The novel synaptonemal complex central element SCEP3 interlinks synapsis initiation and crossover formation in Arabidopsis thaliana

The novel synaptonemal complex central element SCEP3 interlinks synapsis initiation and crossover formation in Arabidopsis thaliana | 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 The novel synaptonemal complex central element SCEP3 interlinks synapsis initiation and crossover formation in Arabidopsis thaliana Stefan Heckmann, Chao Feng, Jana Lorenz, Steven Dreissig, Veit Schubert, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5394998/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jun, 2025 Read the published version in Nature Plants → Version 1 posted You are reading this latest preprint version Abstract The proteinaceous synaptonemal complex (SC) structure forms between meiotic homologous chromosomes. Its central region (CR) consists of transverse filament and central element proteins, in Arabidopsis ZYP1 and SCEP1/SCEP2, respectively. We describe a novel CR protein in Arabidopsis. SCEP3 spatiotemporally overlaps with other CR components and is conserved in plants. In scep3 , SC formation, crossover (CO) assurance (minimum one CO per chromosome pair), CO interference (limited closely-spaced CO) and heterochiasmy (male/female CO rate difference) vanish while genome-wide and particularly female CO increase. Compared with other CR proteins, SCEP3 is also critical for some synapsis-independent CO. SCEP3 interacts with ZYP1 but loads onto recombination intermediates independent of other CR proteins. We propose SCEP3’s loading onto recombination intermediates may stabilize and/or recruit further factors such as ZYP1 to a subset of these intermediates designated to form CO. Hence, SCEP3 interlinks SC and CO formation, being structurally likely the plant ortholog of yeast Ecm11. Biological sciences/Plant sciences/Plant cell biology Biological sciences/Plant sciences/Plant reproduction Biological sciences/Genetics/Plant genetics Biological sciences/Plant sciences/Plant development Arabidopsis thaliana Meiosis Synaptonemal complex SCEP3 ZYP1 Meiotic recombination Crossover interference Heterochiasmy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Meiotic homologous recombination assures genetic diversity in gametes 1 . Repair of programmed DNA double-strand breaks (DSBs) into interhomolog crossover (CO) involves numerous proteins and several consecutive steps 2,3 . In most species, CO are divided into two classes. Class I CO promoted by ZMM proteins (Zip1-4, Mer3, Msh4/5; 4,5 and MutL-γ (Mlh1/3) are interference-sensitive (one CO limits the probability of other CO nearby 6 ). Class II CO are insensitive to interference forming a minority in most species including Arabidopsis thaliana where their formation depends in part on MUS81 7,8 . Meiotic recombination occurs during prophase I. Sister chromatids are initially organized into a linear loop-base array by a proteinaceous structure called meiotic chromosome axis 1 . In Arabidopsis, the chromosome axis consists of ASY1 9,10 , ASY3 11 , ASY4 12 and cohesion-associated proteins including REC8 13 . Initial repair of a large number of DSBs (~200 in Arabidopsis) leads to numerous meiotic recombination intermediates including early interhomolog associations 14 . As a result, loose alignment (~400 nm distance) between two homologous chromosomes occurs. Upon installation of the transverse filament (TF) protein ZYP1, aligned chromosomes get physically connected at ~200 nm 15 , leading to the formation of the tripartite synaptonemal complex (SC) 1 . In most species including Arabidopsis, homolog alignment is independent of SC formation 15 . The SC structure is conserved across species, with two lateral elements (LEs) flanking a CR. Across species, the CR is composed of TF (e.g. Zip1 in Saccharomyces cerevisiae 16 , C(3)G in Drosophila melanogaster 17 or SYCP1 in Mus musculus 18 ) and central element (CE) proteins (e.g. Ecm11, Gmc2 in budding yeast 19 , Corona, Corolla in Drosophila 20,21 or SYCE1/2/3, TEX12, SIX6OS1 in mice 22,23,24,25,26 ). In Arabidopsis, ZYP1 (duplicated genes ZYP1a and ZYP1b ) is known as TF 27 while SCEP1 and SCEP2 as CEs 28 . CR proteins are required for SC assembly 19, 25, 28, 29 . In Drosophila, mice or Sordaria, the SC is required for CO formation 1 , while not in budding yeast 30,31,32 . In plants, the SC is dispensable for CO formation in Arabidopsis and rice 15,28,33,34 , but likely required in barley 35 . ArabidopsisCR mutants suggest that the SC is critical for CO assurance, heterochiasmy and CO interference 15,28,33,36 . Based on Arabidopsis data, coarsening of the ZMM protein HEI10 in frame of the SC was proposed as basis for CO interference 36,37 . However, the SC is not required for implementing CO interference in budding yeast or Sordaria 1 . Interactions among CR proteins are linked to SC assembly, e.g. in budding yeast, Ecm11-Gmc2 promotes Zip1 polymerization 19 , or in mice, SYCP1 tetramers remodeled by SYCE3 form a SYCP1-SYCE3 complex and SYCE3 also interacts with TEX12-SYCE2 and SYCE1-SIX6OS1 38 . In budding yeast, SC assembly and CO formation are coupled via the interaction of Ecm11-Zip4 (in mouse via orthologs TEX12-TEX11) 39 . In Arabidopsis, SCEP1-SCEP2 directly interact, while none interacts with ZYP1 or ZIP4 28 . Whether in Arabidopsis further CE proteins exist that form a complex with ZYP1 and/or interconnect SC assembly and CO formation is unclear. We identified proteins in proximity of ASY1 and ASY3 via proximity-proteomics 40 . Here, we functionally characterize the candidate ATC21 re-named SCEP3. SCEP3 is a novel CE protein conserved across plants and the likely structural ortholog of yeast Ecm11 and possibly also of SYP-4 (worms), Corolla (flies) or SIX6OS1 (mammals). Results SCEP3 is required for synapsis, chiasma formation and CO assurance SCEP3 has 16 exons, encodes a protein of 803 AA (Fig. 1a), and is highly expressed in young flower buds 41 . According to structural prediction by AlphaFold2, the C-terminal 70 AA form an α-helical domain, while the remaining 733 AA are disordered (Fig. S1a). No obvious developmental differences when compared with the WT were found in any scep3 allele (Fig. 1a and Fig. S1b), except slightly shorter siliques with seed gaps (Fig. S1c). In all scep3 alleles including scep3-1scep3-2 , seed fertility was reduced by ~30% suggesting allelism of mutations (Fig. 1b). Pollen viability was also significantly reduced in scep3 (Fig. S1d). In the WT, synapsed chromosomes at pachytene, five bivalents at metaphase I and balanced chromosome segregation are found (Fig. 1c). In scep3 , typical pachytene chromosomes with thick chromatin threads suggesting synapsis were not found but regions with chromosome alignment (Fig. 1c). Indeed, based on spatial structural illumination microscopy (3D-SIM) inter-axes measurements (Fig. S1e), scep3 chromosomes align but do not get in close apposition during pachytene. In scep3 , cells with pair(s) of univalents (absence of CO assurance) and unequal chromosome segregation were found (Fig. 1c). While in the WT invariably five bivalents were found, in all scep3 alleles, the average bivalent number was reduced with ~50% of cells displaying one to three pairs of univalents (Fig. 1d). Minimum chiasmata numbers (MCN) in all scep3 alleles are significantly reduced to ~70-75% of WT-levels (Table 1). No difference was found in scep3-1scep3-2 whencompared with single mutants (Fig. 1d and Table 1; P = 0.2776; one-way ANOVA). Similar to male meiosis, scep3 female meiosis revealed chromosome alignment at larger distances and the absence of CO assurance (Fig. S1f). SCEP3 is found at the central region of the SC To dissect the spatiotemporal localization of SCEP3, antibodies raised against its N- (SCEP3-N) or C-terminus (SCEP3-C) were employed. SCEP3 is initially detected during early zygotene forming a limited number of foci (Fig. 2a). Progressively foci numbers increase with initial SCEP3 stretches during zygotene and full polymerization at pachytene. During all stages, SCEP3 co-localizes with ZYP1. SCEP3 also overlaps at pachytene with the CE proteins SCEP1 and SCEP2 (Fig. 2b). Using 3D-SIM, SCEP3-C overlaps with SCEP3-N albeit less continuous (Fig. 2c). Absence of chromatin-associated signals in scep3 confirms the specificity of both antibodies (Fig. 2d). Via 3D-SIM, the following SC organization was detected at pachytene: REC8 and ASY4 form two parallel lines separated by ~188 and ~176 nm (Fig. S1e and Fig. 2f), respectively, and in between those ZYP1-C forms either two lines or a single bright line. Both SCEP3-N and SCEP3-C are located centrally of the bright/two ZYP1-C line(s) (Fig. 2e,f). Note, a similar Arabidopsis SC organization, i.e. REC8 axes at a distance of 175-213 nm, in between two lines of ZYP1-C and in between those centrally CEs SCEP1 and SCEP2 was found using Stimulated Emission Depletion (STED) microscopy 15,28 . Together, SCEP3 is a CE protein. SCEP3 is critical for SC formation and loads onto recombination intermediates independent of other CR proteins In WT, polymerization of all CR proteins is found at pachytene, while ASY1 gets depleted from synapsed regions (Fig. 2a and Fig. S2a). In scep3-1 pachytene(-like) nuclei, neither chromosome-associated ZYP1, SCEP1 or SCEP2 nor their polymerization is found (Fig. 3a), suggesting absence of SC assembly in scep3-1 and chromosomal loading of ZYP1, SCEP1 and SCEP2 being SCEP3-dependent. Absence of synapsis is also reflected by ASY1 persistence at pachytene(-like) stages 15,28 (Fig. 3a). In zyp1-2 , scep1-1 or scep2-1 pachytene(-like) nuclei, axis-associated SCEP3 foci are found (Fig. 3b). Hence, SCEP3 loads independent of SC formation and other CR proteins. To test whether SCEP3 loads onto recombination intermediates, HEI10 (signal absence in hei10 ; Fig. S2b) and SCEP3 were immunolocalized. In pachytene(-like) nuclei of zyp1-2 , scep1-1 and scep2-1 , SCEP3 associates with HEI10-dependent recombination intermediates (~80% of HEI10 foci overlap with SCEP3 foci and ~55% of SCEP3 foci overlap with HEI10 foci) (Fig. 3c). In WT, SCEP3 and HEI10 foci also co-localize at zygotene, however, due to synapsis-progression, SCEP3 foci gradually turn into uniformly linear signals decorated by bright HEI10 foci (Fig. S2c). In scep3-1 , no ZYP1, SCEP1 or SCEP2 foci overlap with HEI10 (Fig. 3d). Note, no CR component is required for HEI10 loading (Fig 3c,d). ZYP1 co-localizes with SCEP3 in scep1-1 or scep2-1 early pachytene(-like) nuclei (Fig. 3e) and while SCEP3 overlaps with HEI10 foci in zyp1-2 , neither SCEP1 nor SCEP2 does (Fig. 3f). Together, all CR proteins are required for SC assembly but SCEP3 loads onto recombination intermediates (HEI10-marked) independent of other CR components/SC formation and recruits ZYP1, but neither SCEP1 nor SCEP2. SCEP3 physically interacts with ZYP1 Given SCEP3-ZYP1 co-localization even in CE mutants, their relationship was addressed in mutants with full SC polymerization despite impaired ZMM-dependent CO formation ( msh5-2 , hei10-2 , zip4-2 , mer3-1 , shoc1-1 ) or with SC polymerization impaired to different degrees ( dmc1-2 , asy3-1 , rec8-1 , pch2-1 , asy1-4 ). In all cases, SCEP3-ZYP1 co-localization was found (Fig. S3). Thus, SCEP3-ZYP1 form similar complexes even SC-independent. In yeast two-hybrid (Y2H) assays, a strong interaction of SCEP3-ZYP1 (both ZYP1a and ZYP1b) was found mediated by the N-terminus of ZYP1 (49-400 AA, ZYP1b) and SCEP3’s C-terminus (734-803 AA) (Fig. 4a, b). Notably, AlphaFold3 also predicts an interaction of SCEP3’s C-terminus (α-helical domain) and ZYP1’s N-terminus (Fig. 4c). No Y2H interaction of SCEP3 with axis proteins ASY1, ASY3 and REC8 was described 40 . We also found neither an interaction of SCEP3 with axis(-associated) proteins ASY4, COMET and PRD3 nor with ZMM proteins ZIP4, HEI10, MER3 and PTD (Fig. 4d). SCEP3 and its interaction with ZYP1 are evolutionarily conserved in plants Using PSI-BLAST, SCEP3 is conserved across green plants including many lower plants but no homolog was found outside streptophyta (Fig. S4). High sequence similarity at both the N- and C-terminal regions of SCEP3 is found (Fig. S5). Across most plants, homologues of all four CR components are found but in lower plants such as Taxus chinensis or Marchantia polymorpha homologues of SCEP1/SCEP2 are absent (Fig. S4). The evolutionarily closer relationship between SCEP3 and ZYP1 may reflect their detected direct interaction in A. thaliana . In Y2H full-length barley SCEP3-ZYP1 interact (Fig. S6a) and AlphaFold3 predicts SCEP3-ZYP1 interaction in various plants involving similar regions as in Arabidopsis (Fig. S6b). Hence, a SCEP3-ZYP1 interface seems highly conserved across plants. SCEP3 is structurally likely the plant ortholog of yeast Ecm11 Given no SCEP3 homologues outside of plants based on PSI-BLAST, we searched for structural orthologues focusing on known SC proteins (Fig. S7). The structure of plant SCEP3 was similar to budding yeast (or Valsa malicola ) Ecm11, with a typical short alpha-helical domain (70-80 AA) at the C-terminus and a long variable disordered region at the N-terminus. Structural similarities were also found for SYP-4 (worms), Corolla (flies)or SIX6OS1 (mammals). SCEP3 is required for some CO arising upon abolished or impaired SC formation Based on γH2AX foci numbers as a proxy for the number of DSB sites 42,43 , no difference was found in scep3-2 when compared with WT (Fig. S8a, b). Hence, shortage of chiasmata in scep3 is unlikely due to reduced DSB numbers. Compared to zyp1 withincreased HEI10 foci numbers 15 , in scep3-1 similar HEI10 foci numbers were found (9.94 ± 2.43) as in the WT (10.34 ± 1.74; P = 0.36, two-sided Student’s t -test) (Fig. 5a, b). In scep3-1zyp1-2 the MCN is significantly (two-sided Student’s t -test) reduced to 5.62 ± 1.91 when compared with 7.69 ± 1.54 in zyp1-2 ( P = 1.05x10 -9 ), while not when compared with 5.93 ± 1.56 in scep3-1 ( P = 0.25) (Table 1). Univalent frequency in scep3-1zyp1-2 (58.7%) is similar to scep3-1 (55.3%) while higher than in zyp1-2 (14.3%) (Fig. 5c). Moreover, HEI10 foci numbers in scep3-1zyp1-2 (10.03 ± 2.80)are similar to scep3-1 ( P = 0.85, two-sided Student’s t -test) but significantly reduced when compared with zyp1-2 (13.18 ± 3.28; P = 7.07x10 -8 , two-sided Student’s t -test) (Fig. 5a, b) . Hence, scep3 is genetically upstream of zyp1 in terms of CO formation. In asy1 and asy3 with impaired SC formation, genetically upstream of ZYP1 33 , upon SCEP3 depletion, chiasma/bivalent numbers are reduced further (Fig. 5c and Table 1). Thus, SCEP3 is critical for some CO arising upon impaired SC formation. SCEP3 is required for some synapsis-independent class I and class II CO In mutants impaired in class I CO formation ( msh5-2 , hei10-2 , mlh3-1 ), upon SCEP3 depletion chiasma/bivalent numbers are further reduced (Fig. 5c and Table 1). In mus81-2 , deficient in some class II CO, five bivalents are found. In scep3-1mus81-2 , chiasma/bivalent numbers are significantly reduced when compared with scep3-1 (Fig. 5c, Table 1). These data suggest that (i) the majority of CO are ZMM-dependent class I CO in scep3 , (ii) SCEP3 is required for some class II CO, and (iii) SCEP3-independent class II CO in part depend on MUS81 (0.87 bivalents in scep3msh5 vs a reduction of 0.23 bivalents in scep3 upon MUS81 depletion). Genome-wide and particularly female CO are increased while heterochiasmy and CO interference are abolished in scep3 To dissect genome-wide male and female CO, we isolated scep3-4 in Ler-0, with similar phenotypes as scep3 in Col-0 (Fig. 1 and Fig. S9). By crossing scep3-2+/- with scep3-4+/- , we generated F1 hybrids of WT (Col-0 x Ler-0) and scep3 (Col-0 x Ler-0). These were backcrossed with Col-0 either as female ormale and the four offspring groups (WT female/male, scep3 female/male) were sequenced. In scep3 males (25%) and particularly females (105%) CO numbers significantly increased compared to WT (Fig. 6a). Heterochiasmy found in WT vanished in scep3 (Fig. 6a). CO frequencies increased along chromosome arms especially towards chromosome ends and decreased in pericentromeric regions (Fig. 6b). In both sexes, CO interference found in the WT, was abolished in scep3 (Fig. 6c). Together, scep3 CO increased genome-wide as well as heterochiasmy and CO interference vanished similar to other CR mutants 15,28 . However, despite comparable CO distributions (Fig. 6b), male and female scep3 CO are ~15% lower compared to zyp1 or scep1 (Fig. 6a) 15,28 . In scep3 males, this is consistent with the absence of increased HEI10 foci, and reduced chiasmata when compared with zyp1 (Fig. 5b and Table 1). In scep3 females, HEI10 foci strikingly increased by ~92% (10.17, n = 6) when compared with WT (5.29, n = 7) (Fig. 6d) thus largely accounting for the female CO increase. Discussion Two plant CE proteins, SCEP1/2, were identified by transcriptomics 28 . We identified SCEP3 by TbID-based proteomics 40 . SCEP3 spatiotemporally overlaps with CR components, is found within the CR of the SC, is critical for SC assembly, and interacts with ZYP1’s N-terminus that localizes to the SC center. Together, SCEP3 is a novel plant CE protein. Spatiotemporal and functional overlap suggests interactions among CR proteins. SCEP1-SCEP2 form a complex, while none of them interacts with ZYP1 28 . SCEP3 loads at HEI10-marked recombination intermediates independent of SC formation/other CR proteins. Its loading is sufficient for the recruitment of ZYP1, but neither of SCEP1 nor SCEP2. SCEP3 also directly interacts with ZYP1 and this interaction seems conserved across plants. Thus, SCEP3 likely acts as a synapsis initiation factor recruiting ZYP1 to recombination intermediates for synapsis initiation. Notably, SCEP3 and HEI10 foci also form exclusively, e.g. SCEP3 may also localize at non-ZMM intermediates as suggested by its requirement for some class II CO. Whether SCEP3-ZYP1 load as complex or SCEP3 recruits ZYP1 is unclear. We prefer the latter, as SCEP3 loads in zyp1 . Which factor recruits SCEP3 is unclear, as none of the tested axis or ZMM candidates directly interacts with SCEP3 or is critical for SCEP3 localization. In budding yeast, Zip4 links recombination intermediates and SC assembly by recruiting and interacting with Ecm11 39 . While this seems conserved in mammals, in Arabidopsis neither the CE proteins SCEP1, SCEP2 28 or SCEP3 directly interact with ZIP4 nor is SC formation impaired in zip4 . SCEP1 and/or SCEP2 do not form a complex with SCEP3 and/or ZYP1 at least SC independent, i.e. only SCEP3 loads in zyp1 and only SCEP3-ZYP1 load in scep1 and scep2 28 . Whether further proteins/modifications and/or the SC context are required for complex formation is unclear. Together, the Arabidopsis SC is composed of at least two subdomains, SCEP3-ZYP1 and SCEP1-SCEP2 but how they are functionally linked remains to be addressed. In scep3 , CO interference, CO assurance and heterochiasmy vanish but CO numbers increase, suggesting these are the common phenotypes after SC abolishment in Arabidopsis 15,28,33 . However, scep3 CO numbers are lower compared with other CR mutants but increased compared with WT. In scep3 , surplus CO seem largely ZMM-dependent in females, while not in males (no additional HEI10 foci). In both scep3 sexes, HEI10 foci represent ~83% of the total CO, suggesting ~17% ZMM-independent class II CO. However, scep3 offspring CO rates might be overestimated as only meiotic cells with comparatively high CO numbers may form viable gametes. The majority of CO are class I CO in scep3 but SCEP3 is required for some synapsis-independent class I and II CO. In males, SCEP3 is even critical for the surplus HEI10-dependent CO in zyp1 and for some chiasmata in asy1 or asy3 (impaired SC formation). We speculate that SCEP3’s loading at recombination intermediates prior and independent of synapsis/other CR proteins may stabilize and/or recruit further factors such as ZYP1 to a subset of these intermediates for synapsis initiation designated to form class I and/or II CO. In Arabidopsis CR mutants including scep3 , CO interference vanishes 15,28,33 , suggesting the SC per se and not individual components is required for CO interference implementation. Our data are compatible with the HEI10 coarsening model in which the SC (scaffold for HEI10 diffusion and condensation) imposes CO interference 36,37,44 . Two non-exclusive scenarios may also contribute to increased CO numbers without signatures of CO interference in CR mutants, i.e. longer axis persistence of ASY1 and/or lack of PCH2 45 or absence of SC polymerization-mediated local downregulation of de novo DSB 30,46 . The direct interaction of SCEP3’s C-terminus with ZYP1 is likely conserved across plants. The functional significance of the conserved N-terminus remains unclear. We speculate that both conserved termini mediate SCEP3’s role in interlinking meiotic recombination and SC initiation/formation. SCEP3 is likely the plant structural ortholog of fungus Ecm11. Functional similarities, e.g. Ecm11 is a CE, limits CO, or co-localizes with Zip3 (HEI10) independent of Zip1 19 , but also differences, e.g. Ecm11 interacts with Zip4 but not with Zip1 39 , are found. Structural similarities are also found for SYP-4 in C. elegans , COROLLA in D. melanogaster or SIX6OS1 in mammals. We propose a dual role for SCEP3, one as a CR component of the SC required for its assembly and the other as a synapsis initiation factor associated with recombination intermediates, together interlinking SC and CO formation. Methods Plant materials and growth conditions A. thaliana plants were grown under short-day conditions (8/16 h light/dark) for four weeks followed by long-day conditions (16/8 h light/dark) until maturity at constant 22°C. Col-0 was used as WT except where indicated. T-DNA insertion alleles, provided by the Nottingham Arabidopsis Stock Centre 47 , used in this study are: scep3-1 ( AT4G18490 ; SAILseq_210_G05), scep3-2 ( AT4G18490 ; SALK_098044), msh5-2 ( AT3G20475 ; SALK_026553) 48 , mus81-2 ( AT4G30870 ; SALK_107515) 8 , asy1-4 ( AT1G67370 ; SALK_046272) 49 , dmc1-2 ( AT3G22880 ; SAIL_170_F08) 50 , asy3-1 ( AT2G46980 ; SALK_143676) 11 , rec8-1 ( AT5G05490 ; SALK_137095) 51 , pch2-1 ( AT4G24710 ; SAIL_1187_C06) 49 , zip4-2 ( AT5G48390 ; SALK_068052) 52 , hei10-2 ( AT1G53490 ; SALK_014624) 53 , mer3-1 ( AT3G27730 ; Salk_091560) 54 , shoc1-1 ( AT5G52290 ; SALK_057589) 55 and mlh3-1 ( AT4G35520 ; SALK_015849) 56 . zyp1-2 33 , scep1-1 and scep2-1 28 were described. The alleles scep3-3 and scep3-4 were isolated using CRISPR/Cas9 in this study. Details of primers used for genotyping are found in Suppl. Table 1. Isolation of scep3-3 and scep3-4 using CRISPR/Cas9 Targeted mutagenesis in Arabidopsis via CRISPR/Cas9 was performed according 57 . pMOD_A0503, pMOD_B2103, pMOD_C0000 and pTRANS_260d (Addgene #91013, #91061, #91081 and #91126) were used for assembling CRISPR constructs with Cas9 and gRNAs both driven by CmYLCV promoter. Two gRNAs (#1: 5’-GAGCCAAAGCCAAAATCCATTGG-3’; #2: 5’-AACTAGACAAGTTCCCTCCAAGG-3’) addressing SCEP3 were Golden Gate assembled in pMOD_B2103. The final expression cassettes assembled in pTRANS_260d were Agrobacterium-mediated transformed in ecotypes Col-0 and Ler-0 by floral dip 58 . Based on Sanger-sequencing of target sites, scep3-3 and scep3-4 were isolated in transgenic lines in the Col-0 and Ler-0 background, respectively. For primer details see Suppl. Table 1. Yeast two-hybrid (Y2H) assays Full-length or truncated coding sequences of Arabidopsis SCEP3 , ZYP1a , ZYP1b , SCEP1 , SCEP2 , C-terminus of ZIP4 according to 28 , HEI10 , MER3 , PTD , COMET , ASY4 and PRD3 were PCR-amplified using Col-0 flower bud cDNA as template and cloned into pGBKT7 and/or pGADT7 vectors (Takara) by Gibson Assembly (NEB). Cloning of barley SCEP3 and ZYP1 was done accordingly, but using cDNA prepared from barley anthers (cultivar Golden Promise). For primer details, see Suppl. Table 1. Y2H assays were performed according to the manufacturer’s instructions (Takara). Bait and prey plasmids (empty vectors as controls) were co-transformed into the yeast strain Y2HGold (Takara, 630489) and grown at 30°C for 3-5 days on plates with SD Base medium supplemented with DO Supplement –Leu/–Trp (DDO, 630417). Transformed clones underwent selection assays for 5 days on plates with Minimal SD Base medium supplemented with DO Supplement –His/–Leu/–Trp (TDO, 630419) or DO Supplement –Ade/–His/–Leu/–Trp (QDO, 630428). AlphaFold protein structure modeling The protein structure models of SCEP3 homologues (Fig. S7) are from the AlphaFold Protein Structure Database 59, 60 . The Arabidopsis SCEP3 protein structure (Fig. S1a) was modeled by AlphaFold2 using ColabFold 61 . Protein-protein interaction modeling (Fig. 4c and Fig. S6b) was performed with AlphaFold3 62 and depicted with UCSF ChimeraX 63 . Generation of polyclonal antibodies Peptide synthesis and antibody production were performed by LifeTein LLC (New Jersey, USA). The following peptides were selected for respective Arabidopsis proteins and used for immunization: SCEP3-N (69-87 AA; C-GSSFKMDMPDFDFSSPAKK) in rat, SCEP3-C (756-775 AA, C-KKKHEEAKELLVRAVVDNNK) in rabbit, HEI10 (C-PKDEIWPARQNS, according 64 ) in rabbit and guinea pig, ZYP1-C (833-851 AA in ZYP1b; C-SANIGDLFSEGSLNPYADD; peptides identical in ZYP1a/b) in rat and guinea pig, ASY4 (C-AKLPDELDVDVSSDFKGI) and ASY1 (C-SKAGNTPISNKAQPAASRES, according 65 ) in rabbit and rat. All antibodies were affinity-purified against the synthetic peptide. Cytological procedures Pollen viability was assessed using Alexander’s stain 66 as described 40 . Male and female meiotic chromosome spread preparations and MCN counting were performed as described 67 . Immunolocalization was performed as described 67, 68 using fresh Arabidopsis flower buds. Following primary antibodies and dilutions were used: anti-ASY1 (rabbit, 9 ; 1:2,000), anti-ASY1 (rabbit or rat, this study; 1:200), anti-ZYP1-C (guinea pig, 27 ; 1:2,000), anti-ZYP1-C (rat or guinea pig, this study; 1:200), anti-REC8 (rabbit, 69 ; 1:1,000), anti-γH2Ax (mouse, Sigma-Aldrich #05-636; 1:200), anti-SCEP3-N (rat, this study; 1:100), anti-SCEP3-C (rabbit, this study; 1:100), anti-HEI10 (guinea pig, this study; 1:200), anti-SCEP1 (rabbit, 28 ; 1:200) and anti-SCEP2 (rat, 28 ; 1:200). The following secondary antibodies were used (all diluted 1:500): anti-guinea pig Cy5 (Abcam, ab102372), anti-guinea pig Alexa 594 (Invitrogen, A11076), anti-guinea pig Alexa 488 (Invitrogen, A11073), anti-rabbit Alexa 594 (Abcam, ab150076), anti-rabbit Alexa 488 (Abcam, ab150073), anti-rabbit Cy3 (Jackson ImmunoResearch, 111-165-003), anti-rat Alexa 488 (Jackson ImmunoResearch, 112-545-167) and anti-rat alexa 594 (Abcam, ab150160). Microscopy Epifluorescence images were acquired using a Nikon Eclipse Ni-E microscope equipped with a Nikon DS-Qi2 camera and NIS-Elements-AR version 4.60 software (Nikon). SCEP3-HEI10 co-localization analysis was performed using Imaris (Bitplane, Switzerland) version 10.1.0. Automatic spot detection with a size of 0.33 µm and background subtraction was applied for the red and the green channel independently. Afterwards, a filter was chosen on the red channel to identify spots overlapping with spots in the green channel, allowing a maximum distance of 0.33 µm. 3D-SIM image stacks were acquired using an Elyra 7 microscope system and the software ZEN Black (Carl Zeiss GmbH) 70 . Inter-axis distances were measured from center to center of each parallel axis in maximum intensity projection images (positions randomly picked). Images were processed with ZEN 3.1 (blue edition), Fiji (open source) 71 and Adobe Photoshop CS5 (Adobe). Genome-wide mapping of male and female COs in WT and scep3 To generate male and female CO mapping populations, scep3-2 +/- (Col-0) was crossed with scep3-4 +/- (Ler-0) and F1 hybrids WT or biallelic for scep3 were crossed as a male or female with the WT Col-0. Total DNA samples were prepared from the four-resulting backcross populations (WT male, 143 plants; WT female, 237 plants; scep3-2scep3-4 male, 142 plants; scep3-2scep3-4 female, 238 plants) using ~150 mg leaf sample per individual plant and Econospin columns (96 well, Epoch Life Sciences) according to manufacturer’s instructions adapted to plant samples. Whole Genome Shotgun (WGS) sequencing library preparation (Illumina DNA PCR-Free Library Prep, Tagmentation with standard DNA input amount) involved protocols from the manufacturer (Illumina, Inc., San Diego, CA, USA). The library was quantified by qPCR (KAPA Library Quant Kit; Roche Molecular Systems, Inc.) and sequenced according to manufacturer’s instructions using the NovaSeq 6000 device (Illumina, Inc., San Diego, CA, USA; run type: SP flowcell with XP workflow and paired-end sequencing: 151 cycles (read 1) 10 cycles (index read 1), 10 cycles (index read 2) and 151 cycles (read 2)) at IPK Gatersleben. Raw sequence reads were aligned to the A. thaliana Col TAIR10 reference genome 72 using BWA-MEM 73 , and converted to Binary/Alignment Map format and sorted using SAMtools 74 . Variant calling was done using BCFtools 75 filtering for a minimum mapping quality and minimum base quality of 30. The resulting variants matrix in Variant Call format (VCF) was filtered using VCFtools 76 for bi-allelic single nucleotide variants (SNV), minor allele frequency ranging between 0.2 - 0.3, minimum read depth per site of 4, maximum read depth per site of 100, minimum mean read depth of 1 across all samples, and a maximum mean read depth of 1.5. The resulting variant matrix contained 350,575 high-quality single nucleotide variants. Individuals with more than 75% missing data were removed (9 out of 760 samples). Genotype calls homozygous for the reference allele (Ler) were removed from further analysis (average of 8% per sample). Samples with SNV numbers below the 5% percentile or above the 95% percentile were removed from further analysis to avoid potential biases caused by extreme marker number deviations. To measure recombination events, SNV information was first aggregated in sliding windows of 20 consecutive SNVs with a step size of 1 by determining the modal SNV. Secondly, smoothed SNVs were further aggregated in non-overlapping windows of 1 Mb. For CO interference analysis, only chromosomes with exactly 2 CO were used. Observed inter-CO distances were compared against random inter-CO distances obtained via 500 permutations of the respective dataset (i.e. male/female WT, male/female scep3 ). CO positions of zyp1 and scep1 were retrieved from 15, 28 . Statistical analysis of CO count and inter-CO distance was done in R by nested analysis of variance (ANOVA) (aov(CO count or inter-CO distance ~ sex/genotype)) followed by a Tukey HSD test. Recombination landscapes were analyzed via χ 2 -test. Declarations Data availability Data supporting the findings of this research are presented in the main text, figures and supplementary information. Generated materials are available from the corresponding author upon reasonable request. Whole-genome re-sequencing raw data underlying Fig 6 are deposited to the European Nucleotide Archive (ENA) under accession number PRJEB81799 (http://www.ebi.ac.uk/ena/data/view/PRJEB81799). Gene/protein sequences and accession codes used in this study are found in databases TAIR (https://www.arabidopsis.org/) and Ensembl Plants (http://plants.ensembl.org/index.html). Acknowledgements We thank M. Grelon (IJPB, France) for kindly providing REC8, SCEP1 and SCEP2 antibodies as well as scep1-1 and scep2-1 seeds, J. Higgins (University of Leicester, UK) and H. Puchta (JKIP and KIT, Germany) for sharing zyp1-2 seeds and C. Franklin (University of Birmingham, UK) for sharing ASY1 and ZYP1 antibodies. We are grateful to all lab members and colleagues at IPK for fruitful discussions, N. Bruhne for help with DNA sample collection, J. Pohl for help with WGS library preparation and sequencing, A. Fiebig for uploading of sequencing data and K. Weisshart (Carl Zeiss GmbH) for 3D-SIM image analysis. This work has received funding from the Deutsche Forschungsgemeinschaft (DFG) within the framework of two projects (grant agreement 354617974 and 543670370.) to S.H. and from the IPK Gatersleben. S.H. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 949618). B.W. is a holder of a China Scholarship Council (CSC) fellowship (CSC202103250012). M.C. and F.H. acknowledge funding from the state of Sachsen-Anhalt (ZB I 180). Author contributions C.F. with assistance from J.L., B.W. and F.H. conducted most of the research. V.S performed 3D-SIM imaging. A.C. and C.F. performed AlphaFold predictions and interpretations. A.H. and S.D performed whole-genome offspring re-sequencing and data analysis. N.F. and M.C. contributed to immunolocalization procedures and image analysis. S.H. acquired funding. C.F. and S.H. designed the experiments, analyzed the data and wrote the manuscript. 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13:48:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSCEP3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and phenotypic analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003escep3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e alleles.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Gene model of \u003cem\u003eSCEP3\u003c/em\u003e (\u003cem\u003eAT4G18490\u003c/em\u003e; confirmed by Sanger sequencing of flower bud cDNA) including exons (black boxes) and introns (black lines) and below schematic depiction of SCEP3 protein. Mutant alleles are indicated: \u003cem\u003escep3-1\u003c/em\u003e (initially named \u003cem\u003eatc21-1\u003c/em\u003e \u003csup\u003e40\u003c/sup\u003e) and \u003cem\u003escep3-2 \u003c/em\u003e(T-DNA insertions within exon 13 and intron 8, respectively) as well as \u003cem\u003escep3-3\u003c/em\u003e and \u003cem\u003escep3-4\u003c/em\u003e (CRISPR/Cas9-based mutagenesis); \u003cem\u003escep3-4\u003c/em\u003e is in Ler-0 while all other alleles are in Col-0 background. \u003cstrong\u003eb,\u003c/strong\u003e Seeds per silique in the WT, \u003cem\u003escep3\u003c/em\u003e alleles and allelic cross. Data are presented as mean ± standard error of the mean (s.e.m.). \u003cstrong\u003ec,\u003c/strong\u003e Male meiotic chromosome behavior (scale bar, 10 μm; DNA counterstained with DAPI in gray) and \u003cstrong\u003ed,\u003c/strong\u003e frequency of cells with 0-3 pair of univalents including the average bivalent number per cell (n = number of cells analyzed) in the WT, \u003cem\u003escep3\u003c/em\u003e alleles and allelic cross.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/d0d149925bf55304c45f611f.png"},{"id":69547249,"identity":"eded3a7a-0ff9-4359-add1-75dfac16c9ce","added_by":"auto","created_at":"2024-11-21 13:56:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization of SCEP3 at the CR of the SC\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eImmunolocalization in the WT of \u003cstrong\u003ea,\u003c/strong\u003e SCEP3-N, ZYP1-C and ASY1 during prophase I, \u003cstrong\u003eb,\u003c/strong\u003e SCEP3-C and ZYP1-C or SCEP2 as well as SCEP3-N and SCEP1 at pachytene, and \u003cstrong\u003ec,\u003c/strong\u003e SCEP3-C and SCEP3-N in a pachytene nucleus visualized via 3D-SIM. \u003cstrong\u003ed,\u003c/strong\u003eImmunolocalization of SCEP3-C or SCEP3-N and ASY1 in \u003cem\u003escep3-2\u003c/em\u003e. DAPI-stained DNA in gray (a-d).\u003cstrong\u003e \u003c/strong\u003e3D-SIM of a pachytene nucleus in the WT immunolabelled with \u003cstrong\u003ee, \u003c/strong\u003eREC8, ZYP1-C and SCEP3-N or \u003cstrong\u003ef,\u003c/strong\u003e ASY4, ZYP1-C and SCEP3-N. Scale bar in \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e, 10 μm. Scale bar in \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e, 2 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/17a24b690ba253384bb3e54c.png"},{"id":69545792,"identity":"b4e8be30-cc06-4abf-a7e8-476204077669","added_by":"auto","created_at":"2024-11-21 13:48:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":268477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZYP1, SCEP1, SCEP2 and SCEP3 are interdependent for SC assembly but SCEP3 localization is independent of other CR proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunolocalization of \u003cstrong\u003ea,\u003c/strong\u003e ZYP1-C, SCEP1 or SCEP2 and ASY1 in \u003cem\u003escep3-1\u003c/em\u003e, of \u003cstrong\u003eb, \u003c/strong\u003eSCEP3-N and ASY1 and \u003cstrong\u003ec,\u003c/strong\u003e SCEP3-N and HEI10 in \u003cem\u003ezyp1-2\u003c/em\u003e, \u003cem\u003escep1-1\u003c/em\u003e or \u003cem\u003escep2-1\u003c/em\u003e, of \u003cstrong\u003ed,\u003c/strong\u003e ZYP1-C, SCEP1 or SCEP2 and HEI10 in \u003cem\u003escep3-1\u003c/em\u003e, of \u003cstrong\u003ee,\u003c/strong\u003e SCEP3-N and ZYP1-C in \u003cem\u003escep1-1\u003c/em\u003eor \u003cem\u003escep2-1\u003c/em\u003e, and of \u003cstrong\u003ef,\u003c/strong\u003e SCEP1 or SCEP2 and HEI10 in \u003cem\u003ezyp1-2\u003c/em\u003e. DAPI-stained DNA in gray.\u003cstrong\u003e \u003c/strong\u003eScale\u003cstrong\u003e \u003c/strong\u003ebar, 10 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/e5355e6def1a3a6c35bb318e.png"},{"id":69545793,"identity":"206d1a9b-0752-4f52-8489-e4fdd4e304fc","added_by":"auto","created_at":"2024-11-21 13:48:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":179572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCEP3 physically interacts with ZYP1 but neither with axis(-associated) nor with ZMM proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Schematic depiction of ZYP1b (left) and SCEP3 (right) including fragments tested by Y2H assays. ZYP1b: central α-helical domain (orange) flanked by two flexible regions (pink and green). SCEP3: C-terminal α-helical domain (blue) and N-terminal disordered region (red). Green, gray, and black lines indicate positive interaction, negative interaction, and self-activation, respectively, in Y2H experiments depicted below. \u003cstrong\u003eb,\u003c/strong\u003e Y2H interaction studies of ZYP1 and SCEP3: Both ZYP1a and ZYP1b interact with SCEP3 (full-length proteins). To determine the sub-region within ZYP1 and SCEP3 responsible for their interaction, ZYP1 (ZYP1b was used) was divided into four fragments and SCEP3 into two. The N-terminal region of ZYP1 (49-400 AA) and the C-terminal region of SCEP3 (734-803 AA) were found responsible for the interaction between SCEP3 and ZYP1. \u003cstrong\u003ec,\u003c/strong\u003e AlphaFold3 complex prediction using C-terminus of SCEP3 and full length ZYP1b in Arabidopsis. Predicted aligned error (PAE) values shown on the right; the interface predicted template modelling (ipTM) score, 0.48; pTM, 0.24. \u003cstrong\u003ed, \u003c/strong\u003eInteractions tested between SCEP3 and CE (SCEP1 or SCEP2; due to self-activation of SCEP1 and SCEP2 in our Y2H system their interaction with SCEP3 could not be addressed), axis(-associated) (ASY4, COMET or PRD3) or ZMM proteins (ZIP4, HEI10, MER3 or PTD) in Y2H. TDO (SD/-LTH) is a less stringent and QDO (SD/-LTHA) is a more stringent medium used for selection.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/c26ad0012418dacce0d7e65e.png"},{"id":69545796,"identity":"485d22b5-3304-4c39-a0a9-d01e86403cc5","added_by":"auto","created_at":"2024-11-21 13:48:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":146381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCEP3 is critical for class I and class II CO formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Immunolocalization of ASY1 (green) and HEI10 (magenta) and \u003cstrong\u003eb,\u003c/strong\u003e quantification of HEI10 foci number per diplotene/diakinesis cell in WT, \u003cem\u003escep3-1\u003c/em\u003e, \u003cem\u003ezyp1-2\u003c/em\u003e and \u003cem\u003escep3-1zyp1-2\u003c/em\u003e meiocytes. DNA counterstained with DAPI shown in merge and single channel image in blue and gray, respectively. Scale bar, 10 μm. \u003cstrong\u003ec,\u003c/strong\u003eFrequency of cells with 0-5 pair of univalents including the average bivalent number per cell in a series of single or double mutants.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/e371ced00c2a6226b8dfb823.png"},{"id":69547250,"identity":"57fb540b-792d-45ad-9e3f-33d6a5f32b98","added_by":"auto","created_at":"2024-11-21 13:56:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":146221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCO interference and heterochiasmy vanish in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003escep3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e while genome-wide CO increase particularly in females.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Number of COs (data are presented as mean ± standard error of the mean (s.e.m.)) detected by sequencing of recombinant offspring of WT male (4.80 ± 0.17), WT female (3.00 ± 0.11), \u003cem\u003escep3-2\u003c/em\u003e male (6.00 ± 0.24) and \u003cem\u003escep3-2\u003c/em\u003e female (6.15 ± 0.23). The sample sizes used for analysis are indicated in parentheses. Significant difference (Tukey's HSD test) was detected between WT male and female (\u003cem\u003eP\u003c/em\u003e \u0026lt; 1x10\u003csup\u003e-7\u003c/sup\u003e), but not between \u003cem\u003escep3-2\u003c/em\u003e male and female (\u003cem\u003eP\u003c/em\u003e = 0.942). Compared with WT, in \u003cem\u003escep3-2\u003c/em\u003e CO numbers significantly increased in both males (\u003cem\u003eP\u003c/em\u003e = 5.09 x 10\u003csup\u003e-4\u003c/sup\u003e) and females (\u003cem\u003eP\u003c/em\u003e \u0026lt; 1x10\u003csup\u003e-7\u003c/sup\u003e). \u003cstrong\u003eb\u003c/strong\u003e, CO distribution along all five chromosomes in male and female WT, \u003cem\u003ezyp1\u003c/em\u003e \u003csup\u003e15\u003c/sup\u003e, \u003cem\u003escep1-1\u003c/em\u003e \u003csup\u003e28\u003c/sup\u003e and \u003cem\u003escep3-2\u003c/em\u003e. Centromere and pericentromere regions are indicated in gray and blue, respectively. CO data presented with 1 Mb windows. Significant differences (based on χ\u003csup\u003e2\u003c/sup\u003e-test) indicated between WT and \u003cem\u003escep3-2\u003c/em\u003e (green dots), \u003cem\u003escep3-2\u003c/em\u003e and \u003cem\u003ezyp1\u003c/em\u003e (red dots) and \u003cem\u003escep3-2\u003c/em\u003e and \u003cem\u003escep1-1\u003c/em\u003e (orange dots). \u003cstrong\u003ec\u003c/strong\u003e, Distribution of inter-CO distances (only chromosomes with exactly two COs included for analysis) in male and female WT and \u003cem\u003escep3-2\u003c/em\u003e. Random distribution calculated and shown in gray. The statistical significance is indicated in parentheses (Tukey's HSD test). \u003cstrong\u003ed\u003c/strong\u003e, Immunolocalization of ASY1 (green) and HEI10 (magenta) in WT and \u003cem\u003escep3-1\u003c/em\u003e female meiocytes. DAPI-stained DNA in blue. Scale bar, 10 μm.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/068df63727fffadd44e0d5ab.png"},{"id":85586445,"identity":"447024ac-03a6-43c3-9e3e-9c14b5500cf2","added_by":"auto","created_at":"2025-06-28 07:09:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2583297,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/36027d90-42b3-4193-8d3f-f5e7e258de3a.pdf"},{"id":69547375,"identity":"7f554333-4079-41aa-b974-ef64cca07ea8","added_by":"auto","created_at":"2024-11-21 14:04:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppl. Table 1\u003c/strong\u003e:\u003cstrong\u003e Oligonucleotides used in this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"202411FengetalSupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/9e2e2c8650fc9dfc6cb14322.xlsx"},{"id":69545798,"identity":"8dbaabd5-4eab-4796-8d21-fe0f6483efb0","added_by":"auto","created_at":"2024-11-21 13:48:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":188449,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/4a40d6a5ec1a2858f7f20b62.docx"},{"id":69545797,"identity":"3e729f52-50d7-4756-b164-c976535b2d33","added_by":"auto","created_at":"2024-11-21 13:48:46","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4274627,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigs.docx","url":"https://assets-eu.researchsquare.com/files/rs-5394998/v1/29cac252c03e5d35256d7949.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The novel synaptonemal complex central element SCEP3 interlinks synapsis initiation and crossover formation in Arabidopsis thaliana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMeiotic homologous recombination assures genetic diversity in gametes\u003csup\u003e1\u003c/sup\u003e. Repair of programmed DNA double-strand breaks (DSBs) into interhomolog crossover (CO) involves numerous proteins and several consecutive steps\u003csup\u003e2,3\u003c/sup\u003e. In most species, CO are divided into two classes. Class I CO promoted by ZMM proteins (Zip1-4, Mer3, Msh4/5;\u003csup\u003e4,5\u003c/sup\u003e and MutL-γ (Mlh1/3) are interference-sensitive (one CO limits the probability of other CO nearby\u003csup\u003e6\u003c/sup\u003e). Class II CO are insensitive to interference forming a minority in most species including \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003ewhere their formation depends in part on MUS81\u003csup\u003e7,8\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMeiotic recombination occurs during prophase I. Sister chromatids are initially organized into a linear loop-base array by a proteinaceous structure called meiotic chromosome axis\u003csup\u003e1\u003c/sup\u003e. In Arabidopsis, the chromosome axis consists of ASY1\u003csup\u003e9,10\u003c/sup\u003e, ASY3\u003csup\u003e11\u003c/sup\u003e, ASY4\u003csup\u003e12\u003c/sup\u003e and cohesion-associated proteins including REC8\u003csup\u003e13\u003c/sup\u003e. Initial repair of a large number of DSBs (~200 in Arabidopsis) leads to numerous meiotic recombination intermediates including early interhomolog associations\u003csup\u003e14\u003c/sup\u003e. As a result, loose alignment (~400 nm distance) between two homologous chromosomes occurs. Upon installation of the transverse filament (TF) protein ZYP1, aligned chromosomes get physically connected at ~200 nm\u003csup\u003e15\u003c/sup\u003e, leading to the formation of the tripartite synaptonemal complex (SC)\u003csup\u003e1\u003c/sup\u003e. In most species including Arabidopsis, homolog alignment is independent of SC formation\u003csup\u003e15\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe SC structure is conserved across species, with two lateral elements (LEs) flanking a CR. Across species, the CR is composed of TF (e.g. Zip1 in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003csup\u003e16\u003c/sup\u003e, C(3)G in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e or SYCP1 in \u003cem\u003eMus musculus\u003c/em\u003e\u003csup\u003e18\u003c/sup\u003e) and central element (CE) proteins (e.g. Ecm11, Gmc2 in budding yeast\u003csup\u003e19\u003c/sup\u003e, Corona, Corolla in Drosophila\u003csup\u003e20,21\u003c/sup\u003e or SYCE1/2/3, TEX12, SIX6OS1 in mice\u003csup\u003e22,23,24,25,26\u003c/sup\u003e). In Arabidopsis, ZYP1 (duplicated genes \u003cem\u003eZYP1a\u003c/em\u003e and \u003cem\u003eZYP1b\u003c/em\u003e) is known as TF\u003csup\u003e27\u003c/sup\u003e while SCEP1 and SCEP2 as CEs\u003csup\u003e28\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCR proteins are required for SC assembly\u003csup\u003e19, 25, 28, 29\u003c/sup\u003e. In Drosophila, mice or Sordaria, the SC is required for CO formation \u003csup\u003e1\u003c/sup\u003e, while not in budding yeast\u003csup\u003e30,31,32\u003c/sup\u003e.\u0026nbsp;In plants, the SC is dispensable for CO formation in Arabidopsis and rice\u003csup\u003e15,28,33,34\u003c/sup\u003e, but likely required in barley\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;ArabidopsisCR mutants suggest that the SC is critical for CO assurance, heterochiasmy and CO interference\u003csup\u003e15,28,33,36\u003c/sup\u003e. Based on Arabidopsis data, coarsening of the ZMM protein HEI10 in frame of the SC was proposed as basis for CO interference\u003csup\u003e36,37\u003c/sup\u003e. However, the SC is not required for implementing CO interference in budding yeast or Sordaria\u003csup\u003e1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInteractions among CR proteins are linked to SC assembly, e.g. in budding yeast, Ecm11-Gmc2 promotes Zip1 polymerization\u003csup\u003e19\u003c/sup\u003e, or in mice, SYCP1 tetramers remodeled by SYCE3 form a SYCP1-SYCE3 complex and SYCE3 also interacts with TEX12-SYCE2 and SYCE1-SIX6OS1\u003csup\u003e38\u003c/sup\u003e. In budding yeast, SC assembly and CO formation are coupled via the interaction of Ecm11-Zip4 (in mouse via orthologs TEX12-TEX11)\u003csup\u003e39\u003c/sup\u003e. In Arabidopsis, SCEP1-SCEP2 directly interact, while none interacts with ZYP1 or ZIP4\u003csup\u003e28\u003c/sup\u003e. Whether in Arabidopsis further CE proteins exist that form a complex with ZYP1 and/or interconnect SC assembly and CO formation is unclear.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe identified proteins in proximity of ASY1 and ASY3\u0026nbsp;via proximity-proteomics\u003csup\u003e40\u003c/sup\u003e. Here, we functionally characterize the candidate ATC21 re-named SCEP3. SCEP3 is a novel CE protein conserved across plants and the likely structural ortholog of yeast Ecm11 and possibly also of SYP-4 (worms), Corolla (flies) or SIX6OS1 (mammals).\u0026nbsp;\u003c/p\u003e"},{"header":"Results ","content":"\u003cp\u003e\u003cstrong\u003eSCEP3 is required for synapsis, chiasma formation and CO assurance\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSCEP3\u003c/em\u003e has 16 exons, encodes a protein of 803 AA (Fig. 1a), and is highly expressed in young flower buds\u003csup\u003e41\u003c/sup\u003e. According to structural prediction by AlphaFold2, the C-terminal 70 AA form an α-helical domain, while the remaining 733 AA are disordered (Fig. S1a). No obvious developmental differences when compared with the WT were found in any \u003cem\u003escep3\u003c/em\u003e allele (Fig. 1a and Fig. S1b), except slightly shorter siliques with seed gaps (Fig. S1c). In all \u003cem\u003escep3\u003c/em\u003e alleles including \u003cem\u003escep3-1scep3-2\u003c/em\u003e, seed fertility was reduced by\u0026nbsp;~30% suggesting allelism of mutations (Fig. 1b). Pollen viability was also significantly reduced in \u003cem\u003escep3\u003c/em\u003e (Fig. S1d). In the WT, synapsed chromosomes at pachytene, five bivalents at metaphase I and balanced chromosome segregation are found (Fig. 1c). In \u003cem\u003escep3\u003c/em\u003e, typical pachytene chromosomes with thick chromatin threads suggesting synapsis were not found but regions with chromosome alignment (Fig. 1c). Indeed, based on spatial structural illumination microscopy (3D-SIM) inter-axes measurements (Fig. S1e), \u003cem\u003escep3\u003c/em\u003e chromosomes align but do not get in close apposition during pachytene. In \u003cem\u003escep3\u003c/em\u003e, cells with pair(s) of univalents (absence of CO assurance) and unequal chromosome segregation were found (Fig. 1c). While in the WT invariably five bivalents were found, in all \u003cem\u003escep3\u003c/em\u003e alleles, the average bivalent number was reduced with ~50% of cells displaying one to three pairs of univalents (Fig. 1d). Minimum chiasmata numbers (MCN) in all \u003cem\u003escep3\u003c/em\u003e alleles are significantly reduced to\u0026nbsp;~70-75% of WT-levels (Table 1). No difference was found in \u003cem\u003escep3-1scep3-2\u0026nbsp;\u003c/em\u003ewhencompared with single mutants (Fig. 1d and Table 1; \u003cem\u003eP\u003c/em\u003e = 0.2776; one-way ANOVA). Similar to male meiosis, \u003cem\u003escep3\u003c/em\u003e female meiosis revealed chromosome alignment at larger distances and the absence of CO assurance (Fig. S1f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCEP3 is found at the central region of the SC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo dissect the spatiotemporal localization of SCEP3, antibodies raised against its N- (SCEP3-N) or C-terminus (SCEP3-C) were employed. SCEP3 is initially detected during early zygotene forming a limited number of foci (Fig. 2a). Progressively foci numbers increase with initial SCEP3 stretches during zygotene and full polymerization at pachytene. During all stages, SCEP3 co-localizes with ZYP1. SCEP3 also overlaps at pachytene with the CE proteins SCEP1 and SCEP2 (Fig. 2b). Using 3D-SIM, SCEP3-C overlaps with SCEP3-N albeit less continuous (Fig. 2c). Absence of chromatin-associated signals in \u003cem\u003escep3\u003c/em\u003e confirms the specificity of both antibodies (Fig. 2d).\u003c/p\u003e\n\u003cp\u003eVia 3D-SIM, the following SC organization was detected at pachytene: REC8 and ASY4 form two parallel lines separated by\u0026nbsp;~188 and\u0026nbsp;~176 nm (Fig. S1e and Fig. 2f), respectively, and in between those ZYP1-C forms either two lines or a single bright line. Both SCEP3-N and SCEP3-C are located centrally of the bright/two ZYP1-C line(s) (Fig. 2e,f). Note, a similar Arabidopsis SC organization, i.e. REC8 axes at a distance of 175-213 nm, in between two lines of ZYP1-C and in between those centrally CEs SCEP1 and SCEP2 was found using Stimulated Emission Depletion (STED) microscopy\u003csup\u003e15,28\u003c/sup\u003e. Together, SCEP3 is a CE protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCEP3 is critical for SC formation and loads onto recombination intermediates independent of other CR proteins\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn WT, polymerization of all CR proteins is found at pachytene, while ASY1 gets depleted from synapsed regions (Fig. 2a and Fig. S2a). In \u003cem\u003escep3-1\u003c/em\u003e pachytene(-like) nuclei, neither chromosome-associated ZYP1, SCEP1 or SCEP2 nor their polymerization is found (Fig. 3a), suggesting absence of SC assembly in \u003cem\u003escep3-1\u003c/em\u003e and chromosomal loading of ZYP1, SCEP1 and SCEP2 being SCEP3-dependent. Absence of synapsis is also reflected by ASY1 persistence at pachytene(-like) stages\u003csup\u003e15,28\u003c/sup\u003e (Fig. 3a). In \u003cem\u003ezyp1-2\u003c/em\u003e, \u003cem\u003escep1-1\u003c/em\u003e or \u003cem\u003escep2-1\u0026nbsp;\u003c/em\u003epachytene(-like) nuclei, axis-associated SCEP3 foci are found (Fig. 3b). Hence, SCEP3 loads independent of SC formation and other CR proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test whether SCEP3 loads onto recombination intermediates, HEI10 (signal absence in \u003cem\u003ehei10\u003c/em\u003e; Fig. S2b) and SCEP3 were immunolocalized. In pachytene(-like) nuclei of \u003cem\u003ezyp1-2\u003c/em\u003e, \u003cem\u003escep1-1\u003c/em\u003e and \u003cem\u003escep2-1\u003c/em\u003e, SCEP3 associates with HEI10-dependent recombination intermediates (~80% of HEI10 foci overlap with SCEP3 foci and ~55% of SCEP3 foci overlap with HEI10 foci) (Fig. 3c). In WT, SCEP3 and HEI10 foci also co-localize at zygotene, however, due to synapsis-progression, SCEP3 foci gradually turn into uniformly linear signals decorated by bright HEI10 foci (Fig. S2c). In \u003cem\u003escep3-1\u003c/em\u003e, no ZYP1, SCEP1 or SCEP2 foci overlap with HEI10 (Fig. 3d). Note, no CR component is required for HEI10 loading (Fig 3c,d). ZYP1 co-localizes with SCEP3 in \u003cem\u003escep1-1\u003c/em\u003e or \u003cem\u003escep2-1\u0026nbsp;\u003c/em\u003eearly pachytene(-like) nuclei (Fig. 3e) and while SCEP3 overlaps with HEI10 foci in \u003cem\u003ezyp1-2\u003c/em\u003e, neither SCEP1 nor SCEP2 does (Fig. 3f). Together, all CR proteins are required for SC assembly but SCEP3 loads onto recombination intermediates (HEI10-marked) independent of other CR components/SC formation and recruits ZYP1, but neither SCEP1 nor SCEP2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCEP3 physically interacts with ZYP1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven SCEP3-ZYP1 co-localization even in CE mutants, their relationship was addressed in mutants with full SC polymerization despite impaired ZMM-dependent CO formation (\u003cem\u003emsh5-2\u003c/em\u003e, \u003cem\u003ehei10-2\u003c/em\u003e, \u003cem\u003ezip4-2\u003c/em\u003e, \u003cem\u003emer3-1\u003c/em\u003e, \u003cem\u003eshoc1-1\u003c/em\u003e) or with SC polymerization impaired to different degrees (\u003cem\u003edmc1-2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;asy3-1\u003c/em\u003e, \u003cem\u003erec8-1\u003c/em\u003e, \u003cem\u003epch2-1\u003c/em\u003e, \u003cem\u003easy1-4\u003c/em\u003e). In all cases, SCEP3-ZYP1 co-localization was found (Fig. S3). Thus, SCEP3-ZYP1 form similar complexes even SC-independent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn yeast two-hybrid (Y2H) assays, a strong interaction of SCEP3-ZYP1 (both ZYP1a and ZYP1b) was found mediated by the N-terminus of ZYP1 (49-400 AA, ZYP1b) and SCEP3’s C-terminus (734-803 AA) (Fig. 4a, b). Notably, AlphaFold3 also predicts an interaction of SCEP3’s C-terminus (α-helical domain) and ZYP1’s N-terminus (Fig. 4c). No Y2H interaction of SCEP3 with axis proteins ASY1, ASY3 and REC8 was described\u003csup\u003e40\u003c/sup\u003e. We also found neither an interaction of SCEP3 with axis(-associated) proteins ASY4, COMET and PRD3 nor with ZMM proteins ZIP4, HEI10, MER3 and PTD (Fig. 4d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCEP3 and its interaction with ZYP1 are evolutionarily conserved in plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing PSI-BLAST, SCEP3 is conserved across green plants including many lower plants but no homolog was found outside streptophyta (Fig. S4). High sequence similarity at both the N- and C-terminal regions of SCEP3 is found (Fig. S5). Across most plants, homologues of all four CR components are found but in lower plants such as \u003cem\u003eTaxus chinensis\u003c/em\u003e or \u003cem\u003eMarchantia polymorpha\u003c/em\u003e homologues of SCEP1/SCEP2 are absent (Fig. S4). The evolutionarily closer relationship between SCEP3 and ZYP1 may reflect their detected direct interaction in \u003cem\u003eA. thaliana\u003c/em\u003e. In Y2H full-length barley SCEP3-ZYP1 interact (Fig. S6a) and AlphaFold3 predicts SCEP3-ZYP1 interaction in various plants involving similar regions as in Arabidopsis (Fig. S6b). Hence, a SCEP3-ZYP1 interface seems highly conserved across plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCEP3 is structurally likely the plant ortholog of yeast Ecm11\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven no SCEP3 homologues outside of plants based on PSI-BLAST, we searched for structural orthologues focusing on known SC proteins (Fig. S7). The structure of plant SCEP3 was similar to budding yeast (or \u003cem\u003eValsa malicola\u003c/em\u003e) Ecm11, with a typical short alpha-helical domain (70-80 AA) at the C-terminus and a long variable disordered region at the N-terminus. Structural similarities were also found for SYP-4 (worms), Corolla (flies)or SIX6OS1 (mammals).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCEP3 is required for some CO arising upon abolished or impaired SC formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on γH2AX foci numbers as a proxy for the number of DSB sites\u003csup\u003e42,43\u003c/sup\u003e, no difference was found in \u003cem\u003escep3-2\u003c/em\u003e when compared with WT (Fig. S8a, b). Hence, shortage of chiasmata in \u003cem\u003escep3\u003c/em\u003e is unlikely due to reduced DSB numbers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompared to \u003cem\u003ezyp1\u0026nbsp;\u003c/em\u003ewithincreased HEI10 foci numbers\u003csup\u003e15\u003c/sup\u003e, in \u003cem\u003escep3-1\u003c/em\u003e similar HEI10 foci numbers were found (9.94 ± 2.43) as in the WT (10.34 ± 1.74; \u003cem\u003eP\u003c/em\u003e = 0.36, two-sided Student’s \u003cem\u003et\u003c/em\u003e-test) (Fig. 5a, b). In \u003cem\u003escep3-1zyp1-2\u0026nbsp;\u003c/em\u003ethe MCN is significantly (two-sided Student’s \u003cem\u003et\u003c/em\u003e-test) reduced to 5.62 ± 1.91 when compared with 7.69 ± 1.54 in \u003cem\u003ezyp1-2\u0026nbsp;\u003c/em\u003e(\u003cem\u003eP\u003c/em\u003e = 1.05x10\u003csup\u003e-9\u003c/sup\u003e), while not when compared with 5.93 ± 1.56 in \u003cem\u003escep3-1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e = 0.25) (Table 1). Univalent frequency in \u003cem\u003escep3-1zyp1-2\u003c/em\u003e (58.7%) is similar to \u003cem\u003escep3-1\u003c/em\u003e (55.3%) while higher than in \u003cem\u003ezyp1-2\u003c/em\u003e (14.3%) (Fig. 5c). Moreover, HEI10 foci numbers in \u003cem\u003escep3-1zyp1-2\u003c/em\u003e (10.03 ± 2.80)are similar to \u003cem\u003escep3-1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e = 0.85, two-sided Student’s \u003cem\u003et\u003c/em\u003e-test) but significantly reduced when compared with \u003cem\u003ezyp1-2\u0026nbsp;\u003c/em\u003e(13.18 ± 3.28; \u003cem\u003eP\u003c/em\u003e = 7.07x10\u003csup\u003e-8\u003c/sup\u003e, two-sided Student’s \u003cem\u003et\u003c/em\u003e-test) (Fig. 5a, b)\u003cem\u003e.\u003c/em\u003e Hence, \u003cem\u003escep3\u003c/em\u003e is genetically upstream of \u003cem\u003ezyp1\u003c/em\u003e in terms of CO formation. In \u003cem\u003easy1\u003c/em\u003e and \u003cem\u003easy3\u003c/em\u003e with impaired SC formation, genetically upstream of \u003cem\u003eZYP1\u003c/em\u003e\u003csup\u003e33\u003c/sup\u003e, upon \u003cem\u003eSCEP3\u0026nbsp;\u003c/em\u003edepletion, chiasma/bivalent numbers are reduced further (Fig. 5c and Table 1). Thus, \u003cem\u003eSCEP3\u003c/em\u003e is critical for some CO arising upon impaired SC formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCEP3 is required for some synapsis-independent class I and class II CO\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn mutants impaired in class I CO formation (\u003cem\u003emsh5-2\u003c/em\u003e, \u003cem\u003ehei10-2\u003c/em\u003e, \u003cem\u003emlh3-1\u003c/em\u003e), upon SCEP3 depletion chiasma/bivalent numbers are further reduced (Fig. 5c and Table 1). In \u003cem\u003emus81-2\u003c/em\u003e, deficient in some class II CO, five bivalents are found. In \u003cem\u003escep3-1mus81-2\u003c/em\u003e, chiasma/bivalent numbers are significantly reduced when compared with \u003cem\u003escep3-1\u003c/em\u003e (Fig. 5c, Table 1). These data suggest that (i) the majority of CO are ZMM-dependent class I CO in \u003cem\u003escep3\u003c/em\u003e, (ii) SCEP3 is required for some class II CO, and (iii) SCEP3-independent class II CO in part depend on MUS81 (0.87 bivalents in \u003cem\u003escep3msh5\u003c/em\u003e vs a reduction of 0.23 bivalents in \u003cem\u003escep3\u0026nbsp;\u003c/em\u003eupon MUS81 depletion).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome-wide and particularly female CO are increased while heterochiasmy and CO interference are abolished in \u003cem\u003escep3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo dissect genome-wide male and female CO, we isolated \u003cem\u003escep3-4\u003c/em\u003e in Ler-0, with similar phenotypes as \u003cem\u003escep3\u003c/em\u003e in Col-0 (Fig. 1 and Fig. S9). By crossing \u003cem\u003escep3-2+/-\u003c/em\u003e with \u003cem\u003escep3-4+/-\u003c/em\u003e, we generated F1 hybrids of WT (Col-0 x Ler-0) and \u003cem\u003escep3\u003c/em\u003e (Col-0 x Ler-0). These were backcrossed with Col-0 either as female ormale and the four offspring groups (WT female/male, \u003cem\u003escep3\u003c/em\u003e female/male) were sequenced.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003escep3\u003c/em\u003e males (25%) and particularly females (105%) CO numbers significantly increased compared to WT (Fig. 6a). Heterochiasmy found in WT vanished in \u003cem\u003escep3\u003c/em\u003e (Fig. 6a). CO frequencies increased along chromosome arms especially towards chromosome ends and decreased in pericentromeric regions (Fig. 6b). In both sexes, CO interference found in the WT, was abolished in \u003cem\u003escep3\u003c/em\u003e (Fig. 6c). Together, \u003cem\u003escep3\u003c/em\u003e CO increased genome-wide as well as heterochiasmy and CO interference vanished similar to other CR mutants\u003csup\u003e15,28\u003c/sup\u003e. However, despite comparable CO distributions (Fig. 6b), male and female \u003cem\u003escep3\u0026nbsp;\u003c/em\u003eCO are ~15% lower compared to \u003cem\u003ezyp1\u003c/em\u003e or \u003cem\u003escep1\u003c/em\u003e (Fig. 6a)\u003csup\u003e15,28\u003c/sup\u003e. In \u003cem\u003escep3\u003c/em\u003e males, this is consistent with the absence of increased HEI10 foci, and reduced chiasmata when compared with \u003cem\u003ezyp1\u003c/em\u003e (Fig. 5b and Table 1). In \u003cem\u003escep3\u003c/em\u003e females, HEI10 foci strikingly increased by ~92% (10.17, n = 6) when compared with WT (5.29, n = 7) (Fig. 6d) thus largely accounting for the female CO increase.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTwo plant CE proteins, SCEP1/2, were identified by transcriptomics\u003csup\u003e28\u003c/sup\u003e. We identified SCEP3 by TbID-based proteomics\u003csup\u003e40\u003c/sup\u003e. SCEP3 spatiotemporally overlaps with CR components, is found within the CR of the SC, is critical for SC assembly, and interacts with ZYP1’s N-terminus that localizes to the SC center. Together, SCEP3 is a novel plant CE protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpatiotemporal and functional overlap suggests interactions among CR proteins. SCEP1-SCEP2 form a complex, while none of them interacts with ZYP1\u003csup\u003e28\u003c/sup\u003e. SCEP3 loads at HEI10-marked recombination intermediates independent of SC formation/other CR proteins. Its loading is sufficient for the recruitment of ZYP1, but neither of SCEP1 nor SCEP2. SCEP3 also directly interacts with ZYP1 and this interaction seems conserved across plants. Thus, SCEP3 likely acts as a synapsis initiation factor recruiting ZYP1 to recombination intermediates for synapsis initiation. Notably, SCEP3 and HEI10 foci also form exclusively, e.g. SCEP3 may also localize at non-ZMM intermediates as suggested by its requirement for some class II CO. Whether SCEP3-ZYP1 load as complex or SCEP3 recruits ZYP1 is unclear. We prefer the latter, as SCEP3 loads in \u003cem\u003ezyp1\u003c/em\u003e. Which factor recruits SCEP3 is unclear, as none of the tested axis or ZMM candidates directly interacts with SCEP3 or is critical for SCEP3 localization. In budding yeast, Zip4 links recombination intermediates and SC assembly by recruiting and interacting with Ecm11\u003csup\u003e39\u003c/sup\u003e. While this seems conserved in mammals, in Arabidopsis neither the CE proteins SCEP1, SCEP2\u003csup\u003e28\u003c/sup\u003e or SCEP3 directly interact with ZIP4 nor is SC formation impaired in \u003cem\u003ezip4\u003c/em\u003e. SCEP1 and/or SCEP2 do not form a complex with SCEP3 and/or ZYP1 at least SC independent, i.e. only SCEP3 loads in \u003cem\u003ezyp1\u003c/em\u003e and only SCEP3-ZYP1 load in \u003cem\u003escep1\u003c/em\u003e and \u003cem\u003escep2\u003c/em\u003e\u003csup\u003e28\u003c/sup\u003e. Whether further proteins/modifications and/or the SC context are required for complex formation is unclear. Together, the Arabidopsis SC is composed of at least two subdomains, SCEP3-ZYP1 and SCEP1-SCEP2 but how they are functionally linked remains to be addressed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003escep3\u003c/em\u003e, CO interference, CO assurance and heterochiasmy vanish but CO numbers increase, suggesting these are the common phenotypes after SC abolishment in Arabidopsis\u003csup\u003e15,28,33\u003c/sup\u003e. However, \u003cem\u003escep3\u003c/em\u003e CO numbers are lower compared with other CR mutants but increased compared with WT. In \u003cem\u003escep3\u003c/em\u003e, surplus CO seem largely ZMM-dependent in females, while not in males (no additional HEI10 foci). In both \u003cem\u003escep3\u003c/em\u003e sexes, HEI10 foci represent\u0026nbsp;~83% of the total CO, suggesting\u0026nbsp;~17% ZMM-independent class II CO. However, \u003cem\u003escep3\u003c/em\u003e offspring CO rates might be overestimated as only meiotic cells with comparatively high CO numbers may form viable gametes. The majority of CO are class I CO in \u003cem\u003escep3\u003c/em\u003e but SCEP3 is required for some synapsis-independent class I and II CO. In males, SCEP3 is even critical for the surplus HEI10-dependent CO in \u003cem\u003ezyp1\u003c/em\u003e and for some chiasmata in \u003cem\u003easy1\u003c/em\u003e or \u003cem\u003easy3\u003c/em\u003e (impaired SC formation). We speculate that SCEP3’s loading at recombination intermediates prior and independent of synapsis/other CR proteins may stabilize and/or recruit further factors such as ZYP1 to a subset of these intermediates for synapsis initiation designated to form class I and/or II CO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Arabidopsis CR mutants including \u003cem\u003escep3\u003c/em\u003e, CO interference vanishes\u003csup\u003e15,28,33\u003c/sup\u003e, suggesting the SC \u003cem\u003eper se\u003c/em\u003e and not individual components is required for CO interference implementation. Our data are compatible with the HEI10 coarsening model in which the SC (scaffold for HEI10 diffusion and condensation) imposes CO interference\u003csup\u003e36,37,44\u003c/sup\u003e. Two non-exclusive scenarios may also contribute to increased CO numbers without signatures of CO interference in CR mutants, i.e. longer axis persistence of ASY1 and/or lack of PCH2\u003csup\u003e45\u003c/sup\u003e or absence of SC polymerization-mediated local downregulation of de novo DSB\u003csup\u003e30,46\u003c/sup\u003e. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe direct interaction of SCEP3’s C-terminus with ZYP1 is likely conserved across plants. The functional significance of the conserved N-terminus remains unclear. We speculate that both conserved termini mediate SCEP3’s role in interlinking meiotic recombination and SC initiation/formation.\u003c/p\u003e\n\u003cp\u003eSCEP3 is likely the plant structural ortholog of fungus Ecm11. Functional similarities, e.g. Ecm11 is a CE, limits CO, or co-localizes with Zip3 (HEI10) independent of Zip1\u003csup\u003e19\u003c/sup\u003e, but also differences, e.g. Ecm11 interacts with Zip4 but not with Zip1\u003csup\u003e39\u003c/sup\u003e, are found. Structural similarities are also found for SYP-4 in \u003cem\u003eC. elegans\u003c/em\u003e, COROLLA in \u003cem\u003eD. melanogaster\u003c/em\u003e or SIX6OS1 in mammals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe propose a dual role for SCEP3, one as a CR component of the SC required for its assembly and the other as a synapsis initiation factor associated with recombination intermediates, together interlinking SC and CO formation.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods ","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA. thaliana\u003c/em\u003e plants were grown under short-day conditions (8/16 h light/dark) for four weeks followed by long-day conditions (16/8 h light/dark) until maturity at constant 22\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eCol-0 was used as WT except where indicated. T-DNA insertion alleles, provided by the Nottingham Arabidopsis Stock Centre \u003csup\u003e47\u003c/sup\u003e, used in this study are: \u003cem\u003escep3-1\u003c/em\u003e (\u003cem\u003eAT4G18490\u003c/em\u003e; SAILseq_210_G05), \u003cem\u003escep3-2\u003c/em\u003e (\u003cem\u003eAT4G18490\u003c/em\u003e; SALK_098044), \u003cem\u003emsh5-2\u003c/em\u003e (\u003cem\u003eAT3G20475\u003c/em\u003e; SALK_026553) \u003csup\u003e48\u003c/sup\u003e, \u003cem\u003emus81-2\u003c/em\u003e (\u003cem\u003eAT4G30870\u003c/em\u003e; SALK_107515) \u003csup\u003e8\u003c/sup\u003e, \u003cem\u003easy1-4\u003c/em\u003e (\u003cem\u003eAT1G67370\u003c/em\u003e; SALK_046272) \u003csup\u003e49\u003c/sup\u003e, \u003cem\u003edmc1-2\u003c/em\u003e (\u003cem\u003eAT3G22880\u003c/em\u003e; SAIL_170_F08) \u003csup\u003e50\u003c/sup\u003e, \u003cem\u003easy3-1\u003c/em\u003e (\u003cem\u003eAT2G46980\u003c/em\u003e; SALK_143676) \u003csup\u003e11\u003c/sup\u003e, \u003cem\u003erec8-1\u003c/em\u003e (\u003cem\u003eAT5G05490\u003c/em\u003e; SALK_137095) \u003csup\u003e51\u003c/sup\u003e, \u003cem\u003epch2-1\u003c/em\u003e (\u003cem\u003eAT4G24710\u003c/em\u003e; SAIL_1187_C06) \u003csup\u003e49\u003c/sup\u003e, \u003cem\u003ezip4-2\u003c/em\u003e (\u003cem\u003eAT5G48390\u003c/em\u003e; SALK_068052) \u003csup\u003e52\u003c/sup\u003e, \u003cem\u003ehei10-2\u003c/em\u003e (\u003cem\u003eAT1G53490\u003c/em\u003e; SALK_014624) \u003csup\u003e53\u003c/sup\u003e, \u003cem\u003emer3-1\u003c/em\u003e (\u003cem\u003eAT3G27730\u003c/em\u003e; Salk_091560) \u003csup\u003e54\u003c/sup\u003e, \u003cem\u003eshoc1-1\u003c/em\u003e (\u003cem\u003eAT5G52290\u003c/em\u003e; SALK_057589) \u003csup\u003e55\u003c/sup\u003e and \u003cem\u003emlh3-1\u003c/em\u003e (\u003cem\u003eAT4G35520\u003c/em\u003e; SALK_015849) \u003csup\u003e56\u003c/sup\u003e. \u003cem\u003ezyp1-2\u003c/em\u003e \u003csup\u003e33\u003c/sup\u003e, \u003cem\u003escep1-1\u003c/em\u003e and \u003cem\u003escep2-1\u003c/em\u003e \u003csup\u003e28\u003c/sup\u003e were described. The alleles \u003cem\u003escep3-3\u003c/em\u003e and \u003cem\u003escep3-4\u003c/em\u003e were isolated using CRISPR/Cas9 in this study. Details of primers used for genotyping are found in\u0026nbsp;Suppl. Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of \u003cem\u003escep3-3\u003c/em\u003e and \u003cem\u003escep3-4\u003c/em\u003e using CRISPR/Cas9\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTargeted mutagenesis in Arabidopsis via CRISPR/Cas9 was performed according \u003csup\u003e57\u003c/sup\u003e. pMOD_A0503, pMOD_B2103, pMOD_C0000 and pTRANS_260d (Addgene #91013, #91061, #91081 and #91126) were used for assembling CRISPR constructs with \u003cem\u003eCas9\u003c/em\u003e and gRNAs both driven by \u003cem\u003eCmYLCV\u003c/em\u003e promoter. Two gRNAs (#1: 5\u0026rsquo;-GAGCCAAAGCCAAAATCCATTGG-3\u0026rsquo;; #2: 5\u0026rsquo;-AACTAGACAAGTTCCCTCCAAGG-3\u0026rsquo;) addressing \u003cem\u003eSCEP3\u003c/em\u003e were Golden Gate assembled in pMOD_B2103. The final expression cassettes assembled in pTRANS_260d were Agrobacterium-mediated transformed in ecotypes Col-0 and Ler-0 by floral dip \u003csup\u003e58\u003c/sup\u003e. Based on Sanger-sequencing of target sites, \u003cem\u003escep3-3\u003c/em\u003e and \u003cem\u003escep3-4\u003c/em\u003e were isolated in transgenic lines in the Col-0 and Ler-0 background, respectively. For primer details see\u0026nbsp;Suppl. Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast two-hybrid (Y2H) assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull-length or truncated coding sequences of Arabidopsis \u003cem\u003eSCEP3\u003c/em\u003e, \u003cem\u003eZYP1a\u003c/em\u003e, \u003cem\u003eZYP1b\u003c/em\u003e, \u003cem\u003eSCEP1\u003c/em\u003e, \u003cem\u003eSCEP2\u003c/em\u003e, C-terminus of \u003cem\u003eZIP4\u003c/em\u003e according to \u003csup\u003e28\u003c/sup\u003e, \u003cem\u003eHEI10\u003c/em\u003e, \u003cem\u003eMER3\u003c/em\u003e, \u003cem\u003ePTD\u003c/em\u003e, \u003cem\u003eCOMET\u003c/em\u003e, \u003cem\u003eASY4\u003c/em\u003e and \u003cem\u003ePRD3\u003c/em\u003e were PCR-amplified using Col-0 flower bud cDNA as template and cloned into pGBKT7 and/or pGADT7 vectors (Takara) by Gibson Assembly (NEB). Cloning of barley \u003cem\u003eSCEP3\u003c/em\u003e and \u003cem\u003eZYP1\u003c/em\u003e was done accordingly, but using cDNA prepared from barley anthers (cultivar Golden Promise). For primer details, see\u0026nbsp;Suppl. Table 1.\u003c/p\u003e\n\u003cp\u003eY2H assays were performed according to the manufacturer\u0026rsquo;s instructions (Takara). Bait and prey plasmids (empty vectors as controls) were co-transformed into the yeast strain Y2HGold (Takara, 630489) and grown at 30\u0026deg;C for 3-5 days on plates with SD Base medium supplemented with DO Supplement \u0026ndash;Leu/\u0026ndash;Trp (DDO, 630417). Transformed clones underwent selection assays for 5 days on plates with Minimal SD Base medium supplemented with DO Supplement \u0026ndash;His/\u0026ndash;Leu/\u0026ndash;Trp (TDO, 630419) or DO Supplement \u0026ndash;Ade/\u0026ndash;His/\u0026ndash;Leu/\u0026ndash;Trp (QDO, 630428).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlphaFold protein structure modeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein structure models of SCEP3 homologues (Fig. S7) are from the AlphaFold Protein Structure Database \u003csup\u003e59, 60\u003c/sup\u003e. The Arabidopsis SCEP3 protein structure (Fig. S1a) was modeled by AlphaFold2 using ColabFold \u003csup\u003e61\u003c/sup\u003e. Protein-protein interaction modeling (Fig. 4c and Fig. S6b) was performed with AlphaFold3 \u003csup\u003e62\u003c/sup\u003e and depicted with UCSF ChimeraX \u003csup\u003e63\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of polyclonal antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeptide synthesis and antibody production were performed by LifeTein LLC (New Jersey, USA). The following peptides were selected for respective Arabidopsis proteins and used for immunization: SCEP3-N (69-87 AA; C-GSSFKMDMPDFDFSSPAKK) in rat, SCEP3-C (756-775 AA, C-KKKHEEAKELLVRAVVDNNK) in rabbit, HEI10 (C-PKDEIWPARQNS, according \u003csup\u003e64\u003c/sup\u003e) in rabbit and guinea pig, ZYP1-C (833-851 AA in ZYP1b; C-SANIGDLFSEGSLNPYADD; peptides identical in ZYP1a/b) in rat and guinea pig, ASY4 (C-AKLPDELDVDVSSDFKGI) and ASY1 (C-SKAGNTPISNKAQPAASRES, according \u003csup\u003e65\u003c/sup\u003e) in rabbit and rat. All antibodies were affinity-purified against the synthetic peptide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytological procedures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePollen viability was assessed using Alexander\u0026rsquo;s stain \u003csup\u003e66\u003c/sup\u003e as described \u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMale and female meiotic chromosome spread preparations and MCN counting were performed as described \u003csup\u003e67\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunolocalization was performed as described \u003csup\u003e67, 68\u003c/sup\u003e using fresh Arabidopsis flower buds. Following primary antibodies and dilutions were used: anti-ASY1 (rabbit, \u003csup\u003e9\u003c/sup\u003e; 1:2,000), anti-ASY1 (rabbit or rat, this study; 1:200), anti-ZYP1-C (guinea pig, \u003csup\u003e27\u003c/sup\u003e; 1:2,000), anti-ZYP1-C (rat or guinea pig, this study; 1:200), anti-REC8 (rabbit, \u003csup\u003e69\u003c/sup\u003e; 1:1,000), anti-\u0026gamma;H2Ax (mouse, Sigma-Aldrich #05-636; 1:200), anti-SCEP3-N (rat, this study; 1:100), anti-SCEP3-C (rabbit, this study; 1:100), anti-HEI10 (guinea pig, this study; 1:200), anti-SCEP1 (rabbit, \u003csup\u003e28\u003c/sup\u003e; 1:200) and anti-SCEP2 (rat, \u003csup\u003e28\u003c/sup\u003e; 1:200). The following secondary antibodies were used (all diluted 1:500): anti-guinea pig Cy5 (Abcam, ab102372), anti-guinea pig Alexa 594 (Invitrogen, A11076), anti-guinea pig Alexa 488 (Invitrogen, A11073), anti-rabbit Alexa 594 (Abcam, ab150076), anti-rabbit Alexa 488 (Abcam, ab150073), anti-rabbit Cy3 (Jackson ImmunoResearch, 111-165-003), anti-rat Alexa 488 (Jackson ImmunoResearch, 112-545-167) and anti-rat alexa 594 (Abcam, ab150160). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEpifluorescence images were acquired using a Nikon Eclipse Ni-E microscope equipped with a Nikon DS-Qi2 camera and NIS-Elements-AR version 4.60 software (Nikon). SCEP3-HEI10 co-localization analysis was performed using Imaris (Bitplane, Switzerland) version 10.1.0. Automatic spot detection with a size of 0.33 \u0026micro;m and background subtraction was applied for the red and the green channel independently. Afterwards, a filter was chosen on the red channel to identify spots overlapping with spots in the green channel, allowing a maximum distance of 0.33 \u0026micro;m. 3D-SIM image stacks were acquired using an Elyra 7 microscope system and the software ZEN Black (Carl Zeiss GmbH) \u003csup\u003e70\u003c/sup\u003e. \u0026nbsp; Inter-axis distances were measured from center to center of each parallel axis in maximum intensity projection images (positions randomly picked). Images were processed with ZEN 3.1 (blue edition), Fiji (open source)\u003csup\u003e71\u003c/sup\u003e and Adobe Photoshop CS5 (Adobe).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome-wide mapping of male and female COs in WT and \u003cem\u003escep3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate male and female CO mapping populations, \u003cem\u003escep3-2\u003c/em\u003e+/- (Col-0) was crossed with \u003cem\u003escep3-4\u003c/em\u003e+/- (Ler-0) and F1 hybrids WT or biallelic for \u003cem\u003escep3\u003c/em\u003e were crossed as a male or female with the WT Col-0. Total DNA samples were prepared from the four-resulting backcross populations (WT male, 143 plants; WT female, 237 plants; \u003cem\u003escep3-2scep3-4\u003c/em\u003e male, 142 plants; \u003cem\u003escep3-2scep3-4\u003c/em\u003e female, 238 plants) using ~150 mg leaf sample per individual plant and Econospin columns (96 well, Epoch Life Sciences) according to manufacturer\u0026rsquo;s instructions adapted to plant samples. Whole Genome Shotgun (WGS) sequencing library preparation (Illumina DNA PCR-Free Library Prep, Tagmentation with standard DNA input amount) involved protocols from the manufacturer (Illumina, Inc., San Diego, CA, USA). The library was quantified by qPCR (KAPA Library Quant Kit; Roche Molecular Systems, Inc.) and sequenced according to manufacturer\u0026rsquo;s instructions using the NovaSeq 6000 device (Illumina, Inc., San Diego, CA, USA; run type: SP flowcell with XP workflow and paired-end sequencing: 151 cycles\u0026nbsp;(read 1) 10 cycles (index read 1), 10 cycles (index read 2) and 151 cycles (read 2)) at IPK Gatersleben.\u003c/p\u003e\n\u003cp\u003eRaw sequence reads were aligned to the \u003cem\u003eA. thaliana\u0026nbsp;\u003c/em\u003eCol TAIR10 reference genome \u003csup\u003e72\u003c/sup\u003e using BWA-MEM \u003csup\u003e73\u003c/sup\u003e, and converted to Binary/Alignment Map format and sorted using SAMtools \u003csup\u003e74\u003c/sup\u003e. Variant calling was done using BCFtools \u003csup\u003e75\u003c/sup\u003e filtering for a minimum mapping quality and minimum base quality of 30. The resulting variants matrix in Variant Call format (VCF) was filtered using VCFtools \u003csup\u003e76\u003c/sup\u003e for bi-allelic single nucleotide variants (SNV), minor allele frequency ranging between 0.2 - 0.3, minimum read depth per site of 4, maximum read depth per site of 100, minimum mean read depth of 1 across all samples, and a maximum mean read depth of 1.5. The resulting variant matrix contained 350,575 high-quality single nucleotide variants. Individuals with more than 75% missing data were removed (9 out of 760 samples). Genotype calls homozygous for the reference allele (Ler) were removed from further analysis (average of 8% per sample). Samples with SNV numbers below the 5% percentile or above the 95% percentile were removed from further analysis to avoid potential biases caused by extreme marker number deviations. To measure recombination events, SNV information was first aggregated in sliding windows of 20 consecutive SNVs with a step size of 1 by determining the modal SNV. Secondly, smoothed SNVs were further aggregated in non-overlapping windows of 1 Mb. For CO interference analysis, only chromosomes with exactly 2 CO were used. Observed inter-CO distances were compared against random inter-CO distances obtained via 500 permutations of the respective dataset (i.e. male/female WT, male/female \u003cem\u003escep3\u003c/em\u003e). CO positions of \u003cem\u003ezyp1\u003c/em\u003e and \u003cem\u003escep1\u003c/em\u003e were retrieved from \u003csup\u003e15, 28\u003c/sup\u003e. Statistical analysis of CO count and inter-CO distance was done in R by nested analysis of variance (ANOVA) (aov(CO count or inter-CO distance ~ sex/genotype)) followed by a Tukey HSD test. Recombination landscapes were analyzed via\u0026nbsp;\u0026chi;\u003csup\u003e2\u003c/sup\u003e-test.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this research are presented in the main text, figures and supplementary information. Generated materials are available from the corresponding author upon reasonable request. Whole-genome re-sequencing raw data underlying Fig 6 are deposited to the European Nucleotide Archive (ENA) under accession number PRJEB81799 (http://www.ebi.ac.uk/ena/data/view/PRJEB81799). Gene/protein sequences and accession codes used in this study are found in databases TAIR (https://www.arabidopsis.org/) and Ensembl Plants (http://plants.ensembl.org/index.html).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank M. Grelon (IJPB, France) for kindly providing REC8, SCEP1 and SCEP2 antibodies as well as \u003cem\u003escep1-1\u003c/em\u003e and \u003cem\u003escep2-1\u003c/em\u003e seeds, J. Higgins (University of Leicester, UK) and H. Puchta (JKIP and KIT, Germany) for sharing \u003cem\u003ezyp1-2\u003c/em\u003e seeds and C. Franklin (University of Birmingham, UK) for sharing ASY1 and ZYP1 antibodies. We are grateful to all lab members and colleagues at IPK for fruitful discussions, N. Bruhne for help with DNA sample collection, J. Pohl for help with WGS library preparation and sequencing, A. Fiebig for uploading of sequencing data and K. Weisshart (Carl Zeiss GmbH) for 3D-SIM image analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work has received funding from the Deutsche Forschungsgemeinschaft (DFG) within the framework of two projects (grant agreement 354617974 and 543670370.) to S.H. and from the IPK Gatersleben. S.H. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 949618). B.W. is a holder of a China Scholarship Council (CSC) fellowship (CSC202103250012). M.C. and F.H. acknowledge funding from the state of Sachsen-Anhalt (ZB I 180).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.F. with assistance from J.L., B.W. and F.H. conducted most of the research. V.S performed 3D-SIM imaging. A.C. and C.F. performed AlphaFold predictions and interpretations. A.H. and S.D performed whole-genome offspring re-sequencing and data analysis. N.F. and M.C. contributed to immunolocalization procedures and image analysis. S.H. acquired funding. C.F. and S.H. designed the experiments, analyzed the data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZickler D, Kleckner N. Meiosis: Dances Between Homologs. Annu. Rev. Genet. 57, 1\u0026ndash;63 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Massy B. Initiation of meiotic recombination: how and where? Conservation and specificities among eukaryotes. Annu. Rev. Genet. 47, 563\u0026ndash;599 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMercier R, Mezard C, Jenczewski E, Macaisne N, Grelon M. The molecular biology of meiosis in plants. Annu. Rev. Plant Biol. 66, 297\u0026ndash;327 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026ouml;rner GV, Kleckner N, Hunter N. 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Bioinformatics 27, 2156\u0026ndash;2158 (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Arabidopsis thaliana, Meiosis, Synaptonemal complex, SCEP3, ZYP1, Meiotic recombination, Crossover interference, Heterochiasmy","lastPublishedDoi":"10.21203/rs.3.rs-5394998/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5394998/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe proteinaceous synaptonemal complex (SC) structure forms between meiotic homologous chromosomes. Its central region (CR) consists of transverse filament and central element proteins, in Arabidopsis\u003cem\u003e \u003c/em\u003eZYP1 and SCEP1/SCEP2, respectively.\u003c/p\u003e\n\u003cp\u003eWe describe a novel CR protein in Arabidopsis. SCEP3 spatiotemporally overlaps with other CR components and is conserved in plants. In \u003cem\u003escep3\u003c/em\u003e, SC formation, crossover (CO) assurance (minimum one CO per chromosome pair), CO interference (limited closely-spaced CO) and heterochiasmy (male/female CO rate difference) vanish while genome-wide and particularly female CO increase. Compared with other CR proteins, SCEP3 is also critical for some synapsis-independent CO. SCEP3 interacts with ZYP1 but loads onto recombination intermediates independent of other CR proteins. We propose SCEP3’s loading onto recombination intermediates may stabilize and/or recruit further factors such as ZYP1 to a subset of these intermediates designated to form CO. Hence, SCEP3 interlinks SC and CO formation, being structurally likely the plant ortholog of yeast Ecm11.\u003c/p\u003e","manuscriptTitle":"The novel synaptonemal complex central element SCEP3 interlinks synapsis initiation and crossover formation in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-21 13:48:41","doi":"10.21203/rs.3.rs-5394998/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":"bca85470-579e-4264-8b24-b9dc1211b330","owner":[],"postedDate":"November 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40373062,"name":"Biological sciences/Plant sciences/Plant cell biology"},{"id":40373063,"name":"Biological sciences/Plant sciences/Plant reproduction"},{"id":40373064,"name":"Biological sciences/Genetics/Plant genetics"},{"id":40373065,"name":"Biological sciences/Plant sciences/Plant development"}],"tags":[],"updatedAt":"2025-06-28T07:09:37+00:00","versionOfRecord":{"articleIdentity":"rs-5394998","link":"https://doi.org/10.1038/s41477-025-02030-9","journal":{"identity":"nature-plants","isVorOnly":false,"title":"Nature Plants"},"publishedOn":"2025-06-27 04:00:00","publishedOnDateReadable":"June 27th, 2025"},"versionCreatedAt":"2024-11-21 13:48:41","video":"","vorDoi":"10.1038/s41477-025-02030-9","vorDoiUrl":"https://doi.org/10.1038/s41477-025-02030-9","workflowStages":[]},"version":"v1","identity":"rs-5394998","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5394998","identity":"rs-5394998","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}