Ancient peptide–redox signaling underlies sperm motility in Marchantia

Ancient peptide–redox signaling underlies sperm motility in Marchantia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ancient peptide–redox signaling underlies sperm motility in Marchantia Youze Zhang, Jingya Zhang, Haixiu Li, Liuliu Qiu, Xiang Sun, Xiaoyou Lu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7888116/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Sexual reproduction, a nearly universal feature of eukaryotes, begins with the fertilization of gametes 1,2 . Zoidogamy, or fertilization mediated by motile spermatozoids, represents an ancestral mode predating the evolution of pollen tube-based fertilization in angiosperms 3,4 . This mode dominated a long span of evolutionary history and remains widespread among early-diverging plants and animals 5–7 . However, the molecular mechanisms underlying this ancient fertilization process in plants remains poorly understood. In the liverwort Marchantia polymorpha , we identify a mechanism by which the peptide hormone MpRALF1 regulates sperm motility through reactive oxygen species (ROS). The sperm flagella-derived MpRALF1 is perceived by a receptor complex composed of the MpFERONIA receptor kinase and its co-receptor MpLLG. This signaling module activates NADPH oxidase-mediated ROS production, thereby enhancing sperm velocity and progressive motility to ensure fertilization. Our study reveals the ancient origin of RALF peptide signaling in land plants and establishes ROS as a conserved mechanism governing sperm motility across kingdoms. Biological sciences/Plant sciences/Plant reproduction/Fertilization Biological sciences/Plant sciences/Plant signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Text Sexual reproduction, the predominant reproductive strategy across eukaryotes, enhances genetic diversity and adaptability in changing environments 1,2 . Fertilization via motile sperm (zoidogamy) represents an evolutionarily ancestral mode, universally conserved across both plant and animal phyla 3–5 . In land plants, motile spermatozoids with a biflagellate structure inherited from algal ancestors are preserved in bryophytes, the most basal lineage, and became multiflagellate in early vascular plants 6,7 . Zoidogamy prevailed until siphonogamy rose much later in angiosperms, which substituted motile spermatozoids with pollen tube delivery of non-motile sperm 8,9 . Yet, despite its profound evolutionary importance, the molecular basis of zoidogamy remain largely unexplored. The liverwort Marchantia polymorpha is a typical bryophyte reproducing with biflagellate spermatozoids. Dispersed by raindrops from the antheridium, spermatozoids of Marchantia travel long distances to reach the umbrella-shaped archegoniophore and subsequently swim up into the overhanging archegonium against gravity to accomplish fertilization 10 . With described sperm morphology and established molecular tools 11 , Marchantia has emerged as a species suitable for studies on zoidogamy. The signaling framework regulating sperm function has been extensively elucidated in animals. For mammal sperm, reactive oxygen species (ROS) under physiological concentration are indispensable for fertilizing capacity 12,13 . In human beings, a crucial source of sperm ROS is the flagella-localized NADPH oxidase NOX5 activated by Ca 2+ influx through CatSper channels 14–17 . Sperm ROS further stimulate cyclic adenosine monophosphate (cAMP) production, which drives downstream protein tyrosine phosphorylation 18,19 . A comparable role of cAMP has also been reported in Marchantia 20 . In parallel, studies on the molecular basis of angiosperm siphonogamy have established the role of diverse peptide hormones that regulate pollen-stigma interactions and control pollen tube growth, guidance and burst 21–24 . Notably, certain peptide families and related signaling components are conserved in early-divergent lineages including bryophytes 25 . In this work, we performed a peptide hormone screening in antheridia and identified a RAPID ALKALINIZATION FACTOR (RALF) family peptide MpRALF1, that controls ROS generation in Marchantia sperm, revealing a novel signaling pathway that promotes sperm locomotion. Sperm specific Marchantia RALF1 functions in male fertility As peptide hormones actively participate in various fertilization processes in angiosperms, we conducted peptide screening targeting those highly expressed from the published antheridia transcriptome data 26 . From the top 1,000 antheridia-expressed genes, we identified 17 candidates, among which seven genes were annotated or predicted as secreted or signaling peptides and ten polypeptides were unannotated (Fig. 1a and Supplementary Table 1). Notably, Mp RALF1 (Mp7g07270) was the only candidate assigned a specific gene name, derived from the well-established RALF peptide hormone family, while other candidates most lacked clear functional annotations or supporting information (Supplementary Table 1). Mp RALF1 also showed a markedly high expression level among all screened peptide-encoding genes, as it ranked second in expression among all 7 predicted peptides and ranked third among all 17 screened genes (Fig. 1a). The RALF family peptides are well characterized in reproduction, with distinct members coordinating stigma gating, pollen tube penetration, growth and rupture 22,24,27,28 . Given the importance of RALF peptides, we quantified the expression of RALF genes in male and female wild-type Marchantia strains, Uppsala-5 (Upp-5) and Upp-14, respectively. Similar to the transcriptome data, our quantitative reverse transcription PCR (qRT-PCR) analysis showed that Mp RALF1 was particularly highly expressed in the sperm-producing organ antheridia (Extended Data Fig. 1a). We also confirmed that Mp RALF1 was specifically highly expressed in mature antheridia using the pro Mp RALF1 :GUS transgenic plants (Fig. 1b and Extended Data Fig. 1b). Therefore, we focused our study on Mp RALF1 and explored its functionality in spermatozoids. The CRISPR/Cas9 system was used to generate Mp RALF1 knock-out mutants in Upp-5 29 . All three acquired Mp RALF1 mutant lines (#1, 9, 12) carried frameshift mutations in the mature peptide coding region, resulting in premature termination. Specifically, line #1 had a 2-bp deletion, line #9 a 1-bp deletion, and line #12 a 1-bp insertion (Fig. 1c). Under sexual induction, Mp ralf1 mutants developed smaller antheridiophores and produced fewer motile sperm, exhibiting a quantitative deficiency in gametogenesis (Extended Data Fig. 1c-f). We then applied equivalent sperm suspension from either WT males or Mp ralf1 #12 mutants onto archegoniophores of female WT Upp-14 for crossing. The Mp ralf1 crosses produced significantly fewer sporophytes compared with WT controls, suggesting that the Mp RALF1 mutation impaired male fertility through mechanisms beyond simply reducing sperm production (Fig. 1d). By scanning electron microscopy analysis, we found that Mp ralf1 spermatozoids showed no obvious structural defects in the cell body or two flagella, indicating that the Mp RALF1 mutation did not cause major morphological abnormalities of sperm (Fig. 1e and Extended Data Fig. 1g). Moreover, we generated pro Mp RALF1: Mp RALF1-flag transgenic lines and selected lines with higher MpRALF1-flag protein levels, as determined by immunoblotting (Extended Data Fig. 1h). Using these acquired transgenic lines, we conducted immunofluorescence analysis of spermatids at different developmental stages in antheridia and found that MpRALF1-flag initially appeared in the cell body at stage 1, began to localize to the flagella at stage 3, and already became strongly enriched on the flagella in stage 5 mature sperm prior to release (Extended Data Fig. 1i). In released spermatozoids, the MpRALF1-flag fusion protein was enriched in the flagella and the anterior end of the cell body, with a weak signal occasionally detected in the posterior end (Fig. 1f). These collective observations indicated the positive function of Mp RALF1 in fertility regulation of Marchantia sperm, possibly related to their flagella functions. MpRALF1 promotes the kinematic activity of sperm in Marchantia Next, we set up the sperm motility investigation assay. Among sperm released from Marchantia antheridia, four distinct motility types were detected. The predominant group exhibited rapidly progressive (RP) motility, characterized by efficient directional swimming. A smaller subpopulation showed defective motility patterns, including: slow progressive (SP) sperm advancing at velocities below 20 μm/s (< 0.5 flagella per second), non-progressive (NP) sperm displaying trembling movement and distorted directionality with negligible net displacement, rare circular-motility (CM) sperm that traced orbital trajectories due to an immobile anterior flagellum (Fig. 2a). With these sperm populations described, we then systematically evaluated the sperm motility in Mp RALF1 -related mutants and overexpression lines. Apart from Mp ralf1 knock-out mutants acquired earlier, we generated amiR- Mp RALF1 knock-down lines (referred to as Ri- Mp RALF1 lines hereafter) using artificial microRNA (amiRNA)-induced gene silencing (Extended Data Fig. 2a). From more than ten established Ri- Mp RALF1 transgenic lines, we selected three representative lines (#1, 2, and 3) exhibiting mild (#1) to moderate (#2, 3) knockdown efficiency for subsequent analyses (Extended Data Fig. 2b). We also generated 35S: Mp RALF1 overexpression lines (Mp RALF1 OX ), displaying > 100-fold higher Mp RALF1 transcript levels relative to WT controls (Extended Data Fig. 2c). We then analyzed sperm trajectories using two specialized tracking platforms—the ImageJ plugin TrackMate and Dia-Track software—to quantify velocity parameters and classified motility patterns 30,31 . In comparison with WT controls, both Mp ralf1 and Ri- Mp RALF1 mutants exhibited significantly shorter trajectories with higher curvature, whereas Mp RALF1 OX sperm demonstrated enhanced linearity (Fig. 2b). Kinematic profiling using TrackMate demonstrated that sperm from Mp ralf1 and Ri- Mp RALF1 mutants displayed significantly reduced curvilinear velocity (VCL), while Mp RALF1 OX sperm showed elevated VCL compared to WT (Fig. 2b, Extended Data Fig. 2d-f, and Supplementary Video 1). Consistently, Dia-Track analysis revealed comparable VCL profile across transgenic plants, confirming that Mp RALF1 deficiency impairs sperm motility while its overexpression enhanced swimming velocity (Extended Data Fig. 2g). Moreover, we systematically categorized sperm motility in these transgenic plants. The NP sperm category included those with < 6 μm/s displacement (equivalent to the diameter of unhatched sperm cells) and the CM-type sperm, which was incorporated due to their exceptional low occurrence frequency. Both Mp ralf1 and Ri- Mp RALF1 mutants showed significant redistribution of sperm populations from RP to SP category, while Mp RALF1 OX showed the opposite trend, with RP sperm proportion increasing concomitant with NP population decreasing (Fig. 2c and Extended Data Fig. 2d, e). To determine the effect of MpRALF1 on sperm motility, we applied different concentrations of synthetic MpRALF1 peptides on Mp ralf1 sperm samples and analyzed their kinematic parameters. Application of MpRALF1 peptides in Mp ralf1 mutants produced a concentration-dependent rescue of sperm motility as 10 nM MpRALF1 peptides significantly enhanced sperm VCL and 50 nM sufficiently restored both sperm trajectory patterns and VCL to WT levels (Fig. 2d, Extended Data Fig. 2h, and Supplementary Video 2). Moreover, the proportion of RP sperm in Mp ralf1 samples treated with 10 nM MpRALF1 peptide was already equivalent to that in WT samples while SP and NP sperm significantly reduced under 50 nM treatment compared with untreated Mp ralf1 samples (Fig. 2e). Together, our results evidenced that the sperm-derived MpRALF1 peptide was crucial for sperm locomotion in Marchantia . Flagella-localized MpFER receptor kinase and MpLLG GPI-anchored protein control sperm motility It has been disclosed in angiosperms that RALF peptides are perceived by Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) receptor kinases and their co-receptors LORELEI-like glycosylphosphatidylinositol-anchored proteins (LLGs) 32–34 . Especially in plant reproduction processes, different RALF peptides complex with CrRLK1L family members including FERONIA (FER), ANJEA, ANXUR (ANX) and Buddha’s Paper Seal (BUPS) with their co-receptor LLGs to perform distinct functions determining reproductive success 22,27,35,36 . However, as with other peptide-receptor pairs in angiosperm, both RALF peptides and CrRLK1L receptor kinases comprise large gene families (e.g., 37 RALF and 17 CrRLK1L in Arabidopsis thaliana ; 41 RALF and 23 CrRLK1L in Oryza sativa ). This genetic redundancy makes it particularly challenging to decipher the distinct and collaborative functions of individual members 37 . Surprisingly, the Marchantia genome only contains a single CrRLK1L gene, Mp FER and two Mp LRE genes 37–39 . MpLRE2 contained all conserved domains, whereas MpLRE1 lacked the typical GPI-anchoring site found in angiosperms 39 ; owing to this, we focused subsequent analysis on MpLRE2, renaming it as M. polymorpha LORELEI-like GPI-anchored protein (MpLLG). The transcriptome data showed that the Mp FER and Mp LLG were highly enriched in antheridiophores, which was confirmed by the analysis of pro Mp FER:GUS transgenic plants (Extended Data Fig. 3a, b). We then generated pro Mp FER: Mp FER-flag and pro Mp LLG:SP-flag- Mp LLG transgenic plants and used them for protein localization analysis (Extended Data Fig. 3c). Similar to MpRALF1, the immunofluorescence assay showed that the majority of both MpFER-flag and MpLLG-flag fusion proteins accumulated in sperm flagella and the anterior end while occasionally at the posterior end (Fig. 3a). Comparatively, MpLLG signal detected in the posterior end was generally a bit stronger than MpFER. The consistent flagella-localization of MpFER and MpLLG led us to further pursue functional investigations on Mp FER and Mp LLG in motile sperm. Utilizing the CRISPR/Cas9 system, we obtained two knockout lines of Mp FER mutants (#1 & 5). The Mp fer #1 carried a single-base deletion at the 42-bp position, whereas the Mp fer #5 contained an insertion introducing a stop codon after 42-bp. Both mutations resulted in 21-aa truncated proteins albeit with distinct sequences (Extended Data Fig. 3d). Notably, these Mp fer mutants completely failed to develop antheridiophores, and exhibited reduced thallus size and severely impaired rhizoid development (Extended Data Fig. 3e). Their phenotypes of reduced thallus size and defective rhizoid formation were consistent with those previously reported for T-DNA insertion Mp fer-1 mutant and CRISPR/Cas9-based Mp fer-2 and Mp fer-3 knock-out mutants 39,40 . To acquire plant materials suitable for functional study of Mp FER in fertilization, we adopted amiRNA-induced gene silencing and generated six amiR- Mp FER (referred to as Ri- Mp FER hereafter) transgenic lines. Two of the six Ri- Mp FER lines (#8 & 11) exhibited over 50% transcriptional suppression and were selected for the following studies (Fig. 3b and Extended Data Fig. 4a). These lines remained competent for antheridiophore organogenesis but produced receptacles of reduced size, a phenotype consistent with that previously reported for amiR- Mp FER lines 39 (Extended Data Fig. 4b). Despite seemingly unaffected sperm yield and ultrastructure per antheridiophore in the Ri- Mp FER transgenic plants, their fertilization competence was impaired, leading to a reduced number of sporophytes in crosses with wild-type females (Fig. 3c and Extended Data Fig. 4c-e). Moreover, the sperm VCL was significantly reduced in these Ri- Mp FER lines, and an over 10% decrease was observed in RP sperm population with a respondent increase in that of SP sperm, which was comparable to sperm kinetic defects detected in Mp ralf1 and Ri- Mp RALF1 mutants (Fig. 3d, e). Given the experience in the study of Mp FER , we applied both CRISPR/Cas9 system and amiRNA-induced gene silencing to generate knock-out and knock-down mutants for functional analyses on Mp LLG . As expected, five independent Mp llg knock-out lines all exhibited abolished development of antheridiophores and disrupted growth (Extended Data Fig. 3d, e). Meanwhile, three out of ten amiR- Mp LLG ( Ri- Mp LLG hereafter) transgenic lines (#1, 3 and 4) were selected due to high knock-down efficiency, which exhibited disturbed fertility in crosses with female WT, while none displayed obvious deficiency in sperm yield and sperm ultrastructure (Fig. 3b, c and Extended Data Fig. 4b-e). In these lines, we observed significantly reduced sperm VCL and a shift of sperm proportion from RP population to SP population in all Ri- Mp LLG lines, while in particular, an additional increase in the proportion of NP sperm in #3 (Fig. 3d and Extended Data Fig. 4f). Collectively, these results suggest that MpFER and MpLLG are both flagella-localized proteins indispensable for effective sperm motility. MpFER–MpLLG receptor complex promotes sperm motility through MpRALF1 perception We subsequently tested the protein-protein interaction between MpFER and MpLLG to test whether they formed a receptor complex in sperm motility regulation. Interactions of both MpFER ecd and MpFERΔC with MpLLG were detected in pull-down and luciferase complementation assays (Extended Data Fig. 4g, h). We transiently expressed MpLLG-eGFP and MpFERecd-flag proteins by agroinfiltration in N. benthamiana leaves and conducted Co-IP assay. The result confirmed that MpLLG-eGFP could be co-immunoprecipitated by MpFERecd-flag, further supporting the binding of MpFER and MpLLG (Fig. 3f). After determining the binding of MpFER and MpLLG, we treated Ri- Mp FER sperm samples with varying concentrations of MpRALF1 peptides and measured sperm kinematic parameters to further examine whether this receptor complex functioned through ligand perception of the MpRALF1 peptide. Under MpRALF1 peptide treatment at concentrations ranging from 10 nM to 50 nM, the sperm VCL in Ri- Mp FER samples remained consistently low, equivalent with that in untreated Ri- Mp FE R samples (Fig. 3g). Along with this was the identical profile of sperm populations with different motility forms, where Ri- Mp FER samples displayed significantly decreased proportions of RP sperm and the opposite trend in SP population whether MpRALF1 treatment was applied (Extended Data Fig. 5a). This indicated that MpRALF1 peptides enhanced sperm locomotion via the MpFER receptor kinase. We therefore conducted pull-down assay and found the interactions between E. coli -derived GST-MpRALF1 and MBP-MpFERecd, as well as MBP-MpRALF1 and GST-MpLLG (Fig. 3h). The luciferase complementation assay also confirmed the interaction of MpRALF1-nLuc with cLuc-MpFERΔC, as well as with cLuc-MpLLG (Fig. 3i and Extended Data Fig. 5b). Moreover, the interaction between MBP-MpFERecd and GST-MpLLG was enhanced by MpRALF1 peptides in a dose-dependent manner (Fig. 3j). These biochemical evidences and their flagellar localization demonstrated by immunostaining together suggest that MpFER and MpLLG assemble into a receptor–co-receptor complex in sperm flagella to perceive the MpRALF1 peptide, thereby regulating sperm motility. In Arabidopsis , two distinct binding mechanisms of the RALF–CrRLK1L–LLG complex have been characterized. As for RALF23, its N-terminal helix interacts with LLG2 to induce the complexing of FER and LLG2, while its C-terminus displays exclusive and stronger interaction with FER but not LLG2. Structural analyses have further revealed critical residues on FER (G257 and N303) and LLG1 (N91, T99, A117 and N118) that determine their complexing with RALF23 41 . By contrast, RALF4 binds to ANX1/BUPS1 through its N-terminus and the region between the second and the third cysteines, whereas its C-terminus is sufficient for LLG3 binding 35 . To examine the mechanism governing the MpRALF1–MpFER–MpLLG complex assembly, we partitioned MpRALF1 into an 18 aa N-terminal region (MpRALF1 N18 ) terminating before the first Cys and a 38 aa C-terminal region (MpRALF1 C38 ) consisting of the rest of the mature peptide (Extended Data Fig. 5c). Concerning interaction-determinant residues, sequence analyses showed that MpFER G282 aligned with FER G257 , while MpLLG N100 and MpLLG A126 aligned with LLG1 N91 and LLG1 A117 (Extended Data Fig. 5d, e). We therefore constructed point mutation variants corresponding to these sites. Applying pull-down assay, we found that MpLLG displayed a stronger interaction with MpRALF1 C38 , resembling features found in RALF4–LLG3 interaction 35 . Meanwhile, both MpLLG N100A and MpLLG A126Y exhibited disturbed binding to MpRALF1, which was more comparable to the RALF23–LLG2 interaction pattern 41 (Extended Data Fig. 6a). In contrast, MpRALF1 N18 pulled down MpFER more efficiently than MpRALF1 C38 , and MpFER G282A point mutation showed no effect on the interaction with MpRALF1, or its variants, suggesting an interaction pattern distinct from either of the previously reported mechanisms 35,41 (Extended Data Fig. 6b). Taken together, the MpRALF1–MpFER–MpLLG complex may incorporate mechanistic features from both RALF23–FER–LLG2 and RALF4–ANX1/BUPS1–LLG3, also exhibiting distinct structural properties not observed in either. RBOH-derived ROS function downstream of the evolutionarily conserved RALF signaling pathway for sperm motility stimulation In angiosperms, the plasma membrane-associated NADPH oxidase respiratory burst oxidase homolog (RBOH) functions downstream of the RALF–CrRLK1L–LLG signaling pathway for developmental regulations 22,42 . Activated through Rho-like GTPases from plants, RBOH produces superoxide anion, which can be converted into hydrogen peroxide before entering the cell, constituting a key source of cellular ROS 43–45 . Coinciding, mammalian studies have shown that ROS produced by flagella-localized NADPH oxidase NOX5 are essential for sperm capacitation 12,14 . We therefore investigated whether the MpRALF1–MpFER–MpLLG complex formed in sperm flagella also activated ROS production through NADPH oxidase RBOHs. Using redox-sensitive dyes H 2 DCF-DA 22 and CellROX Orange 46 , we detected widespread ROS signals throughout the entire wild-type spermatozoid, including both flagella and the cell body. Compared with wild-type spermatozoids, those from Mp ralf1 and Ri- Mp RALF1 mutants exhibited significantly reduced ROS levels. Interestingly, treatment with as little as 10 nM chemically synthesized MpRALF1 peptide was able to rescue the low ROS levels in Mp ralf1 and Ri- Mp RALF1 mutant sperm (Fig. 4a, b and Extended Data Fig. 7a). Similarly, sperm from Ri- Mp FER and Ri- Mp LLG mutants exhibited lower ROS levels compared with WT samples (Fig. 4c, d and Extended Data Fig. 7b). Moreover, MpRALF1 treatment failed to trigger ROS generation in Ri- Mp FER and Ri- Mp LLG sperm, consistent with the ligand–receptor mechanism (Fig. 4d and Extended Data Fig. 7b). These results demonstrate that the MpRALF1–MpFER–MpLLG signaling module regulates sperm ROS production. Two Mp RBOHs were identified in Marchantia genome, both highly expressed in antheridiophores and antheridia (Extended Data Fig. 8a). We then generate amiR- Mp RBOH1 ( Ri- Mp RBOH1 ) and amiR- Mp RBOH2 ( Ri- Mp RBOH2 ) transgenic plants and acquired lines with moderate knock-down efficiencies (Extended Data Fig. 8b, c). In both Ri- Mp RBOH1 and Ri- Mp RBOH2 , a significant reduction in sperm ROS levels was observed, with sperm from Ri- Mp RBOH1 displaying relatively lower ROS levels than Mp RBOH1 (Fig. 5a). Similar to observations in Mp ralf , Ri- Mp FER and Ri- Mp LLG mutant sperm, sperm motility in the Ri- Mp RBOH1 and Ri- Mp RBOH2 plants revealed various kinetic defects including decreased VCL, a lower proportion of RP sperm and a higher proportion of SP sperm (Fig. 5b). Moreover, pharmacological inhibition of NADPH oxidase using diphenyleneiodonium (DPI) 42 resulted in motility defects similar to the Ri- Mp RBOH1 mutant, accompanied by a significant increase in both SP and NP sperm populations (Fig. 5c, d). Likewise, scavenging of ROS with Tiron or Na-pyruvate treatments 41 reproduced kinematic defects comparable to those observed with DPI treatment (Extended Data Fig. 8d, e). On the contrary, the application of H 2 O 2 could restore sperm motility defects in Mp ralf1 and Ri- Mp FER mutant sperm at the concentration as low as 70 nM. Interestingly, H 2 O 2 treatment at 100 nM further enhanced sperm kinematic parameters beyond WT (Fig. 5e and Supplementary Video 3). In line with this was the similar H 2 O 2 treatment-induced recovery in sperm population proportions in both Mp ralf1 and Ri- Mp FER mutants (Extended Data Fig. 8f, g). Discussion In this work, we reveal a novel mechanism in Marchantia sperm: MpRALF1 peptide activates the MpFER–MpLLG receptor–co-receptor complex to induce ROS generation via the RBOH-dependent pathway. This MpRALF1-regulated redox signaling enhances progressive sperm locomotion, thereby enabling successful fertilization in Marchantia (Fig. 5f). Compared with pollen tube-mediated siphonogamy in flowering plants, zoidogamy is a more ancient and widespread strategy, mediating gamete delivery via motile spermatozoids. Our identification of a sperm-derived MpRALF1 peptide that regulates sperm locomotion highlights a potentially conserved role of secreted peptide signaling in zoidogamy across kingdoms. This finding reveals a novel regulatory function of secreted peptides governing sperm behavior, potentially via an autocrine mechanism, and warrants further investigation in other plant and animal systems. Moreover, we establish that RBOH-generated ROS act as key stimulators of sperm motility in Marchantia . Echoing the important function of NOX5-derived ROS in human sperm capacitation 14,19,47 , the identification of a peptide-triggered ROS pathway represents a major advance in understanding the molecular basis of zoidogamy. Throughout plant evolution, gene families have diversified through duplication events and selection for functional specialization, facilitating adaptation to developmental requirements and environment changes 48,49 . Notably, core components of the RALF–CrRLK1L–LLG–RBOH signaling pathway are conserved across plant lineages, while the diversity of ligand–receptor pairs expand markedly in angiosperms (Extended Data Fig. 9a). Bayesian inference tree analysis suggests that the RALF , CrRLK1L , and LLG gene families share a monophyletic origin. Among them, CrRLK1Ls are estimated to have emerged approximately 1,038.7 million years ago (MYA), followed by LLGs at ~551 MYA, and RALFs at ~458 MYA (Extended Data Fig. 9b-d). These findings indicate that the RALF–CrRLK1L–LLG signaling module likely originated and assembled near the Ordovician Period in early land plants and was subsequently co-opted to support distinctive reproductive processes during the evolution of pollen tube-mediated siphonogamy. The findings of our work established the role of the MpRALF1–MpFER–MpLLG–ROS pathway in the regulation of motile sperm in Marchantia polymorpha . Meanwhile, the function of this signaling pathway has been disclosed in several developmental processes in Brassicaceae model plant Arabidopsis , Poaceae plant rice and several other angiosperm species 22,32,50,36 . It seems possible that this signaling pathway retains conserved molecular functions during evolution. Based on previous phylogenetic analysis and further sequence alignment, all MpRALFs clustered with several representative AtRALFs including AtRALF1, 22, 23, 33, 24, 31, 4, 19, 34 in a named AtRALF1 clade (Extended Data Fig. 10a) 39 . Therefore, we investigated whether these AtRALF peptides could rescue sperm motility defects of Mp ralf1 mutant plants in Marchantia . The functionally well-characterized AtRALFs that clustered with MpRALF1 were selected as representatives and applied to Mp ralf1 mutant sperm to assess their ability to stimulate sperm motility, which included RALF34, RALF4, RALF23 and RALF1 peptides. Treatment with RALF34, RALF4 or RALF1 all increased sperm motility, and could restore the sperm velocity and motility forms in Mp ralf1 mutants to WT levels (Extended Data Fig. 10b to d). Meanwhile, we did not detect a significant recovery in the AtRALF1 peptide treatment samples, which appeared relatively distant in the protein sequence alignment assay (Extended Data Fig. 10e). These findings suggest that RALF peptides plausibly preserved their ancestral functions from bryophytes to angiosperms during plant evolution. In the meantime, these RALF family genes have possibly been obtaining new functions for the adaptation to diverged developmental processes and environmental challenges. Regarding the CrRLK1L receptor kinases, previous studies have reported that the only CrRLK1L gene in Marchantia , Mp FER plays essential roles in the development of thalli, antheridiophores and rhizoids 39,40 . Here in this work, we discovered that Mp FER functions in the regulation of sperm motility in the ancestral motile sperm sexual reproduction processes. Interspecific complementation of At FER and Mp FER has been attempted between Arabidopsis and Marchantia . The approach of Mp FER over-expression in the At fer-1 mutant plants failed to rescue defective vegetative phenotypes, including bursting root hairs and reduced rosette sizes 39 . Similarly, over-expression of At FER did not rescue the vegetative phenotypes in amiR- Mp FER transgenic lines 39 . Further investigations on the amino acid sequences showed that the kinase domains of MpFER and AtFER displayed around 75% similarity, while that of extracellular domains was around 31%. In particular, almost identical sequences were detected in the activation loop and the key phosphorylation sites in the kinase domain of AtFER and MpFER. In contrast, our biochemistry assays demonstrated that the conserved G282 in the extracellular domain was not responsible for the interaction with MpRALF1, even though this only conserved amino acid was proven crucial for AtFER–AtRALF23 interaction, revealing their vast differences in the extracellular domains. Altogether, it is plausible that during plant evolution history, the extracellular domain of CrRLK1L family receptor kinases have been undergoing the process of fine-tuning for the perception of evolving peptide ligands in response to developmental requirements in specific tissues and environmental stimuli. Methods Plant materials and growth conditions Wild-type Marchantia polymorpha L. accessions Uppsala-5 (Upp-5, male) and Uppsala-14 (Upp-14, female) were maintained on 1/2× Gamborg's B5 basal medium supplemented with 1% (w/v) sucrose and solidified with 1% (w/v) agar. For vegetative propagation, plants were cultured at 22°C under continuous white light (50–80 µmol·m -2 ·s -1 ) in a PT-G800-2 growth chamber (XUNON). To induce sexual reproduction, gemmae grown on 1/2× Gamborg's B5 medium solidified with 0.8% (w/v) agar or on vermiculite were exposed to continuous far-red light (λmax = 730 nm, 70 µmol · m -2 · s -1 ), following the photoperiodic induction protocol 51 . Transgenic plants were generated through Agrobacterium tumefaciens (strain GV2260) mediated transformation according to established protocols, and were selected on medium containing 10 mg/L hygromycin B and 100 mg/L cefotaxime 52 . Screening for peptide-encoding genes The original antheridia transcriptome data (MpTak1_v7.1, PRJDB16711) of Marchantia were retrieved from ENA database ( https://www.ebi.ac.uk/ena/ browser/home ), with SRA numbers DRR050349 and DRR050350. FPKM values (fragments per kilobase of transcript per million mapped reads) were calculated through Hisat2 and StringTie tools 53,54 . The top 1,000 expressed genes were identified based on FPKM values for further analysis. Small peptide candidates shorter than 150 amino acids were initially selected and filtered through functional annotation, excluding genes related to histone, transcription, ribosome function, ubiquitin, or enzyme subunits. The remaining polypeptide-encoding genes were subsequently analyzed for signal peptide prediction using SignalP-5.0 ( https://services.healthtech.dtu.dk/services/SignalP-5.0/ ). For candidates lacking a predicted signal peptide, the length threshold was reduced to 100 amino acids to further prioritize potential signaling peptides. All putative peptide-encoding genes identified through this process are listed in Supplementary Table 1. RNA isolation, RT-PCR, and qRT-PCR Sterile Marchantia specimens were cultivated in axenic conditions on 1/2× Gamborg's B5 basal medium. Vegetative-stage samples consisted of 14-day-old thalli harvested during exponential growth phase, while reproductive tissues were collected 28 days post-induction of sexual organs. Vegetative thalli and mature gametophores including anteridiophores and archegoniophores were flash-frozen in liquid nitrogen. Antheridia and archegonia were micro-dissected from freshly harvested gametophores under stereomicroscopy (Leica S8AP0). All dissections were performed under RNase-free conditions with pre-chilled instruments. Total RNA was extracted from cryopreserved samples using FastPure Universal Plant Total RNA Isolation Kit (Vazyme, China) following manufacturer's protocols. First-strand cDNA synthesis was performed using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). To detect changes in the transcript abundance of selected genes, qPCR analysis was performed with primer pairs listed in Supplementary Table 2 using SYBR qPCR Master Mix (Vazyme, China) on a CFX96 Real-Time System (Bio-RAD) with the following thermal profile: 95℃ for 2 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 20 s (with fluorescence acquisition at each cycle); followed by a melt curve analysis from 65°C to 95°C (0.5°C increments). Relative transcript levels were calculated using the 2 −ΔΔCt method normalized to the housekeeping gene Mp EF1α ( Mp3g23400 ). Vector constructions Open reading frames and promoter sequences of Marchantia genes were amplified by PCR using KOD One PCR Master Mix (TOYOBO, China) from cDNA and genomic DNA prepared from Upp-5. For promoter GUS constructs, native promoters of Mp RALF1 (3.3 kb) and Mp FER (3.2 kb) were amplified and subcloned into pCAMBIA1305-GUSplus binary vector upstream of the GUSplus sequence using 2x MultiF Seamless Assembly Mix (ABclonal, China). For pro Mp RALF1: Mp RALF1-flag and pro Mp FER: Mp FER-flag , the coding sequence of MpRALF1 or MpFER was fused with flag-coding sequence and cloned into pCAMBIA1300 binary vector driven by respective native promoter. For pro Mp LLG:SP-flag- Mp LLG , sequences coding the signal peptide of MpLLG (residues 1–22), 3xFlag and MpLLG (residues 23–176) were fused and cloned into pCAMBIA1300 vector driven by its native promoter (3.7 kb).­­ For CRISPR-Mp RALF1 construct, Single-guide RNA (sgRNA) targeting the MpRALF1 coding sequence was designed using CRISPOR algorithm (v5.2) ( https://crispor.gi.ucsc.edu/ ) 55 . The sgRNA scaffold was synthesized as annealed oligos (Sangon Biotech, China) and cloned into BsaI-digested pMpGE_En03 vector 29 using Hief Gold T4 DNA Ligase (Yeasen, China). The CRISPR cassette was subsequently transferred into destination vector pMpGE010 using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific). For artificial microRNA (amiRNA) related constructs, amiRNA sequences were designed using the WMD3 algorithm ( http://wmd3.weigelworld.org/ ) 56 . The 21-nt amiRNA sequences were amplified from pRS1300 vector with WMD3-generated primers and cloned into the pCAMBIA1300 vector using Hief Gold T4 DNA Ligase. The 1.7 kb promoter sequence of Mp EF1α was amplified and fused upstream of the amiRNA cassette. For 35S: Mp RALF1 construct, the coding sequence of MpRALF1 was amplified and cloned into the pHB vector downstream of the CaMV35S promoter sequence. Histochemical GUS staining Histochemical GUS assays were performed by incubating intact antheridiophores and dissected antheridia in X-Gluc solution (100 mM sodium phosphate buffer pH 7.0, 0.1% Triton X-100 [v/v], 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mM X-Gluc) at 37℃ in darkness for 14 h (for pro Mp FER:GUS materials, the concentration of X-Gluc was reduced to 0.1 mM). Tissue destaining was achieved through sequential incubation in 75% ethanol (12 h at room temperature; the ethanol was replaced twice) followed by chloral hydrate-glycerol clearing solution (70% chloral hydrate [w/v], 10% glycerol [v/v]) under gentle agitation. Samples were photographed under a stereomicroscope (Leica, M205 FCA). Sperm preparation and sperm motility observation Mature antheridiophores were dissected and incubated in 20 μL ddH 2 O for 2 min to release motile spermatozoa. 10 μL aliquots of the sperm suspension were transferred to glass slides and covered with 22 × 22 mm coverslips. Samples were observed using an inverted dark-field microscope (Olympus IX83) equipped with U-TV0.63XC optics, capturing videos at 39 frames per second (fps) for 8 s; or using a differential interference contrast (DIC) microscope (Leica DM6-B) with DFC550 camera, recording at 5 fps for 5 s. Sperm motility parameters were quantitatively analyzed from the movies using ImageJ (v1.54f) with the TrackMate plugin (v7.9.2), applying a spot diameter setting of 3 μm and a linking max distance of 17 μm. Swimming trajectories were reconstructed using the LAP tracker at a frame rate of 5 fps, with the average path velocity (VAP) calculated and expressed in standardized units as μm/s. For another tracking software, Diatrack, particles were identified from the movie under subtract background and filtered with the remove dim function, and trajectories were generated from identified particles with a maximum jump of 60 pixels, allowing gaps closure using the close gaps in trajectories function. Peptide and Chemical treatments Chemicals including synthetic MpRALF1 peptide, H 2 O 2 and ROS inhibitors were applied to sperm suspensions in respective experiments. The MpRALF1 (residues 59–114) peptide (DGpeptides Co., Ltd, China; purity > 95%) was reconstituted in ddH 2 O to prepare the 1 mM stock solution. A 30% (w/v) H₂O₂ stock solution was initially diluted with ddH 2 O to prepare a 1 mM intermediate stock, which was subsequently serially diluted to experimental working concentrations immediately prior to application. For ROS inhibitor treatments, 200 μM DPI (NADPH oxidase inhibitor), 10 mM Tiron (superoxide scavenger) and 10 mM sodium pyruvate (H 2 O 2 scavenger) were prepared as stock solutions and diluted before applied to the sample. In treatment for motile spermatozoa, the antheridiophore was soaked in 20 μL of work solution with the concentration indicated in the graph to acquire the sperm suspension in certain treating solution. The sperm motility observation or ROS staining procedures were performed after treatment for 2 min. Detection of Marchantia -expressed fusion proteins Immunoblotting was conducted to examine the expression of pro Mp RALF1: Mp RALF1-flag , pro Mp FER: Mp FER-flag and pro Mp LLG:SP-flag- Mp LLG fusion proteins in Marchantia materials. The mature antheridiophore was harvested, ground in liquid nitrogen and resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 1% Triton NP-40 [v/v], 1 mM PMSF, 1 mM DTT, 50 μM MG132 [MCE] and 1×protease inhibitor cocktail [Roche]). Obtained crude protein extracts were aliquoted, added with SDS-PAGE loading buffer and boiled for 5 min to prepare samples for immunoblot analysis. Flag antibody (1:5,000 in 5% milk, Sigma-Aldrich) and secondary antibody (1:5,000 in 5% milk, Sangon Biotech) were incubated on the immunoblotted membranes and were subject to HRP detection kit (Yeasen). Signals were subsequently acquired using the Tanon-5200 Multi Imaging System (Tanon). Immunofluorescence staining Indirect immunostaining was performed on spermatids in antheridia and spermatozoid samples with modifications 57 . For spermatids, antheridia of pro Mp FER: Mp FER-flag or pro Mp LLG:SP-flag- Mp LLG plant materials were dissected and fixed with 4% (w/v) PFA for over 1 h, followed by cell-wall digestion and permeabilization as described to release spermatids. Samples were then mounted on adhesive silane-coated glass slides (LIU SHENG, China). As for spermatozoid samples, the sperm suspension was air-dried on adhesive silane-coated glass slides (LIU SHENG, China) and fixed with 4% (w/v) PFA for over 60 min. Primary antibody monoclonal ANTI-FLAG M2 (Sigma-Aldrich; 1:1,000 dilution) was incubated at 4℃ overnight and secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Starter; 1:500 dilution) was incubated at 37 ℃ for 1 h. The nucleus was stained with DAPI (4',6-diamidino-2-phenylindole, Sangon Biotech) for 10 min. After washed with PBS, samples were mounted using Antifade Mounting Medium (Absin, China). Prepared samples were observed using a Leica STELLARIS 8 confocal laser scanning microscope (Ex 488 nm, Em 514 nm) under an oil immersion lens (×63). Images were taken with z-stack following process of maximum intensity projection. ROS staining For H 2 DCF-DA (2’,7’-dichlorofluorescein diacetate, Sigma-Aldrich) staining of sperm ROS, 10 μL of the sperm suspension was dyed with 10 μL of 100 μM H 2 DCF-DA for 3 min, and was fixed with 10% melted gelatin solution on the slide. For CellROX Orange (Yeasen) staining, 20 μL of the sperm suspension was dyed with CellROX Orange under a final concentration of 25 μM for 20 min. The stained samples were transient centrifugation under 12,000 rpm to remove the liquid, were carefully washed with ddH 2 O without resuspension. After subsequent transient centrifugation, remnant liquid was removed and resultant samples were resuspended with 10 μL ddH 2 O. Fluorescence imaging was performed using a Leica STELLARIS 8 confocal laser scanning microscope under consistent laser intensity and gain value for either H 2 DCF-DA stained samples (Ex 488 nm, Em 529 nm) or CellROX stained samples (Ex 545 nm, Em 565 nm). The fluorescence intensity of imaged spermatozoa was measured linearly along the flagella using ImageJ (v1.54f) to represent the ROS level. Scanning electronic microscopy Antheridiophores from WT and mutant lines were hydrated in 20 μL ddH 2 O to release spermatozoa. Suspended spermatozoa were adhered to poly-L-lysine-coated slides (LIU SHENG), air-dried and fixed with 2.5% glutaraldehyde in PBS overnight at 4°C. Post-fixation in 1% osmium tetroxide was conducted for 1 h at 4°C, followed by stepwise ethanol dehydration and critical point drying using Leica EM CPD300. Samples were sputter-coated with gold–palladium (Leica EM ACE600) and imaged using a Hitachi S-4800 SEM. Flagellar ultrastructure quantification was performed by measuring the length of cell body, anterior flagellum, and posterior flagellum using ImageJ (v1.54f) with Segmented Line Tool calibration against scale bars. Protein expression and purification The cDNA encoding the extracellular domain of MpFER (residues 54–461), the mature region of MpRALF1 (residues 59–114), and the core region of MpLLG (residues 22–149) were cloned into the pMAL-c5X vector for fusion with MBP-tag. Similarly, point mutated MBP fusion proteins, MBP-MpFERecd G282A , MBP-MpLLG N100A and MBP-MpLLG A126Y were constructed with indicated primers. The cDNA encoding the core region of MpLLG, the mature region of MpRALF1, MpRALF1 N18 (residues 59–76), MpRALF1 C38 (residues 77–114) were cloned into the pGEX-6P-1 vector. Primers used for these constructions were listed in Supplementary Table 2. Constructed plasmids were transformed into E. coli BL21 Chemically Competent Cells (Weidi, China) for expression. For protein expression, cells were incubated at 37°C until OD 600 reached 0.6. After induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 8 h at 25°C, cells were harvested by centrifugation at 4,000 rpm for 10 min at 4°C. Pellets were lysed by sonication on ice in lysis buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM Na 2 -EDTA, 5% glycerol (v/v), 1 mM PMSF and 1 mM DTT. Following centrifugation at 8,500 rpm for 40 min, the supernatant was purified using Dextine Agarose Resin (Yeasen) for MBP fusion protein and Glutathione Agarose Resin (Yeasen) for GST fusion protein. The resin with bound proteins was washed five times with PBS containing 0.1% Triton X-100. The MBP fusion protein was eluted using an elution buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM Na 2 -EDTA, 10 mM maltose). Pull-down assay Purified MBP fusion protein extracts were incubated with resins containing GST fusion proteins at 4℃ for 1.5 h. After washed five times with the washing buffer described above, resins were added with SDS-PAGE loading buffer and boiled for 5 min to prepare samples for immunoblot analysis. To examine whether MpRALF1 peptides enhanced the binding of MpFERecd and MpLLG, MBP-FERecd proteins and resins bound with GST-LLG were prepared as described above. These components were mixed in binding buffer (50 mM Tris-HCl [pH 6.5], 100 mM NaCl, 1 mM Na 2 -EDTA, 5% glycerol, 1 mM PMSF, 0.1% Triton X-100) before aliquoted. Samples were incubated with indicated concentrations of synthesized MpRALF1 peptides at 4℃ for 2 h. Resultant resins were collected, washed with PBS containing 0.1% Triton for five times and sampled for immunoblot analysis. MBP antibody (1:5,000 in 5% milk, Abmart, China) or GST antibody (1:3,000 in 5% milk, Santa Cruz Biotechnology, USA), and secondary antibody (1:5,000 in 5% milk, Sangon Biotech) were used to detect pulled down fusion proteins, MBP-FERecd or GST-LLG in immunoblotting. Luciferase Complementation Assay SP-cLuc-MpFERΔC, SP-cLuc-MpLLG, SP-nLuc-MpLLG, and MpRALF1-nLuc were cloned into the pHB vector, driven by the 35S promoter. Different constructs were transformed into A. tumefaciens (strain GV3101). Transformed strains were cultured overnight and centrifuged at 4,000 rpm. Pellets were resuspended in 10 mM MES (pH 7.5), 10 mM MgCl 2 , 100 μM acetosyringone, 0.015% Tween-20 buffer to an OD 600 of 0.6. Equal volumes of two cultures carrying indicated nLuc and cLuc were mixed and injected into Nicotiana benthamiana leaves. After 12 h of dark incubation, plants were exposed to 16 h/8 h light/dark cycle for 60 h. The 1 mM luciferin was infiltrated into the tobacco leaves and incubated in the dark for 5 min. Images were captured in Tanon-5200 Multi Imaging System with a cooled CCD camera (Tanon). Co-Immunoprecipitation Assay The 35s: Mp FERecd-flag and 35s: Mp LLG-eGFP constructs were transformed into GV3101 and infiltrated into N. benthamiana leaves simultaneously for transient protein expression. Leaves were harvested two days after injection, ground in liquid nitrogen, and then resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 5 mM MgCl 2 , 5% glycerol [v/v], 0.75% Triton X-100 [v/v], 1 mM PMSF, 1 mM DTT, 50 μM MG132 [MCE] and 1×protease inhibitor cocktail [Roche]). Samples were incubated with rotation at 4°C for 30 min, followed by centrifugation at 12,000 rpm for 30 min at 4°C. The crude protein extract was incubated with Anti-GFP Nanobody Magarose Beads (AlpaLifeBio) at 4°C for 2 h. Beads were then washed with PBS containing 0.1% Triton X-100 for three times and aliquoted to prepare samples for immunoblot analysis. Flag antibody (1:5,000 in 5% milk, Sigma-Aldrich) or GFP antibody (1:5,000 in 5% milk, Santa Cruz Biotechnology), and secondary antibody (1:5,000 in 5% milk, Sangon Biotech) were used to detect MpFERecd-flag or MpLLG-eGFP. Bayesian analysis Bayesian phylogenetic trees for proteins encoded by representative RALFs , CrRLK1Ls , and LRE/LLGs genes listed in Supplementary Table 3 were constructed using Beast2 58 . Gene sequences of RALFs , CrRLK1Ls , and LRE/LLGs were first aligned with MAFFT 59 and then performed by PAL2NAL 60 . Gene divergence times were estimated using GTR substitution models for RALFs , CrRLK1Ls , and HKY substitution model for LRE/LLG genes, with model selection done via MEGA12. The analysis incorporated empirical frequencies, a strict molecular clock, and a Calibrated Yule Model. Calibration points for divergence times were derived from timetree ( https://timetree.org/ ). The MCMC chain length was set at 100,000,000, with a 10% burn-in applied using TreeAnnotator, and results were visualized with Figtree. Statistical analysis Statistical analysis was performed using independent-sample two-tailed t -tests, one-way analysis of variance (ANOVA) and two-way ANOVA, as implemented in GraphPad Prism v.8.0.2. The results were considered to be statistically significant as indicated in figure legends. Unless otherwise indicated, the center line of box plots within violin plots denotes the median, the box limits denote the lower and upper quartiles, and the whiskers denote the lowest and highest data points. The means and standard deviation are shown in bar charts with error bars. Reporting summary Further information on research design is available in the Nature Port-folio Reporting Summary linked to this article. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. We obtained RNA-seq expression data from the ENA database (https://www.ebi.ac.uk/ena/ browser/home), with SRA numbers DRR050349 and DRR050350. Source data are provided with this paper. Methods-only References 51. Chiyoda, S., Ishizaki, K., Kataoka, H., Yamato, K. T. & Kohchi, T. Direct transformation of the liverwort Marchantia polymorpha L. by particle bombardment using immature thalli developing from spores. Plant Cell Rep. 27 , 1467–1473 (2008). 52. Kubota, A., Ishizaki, K., Hosaka, M. & Kohchi, T. Efficient Agrobacterium -Mediated Transformation of the Liverwort Marchantia polymorpha Using Regenerating Thalli. Biosci. Biotechnol. Biochem. 77 , 167–172 (2013). 53. Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37 , 907–915 (2019). 54. Kovaka, S. et al. Transcriptome assembly from long-read RNA-seq alignments with StringTie2. Genome Biol. 20 , 278 (2019). 55. Concordet, J.-P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46 , W242–W245 (2018). 56. Schwab, R., Ossowski, S., Warthmann, N. & Weigel, D. Directed Gene Silencing with Artificial MicroRNAs. in Plant MicroRNAs (eds. Meyers, B. C. & Green, P. J.) vol. 592 71–88 (Humana Press, Totowa, NJ, 2010). 57. Minamino, N., Norizuki, T., Mano, S., Ebine, K. & Ueda, T. Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha . Development 149 , dev200951 (2022). 58. Bouckaert, R. et al. BEAST 2: A Software Platform for Bayesian Evolutionary Analysis. PLoS Comput. Biol. 10 , e1003537 (2014). 59. Katoh, K. & Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30 , 772–780 (2013). 60. Suyama, M., Torrents, D. & Bork, P. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 34 , W609–W612 (2006). Declarations Acknowledgments The project was supported by grants from National Natural Science Foundation of China (32425008, 32230009), Science and Technology Commission of Shanghai Municipality (24N12800100). We thank Lin Xu for providing Marchantia polymorpha materials, Upp-5 and Upp-14; Ruiliang Zhu and Jian Wang for phylogenetic and morphological expertise on M. polymorpha ; Baiyan Lu for assistance in ROS staining; Zhiwei Gong for technical support on scanning electronic microscopy sample preparation and imaging; and Mengmeng Liu for technical assistance with dark-field microscopy. Author contributions C.L. conceived and supervised the project. Y.Z. and J.Z. generated transgenic plants, conducted in-planta functional analyses and protein-protein interaction studies. 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Fertilization via motile sperm\u0026nbsp;(zoidogamy) represents an evolutionarily ancestral mode, universally conserved across both plant and animal phyla\u003csup\u003e3\u0026ndash;5\u003c/sup\u003e. In land plants, motile spermatozoids with a biflagellate structure inherited from algal ancestors are preserved in bryophytes, the most basal lineage, and became multiflagellate in early vascular plants\u003csup\u003e6,7\u003c/sup\u003e.\u0026nbsp;Zoidogamy\u0026nbsp;prevailed until siphonogamy rose much later in angiosperms, which substituted motile spermatozoids with pollen tube delivery of non-motile sperm\u003csup\u003e8,9\u003c/sup\u003e. Yet, despite its profound evolutionary importance, the molecular basis of zoidogamy remain largely unexplored. The liverwort \u003cem\u003eMarchantia polymorpha\u003c/em\u003e is a typical bryophyte reproducing with biflagellate spermatozoids. Dispersed by raindrops from the antheridium, spermatozoids of \u003cem\u003eMarchantia\u003c/em\u003e travel long distances to reach the umbrella-shaped archegoniophore and subsequently swim up into the overhanging archegonium against gravity to accomplish fertilization\u003csup\u003e10\u003c/sup\u003e. With described sperm morphology and established molecular tools\u003csup\u003e11\u003c/sup\u003e, \u003cem\u003eMarchantia\u003c/em\u003e has emerged as a species suitable for studies on zoidogamy.\u003c/p\u003e\n\u003cp\u003eThe signaling framework regulating sperm function has been extensively elucidated in animals. For mammal sperm, reactive oxygen species (ROS) under physiological concentration are indispensable for fertilizing capacity\u003csup\u003e12,13\u003c/sup\u003e. In human beings, a crucial source of sperm ROS is the flagella-localized NADPH oxidase NOX5 activated by Ca\u003csup\u003e2+\u003c/sup\u003e influx through CatSper channels\u003csup\u003e14\u0026ndash;17\u003c/sup\u003e. Sperm ROS further stimulate cyclic adenosine monophosphate (cAMP) production, which drives downstream protein tyrosine phosphorylation\u003csup\u003e18,19\u003c/sup\u003e. A comparable role of cAMP has also been reported in \u003cem\u003eMarchantia\u003c/em\u003e\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;In parallel, studies on the molecular basis of angiosperm siphonogamy have established the role of diverse peptide hormones that regulate pollen-stigma interactions and control pollen tube growth, guidance and burst\u003csup\u003e21\u0026ndash;24\u003c/sup\u003e. Notably, certain peptide families and related signaling components are conserved in early-divergent lineages including bryophytes\u003csup\u003e25\u003c/sup\u003e. In this work, we performed a peptide hormone screening in antheridia and identified a RAPID ALKALINIZATION FACTOR (RALF) family peptide MpRALF1, that controls ROS generation in \u003cem\u003eMarchantia\u003c/em\u003e sperm, revealing a novel signaling pathway that promotes sperm locomotion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm specific \u003cem\u003eMarchantia\u003c/em\u003e RALF1 functions in male fertility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs peptide hormones actively participate in various fertilization processes in angiosperms, we conducted peptide screening targeting those highly expressed from the published antheridia transcriptome data\u003csup\u003e26\u003c/sup\u003e. From the top 1,000 antheridia-expressed genes, we identified 17 candidates, among which seven genes were annotated or predicted as secreted or signaling peptides and ten polypeptides were unannotated (Fig. 1a and Supplementary Table 1). Notably, Mp\u003cem\u003eRALF1\u003c/em\u003e (Mp7g07270) was the only candidate assigned a specific gene name, derived from the well-established RALF peptide hormone family, while other candidates most lacked clear functional annotations or supporting information (Supplementary Table 1).\u0026nbsp;Mp\u003cem\u003eRALF1\u003c/em\u003e also showed a markedly high expression level among all screened peptide-encoding genes,\u0026nbsp;as it ranked second in expression among all 7 predicted peptides and ranked third among all 17 screened genes (Fig. 1a).\u0026nbsp;The RALF family peptides are well characterized in reproduction, with distinct members coordinating stigma gating, pollen tube penetration, growth and rupture\u003csup\u003e22,24,27,28\u003c/sup\u003e. Given the importance of RALF peptides, we quantified the expression of RALF genes in male and female wild-type \u003cem\u003eMarchantia\u003c/em\u003e strains, Uppsala-5 (Upp-5) and Upp-14, respectively. Similar to the transcriptome data, our quantitative reverse transcription PCR (qRT-PCR) analysis showed that Mp\u003cem\u003eRALF1\u003c/em\u003e was particularly highly expressed\u003cem\u003e\u0026nbsp;\u003c/em\u003ein the sperm-producing organ antheridia (Extended Data Fig. 1a). We also confirmed that Mp\u003cem\u003eRALF1\u003c/em\u003e was specifically highly expressed in mature antheridia using the \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e\u003cem\u003e:GUS\u003c/em\u003e transgenic plants (Fig. 1b and Extended Data Fig. 1b). Therefore, we focused our study on Mp\u003cem\u003eRALF1\u003c/em\u003e and explored its functionality in spermatozoids. The CRISPR/Cas9 system was used to generate\u003cem\u003e\u0026nbsp;\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e knock-out mutants in Upp-5\u003csup\u003e29\u003c/sup\u003e. All three acquired Mp\u003cem\u003eRALF1\u0026nbsp;\u003c/em\u003emutant lines (#1, 9, 12) carried frameshift mutations in the mature peptide coding region, resulting in premature termination. Specifically, line #1 had a 2-bp deletion, line #9 a 1-bp deletion, and line #12 a 1-bp insertion (Fig. 1c).\u003c/p\u003e\n\u003cp\u003eUnder sexual induction, Mp\u003cem\u003eralf1\u0026nbsp;\u003c/em\u003emutants developed smaller antheridiophores and produced fewer motile sperm, exhibiting a quantitative deficiency in gametogenesis (Extended Data Fig. 1c-f). We then applied equivalent sperm suspension from either WT males or\u003cem\u003e\u0026nbsp;\u003c/em\u003eMp\u003cem\u003eralf1\u003csup\u003e#12\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emutants onto archegoniophores of female WT Upp-14 for crossing. The Mp\u003cem\u003eralf1\u003c/em\u003e crosses produced significantly fewer sporophytes compared with WT controls, suggesting that the Mp\u003cem\u003eRALF1\u003c/em\u003e mutation impaired male fertility through mechanisms beyond simply reducing sperm production (Fig. 1d). By scanning electron microscopy analysis, we found that\u003cem\u003e\u0026nbsp;\u003c/em\u003eMp\u003cem\u003eralf1\u003c/em\u003e spermatozoids showed no obvious structural defects in the cell body or two flagella, indicating that the Mp\u003cem\u003eRALF1\u003c/em\u003e mutation did not cause major morphological abnormalities of sperm (Fig. 1e and Extended Data Fig. 1g). Moreover, we generated \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eRALF1:\u003c/em\u003eMp\u003cem\u003eRALF1-flag\u003c/em\u003e transgenic lines and selected lines\u0026nbsp;with higher MpRALF1-flag protein levels, as determined by immunoblotting (Extended Data Fig. 1h). Using these acquired transgenic lines, we conducted immunofluorescence analysis of spermatids at different developmental stages in antheridia and found that MpRALF1-flag initially appeared in the cell body at stage 1, began to localize to the flagella at stage 3, and already became strongly enriched on the flagella in stage 5 mature sperm prior to release (Extended Data Fig. 1i). In released spermatozoids, the MpRALF1-flag fusion protein was enriched in the flagella and the anterior end of the cell body, with a weak signal occasionally detected in the posterior end (Fig. 1f). These collective observations indicated the positive function of Mp\u003cem\u003eRALF1\u003c/em\u003e in fertility regulation of \u003cem\u003eMarchantia\u003c/em\u003e sperm, possibly related to their flagella functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMpRALF1 promotes the kinematic activity of sperm in \u003cem\u003eMarchantia\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we set up the sperm motility investigation assay.\u0026nbsp;Among sperm released from \u003cem\u003eMarchantia\u003c/em\u003e antheridia, four distinct motility\u0026nbsp;types were detected. The predominant group exhibited rapidly progressive (RP) motility, characterized by efficient directional swimming. A smaller subpopulation showed defective motility patterns, including: slow progressive (SP) sperm advancing at velocities below 20 \u0026mu;m/s (\u0026lt; 0.5 flagella per second), non-progressive (NP) sperm displaying trembling movement and distorted directionality with negligible net displacement, rare circular-motility (CM) sperm that traced orbital trajectories due to an immobile anterior flagellum (Fig. 2a).\u0026nbsp;With these sperm populations described, we then systematically evaluated the sperm motility in Mp\u003cem\u003eRALF1\u003c/em\u003e-related mutants and overexpression lines. Apart from Mp\u003cem\u003eralf1\u003c/em\u003e knock-out mutants acquired earlier, we generated\u003cem\u003e\u0026nbsp;amiR-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e knock-down lines (referred to as \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e lines hereafter) using artificial microRNA (amiRNA)-induced gene silencing (Extended Data Fig. 2a). From more than ten established\u003cem\u003e\u0026nbsp;Ri-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e transgenic lines, we selected three representative lines (#1, 2, and 3) exhibiting mild (#1) to moderate (#2, 3) knockdown efficiency for subsequent analyses (Extended Data Fig. 2b).\u003cem\u003e\u0026nbsp;\u003c/em\u003eWe also generated \u003cem\u003e35S:\u003c/em\u003eMp\u003cem\u003eRALF1\u0026nbsp;\u003c/em\u003eoverexpression lines (Mp\u003cem\u003eRALF1\u003csub\u003eOX\u003c/sub\u003e\u003c/em\u003e), displaying \u0026gt; 100-fold higher Mp\u003cem\u003eRALF1\u003c/em\u003e transcript levels relative to WT controls (Extended Data Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We then analyzed sperm trajectories using two specialized tracking platforms\u0026mdash;the ImageJ plugin TrackMate and Dia-Track software\u0026mdash;to quantify velocity parameters and classified motility patterns\u003csup\u003e30,31\u003c/sup\u003e. In comparison with WT controls, both Mp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e mutants exhibited significantly shorter trajectories with higher curvature, whereas Mp\u003cem\u003eRALF1\u003csub\u003eOX\u003c/sub\u003e\u003c/em\u003e sperm demonstrated enhanced linearity (Fig. 2b). Kinematic profiling using TrackMate demonstrated that sperm from Mp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e mutants displayed significantly reduced curvilinear velocity (VCL), while Mp\u003cem\u003eRALF1\u003csub\u003eOX\u003c/sub\u003e\u003c/em\u003e sperm showed elevated VCL compared to WT (Fig. 2b,\u0026nbsp;Extended Data Fig. 2d-f, and Supplementary Video 1). Consistently, Dia-Track analysis revealed comparable VCL profile across transgenic plants, confirming that Mp\u003cem\u003eRALF1\u003c/em\u003e deficiency impairs sperm motility while its overexpression enhanced swimming velocity (Extended Data Fig. 2g). Moreover, we systematically categorized sperm motility in these transgenic plants. The NP sperm category included those with \u0026lt; 6 \u0026mu;m/s displacement (equivalent to the diameter of unhatched sperm cells) and the CM-type sperm, which was incorporated due to their exceptional low occurrence frequency. Both\u0026nbsp;Mp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e mutants showed significant redistribution of sperm populations from RP to SP category, while\u0026nbsp;Mp\u003cem\u003eRALF1\u003csub\u003eOX\u003c/sub\u003e\u003c/em\u003e showed the opposite trend, with RP sperm proportion increasing concomitant with NP population decreasing\u0026nbsp;(Fig. 2c and\u0026nbsp;Extended Data Fig.\u0026nbsp;2d, e).\u003c/p\u003e\n\u003cp\u003eTo determine the effect of MpRALF1 on sperm motility, we applied different concentrations of synthetic MpRALF1 peptides on Mp\u003cem\u003eralf1\u003c/em\u003e sperm samples and analyzed their kinematic parameters. Application of MpRALF1 peptides in Mp\u003cem\u003eralf1\u003c/em\u003e mutants\u0026nbsp;produced a concentration-dependent rescue of sperm motility\u0026nbsp;as\u0026nbsp;10 nM\u0026nbsp;MpRALF1 peptides\u0026nbsp;significantly enhanced sperm VCL\u0026nbsp;and 50 nM sufficiently restored both sperm trajectory patterns and VCL to WT levels (Fig. 2d,\u0026nbsp;Extended Data Fig.\u0026nbsp;2h, and Supplementary Video 2). Moreover, the proportion of RP sperm in Mp\u003cem\u003eralf1\u003c/em\u003e samples treated with 10 nM MpRALF1 peptide was already equivalent to that in WT samples while SP and NP sperm significantly reduced under 50 nM treatment compared with untreated Mp\u003cem\u003eralf1\u003c/em\u003e samples (Fig. 2e). Together, our results evidenced that the sperm-derived MpRALF1 peptide was crucial for sperm locomotion in \u003cem\u003eMarchantia\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlagella-localized MpFER receptor kinase and MpLLG GPI-anchored protein control sperm motility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been disclosed in angiosperms that RALF peptides are perceived by \u003cem\u003eCatharanthus roseus\u003c/em\u003e receptor-like kinase 1-like (CrRLK1L) receptor kinases and their co-receptors LORELEI-like glycosylphosphatidylinositol-anchored proteins (LLGs)\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e. Especially in plant reproduction processes, different RALF peptides complex with CrRLK1L family members including FERONIA (FER), ANJEA, ANXUR (ANX) and Buddha\u0026rsquo;s Paper Seal (BUPS) with their co-receptor LLGs to perform distinct functions determining reproductive success\u003csup\u003e22,27,35,36\u003c/sup\u003e. However, as with other peptide-receptor pairs in angiosperm, both RALF peptides and CrRLK1L receptor kinases comprise large gene families (e.g., 37 RALF and 17 CrRLK1L in \u003cem\u003eArabidopsis\u0026nbsp;thaliana\u003c/em\u003e;\u003cem\u003e\u0026nbsp;\u003c/em\u003e41 RALF\u003cem\u003e\u0026nbsp;\u003c/em\u003eand 23 CrRLK1L in\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eOryza sativa\u003c/em\u003e). This genetic redundancy makes it particularly challenging to decipher the distinct and collaborative functions of individual members\u003csup\u003e37\u003c/sup\u003e. Surprisingly, the \u003cem\u003eMarchantia\u003c/em\u003e genome only contains\u003cem\u003e\u0026nbsp;\u003c/em\u003ea single \u003cem\u003eCrRLK1L\u003c/em\u003e gene, Mp\u003cem\u003eFER\u003c/em\u003e and two Mp\u003cem\u003eLRE\u003c/em\u003e genes\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e. MpLRE2 contained all conserved domains, whereas MpLRE1 lacked the typical GPI-anchoring site found in angiosperms\u003csup\u003e39\u003c/sup\u003e; owing to this, we focused subsequent analysis on MpLRE2, renaming it as \u003cem\u003eM. polymorpha\u003c/em\u003e LORELEI-like GPI-anchored protein (MpLLG). The transcriptome data showed that the Mp\u003cem\u003eFER\u003c/em\u003e and Mp\u003cem\u003eLLG\u003c/em\u003e were highly enriched in antheridiophores, which was confirmed by the analysis of \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eFER:GUS\u003c/em\u003e transgenic plants (Extended Data Fig. 3a, b). We then generated \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eFER:\u003c/em\u003eMp\u003cem\u003eFER-flag\u003c/em\u003e and \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eLLG:SP-flag-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e transgenic plants and used them for protein localization analysis (Extended Data Fig. 3c). Similar to MpRALF1, the immunofluorescence assay showed that the majority of both MpFER-flag and MpLLG-flag fusion proteins accumulated in sperm flagella and the anterior end while occasionally at the posterior end (Fig. 3a). Comparatively, MpLLG signal detected in the posterior end was generally a bit stronger than MpFER. The consistent flagella-localization of MpFER and MpLLG led us to further pursue functional investigations on Mp\u003cem\u003eFER\u0026nbsp;\u003c/em\u003eand Mp\u003cem\u003eLLG\u003c/em\u003e in motile sperm.\u003c/p\u003e\n\u003cp\u003eUtilizing the CRISPR/Cas9 system, we obtained two knockout lines of Mp\u003cem\u003eFER\u003c/em\u003e mutants (#1 \u0026amp; 5). The Mp\u003cem\u003efer\u003csup\u003e#1\u003c/sup\u003e\u003c/em\u003e carried a single-base deletion at the 42-bp position, whereas the Mp\u003cem\u003efer\u003csup\u003e#5\u003c/sup\u003e\u003c/em\u003e contained an insertion introducing a stop codon after 42-bp. Both mutations resulted in 21-aa truncated proteins albeit with distinct sequences (Extended Data Fig. 3d). Notably, these Mp\u003cem\u003efer\u003c/em\u003e mutants completely failed to develop antheridiophores, and exhibited reduced thallus size and severely impaired rhizoid development (Extended Data Fig. 3e). Their phenotypes of reduced thallus size and defective rhizoid formation were consistent with those previously reported for T-DNA insertion Mp\u003cem\u003efer-1\u003c/em\u003e mutant and CRISPR/Cas9-based Mp\u003cem\u003efer-2\u003c/em\u003e and Mp\u003cem\u003efer-3\u003c/em\u003e knock-out mutants\u003csup\u003e39,40\u003c/sup\u003e. To acquire plant materials suitable for functional study of Mp\u003cem\u003eFER\u003c/em\u003e in fertilization, we adopted amiRNA-induced gene silencing and generated six\u003cem\u003e\u0026nbsp;amiR-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e (referred to as\u003cem\u003e\u0026nbsp;Ri-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e hereafter) transgenic lines. Two of the six \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e lines (#8\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u0026amp;\u0026nbsp;11) exhibited over 50% transcriptional suppression and were selected for the following studies (Fig. 3b and Extended Data Fig. 4a). These lines remained competent for antheridiophore organogenesis but produced receptacles of reduced size, a phenotype consistent with that previously reported for \u003cem\u003eamiR-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e lines\u003csup\u003e39\u003c/sup\u003e (Extended Data Fig. 4b).\u0026nbsp;Despite seemingly unaffected sperm yield and ultrastructure per antheridiophore in the \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003etransgenic\u003cem\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eplants, their fertilization competence was impaired, leading to a reduced number of sporophytes in crosses with wild-type females (Fig. 3c and Extended Data Fig. 4c-e). Moreover, the sperm VCL was significantly reduced in these\u003cem\u003e\u0026nbsp;Ri-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e lines, and an over 10% decrease was observed in RP sperm population with a respondent increase in that of SP sperm, which was comparable to sperm kinetic defects detected in Mp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e mutants (Fig. 3d, e).\u003c/p\u003e\n\u003cp\u003eGiven the experience in the study of Mp\u003cem\u003eFER\u003c/em\u003e, we applied both CRISPR/Cas9 system and amiRNA-induced gene silencing to generate knock-out and knock-down mutants for functional analyses on Mp\u003cem\u003eLLG\u003c/em\u003e. As expected, five independent Mp\u003cem\u003ellg\u003c/em\u003e knock-out lines all exhibited abolished development of antheridiophores and disrupted growth (Extended Data Fig. 3d, e). Meanwhile, three\u0026nbsp;out of ten \u003cem\u003eamiR-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e (\u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e hereafter) transgenic lines (#1, 3 and 4) were selected due to high knock-down efficiency, which exhibited disturbed fertility in crosses with female WT, while none displayed obvious deficiency in sperm yield and sperm ultrastructure (Fig. 3b, c and Extended Data Fig. 4b-e). In these lines, we observed significantly reduced sperm VCL and a shift of sperm proportion from RP population to SP population in all \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e lines, while in particular, an additional increase in the proportion of NP sperm in\u003cem\u003e\u0026nbsp;\u003c/em\u003e#3 (Fig. 3d and Extended Data Fig. 4f).\u0026nbsp;Collectively, these results suggest that MpFER and MpLLG are both flagella-localized proteins indispensable for effective sperm motility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMpFER\u0026ndash;MpLLG receptor complex promotes sperm motility through MpRALF1 perception\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe subsequently tested the protein-protein interaction between MpFER and MpLLG to test whether they formed a receptor complex in sperm motility regulation. Interactions of both MpFER\u003csub\u003eecd\u003c/sub\u003e and MpFER\u0026Delta;C with MpLLG were detected in pull-down and luciferase complementation assays\u0026nbsp;(Extended Data Fig.\u0026nbsp;4g, h). We transiently expressed MpLLG-eGFP and MpFERecd-flag proteins by agroinfiltration in\u003cem\u003e\u0026nbsp;N. benthamiana\u003c/em\u003e leaves and conducted Co-IP assay. The result confirmed that MpLLG-eGFP could be co-immunoprecipitated by MpFERecd-flag, further supporting the binding of MpFER and MpLLG (Fig. 3f). After determining the binding of MpFER and MpLLG,\u0026nbsp;we treated \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e sperm samples with varying concentrations of MpRALF1 peptides and measured sperm kinematic parameters to further examine whether this receptor complex functioned through ligand perception of the MpRALF1 peptide. Under MpRALF1 peptide treatment at concentrations ranging from 10 nM to 50 nM, the sperm VCL in \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e samples remained consistently low, equivalent with that in untreated \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFE\u003c/em\u003eR samples (Fig. 3g). Along with this was the identical profile of sperm populations with different motility forms, where \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e samples displayed significantly decreased proportions of RP sperm and the opposite trend in SP population whether MpRALF1 treatment was applied (Extended Data Fig. 5a). This indicated that MpRALF1 peptides enhanced sperm locomotion via the MpFER receptor kinase. We therefore conducted pull-down assay and found the interactions between \u003cem\u003eE. coli\u003c/em\u003e-derived GST-MpRALF1 and MBP-MpFERecd, as well as MBP-MpRALF1 and GST-MpLLG\u0026nbsp;(Fig. 3h). The luciferase complementation assay also confirmed the interaction of MpRALF1-nLuc with cLuc-MpFER\u0026Delta;C, as well as with cLuc-MpLLG (Fig. 3i and\u0026nbsp;Extended Data Fig.\u0026nbsp;5b). Moreover, the interaction between MBP-MpFERecd and GST-MpLLG was enhanced by MpRALF1 peptides in a dose-dependent manner (Fig. 3j). These biochemical evidences and their flagellar localization demonstrated by immunostaining together suggest that MpFER and MpLLG assemble into a receptor\u0026ndash;co-receptor complex in sperm flagella to perceive the MpRALF1 peptide, thereby regulating sperm motility.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, two distinct binding mechanisms of the RALF\u0026ndash;CrRLK1L\u0026ndash;LLG complex have been characterized. As for RALF23, its N-terminal helix interacts with LLG2 to induce the complexing of FER and LLG2, while its C-terminus displays exclusive and stronger interaction with FER but not LLG2.\u0026nbsp;Structural analyses have further revealed critical residues on FER (G257 and N303) and LLG1 (N91, T99, A117 and N118) that determine their complexing with RALF23\u003csup\u003e41\u003c/sup\u003e. By contrast, RALF4 binds to ANX1/BUPS1 through its N-terminus and the region between the second and the third cysteines, whereas its C-terminus is sufficient for LLG3 binding\u003csup\u003e35\u003c/sup\u003e. To examine the mechanism governing the MpRALF1\u0026ndash;MpFER\u0026ndash;MpLLG complex assembly, we partitioned MpRALF1 into an 18 aa N-terminal region (MpRALF1\u003csup\u003eN18\u003c/sup\u003e) terminating before the first Cys and a 38 aa C-terminal region (MpRALF1\u003csup\u003eC38\u003c/sup\u003e) consisting of the rest of the mature peptide (Extended Data Fig.\u0026nbsp;5c). Concerning interaction-determinant residues, sequence analyses showed that MpFER\u003csup\u003eG282\u003c/sup\u003e aligned with FER\u003csup\u003eG257\u003c/sup\u003e, while MpLLG\u003csup\u003eN100\u003c/sup\u003e and MpLLG\u003csup\u003eA126\u003c/sup\u003e aligned with LLG1\u003csup\u003eN91\u0026nbsp;\u003c/sup\u003eand LLG1\u003csup\u003eA117\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;5d, e). We therefore constructed point mutation variants corresponding to these sites. Applying pull-down assay, we found that MpLLG displayed a stronger interaction with MpRALF1\u003csup\u003eC38\u003c/sup\u003e, resembling features found in RALF4\u0026ndash;LLG3 interaction\u003csup\u003e35\u003c/sup\u003e. Meanwhile, both MpLLG\u003csup\u003eN100A\u003c/sup\u003e and MpLLG\u003csup\u003eA126Y\u003c/sup\u003e exhibited disturbed binding to MpRALF1, which was more comparable to the RALF23\u0026ndash;LLG2 interaction pattern\u003csup\u003e41\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;6a). In contrast, MpRALF1\u003csup\u003eN18\u0026nbsp;\u003c/sup\u003epulled down MpFER more efficiently than MpRALF1\u003csup\u003eC38\u003c/sup\u003e, and MpFER\u003csup\u003eG282A\u003c/sup\u003e point mutation showed no effect on the interaction with MpRALF1, or its variants, suggesting an interaction pattern distinct from either of the previously reported mechanisms\u003csup\u003e35,41\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;6b). Taken together, the MpRALF1\u0026ndash;MpFER\u0026ndash;MpLLG complex may incorporate mechanistic features from both RALF23\u0026ndash;FER\u0026ndash;LLG2 and RALF4\u0026ndash;ANX1/BUPS1\u0026ndash;LLG3, also exhibiting distinct structural properties not observed in either.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRBOH-derived ROS function downstream of the evolutionarily conserved RALF signaling pathway for sperm motility stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn angiosperms, the plasma membrane-associated NADPH oxidase respiratory burst oxidase homolog (RBOH) functions downstream of the RALF\u0026ndash;CrRLK1L\u0026ndash;LLG signaling pathway for developmental regulations\u003csup\u003e22,42\u003c/sup\u003e. Activated through Rho-like GTPases from plants, RBOH produces superoxide anion, which can be converted into hydrogen peroxide before entering the cell, constituting a key source of cellular ROS\u003csup\u003e43\u0026ndash;45\u003c/sup\u003e. Coinciding, mammalian studies have shown that ROS produced by flagella-localized NADPH oxidase NOX5 are essential for sperm capacitation\u003csup\u003e12,14\u003c/sup\u003e. We therefore investigated whether the MpRALF1\u0026ndash;MpFER\u0026ndash;MpLLG complex formed in sperm flagella also activated ROS production through NADPH oxidase RBOHs. Using redox-sensitive dyes H\u003csub\u003e2\u003c/sub\u003eDCF-DA\u003csup\u003e22\u003c/sup\u003e and CellROX Orange\u003csup\u003e46\u003c/sup\u003e, we detected widespread ROS signals throughout the entire wild-type spermatozoid, including both flagella and the cell body. Compared with wild-type spermatozoids, those from Mp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRALF1\u0026nbsp;\u003c/em\u003emutants exhibited significantly reduced ROS levels. Interestingly, treatment with as little as 10 nM chemically synthesized MpRALF1 peptide was able to rescue the low ROS levels in Mp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e mutant sperm (Fig. 4a, b and\u0026nbsp;Extended Data Fig.\u0026nbsp;7a). Similarly, sperm from \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e mutants exhibited lower ROS levels compared with WT samples (Fig. 4c, d and Extended Data Fig.\u0026nbsp;7b). Moreover, MpRALF1 treatment failed to trigger ROS generation in \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e sperm, consistent with the ligand\u0026ndash;receptor mechanism (Fig. 4d and\u0026nbsp;Extended Data Fig.\u0026nbsp;7b). These results demonstrate that the MpRALF1\u0026ndash;MpFER\u0026ndash;MpLLG signaling module regulates sperm ROS production.\u003c/p\u003e\n\u003cp\u003eTwo Mp\u003cem\u003eRBOHs\u003c/em\u003e were identified in \u003cem\u003eMarchantia\u0026nbsp;\u003c/em\u003egenome, both highly expressed in antheridiophores and antheridia (Extended Data Fig.\u0026nbsp;8a). We then generate \u003cem\u003eamiR-\u003c/em\u003eMp\u003cem\u003eRBOH1\u0026nbsp;\u003c/em\u003e(\u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRBOH1\u003c/em\u003e) and \u003cem\u003eamiR-\u003c/em\u003eMp\u003cem\u003eRBOH2\u0026nbsp;\u003c/em\u003e(\u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRBOH2\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003etransgenic plants and acquired lines with moderate knock-down efficiencies (Extended Data Fig.\u0026nbsp;8b, c). In both \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRBOH1\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Ri-\u003c/em\u003eMp\u003cem\u003eRBOH2\u003c/em\u003e, a significant reduction in sperm ROS levels was observed, with sperm from\u003cem\u003e\u0026nbsp;Ri-\u003c/em\u003eMp\u003cem\u003eRBOH1\u003c/em\u003e displaying relatively lower ROS levels than Mp\u003cem\u003eRBOH1\u003c/em\u003e (Fig. 5a). Similar to observations in Mp\u003cem\u003eralf\u003c/em\u003e, \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e mutant sperm, sperm motility in the \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRBOH1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRBOH2\u0026nbsp;\u003c/em\u003eplants revealed various kinetic defects including decreased VCL, a lower proportion of RP sperm and a higher proportion of SP sperm (Fig. 5b).\u0026nbsp;Moreover, pharmacological inhibition of NADPH oxidase using diphenyleneiodonium (DPI)\u003csup\u003e42\u003c/sup\u003e resulted in motility defects similar to the \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eRBOH1\u003c/em\u003e mutant, accompanied by a significant increase in both SP and NP sperm populations (Fig. 5c, d).\u0026nbsp;Likewise, scavenging of ROS with Tiron or Na-pyruvate treatments\u003csup\u003e41\u003c/sup\u003e reproduced kinematic defects comparable to those observed with DPI treatment (Extended Data Fig.\u0026nbsp;8d, e). On the contrary, the application of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e could restore sperm motility defects in Mp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e mutant sperm at the concentration as low as 70 nM. Interestingly, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003etreatment at 100 nM\u003csub\u003e\u0026nbsp;\u003c/sub\u003efurther enhanced sperm kinematic parameters beyond WT (Fig. 5e and Supplementary Video 3). In line with this was the similar H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment-induced recovery in sperm population proportions in both\u003cem\u003e\u0026nbsp;\u003c/em\u003eMp\u003cem\u003eralf1\u003c/em\u003e and \u003cem\u003eRi-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e mutants (Extended Data Fig.\u0026nbsp;8f, g).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we reveal a novel mechanism in \u003cem\u003eMarchantia\u003c/em\u003e sperm: MpRALF1 peptide activates the MpFER\u0026ndash;MpLLG receptor\u0026ndash;co-receptor complex to induce ROS generation via the RBOH-dependent pathway. This MpRALF1-regulated redox signaling enhances progressive sperm locomotion, thereby enabling successful fertilization in \u003cem\u003eMarchantia\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Fig. 5f). Compared with pollen tube-mediated siphonogamy in flowering plants, zoidogamy is a more ancient and widespread strategy, mediating gamete delivery via motile spermatozoids. Our identification of a sperm-derived MpRALF1 peptide that regulates sperm locomotion highlights a potentially conserved role of secreted peptide signaling in zoidogamy across kingdoms. This finding reveals a novel regulatory function of secreted peptides governing sperm behavior, potentially via an autocrine mechanism, and warrants further investigation in other plant and animal systems. Moreover, we establish that RBOH-generated ROS act as key stimulators of sperm motility in \u003cem\u003eMarchantia\u003c/em\u003e. Echoing the important function of NOX5-derived ROS in human sperm capacitation\u003csup\u003e14,19,47\u003c/sup\u003e, the identification of a peptide-triggered ROS pathway represents a major advance in understanding the molecular basis of zoidogamy.\u003c/p\u003e\n\u003cp\u003eThroughout plant evolution, gene families have diversified through duplication events and selection for functional specialization, facilitating adaptation to developmental requirements and environment changes\u003csup\u003e48,49\u003c/sup\u003e. Notably, core components of the RALF\u0026ndash;CrRLK1L\u0026ndash;LLG\u0026ndash;RBOH signaling pathway are conserved across plant lineages, while the diversity of ligand\u0026ndash;receptor pairs expand markedly in angiosperms (Extended Data Fig. 9a).\u0026nbsp;Bayesian inference tree analysis suggests that the \u003cem\u003eRALF\u003c/em\u003e, \u003cem\u003eCrRLK1L\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;LLG\u003c/em\u003e gene families share a monophyletic origin. Among them, \u003cem\u003eCrRLK1Ls\u003c/em\u003e are estimated to have emerged approximately 1,038.7 million years ago (MYA), followed by \u003cem\u003eLLGs\u003c/em\u003e at ~551 MYA, and \u003cem\u003eRALFs\u003c/em\u003e at ~458 MYA\u0026nbsp;(Extended Data Fig.\u0026nbsp;9b-d). These findings indicate that the RALF\u0026ndash;CrRLK1L\u0026ndash;LLG signaling\u0026nbsp;module likely originated and assembled near the Ordovician Period in early land plants and was subsequently co-opted to support distinctive reproductive processes during the evolution of pollen tube-mediated siphonogamy.\u003c/p\u003e\n\u003cp\u003eThe findings of our work established the role of the MpRALF1\u0026ndash;MpFER\u0026ndash;MpLLG\u0026ndash;ROS pathway in the regulation of motile sperm in \u003cem\u003eMarchantia polymorpha\u003c/em\u003e. Meanwhile, the function of this signaling pathway has been disclosed in several developmental processes in Brassicaceae model plant \u003cem\u003eArabidopsis\u003c/em\u003e, Poaceae plant rice and several other angiosperm species\u003csup\u003e22,32,50,36\u003c/sup\u003e. It seems possible that this signaling pathway retains conserved molecular functions during evolution. Based on previous phylogenetic analysis and further sequence alignment, all MpRALFs clustered with several representative AtRALFs including AtRALF1, 22, 23, 33, 24, 31, 4, 19, 34 in a named AtRALF1 clade (Extended Data Fig. 10a)\u003csup\u003e39\u003c/sup\u003e. Therefore, we investigated whether these AtRALF peptides could rescue sperm motility defects of Mp\u003cem\u003eralf1\u003c/em\u003e mutant plants in \u003cem\u003eMarchantia\u003c/em\u003e. The functionally well-characterized AtRALFs that clustered with MpRALF1 were selected as representatives and applied to Mp\u003cem\u003eralf1\u003c/em\u003e mutant sperm to assess their ability to stimulate sperm motility, which included RALF34, RALF4, RALF23 and RALF1 peptides. Treatment with RALF34, RALF4 or RALF1 all increased sperm motility, and could restore the sperm velocity and motility forms in Mp\u003cem\u003eralf1\u003c/em\u003e mutants to WT levels (Extended Data Fig. 10b to d). Meanwhile, we did not detect a significant recovery in the AtRALF1 peptide treatment samples, which appeared relatively distant in the protein sequence alignment assay (Extended Data Fig. 10e). These findings suggest that RALF peptides plausibly preserved their ancestral functions from bryophytes to angiosperms during plant evolution. In the meantime, these \u003cem\u003eRALF\u003c/em\u003e family genes have possibly been obtaining new functions for the adaptation to diverged developmental processes and environmental challenges.\u003c/p\u003e\n\u003cp\u003eRegarding the CrRLK1L receptor kinases, previous studies have reported that the only \u003cem\u003eCrRLK1L\u003c/em\u003e gene in \u003cem\u003eMarchantia\u003c/em\u003e, Mp\u003cem\u003eFER\u003c/em\u003e plays essential roles in the development of thalli, antheridiophores and rhizoids\u003csup\u003e39,40\u003c/sup\u003e. Here in this work, we discovered that Mp\u003cem\u003eFER\u003c/em\u003e functions in the regulation of sperm motility in the ancestral motile sperm sexual reproduction processes. Interspecific complementation of At\u003cem\u003eFER\u003c/em\u003e and Mp\u003cem\u003eFER\u003c/em\u003e has been attempted between \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eMarchantia\u003c/em\u003e. The approach of Mp\u003cem\u003eFER\u003c/em\u003e over-expression in the At\u003cem\u003efer-1\u003c/em\u003e mutant plants failed to rescue defective vegetative phenotypes, including bursting root hairs and reduced rosette sizes\u003csup\u003e39\u003c/sup\u003e. Similarly, over-expression of At\u003cem\u003eFER\u003c/em\u003e did not rescue the vegetative phenotypes in\u003cem\u003e\u0026nbsp;amiR-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e transgenic lines\u003csup\u003e39\u003c/sup\u003e. Further investigations on the amino acid sequences showed that the kinase domains of MpFER and AtFER displayed around 75% similarity, while that of extracellular domains was around 31%. In particular, almost identical sequences were detected in the activation loop and the key phosphorylation sites in the kinase domain of AtFER and MpFER. In contrast, our biochemistry assays demonstrated that the conserved G282 in the extracellular domain was not responsible for the interaction with MpRALF1, even though this only conserved amino acid was proven crucial for AtFER\u0026ndash;AtRALF23 interaction, revealing their vast differences in the extracellular domains. Altogether, it is plausible that during plant evolution history, the extracellular domain of CrRLK1L family receptor kinases have been undergoing the process of fine-tuning for the perception of evolving peptide ligands in response to developmental requirements in specific tissues and environmental stimuli.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild-type \u003cem\u003eMarchantia polymorpha\u003c/em\u003e L. accessions Uppsala-5 (Upp-5, male) and Uppsala-14 (Upp-14, female) were maintained on 1/2\u0026times; Gamborg\u0026apos;s B5 basal medium supplemented with 1% (w/v) sucrose and solidified with 1% (w/v) agar. For vegetative propagation, plants were cultured at 22\u0026deg;C under continuous white light (50\u0026ndash;80 \u0026micro;mol\u0026middot;m\u003csup\u003e-2\u003c/sup\u003e\u0026middot;s\u003csup\u003e-1\u003c/sup\u003e) in a PT-G800-2 growth chamber (XUNON). To induce sexual reproduction, gemmae grown on 1/2\u0026times; Gamborg\u0026apos;s B5 medium solidified with 0.8% (w/v) agar or on vermiculite were exposed to continuous far-red light (\u0026lambda;max = 730 nm, 70 \u0026micro;mol\u003cstrong\u003e\u0026middot;\u003c/strong\u003em\u003csup\u003e-2\u003c/sup\u003e\u003cstrong\u003e\u0026middot;\u003c/strong\u003es\u003csup\u003e-1\u003c/sup\u003e), following the photoperiodic induction protocol\u003csup\u003e51\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTransgenic plants were generated through \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (strain GV2260) mediated transformation according to established protocols, and were selected on medium containing 10 mg/L hygromycin B and 100 mg/L cefotaxime\u003csup\u003e52\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening for peptide-encoding genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original antheridia transcriptome data (MpTak1_v7.1, PRJDB16711) of \u003cem\u003eMarchantia\u003c/em\u003e were retrieved from ENA database (\u003cu\u003ehttps://www.ebi.ac.uk/ena/ browser/home\u003c/u\u003e), with SRA numbers DRR050349 and DRR050350. FPKM values (fragments per kilobase of transcript per million mapped reads) were calculated through Hisat2 and StringTie tools\u003csup\u003e53,54\u003c/sup\u003e. The top 1,000 expressed genes were identified based on FPKM values for further analysis. Small peptide candidates shorter than 150 amino acids were initially selected and filtered through functional annotation, excluding genes related to histone, transcription, ribosome function, ubiquitin, or enzyme subunits. The remaining polypeptide-encoding genes were subsequently analyzed for signal peptide prediction using SignalP-5.0 (\u003cu\u003ehttps://services.healthtech.dtu.dk/services/SignalP-5.0/\u003c/u\u003e). For candidates lacking a predicted signal peptide, the length threshold was reduced to 100 amino acids to further prioritize potential signaling peptides. All putative peptide-encoding genes identified through this process are listed in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation, RT-PCR, and qRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSterile \u003cem\u003eMarchantia\u003c/em\u003e specimens were cultivated in axenic conditions on 1/2\u0026times; Gamborg\u0026apos;s B5 basal medium. Vegetative-stage samples consisted of 14-day-old thalli harvested during exponential growth phase, while reproductive tissues were collected 28 days post-induction of sexual organs. Vegetative thalli and mature gametophores including anteridiophores and archegoniophores were flash-frozen in liquid nitrogen. Antheridia and archegonia were micro-dissected from freshly harvested gametophores under stereomicroscopy (Leica S8AP0). All dissections were performed under RNase-free conditions with pre-chilled instruments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cryopreserved samples using FastPure Universal Plant Total RNA Isolation Kit (Vazyme, China) following manufacturer\u0026apos;s protocols. First-strand cDNA synthesis was performed using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). To detect changes in the transcript abundance of selected genes, qPCR analysis was performed with primer pairs listed in Supplementary Table 2 using SYBR qPCR Master Mix (Vazyme, China) on a CFX96 Real-Time System (Bio-RAD) with the following thermal profile: 95℃ for 2 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 20 s (with fluorescence acquisition at each cycle); followed by a melt curve analysis from 65\u0026deg;C to 95\u0026deg;C (0.5\u0026deg;C increments). Relative transcript levels were calculated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method normalized to the housekeeping gene Mp\u003cem\u003eEF1\u0026alpha;\u003c/em\u003e (\u003cem\u003eMp3g23400\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVector constructions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen reading frames and promoter sequences of \u003cem\u003eMarchantia\u003c/em\u003e genes were amplified by PCR using KOD One PCR Master Mix (TOYOBO, China) from cDNA and genomic DNA prepared from Upp-5. For promoter GUS constructs, native promoters of Mp\u003cem\u003eRALF1\u003c/em\u003e (3.3 kb) and Mp\u003cem\u003eFER\u003c/em\u003e (3.2 kb) were amplified and subcloned into pCAMBIA1305-GUSplus binary vector upstream of the GUSplus sequence using 2x MultiF Seamless Assembly Mix (ABclonal, China). For \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eRALF1:\u003c/em\u003eMp\u003cem\u003eRALF1-flag\u003c/em\u003e and \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eFER:\u003c/em\u003eMp\u003cem\u003eFER-flag\u003c/em\u003e, the coding sequence of MpRALF1 or MpFER was fused with flag-coding sequence and cloned into pCAMBIA1300 binary vector driven by respective native promoter. For \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eLLG:SP-flag-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e, sequences coding the signal peptide of MpLLG (residues 1\u0026ndash;22), 3xFlag and MpLLG (residues 23\u0026ndash;176) were fused and cloned into pCAMBIA1300 vector driven by its native promoter (3.7 kb).\u0026shy;\u0026shy;\u003c/p\u003e\n\u003cp\u003eFor CRISPR-Mp\u003cem\u003eRALF1\u003c/em\u003e construct, Single-guide RNA (sgRNA) targeting the MpRALF1 coding sequence was designed using CRISPOR algorithm (v5.2) (\u003cu\u003ehttps://crispor.gi.ucsc.edu/\u003c/u\u003e)\u003csup\u003e55\u003c/sup\u003e. The sgRNA scaffold was synthesized as annealed oligos (Sangon Biotech, China) and cloned into BsaI-digested pMpGE_En03 vector\u003csup\u003e29\u003c/sup\u003e using Hief Gold T4 DNA Ligase (Yeasen, China). The CRISPR cassette was subsequently transferred into destination vector pMpGE010 using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific). For artificial microRNA (amiRNA) related constructs, amiRNA sequences were designed using the WMD3 algorithm (\u003cu\u003ehttp://wmd3.weigelworld.org/\u003c/u\u003e)\u003csup\u003e56\u003c/sup\u003e. The 21-nt amiRNA sequences were amplified from pRS1300 vector with WMD3-generated primers and cloned into the pCAMBIA1300 vector using Hief Gold T4 DNA Ligase. The 1.7 kb promoter sequence of Mp\u003cem\u003eEF1\u0026alpha;\u003c/em\u003e was amplified and fused upstream of the amiRNA cassette. For \u003cem\u003e35S:\u003c/em\u003eMp\u003cem\u003eRALF1\u003c/em\u003e construct, the coding sequence of MpRALF1 was amplified and cloned into the pHB vector downstream of the \u003cem\u003eCaMV35S\u003c/em\u003e promoter sequence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistochemical GUS staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistochemical GUS assays were performed by incubating intact antheridiophores and dissected antheridia in X-Gluc solution (100 mM sodium phosphate buffer pH 7.0, 0.1% Triton X-100 [v/v], 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mM X-Gluc) at 37℃ in darkness for 14 h (for \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eFER:GUS\u003c/em\u003e materials, the concentration of X-Gluc was reduced to 0.1 mM). Tissue destaining was achieved through sequential incubation in 75% ethanol (12 h at room temperature; the ethanol was replaced twice) followed by chloral hydrate-glycerol clearing solution (70% chloral hydrate [w/v], 10% glycerol [v/v]) under gentle agitation. Samples were photographed under a stereomicroscope (Leica, M205 FCA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm preparation\u0026nbsp;and sperm motility observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature antheridiophores were dissected and incubated in 20 \u0026mu;L ddH\u003csub\u003e2\u003c/sub\u003eO for 2 min to release motile spermatozoa. 10 \u0026mu;L aliquots of the sperm suspension were transferred to glass slides and covered with 22 \u0026times; 22 mm coverslips. Samples were observed using an inverted dark-field microscope (Olympus IX83) equipped with U-TV0.63XC optics, capturing videos at 39 frames per second (fps) for 8 s; or using a differential interference contrast (DIC) microscope (Leica DM6-B) with DFC550 camera, recording at 5 fps for 5 s.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSperm motility parameters were quantitatively analyzed from the movies using ImageJ (v1.54f) with the TrackMate plugin (v7.9.2), applying a spot diameter setting of 3 \u0026mu;m and a linking max distance of 17 \u0026mu;m. Swimming trajectories were reconstructed using the LAP tracker at a frame rate of 5 fps, with the average path velocity (VAP) calculated and expressed in standardized units as \u0026mu;m/s. For another tracking software, Diatrack, particles were identified from the movie under subtract background and filtered with the remove dim function, and trajectories were generated from identified particles with a maximum jump of 60 pixels, allowing gaps closure using the close gaps in trajectories function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide and Chemical treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemicals including synthetic MpRALF1 peptide, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and ROS inhibitors were applied to sperm suspensions in respective experiments. The MpRALF1 (residues 59\u0026ndash;114) peptide (DGpeptides Co., Ltd, China; purity \u0026gt; 95%) was reconstituted in ddH\u003csub\u003e2\u003c/sub\u003eO to prepare the 1 mM stock solution. A 30% (w/v) H₂O₂ stock solution was initially diluted with ddH\u003csub\u003e2\u003c/sub\u003eO to prepare a 1 mM intermediate stock, which was subsequently serially diluted to experimental working concentrations immediately prior to application. For ROS inhibitor treatments, 200 \u0026mu;M DPI (NADPH oxidase inhibitor), 10 mM Tiron (superoxide scavenger) and 10 mM sodium pyruvate (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger) were prepared as stock solutions and diluted before applied to the sample.\u003c/p\u003e\n\u003cp\u003eIn treatment for motile spermatozoa, the antheridiophore was soaked in 20 \u0026mu;L of work solution with the concentration indicated in the graph to acquire the sperm suspension in certain treating solution. The sperm motility observation or ROS staining procedures were performed after treatment for 2 min.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of \u003cem\u003eMarchantia\u003c/em\u003e-expressed fusion proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunoblotting was conducted to examine the expression of \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eRALF1:\u003c/em\u003eMp\u003cem\u003eRALF1-flag\u003c/em\u003e, \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eFER:\u003c/em\u003eMp\u003cem\u003eFER-flag\u003c/em\u003e and \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eLLG:SP-flag-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e fusion proteins in \u003cem\u003eMarchantia\u003c/em\u003e materials. The mature antheridiophore was harvested, ground in liquid nitrogen and resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM EDTA, 1% Triton NP-40 [v/v], 1 mM PMSF, 1 mM DTT, 50 \u0026mu;M MG132 [MCE] and 1\u0026times;protease inhibitor cocktail [Roche]). Obtained crude protein extracts were aliquoted, added with SDS-PAGE loading buffer and boiled for 5 min to prepare samples for immunoblot analysis. Flag antibody (1:5,000 in 5% milk, Sigma-Aldrich) and secondary antibody (1:5,000 in 5% milk, Sangon Biotech) were incubated on the immunoblotted membranes and were subject to HRP detection kit (Yeasen). Signals were subsequently acquired using the Tanon-5200 Multi Imaging System (Tanon).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndirect immunostaining was performed on spermatids in antheridia and spermatozoid samples with modifications\u003csup\u003e57\u003c/sup\u003e. For spermatids, antheridia of \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eFER:\u003c/em\u003eMp\u003cem\u003eFER-flag\u003c/em\u003e or \u003cem\u003epro\u003c/em\u003eMp\u003cem\u003eLLG:SP-flag-\u003c/em\u003eMp\u003cem\u003eLLG\u003c/em\u003e plant materials were dissected and fixed with 4% (w/v) PFA for over 1 h, followed by cell-wall digestion and permeabilization as described to release spermatids. Samples were then mounted on adhesive silane-coated glass slides (LIU SHENG, China). As for spermatozoid samples, the sperm suspension was air-dried on adhesive silane-coated glass slides (LIU SHENG, China) and fixed with 4% (w/v) PFA for over 60 min. Primary antibody monoclonal ANTI-FLAG M2 (Sigma-Aldrich; 1:1,000 dilution) was incubated at 4℃ overnight and secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Starter; 1:500 dilution) was incubated at 37 ℃ for 1 h. The nucleus was stained with DAPI (4\u0026apos;,6-diamidino-2-phenylindole, Sangon Biotech) for 10 min. After washed with PBS, samples were mounted using Antifade Mounting Medium (Absin, China). Prepared samples were observed using a Leica STELLARIS 8 confocal laser scanning microscope (Ex 488 nm, Em 514 nm) under an oil immersion lens (\u0026times;63). Images were taken with z-stack following process of maximum intensity projection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor H\u003csub\u003e2\u003c/sub\u003eDCF-DA (2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein diacetate, Sigma-Aldrich) staining of sperm ROS, 10 \u0026mu;L of the sperm suspension was dyed with 10 \u0026mu;L of 100 \u0026mu;M H\u003csub\u003e2\u003c/sub\u003eDCF-DA for 3 min, and was fixed with 10% melted gelatin solution on the slide. For CellROX Orange (Yeasen) staining, 20 \u0026mu;L of the sperm suspension was dyed with CellROX Orange under a final concentration of 25 \u0026mu;M for 20 min. The stained samples were transient centrifugation under 12,000 rpm to remove the liquid, were carefully washed with ddH\u003csub\u003e2\u003c/sub\u003eO without resuspension. After subsequent transient centrifugation, remnant liquid was removed and resultant samples were resuspended with 10 \u0026mu;L ddH\u003csub\u003e2\u003c/sub\u003eO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFluorescence imaging was performed using a Leica STELLARIS 8 confocal laser scanning microscope under consistent laser intensity and gain value for either H\u003csub\u003e2\u003c/sub\u003eDCF-DA stained samples (Ex 488 nm, Em 529 nm) or CellROX stained samples (Ex 545 nm, Em 565 nm). The fluorescence intensity of imaged spermatozoa was measured linearly along the flagella using ImageJ (v1.54f) to represent the ROS level.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electronic microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntheridiophores from WT and mutant lines were hydrated in 20 \u0026mu;L ddH\u003csub\u003e2\u003c/sub\u003eO to release spermatozoa. Suspended spermatozoa were adhered to poly-L-lysine-coated slides (LIU SHENG), air-dried and fixed with 2.5% glutaraldehyde in PBS overnight at 4\u0026deg;C. Post-fixation in 1% osmium tetroxide was conducted for 1 h at 4\u0026deg;C, followed by stepwise ethanol dehydration and critical point drying using Leica EM CPD300.\u003c/p\u003e\n\u003cp\u003eSamples were sputter-coated with gold\u0026ndash;palladium (Leica EM ACE600) and imaged using a Hitachi S-4800 SEM. Flagellar ultrastructure quantification was performed by measuring the length of cell body, anterior flagellum, and posterior flagellum using ImageJ (v1.54f) with Segmented Line Tool calibration against scale bars.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cDNA encoding the extracellular domain of MpFER (residues 54\u0026ndash;461), the mature region of MpRALF1 (residues 59\u0026ndash;114), and the core region of MpLLG (residues 22\u0026ndash;149) were cloned into the pMAL-c5X vector for fusion with MBP-tag. Similarly, point mutated MBP fusion proteins, MBP-MpFERecd\u003csup\u003eG282A\u003c/sup\u003e, MBP-MpLLG\u003csup\u003eN100A\u003c/sup\u003e and MBP-MpLLG\u003csup\u003eA126Y\u003c/sup\u003e were constructed with indicated primers. The cDNA encoding the core region of MpLLG, the mature region of MpRALF1, MpRALF1\u003csup\u003eN18\u003c/sup\u003e (residues 59\u0026ndash;76), MpRALF1\u003csup\u003eC38\u003c/sup\u003e (residues 77\u0026ndash;114) were cloned into the pGEX-6P-1 vector. Primers used for these constructions were listed in Supplementary Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConstructed plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 Chemically Competent Cells (Weidi, China) for expression. For protein expression, cells were incubated at 37\u0026deg;C until OD\u003csub\u003e600\u003c/sub\u003e reached 0.6. After induction with 0.5 mM isopropyl-\u0026beta;-D-thiogalactopyranoside (IPTG) for 8 h at 25\u0026deg;C, cells were harvested by centrifugation at 4,000 rpm for 10 min at 4\u0026deg;C. Pellets were lysed by sonication on ice in lysis buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM Na\u003csub\u003e2\u003c/sub\u003e-EDTA, 5% glycerol (v/v), 1 mM PMSF and 1 mM DTT. Following centrifugation at 8,500 rpm for 40 min, the supernatant was purified using Dextine Agarose Resin (Yeasen) for MBP fusion protein and Glutathione Agarose Resin (Yeasen) for GST fusion protein. The resin with bound proteins was washed five times with PBS containing 0.1% Triton X-100. The MBP fusion protein was eluted using an elution buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM Na\u003csub\u003e2\u003c/sub\u003e-EDTA, 10 mM maltose).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePull-down assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePurified MBP fusion protein extracts were incubated with resins containing GST fusion proteins at 4℃ for 1.5 h. After washed five times with the washing buffer described above, resins were added with SDS-PAGE loading buffer and boiled for 5 min to prepare samples for immunoblot analysis. To examine whether MpRALF1 peptides enhanced the binding of MpFERecd and MpLLG, MBP-FERecd proteins and resins bound with GST-LLG were prepared as described above. These components were mixed in binding buffer (50 mM Tris-HCl [pH 6.5], 100 mM NaCl, 1 mM Na\u003csub\u003e2\u003c/sub\u003e-EDTA, 5% glycerol, 1 mM PMSF, 0.1% Triton X-100) before aliquoted. Samples were incubated with indicated concentrations of synthesized MpRALF1 peptides at 4℃ for 2 h. Resultant resins were collected, washed with PBS containing 0.1% Triton for five times and sampled for immunoblot analysis. MBP antibody (1:5,000 in 5% milk, Abmart, China) or GST antibody (1:3,000 in 5% milk, Santa Cruz Biotechnology, USA), and secondary antibody (1:5,000 in 5% milk, Sangon Biotech) were used to detect pulled down fusion proteins, MBP-FERecd or GST-LLG in immunoblotting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuciferase Complementation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSP-cLuc-MpFER\u0026Delta;C, SP-cLuc-MpLLG, SP-nLuc-MpLLG, and MpRALF1-nLuc were cloned into the pHB vector, driven by the \u003cem\u003e35S\u003c/em\u003e promoter. Different constructs were transformed into \u003cem\u003eA. tumefaciens\u003c/em\u003e (strain GV3101). Transformed strains were cultured overnight and centrifuged at 4,000 rpm. Pellets were resuspended in 10 mM MES (pH 7.5), 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 100 \u0026mu;M acetosyringone, 0.015% Tween-20 buffer to an OD\u003csub\u003e600\u003c/sub\u003e of 0.6. Equal volumes of two cultures carrying indicated nLuc and cLuc were mixed and injected into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. After 12 h of dark incubation, plants were exposed to 16 h/8 h light/dark cycle for 60 h. The 1 mM luciferin was infiltrated into the tobacco leaves and incubated in the dark for 5 min. Images were captured in Tanon-5200 Multi Imaging System with a cooled CCD camera (Tanon).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-Immunoprecipitation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003e35s:\u003c/em\u003eMp\u003cem\u003eFERecd-flag\u003c/em\u003e and \u003cem\u003e35s:\u003c/em\u003eMp\u003cem\u003eLLG-eGFP\u003c/em\u003e constructs were transformed into GV3101 and infiltrated into\u003cem\u003e\u0026nbsp;N.\u003c/em\u003e \u003cem\u003ebenthamiana\u003c/em\u003e leaves simultaneously for transient protein expression. Leaves were harvested two days after injection, ground in liquid nitrogen, and then resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5% glycerol [v/v], 0.75% Triton X-100 [v/v], 1 mM PMSF, 1 mM DTT, 50 \u0026mu;M MG132 [MCE] and 1\u0026times;protease inhibitor cocktail [Roche]). Samples were incubated with rotation at 4\u0026deg;C for 30 min, followed by centrifugation at 12,000 rpm for 30 min at 4\u0026deg;C. The crude protein extract was incubated with Anti-GFP Nanobody Magarose Beads (AlpaLifeBio) at 4\u0026deg;C for 2 h. Beads were then washed with PBS containing 0.1% Triton X-100 for three times and aliquoted to prepare samples for immunoblot analysis. Flag antibody (1:5,000 in 5% milk, Sigma-Aldrich) or GFP antibody (1:5,000 in 5% milk, Santa Cruz Biotechnology), and secondary antibody (1:5,000 in 5% milk, Sangon Biotech) were used to detect MpFERecd-flag or MpLLG-eGFP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBayesian analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBayesian phylogenetic trees for proteins encoded by representative \u003cem\u003eRALFs\u003c/em\u003e, \u003cem\u003eCrRLK1Ls\u003c/em\u003e, and \u003cem\u003eLRE/LLGs\u003c/em\u003e genes listed in Supplementary Table 3 were constructed using Beast2\u003csup\u003e58\u003c/sup\u003e. Gene sequences of \u003cem\u003eRALFs\u003c/em\u003e, \u003cem\u003eCrRLK1Ls\u003c/em\u003e, and \u003cem\u003eLRE/LLGs\u0026nbsp;\u003c/em\u003ewere first aligned with MAFFT\u003csup\u003e59\u003c/sup\u003e and then performed by PAL2NAL\u003csup\u003e60\u003c/sup\u003e. Gene divergence times were estimated using GTR substitution models for \u003cem\u003eRALFs\u003c/em\u003e, \u003cem\u003eCrRLK1Ls\u003c/em\u003e, and HKY substitution model for \u003cem\u003eLRE/LLG\u003c/em\u003e genes, with model selection done via MEGA12. The analysis incorporated empirical frequencies, a strict molecular clock, and a Calibrated Yule Model. Calibration points for divergence times were derived from timetree (\u003cu\u003ehttps://timetree.org/\u003c/u\u003e). The MCMC chain length was set at 100,000,000, with a 10% burn-in applied using TreeAnnotator, and results were visualized with Figtree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using independent-sample two-tailed \u003cem\u003et\u003c/em\u003e-tests, one-way analysis of variance (ANOVA) and two-way ANOVA, as implemented in GraphPad Prism v.8.0.2. The results were considered to be statistically significant as indicated in figure legends.\u0026nbsp;Unless otherwise indicated, the center line of box plots within violin plots denotes the median, the box limits denote the lower and upper quartiles, and the whiskers denote the lowest and highest data points. The means and standard deviation are shown in bar charts with error bars.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReporting summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Port-folio Reporting Summary linked to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. We obtained RNA-seq expression data from the ENA database (https://www.ebi.ac.uk/ena/ browser/home), with SRA numbers DRR050349 and DRR050350. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods-only References\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e51. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Chiyoda, S., Ishizaki, K., Kataoka, H., Yamato, K. T. \u0026amp; Kohchi, T. Direct transformation of the liverwort Marchantia polymorpha L. by particle bombardment using immature thalli developing from spores. \u003cem\u003ePlant Cell Rep.\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1467\u0026ndash;1473 (2008).\u003c/p\u003e\n\u003cp\u003e52. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Kubota, A., Ishizaki, K., Hosaka, M. \u0026amp; Kohchi, T. Efficient \u003cem\u003eAgrobacterium\u003c/em\u003e -Mediated Transformation of the Liverwort \u003cem\u003eMarchantia polymorpha\u003c/em\u003e Using Regenerating Thalli. \u003cem\u003eBiosci. Biotechnol. Biochem.\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 167\u0026ndash;172 (2013).\u003c/p\u003e\n\u003cp\u003e53. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Kim, D., Paggi, J. M., Park, C., Bennett, C. \u0026amp; Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 907\u0026ndash;915 (2019).\u003c/p\u003e\n\u003cp\u003e54. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Kovaka, S. \u003cem\u003eet al.\u003c/em\u003e Transcriptome assembly from long-read RNA-seq alignments with StringTie2. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 278 (2019).\u003c/p\u003e\n\u003cp\u003e55. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Concordet, J.-P. \u0026amp; Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, W242\u0026ndash;W245 (2018).\u003c/p\u003e\n\u003cp\u003e56. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Schwab, R., Ossowski, S., Warthmann, N. \u0026amp; Weigel, D. Directed Gene Silencing with Artificial MicroRNAs. in \u003cem\u003ePlant MicroRNAs\u003c/em\u003e (eds. Meyers, B. C. \u0026amp; Green, P. J.) vol. 592 71\u0026ndash;88 (Humana Press, Totowa, NJ, 2010).\u003c/p\u003e\n\u003cp\u003e57. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Minamino, N., Norizuki, T., Mano, S., Ebine, K. \u0026amp; Ueda, T. Remodeling of organelles and microtubules during spermiogenesis in the liverwort \u003cem\u003eMarchantia polymorpha\u003c/em\u003e. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e149\u003c/strong\u003e, dev200951 (2022).\u003c/p\u003e\n\u003cp\u003e58. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Bouckaert, R. \u003cem\u003eet al.\u003c/em\u003e BEAST 2: A Software Platform for Bayesian Evolutionary Analysis. \u003cem\u003ePLoS Comput. Biol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e1003537 (2014).\u003c/p\u003e\n\u003cp\u003e59. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Katoh, K. \u0026amp; Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 772\u0026ndash;780 (2013).\u003c/p\u003e\n\u003cp\u003e60. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Suyama, M., Torrents, D. \u0026amp; Bork, P. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, W609\u0026ndash;W612 (2006).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was supported by grants from National Natural Science Foundation of China (32425008, 32230009), Science and Technology Commission of Shanghai Municipality (24N12800100). We thank Lin Xu for providing \u003cem\u003eMarchantia polymorpha\u003c/em\u003e materials, Upp-5 and Upp-14; Ruiliang Zhu and Jian Wang for phylogenetic and morphological expertise on \u003cem\u003eM. polymorpha\u003c/em\u003e; Baiyan Lu for assistance in ROS staining; Zhiwei Gong for technical support on scanning electronic microscopy sample preparation and imaging; and Mengmeng Liu for technical assistance with dark-field microscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.L. conceived and supervised the project. Y.Z. and J.Z. generated transgenic plants, conducted in-planta functional analyses and protein-protein interaction studies. C.L., Y.Z. and J.Z. prepared the manuscript and figures. H.L. conducted bioinformatics analyses. L.Q. contributed to \u003cem\u003eamiR-\u003c/em\u003eMp\u003cem\u003eFER\u003c/em\u003e constructs. X.S. genetically characterized the genes studied in this project. M.X. and X.L. contributed to the construct and imaging of Mp\u003cem\u003efer\u003c/em\u003e mutants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data\u003c/strong\u003e are available for this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e is available for this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u0026nbsp;\u003c/strong\u003eshould be addressed to Chao Li.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGoodenough, U. \u0026amp; Heitman, J. Origins of Eukaryotic Sexual Reproduction. \u003cem\u003eCold Spring Harb. Perspect. 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Plant Physiol.\u003c/em\u003e \u003cstrong\u003e305\u003c/strong\u003e, 154421 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7888116/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7888116/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Sexual reproduction, a nearly universal feature of eukaryotes, begins with the fertilization of gametes\u003csup\u003e1,2\u003c/sup\u003e. Zoidogamy, or fertilization mediated by motile spermatozoids, represents an ancestral mode predating the evolution of pollen tube-based fertilization in angiosperms\u003csup\u003e3,4\u003c/sup\u003e. This mode dominated a long span of evolutionary history and remains widespread among early-diverging plants and animals\u003csup\u003e5–7\u003c/sup\u003e. However, the molecular mechanisms underlying this ancient fertilization process in plants remains poorly understood. In the liverwort \u003ci\u003eMarchantia polymorpha\u003c/i\u003e, we identify a mechanism by which the peptide hormone MpRALF1 regulates sperm motility through reactive oxygen species (ROS). The sperm flagella-derived MpRALF1 is perceived by a receptor complex composed of the MpFERONIA receptor kinase and its co-receptor MpLLG. This signaling module activates NADPH oxidase-mediated ROS production, thereby enhancing sperm velocity and progressive motility to ensure fertilization. Our study reveals the ancient origin of RALF peptide signaling in land plants and establishes ROS as a conserved mechanism governing sperm motility across kingdoms.","manuscriptTitle":"Ancient peptide–redox signaling underlies sperm motility in Marchantia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 06:15:43","doi":"10.21203/rs.3.rs-7888116/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-plants","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nplants","sideBox":"Learn more about [Nature Plants](http://www.nature.com/nplants/)","snPcode":"","submissionUrl":"","title":"Nature Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"07a86a0b-6ec7-40bd-80c0-e20a1b813ee1","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65133130,"name":"Biological sciences/Plant sciences/Plant reproduction/Fertilization"},{"id":65133131,"name":"Biological sciences/Plant sciences/Plant signalling"}],"tags":[],"updatedAt":"2026-05-11T04:41:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 06:15:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7888116","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7888116","identity":"rs-7888116","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}