Dual role of Arabidopsis SRS2 helicase in meiotic recombination

Dual role of Arabidopsis SRS2 helicase in meiotic recombination | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Dual role of Arabidopsis SRS2 helicase in meiotic recombination View ORCID Profile Valentine Petiot , View ORCID Profile Floriane Chéron , View ORCID Profile Charles I. White , View ORCID Profile Olivier Da Ines doi: https://doi.org/10.1101/2025.02.26.640294 Valentine Petiot 1 Institut Génétique Reproduction et Développement (iGReD), Université Clermont Auvergne , UMR 6293 CNRS, U1103 INSERM, F-63000 Clermont-Ferrand, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Valentine Petiot Floriane Chéron 1 Institut Génétique Reproduction et Développement (iGReD), Université Clermont Auvergne , UMR 6293 CNRS, U1103 INSERM, F-63000 Clermont-Ferrand, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Floriane Chéron Charles I. White 1 Institut Génétique Reproduction et Développement (iGReD), Université Clermont Auvergne , UMR 6293 CNRS, U1103 INSERM, F-63000 Clermont-Ferrand, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Charles I. White Olivier Da Ines 1 Institut Génétique Reproduction et Développement (iGReD), Université Clermont Auvergne , UMR 6293 CNRS, U1103 INSERM, F-63000 Clermont-Ferrand, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Olivier Da Ines For correspondence: olivier.da_ines{at}uca.fr Abstract Full Text Info/History Metrics Preview PDF Abstract Helicases are enzymes that use the energy derived from ATP hydrolysis to translocate along and unwind nucleic acids. Accordingly, helicases are instrumental in maintaining genomic integrity and ensuring genetic diversity. Srs2 is a multi-functional DNA helicase that dismantles Rad51 nucleofilaments and regulates DNA strand invasion to prevent excessive or inappropriate homologous recombination in yeast. Consistently, the deletion of Srs2 has significant consequences for the maintenance of genome integrity in mitotic cells. In contrast, its role in meiotic recombination remains less clear. We present here substantial evidence that SRS2 plays an important role in meiotic recombination in the model plant Arabidopsis thaliana . Arabidopsis srs2 mutants exhibit moderate defects in DNA damage-induced RAD51 focus formation, but SRS2 is dispensable for DNA repair and RAD51-dependent recombination in somatic cells. Meiotic progression and fertility appear unaffected in srs2 plants but strikingly, the absence of SRS2 leads to increased genetic interference accompanied by increased numbers of Class I COs and a reduction in MUS81-dependent Class II COs. SRS2 thus has both anti- and pro-CO roles during meiosis in Arabidopsis thaliana - acting as an anti-CO factor by influencing the stability and/or the dissociation of early recombination intermediates, while playing a pro-CO role by promoting MUS81-mediated resolution of meiotic recombination intermediates. Author Summary Helicases are enzymes that use ATP to unwind DNA. They play a crucial role in maintaining genomic stability. One such helicase, Srs2, is known to regulate homologous recombination in yeast by preventing excessive or inappropriate recombination events. While its function in mitotic cell division is well understood, its role in meiosis - the process that generates reproductive cells - remains less clear. We show here that SRS2 plays a significant role in meiotic recombination in the plant Arabidopsis thaliana . Although Arabidopsis plants lacking SRS2 do not exhibit major defects in DNA repair or fertility, they show modifications in meiotic crossover recombination patterns. Specifically, the absence of SRS2 shifts the balance between Class I and Class II crossovers, leading to an increase in Class I crossovers and a decrease in MUS81-dependent Class II crossovers. These findings provide new insights into the role of SRS2 in meiosis, suggesting that it has a dual role in the regulation of crossover formation. Introduction Homologous recombination (HR) is a high-fidelity mechanism of repair of DNA double-strand breaks (DSBs) that plays a vital role in preserving genomic stability and promoting genetic diversity [ 1 – 3 ]. In mitotically dividing cells, HR repairs DNA breaks caused by both environmental and endogenous factors and is crucial for the recovery of stalled or collapsed replication forks. In meiotic cells, HR is essential for proper chromosome segregation and creates genetic diversity among meiotic products [ 2 – 5 ]. The central feature of HR is the use of a homologous DNA molecule as a template to restore the original DNA sequence. This process begins with the formation of DNA DSBs followed by the resection of the 5’-ended strands of the DSBs, creating long 3’ single-stranded DNA (ssDNA) overhangs. These ssDNA overhangs are then coated by the recombinase RAD51 in somatic cells, or by RAD51 and DMC1 in meiotic cells, forming a right-handed helical nucleofilament [ 6 ]. This nucleofilament initiates a homology search and promotes strand invasion of a homologous DNA template by the 3’-ended DNA strand(s), which are then extended through DNA synthesis. The resulting joint recombination intermediates can finally be resolved through different pathways to complete the process and restore chromosome integrity [ 1 , 2 , 7 , 8 ]. The DNA-strand invasion step catalyzed by the DMC1 and/or RAD51 nucleofilament is an important point of regulation for the fate of DSBs [ 6 , 9 ]. The stability of the intermediates thus formed and their subsequent resolution can strongly influence the formation of crossovers (COs) or non-crossovers (NCOs). The assembly/disassembly, stability and activity of the nucleoprotein filament are highly dynamic processes, tightly regulated by the coordinated actions of various positive and negative factors [ 6 , 9 , 10 ]. In particular, a number of ATP-dependent helicases are instrumental by disrupting various recombination intermediates. In yeast, one such helicase is Srs2, which has multifunctional roles in DNA metabolism processes and plays a critical role in maintaining genome stability by resolving DNA structures that can lead to recombination or repair errors [ 11 – 14 ]. Playing both anti-recombination and pro-recombination roles, the absence of Srs2 induces pleiotropic recombination phenotypes [ 11 – 14 ]. The Srs2 helicase ( S uppressor of R AD S ix-screen mutant 2 ) belongs to the superfamily 1 (Sf1) of helicases and shows structural and functional similarities to the bacterial UvrD helicase. The Saccharomyces cerevisiae Srs2 protein is well-characterized, exhibiting 3′ to 5′ helicase activity and is considered a prototypical anti-recombinase due to its ability to remove Rad51 from ssDNA, thereby preventing excessive or inappropriate HR events [ 15 – 17 ]. Consequently, srs2 mutants exhibit a hyper-recombination phenotype as well as synthetic lethality with a number of other mutations affecting proteins involved in homologous recombination [ 12 , 13 , 15 , 16 , 18 – 24 ]. One current hypothesis proposes that Srs2 disrupt Rad51 filaments to channel recombination intermediates into non-crossover pathways (through SDSA), thus preventing CO in mitotic cells. Additionally, Srs2 has roles in other DNA metabolism processes, such as replication fork repair, post-replication repair via the template-switching pathway, non-homologous end joining [ 11 – 13 ] and even plays a role in DNA damage checkpoint-mediated cell cycle arrest [ 25 , 26 ]. Understanding of the role of Srs2 in meiosis is however much less complete. S. cerevisiae Srs2 is upregulated during meiosis [ 27 ] and its absence leads to delay in DSB repair and meiotic progression, reduced formation of both NCOs and COs, and a reduction in spore viability [ 18 , 27 – 31 ]. Overexpression of Srs2 also decreases CO and NCO formation, and reduces spore viability [ 27 ]. Interestingly, overexpression of Srs2 specifically disrupts the formation of RAD51 filaments, whereas DMC1 filaments remain unaffected [ 27 ]. This is in accordance with the in vitro demonstration that DMC1 directly inhibits the ATPase activity of Srs2, preventing it from translocating on single-stranded DNA (ssDNA) [ 14 , 32 ]. This inhibition could help promoting the formation of COs by DMC1, which are essential for the faithful segregation of homologous chromosomes. Finally, Srs2 has recently been proposed to protect against the accumulation of aberrant recombination intermediates at the end of meiotic Prophase I [ 30 , 31 ]. Indeed, srs2 mutants exhibit DNA damage accumulation at the end of Prophase I that are associated with the aggregation of RAD51 visible after the completion of meiotic recombination [ 30 , 31 ]. Thus, fine regulation of Srs2 is necessary for homologous recombination to proceed normally during meiosis in S. cerevisiae . No meiotic phenotype of Srs2 is apparent in Schizosaccharomyces pombe [ 33 – 35 ]. However, this is likely the consequence of the presence of a second UvrD-type DNA helicase, called Fbh1 (lacking in S. cerevisiae ). Like S. cerevisiae Srs2, Fbh1 is capable of removing Rad51 from DNA in vitro , and fbh1 mutants exhibit Rad51 accumulation in meiotic cells and a strong reduction in spore viability [ 36 , 37 ]. Furthermore, srs2 fbh1 double mutants are synthetic lethal supporting their redundant roles in recombination [ 38 ]. Surprisingly given its importance, studies on the role of Srs2 helicase in homologous recombination in multicellular eukaryotes are scarce. Srs2 appears to be conserved through evolution with homologs found in Viridiplantae, Heterokonts, and Metazoa [ 39 , 40 ]. Interestingly however, no SRS2 ortholog is found in fish and mammals, although functional homologs have been suggested [ 11 – 13 , 40 ]. A homolog of Srs2 has been identified in plant and in particular in the model plant Arabidopsis thaliana [ 39 , 40 ]. The AtSRS2 gene is located on chromosome 4 (At4g25120) and encodes a protein of 1147 amino acids. The two functional domains of the protein (the ATP-binding domain and the C-terminal domain) are highly conserved [ 39 , 40 ]. in vitro characterization of Arabidopsis SRS2 has confirmed that it is a functional 3’-5’ helicase capable of unwinding DNA [ 39 ]. Specifically, Arabidopsis SRS2 is capable of processing branched structures generated during SDSA, and also exhibits strand annealing activity, indicating a potential role during HR. However, although Arabidopsis thaliana SRS2 shows clear helicase activity in vitro , its function in vivo remains to be determined. To date, the SRS2 gene from the moss Physcomitrella patens is the only plant ortholog studied in vivo [ 41 , 42 ]. Moss srs2 mutants do not exhibit major defects in homologous recombination, show no hypersensitivity to DNA damage induced by bleomycin, no effect on gene-targeting events and no fertility defects [ 41 , 42 ]. Thus, the role of the SRS2 helicase in homologous recombination in plants remains to be demonstrated. We present here an analysis of SRS2 function in homologous recombination in the flowering plant, Arabidopsis thaliana . Our data show that SRS2 is dispensable for DNA repair and RAD51-dependent recombination in somatic cells, although srs2 mutants exhibit moderate defects in RAD51 focus formation. No effects on fertility and meiotic progression are seen, but strikingly, absence of SRS2 leads to both increased genetic interference and increased number of Class I COs. This is accompanied by a concomitant reduction of MUS81-dependent Class II COs. SRS2 has both anti- and pro-recombination roles during meiosis in Arabidopsis thaliana . Results Isolation and Molecular Characterization of SRS2 T-DNA Insertion Mutants As mentioned above, a homolog of the yeast Srs2 is found in the Arabidopsis genome [ 39 ]. Srs2 is a homolog of the bacterial UvrD helicase. It is a Sf1a helicase translocating in the 3’-5’ direction along ssDNA. Sf1 helicases contain several archetypical helicase motifs and these are conserved in the Arabidopsis thaliana SRS2 (SRS2) homolog (Figure S1). Remarkably, all amino acids recently identified as essential for budding yeast Srs2 activities, but one, are conserved in SRS2 (Figure S1; [ 43 ]). Notably, K41, F285, and H650, all instrumental for helicase activity [ 43 ] are conserved in the Arabidopsis protein (K273, F518, and H856, respectively; Figure S1). This is in accordance with conserved helicase activity of Arabidopsis SRS2. We also note that Srs2 Y775 (V963 in Arabidopsis), recently shown to be essential for disrupting D-loops [ 43 ], is not preserved in Arabidopsis SRS2 - suggesting a potential functional divergence/loss (Figure S1). In order to study SRS2 function in planta , we searched for and characterized 3 T-DNA insertion mutant lines, thereafter named srs2-1 (GABI-637C01), srs2-2 (GABI_647B04), and srs2-3 (SALK_039766; Figure 1A ). The mutant alleles were verified by PCR and sequencing to determine the exact genomic position of the insertions ( Figure 1A-B ). In srs2-1 , the T-DNA is inserted in exon 7. This insertion is associated with a deletion of 32 bp and is flanked by two T-DNA left borders in opposite orientations ( Figure 1A ). Sequence analysis of the T-DNA junction indicated that an in-frame stop codon is present immediately at the T-DNA left border ( Figure 1A ). In srs2-2 and srs2-3 , the T-DNA is inserted in introns 3 and 15, respectively. In srs2-3 , the insertion is associated with a deletion of 29 bp and is flanked by two left borders ( Figure 1A-B ). Sequence analysis of the T-DNA junction indicated that an in-frame stop codon is present in intron 15 before the T-DNA integration ( Figure 1B ). Homozygous mutant lines were further analyzed by RT-PCR to confirm the absence of the respective transcript ( Figure 1C ). For all three mutants, no transcript was detected with primers surrounding the T-DNA insertion site, confirming the absence of full-length transcript and that no functional SRS2 protein can be produced ( Figure 1C ). Download figure Open in new tab Figure 1. Arabidopsis SRS2 T-DNA Insertion Mutants (A) Structure of SRS2 and T-DNA insertion mutant alleles. Dark gray boxes show exons and light grey boxes indicate 5’ and 3’ untranslated regions. Triangles (purple, blue, or green triangles). The position of the T-DNA insertions is indicated (purple, blue, or green triangles) with arrows showing the orientation of the left border and sequences of the T-DNA/ chromosome junctions below (SRS2 sequence in black and T-DNA sequence in red). In srs2-1 , insertion is accompanied by a 32bp deletion in exon 7. A putative in-frame TGA codon is underlined. Numbering under the sequences is relative to the SRS2 start codon. ( B) Sequences of the T-DNA/ chromosome junctions in srs2-2 and srs2-3 . Putative in-frame stop codon is underlined. Numbering under the sequences is relative to the SRS2 start codon. (C) RT-PCR analyses of transcripts of srs2 insertion mutants. Amplification of the actin transcript (ACT) was used as a control for RT-PCR. The positions and orientations of the PCR primers are shown with capital letters in the diagram. SRS2 is dispensable for DNA repair and RAD51-dependent recombination in somatic cells To assess the involvement of SRS2 in DNA repair, we tested the sensitivity of the srs2-1, srs2-2 and srs2-3 mutants to the DNA-damaging agent Mitomycin C (MMC). MMC forms DNA interstrand crosslink adducts and subsequently DNA strand breaks that are repaired by RAD51-dependent homologous recombination. No hypersensitivity to MMC was observed in the mutants ( Figure 2A-B ). Download figure Open in new tab Figure 2. Mitomycin C sensitivity and somatic HR in srs2 mutants (A) Representative pictures of 2-week-old seedlings grown without (left), or with MMC (middle: 30 µM; right: 40 µM). (B) Mean number of true leaves per seedling counted by direct visual inspection. Data are represented as mean ± SEM of 3 independent experiments. P values were calculated using nonparametric statistical analysis (Kruskal–Wallis test). ** p-value < 0.01. rad51 is RAD51-GFP transgenic line described in [ 73 ] (C) Mean number of true leaves per seedling of several helicase mutants and double mutant lines counted under a binocular microscope. Data are represented as mean ± SEM. Statistical analysis was performed using Kruskal-Wallis test; * p-value < 0.05. (D) Quantification of the number of spontaneous HR events (blue sectors) in somatic cells using IU-GUS assay, with n indicating the number of seedlings analysed. Each mutant was compared to wild-type (SRS2+/+, grey boxes) sister plants. Data are represented as mean ± SEM. P values were calculated using nonparametric statistical analysis (Kruskal–Wallis test). In budding yeast, srs2 mutants show synthetic lethality with mutation of other helicases such as Rad54, Sgs1/Recq4a, and Mus81 (reviewed in [ 44 ]). We thus generated the corresponding double mutants in Arabidopsis ( srs2 rad54 , srs2 recq4a , srs2 mus81 , and srs2 rtel1 ) and tested their sensitivity to MMC. Neither the single nor the double mutants showed visible growth phenotypes without treatment. As previously reported, rad54 and mus81 mutants exhibit hypersensitivity to MMC ( Figure 2C ). However, no further hypersensitivity to DNA damage was observed in the double mutants compared to the single helicase mutants ( Figure 2C ). This contrasts strikingly with yeast double mutants [ 45 ]. SRS2 is thus dispensable for RAD51-dependent DNA repair in Arabidopsis. To take this further, we directly assessed RAD51-dependent somatic homologous recombination in the absence of SRS2 using the previously described IU.GUS recombination assay, specifically measuring RAD51-dependent recombination [ 46 , 47 ]. This assay system makes use of an interrupted, nonfunctional beta-glucuronidase ( GUS ) gene, and a template GUS sequence which serves as a substrate for homologous recombinational repair. In cells where homologous recombination events occur, the GUS gene is restored, and subsequently, GUS activity can be histochemically detected as blue tissue sectors. The line with the disrupted GUS gene was crossed to srs2 mutants and somatic HR frequencies were monitored in progeny homozygous for IU.GUS reporter. As anticipated, we did not observe any significant difference in the number of recombination events per seedling between WT and srs2 mutants ( Figure 2D ), further supporting the notion that SRS2 does not play a key role - or is redundant with another helicase - in RAD51-dependent somatic HR. RAD51 focus formation in somatic cells upon DNA damage A major role of Srs2 is dismantling Rad51 nucleofilaments through its ability to translocate along ssDNA. To check this, we performed RAD51 immunofluorescence on root apex nuclei of 5-day-old seedlings, treated or not with Mitomycin C (30 μM for 6 hours; Figure 3 ). As expected, no RAD51 foci were detected without treatment ( Figure 3A and C ) and numerous RAD51 foci were detected after MMC treatment in both wild-type and srs2 mutants. Interestingly, a moderate but significant increase in the mean number of RAD51 foci per nucleus was detected in srs2 mutants ( Figure 3C ). In wild-type plants, we observed an average of 3.5 RAD51 foci per nucleus (± 0.5; n = 404), while srs2-1 and srs2-3 mutants had means of 4 (± 0.6; n = 345) and 4.1 (± 0.5; n = 391) foci per nucleus, respectively ( Figure 3C ). In accordance with this increase, the proportion of cells exhibiting RAD51 foci increases in srs2 mutants ( Figure 3D ). This moderate increase confirms a (minor) role for SRS2 in stripping RAD51 nucleofilaments and/or avoiding non-specific binding of RAD51 in somatic cells, without however having a major impact on DNA repair and RAD51-mediated recombination. Download figure Open in new tab Figure 3. RAD51 foci in somatic cells in srs2 mutants (A-B) Immunolocalization of RAD51 in root tip nuclei of 5-days-old seedlings untreated (A) or treated with 30 µM MMC for 6h (B) . Experiments were performed on srs2-1 and srs2-3 mutant lines. DNA is stained with DAPI (blue) and RAD51 foci (detected using an antibody against RAD51) are colored in green. Images are collapsed Z-stack projections of 3D image stacks. Scale bar: 5 µm. (C) Quantification of RAD51 foci in root tip nuclei of WT, srs2-1, and srs2-3 mutant lines before and after MMC treatment. Data are shown as mean ± SD, with n indicating the number of cells analyzed. More than 300 nuclei from at least 3 seedlings were analyzed per genotype. P values were calculated using nonparametric statistical analysis (Kruskal–Wallis test); * p-value < 0.05; **** p-value 50 RAD51 foci for each genotype, before and after MMC treatment. Cells analyzed are the same as in (C) . Normal fertility and meiotic progression in the absence of SRS2 Homozygous srs2 mutant plants develop normally and exhibit no fertility defects ( Figure 4A ). This is consistent with the apparent normal meiotic progression observed in srs2 mutants, which show fully synapsed chromosomes at pachytene, 5 bivalents at metaphase I, normal chromosome segregation at anaphase I, and four balanced nuclei at the tetrad stage ( Figure 4B ). RAD51 and the synaptonemal complex transverse filament protein ZYP1 appear to load normally in srs2 mutants ( Figure 4C-D ). Finally, we estimated the chiasma number by counting the number of ring and rod bivalents at Metaphase I, assuming 2 or 3 crossovers, or 1 crossover for ring and rod bivalents, respectively. We counted an average of 9.2 chiasmata per cell in srs2 mutants, not significantly different from the 8.8 chiasmata per cell in wild-type plants ( Figure 4E ). Thus, the absence of SRS2 does not detectably affect meiotic progression, nor total chiasma numbers. Download figure Open in new tab Figure 4. Fertility and meiotic progression in srs2 mutants (A) Plant fertility was measured by counting the number of seeds per silique, with n indicating the number of siliques analyzed. Each dot represents one silique. Mean is represented as a red line. Statistical analysis was performed using nonparametric Kruskal-Wallis test; ns : not significant (p-value > 0.05). (B) DAPI-stained male meiotic nuclei. Normal meiotic progression occurs in srs2 mutants, with complete synapsis at pachytene, five intact bivalents at metaphase I, two pools of five chromosomes at Anaphase I, and tetrads with four balanced meiotic products. Scale bar: 10 µm. (C) Co-immunolocalization of RAD51 (magenta) and the chromosome axis protein ASY1 (green) on leptotene/zygotene meiotic chromosome spreads. Scale bar: 5 µm. (D) Co-immunolocalization of RAD51 (magenta) and ZYP1 (green) on zygotene/pachytene meiotic chromosome spreads. Scale bar: 5 µm. (E) Chiasma number per cell at metaphase I stage, with n indicating the number of cells analyzed. The number of chiasmata per cell was estimated based on the shape of the bivalents. Statistical analysis was performed using parametric unpaired t-test; ns : not significant (p-value > 0.05). In S. cerevisiae , Dmc1 is a potent inhibitor of Srs2 activity during meiosis and Srs2 specifically dismantles Rad51 nucleofilament whereas recombination intermediates containing Dmc1 are not affected [ 27 , 32 ]. To test the hypothesis that Arabidopsis SRS2 specifically acts on RAD51-dependent recombination intermediates, we analyzed meiotic progression in the absence of both DMC1 (RAD51 becomes active) and SRS2. Meiotic progression of the dmc1 srs2-1 double mutant closely resembles that of the dmc1 mutant, in which RAD51 repairs meiotic DSB without forming interhomolog CO (Figure S2). This suggests that SRS2 has no major effect on RAD51-mediated meiotic DSB repair in Arabidopsis meiosis, even in the absence of DMC1 (Figure S2). Increased genetic interference in the absence of SRS2 We next sought to analyze more closely the impact of SRS2 on meiotic recombination by measuring meiotic CO rates in genetic intervals defined by markers conferring fluorescence in pollen grains (Fluorescent-Tagged-Lines, FTLs, [ 48 ]). When combined with mutation of the QUARTET1 gene ( qrt ), which prevents separation of the four pollen grains, these FTL lines allow direct measurement of recombination between the linked fluorescent markers, and estimation of genetic interference by scoring pollen fluorescence in tetrads. We determined CO rates in two adjacent intervals on chromosome 1 (I1b and I1c) in wild-type and srs2 mutant plants. No difference in recombination frequency was observed for either interval in srs2 mutants ( Figure 5A and Table S1). Remarkably however, genetic interference is significantly increased in both srs2 mutants tested ( srs2-1 and srs2-3 ), increasing from 0.53 in wild-type to 0.65 and 0.63 in srs2-1 and srs2-3 , respectively ( Figure 5B ). The number of double CO in these intervals is thus significantly lower in the srs2 mutants. Analyses of two additional intervals on chromosome 2 (I2f and I2g) confirmed this result, with no impact on CO frequency but an increased genetic interference in srs2-1 mutants (although not statistically significant likely due to the very short size of the I2fg interval which in practice limits the detection of a sufficiently large number of double CO events; Figure S3 and Table S2). Thus, absence of SRS2 affects genetic interference in the studied intervals, while CO frequency remains unchanged. Download figure Open in new tab Figure 5. Increased genetic interference and Class I CO in srs2 mutants. (A) FTL crossover frequency (I1bc interval) in wild-type (grey), srs2-1 (blue) and srs2-3 (green). CO frequency within I1bc interval is presented as the genetic distance. Each mutant is compared to wild-type (WT) sister plants (SRS2 +/+, in grey). Each dot represents one plant. Means are indicated by red horizontal lines. (B) Crossover Interference within I1bc interval in WT, srs2-1 and srs2-3 mutants. Each mutant is compared to wild-type sister plants that are SRS2 +/+ (in grey). Each dot represents one plant. Means are indicated by red horizontal lines. Statistical analysis was performed using Z-test. ** p-value < 0.01; *** p-value < 0.001. (C) Representative images of MLH1 (magenta) immunolocalization on diakinesis-staged male meiocytes in wild-type and srs2 mutants. Scale bar: 10 µm. (D) Number of MLH1 foci per cell, with n indicating the number of cells analyzed. Each dot represents the total number of MLH1 foci in an individual cell. Mean is presented as a red bar. Statistical analysis was performed with unpaired Kruskal-Wallis test; * p-value < 0.05. (E) Representative images of MLH1/HEI10 co-immunolocalization on diakinesis-staged male meiocytes in wild-type and srs2 mutants. Scale bar: 10 µm. (F) Number of MLH1/HEI10 co-foci per cell, with n indicating the number of cells analyzed. Each dot represents the total number of MLH1/HEI10 co-foci in an individual cell. Mean is presented as a red bar. Statistical analysis was performed with unpaired Kruskal-Wallis test; * p-value < 0.05. Altered balance of Class I and Class II CO in srs2 mutants Class I CO are sensitive to interference, while Class II CO are not [ 49 ]. Given that the number of CO does not significantly differ between WT and srs2 plants, a possible explanation for the increased CO interference in the absence of SRS2 would be an increase in the proportion of CO that are subject to interference (Class I). To test this hypothesis, we performed MLH1 immunofluorescence to quantify Class I CO on diakinesis-staged meiocytes ( Figure 5C-D ). A significant increase of MLH1 foci is seen in both srs2-1 (12.2 foci, n = 55) and srs2-3 (12.3 foci, n = 23) mutants compared to the wild-type (11.4 foci, n = 50; Figure 5C-D ). This result was confirmed by co-immunostaining MLH1 and HEI10 on diakinesis-staged meiocytes. We counted an average of 9.2 MLH1-HEI10 co-foci in s rs2-1 mutants (n = 35), significantly more than the 8.4 MLH1-HEI10 co-foci in wild-type meiocytes (n = 30; Figure 5E-F ). The absence of SRS2 thus leads to an increase in Class I CO numbers, confirming an anti-Class I-CO role for SRS2 in Arabidopsis. This being so, the absence of an overall increase in the number of CO would imply a corresponding reduction in the number of Class II CO in the absence of SRS2. It is possible to quantify numbers of Class II CO in zmm mutants (no Class I CO). We thus crossed srs2 plants with the zmm mutants zip4 and msh5 and counted the number of bivalents at Metaphase I. In accordance with expectation, we observed an average of 1.5 bivalents per cell in zip4 and msh5 mutant meioses (n = 49 and 37, respectively; Figure 6A-C ). Remarkably, both the zip4 srs2 and msh5 srs2 double mutants exhibited significantly reduced numbers of bivalents per cell with 1.05 (n = 42) and 1.04 (n = 68) bivalents respectively per meiosis ( Figure 6A-C ). Numbers of Class II CO are thus reduced in the absence of SRS2. Download figure Open in new tab Figure 6. Mutation of srs2 reduces Class II CO (A) Representative images of metaphase I-staged male meiocytes in zip4 , zip4 srs2-1 , msh5 , and msh5 srs2-1 mutants. Scale bar: 10 µm. (B) Number of bivalents per cell in zip4 , zip4 srs2-1 , msh5 , and msh5 srs2-1 mutants. Data are represented as mean ± SEM with n indicating the number of cells analyzed. Statistical analysis was performed using unpaired Mann-Whitney test; ** p-value < 0.01. (C) Percentage of cells with 0, 1, 2, 3, or 4 bivalents in zip4 , zip4 srs2-1 , msh5 , and msh5 srs2-1 mutants. Number of cells analyzed is the same as in (B) . (D) Representative pictures of metaphase I-staged male meiocytes in zip4 , zip4 mus81 , zip4 srs2-1 , and zip4 srs2-1 mus81 mutants. Scale bar: 10 µm. (E) Number of bivalents per cell meiocytes in zip4 , zip4 mus81, zip4 srs2-1 , and zip4 srs2-1 mus81 mutants. Data are represented as mean ± SEM, with n indicating the number of cells analyzed. Statistical analysis was performed using unpaired Mann-Whitney test; * p-value < 0.05; ** p-value < 0.01. (F) Percentage of cells with 0, 1, 2, or 3 bivalents in zip4 , zip4 mus81, zip4 srs2-1 , and zip4 srs2-1 mus81 mutants. Number of cells analyzed is the same as in (E) . The absence of SRS2 affects MUS81-dependent Class II COs The formation of Class II CO involves the action of structure-specific endonucleases, notably MUS81. To further confirm this impact of the absence of SRS2 on Class II CO, we tested its dependence on MUS81. We thus sought to assess whether the absence of SRS2 could affect MUS81-dependent Class II CO. zip4 srs2-1 plants were crossed with mus81 plants to produce zip4 , zip4 srs2-1 , zip4 mus81 , and zip4 srs2-1 mus81 mutants, and numbers of bivalents were counted at Metaphase I in these plants ( Figure 6D-F ). As expected, zip4 mus81 have significantly fewer bivalents than the zip4 single mutant (0.84 bivalent/cell, n = 77; versus 1.34 bivalent/cell, n = 56; [ 50 – 52 ]). We also confirmed that zip4 srs2 exhibits fewer bivalents per cell (0.92, n = 56). Strikingly, no difference in the numbers of bivalents was seen between zip4 srs2 mus81 triple mutant, zip4 mus81, and zip4 srs2 double mutants ( Figure 6D-F ). SRS2 and MUS81 are thus epistatic for this phenotype, in agreement with a role for SRS2 in the Class II CO pathway. Discussion Plants possess a SRS2 homolog and in vitro studies of Arabidopsis SRS2 show that it is a functional 3’-5’ helicase capable of unwinding DNA [ 39 ]. In accordance with this, key amino acids essential for Srs2 helicase activities (e.g. Rad51 stripping) in budding yeast are conserved in Arabidopsis SRS2 [ 43 ], however, we note that Srs2 Y775 (V963 in Arabidopsis), essential for disrupting D-loops in vitro [ 43 ], is not conserved in Arabidopsis. This suggests a potential functional divergence or loss, and raises questions about the in vivo role of SRS2. To clarify this, we isolated Arabidopsis mutants lacking SRS2 and analyzed its role in mitotic and meiotic recombination. SRS2 is dispensable for RAD51-dependent recombination in somatic cells Our data show that absence of Arabidopsis SRS2 has no detectable effect on sensitivity to DNA damaging agent Mitomycin C and RAD51-dependent recombination. This is in agreement with data in moss Physcomitrella patens where srs2 mutants do not exhibit major defects in somatic homologous recombination [ 41 , 42 ] but in striking contrast with data in yeast where srs2 mutants are highly sensitive to DNA-damaging agents and display a hyper-recombination phenotype [ 12 , 13 , 15 , 16 , 18 – 24 ]. Moreover, yeast srs2 mutants are synthetic lethal when combined with other mutations that affect DNA repair and recombination [ 12 , 13 , 19 , 20 , 22 ]. Synthetic lethality of such srs2 double mutants is suppressed by eliminating homologous recombination, suggesting that accumulation of toxic recombination intermediates is the cause of the lethality [ 11 , 13 , 43 , 44 , 53 , 54 ]. We thus tested synthetic genetic interaction between Arabidopsis SRS2 and several other helicases identified in yeast, such as RAD54, MUS81, or RECQ4A (Sgs1), as well as RTEL1 (putative functional homolog of SRS2 in mammals, also present in plants). However, none of the combinations tested led to an obvious phenotype, either under standard growth conditions or in response to induced DNA damage. This suggests that the absence of Arabidopsis SRS2 does not lead to the accumulation of toxic recombination intermediates in the background tested, or alternatively, that this is compensated for by another helicase (or mechanism) that has not been tested in this study. Interestingly however, we observed a slight increase in DNA damage-induced RAD51 foci in absence of SRS2. This moderate increase suggests that SRS2 could play a minor role in dismantling RAD51 nucleofilaments in somatic cells following DNA damage induction. This is in accordance with data in yeast where Srs2 is well-known for its anti-recombination function. It disrupts the RAD51 nucleofilament to promote the SDSA pathway and limit the number of CO in somatic cells, and also to prevent toxic or untimely HR events [ 15 – 17 , 27 , 55 – 57 ]. A recent study using a functionally tagged RAD51 to track RAD51 nucleofilament formation in living cells showed a moderate effect of srs2 deletion on RAD51 nucleofilament formation [ 58 ]. Indeed, more functionally tagged RAD51 structures could be detected in a S. cerevisiae srs2 mutant 2 hours after induction of a unique irreparable DSB in haploid cells. Interestingly, this increase was associated with brighter and longer RAD51 filaments in srs2 mutants and (nearly) disappeared 4 hours after induction of the DSB [ 58 ]. Thus, in agreement with the known role of Srs2 in yeast, we hypothesized that Arabidopsis SRS2 could regulate RAD51 nucleofilaments. In the absence of SRS2, the nucleofilament is still formed, but more RAD51 could assemble along the filament leading to brighter, more readily detectable RAD51 foci. Although this does not appear to affect repair of MMC-induced DNA damage or spontaneous RAD51-dependent HR events, it could impact the channeling of recombination events into different repair pathways. Alternatively, the absence of SRS2 could lead to non-specific accumulation of RAD51 at undamaged sites. However, we do not favor the latter hypothesis since we did not detect RAD51 foci in srs2 mutants in absence of DNA damage. SRS2 plays both an anti-CO and a pro-CO role in meiotic recombination Studies in budding and fission yeast have shown that Srs2 is highly expressed in meiosis and that absence of Srs2 reduces spore viability and decreases both CO and NCO in meiosis [ 18 , 27 – 31 , 45 ]. Moreover, Srs2 protects against the accumulation of aberrant recombination intermediates at the end of meiotic Prophase I, as seen in the aggregation of RAD51 at late Prophase I in srs2 mutants [ 30 , 31 ]. Interestingly, Srs2 appears to specifically affect RAD51-bound recombination intermediates since (i) DMC1-bound intermediates are not affected, and (ii) DMC1 inhibits Srs2 ATP hydrolysis activity [ 29 , 32 ]. These data suggest important functions of Srs2 in meiotic recombination. In multicellular eukaryotes, evidence for a role of SRS2 homologs in meiosis remained to be demonstrated. Mammals have no ortholog of SRS2, although a number of functional homologs are known (RECQ5, PARI, RTEL1, BLM, FBH1) [ 2 , 12 , 40 ]. Our study highlights multiple roles for SRS2 in meiotic recombination in Arabidopsis and suggests that SRS2 plays both an anti-CO and pro-CO role in Arabidopsis meiosis. Arabidopsis SRS2 is more strongly expressed in meiotic tissues than in somatic tissues [ 59 , 60 ] and we show here that the absence of SRS2 leads to a shift in the ratio of Class I to Class II COs (more Class I COs and less Class II COs), without affecting total chiasma number. This shift in the ratio of Class I to Class II COs is accompanied by a corresponding increase in genetic interference measured in two paired chromosome intervals. We propose a model in which SRS2 could play an anti-CO role by influencing the stability and/or the dissociation of some early RAD51/DMC1-bound recombination intermediates to promote SDSA ( Figure 7 ). In turn, the absence of SRS2 would lead to more DMC1-bound interhomolog intermediates generating more precursor that are channeled into the ZMM pathway, ultimately increasing Class I COs (supported by the moderate increase in MLH1/HEI10 foci in meiosis; Figure 7 ). Interestingly, such increase in MLH1 foci in UvrD-type mutants is not unique to Arabidopsis SRS2. In mouse, deletion of PARI, an antirecombinase related to yeast Srs2 and bacterial UvrD, also leads to increased MLH1 foci [ 61 , 62 ]. Download figure Open in new tab Figure 7. Model for the dual role of SRS2 in meiotic recombination We hypothesize that SRS2 acts at both the invasion step and at a later stage during resolution of recombination intermediates (left panel). First, SRS2 shows an anti-CO role, possibly through an anti-recombinase effect. Accordingly, absence of SRS2 could regulate RAD51/DMC1 nucleofilament assembly/disassembly/stability at ssDNA and/or D-loop stage (right panel). This moderate effect on nucleofilaments has little - if any - effect on somatic DNA repair. However, during meiosis, some recombination intermediates normally channeled to the NCO (for instance through SDSA) pathway could be redirected to the ZMM pathway thus generating more Class I CO. At the same time, SRS2 may promote MUS81 resolution of specific recombination intermediates (left panel), thus showing pro-CO activity. In srs2 mutant, MUS81 resolution is then compromised and less Class II CO are formed (right panel). Additionally, SRS2 may also play a pro-CO role by promoting MUS81-mediated resolution of recombination intermediates in meiosis ( Figure 7 ). Previous studies in yeast have demonstrated that Srs2 interacts with and promotes Mus81-mediated resolution of recombination intermediates in somatic cells [ 57 , 63 ]. Interestingly, the stimulation of Mus81-Mms4 activity by Srs2 is independent of its helicase activity [ 63 ]. Moreover, Mus81 appears to prevent Srs2 from unwinding recombination intermediates, suggesting a tight coordination of the activities of both proteins for resolution of recombination intermediates. In meiosis, the non-interfering Class II CO pathway relies primarily on MUS81 in budding yeast, plants, and animals [ 4 , 5 , 50 , 51 , 64 – 66 ]. Accordingly, the absence of MUS81 leads to a 15-20% reduction in CO. Similarly to our observation in srs2 mutants, Arabidopsis mus81 mutants exhibit reduced class II CO and increased genetic interference [ 50 , 51 ]. If SRS2 facilitates MUS81-mediated resolution, the loss of SRS2 would impair MUS81 activity, leading to reduced Class II CO and increased genetic interference, as observed in Arabidopsis srs2 mutants in this work ( Figure 7 ). Interestingly, similar findings were reported for MUS81-deficient mice, where class II CO suppression was associated with a significant increase in MLH1 foci, while the number of chiasmata per cell remained unchanged [ 63 ]. Another simple alternative exists where SRS2 acts on a specific pool of recombination intermediates channeled toward MUS81-dependent Class II CO – potentially by removing RAD51 to facilitate MUS81 access and/or promoting MUS81-mediated resolution. In this scenario, the absence of SRS2 would redirect these intermediates into the ZMM pathway, increasing Class I COs while reducing Class II COs without altering the total number of CO precursors. In conclusion, our data clearly point to a dual role for SRS2 in meiotic recombination, controlling the balance of Class I versus Class II CO in Arabidopsis meiosis. Further studies are nevertheless required to better understand the role of SRS2 in meiotic recombination and in particular in MUS81-dependent Class II CO establishment. Materials and Methods Plant growth and in vitro culture conditions In this study, the following mutant lines were used (all in Col0 background): srs2-1 (GABI_637C01), srs2-2 (GABI_647B04), srs2-3 (SALK_039766), zip4 (SALK_068052; [ 67 ]), msh5 (SALK_110240; [ 68 ]), mus81-2 (SALK_107515; [ 51 ]), I1bc FTL (FTL567-YFP/ FTL1262-DsRed2/FTL992-AmCyan/ qrt1-2 ) [ 69 ], I2fg FTL (FTL800-DsRed2/FTL3411-YFP/ FTL3263-AmCyan/ qrt1-2 ) [ 69 ], recq4a-4 (GABI_203C07 ; [ 70 ]), rtel1-1 (SALK_ 113285 ; [ 71 ]), rad54-1 (SALK_088057; [ 72 ]), RAD51-GFP [ 73 ]. Plants were grown on soil or in vitro (on 0.5X MS medium [M0255; Duchefa Biochemie] with 0.8% [m/v] agar, 1% sucrose) and stratified for 2 days at 4°C then grown in a growth chamber at 23°C under a 16h:8h light:dark photoperiod with 60% relative humidity. Seeds grown in vitro were first surface sterilized in 70% ethanol/0.05% SDS for 5 minutes, washed in 95% ethanol and then air-dried under a laminar flux hood. Molecular characterization of srs2 T-DNA insertion mutants The srs2-1 mutant was genotyped using primers A and B to detect the wild-type loci and primers A and I or B and I (GABI-Kat T-DNA left border-specific primer) to detect the T-DNA insertion allele. For the srs2-3 mutant, genotyping was performed using primers G and H to detect the wild-type loci and primers G (or H) and L (SALK T-DNA left border-specific primer) to detect the T-DNA allele. Left and/or right boundaries of T-DNAs have been sequenced by Sanger sequencing using the following primer pairs A+K and B+K for srs2-1 , K+J for srs2-2 , and G+L and L+H for srs2-3 plants. Sequences of all primers used for genotyping and sequencing are listed in S3 Table . RT-PCR analyses Total RNA was extracted on 7-days-old seedlings using RNeasy® Plant mini kit (Qiagen). Two µg of RNA was then treated with RQ1 RNase-free DNase (Promega). Random hexamers were added to treated RNA and incubated 5 minutes at 70°C and then immediately placed on ice. Finally, M-MLV reverse transcriptase (Promega), 5X buffer, 10 mM dNTPs, 30U RNasin and ultrapure water were added and incubated at 37°C for 1h. PCR amplification were then performed using primers listed in S3 Table . MMC sensitivity assay Surface sterilized seeds were sown onto half-strength MS medium (0.8% {m/v} agar, 1% sucrose) supplemented or not with 30µM MMC (Sigma-Aldrich). Dishes were stratified for 2 days at 4°C then grown at 23°C for 2 weeks. As described in [ 74 ], the number of true leaves per seedling was counted either by direct visual inspection or under a binocular microscope to measure MMC sensitivity. Statistical analysis was performed with Kruskal-Wallis test (GraphPad Prism v10.4.0 software). Somatic homologous recombination assay using histochemical GUS staining The frequency of somatic HR events was assessed using the IU-GUS.8 line containing an interrupted ß-Glucuronidase ( GUS ) gene [ 75 ]. Surface sterilized seeds were grown in vitro on MS medium (2 days of stratification then 2 weeks at 23°C), then incubated in GUS staining buffer (0.2% Triton X-100, 50 mM sodium phosphate buffer, pH 7.2, and 2 mM X-Gluc [Biosynth] dissolved in N,N-dimethylformamide). Plants were then vacuum infiltrated for 15 minutes and then incubated at 37°C for 24h. Staining buffer was then replaced with 70% EtOH to remove leaves pigmentation. Finally, blue spots were counted under a binocular microscope. Statistical analysis was performed with Kruskal-Wallis test (GraphPad Prism v10.4.0 software). RAD51 foci on root apex nuclei Plants were grown for 5 days on half-strength MS medium and fixed in 4% PFA (in 1X PME) for 45 min. Immunostaining in root tip nuclei was then performed as previously described [ 76 ]. Slides were incubated with rat α-RAD51 (1/500 in 3% BSA, 0.05% Tween-20 in 1X PBS) in a moist chamber at 4°C overnight. Slides were washed 3 times in 1X PBS-0.05% Tween-20, air-dried, then incubated with secondary antibody solution (chicken anti-rat Alexa 488 (Invitrogen) diluted 1/1000 in 3% BSA, 0.05% Tween-20 in 1X PBS) in a moist chamber for 3h at room temperature in the dark. Slides were finally washed 3 times in 1X PBS-0.05% Tween-20, air-dried, and mounted in VECTASHIELD mounting medium containing DAPI (1.5 μg/ml; Vector Laboratories Inc.). Z-stacks images were acquired with a Zeiss Cell Observer Spinning Disk microscope and analyzed using Imaris software v9.8.2 as previously described [ 77 ]. Briefly, 3D root nuclei were segmented and a mask was created on the segmented surfaces to display EGFP (Alexa488, marking RAD51 foci) only in the surfaces. A random color mask was applied on the DAPI channel to assign a unique color ID to each surface. Finally, spots were created with the “Spots” tool on RAD51 foci using Sum Square of Alexa488 as a quality control. Statistics (Surface: Volume, Median Intensity; Spots: Intensity Min, Max, Mean, Median, Sum, SD, Sum Square, Median Intensity) were exported and data were plotted using GraphPad Prism v10.4.0. Statistical analysis of the number of RAD51 foci per nucleus was performed using Kruskal Wallis test (GraphPad Prism v10.4.0 software). Plant fertility assessment Plant fertility was assessed by counting the number of seeds per silique. About 30 siliques from the primary stem were collected per plant and bleached in 95% EtOH at 70°C for several hours. The number of seeds per silique was counted manually under a binocular microscope. All analyzed plants were grown in the same conditions. Statistical analysis was performed using Kruskal-Wallis test (GraphPad Prism software v10.4.0). Meiotic chromosome spreads Meiotic chromosome spreads were prepared as previously described [ 78 ]. Inflorescences were collected from secondary stems and fixed in Carnoy’s fixative (3:1 EtOH : acetic acid). Fixed inflorescences were washed once in ultrapure water then twice in 10 mM citrate buffer (pH 4.5). Flower buds were then digested in enzyme mixture (0.3% cellulase, 0.3% pectolyase, and 0.3% cyclohelicase; Sigma-Aldrich) for 3h at 37°C in a moist chamber. The digestion reaction was stopped by placing the slides on ice and replacing enzyme mix with ice-cold 10 mM citrate buffer (pH 4.5). Immature flower buds of appropriate stage (0-3-0.6 mm) were selected under a binocular microscope, placed individually on a clean microscope slide, and crushed with a dissection needle. Chromosomes were spread by stirring for 1min in 20μl 60% acetic acid at 45°C, fixed with Carnoy’s fixative, and air-dried. Finally, slides were mounted in VECTASHIELD mounting medium containing DAPI (1.5 μg/ml DAPI; Vector Laboratories Inc.) and covered with 24×32 mm coverslip. Images were acquired with a Zeiss AxioImager.Z1 epifluorescence microscope equipped with an Axio-Cam Mrm camera and DAPI filter. Immunolocalization of MLH1/HEI10 on meiotic chromosome spreads All slides used in this experiment were washed in acetone, water, and EtOH then airdried before use. Immunolocalization of MLH1 and HEI10 proteins was performed on meiotic chromosome spreads slides containing diakinesis-staged meiocytes as previously described [ 79 ]. Selected slides were placed in room-temperature PBST (1X PBS – 0,1% Triton), transferred for 45 seconds in (barely) boiling Tris-sodium citrate solution, then transferred back in room-temperature PBST. Slides were then incubated with the primary antibody solution (Rabbit MLH1: 1/200; Chicken HEI10: 1/100; diluted in 1% BSA-PBST) for 2 days in a moist chamber at 4°C. Slides were then washed 3 times in PBST and incubated with the secondary antibody solution (Donkey anti-Rabbit Cy3: 1/1000; goat anti-chicken Alexa488: 1/1000; diluted in 1% BSA-PBST) for 30 min in a dark moist chamber at 37°C. Finally, the slides were washed 3 times in PBST and air-dried in the dark for 5 minutes. Slides were mounted in Vectashield mounting medium containing DAPI (1.5 μg/ml DAPI; Vector Laboratories Inc.) and covered with a coverslip. The coverslip was sealed to the slide using clear nail polish. RAD51/ASY1 and RAD51/ZYP1 immunolocalization on meiocytes All slides used in this experiment were washed in acetone, water, and EtOH then air-dried before use. Inflorescences were harvested from secondary stems and placed in an ice-cold Petri dish with moist filter paper. Buds ranging from 0.3 mm to 0.5 mm were selected using a binocular microscope. Approximately 6-8 buds were dissected in 10 µl Enzyme Mix (0.4% Cytohelicase, 1.5% sucrose, 1% PVP, in H2O) to retrieve as many anthers as possible. After a 5 minutes digestion at 37°C in a moist chamber, anthers were macerated with a dissection needle, 10 µl Enzyme Mix was added and slides were again incubated at 37°C for 10 minutes in a moist chamber. Then, 10 µl of 1% Lipsol was added to the slide and the droplet was stirred vigorously for 2 minutes, then incubated for 3 minutes. Under a fume hood, 40 µl of 4% PFA was added and spread with a needle. Slides were air-dried for 2-3 hours. For the immunostaining, slides were washed 3 times in PBST (1X PBS, 0.1% Triton X-100), and blocked with 1% BSA (in PBST) at room temperature for 30 minutes. The slides were then incubated with the primary antibody solution (rat anti-RAD51: 1/500; Guinea Pig anti-ASY1: 1/500; Rabbit anti-ZYP1: 1/500; diluted in 1% BSA in PBST) for 2 days in a moist chamber at 4°C. Then, the slides were washed 3 times in PBST and incubated with the secondary antibody solution (donkey anti-rat Cy3: 1/1000; goat anti-G. Pig Alexa488: 1/1000; diluted in 1% BSA in PBST) for 1 hour at 37°C in a dark moist chamber. Finally, slides were washed 3 times in PBST and air-dried, and mounted in Vectashield mounting medium containing DAPI (1.5 μg/ml DAPI; Vector Laboratories Inc.) and covered with a coverslip. The coverslip was sealed to the slide using clear nail polish. Recombination measurement and interference using FTLs We used Fluorescent Tagged Lines (FTLs) to estimate male meiotic recombination rates at two genetic intervals: I1bc on chromosome 1 and I2fg on chromosome 2. Heterozygous plants for the linked fluorescent markers were generated and siblings from the same segregating progeny were used to compare the recombination frequency between different genotypes. Slides and fluorescent tetrad analysis were performed as previously described [ 48 ]. Tetrads were counted and attributed to specific classes (A to L) (see Tables S1 and S2 for the classification and raw data). Genetic distances of each interval were calculated using Perkins equation as follows: X =100 [(1/2 Tetratype + 3 Non-Parental Ditype)/n] in cM [ 48 ]. Interference was estimated using the Coefficient of Coincidence (CoC (I1,I2) = , and interference calculated as 1-CoC. Thus, a calculated interference of 0 reveals the absence of interference in the interval. Statistical analysis was performed using Z test, and data plotted using GraphPad Prism software v.10.4.0. Metaphase I Image analysis Chiasma count of metaphase I-stage meiocytes was performed as previously described in [ 80 ]. Statistical analysis of chiasmata count of metaphase I-staged meiocytes was performed using an unpaired t-test (GraphPad Prism software v. 10.4.0). Meiotic chromosome spreads pictures obtained for the counting of bivalents in ZMM mutant background were assigned a random name to allow an unbiased analysis using the Blind Analysis Tool (Fiji). Statistical analysis of the number of bivalents was then performed using unpaired Mann-Whitney test (GraphPad Prism software v.10.4.0). Supporting information Supplemental Figure 1. Sequence alignment of Srs2 and SRS2 and alignment of UvrD, Srs2 and SRS2 conserved helicase motifs (A) Identical (red), conserved (green) and semi-conserved (yellow) amino acids are depicted. Magenta and turquoise arrows show UvrD-like helicase ATP-binding and UvrD-like helicase C-terminal domains, respectively. Identity 26,26% (219 / 834), similarity 43,53% (363 / 834). (B) Amino acid residues shown to be essential for Srs2 activities are underlined in bold. Three of these amino acids are not conserved in Arabidopsis (neither identical nor similar) and are marked with an asterisk. Supplemental Figure 2. Meiotic progression in dmc1 srs2 double mutant Meiotic progression of male meiocytes stained with DAPI in WT, dmc1 , and dmc1 srs2-1 plants. Absence of DMC1 leads to asynapsis (late Prophase I) and lack of inter-homologue CO. Intact univalents are thus observed at Metaphase I owing to DSB repair by RAD51, most probably using sister chromatids. Univalents then segregate randomly at Anaphase I and ultimately this produces unbalanced Tetrads. A similar meiotic phenotype is observed in dmc1 srs2 mutant. Scale bar: 10 µm. Supplemental Figure 3. CO frequency and interference in I2fg interval (A) CO frequency within I2fg interval represented as the genetic distance (cM). Each dot represents one plant, with 400 to 800 tetrads analyzed per plant. Mean is presented as a red bar. (B) Interference within I2fg interval of WT and srs2-1 plants. Each dot represents one plant. Mean is presented as a red bar. Statistical analysis was performed using Z-test. S1 Table. FTLs raw data and Interference ratio calculation for I1bc interval. Tetrad count for all tetrad categories for I1bc interval. Tetrad categories (a to l) were classified as described previously by [ 48 ]. S2 Table. FTLs raw data and Interference ratio calculation for I2fg interval. Tetrad count for all tetrad categories for I2fg interval. Tetrad categories (a to l) were classified as described previously by [ 48 ]. S1 Data. Raw data for all countings presented in this study. View this table: View inline View popup Download powerpoint S3 Table. List of primers used for characterizing Atsrs2 T-DNA mutants Acknowledgments We thank Mathilde Grelon for ZYP1, MLH1 and HEI10 antibodies, Peter Schlögelhofer for RAD51 antibody and Eugenio Sanchez-Moran for the ASY1 antibody. 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Evaluation of Crossover Number , Distribution, and Interference Using Cytological Assays in Arabidopsis. Curr Protoc . 2022 ; 2 . doi: 10.1002/cpz1.599 OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted February 28, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Dual role of Arabidopsis SRS2 helicase in meiotic recombination Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Dual role of Arabidopsis SRS2 helicase in meiotic recombination Valentine Petiot , Floriane Chéron , Charles I. White , Olivier Da Ines bioRxiv 2025.02.26.640294; doi: https://doi.org/10.1101/2025.02.26.640294 Share This Article: Copy Citation Tools Dual role of Arabidopsis SRS2 helicase in meiotic recombination Valentine Petiot , Floriane Chéron , Charles I. 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