of light-signalling pathways that integrate far-red irradiance and photoperiodic cues( Inoue et al. 2019 ). Key genes include Mp BNB and Mp GLID , which drive the formation of male gamete precursor cells( Ren et al. 2024 ). In swi3 OE-♂, both these genes are significantly upregulated, yet the plants developed fewer gametangiophores. It suggests that Mp SWI3 overexpression may cause premature or ectopic activation of these two genes in vegetative tissues, producing – as previously reported( Ren et al. 2024 ) – a transcriptional signature of reproductive induction without proper organ differentiation. Such an uncoupling between germline specification and organogenesis in swi3 OE -♂ likely reflects impaired FR-phytochrome-PIF signalling and reduced gibberellin (GA) biosynthesis, consistent with the observed downregulation of Mp PIF , Mp CPS , and Mp KOL . PIF- and GA-dependent pathways act upstream of the Mp BNB– Mp GLID module to promote organ initiation and maturation; their downregulation would therefore weaken gametangiophore formation, even when germline markers remain elevated( Sun et al. 2023 ). Similarly, GA-deficient mutants produce fewer and morphologically abnormal gametangiophores( Sun et al. 2023 ). Together, these observations suggest that SWI3 overexpression disrupts the coordination between germline activation and organ differentiation. Beyond its role in male reproductive development, our results uncover a striking reversal of developmental fate, as excess SWI3 reinforces vegetative propagation through excessive gemmae production in swi 3OE-♂ under conditions that normally induce reproductive growth. A key regulator of gemmae production and vegetative growth is Mp KAI2, which drives gemma cup formation and gemma initiation( Komatsu et al. 2023 ). Strong overexpression of Mp KAI2B in swi3 OE-♂ lines underlies this reversal towards vegetative growth despite reproduction-inducing cues. This finding highlights a broader role for SWI3 in balancing vegetative and reproductive programs as an epigenetic modulator of phase transitions in the bryophyte. Such a shift toward vegetative propagation, particularly in lines where reproductive growth is compromised, may reflect a bet-hedging strategy in which vegetative reproduction ensures survival when sexual reproduction is impaired. This regulatory flexibility could allow plants in nature to abort or delay reproductive development, when conditions are suboptimal for fertilization, maintaining persistence through vegetative growth. Such environmentally driven developmental plasticity might have been crucial for the survival of early land plants adapting to novel terrestrial stresses and continues to contribute to the resilience of extant bryophytes that thrive across diverse habitats. Altogether, our findings position SWI3 as a key chromatin regulator orchestrating the balance between vegetative propagation and sexual reproduction in Marchantia males. This is the first report of sex-specific roles of SWI3, and during the vegetative stage (before gametaniophore formation), in contrast to other species ( Genau et al. 2021 ) ( Sarnowski et al. 2005 ). By modulating the accessibility of key regulatory loci, Mp SWI3 likely integrates environmental and developmental cues to determine whether cells commit to vegetative or reproductive fates. These results underscore that epigenetic mechanisms were essential for synchronizing gene expression with developmental contex during the origin of embryophytes. On the molecular level, SWI3 via modulation of H3 K27 acetylation probably acts as a counterpart of PRC2, a writer of H3K27me3 repressive marks( Sarnowski et al. 2005 ; Zheng and Chen 2011 ; Wu et al. 2012 ). Factors such as far red light apparently influence this balance, and an unbalance such as by the overexpression in swi3 OE-♂ is able to tip the system into different states. Understanding how SWI3-mediated chromatin remodeling governs these ancestral phase transitions reveals the molecular basis of reproductive plasticity in bryophytes and provides first evolutionary insight into how chromatin dynamics and diversification of factors such as SWI3 facilitated the shift from reversible vegetative–reproductive balance in early land plants to fixed floral commitment in angiosperms. The mechanisms by which chromatin regulation integrates environmental and developmental signals to determine reproductive fate remain poorly understood outside of angiosperms. Bryophytes, particulary dioecious species, provide a powerful model to uncover how these ancestral processes evolved into the complex reproductive strategies of terrestrial plants. Materials and methods Plant growth conditions Marchantia polymorpha (BoGa ecotype; Botanical Garden, Osnabrück University, Germany) was cultivated on half-strength Gamborg’s B5 medium containing 1% glucose and 1% agar under continuous full light spectrum (450-700nm) 70 μmol m -2 s -1 at 22°C. To induce the reproductive stage, the light conditions were supplemented with far-red light (700-750nm) 40 μmol m -2 s -1 at 22°C in ECO 2 boxes (oval, 80 mm) with a green filter #40 (Duchefa Biochemie). Generation of mutant plants CRISPR/Cas9-based genome editing of MpSW3a/b (Mp8g15610) was performed as previously described ( Sugano et al. 2018 ). Three sgRNAs (sgRNA1,2,3) were designed, sgRNA1 targeted the promoter region, sgRNA2 and sgRNA3 targeted the coding sequence of Mp SWI3 (Fig. S1). Cloning of sgRNA was performed using pMpGE_En03 as the entry- and pMpGE010 as a destination vector using LR Clonase II enzyme mix (Thermo Fisher Scientific). Destination vectors were introduced into electrocompetent Agrobacterium tumefaciens (GV301 without pSOUP) by electroporation (Bio-Rad GenePulser Xcell, 1.44 kV). Agrobacterium -mediated sporeling transformation was performed as described previously( Ishizaki et al. 2008 ). After three days of spore co-cultivation with Agrobacterium transfectants, sporelings were washed three times with liquid ½ Gamborg’s B5 medium to remove Agrobacteria and plated on agar plates with half-strength Gamborg’s B5 medium with 1% Glucose supplemented with 10 μg/mL Hygromycin and 100 μg/mL Cefotaxime for selection. Macroscopic phenotyping To assay gametangiophore numbers, 15 plants per line were grown in ECO 2 boxes that allow the upwards growth of gametangiophores. Each gametangiophore and gemma cup was counted as soon as it could be seen by sight from the top of the thallus. To quantify gemmae per cup, all gemmae from gemmae cups closer to the thallus base were harvested by a toothpick, gemmae were spread on a plastid sheet, imaged and counted using ImageJ(1.54f). Sperm phenotyping Sperms were harvested by applying water on top of the antheridia from 30 days old plants cultivated under far red light. After 5 min of incubation, the water was collected and sperm morphology monitored with a Nikon eclipse Ti imaging platform. For quantitative motility analysis, sperms were video-recorded at the resolution of 1280 x 1024 pixels and at the rate of 33 frames per second (fps) by a Nikon Digital Sight DS-U3. Movies were converted into a sequence of individual frames and sperm movement tracked by the ImageJ plugin TrackMate v7.11.1. Two movies were analyzed to obtain sperm swimming motility parameters for each line. RNA-seq Total RNA was extracted from WT and swi3 OE-♂ plants incubated under far red light for 14 days, using the RNeasy Plant Mini Kit for RNA Extraction (Qiagen). Three biological replicates were prepared for each genotype and mRNA isolated via their poly(A) tail. RNA integrity and concentration were assessed using a NanoDrop spectrophotometer. Libraries were prepared by Eurofins Genomics (Ebersberg, Germany). Sequencing was performed on an Illumina NovaSeq 6000 platform, generating paired-end reads (2 × 150 bp), with an average of ∼30 million read pairs per sample. Sequencing data was quality checked and analyzed as described previously( Frangedakis et al. 2024 ). Briefly, high quality reads were retained using FASTQC and TrimGalore, aligned to Marchantia polymorpha genome (v.7.1) using Kallisto( Bray et al. 2016 ; Bowman et al. 2017 ) and DEG were obtained through DESeq2( Love et al. 2014 ). Data availability Supplementary figures and tables are available with this submission. Transcriptome data are available in the NCBI Sequence Read Archive: PRJNA1353859. Author contributions MLQ: supervision; experimental design, investigation, data curation, formal analysis, validation and visualization, writing original draft, review and editing. PKR: formal analysis, visualization, writing original draft, review and editing. SR: conceptualization; funding and resource acquisition. SBG: conceptualization; project administration; supervision; experimental design; funding and resource acquisition; data visualization; writing original draft, review and editing. Funding This project was carried out in the framework of MAdLand ( https://madland.science , DFG priority programme 2237), SBG and SAR are grateful for funding by the DFG (SPP2237–440043394). Acknowledgments We acknowledge support from the high-performance computing cluster (HILBERT) and thank Daniel Wasim Djamriani, Lilly Möbus, Franklin Cooper, and Margarete Stracke for their help. We also thank Sabine Zachgo and Nora Gutsche from the University of Osnabrück (Germany) for providing us with spores, and Zhanghai Li for providing us with pMpGE010_sgRNA1. PKR is grateful to Bill Martin for providing financial support. Funder Information Declared Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64 , SPP2237–440043394 Footnotes https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1353859/ References ↵ Andrés F , Coupland G ( 2012 ) The genetic basis of flowering responses to seasonal cues . Nat Rev Genet 13 : 627 – 639 . doi: 10.1038/nrg3291 OpenUrl CrossRef PubMed ↵ Becker A , Chen X , Dresselhaus T , et al. ( 2025 ) Sexual reproduction in land plants: an evolutionary perspective . Plant Reprod 38 doi: 10.1038/nrg3291 OpenUrl CrossRef PubMed Blázquez MA , Weigel D ( 2000 ) Integration of floral inductive signals in Arabidopsis . Nature 404 : 889 – 892 . doi: 10.1038/35009125 OpenUrl CrossRef PubMed Web of Science ↵ Borg M , Berger F ( 2015 ) Chromatin remodelling during male gametophyte development . Plant J 83 : 177 – 188 . doi: 10.1111/tpj.12856 OpenUrl CrossRef PubMed ↵ Bowman JL ( 2016 ) A brief history of Marchantia: from Greece to genomics . Plant Cell Physiol 57 : 210 – 229 . doi: 10.1093/pcp/pcv044 OpenUrl CrossRef PubMed ↵ Bowman JL , Kohchi T , Yamato KT , et al. ( 2017 ) Insights into land plant evolution garnered from the Marchantia polymorpha genome . Cell 171 : 287 – 304.e15 . doi: 10.1016/j.cell.2017.09.030 OpenUrl CrossRef PubMed Bray NL , Pimentel H , Melsted P , Pachter L ( 2016 ) Near-optimal probabilistic RNA-seq quantification . Nat Biotechnol 34 : 525 – 527 . doi: 10.1038/nbt.3519 OpenUrl CrossRef PubMed Clapier CR , Cairns BR ( 2009 ) The biology of chromatin remodeling complexes . Annu Rev Biochem 78 : 273 – 304 . doi: 10.1146/annurev.biochem.77.062706.153223 OpenUrl CrossRef PubMed Web of Science de Vries J , Archibald JM ( 2018 ) Plant evolution: landmarks on the path to terrestrial life . New Phytol 217 : 1428 – 1434 . doi: 10.1111/nph.14975 OpenUrl CrossRef PubMed ↵ Donoghue PCJ , Harrison CJ , Paps J , Schneider H ( 2021 ) The evolutionary emergence of land plants . Curr Biol 31 : R1281 – R1298 . doi: 10.1016/j.cub.2021.07.038 OpenUrl CrossRef PubMed ↵ Bray NL , Pimentel H , Melsted P , Pachter L ( 2016 ) Near-optimal probabilistic RNA-seq quantification . Nat Biotechnol 34 : 525 – 527 . doi: 10.1038/nbt.3519 OpenUrl CrossRef PubMed ↵ Clapier CR , Cairns BR ( 2009 ) The biology of chromatin remodeling complexes . Annu Rev Biochem 78 : 273 – 304 . doi: 10.1146/annurev.biochem.77.062706.153223 OpenUrl CrossRef PubMed Web of Science ↵ de Vries J , Archibald JM ( 2018 ) Plant evolution: landmarks on the path to terrestrial life . New Phytol 217 : 1428 – 1434 . doi: 10.1111/nph.14975 OpenUrl CrossRef PubMed Donoghue PCJ , Harrison CJ , Paps J , Schneider H ( 2021 ) The evolutionary emergence of land plants . Curr Biol 31 : R1281 – R1298 . doi: 10.1016/j.cub.2021.07.038 OpenUrl CrossRef PubMed ↵ Frangedakis E , Yelina NE , Billakurthi K , et al. ( 2024 ) MYB-related transcription factors control chloroplast biogenesis . Cell 187 : 3601 – 3621.e19 . doi: 10.1016/j.cell.2024.06.039 OpenUrl CrossRef ↵ Garg SG , Martin WF ( 2016 ) Mitochondria, the cell cycle, and the origin of sex via a syncytial eukaryote common ancestor . Genome Biol Evol 8 : 1950 – 1970 . doi: 10.1093/gbe/evw136 OpenUrl CrossRef PubMed ↵ Genau AC , Li Z , Renzaglia KS , et al. ( 2021 ) HAG1 and SWI3A/B control of male germ line development in Physcomitrium patens suggests conservation of epigenetic reproductive control across land plants . Plant Reprod 34 : 149 – 173 . doi: 10.1007/s00497-021-00409-0 OpenUrl CrossRef ↵ Goodenough U , Heitman J ( 2014 ) Origins of eukaryotic sexual reproduction . Cold Spring Harb Perspect Biol 6 : a016154 . doi: 10.1101/cshperspect.a016154 OpenUrl Abstract / FREE Full Text ↵ Han SK , Sang Y , Rodrigues A , et al. ( 2013 ) The SWI2/SNF2 chromatin remodeling ATPase BRAHMA represses abscisic acid responses in the absence of the stress stimulus in Arabidopsis . Plant Cell 25 : 4892 – 4906 . doi: 10.1105/tpc.112.105114 OpenUrl CrossRef ↵ Harris BJ , Clark JW , Schrempf D , et al. ( 2022 ) Divergent evolutionary trajectories of bryophytes and tracheophytes from a complex common ancestor of land plants . Nat Ecol Evol 6 : 1634 – 1643 . doi: 10.1038/s41559-022-01885-x OpenUrl CrossRef ↵ He Y ( 2012 ) Chromatin regulation of flowering . Trends Plant Sci 17 : 556 – 562 . doi: 10.1016/j.tplants.2012.05.001 OpenUrl CrossRef PubMed Web of Science ↵ Heitman J ( 2015 ) Evolution of sexual reproduction: a view from the fungal kingdom supports an evolutionary epoch with sex before sexes . Fungal Biol Rev 29 : 108 – 117 . doi: 10.1016/j.fbr.2015.08.002 OpenUrl CrossRef PubMed ↵ Higo A , Kawashima T , Borg M , et al. ( 2018 ) Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants . Nature Communications 9 : 5283 . doi: 10.1038/s41467-018-07728-3 OpenUrl CrossRef PubMed ↵ Hisanaga T , Okahashi K , Yamaoka S , et al. ( 2019 ) A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort Marchantia polymorpha . EMBO J 38 : e100240 . doi: 10.15252/embj.2018100240 OpenUrl Abstract / FREE Full Text ↵ Inoue K , Nishihama R , Araki T , Kohchi T ( 2019 ) Reproductive induction is a far-red high-irradiance response that is mediated by phytochrome and phytochrome-interacting factor in Marchantia polymorpha . Plant Cell Physiol 60 : 1136 – 1145 . doi: 10.1093/pcp/pcz029 OpenUrl CrossRef ↵ Ishizaki K , Chiyoda S , Yamato KT , Kohchi T ( 2008 ) Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology . Plant Cell Physiol 49 : 1084 – 1091 . doi: 10.1093/pcp/pcn085 OpenUrl CrossRef PubMed Web of Science ↵ Jeffries D , Benvenuto C , Böhne A , et al. ( 2025 ) The Tree of Sex Consortium: a global initiative for studying the evolution of reproduction in eukaryotes . J Evol Biol 38 : 861 – 886 . doi: 10.1093/jeb/voaf053 OpenUrl CrossRef ↵ Jiang D , Wang Y , Wang Y , He Y ( 2013 ) Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb Repressive Complex 2 components . PLoS One 8 : e3404 . doi: 10.1371/journal.pone.0003404 OpenUrl CrossRef ↵ Kalshoven HW , Musgrave A , van den Ende H ( 1990 ) Sexual plant reproduction in Chlamydomonas eugametos gametes . Sex Plant Reprod 3 : 98 – 107 . doi: 10.1007/BF00189839 OpenUrl CrossRef ↵ Komatsu A , Kodama K , Mizuno Y , et al. ( 2023 ) Control of vegetative reproduction in Marchantia polymorpha by the KAI2-ligand signaling pathway . Curr Biol 33 : 1196 – 1210.e4 . doi: 10.1016/j.cub.2023.02.022 OpenUrl CrossRef PubMed ↵ Koornneef M , Meinke D ( 2010 ) The development of Arabidopsis as a model plant . Plant J 61 : 909 – 921 . doi: 10.1111/j.1365-313X.2009.04086.x OpenUrl CrossRef PubMed Web of Science ↵ Linde AM , Singh S , Bowman JL , et al. ( 2023 ) Genome evolution in plants: complex thalloid liverworts (Marchantiopsida) . Genome Biol Evol 15 : evad014 . doi: 10.1093/gbe/evad014 OpenUrl CrossRef PubMed ↵ Love MI , Huber W , Anders S ( 2014 ) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biol 15 : 550 . doi: 10.1186/s13059-014-0550-8 OpenUrl CrossRef PubMed ↵ McCourt RM , Delwiche CF , Karol KG ( 2004 ) Charophyte algae and land plant origins . Trends Ecol Evol 19 : 661 – 666 . doi: 10.1016/j.tree.2004.09.013 OpenUrl CrossRef PubMed Web of Science ↵ Meyberg R , Perroud PF , Haas FB , et al. ( 2020 ) Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model . New Phytol 227 : 440 – 454 . doi: 10.1111/nph.16486 OpenUrl CrossRef ↵ Molitor AM , Bu Z , Yu Y , Shen WH ( 2014 ) Arabidopsis AL PHD–PRC1 complexes promote seed germination through H3K4me3-to-H3K27me3 chromatin state switch in repression of seed developmental genes . PLoS Genet 10 : e1004091 . doi: 10.1371/journal.pgen.1004091 OpenUrl CrossRef PubMed ↵ Mori T , Kawai-Toyooka H , Igawa T , Nozaki H ( 2015 ) Gamete dialogs in green lineages . Mol Plant 8 : 1442 – 1454 . doi: 10.1016/j.molp.2015.05.011 OpenUrl CrossRef PubMed ↵ Niklas KJ , Kutschera U ( 2010 ) The evolution of the land plant life cycle . New Phytol 185 : 27 – 41 . doi: 10.1111/j.1469-8137.2009.03054.x OpenUrl CrossRef PubMed Web of Science ↵ Pandey S , Moradi AB , Dovzhenko O , et al. ( 2022 ) Molecular control of sporophyte–gametophyte ontogeny and transition in plants . Front Plant Sci 12 : 796570 . doi: 10.3389/fpls.2021.796570 OpenUrl CrossRef ↵ Perroud PF , Haas FB , Hiss M , et al. ( 2018 ) The Physcomitrella patens gene atlas project: large-scale RNA-seq-based expression data . Plant J 95 : 168 – 182 . doi: 10.1111/tpj.13940 OpenUrl CrossRef PubMed ↵ Raval PK , Garg SG , Gould SB ( 2022 ) Endosymbiotic selective pressure at the origin of eukaryotic cell biology . Elife 11 : e77004 . doi: 10.7554/eLife.77004 OpenUrl CrossRef ↵ Ren X , Zhang X , Qi X , et al. ( 2024 ) The BNB–GLID module regulates germline fate determination in Marchantia polymorpha . Plant Cell 36 : 3824 – 3837 . doi: 10.1093/plcell/koae206 OpenUrl CrossRef ↵ Rensing SA , Goffinet B , Meyberg R , et al. ( 2020 ) The moss Physcomitrium (Physcomitrella) patens: a model organism for non-seed plants . Plant Cell 32 : 1361 – 1376 . doi: 10.1105/tpc.00732.2019 OpenUrl Abstract / FREE Full Text ↵ Renzaglia K ( 2001 ) Motile gametes of land plants: diversity, development, and evolution . Crit Rev Plant Sci 20 : 107 – 213 . doi: 10.1080/20013591099209 OpenUrl CrossRef Web of Science ↵ Rizos I , Frada MJ , Bittner L , Not F ( 2024 ) Life cycle strategies in free-living unicellular eukaryotes: diversity, evolution, and current molecular tools to unravel the private life of microorganisms . J Eukaryot Microbiol 71 : e13057 . doi: 10.1111/jeu.13057 OpenUrl CrossRef ↵ Sarnowski TJ , Rios G , Jásik J , et al. ( 2005 ) SWI3 subunits of putative SWI/SNF chromatin-remodeling complexes play distinct roles during Arabidopsis development . Plant Cell 17 : 2454 – 2472 . doi: 10.1105/tpc.105.031203 OpenUrl Abstract / FREE Full Text ↵ She W , Baroux C ( 2014 ) Chromatin dynamics during plant sexual reproduction . Front Plant Sci 5 : 354 . doi: 10.3389/fpls.2014.00354 OpenUrl CrossRef ↵ Speijer D , Lukeš J , Eliáš M ( 2015 ) Sex is a ubiquitous, ancient, and inherent attribute of eukaryotic life . Proc Natl Acad Sci U S A 112 : 8827 – 8834 . doi: 10.1073/pnas.1501725112 OpenUrl Abstract / FREE Full Text ↵ Sugano SS , Nishihama R , Shirakawa M , et al. ( 2018 ) Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha . PLoS One 13 : e0205117 . doi: 10.1371/journal.pone.0205117 OpenUrl CrossRef ↵ Sun R , Okabe M , Miyazaki S , et al. ( 2023 ) Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha . Plant Cell 35 : 4111 – 4132 . doi: 10.1093/plcell/koad216 OpenUrl CrossRef PubMed ↵ Wu MF , Sang Y , Bezhani S , et al. ( 2012 ) SWI2/SNF2 chromatin remodeling ATPases overcome Polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors . Proc Natl Acad Sci U S A 109 : 3576 – 3581 . doi: 10.1073/pnas.1113409109 OpenUrl Abstract / FREE Full Text ↵ Yadav V , Sun S , Heitman J ( 2023 ) On the evolution of variation in sexual reproduction through the prism of eukaryotic microbes . Proc Natl Acad Sci U S A 120 : e2221505120 . doi: 10.1073/pnas.2221505120 OpenUrl CrossRef ↵ Yamamoto C , Takahashi F , Suetsugu N , et al. ( 2024 ) The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha . Proc Natl Acad Sci U S A 121 : e2322211121 . doi: 10.1073/pnas.2322211121 OpenUrl CrossRef PubMed ↵ Yan W , Chen D , Schumacher J , et al. ( 2019 ) Dynamic control of enhancer activity drives stage-specific gene expression during flower morphogenesis . Nat Commun 10 : 4789 . doi: 10.1038/s41467-019-09513-2 OpenUrl CrossRef ↵ Zhang J , Fu XX , Li RQ , et al. ( 2020 ) The hornwort genome and early land plant evolsssution . Nat Plants 6 : 107 – 118 . doi: 10.1038/s41477-019-0588-4 OpenUrl CrossRef PubMed ↵ Zheng B , Chen X ( 2011 ) Dynamics of histone H3 lysine 27 trimethylation in plant development . Curr Opin Plant Biol 14 : 123 – 129 . doi: 10.1016/j.pbi.2010.11.004 OpenUrl CrossRef PubMed ↵ Zheng S , Hu H , Ren H , et al. ( 2019 ) The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature- and photoperiod-dependent flowering repressor . Nat Commun 10 : 1303 . doi: 10.1038/s41467-019-09310-x OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 30, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following SWI3 regulates male sex determination in Marchantia polymorpha Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share SWI3 regulates male sex determination in Marchantia polymorpha Maria Lozano-Quiles , Parth K. Raval , Stefan A Rensing , Sven B. Gould bioRxiv 2025.10.29.685095; doi: https://doi.org/10.1101/2025.10.29.685095 Share This Article: Copy Citation Tools SWI3 regulates male sex determination in Marchantia polymorpha Maria Lozano-Quiles , Parth K. Raval , Stefan A Rensing , Sven B. Gould bioRxiv 2025.10.29.685095; doi: https://doi.org/10.1101/2025.10.29.685095 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17681) Bioengineering (13890) Bioinformatics (41929) Biophysics (21446) Cancer Biology (18586) Cell Biology (25492) Clinical Trials (138) Developmental Biology (13374) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22497) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88584) Paleontology (666) Pathology (2831) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)