A PLETHORA transcription factor shapes cucumber shoot architecture

A PLETHORA transcription factor shapes cucumber shoot architecture | 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 A PLETHORA transcription factor shapes cucumber shoot architecture View ORCID Profile Merijn Kerstens , Florian Müller , Kelvin Adema , Olga Kulikova , Magdalena Lastdrager , Ben Scheres , View ORCID Profile Viola Willemsen doi: https://doi.org/10.1101/2025.09.18.677013 Merijn Kerstens 1 Cell and Developmental Biology, Wageningen University and Research , Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Merijn Kerstens For correspondence: merijn.kerstens{at}wur.nl viola.willemsen{at}wur.nl Florian Müller 2 Rijk Zwaan Breeding BV , de Lier, Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kelvin Adema 1 Cell and Developmental Biology, Wageningen University and Research , Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Olga Kulikova 3 Molecular Biology, Wageningen University and Research , Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Magdalena Lastdrager 2 Rijk Zwaan Breeding BV , de Lier, Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ben Scheres 1 Cell and Developmental Biology, Wageningen University and Research , Netherlands 2 Rijk Zwaan Breeding BV , de Lier, Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Viola Willemsen 1 Cell and Developmental Biology, Wageningen University and Research , Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Viola Willemsen For correspondence: merijn.kerstens{at}wur.nl viola.willemsen{at}wur.nl Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary PLETHORA transcription factors (PLTs) are master regulators of plant development. Loss of shoot meristematic PLTs leads to reduced phyllotactic regularity and robustness in Arabidopsis and increased inflorescence branching in tomato. Whether these factors have similar functions in other species is not known. To address this knowledge gap, we integrated phylogenetic, transcriptomic, and genome-wide in vitro binding strategies with quantitative shoot architecture phenotyping of a panel of cucumber TILLING mutants of each CsPLT homolog. We determined that CsPLT3/7 and CsPLT are expressed in complementary domains in the cucumber shoot apex. DAP-seq data indicated that both transcription factors recognised an ANT-like consensus motif and bound to SAM organising genes. We identified strong phenotypic defects in multiple Csplt3/7 mutants. In mature seeds, Csplt3/7 mutants formed a flat apex instead of a shoot apical meristem, from which secondary meristems emerged during subsequent seedling development. Mature Csplt3/7 shoots exhibited defects in internode length regularity, stem architecture, flower morphology, and axillary organ initiation. Moreover, phyllotactic patterns were slightly shifted from a spiral towards a distichous orientation. We present one of the first pieces of evidence that the PLT3/7 clade fulfils both conserved and diversified roles in shoot development across taxonomic family boundaries. Introduction In plants, the shaping, spacing and orientation of aboveground tissues collectively establish a certain shoot architecture. Shoot growth is a modular process, in which nodes harbouring lateral organs such as leaves, flowers and axillary shoots are separated by organ-free internodes ( Wang et al ., 2018 ), the total product of which determines optimal cultivation strategies in agriculture. For instance, the economically relevant species cucumber ( Cucumis sativus ) grows long, vining stems that in modern high-wire cultivation methods are guided upwards along strings. Cucumber plants form one leaf per node, while branches, tendrils and flowers emerge from the leaf axils, thus combining reproductive and vegetative growth in a single shoot axis ( Liu et al ., 2021 ). Cucumber leaves are formed at the shoot apical meristem (SAM), a dome-shaped structure from which primordia emerge rhythmically. SAM anatomy and primordium initiation have been studied extensively in Arabidopsis ( Arabidopsis thaliana ). In this species, the vegetative SAM and inflorescence meristem (IM) domes can be subdivided into distinct domains. At the dome summit, the central zone (CZ) contains slowly dividing stem cells whose progeny move outward to the peripheral zone (PZ), gradually differentiating to allow production of new organs ( Reddy et al ., 2004 ). Spatiotemporal integration of auxin signalling is a major factor in specifying the sites at which such organ primordia can be formed, giving rise to highly non-random phyllotactic patterns ( Reinhardt et al ., 2003 ; Heisler et al ., 2005 ; Smith et al ., 2006 ; de Reuille et al ., 2006 ; Jönsson et al ., 2006 ; Galvan-Ampudia et al ., 2020 ). Stem-cell identity of the CZ is maintained by the underlying organizing centre (OC), a group of cells that prevents cell differentiation through a negative feedback loop between the CZ-secreted CLAVATA3 (CLV3) peptide and the OC-produced mobile WUSCHEL (WUS) transcription factor ( Laux et al ., 1996 ; Mayer et al ., 1998 ; Schoof et al ., 2000 ; Brand et al ., 2000 ; Müller et al ., 2006 ). Whereas loss of CLV3 or its receptor CLV1 leads to increased meristem size and fasciation ( Clark et al ., 1993 , 1995 ), wus mutants fail to maintain the stem cell pool in the CZ ( Laux et al ., 1996 ), highlighting the dynamic interplay of the CLV3-WUS module in shaping SAM function. In cucumber, it was shown that the orthologs of WUS and CLV3 antagonistically control carpel numbers, but they are both expressed exclusively in the zone underneath the stem cells ( Che et al ., 2020 ). This hints that maintenance of cucumber stem cells might be differently regulated compared to Arabidopsis. PLETHORA (PLT) transcription factors (TFs) of the euAINTEGUMENTA (euANT) family are master regulators of plant development. PLTs are conserved across angiosperms and can be phylogenetically separated into four homology groups, i.e. PLT1/2, PLT3/7, BABY BOOM (BBM)/PLT4 and PLT5, named according to the grouping of the Arabidopsis homologs ( Kerstens et al ., 2020 ). In this species, PLTs are thought to confer meristematic potential to critical developmental processes, including (lateral) root development, embryogenesis and regeneration, promoting inception and maintenance of growth apices ( Aida et al ., 2004 ; Galinha et al ., 2007 ; Kareem et al ., 2015 ; Du & Scheres, 2017 ; Kerstens et al ., 2024 ). In the SAM and IM, PLT3, PLT5 and PLT7 are expressed in partially overlapping domains and promote robustness of the phyllotactic spiral ( Prasad et al ., 2011 ; Pinon et al ., 2013 ; Kerstens et al ., 2025a ). Meristems devoid of all three PLTs remain functional, but exhibit a delayed convergence from a decussate configuration to the golden angle (137.5°) in the rosette. Additionally, plt3 plt5 plt7 mutants display rare phyllotactic shifts to 180° and 90° angles in the IM, which was linked to reduced auxin and cytokinin signalling, and transport ( Prasad et al ., 2011 ; Pinon et al ., 2013 ; Kerstens et al ., 2025a ). In tomato ( Solanum lycopersicum ), SlPLT3 and SlPLT7 control branching of the inflorescence, suggesting that PLT3/7 TFs are involved in shoot development across eudicots, but regulate different processes ( Zebell et al ., 2025 ). Outside of Arabidopsis and tomato, however, no role for PLTs in shaping shoot architecture have been described. In this study, we investigated whether PLTs are also involved in shoot development in cucumber. We uncovered that only two of the four cucumber PLT s ( CsPLT ) are expressed in the SAM, of which only CsPLT3/7 localises to the stem cell niche. Plants containing a loss-of-function Csplt3/7 TILLING allele lack SAMs in young seedlings, and the combinatorial loss of CsPLT1/2 and CsPLT3/7 proved embryonic lethal. Although spontaneous SAM emergence occurred at the shoot apex in older Csplt3/7 seedlings, mature shoots exhibited extreme internode lengths, axillary organ outgrowth defects, abnormal flower configurations and disturbed phyllotaxis. We thus establish CsPLT3/7 as a key developmental TF in shaping cucumber shoot architecture and, more generally, demonstrate shared and distinct developmental roles for PLTs across species boundaries. Results The cucumber genome contains four PLT homologs expressed in root and shoot apices In order to identify shoot developmental roles of PLTs in cucumber, we first generated a phylogenetic tree of the euANT TF family within 13 species of the cucurbit lineage. In contrast to Arabidopsis, which contains six PLT homologs divided over four gene clades ( Kerstens et al ., 2020 ), cucumber and its close relatives harboured only four PLT homologs, one in each clade ( Fig. 1a,b ). Per-clade PLT copy number was generally increased to two in the genus Cucurbita , which aligns with the predicted whole-genome duplication event in the tribe Cucurbitae ( Sun et al ., 2017 ; Montero-Pau et al ., 2018 ; Barrera-Redondo et al ., 2019 ). Download figure Open in new tab Figure 1. CsPLTs are expressed in root and shoot apices. ( a ) Maximum-likelihood phylogenetic tree of euANT transcription factors with >1 AP2 domain in 13 cucurbit species, Arabidopsis and tomato. The tree is rooted at the split between the ANT/AIL1 (collapsed) and PLT clades. Black circles correspond to bootstrap values > 70. Cucumber homologs are indicated with *. ( b ) Phylogeny and whole-genome duplication events (stars, ( Ma et al ., 2022 )) in the analysed cucurbit species. Copy numbers per PLT clade per species are indicated in grey (light = 1, dark = 2). Squares with red borders indicate that the count includes one homolog with < 2 AP2 domains, inferred from synteny with the cucumber homolog ( Yu et al ., 2023 ), possibly because of wrong annotation. ( c ) Schematic drawing of a 6 dpg cucumber seedling. sam = shoot apical meristem, co = cotyledon, hy = hypocotyl, ram = root apical meristem. ( d ) Heatmap of cucumber seedling CsPLT expression in tissues demarcated by dashed rectangles in ( c ). Intensity corresponds to the log 2 expression value (+ 1e-5). Subsequently, we aimed to study expression patterns of these four cucumber PLTs (CsPLT). We germinated seeds of Chinese long inbred line ‘9930’, a wild type monoecious cucumber with a well-annotated genome ( Li et al ., 2019 ), and dissected various tissues at 6 days post germination (dpg). Like in Arabidopsis, CsPLTs were most strongly expressed in meristematic tissues ( Fig. 1c,d ). Whereas we detected expression of all four genes in the root tip, only CsPLT3/7 and CsPLT5 were expressed in shoot tissues ( Fig. 1d ). Transcript levels were particularly elevated in the shoot apex, containing both the shoot apical meristem and early leaf primordia. We thus inferred that only CsPLT3/7 and CsPLT5 were potentially involved in cucumber shoot development. CsPLT3/7 and CsPLT5 are expressed in partially overlapping domains To increase the resolution of transcript localisation, we performed in situ hybridisations on longitudinal sections of 5 dpg ‘9930’ shoot apices with CsPLT3/7 and CsPLT5 probe sets that form a highly specific red/pink precipitate emitting fluorescence upon transcript detection. Compared to the no probe control, CsPLT3/7 mRNA localised primarily to the summit of the SAM dome (i.e. the CZ, containing the stem cell niche), the axillary meristems, and weakly in developing leaf primordia ( Fig. 2a,b,d,e ). On the other hand, CsPLT5 was expressed strongly in leaf primordia and moderately throughout the SAM, but not clearly in the CsPLT3/7 domain ( Fig. 2a,c,d,f ). To verify this observation, we made cross sections through the stem cell niche and again observed that CsPLT5 was not expressed in this region, unlike CsPLT3/7 ( Fig. 2g-l ). Thus, CsPLT3/7 and CsPLT5 are expressed in partially overlapping expression domains and likely influence distinct processes in the SAM and leaf primordia. Download figure Open in new tab Figure 2. CsPLT3/7 and CsPLT5 are expressed the cucumber SAM. ( a-f ) Longitudinal sections of 5 dpg cucumber SAMs after in situ hybridisation with CsPLT3/7 or CsPLT5 probe sets, with a no-probe control. The brightfield images ( a-c ) correspond to the equal-exposure TRITC fluorescent images in ( d-f ). Cross sections are displayed in ( g-l ). Sections are counterstained with Gill’s Hematoxylin I. L = leaf primordium, ax = axillary meristem. Scale bars are 100 μm. CsPLT3/7 and CsPLT5 bind to SAM patterning genes To gain more insight in the function of CsPLT3/7 and CsPLT5, we performed DNA Affinity Purification sequencing (DAP-seq) of genomic DNA from cucumber shoot apices. We identified 3937 and 1947 regions bound by CsPLT3/7 and CsPLT5, respectively, with strong congruence between both datasets; 1599 of 1947 (82%) CsPLT5 peak coordinates overlapped for at least 50% with a CsPLT3/7 peak ( Fig. 3a , Table S1). Across two replicates, the CsPLT5 DAP seemed to have a slightly lower signal-to-noise ratio than CsPLT3/7, reflected by its smaller number of significant peaks and lower FRiP score (Table S2). Motif analysis of the summit sequences revealed that both TFs recognize a centrally enriched ANT-like consensus motif, which is very similar to that of Arabidopsis PLT3, PLT5 and PLT7 ( Fig. 3b,c ). The high similarity of the binding motifs, together with the extensively shared DAP peaks, suggests that CsPLTs bind the same target sequences, as is the case in Arabidopsis ( Santuari et al ., 2016 ; Kerstens et al ., 2025a ). Download figure Open in new tab Figure 3. CsPLTs bind SAM-expressed genes in vitro . ( a ) CsPLT3/7 and CsPLT5 DAP-seq peaks identified from two replicates. Overlapping peaks are CsPLT5 peaks shared for at least 50% (of the CsPLT5 peak) with CsPLT3/7. ( b ) De novo motif discovery of CsPLT3/7 and CsPLT5, in comparison to the motifs of AtPLT3, AtPLT5 and AtPLT7 generated from inflorescence meristem DNA in Arabidopsis ( Kerstens et al ., 2025a ). ( c ) Central enrichment of the motifs in (b) as calculated by CentriMo. ( d ) Distance of CsPLT DAP peaks from the nearest TSS. The red dashed line represents the by chance expected distribution of distances to the nearest TSS based on 10000 randomly selected genomic regions. ( e ) Coverage tracks of CsPLT3/7 (top) and CsPLT5 (bottom) over annotated SAM organising genes. CsSTM2 is one of two AtSTM co-orthologs. * Denote significant peaks (IDR < 0.05). We then identified CsPLT-regulated candidate genes by attributing peaks to the nearest transcription start site (TSS). Both CsPLTs showed enriched binding near TSSs over a simulated background, primarily within 5 kbp from the TSS and peaking around the central coordinate ( Fig. 3d , Table S1). To narrow down relevant target genes, we considered only those genes bound by either CsPLT within a [-5 kbp, 5 kbp] range from the TSS. Additionally, we performed RNA-seq on cucumber shoot apices to select for candidate genes expressed within the SAM (Table S3). Within the curated list of 1446 candidate target genes (Table S4), we found the cucumber orthologs of WUS ( CsaV3_6G047050 ), the Class I KNOTTED-LIKE FROM ARABIDOPSIS THALIANA ( KNAT ) genes SHOOT MERISTEMLESS ( STM ; CsaV3_7G003300 ) and KNAT2/KNAT6 ( CsaV3_2G024800 ), CUP-SHAPED COTYLEDON1/2 ( CUC1/CUC2; CsaV3_4G033430 ), BARELY ANY MERISTEM1/2 ( BAM1/BAM2; CsaV3_6G044530 ), and SCARECROW ( SCR; CsaV3_4G013520 ), all of which are (redundantly) required for SAM formation, maintenance or size in Arabidopsis ( Fig. 3e ; ( Barton & Poethig, 1993 ; Laux et al ., 1996 ; Aida et al ., 1997 ; DeYoung et al ., 2006 ; Belles-Boix et al ., 2006 ; Bahafid et al ., 2023 )). Additionally, a peak close to the CsPLT5 TSS was observed, indicating the potential existence of an autoregulatory feedback loop ( Fig. 3e ). SAM PLTs thus bind to - and potentially regulate - key meristematic organizing genes in cucumber. CsPLT1/2 and CsPLT3/7 are required during embryogenesis We then set out to determine the functional role of CsPLTs in shoot architecture. Using EMS mutagenesis and TILLING, we generated a library of Csplt mutant alleles in a gynoecious parental line (Fig. S1a; Table S5). For CsPLT1/2 , we obtained two premature stop codon alleles at tryptophan 52 and glutamine 58, which lack both AP2 DNA-binding domains and are thus predicted to be loss-of-function alleles. For CsPLT3/7 , we obtained the two premature stop codon alleles Csplt3/7 W48* and Csplt3/7 Q156* , both lacking the AP2 domains, and Csplt3/7 R355H , harbouring an arginine to histidine amino acid substitution within the second AP2 domain. We also obtained the Csbbm W317* , Csplt5 R289* and Csplt5 L189F alleles, in which the proteins are truncated at the end of the first or second AP2 domain or harbouring a leucine to phenylalanine substitution in the first AP2 domain, respectively. During propagation of the TILLING mutants, we obtained lower frequencies of mutants than expected, suggesting a role for CsPLTs during embryogenesis. Indeed, homozygous Cs plt3/7 W48* and Csplt3/7 Q156* mutants occurred at significantly lower frequencies in the segregating offspring from a selfed heterozygous plant than heterozygous or wild type genotypes (Fig. S1b), which we did not observe for any of the other genotypes in this study. To uncover whether specific combinations of Csplt alleles are fully embryonic lethal, we generated various Csplt double mutant combinations through crossing. Segregation of single Csplt alleles in a wild type background in this experiment again demonstrated that only Csplt3/7 Q156* mutants were underrepresented in the progeny ( Fig. 4a ). Strikingly, we did not find a single homozygous Csplt3/7 Q156* seedling in 337 plants with a homozygous Csplt1/2 W52* mutation, nor a single homozygous Csplt1/2 W52* seedling in 139 plants homozygous for Csplt3/7 Q156* ( Fig. 4b ). Moreover, out of all double mutants, only the frequencies of Csplt3/7 Q156* in the Csplt1/2 W52* background and vice versa were significantly reduced in comparison to the respective single mutant allele frequencies ( Fig. 4b ). The occurrence of the homozygous Csbbm W317* genotype in the Csplt3/7 Q156* background and vice versa also seemed to be slightly underrepresented, albeit not significantly ( Fig. 4b ). Corroborating these findings, we found that expression of GUS from the CsPLT1/2 and CsPLT3/7 promoters in Arabidopsis occurred during early embryogenesis (Fig. S2). We conclude that the combined loss of CsPLT1/2 and CsPLT3/7 is embryonic lethal. Download figure Open in new tab Figure 4. Csplt1/2 W52* Csplt3/7 Q156* double mutants are embryonic lethal. ( a ) Pie charts displaying the proportion of homozygous mutants (red) and heterozygous + wild type seedlings (green) per analysed TILLING allele, with counts indicated. The dashed line indicates the expected frequency of 0.25 based on Mendelian segregation. ( b ) Heat map matrix showing homozygous Csplt genotype frequency in different homozygous Csplt backgrounds, relative to the frequency of the respective single mutant genotype in ( a ). For example, the upper left square is the occurrence frequency of the homozygous Csplt5 R289* genotype in a homozygous Csplt1/2 W52* background, relative to the frequency of the homozygous Csplt5 R289* single mutant. Self-comparisons are plotted in white. Statistics are Benjamini-Hochberg (BH) and Yates(Y)-corrected z-tests (freq. < 0.25 ( a ) or freq. < freq(homozygous single mutant) ( b ); ** p = 0.003; *** p = 3.8e-4). Csplt3/7 mutants lack a functional SAM at germination Given the role of CsPLT3/7 during embryogenesis and its expression in the stem cell niche, we next asked if shoot development in young seedlings with a loss-of-function Csplt3/7 allele (i.e. Csplt3/7 Q156* ) was affected. In mature embryos dissected from wild type seeds, a single leaf primordium was visible ( Fig. 5a ). At 6 dpg, this primordium grew into a distinct first true leaf (L1), which after further dissection revealed a much smaller second leaf (L2) and recently initiated primordium (L3), with a dome-shaped SAM at the centre ( Fig. 5b-d ). Contrarily, Csplt3/7 embryos already had two primordia before germination, which developed into two equally sized leaves oriented at 180° from each other at 6 dpg ( Fig. 5e-g ). Strikingly, the two primordia were separated by a flat apex bearing no resemblance to a SAM ( Fig. 5h ). Microscopic analysis revealed the presence of trichomes across the entirety of the flat apex - a hallmark of differentiation, which in wild types occurred only in the tissue surrounding the SAM ( Fig. 5i,j ). In 15 dpg Csplt3/7 seedlings, numerous leaf-like structures emerged from the from the apex, at which point wild type plants possessed one large and one small leaf ( Fig. 5o-n ). This indicates that the Csplt3/7 apex retains meristematic properties despite the initial absence of a functional SAM. The Csplt3/7 leaves displayed roughly synchronous development at later time points, eventually establishing new functional secondary meristems at leaf junctions in 29 of 30 plants at 45 dpg ( Fig. 5p,q ). We conclude that CsPLT3/7 is required for embryonic SAM establishment or maintenance during the formation of the first leaf primordia. Download figure Open in new tab Figure 5. CsPLT3/7 regulates leaf initiation in seedlings. ( a ) 4 hours past imbibition WT embryo, ( b ) 6 dpg seedling with top-view inset ( b ), shoot apex closeup side view ( c ) and top view ( d ) compared to same-age Csplt3/7 Q156* ( e - h ). Arrowheads in point to initiated leaves. The dotted lines indicate the site where a SAM would have been expected. ( i,j ) Maximum projections of shoot apices, with leaves (scars) indicated in yellow. The SAM is coloured green. Cell wall staining is SR2200. ( k - n ) 15 dpg seedlings, with a closeup ( o) showing de novo leaf formation in Csplt3/7 Q156* (arrowheads). ( p ) 22 dpg Csplt3/7 Q156* plant with newly formed leaves (arrowheads) and appearance of a new SAM at 45 dpg ( q , arrowhead). Abbreviations: h = hypocotyl, c = cotyledon, c’ = resected cotyledon, Lx = leaf (order of initiation), * = SAM. Scale bars are 200 μm in ( a - h ), 100 μm in ( i , j ), 75 μm in ( o ) and 1 cm in ( k - n , p, q ). CsPLT3/7 shapes mature shoot architecture We then grew all obtained TILLING mutant lines, including Csplt3/7 Q156* , in the greenhouse in order to evaluate shoot architecture traits in mature plants. At 6 weeks after planting, homozygous Csplt1/2 W52* , Csplt1/2 Q58* and Csbbm W317* mutants did not display shoot phenotypes and had a similar internode length compared to wild type plants, in line with absence of expression in the SAM (Fig. S3). Likewise, Csplt5 L189F and Csplt5 R289* mutants exhibited no conspicuous phenotypes, although internodes in the latter line were slightly (4%; 9.3 vs. 9.7 cm) shorter than the wild type (Fig. S3). To the contrary, the three Csplt3/7 mutants each exhibited clear defects in shoot development. In all three lines we observed aberrant internode length, ranging from near absence to lengths up to 20 cm, in contrast to highly regular lengths between 5 and 10 cm in wild type plants ( Fig. 6a-g ). Occasionally, Csplt3/7 Q156* plants formed temporary stretches of extremely short internodes succeeded by extremely long ones, resulting in pairs of leaves separated by long stem sections ( Fig. 6c ). Since these stem arrangements occurred spontaneously amidst medium-sized internodes, we wondered if certain internode sizes occurred preferentially in succession. We therefore used a Markov chain framework to determine the probabilistic transitions between three internode length classes in Csplt3/7 Q156* , i.e. short, medium-length, and long internodes. Whereas medium-length internodes - constituting the majority of measured internodes (Fig. S4a) - were most frequently (81%) succeeded by medium-length internodes, short internodes were preferentially (62%) followed by long internodes, and long internodes were succeeded equally likely (48% each) by short internodes or medium-sized internodes (Fig. S4b). Short internodes occurred sporadically (19%) in succession, but this only happened very rarely (3%) for long internodes (Fig. S4). These data suggest that after an aberrant internode length is generated, subsequent internodes generally remain extreme in size. Download figure Open in new tab Figure 6. CsPLT3/7 regulates shoot architecture and flower development. ( a ) Gene model displaying Csplt3/7 alleles. AP2 domains are indicated in grey. ( b - c ) 6-week-old WT or Csplt3/7 Q156* shoots, after removal of lower axillary shoots and tendrils. Arrowheads point towards nodes. ( d - f ) Near-co-initiated nodes of Csplt3/7 W48* , Csplt3/7 Q156* and Csplt3/7 R355H . ( g ) Internode length of the three alleles, with each dot representing individual internodes. Note that fasciated regions of Csplt3/7 W48* were not counted. ( h - j ) Axil morphology of WT, Csplt3/7 W48* and Csplt3/7 Q156* plants. Arrowheads point to initiated flowers from the callus-like tissue. ( k ) Older axil of Csplt3/7 Q156* showing extreme flower production and two male flowers. ( l ) Fasciation in Csplt3/7 W48* and a cross section through the stem ( m ). ( n ) Shoot height of WT and Csplt3/7 mutants. Statistics are two-tailed Wilcoxon rank-sum tests (left to right: p = 4.1e-4, p = 0.03, p = 0.26). ( o - r ) Stem cross sections of WT and Csplt3/7 lines. * and ** denote outer and inner vascular bundles, respectively. ( s ) Average leaf formation per week of WT and Csplt3/7 mutants. Statistics are two-tailed Wilcoxon rank-sum tests (left to right: p = 0.03, p = 0.71, p = 0.03). ( t - w ) Flower morphology of WT and Csplt3/7 lines. Scale bars in ( b - f , h - l , t - w ) are 1 cm, and 0.1 mm in ( m , o - r ). Abbreviations: l = leaf, ax = axillary shoot, f = flower, t = tendril. We observed that in the two presumed loss-of-function mutants, Csplt3/7 W48* and Csplt3/7 Q156* , callus-like growths consistently emerged from the axils, from which large numbers of female and sometimes male flowers emerged later, despite the background line being gynoecious and normally producing only 1 female flower per node ( Fig. 6h-k ). While axillary shoots could still form, their emergence appeared inconsistent ( Fig. 6h-j ). In wild type plants, the first axillary shoots generally developed from the axils of the 8 th to 10 th node (from the shoot apex) and by the 14 th node were consistently present (Fig. S5). Conversely, in same-age Csplt3/7 Q156* plants, axillary shoots were routinely absent and were formed infrequently (Fig. S5). Such shoots also exhibited the internode phenotype of the main shoot (Fig. S6). Tendril development appeared unaffected. We also observed that Csplt3/7 W48* stems often (10/17) underwent fasciation during growth ( Fig. 6l,m ), which never occurred in Csplt3/7 Q156* and Csplt3/7 R355H . Furthermore, shoot height of the loss-of-function mutants was reduced ( Fig. 6n ), and leaf production - a proxy for plastochron - was increased in the fasciating Csplt3/7 W48* line, but marginally decreased in Csplt3/7 R355H ( Fig. 6s ). Since Csplt3/7 W48* mutants experienced fasciation, we wondered whether stem architecture of Csplt3/7 Q156* and Csplt3/7 R355H was more subtly affected. In cross sections of internodes of Csplt3/7 Q156* and Csplt3/7 R355H mutants, as well as in non-fasciating Csplt3/7 W48* stems, supernumerary ridges and vascular bundles were present compared to 5-ridged wild type stems with 4 inner and 5 outer vascular bundles ( Fig. 6o-r ). Finally, flower morphology of the two presumed loss-of-function mutants was disturbed, exhibiting alternative sepal/petal numbers and shape ( Fig. 6t-w ). We measured this phenotype in more detail in Csplt3/7 Q156* . Whereas wild type flowers (n = 120) invariably formed 5 sepals, 5 petals and 3 carpels, Csplt3/7 Q156* flowers developed a wider range (between 3 and 9) of sepals and petals (Fig. S7). Moreover, we observed partial sepal-sepal or sepal-petal fusions in ∼40% (13/32) of flowers, and ∼6% (2/32) of flowers exhibited formation of 4 carpels instead of 3 (Fig. S7). We conclude that CsPLT3/7 regulates internode length, axillary shoot and flower emergence, and floral morphology in mature plants. CsPLT3/7 regulates phyllotaxis Since loss of Arabidopsis SAM- and IM-expressed PLTs, i.e. PLT3, PLT5 and PLT7, reduces robustness of the phyllotactic spiral ( Prasad et al ., 2011 ; Pinon et al ., 2013 ; Kerstens et al ., 2025a ), we asked if Csplt3/7 mutants experienced more irregular circumferential leaf patterning. Using a custom-made hinged protractor ( Robertson et al ., 2025 ), we manually quantified divergence angles in 22.5° bins between successive leaves in Csplt3/7 W48* , Csplt3/7 Q156* and Csplt3/7 R355H , and the respective wild type sister progeny per line. Unlike Arabidopsis, in which spiral phyllotaxis is characterised by pattern convergence around the ‘golden angle’ (∼137.5°; within a 135±22.5° angle bin), the most frequently occurring divergence angle bin in all three wild type cucumber lines was 157.5±22.5° ( Fig. 7a ). These data suggest that spiral phyllotaxis does not typically conform to the golden angle on mature cucumber stems. In Csplt3/7 W48* , in which we could only analyse a handful of divergence angles from non-fasciating stems with a clear initiation order, divergence angle distribution was much broader than in its wild type sister progeny, suggesting reduced robustness of the phyllotactic spiral ( Fig. 7a ). On the other hand, in both Csplt3/7 Q156* and Csplt3/7 R355H the overall patterning regularity seemed unaffected. We instead found a significant shift towards a distichous pattern, in which leaves are consecutively positioned at 180±22.5° angles from each other ( Fig. 7a,b ). The distichous pattern only manifested occasionally; a large proportion of divergence angles also approached 157.5±22.5°. Download figure Open in new tab Figure 7. Csplt3/7 mutants exhibit altered phyllotaxis. ( a ) Histograms and density plots displaying the occurrence of divergence angles within 22.5° bins. The dashed line indicates the golden angle (137.5°), and arrowhead points towards the bin containing 180°. Significance from BHY-corrected pairwise z-tests (top to bottom: p = 0.71, p = 5.0e-5, p = 5-4e-4). ( b ) Example sections of cucumber stems displaying “spiral” (upper; counterclockwise) and distichous (lower; clockwise) phyllotactic patterning. Scale bar is 1 cm. Given that Csplt3/7 Q156* mutants formed extreme internodes and we previously showed that Arabidopsis phyllotaxis is strongly chirality-dependently modified by stem torsion acting on internode length ( Kerstens et al ., 2025a ), we investigated if its shift towards distichous phyllotaxis is caused by stem torsion. We first quantified the degree to which torsion occurred and affected wild type phyllotaxis in our growth conditions. Unlike Arabidopsis, in which stems twisted persistently in a counterclockwise manner ( Kerstens et al ., 2025a ), cucumber stems twisted almost equally in clockwise and counterclockwise directions with on average a minute preference to clockwise twisting (μ = -0.17°; Fig. S8a,b). Using a trigonometric representation of the stem that could previously predict the effect of torsion on phyllotaxis ( Landrein et al., 2013 ), we predicted that in an average internode with an average stem radius, the divergence angle would be displaced 7° in the clockwise direction (Fig. S8c,d; Equation 1 ). We next plotted divergence angles against internode length separately for clockwise and counterclockwise-turning meristems. Divergence angles of clockwise and counterclockwise-turning meristems increased and decreased with longer internodes, albeit insignificantly, as a consequence of (on average) slight clockwise stem torsion (Fig. S8e). Accordingly, the average divergence angle of clockwise-turning meristems was slightly larger than that of counterclockwise-turning meristems (Fig. S8f). When we repeated this analysis for Csplt3/7 Q156* and its wild type sister progeny in a different growth season, however, the relationship between internode length and divergence angle appeared inverted and was again not significant (Fig. S8g). Moreover, in internodes of equal length (9-11 cm), the proportion of 180° angles in the mutant was still enriched (Fig. S8h). We conclude that the phyllotaxis defect of Csplt3/7 Q156* , and likely per extension, that of Csplt3/7 R355H , cannot be explained by a cooperative effect of stem torsion and altered internode length. Thus, loss of functional CsPLT3/7 shifts phyllotaxis to larger divergence angles. Discussion In this study, we have shown that PLT genes in cucumber are expressed in meristematic tissues, with CsPLT3/7 and CsPLT5 being the only two detected in the SAM ( Fig. 1 ). Within the SAM, both TFs recognised the same consensus motif and bound to SAM organising genes, but only CsPLT3/7 was expressed in the stem cell niche ( Fig. 2 , 3 ). Lack of CsPLT3/7 distorted embryogenesis ( Fig. 4 ), as well as embryonic SAM establishment or maintenance ( Fig. 5 ). Mature Csplt3/7 shoots occasionally displayed irregular internode lengths and axillary organ development and shifted phyllotactic patterning ( Fig. 6 , 7 ). Altogether, we firmly establish CsPLT3/7 as a major regulator of cucumber shoot development and architecture. Despite the use of TILLING lines, several lines of evidence support that the observed phenotypes are caused by mutations in CsPLT3/7 . Lack of initial SAMs, leaf initiation and axil defects in Csplt3/7 W48* and Csplt3/7 Q156* are in congruence with CsPLT3/7 expression domains based on our in situ hybridizations. The fact that internode length and stem architecture aberrancies occurred in three independent TILLING alleles, and the same axil and flower defects occurred in the premature stop alleles Csplt3/7 W48* and Csplt3/7 Q156* , reinforces the validity of CsPLT3/7 being the causal gene. Why only Csplt3/7 W48* mutant stems fasciate despite both stop alleles truncating the transcript in the first exon, remains unclear; perhaps another TILLING lesion in close proximity to CsPLT3/7 co-segregated with Csplt3/7 W48* , further exacerbating the phenotype. Notably, a similar phenotype has been described for an amino acid substitution allele of Csclv1 , in which stem fasciation occurred and flowers exhibited supernumerary petals and carpels ( Cheng et al ., 2022 ). Supernumerary floral organs, but not fasciation, was also described for Csclv3 mutants and RNAi lines ( Che et al ., 2020 ; Han et al ., 2024 ). We did not identify notable shoot phenotypes for other Csplt mutants, which was expected for the non-SAM-expressed CsPLT1/2 and CsBBM , but not CsPLT5 . Strictly, we cannot exclude that CsPLT5 plays an additional role in the shaping shoot architecture due to the nature of the TILLING alleles, i.e. the weak Csplt5 L189F and the Csplt5 R289* alleles, which lacks only the most terminal portion of the second AP2 domain. While targeted gene editing strategies such as CRISPR/Cas9 would be required to resolve such ambiguities, stable transformation of the recalcitrant cucumber remains a persistent bottleneck that only select laboratories have been able to overcome. Even though Csplt3/7 mutant shoots resumed indeterminate growth, the regularity of wild type internode length was lost. The mechanism behind the formation of extremely short and long internodes remains a matter of speculation at this point. One possible scenario is that Csplt3/7 SAMs are unable to rhythmically develop leaf primordia due to disruption of auxin signalling and transport. Short internodes would then result from the near simultaneous formation of two primordia, for example through increased meristem size ( Landrein et al ., 2015 ) or permissive inhibitory fields ( Besnard et al ., 2014 ). Extremely long internodes could arise from the inability to establish auxin maxima, as we previously observed in inflorescences of the Arabidopsis plt3 plt5 plt7 pin1 T600I quadruple mutant ( Kerstens et al ., 2025a ). Our observations that extreme internodes preferentially occur in succession and that phyllotactic patterns are shifted, lends additional support to the scenario that the internode phenotype results from primordium initiation defects. Importantly, this would fit with the proposed hypothesis that shoot meristematic PLTs confer patterning robustness to the SAM ( Kerstens et al ., 2025a ), acting within the CZ ( Pinon et al ., 2013 ), in which CsPLT3/7 was expressed. Nevertheless, we strictly cannot exclude that extreme internodes result instead from variable inclusion of cells in internodes due to increased and decreased activity of the rib meristem. Translating findings from the well-studied Arabidopsis to more distantly related species is a contemporary challenge with great impact on application potential. The nature of PLT TFs, both in regard to their conserved phylogenomic traits and functions throughout development, makes them attractive candidates to put translational hypotheses to the test. Our study shows that PLTs control meristematic processes in cucumber, as in Arabidopsis. On a whole-tissue scale, we additionally observed involvement of the same PLT clades on the root-shoot axis: CsPLT1/2 and CsBBM exhibited root-specific expression, while CsPLT3/7 and CsPLT5 were also expressed in the shoot apex, which conforms to the expression patterns of orthologous AtPLTs ( Fig. 1d ) ( Galinha et al ., 2007 ; Prasad et al ., 2011 ). Nevertheless, we observed clear distinctions between Arabidopsis and cucumber. Within the shoot apex, CsPLT3/7 and CsPLT5 were expressed in partially overlapping domains, with only CsPLT3/7 transcripts localising to the stem cell niche. This contrasts with Arabidopsis, in which AtPLT3 , AtPLT5 and AtPLT7 are all expressed in this zone ( Prasad et al ., 2011 ). Additionally, the observed expression of CsPLT3/7 in axils was never reported for any PLT in Arabidopsis, perhaps reflecting its different growth habit or lower complexity compared to the cucumber axil. Distinctions extend to the developmental processes CsPLTs are involved in. Csplt1/2 Csplt3/7 double mutants proved embryonic lethal, whereas in Arabidopsis, embryo lethality is only exhibited by plt2 bbm mutants ( Chen et al ., 2022 ; Kerstens et al ., 2024 ). In the related cucurbit watermelon ( Citrullus lanatus ), homozygous seeds of Clplt1/2 Clbbm mutants could also be recovered ( Liu et al ., 2025 ). It was previously described that Brassicaceae BBM orthologs are positioned in a distinctive synteny context different from other eudicots ( Kerstens et al ., 2020 ), which might indicate that zygotic expression of BBM is not ancestral and might be controlled by different PLTs in other lineages, for instance in Cucurbitaceae. Another distinction is that we observed a broad requirement for CsPLT3/7 during seedling and shoot development, which is much more extreme than in Arabidopsis Atplt3 Atplt7 ( Kerstens et al ., 2025a ). In young Csplt3/7 Q156* seedlings, leaves emerge de novo from the tissue where the SAM should have formed, which harbours striking similarities to wus mutants in Arabidopsis and Petunia ( Petunia hybrida ) ( Laux et al ., 1996 ; Stuurman et al ., 2002 ). Mature Atwus embryos and Phwus seedlings lack SAMs, possess a flat apex, and eventually produce multiple leaf primordia and secondary SAMs across the apex in a stop-and-grow fashion ( Laux et al ., 1996 ; Stuurman et al ., 2002 ). Secondary Csplt3/7 Q156* SAMs did not terminate, however, suggesting that CsPLT3/7 is only required in the establishment or maintenance of the embryonic SAM. Since we observed binding of CsPLT3/7 near CsWUS , we hypothesise that the phenotype results from misregulation of CsWUS during early development. Although the AtPLT3 and AtWUS genetic pathways interact in Arabidopsis ( Mudunkothge & Krizek, 2012 ), SAM termination as a consequence of PLT absence does not occur in this species ( Prasad et al ., 2011 ). This suggests that the regulation of SAM cell fate in cucumber is wired differently. In line with this, CsCLV3 is specifically expressed underneath the stem cell niche together with CsWUS ( Che et al ., 2020 ), which is incompatible with the canonical CLV3-WUS negative feedback loop in stem cell maintenance. Although perhaps another CsCLE family member could fulfil the function of CLV3, it is also possible that CsPLTs are responsible for stem cell maintenance, as is the case in the Arabidopsis root apical meristem ( Aida et al ., 2004 ). Another example that advocates differential SAM wiring in cucumber, is that the TF LEAFY, which localises to floral meristems in Arabidopsis ( Weigel et al ., 1992 ), is expressed in the cucumber SAM and is required for SAM maintenance in a potentially CsWUS-dependent manner ( Zhao et al ., 2018 ). A broader requirement for PLT3/7 TFs than in Arabidopsis was also recently reported in tomato inflorescences. Lack of SlPLT3 and SlPLT7 caused extreme branching through the disturbed regulation of SEPALLATA homologs ( Zebell et al ., 2025 ). Moreover, these inflorescences exhibited meristem overproliferation ( Zebell et al ., 2025 ), which we also observed in cucumber axils ( Fig. 6i,j ). Altogether, our findings highlight that the processes controlled by orthologous genes can shift across species, even in functionally redundant gene families such as PLTs . It thus becomes evident that our understanding of SAM regulatory networks across angiosperms, including cucumber, remains limited. Methods Plant material and gene identifiers ’Chinese long 9930’ and ‘long’-type parental inbred cucumber plants were propagated at Rijk Zwaan Breeding B.V. The generated mutants in the parental inbred line background and wild type sister progeny were analysed in the following generations: Csplt1/2 W52* (F 2 BC 3 ), Csplt1/2 Q58* (F 2 BC 3 ), Csplt3/7 W48* (F 2 from crossed M 2 ), Csplt3/7 Q156* (mature plants: M 2 and F 2 BC 3 ; seedlings: F 3 BC 4 ), Csplt3/7 R355H (M 2 ), Csbbm W317* (F 2 BC 3 ), Csplt5 L189F (M 2 ), Csplt5 R289* (F 2 BC 3 ). Double mutant genotyping was performed on F 2 progeny obtained from two independent lines, segregating either for Csplt1/2 W52* , Csplt3/7 Q156* and Csbbm W317* , or for Csplt1/2 W52* , Csplt3/7 Q156* and Csplt5 R289* . These segregating lines were obtained through repeated crossing of fixed F 3 BC 3 lines. The F 2 progeny segregating for Csbbm W317* and Csplt5 R289* was obtained through crossing of M 2 lines. CsPLT1/2 is CsaV3_4G024120 , CsPLT3/7 is CsaV3_3G006900 , CsBBM is CsaV3_2G010320 , and CsPLT5 is CsaV3_1G031650 . Arabidopsis plants were of ecotype Col-0. Phylogenetics Representative PLT protein sequences of Citrullus lanatus (97103 v2.5), Lagenaria siceraria (Hangzhou Gourd v1), Benincasa hispida (B227 v1), cucumber (Chinese long v3 cv 9930), Cucumis melo (DHL92 v4), Cucurbita argyrosperma (SMH-JMG-627 v2), Cucurbita moschata (Rifu v1), Cucurbita pepo (MU-CU-16 v4.1), Cucurbita maxima (Rimu v1.1), Trichosanthes anguina (v1), Sechium edule (v1), Luffa cylindrica (v1) and Siraitia grosvenorii (Qingpiguo v1) were retrieved from CuGenDBv2 ( Yu et al ., 2023 ). Arabidopsis (Araport 11) and tomato (ITAG4.0) sequences were obtained from PLAZA 5.0 ( Van Bel et al ., 2022 ). Proteomes were screened for sequences with > 1 AP2 domain using HMMER v3.2.1 ( hmmer.org ), then aligned with MAFFT v7 ( Katoh et al ., 2017 ), using a BLOSUM80 substitution matrix and FFT-NS-i strategy, then trimmed with trimAl v1.2rev59 ( Capella-Gutiérrez et al ., 2009 ), using the ‘-gt 0.5’ and ‘-cons 0.7’ flags. The trimmed sequences were aligned with IQ-TREE v1.6.10 with 1000 ultrafast bootstraps ( Nguyen et al ., 2015 ) and visualised with iTOL v6.8.2 ( Letunic & Bork, 2019 ). PLT clades were defined based on the presence of Arabidopsis orthologs. Quantitative PCR of CsPLTs RNA samples were extracted ( Yaffe et al ., 2012 ) from hand-dissected 6 dpg ‘9930’ cucumber seedlings grown in square petri dishes on top of a nylon mesh covering a layer of wet potting soil in a long day growth chamber (16 h light, 8 h dark) at 22 °C under white ∼100 μmol/s LED lights. Each of the four biological replicates consisted of pooled tissues of 7 seedlings. Roots were subdivided in four regions, i.e. the bottom ∼ 2 mm containing the apical meristem and the lower elongation zone, and three subsequent ∼3 mm segments encompassing the remaining elongation zone, maturation zone with emerging root hairs, and mature root tissue. A ∼ 3 mm central segment was retrieved from the hypocotyl, and a ∼ 3 x 3 mm piece was taken from one of the cotyledons. The first true leaf was removed from the shoot apices. cDNA was synthesized with the RevertAid Reverse Transcriptase system (Fermentas) using oligo(dT)18 primers. qPCRs were performed with SYBR Green in the Bio-Rad CFX Connect Real-Time PCR Detection System using 3 technical replicates per sample. Primers are listed in Table S6. Cq values were normalised against the CsUBC gene (CsaV3_2G031000). In situ hybridisation ’9930’ cucumbers were grown for 5 days in wet germination pouches (CYG) in a long day growth chamber. Shoot apices were collected in ice-cold fixative (4% paraformaldehyde with 0.5% EM-grade glutaraldehyde in PBS), exposed to repeated 5-minute vacuum bursts until sinking to the bottom of the vial, then incubated at 4°C overnight. Further dehydration, paraffin infiltration and embedding steps are described in ( Kulikova et al ., 2018 ). The tissues were sectioned longitudinally (20 μm) or transversally (10 μm) prior to in situ hybridisation with the Invitrogen™ ViewRNA™ ISH Tissue Assay kit (ThermoFisher Scientific) according to manufacturer’s user guide. Type 1 RNA ISH probe sets were designed from full-length cDNA and synthesized at ThermoFisher Scientific. Catalogue numbers are VPGZFCU for CsPLT3/7 and VP2W7TZ for CsPLT5. Sections were imaged with a Nikon Eclipse 80i using brightfield and fluorescent (TRITC; excitation 527-552 nm, emission 577-632 nm) channels. EMS mutagenesis and TILLING EMS mutagenesis was performed on the ‘long’-type parental inbred line. Seeds were treated with 0.7% EMS and sown on the following day, transplanted after 5 days to pots and transplanted to soil in an unlighted greenhouse 4 weeks after sowing. Plants were insect pollinated and upon maturity fruits were harvested in bulk. For TILLING screens, 5-day old plants were sampled and genotyped by amplicon sequencing (Illumina MiSeq). For selection and seed production in subsequent generations, allele-specific KASP assays were developed. Confocal microscopy 6 dpg wild type and Csplt3/7 Q156* shoot apices were collected in Renaissance (RS2200) solution ( Musielak et al ., 2015 ) and stored at 4 °C until imaging. Tissues were imaged on a Zeiss LSM710 confocal microscope with an excitation wavelength of 405 nm. Prior to imaging with a water dipping lens, SAMs were positioned upright in 3% agarose and submerged in Milli-Q water. Greenhouse measurements Seeds were sown in a greenhouse compartment on rockwool trays, covered with vermiculite, watered with tap water and incubated at 27 °C under natural lighting for 4 days. At this point, germinated plants were sampled for genotyping if required. Sampled plants were stored until genotype selection at 15 °C or directly transplanted to a greenhouse at 23 °C in rockwool blocks with supplemental lighting (Philips Son-T gas pressure lamps, 150 µmol/m 2 s). Plants were transferred to a 22/18 °C (light/dark hours) greenhouse with supplemental lighting (Philips Son-T gas pressure lamps, 150 µmol/m 2 s) 4 weeks past germination, planted in rock wool mats with an automatic watering system and guided upwards along a vertical wire in an umbrella cultivation system. To minimise stem torsion, shoots were attached to the wire using metal rings. Axillary shoots were pruned weekly from the plants, unless counted. Fruits were maintained intermittently (i.e. one fruit per two nodes). Internode length and plant height were measured with a flexible measuring rope. Leaf production per week was determined by weekly attachment of plastic rings around the highest possible internode, then counting the leaves in between successive rings. Axillary shoot counting was performed from top to bottom to account for the initial SAM loss of Csplt3/7 Q156* . Only distinct axillary shoots (> 3 cm) were considered. Phyllotaxis was measured with 360-degree hinged protractors that can be clipped around the cucumber stem, simultaneously allowing measurement of both meristem chirality and angle in 22.5° bins. Chirality was attributed based on the most frequent direction of turning in the upper five measured internodes. Torsion was measured in internode photographs and defined as the angle deflection to the right (counterclockwise) or left (clockwise) from the vertical internode axis. The predicted effect of torsion was calculated according to equation 1 ( Landrein et al., 2013 ): In which δ f is the final divergence angle, δ i the initial divergence angle at the IM, α the torsion angle, I internode length, and r stem radius. RNA-sequencing RNA-seq was carried out in triplicate on shoot apices dissected from 6 dpg ‘9930’ seedlings grown under long day conditions at 22 °C in soil plates on top of a nylon mesh (7 pooled apices per replicate) and grown in wet germination pouches (20 pooled apices per replicate). The first true leaf from each apex was removed. Tissue was collected in 200 µL RNAlater (Invitrogen) on ice, which was removed after adding 750 µL Milli-Q water prior to freezing in liquid N 2 . RNA was extracted with the LogSpin protocol ( Yaffe et al ., 2012 ), treated with DNaseI (Qiagen), then purified with EtOH purification. The libraries were sequenced on a NovaSeq6000 Plus at GenomeScan. Data analysis was performed by trimming the reads with fastp v0.23.4 ( Chen et al ., 2018 ), then pseudo-aligning to the PLAZA5 instance of the full ‘Chinese long 9930 v3’ transcriptome ( Van Bel et al ., 2022 ) with kallisto v0.46.1 ( Bray et al ., 2016 ). Genes were considered expressed if TPM > 1 in at least three of the six samples. Cloning The DAP-seq construct pSPUTK-GG 3xFLAG-GFP was described previously ( Kerstens et al ., 2024 ). The similar constructs pSPUTK-GG 3xFLAG-CsPLT3/7 and pSPUTK-GG 3xFLAG-CsPLT5 were assembled by combining pSPUTK-GG ( Kerstens et al ., 2024 ), pICSL30005 (Addgene #50299) and a BsaI-cCsPLT3/7 or BsaI-cCsPLT5 amplicon through BsaI cloning. Transcriptional reporters were generated by amplifying 6.1 kb ( CsPLT1/2 ) or 6.3 kb ( CsPLT3/7 ) promoter + 5’UTR fragments from ‘9930’ gDNA and subsequent Gateway cloning into pGEM-T Easy 221 (Invitrogen) and pGWB3 ( Nakagawa et al ., 2007 ). Arabidopsis was transformed by floral dip ( Clough & Bent, 1998 ). DAP-sequencing To obtain genomic DNA with SAM-specific modifications, dissected SAMs without the first true leaves were collected from 6 dpg parental inbred line seedlings grown in wet germination pouches (CYG) in a long day (18h light, 6h dark) greenhouse with fluctuating temperatures (day: 20.5 °C; night: 16.0 °C). Two replicates of 100 SAMs each were collected on ice and snap-frozen in liquid N 2 . Further DAP and analysis steps were carried out as described previously ( Kerstens et al ., 2025a ), using the ‘Chinese long 9930 v3’ genome ( Li et al ., 2019 ) and integrating replicates through IDR analysis ( https://github.com/nboley/idr ). Fragments of reads in peaks (FRiP) was calculated by dividing the number of filtered, mapped reads within peaks by the total number of filtered, mapped reads per sample. (Co-)orthology of target genes was inferred from both collinearity (’anchor point’) and best BLAST hits (’BHIF’) as available from the Integrative Orthology data within the PLAZA5 Dicots database ( Van Bel et al ., 2022 ). GUS staining Arabidopsis Col-0 plants were grown for 5-6 weeks in long day conditions (16 h light, 8 h dark) at 22 °C under white LED lights. Ovules were hand-dissected from young siliques and directly transferred to GUS staining solution (50 mM sodium phosphate buffer pH 7.0, 2 mM potassium ferro/ferricyanide, 0.1% IGEPAL CA-630, 0.5 mg/mL X-GlcA). Submerged ovules were vacuum infiltrated for 15 minutes, then stained overnight at 37 °C. Stained ovules were cleared at 4 °C in chloral hydrate solution (8 g chloral hydrate, 1 g glycerol, 3 mL H 2 O) and imaged using a Zeiss Axio Imager A1. Author contributions Conceptualization: M.K., B.S., V.W.; Methodology: M.K., B.S., V.W.; Investigation: M.K., F.M., K.A., O.K., M.L.; Writing: M.K., V.W.; Editing: B.S.; Visualization: M.K.; Supervision: B.S., V.W.; Project administration: V.W.; Funding acquisition: M.K. Funding This research was funded by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (GSGT.2019.019 to M.K.). Significance statement We reveal that shoot apical meristem-expressed PLETHORA3/7 in cucumber shapes shoot architecture. Csplt3/7 mutant mature embryos initially lack shoot apical meristems, but later form secondary meristems from their apex, resulting in shoots with irregular internode length, phyllotaxis, and aberrant development of organs in leaf axils. Competing interests None declared. Data availability The DAP-seq and RNA-seq data are available in the Gene Expression Omnibus with accession numbers GSE294326 and GSE294327, respectively. Acknowledgements We thank Freek van der Klugt, Rory McCarthy and Jarno van Schijndel for assisting with greenhouse measurements, wet lab experiments, and amplification and size-selection of DAP libraries. Taco Jesse is acknowledged for coordination of RNA and DAP-sequencing, and Kees van Dun for stimulating discussions. Funder Information Declared Dutch Research Council, https://ror.org/04jsz6e67 , GSGT.2019.019 References ↵ Aida M , Beis D , Heidstra R , Willemsen V , Blilou I , Galinha C , Nussaume L , Noh YS , Amasino R , Scheres B . 2004 . The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche . Cell 119 : 109 – 120 . OpenUrl CrossRef PubMed Web of Science ↵ Aida M , Ishida T , Fukaki H , Fujisawa H , Tasaka M . 1997 . Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant . The Plant Cell 9 : 841 – 857 . 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Share A PLETHORA transcription factor shapes cucumber shoot architecture Merijn Kerstens , Florian Müller , Kelvin Adema , Olga Kulikova , Magdalena Lastdrager , Ben Scheres , Viola Willemsen bioRxiv 2025.09.18.677013; doi: https://doi.org/10.1101/2025.09.18.677013 Share This Article: Copy Citation Tools A PLETHORA transcription factor shapes cucumber shoot architecture Merijn Kerstens , Florian Müller , Kelvin Adema , Olga Kulikova , Magdalena Lastdrager , Ben Scheres , Viola Willemsen bioRxiv 2025.09.18.677013; doi: https://doi.org/10.1101/2025.09.18.677013 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 (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41913) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13372) Ecology (19890) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15600) Genomics (22483) Immunology (17728) Microbiology (40365) Molecular Biology (17164) Neuroscience (88540) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15136) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9818) Zoology (2269)