Recessive antimorph alleles reveal novel functions of the OPAQUE1 myosin XI in maize

Recessive antimorph alleles reveal novel functions of the OPAQUE1 myosin XI in maize | 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 Recessive antimorph alleles reveal novel functions of the OPAQUE1 myosin XI in maize View ORCID Profile Brian Zebosi , View ORCID Profile Stephanie E. Martinez , View ORCID Profile Kokulapalan Wimalanathan , View ORCID Profile John Ssengo , View ORCID Profile Gabriela Brown , View ORCID Profile Norman B. Best , View ORCID Profile Michelle Facette , View ORCID Profile Carolyn G. Rasmussen , View ORCID Profile Erik Vollbrecht doi: https://doi.org/10.1101/2025.06.26.661838 Brian Zebosi 1 Department of Genetics, Development and Cell Biology, Iowa State University , Ames, IA 50011 2 Interdepartmental Genetics and Genomics Graduate Program, Iowa State University , Ames, IA 50011 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Brian Zebosi Stephanie E. Martinez 3 Botany and Plant Sciences Department, University of California , Riverside, Riverside, CA 92507 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stephanie E. Martinez Kokulapalan Wimalanathan 1 Department of Genetics, Development and Cell Biology, Iowa State University , Ames, IA 50011 4 Interdepartmental Bioinformatics and Computational Biology Graduate Program, Iowa State University , Ames, IA 50011 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kokulapalan Wimalanathan John Ssengo 2 Interdepartmental Genetics and Genomics Graduate Program, Iowa State University , Ames, IA 50011 5 Department of Plant Pathology, Iowa State University , Ames, IA 50011 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for John Ssengo Gabriela Brown 3 Botany and Plant Sciences Department, University of California , Riverside, Riverside, CA 92507 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabriela Brown Norman B. Best 6 USDA-ARS, Plant Genetics Research Unit , Columbia, MO 65211 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Norman B. Best Michelle Facette 7 Department of Biology, University of Massachusetts , Amherst, Amherst, MA 01003 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michelle Facette Carolyn G. Rasmussen 3 Botany and Plant Sciences Department, University of California , Riverside, Riverside, CA 92507 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carolyn G. Rasmussen Erik Vollbrecht 1 Department of Genetics, Development and Cell Biology, Iowa State University , Ames, IA 50011 2 Interdepartmental Genetics and Genomics Graduate Program, Iowa State University , Ames, IA 50011 4 Interdepartmental Bioinformatics and Computational Biology Graduate Program, Iowa State University , Ames, IA 50011 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Erik Vollbrecht For correspondence: vollbrec{at}iastate.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Ideal plant architecture optimizes canopy structure and increases grain yield in maize. However, its underlying genetic mechanisms remain poorly characterized. Two recessive, EMS-induced maize mutants were identified that have reduced stature, opaque kernels, and abnormal subsidiary cell division, and are allelic to opaque1 (o1) . These two new missense alleles, o1-2995 and o1-tan62 , unlike the loss-of-function alleles previously identified, generate O1 protein but paradoxically generate more severe morphological defects. These defects include reduced internode elongation and partial suppression of excessive tassel and ear branching in ramosa1 (ra1), ra2, and ra3 mutants. We show that o1-2995 and o1-tan62 are novel alleles of o1 , and play a role in plant growth via internode elongation, subsidiary cell division positioning, leaf patterning, inflorescence development, and overall plant architecture. Introduction Altering maize plant architecture traits has led to significant increases in yield ( Tian et al. 2019 ) . Modern hybrids typically have narrow, erect leaves and tassels with few and upright branches that facilitate enhanced photosynthetic efficiency, efficient light interception, and tolerance to plant lodging under high planting densities ( Lambert and Johnson 1978 ; Tian et al. 2019 ; Pendleton et al. 1968 ) . A key aspect of development in grasses is the regulation of branching in grain-producing inflorescences such as the tassel and ear of maize. Inflorescence branching architecture reflects the presence and differential activity of multiple axillary meristems. Prior research has identified several genes required for initiating axillary meristems and maintaining their determinacy. Some of these genes such as Barren inflorescence1 ( Bif1 ), Bif2 , Bif4 , and Barren stalk 1 ( Ba1 ) control auxiliary meristem initiation and their mutants display decreased tassel branching ( Barazesh and McSteen 2008 ; Gallavotti et al. 2004 ; Galli et al. 2015 ; McSteen et al. 2007; McSteen and Hake 2001 ) . In contrast, the ramosa genes ( Ra1 , Ra2 , and Ra3 ) regulate spikelet pair meristem (SPM) determinacy, and their loss of function mutants produce more tassel and ear branches ( Bortiri et al. 2006 ; Satoh-Nagasawa et al. 2006 ; Vollbrecht et al. 2005) . Ra1 encodes a C2H2-type zinc finger transcriptional regulator that likely acts as both an activator and repressor of target genes ( Gallavotti et al. 2010 ; Eveland et al. 2014 ; Vollbrecht et al. 2005) . Ra2 encodes a LATERAL ORGAN BOUNDARIES domain transcription factor ( Bortiri et al. 2006 ) . Ra3 encodes a trehalose-6-phosphate phosphatase whose role in branching is uncoupled from enzymatic activity ( Claeys et al. 2019 ; Satoh-Nagasawa et al. 2006 ) ). Using a genetic approach to identify modifiers of ramosa1 ( ra1 ), we uncovered a novel mutant that suppresses tassel branching independent of the ramosa pathway and that disrupts a gene encoding a myosin XI-I called Opaque1 ( Wang et al. 2012 ) . Myosins are molecular motors that transport cargo along cytoskeletal actin filaments (F-actin) and are required for proper cell function. Myosins are important for processes such as organelle movement, cell shape maintenance, cell polarization, actin organization and signal transduction ( Madison and Nebenführ 2013 ) . The myosin protein structure typically consists of three domains: the head, neck, and tail ( Foth et al. 2006 ; Tominaga and Nakano 2012 ) . The head motor domain functions in actin binding and in ATP binding and hydrolysis for motor activity, the neck domain contains a linker that influences motor step size, while the tail binds cargo and facilitates dimerization ( Nebenführ and Dixit 2018 ) . Myosins are highly conserved across many eukaryotic species, including mammals, fungi, and plants ( Richards and Cavalier-Smith 2005 ) . Two distinct myosin groups (VIII and XI) are specific to plants and present as several subfamilies due to repeated rounds of whole-genome duplications ( Foth et al. 2006 ; Mühlhausen and Kollmar 2013; Nebenführ and Dixit 2018 ) . Genetic studies and functional characterization of mutants of these plant-specific myosins indicate they are involved in many processes including organelle translocation, cytoplasmic streaming, cytokinesis, tip growth, exocytosis and endocytosis ( Nebenführ and Dixit 2018 ; Zhang et al. 2019 ; Chocano-Coralla and Vidali 2024 ; Golomb et al. 2008 ; Peremyslov et al. 2010 ; Olatunji et al. 2023 ; Tominaga and Nakano 2012 ; Ueda et al. 2015 ) . In particular, Arabidopsis myosin XI-I isoforms (myosin XI-Is) are localized to the nuclear envelope and act as linkers between the nucleus and actin cytoskeleton through a linker of nucleoskeleton and cytoskeleton (LINC) complex ( Tamura et al. 2013 ) . Arabidopsis myosin XI-I mutants ( Atxi-1 / kaku1 ) have aberrant nuclear envelopes with impaired nuclear positioning and movement, thus emphasizing the vital role of myosins in nuclear movement and shape maintenance ( Tamura et al. 2013 ; Muroyama et al. 2020 ) . Although important for nuclear movement in Arabidopsis, genetic studies in maize indicate that myosin XI-I is instead required for protein body formation during seed development and endoplasmic reticulum (ER) movement ( Wang et al. 2012 ) . Additionally, the opaque1 mutants have phragmoplast guidance defects leading to asymmetric cell division defects ( Nan et al. 2023 ) . Unlike kaku1 mutants, no nuclear movement abnormalities were observed in o1 asymmetric divisions ( Nan et al. 2023 ) . However, neither growth nor developmental defects were reported in these myosin XI mutants. Here, we functionally characterized two new alleles of opaque1, a myosin XI-I (Zm00001eb193160). One mutant allele, ramosa1 suppressor locus*12.2995 ( rsl*-12.2995 ), was identified from a ramosa1 ethyl methanesulfonate (EMS) enhancer-suppressor screen as a mutant with reduced stature, abnormal subsidiary cell division, opaque kernels and impaired tassel, ear and vegetative shoot development. The rsl*-12.2995 mutation was mapped to the O1 locus using map-based cloning and whole-genome sequencing and subsequently named o1-2995 . The other mutant allele, tangled62 ( o1-tan62 ), was identified from a forward genetics EMS screen for plants with abnormal subsidiary cell divisions. Allelism tests confirmed both as new o1 alleles that in genetic analyses behaved as recessive antimorphs. Thus, o1-2995 and o1-tan62 are novel alleles of Opaque1 (O1) that regulate plant growth, architecture and asymmetric division positioning in maize. Results The o1-2955 mutant shows aberrant shoot and inflorescence architecture Genetic and genomics studies indicate that three Ramosa genes in maize ( Ra1, Ra2 and Ra3 ) regulate branching in grass inflorescences by modulating shared and discrete developmental modules ( Vollbrecht et al. 2005 ; Bortiri et al. 2006 ; Satoh-Nagasawa et al. 2006 ; Eveland et al. 2014) . To identify ramosa1 ( ra1 ) mutant modifiers, we performed an EMS mutagenesis screen of ra1-63 mutants ( Vollbrecht et al. 2005 ) in the Mo17 genetic background. In the M2 generation, we identified a short-statured mutant that suppressed ra1 -induced tassel and ear branching, named it ramosa suppressor locus*-12.2995 ( rsl*-12.2995 ) and later renamed it o1-2995 . In F2 and backcross populations, o1-2995 segregated as a single recessive locus (wildtype: o1-2995 , 48:22 [χ2, P = 0.21] and 22:18 [χ2, P = 0.53], respectively). o1-2995 single mutants also showed reduced stature after backcrossing 5-8 times into B73 and W22 ( Figure 1D and 1E ). The o1-2995 mutant’s effect on plant height was completely penetrant in all three genetic backgrounds, but its expressivity varied. Compared to their normal siblings, field-grown o1-2995 mutants were shortest in B73, suggesting that genetic modifiers interact with O1 in its effect on plant height. All subsequent phenotyping was carried out using the well-introgressed B73 stocks. Download figure Open in new tab Figure 1. Phenotypic characterization of o1-2995 and of its interaction with ramosa ( ra ) pathway genes. (A-C) . o1-2995 and its interaction with ra1 , ra2 and ra3 in B73. (A). Left, tassel branch number (TBN) of normal siblings (WT, n = 75) and o1-2995 single mutants (n = 33). Right, TBN and Ear branch number (EBN) of ra1 single mutants and ra1;o1-2995 double mutants. (B). Left, TBN of ra2 single mutants and ra2;o1-2995 double mutants. Right, TBN and EBN of ra3 single mutants and ra3;o1-2995 double mutants. (C) . Representative tassels and ears as quantified in Panel A, right. (D-E). Plant height across inbred line (B73, Mo17 and W22) introgressions for normal siblings (WT, n = 47, 20, 21 for Mo17, B73, W22 respectively) and o1-2995 mutants (n = 39, 17, 39 for Mo17, B73, W22 respectively). (F) . Peduncle length and whole tassel length of normal siblings (WT, n = 30) and o1-2995 mutants (n = 30) in B73. (G) . Length of several internodes of the main shoot in normal siblings (WT, black, left, successive means connected by red line, n = 20) and o1-2995 mutants (orange, right, successive means connected by blue line, n = 17) in B73. I-1 denotes the internode nearest the tassel and I-12 is 11 internodes away basally, near the soil line. Asterisks (***), significant difference at P < 0.001. In B73, tassel branch number was reduced in the o1-2995 single mutant which produced 51.1% fewer tassel branches than its wildtype siblings ( Figure 1A ). Length of the tassel peduncle and tassel length exhibited a 70% and 78.1% reduction, respectively, in mutants compared to wildtype siblings ( Figure 1F ). Because o1-2995 was isolated from a ra1-63 mutant suppressor genetic screen, we constructed double mutants between o1-2995 and all three ramosa pathway mutants ( ra1 , ra2 , and ra3 ), and also produced one triple mutant ( ra1;ra2;o1-2995 ). We compared the higher order mutant phenotypes to the single mutants to determine if O1 and Ra genes function in an additive, synergistic, or epistatic manner with one another. In ra1 , ra2 or ra3 mutants, additional long branches form in both the ear and in the tassel. As stated earlier, the o1-2995 single mutant reduced tassel branch number (TBN) by 51.1% as compared to its wildtype siblings ( Figure 1A , left; 1C ). o1-2995 single mutants did not reduce ear branch number (EBN) as the normal ear is unbranched. In double mutant populations with ra1-63, the o1-2995 mutant reduced TBN by 36.5% and EBN by 72.3% (P < 0.001, Student’s t-test), as compared to ra1-63 single mutants ( Figure 1A , right; 1C ). Thus, in combination with ra1-63 , the o1-2995 mutant behaved additively by slightly reducing overall branching. The ra2-R;o1-2995 double mutant combination also appeared additive with a 51.4% reduction in TBN in the double mutant as compared to the ra2-R single mutant ( Figure 1B , left ). In mutant combinations with ra3 , o1-2995 also additively suppressed tassel and ear branching; the ra3-R ; o1-2995 double mutants exhibited 33.4% and 76.6% reductions in TBN and EBN, respectively, compared to ra3-R single mutants ( Figure 1B , right ). Thus, for all three double mutants ( ra1-63 ; o1-2995 , ra2-R ; o1-2995 and ra3-R ; o1-2995 ) the inflorescence branching phenotypes provided no clear evidence of epistasis, as if o1-2995 affected growth more generally without specifically affecting the meristem determinacy regulated by the ramosa genes. As a final test we examined ra1;ra2 double mutants which interact synergistically by more completely reducing activity of the ramosa pathway, leading to profusely branched ears ( Vollbrecht et al. 2005 ) . The ( ra1-63;ra2-R;o1-2995 ) triple mutants had substantially less ear branching and smooth patches on the ear axis, which we interpreted as additive or possibly synergistic, since the patches are not seen in either single or double mutant ( Supplementary Figure 1B). This implies that O1 and ramosa genes are in either independent or converging genetic pathways, and are unlikely to participate together in a simple linear pathway to regulate inflorescence branching. Map-based cloning and identification of o1-2995 as a novel allele of Opaque1 To identify the causative mutation responsible for the rsl*-12.2995 ( o1-2995 ) mutant phenotype, we used map-based cloning methods. Bulked segregant analysis using genotyping-by-sequencing (BSA-GBS) mapped the o1-2995 mutation to the long arm of chromosome 4 ( Supplementary Figure 2), within a peak containing 989 genes. Mapping using publicly available molecular markers identified a 1.5Mb interval containing 28 genes ( Figure 2A ) which, in non-repetitive regions, contained no DNA sequence polymorphisms between B73 and Mo17. We therefore performed whole-genome sequencing (WGS) of a single mutant plant and identified EMS-induced SNPs in the interval. Fine-mapping in a second, larger (2289 plants) population indicated that the causal mutation was within a physical distance of ∼ 0.6Mb that contained only one gene, the previously cloned Opaque1 ( O1 ) ( Figure 2B ). We therefore phenotyped kernels from our segregating populations, and found an opaque kernel phenotype co-segregated with the rsl*-12.2995 mutant plant phenotypes. Download figure Open in new tab Figure 2. Map-based cloning, o1-2995 and o1-tan62 complementation tests and antimorph allele relationships. (A-B) . Map based cloning of o1-2995 . (A) . Highlighted interval (pink) is ∼1.5MB with 28 genes and no B73/Mo17 polymorphism. (B). Whole-genome sequencing-aided recombination mapping. E, EMS-like SNP. M, homozygous Mo17. H, heterozygous Mo17/B73. B, homozygous B73. The new interval contained eight genes and only one exonic, EMS-like SNP (G>A), in the o1 gene. (C). o1 intron-exon gene structure (from NCBI) showing relative mutation sites of o1-2995 and o1-tan62 and of null alleles o1-N1242A and o1-ref ( Wang et al. 2012 ) used in this study. (D) Maize O1 protein domain structure and relative locations of o1-2995 and o1-tan62 missense mutations in the myosin head domain. S, SH3-like domain; IQ, region of six IQ domains; CC, coiled-coil domain. (E) . SDS-PAGE loading control (left) and anti-O1 western blot (right) of membrane and membrane-associated proteins isolated from the stomatal division zone of wild type (B73) and o1 mutant plants. Double arrowhead, migration position of the O1 protein. (F, G and H) . Complementation test for plant height and kernel phenotypes. (F) . Kernels of indicated genotypes, illuminated from below on a light box; o1-2995 and o1-tan62 fail to complement endosperm phenotype of null alleles o1-ref and o1-N1242A . (G and H) . Field-grown plants of indicated genotypes in B73; o1-2995 fails to complement plant height phenotype of null alleles o1-ref and o1-N1242A . Panel H, for the genotypes left to right, n = 70, 29, 39, 36, 59, 37, respectively; letters a, b, c, significant groupings by ANOVA with Tukey’s HSD. Sanger sequencing of the O1 locus in o1-2995 confirmed the single nucleotide change (G > A) in the 15 th exon ( Figure 2C ), causing a missense mutation (S602L) within the myosin head domain of O1 ( Figure 2D ). S602 is a nearly-invariant residue near the actin-binding motif: across the 72 myosin VIII and myosin XI genes from flowering and non-flowering plants we surveyed for Figure 3 , 71 genes encode an S in this position and one encodes a T. Download figure Open in new tab Figure 3. Phylogenetic and gene expression analysis of plant myosins XI and VIII. (A) . Phylogenetic analysis of all plant myosins XI and VIII across several species. Zm, maize; Sobic., Sorghum bicolor ; SEVIR, Seteria viridis ; Os, rice; AT, Arabidopsis; Pp, Physcomitrella patens (moss) as an outgroup. ( B). Relative transcript expression levels of myosin XI and VIII genes in different maize tissues; from Walley et al., (2016) . To confirm the genetically linked, EMS-induced mutation in O1 as causal of the o1-2995 phenotype, we performed complementation tests with previously identified null mutants, o1-ref and o1-N1242A ( Wang et al. 2012 ) . o1-2995 failed to complement the o1 null alleles for kernel opaqueness ( Figure 2F ). Interestingly, we also discovered that each null mutant, o1-ref and o1-N1242A , showed slightly reduced plant height ( Figure 2G and 2H ), a previously unreported phenotype. Moreover, the reduced plant height of the compound heterozygous mutants ( o1-ref / o1-2995 and o1-N1242A / o1-2995 ) was similar to o1-ref or o1-N1242A homozygotes. Plant height was greatly reduced for o1-2995 mutants ( Figure 2G and 2H ; see also Figure 4A ). Thus, the o1-2995 allele generates a stronger mutant phenotype when homozygous than do null mutant alleles o1-ref or o1-N1242A . These data imply that o1-2995 functions as a formally recessive but dose-dependent, antimorphic, allele of O1 . Download figure Open in new tab Figure 4. Shoot phenotypes of o1 mutant alleles in B73. (A) . Representative field-grown plants of indicated genotypes and their wild type (N) siblings. (B) . Plant height (left) and tassel branch number (right) of indicated genotypes and wild type (WT) siblings. (C) . Quantification of leaf characters (left) and images of representative leaves (right) of WT (B73) and indicated mutant genotypes. (D) . Ears (top) and quantification of ear size characters of indicated genotypes and wild type (WT) siblings. Error bars, standard error; asterisks (***), significance at P < 0.001 by t-test; letters a, b, c, significant groupings by ANOVA with Tukey’s HSD; ns, no significant difference. To investigate if the non-synonymous point mutation in o1-2995 altered O1 protein levels, we isolated proteins from the stomatal division zone of the leaf and used an O1-specific antibody to compare amounts of protein produced between o1-ref and o1-2995 . As expected, a band was detected in the normal inbred B73 plants and wildtype, heterozygous siblings of o1-ref and o1-2995 , while no band corresponding to the size of the O1 protein was detected in o1-ref mutant plants ( Figure 2I ). In contrast, in o1-2995 mutants, a band of the expected size was detected, indicating the mutant produces a protein ( Figure 2I ). These data indicate that o1-2995 , which expresses myosin XI-I containing a S602L mutation in the motor domain, confers a stronger mutant phenotype than o1 null mutants which express no or very little myosin XI-I protein. o1-tangled62 is another novel allele of opaque1 An additional mutant allele of o1 was identified through a forward genetics screen to identify mutants with division plane orientation defects. A recessive mutant with aberrant subsidiary cells was initially identified as tangled62 ( tan62 ) ( Supplementary Figure 3). Due to the similarity of the subsidiary cell division defect compared to o1 mutants as well as an opaque kernel phenotype ( Nan et al. 2023 ; Wang et al. 2012 ) , complementation tests were performed with o1-N1242A mutants. Across 11 independent crosses, tan62 failed to complement the o1-N1242A opaque kernel phenotype, suggesting that tan62 is allelic to o1 ( Figure 2F ). Sequencing of the tan62 cDNA and DNA extracted from three additional tan62 mutants revealed a single nucleotide change (G > A) in the 4 th exon ( Figure 2C ), causing a missense mutation (G159D) within the myosin motor head domain ( Figure 2D ). G159 is an invariant residue in the head domain of all myosins VIII and XI across flowering and non-flowering plants surveyed in Figure 3 , located in the ATP binding pocket. This type of mutation is predicted to be deleterious but still produce protein, similar to O1-2995. Therefore, tan62 was renamed to o1-tan62 . Genome-wide identification and functional analysis of myosin XIs in maize To consider novel O1 alleles in the context of the genome-wide diversification of myosins in maize and other plants, we constructed a phylogenetic tree and annotated it with expression data using publicly available sources ( Walley et al. 2016 ; Wang et al. 2012 ) . Using blast searches based on Arabidopsis myosin XI and VIII protein sequences, we included all relevant amino acid sequences from Arabidopsis, rice, sorghum, Setaria and as an out-group, Physcomitrium patens (moss) ( Figure 3A ). Within the myosin XI clade, Arabidopsis, maize and rice have 13, 12 and 11 genes, respectively, while Setaria and sorghum have 10 myosin XIs. In general ( Figure 3A ), the myosin XIs are further clustered into five distinct subclades ( XI-I, XI-K, XI-E, XI-F, and XI-G) based on the Arabidopsis nomenclature ( Avisar et al. 2008 ; Reddy and Day 2001 ; Peremyslov et al. 2011 ) . Opaque1 ( O1 ) belongs to the myosin XI-I clade, which comprises two genes in maize, Setaria and sorghum, contrasting to one in Arabidopsis and four in rice. Spatial expression differences among the different maize myosins ( Figure 3B ) were annotated from publically available expression data ( Walley et al. 2016 ; Wang et al. 2012 ) . The two myosin XI-I genes in maize (Zm00001eb223530 and O1 ) exhibited opposite expression patterns. Notably, O1 is expressed more broadly and more highly than Zm00001eb223530 in most tissues surveyed except mature pollen, which implies functional diversification between the two genes and suggests they function non-redundantly. Effects of different o1 mutant alleles on plant shoot phenotypes and in different environmental conditions opaque1 has been studied for over 50 years as a classical endosperm mutant ( Wang et al. 2012 ; Neuffer et al. 1968 ) yet only recently have any plant shoot phenotypes been reported in o1 loss of function mutants, for a role in asymmetric cell divisions in the leaf ( Nan et al. 2023 ) . Because o1-2995 antimorph allele mutants had altered shoot development phenotypes such as tassel branching and plant height ( Figure 1 ) and both o1-2995 and o1-tan62 failed to complement the endosperm phenotype of o1 null alleles ( Figure 2F ), we hypothesized that additional shoot phenotypes would be affected by various alleles, and tested this hypothesis using our mutant stocks in the B73 genetic background. Field-grown plant phenotypes indicate a role for O1 in plant height and ear growth In side-by-side field growth experiments comparing single mutants of the antimorphic o1-2995 allele and the null alleles o1-ref and o1-N1242A , the o1-2995 plants were again dramatically smaller (45.8% height reduction) than o1-2995/+ or homozygous +/+ siblings. The o1-ref and o1-N1242A mutants also showed height reduction compared to their wild type siblings, although the reduction was less severe than in o1-2995 (18.7% and 19.6% reduction, respectively) ( Figure 4A , 4B ). On the other hand, o1-2995 mutants had 3-5 fewer tassel branches but o1-ref and o1-N1242A null mutant alleles had no significant effects on TBN relative to their wildtype siblings ( Figure 4B , right ). Finally, the length and width of ears, and kernel row number, was significantly reduced in all mutants compared to their wild type siblings, with more abnormal phenotypes observed in o1-2995 mutants than in the null allele mutants ( Figure 4D ). These data suggest that O1 plays a greater role in plant height, and ear growth, than tassel branching. Greenhouse-grown plant phenotypes suggest O1 is responsive to environmental conditions Greenhouse growth experiments also showed reductions in plant height in some of o1 mutants compared to wild-type siblings, but with more variability. When o1-2995 mutants were grown in the greenhouse during the shorter days of winter 2018, mutants were significantly shorter than their wildtype siblings (average 179.7 cm and 225.8cm, respectively; P = 0.00042), although proportionally less so (20.2%,) compared to the reduction in field-grown plants (45.8%). In a separate greenhouse experiment performed in 2024, both o1-2995 and o1-tan62 mutants had a significant reduction in plant height of 37.8% and 17.1%, respectively, compared to wild-type siblings, while o1-N1242A null mutants did not, when measured at the whole plant level ( Supplementary Figure 5A). Similarly, peduncle length, tassel length and TBN were unaffected in o1-N1242A but were generally reduced in o1-tan62 and o1-2995 mutants. Thus, environmental conditions may modulate the severity of phenotypic effects but under all conditions tested o1-2995 and o1-tan62 had more reduced stature compared to both wild-type siblings and to null mutants o1-ref and o1-N1242A . Plant height reduction is due to compressed internodes To understand the basis of reduced stature of the o1-2995 mutants, we counted the number of leaves formed and measured the size of internodes. Our results showed that the normal siblings and mutants generated similar numbers of leaves (normal, n=100, 20.2 leaves; o1-2995 , n= 42, 19.1 leaves; P=0.072) but that in mutants, internode lengths and diameters were compressed, with a 26-83% internode length reduction (P<0.0001) compared to wild-type siblings depending on the observed internode ( Figure 1G and Supplementary Figure 4). The reduction in internode length varied but was more pronounced in the lower internodes (with 78.9% and 83.2% reduction) than in the internodes above the ear and towards the tassel. Similarly reduced internode lengths were also observed in o1-tan62 mutants ( Supplementary Figure 5B). Thus, the plant height reduction in the o1-2995 and o1-tan62 mutants is due to impaired internode elongation. O1 functions in leaf cell expansion and leaf development In side-by-side field growth experiments comparing single mutants of the antimorphic o1-2995 allele and the null alleles o1-ref and o1-N1242A, we observed generally smaller and narrower leaves in all mutants as compared to their normal siblings. Mirroring the trend observed for plant height, length of leaf blades and sheaths, and width of leaf blades were slightly reduced in the null mutants, and more so in o1-2995 antimorph mutants ( Figure 4C ). To determine the basis of small leaf size we examined o1-2995 mutants by making abaxial leaf epidermal impressions from leaf 16 using super-glue and observed the impressions under a compound microscope. Compared to their wildtype siblings, o1-2995 mutants had significantly more cells per microscope field of view ( Supplementary Figure 6D ) due to reduced cell size ( Supplementary Figure 6C ) . Thus, the o1-2995 mutation impairs cell expansion and growth. In addition, o1-2995 mutants displayed aberrant leaf patterning. Especially when grown in the field, o1-2995 mutants had ectopic tissue outgrowths extending from the ligule into the leaf midrib ( Supplementary Figure 7A-7C). To further investigate if o1-2995 mutation also affected vein development, we examined acidified phloroglucinol stained cross sections of distal midribs of adult leaf 10 under a dissecting microscope. Mutants had markedly reduced volume of clear cells, the tissue located adaxial to the midvein that comprises the bulk of the midrib, accordingly reduced midribs, and smaller veins compared to their wild type siblings ( Supplementary Figure 7D-7G). The ectopic outgrowths were not observed in o1-2995 or o1-tan62 mutants grown in the greenhouse, or in any o1 null mutants grown under any conditions. These data indicate that the antimorph o1-2995 allele disrupts ligule and midrib patterning and development, and does so more severely under field grown conditions. o1 antimorph alleles show severe subsidiary cell division defects o1 null mutant alleles ( o1-ref and o1-N1243 ) were reported to cause kernel opaqueness, impaired endoplasmic reticulum (ER) motility, irregular protein body shape, and aberrant stomatal subsidiary cell division ( Nan et al., 2023 ; Wang et al., 2012 ). We showed previously that o1 null mutants have abnormal subsidiary cells and defective phragmoplast guidance ( Nan et al., 2023 ). From epidermal glue impressions of leaf 4 the o1-ref allele had 20% abnormal subsidiary cells, similar to previously published results, ( Nan et al., 2023 ), while plants homozygous for the antimorph o1-2995 allele had ∼45% abnormal subsidiary cells ( Figure 5A to 5F ). o1-N1242A mutants had ∼20% abnormal subsidiary cells (n=11 plants), also similar to previous reports (Nan et al. 2023) , while o1-tan62 mutants had ∼30% abnormal subsidiary cells (n=15 plants) in juvenile leaf 2 ( Supplementary Figure 3A). Thus, both antimorph alleles o1-2995 and o1-tan62 more severely disrupt cell divisions for subsidiary cells than o1 null mutants. Download figure Open in new tab Figure 5: o1 mutants exhibit abnormal subsidiary cell division. (A) . Abnormal subsidiary cells were counted in B73 wildtype, o1-2995 , o1-ref and their corresponding wildtype siblings. Genotypes that are not significantly different at p=0.05 via two-sided t-test are joined noted by the same letter. Different letters indicate significantly different. Methacrylate impressions on leaf 4 of ( B ) B73 ( C ) o1-ref/+ and ( D ) o1-ref , ( E ) o1-2995/+ and ( F ) o1-2995 . Abnormal cells are highlighted in brown. To determine when the asymmetric division of the subsidiary mother cell becomes misoriented, o1-2995 and o1-tan62 were crossed to a live cell marker for microtubules (YFP-TUBULIN or CFP-TUBULIN ( Mohanty et al. 2009 ) ). Then, three wild-type siblings and three mutants were dissected to reveal the asymmetric division zone and imaged. In wild-type siblings, premitotic microtubule structures were oriented correctly ( Figure 6A ). Similarly, spindles and preprophase bands in o1-2995 and o1-tan62 were similar to wild type. ( Figure 6B and 6C ). In o1-2995 and o1-tan62 mutants, phragmoplasts were often misoriented (∼60% for both). These data indicate that o1-2995 and o1-tan62 exhibit phragmoplast guidance defects, similar to, but more severe in frequency than, o1 null mutants previously described ( Nan et al., 2023 ). Download figure Open in new tab Figure 6. o1-2995 and o1-tan62 have phragmoplast guidance defects. Representative micrographs of each cell cycle stage in plants expressing a TUBULIN fluorescent marker. Three plants of o1-2995, o1-tan62, and their respective wild-type siblings were analyzed. For prophase, counts indicate total number of cells with normal preprophase bands. Wild type sibling of o1-2995 : n=442/442 cells; wild-type sibling of o1-tan62 : n=501/503 cells. For metaphase, counts indicate the total number of cells with normal spindle morphology. Wild type sibling of o1-2995 : n=80/83 cells; wild-type sibling of o1-tan62 : n=47/47 cells. For telophase, count in wild type indicates the total number of oriented phragmoplasts. Wild type sibling of o1-2995 : n=90/91 cells; wild-type sibling of o1-tan62 : n=72/72 cells. Telophase cell count in o1-2995 and o1-tan62 indicates total number of cells with misoriented phragmoplasts. Yellow arrows = oriented phragmoplast. Red X = misoriented phragmoplast. Scale bar = 10 µm. Discussion Similar to o1 mutants, triple and quadruple mutants in class XI myosins in Arabidopsis generate smaller plants, with reduced cell expansion, short roots, aberrant organelle movement and division plane positioning defects ( Peremyslov et al. 2010 ; Madison et al. 2015 )(Abu-Abied et al. 2018; Huang et al. 2024 ) . Often, myosin XI mutants have shortened root hairs or pollen tubes indicating cell elongation defects ( Madison et al. 2015 ; Peremyslov et al. 2015 ) . In Physcomitrium patens, RNAi directed at the only two myosin XIs generated very small plants with round cells also indicating a critical role in cell elongation ( Vidali et al. 2010 ) . In contrast, single myosin XI mutants ( myosin XI-I ) in Arabidopsis have more subtle phenotypes: nuclear migration defects that generate aberrant division planes during stomatal development that reduce stomatal density ( Muroyama et al. 2020 ) and nuclear shape and movement defects (Tamura et al. 2013) . Defects in nuclear migration/polarization during subsidiary cell development were not detected in the o1 mutant in maize, but both ER organization and subsidiary cell division positioning were aberrant ( Nan et al. 2023 ; Wang et al. 2012 ) . The o1 mutant was originally identified due to the opaque kernel phenotype, which is caused by aberrant protein body accumulation in the endosperm ( Wang et al. 2012 ) . The O1 protein associates with the ER, and also as puncta in the cytoplasm and in the phragmoplast midline ( Nan et al. 2023 ; Wang et al. 2012 ) . Overexpression of the tail domain disrupts ER motility in tobacco cells ( Wang et al., 2012 ). Plant class XI myosins are processive motors that dimerize and transport cargo and promote cytoplasmic streaming ( Tominaga and Nakano 2012 ; Tominaga et al. 2003 ) . Overexpression or ectopic expression of truncated myosins lacking the motor domain has a dominant-negative effect that impairs myosin activity in vivo, potentially due to dimerization ( Stephan et al. 2021 ; Peremyslov et al. 2008 ; Sparkes et al. 2008 ; Avisar et al. 2008 ; Wang et al. 2012 ; Avisar et al. 2009) . As a motor, Arabidopsis Myosin XI-I has unique features, including a high affinity for actin with corresponding low velocity and ATPase activity compared to other myosin XIs ( Haraguchi et al. 2016 ) . However, individual myosins are likely to have different actin binding affinities, cargo, and velocities, and therefore different (but potentially overlapping) cellular functions. These diverse cellular functions may translate into different phenotypes on the tissue, organ and plant level. Previous immunoprecipitation experiments show that O1 interacts with other myosins, including Myosin XI-K, XI-F and XI-G family members as well as multiple myosin VIIIs ( Nan et al. 2023 ) . Notably, O1 has now been identified in four genetic screens, with three distinct phenotypes ( Nan et al. 2023 ; Wang et al. 2012 ) . To the best of our knowledge, no other maize gene encoding a myosin has been identified in a genetic screen. This implies that O1’s function may be unique - either because of its intrinsic properties, such as binding affinity or cargo - or because it heterodimerizes with many different myosins. O1 also interacts with actin binding proteins, heat shock interacting proteins and kinesins that are related to PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 ( Nan et al. 2023 ; Wang et al. 2012 ; Müller et al. 2006 ) . Determining the scope of dimerization and cargos across different myosins in future studies may help elucidate O1 and myosin function in general. Our current hypothesis is that both missense mutant alleles described in this paper generate proteins with impaired function of a motor domain activity, such as actin binding or the ATPase activity required for movement. We predict that O1-2995 and O1-TAN62 mutant proteins, despite predicted defects in motor domain function, would still be capable of interacting with multiple other proteins, including both myosin XIs and myosin VIIIs ( Nan et al. 2023 ) , and cargoes, as outlined in our speculative model ( Figure 7 ). The o1-2995 mutant is recessive: no antimorph effect is seen in either simple ( O1/o1-2995 ) or compound ( o1-ref/o1-2995 ) heterozygous plants. We speculate that the recessive nature of o1-2995 reflects insufficient expression levels of the mutant form of the protein. Thus, the more severe mutant phenotype of homozygous o1-2995 or o1-tan-62 relative to o1 null mutants could be due to O1 mutant proteins binding to and reducing activity of other myosins ( Figure 7 ) or even other binding partners, potentially including cargo. It is plausible that the antimorph effects are only observed in homozygous o1-2295 mutants due to dosage effects, i.e., increased expression of o1-2995 coupled with the absence of functional O1 may sufficiently increase the relative abundance of poisoned O1-containing protein complexes to enhance the plant growth and cell division positioning phenotypes. Download figure Open in new tab Figure 7. Model of O1 cellular function across different genotypes. Myosin VIII (brown) and XI (green) polypeptide monomers contain a motor head, a neck region containing IQ domains and a tail region with coiled coil domains involved in cargo binding. Myosin XI monomers (including O1, light green) also contain a dilute domain in the tail region. The defective motor head of the O1 antimorph monomer is indicated by a misshapen motor with a solid outline. Each main panel illustrates one genotype and its expressed myosin dimers bound to actin microfilaments (gray), and whether the dimers are predicted to have functional motor activity (arrows) or not (double parentheses). Though depicted as such, antimorph-containing dimers may or may not bind to actin. Top Left In wild type plants, O1 homodimerizes and heterodimerizes with other Myosin XI isoforms and with Myosin VIII. Relative numbers of hetero- and homodimers in vivo is unknown; this panel illustrates two “copies” of each dimer type. Each myosin dimer will have associated cargo (not shown), which could be specific or shared with other dimers. Top Center In o1 null heterozygotes, the frequency of dimers containing O1 decreases, but all dimer types are present. The plants are phenotypically wild type. Bottom Center Complete loss of O1 in o1 null mutants eliminates any complex containing O1, leading to fewer dimer types and an intermediate plant phenotype. Top Right In o1 antimorph heterozygotes, O1-2995 and O1-tan62 proteins have defective head domains, leading to “poisoned” complexes. Nonetheless, these alleles behave as fully recessive and not as dominant negatives, i.e. heterozygotes are phenotypically normal, plausibly because some non-poisoned homo- and hetero-dimers form which are sufficient for function. Bottom Right In antimorph homozygotes, there are only non-functional O1 homodimers and more poisoned heterodimers, leading to a stronger phenotype than the null mutants. This may be due in part to disabling of functional complexes (i.e., binding to other myosins) or to sequestering cargo (i.e., binding to other proteins, not shown). Bottom Left Plants that have one antimorph allele and one null allele have an intermediate phenotype similar to the null homozygote, suggesting a dosage requirement for poisoned complexes to manifest a strong phenotype. Severity of the subsidiary cell division defects correlated with defects in plant height, but the relationship between cell division defects and plant height is unclear. One potential hypothesis is that gas exchange defects might lead to short plants. However, while aberrant subsidiary cells disrupt the closure of guard cells, either mild or no gas exchange defects were observed in other mutants with aberrant subsidiary cells including pangloss1 (pan1), pan2, or the pan1 pan2 double mutant ( Liu et al. 2024 ) . Alternative and more plausible hypotheses posit that O1 has multiple functions: one required for cell expansion and another that affects subsidiary cell division. Cell expansion defects could be enhanced by additional mechanical stresses generated via aberrantly shaped cells caused by division positioning defects ( Sampathkumar et al. 2014 ) . Materials and methods Genetic stocks and phenotypic characterization The rsl*-12.2995 ( o1-2995 ) mutant allele originated from a ramosa1 ( ra1 ) modifier screening population generated via ethyl methane sulfonate (EMS) pollen mutagenesis. ra1-63 was backcrossed six times to Mo17 to create a homozygous mutant stock. Pollen from mutants was collected, treated with 0.06% EMS in paraffin oil as described ( Weeks 2013 ) and used to pollinate the same genotype. We screened for dominant modifiers in the M1 generation, and then the remaining plants were self-fertilized. The resulting M2 populations were screened for recessive modifiers, where we identified rsl*-12.2995 ( o1-2995 ) as a mutant that segregated 3:1 and displayed short plant stature with suppressed inflorescence branching. To understand its penetrance and expressivity in diverse genetic backgrounds we backcrossed the o1-2995 allele to B73, Mo17, and W22 inbred lines at least five times. B73-introgressed stocks for other mutants were sourced from the authors’ labs for ra1-R (EV), ra2-R (EV), o1-ref (MF) and o1-N1242A (MF), or from David Jackson (Cold Spring Harbor Laboratory) for ra3-ref . Alleles of o1-ref and o1-N1242A were obtained from the Maize Genetics Cooperation Stock Center, and introgressed into B73 four times before crossing to o1-2995 . o1-tangled62 ( o1-tan62 ) was identified from an EMS mutagenesis performed on B73 kernels. The M0 generation was open-pollinated, followed by self-fertilization of the M1 generation. Glue impressions of leaf 2 or 3 in the M2 generation were examined for division plane positioning defects. o1-tan62 was backcrossed to B73 at least three times before sequencing the O1 locus. Map-based cloning, sequencing, linkage analysis, and complementation test To identify the causative mutation lesion responsible for the rsl*-12.2995 ( o1-2995 ) mutant phenotype ( Zebosi 2022 ), we used map-based cloning methods with an F2 mapping population developed between Mo17 and B73. A bulked segregant analysis using genotyping-by-sequencing (BSA-GBS) method was devised and implemented as follows. A [B73 x Mo17] F2 mapping population was sown and leaf tissue samples collected as two pools bulked from mutant (n=12) and normal sibling (n=26) plants. 150-300 leaf punches (6 mm) were collected for each pool, with an equal number of punches collected from each individual plant in the bulked sample. High molecular weight genomic DNA was extracted using a urea-based protocol ( Chen and Dellaporta 1994 ) . Genotyping-by-sequencing ( Elshire et al. 2011 ) was then adapted and applied to the DNA extracted from the two bulked pools (see ( Kokulapalan 2018 ) for more detail). Briefly, genomic DNAs were quantified using the Promega Quantifluor dsDNA system, and used in the BSA-GBS bench protocol consisting of three main steps. Namely, restriction digestion using ApeKI, ligation of a barcoded and a common adapter, and PCR amplification and cleanup to construct the barcoded sequencing library. Sequencing was performed using 100-bp single-end sequencing with Illumina HiSeq 2500 (Iowa State University, DNA facility). Sequencing Reads were de-multiplexed, separated into independent files for each bulked pool and clipped, all using fastq-mcf ( Aronesty 2013 ) . Reads were aligned to maize B73 RefGen_AGPv4 using minimap2 (Jiao et al. 2017; Li 2018 ) . Euclidean distance (ED) between the normal and mutant pools was calculated using nucleotide composition and used to calculate ED4 to suppress noise. ED4 distances were plotted on a Manhattan plot using ggplot2 to visualize differences between the pools ( Wickham 2016 ) . (BSA-GBS) roughly mapped the o1-2995 mutation to the long arm of chromosome 4 ( Supplementary Figure 2), within a peak containing approximately 989 genes. Using publicly available molecular markers, we further mapped the mutation to a region between markers IDP2 (18 recombinants, 2.24 cM) and IDP8018 (41 recombinants, 5.11 cM), and then with public SNPs to between BZ2995_2 (8 recombinants, 1 cM) and BZ2995_1 (2 recombinants, 0.25 cM), comprising a physical distance of 1.5Mb and 28 genes ( Figure 2A ). To further narrow down the location of the causal mutation, we used two fine-mapping populations (F2 and F1BC1) constructed by crossing o1-2995 mutants in the original Mo17 genetic background to the B73 inbred line. DNA was extracted as previously described ( Zebosi et al. 2025 ) . Using publicly available insertion/deletion polymorphisms between B73 and Mo17, we designed new markers to reduce the fine-mapping to the interval between IDP2 (18/802, 2.24 cM) and IDP8018 (41/802, 5.11 cM). To further narrow the interval, single nucleotide polymorphisms (SNPs) were used to design new markers that localized the mutation to a 1.5Mb interval between BZ2995_6 (8/802, 1 cM) and BZ2995_1 (2/802, 0.25 cM) with 28 genes ( Figure 2A ). In non-repetitive regions between markers BZ2995_6 and BZ2995_1, B73 and Mo17 contained no DNA sequence polymorphisms to advance the fine-mapping process. We therefore performed whole-genome sequencing (WGS) of a single mutant plant to identify EMS-induced SNPs in the interval for marker development and identifying potential candidate genes. The library was prepared and sequenced on an Illumina HiSeq by Novogene Inc., UC Davis, California, to generate 50Gb of 150-bp paired-end sequencing data. The raw reads were subjected to quality control using FASTQC ( Andrews 2010 ) and adapters, and reads with low base quality were trimmed using Trimmomatic version 0.32 ( Bolger et al. 2014 ) . The trimmed reads were aligned to the Mo17 genome using the ‘mem’ algorithm in BWA-MEM version 0.7.17 (Li and Durbin 2010) , and the resulting sequence alignment SAM files were converted to BAM files and filtered for uniquely mapped reads using Samtools ( Li et al. 2009 ) . SNP calling against the Mo17 genome was performed using the Samtools mpileup command, and homozygous non-Mo17 or EMS SNPs (G>A and C > T) in the 1.5Mb interval were identified. We identified 3 genes with exonic EMS SNPs, GRMZM2G521481, GRMZM2G091143, and GRMZM2G449909 ( Opaque1/O1 ). From the EMS-like SNPs we developed new markers and used them in F2 populations consisting of 2289 plants (both wildtype and mutants) to find six recombination events between the three candidate genes. Left-side recombinants (3 and 17) and right-side recombinants (9 and 16) suggested that the causal mutation was within a physical distance of ∼ 0.6Mb that contained only one gene, the previously cloned Opaque1 ( O1 ) ( Figure 2B ). We therefore phenotyped kernels from our segregating populations, and found an opaque kernel phenotype co-segregated with the o1-2995 mutants. We verified the six recombinants by test crossing each with o1-2995 mutants and phenotyping the progeny for kernel opaqueness and plant height. To validate Opaque1 as the causal locus, we performed a complementation test by crossing o1-2995 mutant to two o1 mutant alleles ( o1-ref/+ and o1-N1243/+ ). To sequence the O1 coding sequence in tan62 mutants, RNA was extracted from young leaf tissue of o1-tan62 homozygous mutants and wild-type plants using the RNeasy Plant Mini Kit (QIAGEN) and reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN). Then, the cDNA was amplified into two halves and sequenced using primers in Supplementary Table 1. PCR products were run on a gel and bands were gel purified using the QIAquick Gel Extraction Kit (QIAGEN). Seven PCR products spanning the entire coding sequence were sent for sanger sequencing at the Genomics Core Facility (University of California, Riverside). o1-tan62 and wild-type sequences were compared to each other and GRMZM2G449909 ( O1 ) by using A plasmid Editor ( Davis and Jorgensen 2022 ) and NCBI BLAST ( Altschul et al. 1990 ) . DNA from an additional 3 tan62 mutants was extracted, and sequenced PCR products confirmed the mutation. Complementation test crosses between o1-tan62 and o1-N1242A homozygous mutants were done in the field at the University of California, Riverside in the summer of 2021. Approximately 50 kernels each from 11 independent crosses were assessed for and had opaque kernel phenotype confirming that tan62 was allelic to o1 . Plant growth conditions Field-grown plants were grown in the summer maize nursery at Curtiss Farms, Iowa State University in Ames, Iowa or at University of California, Riverside (UCR) Agricultural Operations. UCR greenhouse growing conditions: maize kernels were planted in 3.8 liter pots with soil (20% peat, 50% bark, 10% perlite, and 20% medium vermiculite) supplemented with magnesium nitrate (50 ppm N and 45 ppm Mg), calcium nitrate (75 ppm N and 90 ppm Ca), and Osmocote Classic 3-4 M (NPK 14-14-14%, AICL SKU #E90550) with standard greenhouse conditions (31-33°C daytime temperature with supplemental lighting from 17:00-21:00 PM at ∼ 400 µEm -2 s -1 ) ( Uyehara et al. 2024 ) Phylogenetic and gene expression analysis Myosin amino acid sequences of Arabidopsis, maize, rice, Setaria, sorghum, and Physcomitrella were obtained from Phytozome ( Goodstein et al. 2012 ) . Sequence alignments were performed using ClustalW2 ( Larkin et al. 2007 ) and Mesquite software ( Maddison and Maddison, 2011 ). Approximate maximum-likelihood phylogenetic trees were constructed as described by (Zebosi et al. 2024) . Raw RNA sequence reads from several tissues were downloaded from the short read archive ( https://www.ncbi.nlm.nih.gov/sra ) and reads were analyzed as described ( Zebosi et al., 2024 ). Protein analysis Membrane proteins were extracted from the basal 0.5-3 cm of leaves from the 4-leaf stage and separated by SDS-PAGE, according to ( Facette et al. 2015 ) . The previously described rabbit antibody was generated by injection of O1-specific peptides ( Nan et al. 2023 ) and used at 0.66 micrograms/ml. Phenotypic characterization Phenotypic characterization was done using the o1-2995 allele or the o1-tan62 allele introgressed in B73 compared to their corresponding wild-type siblings. Agronomic traits such as plant height, total leaf number, leaf length and width, tassel length, tassel, and ear branch number were characterized using segregating populations. Plant height measurements were taken by measuring from the soil surface to the tip of the tassel at maturity. Total leaf number was tracked from germination and measured in mature plants. Peduncle length was measured from the flag leaf node to the lowest tassel branch. Tassel length was measured from the lowest tassel branch to the tip of the tassel. Internode length and diameter, and peduncle lengths were measured with a tape measure. For internode length, measurements were taken starting from the first internode closest to the tassel proceeding basipetally to the 12th internode from the tassel. Phloroglucinol-HCl staining For Phloroglucinol-HCl staining, midribs from adult leaf 10 were hand-sectioned with a double-edge razor, and sections were stained using a Phloroglucinol-HCl staining solution (Strable et al. 2017) . The stained sections were observed under a dissecting microscope (Leica MZ125) and imaged using a digital camera. Leaf epidermal impressions Leaf epidermal impressions of the abaxial surface were produced from mid-length between the ligule and leaf tip and mid-way between the leaf margin and the mid-vein using cyanoacrylate glue as described ( Allsman et al. 2019 ) .. The slides with the epidermal imprints were observed under the Olympus BX60 light microscope and imaged using a digital camera (Jenoptik C5). The size of epidermal cells and the number of aberrant subsidiary cells were quantified from three random viewable fields using ImageJ ( Rueden et al. 2017 ) . For o1-tan62 mutants and wild-type siblings, leaf epidermal impressions were taken from the abaxial side of the second leaf. Epidermal imprints were imaged with a light compound microscope (Nikon) with digital microscope camera attachment (AmScope MD130). Confocal Microscopy Images were taken of the asymmetric divisions of the subsidiary mother cell using a Yokogawa W1 spinning disk on a Nikon Eclipse TE inverted stand microscope with an EM-CCD camera (Hamamatsu 9100c). The ASI Peizo stage and 3 axis DC servo motor controllers were controlled using Micromanager software ( www.micromanager.org ). Solid-state Obis lasers (power from 40 to 100 mW) were used in combination with standard emission filters (Chroma Technology). For CFP-ꞵ-TUBULIN, a 445 laser with emission filter of 480/40 was used. For YFP- ɑ -TUBULIN, a 514 laser with emission filter 540/30 was used. o1-2995 , o1-tan62 , and their wild-type siblings, expressing CFP-ꞵ-TUBULIN or YFP- ɑ -TUBULIN ( Mohanty et al. 2009 ) , were grown in the greenhouse under standard conditions as described ( Uyehara et al. 2024 ). Four week-old plants were dissected to an emerging leaf with ligule height < 2 mm, and the asymmetric dividing zone (within 1.5 cm from the ligule) was mounted in a rose chamber ( Rasmussen 2016 ) . Micrographs of asymmetric divisions of the subsidiary mother cell were captured along the entirety of the section and subsequently analyzed using the FIJI version of ImageJ ( Schindelin et al. 2012 ) . Data Analysis and Figure Preparation Graphs and statistics were done using the R software environment for statistical computing and graphics ( https://www.R-project.org/ ) and RStudio software https://posit.co/ using several packages. Some figures were assembled using the Gnu Image Manipulation Program (Gimp, version 2.10.38, https://www.gimp.org ). Accession numbers Opaque1 (O1): GRMZM2G449909 (B73 RefGen_v3), Zm00001d052110 (Zm-B73-REFERENCE-GRAMENE-4.0), Zm00001eb193160 (Zm-B73-REFERENCE-NAM-5.0) Ramosa1 (Ra1): GRMZM2G361210 (B73 RefGen_v3), Zm00001d034642 (Zm-B73-REFERENCE-GRAMENE-4.0), Zm00001eb062570 (Zm-B73-REFERENCE-NAM-5.0) Ramosa2 (Ra2): AC233943.1_FG002 (B73 RefGen_v3), Zm00001d039694 (Zm-B73-REFERENCE-GRAMENE-4.0), Zm00001eb123060 (Zm-B73-REFERENCE-NAM-5.0) Ramosa3 (Ra3): GRMZM2G014729 (B73 RefGen_v3), Zm00001d022193 (Zm-B73-REFERENCE-GRAMENE-4.0), Zm00001eb327910 (Zm-B73-REFERENCE-NAM-5.0) Author contributions E.V. conceptualized the project; B.Z., S.E.M, and E.V. curated data; B.Z. and S.E.M. performed formal analysis; S.E.M., G.S.B., M.F., C.R. and E.V. acquired funding; B.Z., S.E.M., K.W., J.S., G.S.B., N.B.B., M.F. and E.V. performed experiments and/or collected data; C.R. and E.V. administered and supervised the project; M.F., C.R. and E.V. provided material resources for study; K.W. developed software; B.Z., S.E.M. and K.W. performed validation; B.Z., S.E.M., K.W., N.B.B., M.F., C.R. and E.V. prepared figures for visualization; B.Z. wrote the original draft of the manuscript; B.Z., S.E.M., K.W., M.F., C.R. and E.V. reviewed and edited the manuscript. Acknowledgments We want to thank Connor Hamers (ISU), Jack Schwickerath (ISU), Nicole Essner (ISU), David Wetovic (UCR), and Lindy Allsman (UCR) for their help with the summer fieldwork, and the Curtiss Farms (ISU) and Agricultural Operations (UCR) staff for maize genetics nursery efforts. We thank Colin Finnegan (ISU) and Fred Roger Namanda (ISU) for help with plant phenotyping, Erica Unger-Wallace (ISU) for training in laboratory methods and Professor David Nelson (UCR) for discussing genetics. Thanks to the Maize Genetics Cooperation Stock Center for providing maize kernels. This work was supported by the NSF-PGRP 1238202 to E.V., NSF-CAREER 1942734 and NSF-2426623 to C.G.R., NSF NRT Plants3D (DBI-1922642) to S.E.M. and G.S.S. Funder Information Declared NSF , 1238202 , 1942734 , 2426623 , DBI-1922642 Footnotes bzebosi{at}iastate.edu , smart046{at}ucr.edu , kokul{at}bioinformapping.com , jssengo{at}iastate.edu , gsala014{at}ucr.edu , norman.best{at}usda.gov , mfacette{at}umass.edu , crasmu{at}ucr.edu , Literature Cited Abu-Abied M , Belausov E , Hagay S , Peremyslov V , Dolja V , Sadot E ( 2018 ) Myosin XI-K is involved in root organogenesis, polar auxin transport, and cell division . J Exp Bot 69 : 2869 – 2881 OpenUrl CrossRef PubMed ↵ Allsman LA , Dieffenbacher RN , Rasmussen CG ( 2019 ) Glue impressions of maize leaves and their use in classifying mutants . Bio-protocol 9 : e3209 OpenUrl ↵ Altschul SF , Gish W , Miller W , Myers EW , Lipman DJ ( 1990 ) Basic local alignment search tool . J Mol Biol 215 : 403 – 410 OpenUrl CrossRef PubMed Web of Science ↵ Andrews S ( 2010 ) FastQC: quality control tool high throughput sequence data . 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