SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development

SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development | 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 SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development View ORCID Profile Veronique Hugouvieux , View ORCID Profile Romain Blanc-Mathieu , View ORCID Profile Michel Paul , View ORCID Profile Aline Janeau , View ORCID Profile Xiaocai Xu , Jeremy Lucas , Xuelei Lai , Antonin Galien , View ORCID Profile Wenhao Yan , Max Nanao , View ORCID Profile Kerstin Kaufmann , View ORCID Profile François Parcy , View ORCID Profile Chloe Zubieta doi: https://doi.org/10.1101/2023.05.23.541941 Veronique Hugouvieux 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Veronique Hugouvieux For correspondence: veronique.hugouvieux{at}cea.fr chloe.zubieta{at}cea.fr Romain Blanc-Mathieu 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Romain Blanc-Mathieu Michel Paul 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michel Paul Aline Janeau 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aline Janeau Xiaocai Xu 2 Plant Cell and Molecular Biology, Institute of Biology, Humboldt-Universität zu Berlin , Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xiaocai Xu Jeremy Lucas 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xuelei Lai 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France 3 Current address: National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University , Wuhan, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Antonin Galien 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wenhao Yan 2 Plant Cell and Molecular Biology, Institute of Biology, Humboldt-Universität zu Berlin , Berlin, Germany 4 Current address: College of Plant Science and Technology, Wheat Genetics and regulomics, Huazhong Agricultural University , Wuhan, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wenhao Yan Max Nanao 5 Structural Biology, European Synchrotron Radiation Facility, 71 Avenue des Martyrs , 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kerstin Kaufmann 2 Plant Cell and Molecular Biology, Institute of Biology, Humboldt-Universität zu Berlin , Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kerstin Kaufmann François Parcy 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for François Parcy Chloe Zubieta 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes , CNRS, CEA, INRAE, IRIG-DBSCI, 17 rue des Martyrs, 38000 Grenoble, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chloe Zubieta For correspondence: veronique.hugouvieux{at}cea.fr chloe.zubieta{at}cea.fr Abstract Full Text Info/History Metrics Preview PDF Abstract MADS genes encode transcription factors that act as master regulators of plant reproduction and flower development. The SEPALLATA (SEP) subfamily is required for the development of floral organs and plays roles in inflorescence architecture and development of the floral meristem. The SEPALLTAs act as organizers of MADS complexes, forming both heterodimers and heterotetramers in vitro . To date, the MADS complexes characterized in angiosperm floral organ development contain at least one SEPALLATA protein. Whether DNA-binding by SEPALLATA-containing dimeric MADS complexes are sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization impaired SEPALLATA mutants. Here, we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3/AGAMOUS tetramerization domain, structure-based mutagenesis and complementation experiments in sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers are able to bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild type binding sites genome-wide, tetramerization is not only required for floral meristem determinacy, but also absolutely required for floral organ identity in the second, third and fourth whorls. Introduction MADS genes play central roles in the development of reproductive structures, from the specification of male and female cones in gymnosperms 1 – 5 to the development of inflorescence architecture, 6 , 7 determinacy of the floral meristem 8 and the specification of floral organ identity in angiosperms 2 , 9 , 10 . The encoded MADS transcription factors (MTFs) bind to a highly conserved DNA sequence called a CArG box (CC-“Adenine-rich”-GG) as obligate dimers. The MTFs involved in reproductive development belong to the MADS type II, or MEF2 clade, and have a multidomain structure 11 . These domains consist of the highly conserved eukaryotic-specific DNA-binding MADS domain (M domain), a ∼30 amino acid alpha helical Intervening domain (I domain) critical for dimerization specificity 12 , a plant-specific coiled-coil Keratin-like oligomerization domain (K domain) and a largely unstructured and sequence-variable C-terminal domain (C domain). Based on this conserved domain structure, the type II MADS are also called MIKC and form two main groups which differ in their oligomerization capability. The “classic” MIKC c group form dimers and tetramers and are important for reproductive structure development and organ identity. The MIKC* group, dimeric MTFs, has a more limited role in male gametophyte development 13 , 14 . The well-established ABCE genetic model of floral organ identity requires the combinatorial activity of the MIKC c genes 15 . In floral organ development, the MADS genes are divided into the A ( SQUAMOSA, SQUA-like ), B ( DEFICIENS/GLOBOSA, DEF/GLO-like ), C ( AGAMOUS, AG-like ) and E ( AGL2/AGL6 or SEP-like ) class genes 2 . The most recent common ancestor of seed plants likely contained both an A and E class ancestor which has been lost in gymnosperms 16 . Extant gymnosperms contain only B and C class MADS genes 2 , with B and C class-encoded MTFs directly interacting to specify the formation of male cones and the C-class MADS complexes specifying of female cones 4 , 17 . While gymnosperm B and C-class MTFs are able to directly interact and likely form tetrameric complexes, this property has been lost in flowering plants, which require the angiosperm-specific SEPALLATA (SEP) subfamily (E class) to allow interaction of B and C MTFs for third whorl organ specification (stamen). Likewise, female organ development (carpel) in the fourth whorl of angiosperms also requires the E class SEP subfamily in addition to the C class MADS 5 , 18 , 19 . The identity programs for the perianth organs (sepals and petals) in angiosperms require an A class MADS gene, with sepal formation in Arabidopsis dependent on A and E class MTFs and A, B and E class MTFs required for the determination of second whorl petal identity 15 , 20 . The tetramerization domain was recruited early in seed plant evolution, with more promiscuous tetramerization putatively occurring between different MIKC c MTFs in ancestral species. Loss of direct tetramerization capability between B and C class MTFs occurred after the gymnosperm-angiosperm split, with the E class SEPALLATAs taking over the tetramerization function. Based on these data, tetramer formation has been long hypothesized to be key for reproductive organ development triggered by MTFs. However, direct evidence for this has remained elusive, due in part to the limitations in protein-protein interaction studies which mainly identify binary interactions, the difficulty in characterizing transcriptionally active MADS complexes in vitro and in vivo and the study of loss-of-function mutants which are not sufficient to probe development as a function of different oligomerization states. In Arabidopsis thaliana , which contains four SEP genes, triple ( sep1 sep2 sep3 ) and quadruple ( sep1 sep2 sep3 sep4 ) mutants display strong floral phenotypes, including loss of meristem determinacy and homeotic conversion of floral organs into sepaloid or leaf-like organs, respectively 21 – 23 . Furthermore, 35S -driven expression of B and/or C class MADS genes alone is not sufficient to launch floral organ identity programs and the concurrent expression of a SEP gene is required for the formation of ectopic floral organs 21 , 22 , 24 , 25 . At the molecular level, this suggests that SEP-containing heterodimers or tetramers are required for proper MADS function. Extensive yeast 2-hybrid experiments have demonstrated that SEP MTFs are able to oligomerize with class A, B and C MTFs. Yeast 3-hybrid and in vitro experiments further demonstrate the formation of SEP-containing heterotetrametric complexes, 2-site DNA binding and DNA-looping 26 – 29 . Recent studies using a tetramerization-impaired SEP3 allele, SEP3 Δtet , expressed in the sep1 sep2 sep3 mutant background attempted to decouple DNA binding and oligomerization state. This work demonstrated that robust tetramerization is required for floral meristem determinacy, but left open the question as to the role of tetramerization in floral organ identity as second and third whorl organ identity programs were not affected and fourth whorl organ identity was only partially perturbed compared to the loss-of-function sep1 sep2 sep3 triple mutant 30 . Examination of genome-wide binding using sequential DNA affinity purification and sequencing (seq-DAP-seq) indicated two-site co-operative binding at certain loci by SEP3/AG tetramers, the complex required for fourth whorl organ identity, which was lost in complexes containing SEP3 Δtet , further suggesting that hetero-dimerization of E and C class MTFs may be sufficient for carpel identity 30 . In order to address the fundamental question of the physiological role of tetramerization in flower development, we performed structural, biochemical, and in vivo experiments to correlate oligomerization state with DNA-binding and physiological function. Using structure-based design, we generated SEP3 and AG mutants with strongly abrogated tetramerization capability and compared their DNA-binding and ability to rescue the sep1 sep2 sep3 triple mutant phenotype. These results demonstrate that while SEP3-containing dimeric complexes bind many of the same sites as SEP3-containing tetramers genome-wide, they are unable to restore organ identity in the second, third and fourth whorls. Short-range binding site co-operativity based on intersite spacing enrichment is strongly reduced in the tetramerization mutants in seq-DAP-seq experiments and band-shift assays, pointing to a mechanism of DNA looping as important for proper gene regulation in organ identity. Taken together, these data show the absolute requirement of tetramerization for organ specification and proper cellular identity of petals, stamen and carpels. RESULTS SEP3 oligomerization, structural studies and mutant design The importance of MADS tetramerization in floral organ development has been extensively investigated, most recently in the context of the central role of SEP3 , the only SEP gene able to fully complement organ identity as a single allele in the sep1 sep2 sep3 triple mutant (Supplemental Figure S1). Using a natural splice variant impaired in tetramerization, SEP3 Δtet , in vitro DNA-binding studies demonstrated the loss of co-operative two-site DNA binding for the SEP3/AG heterocomplex, responsible for fourth whorl development and determinacy 31 . Relatively mild effects were observed in vivo in complementation assays, with phenotypes restricted to the fourth whorl and indeterminacy of the floral meristem 30 . Genome-wide binding studies using ChIP-seq (Supplemental Figure S2A) coupled with comparative RNA-seq of SEP3 and SEP3 Δtet expressing plants (Supplemental Figure S2B and C) were consistent with the observed phenotypes and highlighted relatively few differences in DNA-binding or gene regulation between SEP3 and SEP3 Δtet in vivo . This may be due to the residual ability of SEP3 Δtet to tetramerize in vivo with MADS partners, and would account for the rescue of second and third whorl organ identity as well as the partial restoration of fourth whorl identity as previously described 30 . In order to better design SEP3 mutants no longer able to tetramerize, we solved the structure of the physiologically relevant MADS heterotetrameric SEP3/AG K domain complex, using seleno-methionine derivatized protein and single anomalous dispersion (SAD) phasing. A partial structure was autobuilt using ARP/wARP 32 and subsequently used for molecular replacement of a higher resolution native SEP3/AG dataset. The protein complex crystallized in spacegroup C222 1 with 8 molecules per asymmetric unit. The resolution was 2.4Å for the native dataset and the refined model exhibited very good geometry and no residues in disallowed regions of the Ramachandran plot ( Table 1 ). As shown in Figure 1A , the crystal structure of SEP3/AG contains the complete K domains of SEP3 and AG, a small portion of the I domain and several residues of the C domain, with the tetramer adopting a cross-like configuration with outstretched alpha helical “arms”. The overall structure is very similar to the previously described SEP3 homotetramer (PDB 40XO), however the tetramer of SEP3/AG exhibits additional salt bridge interactions along the protein-protein interface ( Figure 1B ) 29 . Structural comparisons between SEP3/AG and SEP3 tetramers ( Figure 1C ) reveals a slight change in orientation of the alpha helical arms, with SEP3/AG exhibiting a more planar orientation of the N-terminal helices. Download figure Open in new tab Figure 1. Structure of SEP3/AG heterotetramer. A. SEP3 (blue) and AG (green) tetramer shown as a cartoon. N-and C-termini are labeled. The red box denotes the zoomed in region in B . B. Close-up of salt bridges at the tetramerization interface of SEP3 and AG. Residues are labeled and salt bridges are shown as dashed yellow lines with distances shown. C. View of SEP3/AG (left) and SEP3 (right) tetramers looking down the C-terminal alpha helices. SEP3 homo-tetramer exhibits a curvature as compared to SEP3/AG heterotetramer. D. Cartoon representation as in A , with the deletion mutations SEP3 Δtet and AG Δtet colored in gray and indicated by red arrows. The circled region is rotated for clarity and shown in E . E. Close-up view of the hydrophobic tetramerization interface between the C-terminal alpha helices of SEP3 and AG. Hydrophobic residues are labeled. The SEP3 Δtet3M mutations target the leucine zipper, with M150A, L154A and L157A and a deletion of residues 161-174 all affecting the tetramerization interface. View this table: View inline View popup Table 1. Data collection and refinement statistics Examination of the highly conserved hydrophobic leucine zipper critical for tetramerization allowed us to design point mutations to generally target the tetramerization interface for SEP3-containing MADS complexes ( Figure 1D and E ). In addition to the SEP3 Δtet mutation that deletes residues 161-174, three additional point mutations were introduced to create a new mutant, SEP3 Δtet3M , carrying the 161-174 amino acid deletion and mutations M150A, L154A and L157A ( Figure 1D and E ). The introduced mutations are all present at the predicted protein-protein interface for the heterotetrametric MADS protein complexes, suggesting that these mutations should universally disrupt SEP3-dependent tetramerization. In addition, the ag-4 allele, which encodes a version of AG lacking residues 159-172, which we refer to as AG Δtet , was mapped to the SEP3/AG structure ( Figure 1D and E ). This deletion mutant affects the N-terminal portion of the SEP3/AG tetramerization interface. Based on the structure of SEP3/AG, the combination of SEP3 Δtet and AG Δtet results in a SEP3/AG complex unable to tetramerize as it completely lacks the interface required for stable tetramer formation. In vitro characterization of SEP3 Δtet and SEP3 Δtet3M protein complexes Electrophoretic mobility shift assays (EMSA) were performed to evaluate the impact of mutations on MADS complex formation and DNA binding. First, we tested different SEP3 mutants with AG and AG Δtet . As shown in Figure 2A , EMSAs performed with SEP3 Δ tet3M /AG or with SEP3 Δ tet /AG Δtet confirmed that the mutations completely abolish heterotetramer formation in vitro , as observed by the complete disappearance of the band corresponding to tetrameric complexes. As previously reported, SEP3 Δ tet was only partially impaired in its ability to form heterotetramers with AG 30 . In all cases, mutations did not impair dimer formation and binding to DNA as confirmed by the presence of a strong band corresponding to migration of a dimer bound to DNA. Download figure Open in new tab Figure 2. Electrophoretic mobility shift assays for MADS complexes using DNA with two CArG-box MADS binding sites. A. Fourth whorl C and E MADS complexes are shown with different SEP3/AG complexes forming dimers and tetramers. The wild-type SEP3/AG complex binds DNA as a tetramer, SEP3 Δtet /AG binds as a mixture of dimeric and tetrameric species. SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet bind DNA as one or two dimers. B. MADS B+C+E complexes important for third whorl organ identity with SEP3/AG shown for comparison. The mixture of SEP3 Δtet3M /AG/AP3/PI shows a reduction in tetramer formation while the mixture of SEP3 Δtet /AG Δtet /AP3/PI does not bind DNA as a tetramer but as one or two dimers. C. MADS A+B+E complexes important for second whorl organ identity. The mixture of SEP3 Δtet3M /AP1/AP3/PI shows a reduction in tetramer formation as compared to SEP3/AP1/AP3/PI or SEP3 Δtet /AP1/AP3/PI. The AP3/PI heterodimer and SEP3/AP1 hetero-tetramer are shown for comparison. SEP3 Δtet is denoted as SEP3 Δ and AG Δtet as AG Δ for simplicity in the figure. Arrows indicate dimeric complexes, * indicates a tetramer and ** indicates two dimers. Next, we evaluated SEP3 and AG mutants for their ability to affect complexes important for third whorl (AP3/PI/AG/SEP3) organ identity and SEP3 mutants for second whorl (AP1/AP3/PI/SEP3) identity. When co-expressed with AG/AP3/PI ( Figure 2B ) or AP1/AP3/PI ( Figure 2C ), SEP3 Δ tet3M was more strongly affected in tetramer formation than SEP3 Δ tet , as indicated by a less intense upper tetramerization band and a more intense lower band corresponding to a dimer-bound DNA complex. Interestingly, heterocomplex formation was completely abolished between AP3/PI/AG Δtet /SEP3 Δ tet , as no band corresponding to the hetero-complex was observed ( Figure 2B ). Taken together, the data show that the SEP3 Δ tet3M mutant, or the combination of SEP3 Δ tet and AG Δtet , provoke much stronger tetramerization defects in vitro than SEP3 Δ tet alone, as effects on co-operative DNA-binding were observable for MADS complexes involved in second, third and fourth whorl, and third and fourth organ identity, respectively. Comparison of DNA-binding by SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet complexes Based on these results, SEP3 Δtet3M containing complexes and the SEP3 Δtet /AG Δtet complex almost completely abolish tetramer formation as compared to SEP3 Δtet on individual binding sites. We used seq-DAP-seq for a genome-wide comparison of the binding of the three complexes (SEP3 Δtet /AG, SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet ) at regions bound by the wild type SEP3/AG. Our previous analysis using seq-DAP-seq showed that the SEP3 Δtet mutation reduced both the binding affinity and the preference for specific CArG-box spacing (36, 46, 56 bp) 31 . We considered a region to be ‘unbound’ by a mutant complex for which the binding intensity is decreased by at least a factor two (Coverage Fold Reduction CFR > 2) as compared to SEP3/AG binding ( Figure 3 ). Most regions (> 4672, 74%) were bound with similar intensity (CFR relative to SEP3/AG < 2) by all complexes ( Figure 3A ). The presence of SEP3 Δtet3M or AG Δtet in the heterocomplex led to an additional binding reduction as compared to the SEP3 Δtet mutation alone with 690 and 686 regions bound by SEP3 Δtet /AG and not by SEP3 Δtet3M /AG or SEP3 Δtet /AG Δtet , respectively. Of these newly lost regions, 377 were shared by SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet complexes. Moreover, genome-wide, the median binding intensity of SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet relative to that of the SEP3 Δtet /AG mutant had a stronger decrease at regions containing a preferred CArG-box intersite spacing as compared to regions with no preferred CArG-box intersite spacing (Wilcoxon test, P =7×10 - 14 and 0.0005 for SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet , respectively), suggesting that SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet are less able to bind interspaced CArG-boxes as compared to SEP3 Δtet /AG ( Figure 3B ). The list of genes associated with at least a two-fold reduction in binding between the wild type and tetramerization mutants is given in Table SI and includes genes such as KANADI2 (KAN2) , which encodes a TF involved in carpel and ovule development and the establishment of polarity of floral organs 33 , 34 , JAGGED (JAG) , which encodes a zinc-finger TF important for stamen and carpel development 35 , 36 and INNER NO OUTER (INO ), a gene encoding a YABBY TF implicated in ovule integument development 37 . Taken together, these in vitro genome-wide binding comparisons demonstrate a small but statistically significant impairment of SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet DNA-binding compared to SEP3 Δtet /AG, and a strong decrease in regions bound by tetrameric SEP3/AG complexes. This suggests a genome-wide quantitative relationship between tetramer formation and access to regions showing specific intersite spacing at certain loci, with these regions putatively acting as important organ identity determinants. Download figure Open in new tab Figure 3. Genome-wide DNA binding comparisons determined by seq-DAP-seq for SEP3/AG wild-type and mutant complexes. A . Venn diagrams showing regions specifically bound by SEP3/AG (blue), SEP3 Δtet /AG (red) and SEP3 Δtet3M /AG (green; diagram on the left) or specifically bound by SEP3/AG (blue), SEP3 Δtet /AG (red) and SEP3 Δtet /AG Δtet (green; diagram on the right) complexes. Regions specifically bound are defined as having a binding intensity at least twice greater for a complex relative to the other complexes. B . Binding intensity ratio of SEP3 Δtet /AG to SEP3 Δtet3M /AG (red) and SEP3 Δtet /AG to SEP3 Δtet /AG Δtet (blue) over the 6,347 regions bound by SEP3/AG. The change in binding intensity is more significant for regions with a specific CArG-box intersite spacing ( n =2270) than for region with no spacing ( n =4077) for both SEP3 Δtet3M /AG and SEP3 Δtet /AG Δtet versus SEP3 Δtet /AG (Wilcoxon test, ***: P <10 - 5 , *: P <10 - 3 ). Impact of MADS tetramerization mutants in floral organ development and cell identity Based on in vitro data, the series of mutations targeting the tetramerization interface were used to assess the importance of MADS tetramerization in the different floral organ development programs. We generated sep1 sep2 sep3 plants expressing SEP3 , SEP3 Δ tet or SEP Δ tet3M , and sep1 sep2 sep3 ag-4 plants expressing SEP3 Δ tet and analyzed the overall morphology of each floral organ and the surface cell identity in the second, third and fourth whorls by scanning electron microscopy (SEM) ( Figures 4 and 5 ). The sep1 sep2 sep3 mutant exhibited conversion of all floral organs to sepaloid structures that showed numerous stomata and typical elongated cells at their surfaces ( Figures 4 and 5 , first columns). The lack of determinacy of the floral meristem results in the continuous generation of a new “flower” made of sepaloid organs in the fourth whorl ( Figure 4 ) 21 , 30 . Flowers of sep1 sep2 sep3 plants expressing SEP3 were fully complemented ( Figure 4 , second column) and showed WT petals, stamens and carpels in whorls 2, 3 and 4, respectively, with conical cells, pollen grains and stigmatic papilla, style and replum cells at the appropriate organ surface ( Figure 5 , second column). As previously described, SEP3 Δ tet expression in sep1 sep2 sep3 was able to fully complement petal and stamen formation in whorls 2 and 3, but only partially complemented whorl 4, which exhibited two unfused carpel-like structures and indeterminacy ( Figures 4 and 5 , third column) 30 . In contrast, flowers of plants expressing SEP3 Δ tet3M ( Figures 4 and 5 , fourth column) showed significant defects in whorls 2 and 3 compared to SEP3 Δ tet expressing plants, and no carpel-like structures in whorl 4. In the second whorl, the petaloid organs were much shorter than WT petals and remained green ( Figure 4 ). No stomata cells were visible and conical cells were only occasionally observed by SEM ( Figure 5 ). In the third whorl, only immature greenish stamen could be observed ( Figure 4 ). Small blisters at the organ margin that resemble developing pollen sacs were also noted but no pollen grains were produced ( Figure 5 ). The number of organs in whorls 2 and 3 was not affected in these plants, with four and six organs in the second and third whorls, respectively. These data show that reducing the ability of SEP3 to tetramerize results in increasingly strong defects in floral organs, including incomplete organ differentiation and cell identity, notably in whorls 2 and 3 that were unaffected in the SEP3 Δ tet expressing plants. Download figure Open in new tab Figure 4. The flower and second, third and fourth whorl floral organs in Arabidopsis expressing wild-type and MADS mutants. A-E. Representative whole flowers in, from left to right, sep1 sep2 sep3 , sep1 sep2 sep3 expressing SEP3, SEP3 Δtet or SEP3 Δtet3M and sep1 sep2 sep3 ag-4 expressing SEP3 Δtet as labeled (top). Representative organs of whorl two (F-J) , whorl three (K-O) and whorl four (P-T) for each genotype described above. sep1 sep2 sep3 expressing SEP3 plants are fully complemented and show WT organs . SEP3 Δtet3M expressing plants exhibit strong floral organ phenotypes in the second, third and fourth whorls with immature green organs. The combination of ag-4 and SEP3 Δtet triggers the complete transformation of stamen into petals in the third whorl and indeterminacy in the fourth whorl. Scale bars indicate 500 µm. Download figure Open in new tab Figure 5. Scanning electron microscopy of epidermal cells for second, third and fourth whorl floral organs in Arabidopsis expressing wild-type and MADS mutants. A-E . SEM of adaxial cell surface of whorl 2, left to right, sep1 sep2 sep3, sep1 sep2 sep3 expressing SEP3, SEP3 Δtet or SEP3 Δtet3M and sep1 sep2 sep3 ag-4 expressing SEP3 Δtet as labeled (top). Typical conical petal cells are observed in the SEP3 ( B ) and SEP3 Δtet ( C, E ) expressing lines, but absent in SEP3 Δtet3M expressing lines ( D ). F-J. SEM of adaxial cell surface of whorl 3. Typical pollen grains are only observed in the SEP3 ( G ) and SEP3 Δtet ( H ) expressing lines. The triple mutant expressing SEP3 Δtet3M ( I ) shows incomplete differentiation of the third whorl organs whereas the quadruple mutant expressing SEP3 Δtet shows characteristic conical petal cells ( J ). K-O. SEM of the abaxial cell surface of whorl 4 in plants as in A . The triple mutant expressing SEP3 Δtet3M ( N ) and the quadruple mutant expressing SEP3 Δtet ( O ) exhibit elongated sepaloid cells and no stigmatic cells, whereas the triple mutant expressing SEP3 Δtet exhibits partial complementation ( M ), with two unfused carpel with stigmatic cells present. Scale bars indicate 100 µm except for E (30 µm). In order to further examine the role of tetramerization, the sep1 sep2 sep3 ag-4 expressing SEP3 Δ tet mutant was generated by crossing sep1 sep2 sep3 expressing SEP3 Δ tet and sep1 sep2 ag-4 +/- . Due to the very low number of seeds produced, a single sep1 sep2 sep3 ag-4 plant expressing SEP3 Δ tet was genotyped and analyzed ( Figures 4 and 5 , fifth column). This mutant showed strong floral organ defects specifically in whorls 3 and 4, as would be expected due to both SEP3 Δ tet and AG Δ tet exhibiting impaired tetramerization. In whorl 3, the stamens were replaced by six petaloid organs with conical cells characteristic of petals ( Figures 4 and 5 ). As AG is required for repressing AP1 expression in the third whorl, the lack of AG function due to impaired tetramerization would be predicted to result in petal formation instead of stamens in whorl 3, as shown in the ag loss-of-function mutants 38 . Whorl 4 was not complemented, showing an indeterminate flower consisting of sepaloid structures with characteristic elongated cells, as in sep1 sep2 sep3 plants or sep1 sep2 sep3 plants expressing SEP3 Δ tet3M ( Figures 4 and 5 ). Conversion of stamens to petaloid organs was also observed in 7 plants genotyped sep1 sep2 sep3 +/- ag-4 expressing SEP3 Δ tet (Supplemental Figure S3). Taken together, these data demonstrate that perturbing MTF tetramerization by introducing structure-based mutations in SEP3 or in SEP3 and AG has a strong effect on floral organ differentiation and cell identity in the second, third and fourth whorls, correlating tetramerization defects characterized in vitro with physiological function. Discussion MIKC c MTFs fulfill important roles in plant reproductive development. Evidence from gymnosperms, angiosperms and ancestral reconstructions of the most recent common ancestor of extant seed plants suggests that tetramerization of MTFs is likely widespread, however whether or not tetramerization is required for specifying reproductive organ identity has been less clear 4 , 10 . In mammals and fungi, for example, MTFs regulate different developmental processes via dimer formation, with no higher order MADS oligomerization states accessible or required for DNA-binding or activity 39 , 40 . While the addition of the Keratin-like tetramerization domain occurred early in evolution, with MIKC c MTFs even present in charophyte green algae, defining the physiological role of tetramerization has been challenging due to the difficulties in fully decoupling DNA binding and dimerization/tetramerization 11 , 41 . In addition, in vitro studies of MADS tetramerization mutants have demonstrated robust DNA-binding of MADS homo-and heterodimers, further raising the question of whether or not tetramer formation is indispensable for physiological functions 30 , 31 , 42 . In angiosperms, B and C class organ identity MADS are not able to tetramerize directly based on in vitro and in vivo studies, with tetramerization requiring a SEPALLATA clade member 42 , 43 . Over-expression of A, B and C class MADS genes is not sufficient to confer organ identity, with conversion of leaves to petaloid or stamenoid organs requiring an E class MADS in addition to A, B and C class 22 , 25 . However, SEP clade member also heterodimerize promiscuously with A, B and C MTFs, raising the possibility that SEP-containing MADS heterodimers are the essential complex for specifying organ identity 28 . Recent studies have further demonstrated that functional identity of MTFs is conferred at least in part by the dimerization I domain which helps determine MADS protein-protein interaction and DNA-binding specificity 12 . Combining structure-based mutagenesis, detailed in vitro characterization of oligomerization state, DNA-binding and comparative transgenic studies allows us to more fully determine the role of MADS tetramer formation in floral organ development. By progressively mutating the tetramerization interface and examining the DNA-binding patterns as well as the ability of SEP3 mutants to complement the homeotic conversion of second, third and fourth whorl organs to sepals in the triple sep1 sep2 sep3 mutant, the role of hetero-dimerisation versus heterotetramerisation of MADS organ identity complexes can be addressed. Based on the data presented here, second, third and fourth whorl organ identity requires tetramer formation of MTF complexes. While dimeric MADS complexes are able to strongly bind DNA in vitro and in vivo based on band shift assays, seq-DAP-seq and ChIP-seq experiments, this is not sufficient for proper gene regulation in the context of organ identity specification. A key outstanding question is the underlying molecular mechanism of gene regulation by MADS tetrameric complexes. SEP3/AG wildtype complexes show an enrichment in 36, 46 and 56 base pair intersite spacing due to concurrent two-site binding of DNA by tetrameric complexes, with these distances present in genes important for meristem determinacy. Seq-DAP-seq studies demonstrate the loss of intersite spacing even for the weakly impaired SEP3 Δ tet /AG tetramerization mutant, whose expression in planta led to an indeterminacy phenotype, correlating well with changes in regulation of genes such as KNU but no defects in second or third whorl organ specification and only limited defects in fourth whorl organ identity 30 . Importantly, however, examination of the genome-wide binding by the strong SEP3 Δ tet3M /AG and SEP3 Δ tet3M /AG Δ tet tetramerization mutants in this study demonstrates the reduction in DNA-binding most strongly affects binding sites in regions enriched for specific intersite distances. These regions contain putatively relevant genes involved in organ development including KAN2, JAG and INO . Expression of strong SEP3 and AG tetramerization mutants in planta results in much more pronounced floral organ defects in addition to the indeterminacy phenotype observed for SEP3 Δ tet -expressing plants. This may indicate that the experimental conditions of seq-DAP-seq are underestimating the ability of the SEP3 Δ tet /AG complex to weakly tetramerize or that dimer binding, even to relatively poor binding sites that may require co-operativity in vivo , are detected in seq-DAP-seq, masking changes in binding at loci important for organ identity specification. In addition, an important limitation to seq-DAP-seq experiments is the use of naked DNA to examine binding patterns, thus neglecting the chromatin landscape, which plays a critical role in gene regulation. Recent studies have sought to address the challenge of deciphering the role of chromatin architecture in MTF gene regulation. In vitro and in vivo experiments for AP1 have shown that tetramerization of AP1 strengthens binding to CArG boxes on nucleosomal DNA and tetramer formation may be required for efficient displacement of histones for clustered MADS binding sites. Thus, optimized intersite spacing and nucleosome positioning may both be key to why tetramerization of MTFs is required in vivo for launching floral organ identity programs. Taken together, the structural, in vitro and in vivo experiments presented here demonstrate the critical importance of MADS tetramer formation in floral organ identity in the second, third and fourth whorls, in addition to the previously described importance of tetramerization in floral meristem determinacy 30 . Interestingly, MIKC c MTFs are present in non-seed plants including algae, mosses and ferns which implies that tetramerization may have occurred early in evolution and may be required for gene regulation for all MIKC c MTFs in the green lineage, although this remains to be determined. Further studies examining the role of oligomerization and mechanisms of gene regulation in diverse species by MADS complexes will shed light on how this TF family has evolved central and diverse roles in development from algae to land plants. Materials and Methods Plant material and growth conditions All experiments were performed using Arabidopsis thaliana WT and MADS mutants in the Col-0 background. The ag-4 mutant, originally generated in the Ler background, 44 was back-crossed 5 times in the Col-0 ecotype. The ag - 4 mutant expresses two variants of AG carrying deletion of 12 or 14 amino acids in the tetramerization interface, due to a splicing site mutation 44 . Seedlings were grown in controlled growth chambers in long day conditions (16h light / 8h dark) at 22 ◦ C for plant transformation and phenotype analysis. Plasmid construction for sep1 sep2 sep3 and plant complementation analysis The originally generated sep1 sep2 sep3, containing a T-DNA insertion in SEP1 and an unstable transposon insertion in SEP2 and SEP3 21 , was replaced in this study by a stable mutant generated using CRISPR-Cas9 genome editing to delete portions of the SEP2 and SEP3 genes. To generate a stable null mutation in SEP2, two guide RNA (gRNA) sequences were designed with no off targets using CHOPCHOP 45 . The two gRNA sequences were first cloned in pATU26:U26gRNA vectors and finally inserted into pCAMBIA together with the cassette containing the Cas9 sequence from pBSK:pUBQ10:CoCas9 46 and transformed into Agrobacterium tumefaciens . The generated sep2 mutant carries a deletion of 795 bp starting at +6 in exon 1 and removing the first 16 bp of exon 2, resulting in a frame shift. A similar strategy was followed to generate two sep3 alleles. The first sep3 mutant ( sep3-3 ) carries a 1081 bp deletion removing the last 38 bp of intron 1 up to the first 83 bp of exon 8. The second generated s ep3 mutant, named sep3-4 , carries a deletion of 963 bp starting from +25 in exon 1. Sequences for generating gRNA are presented in Table SII. Sequences of sep2 and sep3 at the site of deletion are provided in Table SIII. The triple sep1 sep2 sep3 mutants were generated by crosses. The newly generated mutants have the same flower phenotype of the previously described triple sep1 sep2 sep3 transposon mutant, with sepaloid organs in all whorls and flower indeterminacy 21 . For the complementation analysis, p SEP3::SEP3 (ABRC stock number CD3-2708) and p SEP3::SEP3 Δtet (ABRC stock number CD3-2709) were used. p SEP3::SEP3 Δtet3M was constructed as described for the above plasmids using PCR amplified specific sequence of SEP3 Δtet3M cloned into pSP64. These three plasmids allow the expression of SEP3 , SEP3 Δtet and SEP3 Δtet3M under the control of the SEP3 promoter and contain the SEP3 regulatory intron 1 sequence cloned between exon 1 and 2, as described previously 30 . The vector backbone, pFP100, allows GFP expression in seeds for selection of transformants 47 . Plant transformation and floral phenotype analysis For the generation of sep1 sep2 sep3 expressing SEP3 , SEP3 Δtet and SEP3 Δtet3M , heterozygous sep1 sep2 sep3-3 +/− plants were transformed with the pSEP3::SEP3 , pSEP3::SEP3 Δtet , and p SEP3:: SEP3 Δtet3M using the floral dip method 48 . Transformants were selected based on the fluorescence of GFP-positive seeds. For the generation of sep1 sep2 sep3 ag-4 expressing SEP3 Δtet , sep1 sep2 was crossed with the ag-4 mutant to generate the sep1 sep2 ag-4 +/- mutant. Pollen from sep1 sep2 sep3-3 plants expressing SEP3 Δtet was used to fertilize sep1 sep2 ag-4 +/- and sep1 sep2 sep3-3 +/- ag-4 +/- plants expressing SEP3 Δtet could be genotyped after crossing. Manual self-fertilization of these plants generated sep1 sep2 sep3-3 ag - 4 (named sep1 sep2 sep3 ag-4 for simplicity) expressing SEP3 Δtet in the next generation. All the primers used for plant genotyping are listed in Table SII. Floral phenotypic analyses were performed by light microscopy on flower numbers 10–19 based on their order of emergence on T1 plants genotyped sep1 sep2 sep3 expressing SEP3 (3 T1), SEP3 Δtet (2 T1) and SEP3 Δtet3M (5 T1), on control untransformed sep1 sep2 sep3 plants, and on sep1 sep2 sep3 ag-4 expressing SEP3 Δtet (1 line) and sep1 sep2 sep3 +/- ag-4 expressing SEP3 Δtet (7 lines). In Figure 4 , black squares were added to mask magnification and scale marks automatically generated by the software and appropriate scale bars were added manually in white for clarity. Environmental scanning electron microscopy Scanning electron microscopy (SEM) experiments were performed at the Electron Microscopy Facility of the Institut de Chimie Moleculaire of Grenoble Nanobio-Chemistry Platform, as previously described 12 . Untreated flowers were directly placed in the microscope chamber. Care was taken to maintain humidity during the pressure decrease in the chamber in order to prevent tissue drying. Secondary electron images were recorded with a Quanta FEG 250 (FEI) microscope while maintaining the tissue at 2 °C, under a pressure of 500 Pa and a 70% relative humidity. The accelerating voltage was 14 kV and the image magnification ranged from 100 to 800Å. Flowers from three independent lines were observed for each genotype. SEP3-AG K domain construct, protein expression and purification The SEP3 K domain corresponding to residues 75-178 was PCR amplified and inserted by Gibson assembly to the NcoI/HindIII linearized pETDuet vector to generate the pETDuet-SEP3 75–178 construct. A Tobacco Etch Virus (TEV) cleavable 6x histidine-maltose binding protein (His-MBP) tag amplified from the pETM-41 vector followed by the region corresponding to AG 90–189 K domain with an additional TEV cleavage site at the C terminus, were inserted into the pETDuet -SEP3 75–178 linearized by NdeI, using Gibson assembly to create the pETDuet SEP3 75–178 /AG 90–189 construct. Primers are listed in Table SII. E. coli BL21 Rosetta 2 (Novagen) were transformed with the pETDuet SEP3 75–178 /AG 90–189 construct and grown either in LB or minimal medium containing selenomethionine as described 49 . Cells were grown at 37 °C to an OD600 of 0.6 – 0.8 after which time the temperature was reduced to 18 °C and protein expression induced by addition of 1 mM of isopropyl-β-D-1- thiogalactoside for 12 h. Cells were harvested by centrifugation and the cell pellet resuspended in lysis buffer, 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM tris(2- carboxyethyl)phosphine (TCEP), supplemented with 1x complete protease inhibitors (Roche). Cells were lysed by sonication and cell debris pelleted at 25,000 rpm for 40 min. The soluble fraction was applied to a 1 ml Ni-NTA column, washed with lysis buffer + 10 mM imidazole and the protein eluted with lysis buffer + 250 mM imidazole. Cleavage of the His-MBP tag was carried out overnight at 4°C during dialysis against Tris-HCl 50 mM pH 7.5, 300 mM NaCl, 1 mM TCEP in the presence of 1:100 (w:w) His-tagged TEV protease. The protein was then passed over a Ni-NTA column to deplete the TEV and any uncleaved protein. SEP3 75–178 /AG 90–189 complex was further purified by gel filtration using a Superdex 200 10/300 column (GE Healthcare). The protein complex was concentrated to 6-8 mg/ml and used for crystallization trials. Protein crystallization, data collection and refinement SEP3 75–178 /AG 90–189 at a concentration of 6-8 mg/ml was mixed at a 1:1 ratio with Tris-HCl 100 mM pH 8 and 2 M sodium formate. The protein crystallized after 3 days at 4 °C forming rectangle shaped single crystals. Seleno-methionine derivatized crystals were obtained after seeding with WT crystals. Glycerol was added to the drop to ∼20% final concentration as cryoprotectant and the crystals were then flash frozen in N 2(l) . Diffraction data were collected at 100 K at the European Synchrotron Radiation Facility, Grenoble, France, on ID23-2 at a wavelength of 0.873 Å. Indexing was performed using MXCube 50 and the default optimized oscillation range and collection parameters used for data collection. All datasets were integrated and scaled using the programs XDS and XSCALE 51 . For seleno-methionine containing crystals, 6 SeMet data sets were collected from three crystals. Data were automatically processed by XDS within the Grenades pipeline 52 and submitted to CODGAS 53 to group isomorphous datasets. This identified two datasets from the same crystal which were merged and analyzed by SIRAS using the CRANK2 54 phasing program. Diffraction images and XDS input files have been deposited at Zenodo (). The partial model from CRANK2 was used for molecular replacement of the native dataset with Phaser 55 . Model building was performed using Coot 56 and all refinements were carried out in Refmac 57 . The structure quality was assessed using MolProbity 58 . Data collection and refinement statistics are given in Table 1 . The structure is deposited under PDB 8CRA. Plasmid construction and EMSA experiments Vectors containing AG (At4g18960.1), SEP3 (At1g24260.2), SEP3 Δtet (At1g24260.3), AP3 (At3g54340) and PI (At5g20240) cDNAs were used as previously described 30 . AP1 (AT1G69120) cDNA was PCR-amplified using specific primers and inserted into XbaI/BamHI digested pSP64 (Promega) vector. Coding sequences for SEP3 Δtet3M and AG Δtet were generated using the QuikChange (Agilent) protocol according to the manufacturer’s instructions and cloned into pSP64 vector as described for AP1 . Primers used to generate the vectors are listed in Table SII. These vectors were used for in vitro protein production using SP6 High-Yield Wheat Germ Protein Expression System (Promega L3260) according to the manufacturer’s instructions. Electrophoretic mobility shift assay (EMSA) were performed as described 30 . The 103-bp DNA probe from the SEP3 promoter 30 containing two CArG box binding sites was labeled with Cy5 (Eurofins). For each EMSA, a negative control was run corresponding to labelled DNA incubated with in vitro transcription translation mix and empty pSP64 vector. Plasmid construction and seq-DAP-seq experiments For seq-DAP-seq experiments, the following C-terminal-tagged constructs were generated using Gibson assembly and PCR amplified: pTnT -SEP3 Δtet3M -3FLAG and pTnT - AG Δtet - 5Myc as described 31 . pTnT -SEP3-3FLAG, pTnT -SEP3 Δtet -3FLAG, pTnT -AG-5Myc are reported previously 31 . Seq-DAP-seq for SEP3 Δtet3M -AG complex and SEP3 Δtet -AG Δtet complex was performed as described previously 12 , 31 . Briefly, 2 μg of each purified plasmid was used as input in a 50 μl TnT (Promega) reaction incubated at 25 °C for 2 h. The reaction solution was then combined with 50 μl IP buffer (PBS supplemented with 0.005% NP40 and proteinase inhibitors (Roche)) and mixed with 20 μl anti-FLAG magnetic beads (Merck Millipore M8823). Following 1 h incubation at room temperature, the anti-FLAG magnetic beads were immobilized, and washed three times with 100 μl IP buffer. TF complexes were eluted with 100 μl IP buffer supplemented with 200 μg/ml 3xFLAG peptide (Merck Millipore F4799). The eluted protein was then immobilized on anti-c-Myc magnetic beads (Thermo Fisher 88843) and washed three times with 100 μl IP buffer to isolate homogeneous SEP3 Δtet3M -AG or SEP3 Δtet -AG Δtet complexes. The purified protein complexes, while still bound on anti-c-Myc magnetic beads, were incubated with 50 ng DAP-seq input library pre-ligated with Illumina adaptor sequences. The reaction was incubated for 90 min, and then washed six times using 100 μl IP buffer. The bound DNA was heated to 98 °C for 10 min and eluted in 30 μl EB buffer (10 mM Tris-Cl, pH 8.5). The eluted DNA fragments were PCR amplified using Illumina TruSeq primers for 20 cycles, and purified by AMPure XP beads (Beckman). The libraries were quantified by qPCR, pooled and sequenced on Illumina HiSeq (Genewiz) with specification of pairedend sequencing of 150 cycles. Each library obtained 10–20 million reads. The seq-DAP-seq was performed in triplicate. Seq-DAP-seq data analysis For each seq-DAP-seq samples, reads were checked using FastQC 59 and adaptor sequences removed with NGmerge 60 and mapped with bowtie 61 onto the TAIR10 version of the A. thaliana genome ( https://www.arabidopsis.org ), devoid of the mitochondrial and the chloroplast genomes. The duplicated reads were removed using the samtools rmdup program 62 .The resulting alignment files were used to derive the binding intensity of each complex at 6347 regions bound by the SEP3/AG complex 31 . The binding intensity of a given complex at bound regions was computed as the normalized reads coverage, averaged across replicates, and expressed as reads per kilobase per million mapped reads (RPKM). To limit the bias due to differences in the signal-to-noise ratio between seq-DAP-seq samples (Table SIV), the per-million scaling factor was done with the total number of reads mapped in peaks instead of all mapped reads. We made this choice over the classical normalization with all mapped reads because normalizing by total mapped reads flattens the signal for SEP3 Δtet /AG and SEP3 Δtet3M /AG (samples for these two conditions have the lowest fraction of reads in peaks (FRiP) values, Table SIII) 12 , 63 . This artificially makes SEP3 Δtet /AG Δtet more similar to SEP3-AG. This choice assumes that differences in FRiP values are due to differential DAP-seq efficiency. The coverage fold reduction (CFR) was computed as the ratio between the mean normalized coverage of a complex relative to that of another complex. A SEP3/AG position weight matrix was used to search CArG boxes in the 6,367 bound sequences and subsequences with score > −9 were retained. This was used to separate regions harboring a preferred spacing from regions with no preferred spacing in figure 3B . Chip-seq experiments and data analysis sep1 sep2 sep3-4 lines expressing either wildtype SEP3 or the tetramerization deficient, SEP3 Δtet were used to conduct chromatin immunoprecipitation experiments according to previously published protocols 64 . Briefly, 1 g inflorescence (flower stage 1-12) were collected from 4–5-week-old plants. The tissue was fixed for 30 min and the immunoprecipitation performed using a SEP3-specific antibody followed by library preparation using ThruPLEX DNA-Seq Kit (Takara) and deep sequencing 65 , 66 . Experiments were done with two biological replicates and the control sample was generated using pre-immune serum. The two lines were grown in parallel and genotyped (see primer Table SII) prior to sample collection. For each ChIP-seq data, reads were checked as described in the DAP-Seq data analysis section. Peaks were identified using MACS2 67 and merged using MSPC 68 , resulting in 4,369 unique regions. The binding intensity of a given complex at bound regions was computed as the normalized reads coverage, averaged across replicates, and expressed as reads per kilo per million (RPKM). RNA-seq experiments and data analysis Total RNA were extracted from two independent lines for sep1 sep2 sep3 expressing SEP3 and three independent lines for sep1 sep2 sep3 expressing SEP3 Δtet , and in duplicate from sep1 sep2 and sep1 sep2 sep3 lines, with lines as described 30 . All the plants were grown in parallel. Quality of the total RNA was validated by their 260 / 280 absorbance ratio and the integrity of the ribosomal RNA by agarose gel. RNA libraries construction and sequencing were performed by GENEWIZ (USA) using Illumina HiSeq and 2 °ø 150bp configuration as described 31 . Between 25 and 35 million reads were obtained for each library. Mapping onto the Arabidopsis genome (TAIR10), read count per gene and statistical analysis were done using STAR (no multimapping, mismatch number < 10), FeatureCount (default parameters) and EdgeR (default parameters), respectively, available in the Galaxy platform 69 , 70 . Genes were considered differentially expressed (DE) between two genotypes when the log FC was > 1 or < − 1 and the FDR value < 0.05. DE genes were determined for sep1 sep2 sep3 expressing SEP3 Δtet versus sep1 sep2 sep3 and sep1 sep2 sep3 expressing SEP3 vs sep1 sep2 sep3 expressing SEP3 Δtet . DE genes were previously determined for sep1 sep2 sep3 vs sep1sep2 and sep1 sep2 sep3 expressing SEP3 vs sep1 sep2 sep3 31 . Data availability Crystallographic data have been deposited with the PDB under the code 8CRA. RNA-seq, ChIP–seq and seq-Dap-Seq datasets have been deposited in the GEO database and can be download with the following tokens: ylszyocitlabxcx (RNA-seq), mrcricesndczrod (ChIP-seq) and clwncugebvmlrud (seq-DAP-seq). Funding This project received support from the Agence National de la Recherche (ANR-16-CE92- 0023) and GRAL, a program from the Chemistry and Biology Health Graduate School of the University Grenoble Alpes (ANR-17-EURE-0003), with a thesis fellowship to AJ. The X-ray diffraction experiments were performed on beamline ID23-2 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02). The research leading to these results has received funding from the European Community’s Seventh Framework Programme H2020 under iNEXT Discovery (project number 871037). Author contributions V.H. and C.Z. conceived the study. V.H, C.Z and K.K. designed experiments. V.H., X.L., M.P., A.J., A.G., X.X., W.Y. performed the experiments. C.Z. and M.N. solved the 3D structure. R.B.-M, J.L., K.K. and F.P. analyzed the genome wide data. C.Z. and V.H. wrote the manuscript with the help of all authors. Acknowledgment: We thank Franck Wellmer (Smurfit Institute of Genetics, Dublin, Ireland) for providing the original ag - 4 mutant. The authors thank the NanoBio-ICMG Platform (UAR 2607, Grenoble) for granting access to the Electron Microscopy facility. Footnotes Material distribution: The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors ( https://academic.oup.com/plcell/pages/General-Instructions ) are: Véronique Hugouvieux ( veronique.hugouvieux{at}cea.fr ) and Chloe Zubieta ( chloe.zubieta{at}cea.fr ). 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Share SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development Veronique Hugouvieux , Romain Blanc-Mathieu , Michel Paul , Aline Janeau , Xiaocai Xu , Jeremy Lucas , Xuelei Lai , Antonin Galien , Wenhao Yan , Max Nanao , Kerstin Kaufmann , François Parcy , Chloe Zubieta bioRxiv 2023.05.23.541941; doi: https://doi.org/10.1101/2023.05.23.541941 Share This Article: Copy Citation Tools SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development Veronique Hugouvieux , Romain Blanc-Mathieu , Michel Paul , Aline Janeau , Xiaocai Xu , Jeremy Lucas , Xuelei Lai , Antonin Galien , Wenhao Yan , Max Nanao , Kerstin Kaufmann , François Parcy , Chloe Zubieta bioRxiv 2023.05.23.541941; doi: https://doi.org/10.1101/2023.05.23.541941 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7975) Biochemistry (18653) Bioengineering (14781) Bioinformatics (44203) Biophysics (22484) Cancer Biology (19609) Cell Biology (26767) Clinical Trials (138) Developmental Biology (13907) Ecology (20900) Epidemiology (2067) Evolutionary Biology (25338) Genetics (16108) Genomics (23413) Immunology (18625) Microbiology (42282) Molecular Biology (17958) Neuroscience (92997) Paleontology (694) Pathology (2973) Pharmacology and Toxicology (5068) Physiology (8074) Plant Biology (15917) Scientific Communication and Education (2093) Synthetic Biology (4538) Systems Biology (10192) Zoology (2376) window.__CF$cv$params={r:'a3892fda1c9373d5',t:'MTc4ODk4ODMwMg==',u:'01a0880330937a719d3ea6768620bbc2',ut:'UvN.Q..yQ.7Mzl99Q2fqS.mqNFiCyHP5MBmHwqY1WnY-1788988305-1.2.1.1-YLrxoCM.OAm9hB1noaelu45fPoHEGp.ragyHeUejEbxv4ruB.5jmwfXi5Mko4x4Xjzd2dw99YE.ePtBu_HBSEH4P7G4JpwJ1qddSjqhzjpA',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();