Fern and gymnosperm SPCH/MUTE and FAMA can regulate multiple cell fate transitions during stomatal development

Fern and gymnosperm SPCH/MUTE and FAMA can regulate multiple cell fate transitions during stomatal development | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Fern and gymnosperm SPCH/MUTE and FAMA can regulate multiple cell fate transitions during stomatal development View ORCID Profile Miki Zaizen-Iida , View ORCID Profile Kaotar Elhazzime , View ORCID Profile Anne Vatén doi: https://doi.org/10.1101/2025.11.10.687565 Miki Zaizen-Iida 1 Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences and Viikki Plant Science Centre, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Miki Zaizen-Iida Kaotar Elhazzime 1 Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences and Viikki Plant Science Centre, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kaotar Elhazzime Anne Vatén 1 Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences and Viikki Plant Science Centre, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anne Vatén For correspondence: anne.vaten{at}helsinki.fi Abstract Full Text Info/History Metrics Preview PDF Abstract Stomatal development driving bHLHs are conserved — it is likely that the genome of the first plant with stomata already carried a single gene ancestral to these genes. It has been proposed that the ancestral bHLH gene may have duplicated and gained novel functions which lead to the stage-specific functions characterizing three angiosperm stomatal master regulators, SPEECHLESS (SPCH), MUTE, and FAMA. However, it has remained unexplored when and how these stage-specific functions evolved. Here, we perform the first functional analysis of stomatal regulators from non-angiosperm vascular plants by using fern Ceratopteris richardii and gymnosperm Picea abies as model species. We show that the genomes of these species contain a single gene encoding SPCH/MUTE (SM), and another gene encoding FAMA. These factors are likely to share a common ancestral gene with angiosperm SPCH and MUTE , and angiosperm FAMA , respectively. Our work shows that C.fern and spruce SM and FAMA can promote gene functions resembling angiosperm SPCH and MUTE , and FAMA functions, respectively. However, all the four proteins display ability to drive asymmetric entry divisions in the stomatal lineage characteristic previously associated specifically to angiosperm SPCH. Further, PaSM is sufficient to promote stomatal pore formation, suggesting that it can control subset of FAMA target genes. Our work demonstrates that SM and FAMA in early evolved vascular plants already show functional divergence thus resembling angiosperm stomatal regulators, however, they also display multifunctional properties. Introduction Appearance of stomata, tiny adjustable valves locating at the shoot epidermis, allowed early land plants to control evaporation of water and acquisition of CO 2 and thus, likely enabled expansion of vegetation to dry environments. Stomatal development is highly conserved — most likely stomata evolved only once, and the genome of the first plant possessing stomata already contained a single ancestral gene that later gave rise to the genes required for stomatal development in present-day plant species. Although core stomatal development is conserved, stomatal pattern and density are not; these characteristics show rich diversity among plant groups and even closely related species can show distinct stomatal pattern 1 , 2 . On the other hand, stomatal development is plastic; many species can adjust stomatal numbers during organ development according to environmental conditions allowing modulation of the gas-exchange capacity 3 . Since stomata physiology is closely connected to optimized use of resources, regulation of stomatal development can be also used to generate plants with improved water-use efficiency 4 . Stomatal development is driven by basic Helix-loop-helix (bHLH) transcription factors which belong to the bHLH subgroup 1a (bHLH1a). In angiosperm model plant, Arabidopsis thaliana , three genes encoding bHLH transcription factors SPEECHLESS (SPCH), MUTE, and FAMA control stomatal development 5 – 7 . SPCH initiates the stomatal lineage by driving asymmetric meristemoid divisions, MUTE promotes guard mother cell fate, and FAMA establishes guard cell identity. It has been proposed that the ancestral bHLH gene has gained novel function through duplications and functional diversification allowing the stage-specific regulation of stomatal development observed in angiosperms 8 , 9 . Also, an alternative hypothesis has been presented based on phylogenetic analysis; ancient stomatal development toolkit present in early land plants may have been already similar to present-day angiosperms. However, some plant lineages may have lost some of the regulatory genes, resulting in reduced stomatal complexity 10 . Stomatal bHLHs have been experimentally studied in moss Physcomitrium patens 11 , 12 and set of angiosperm model species 13 – 15 , however, their functions in other non-angiosperm species have not been investigated. Thus, when and how the stage-specific regulation of stomatal development evolved has remained elusive. Using Picea abies , a gymnosperm, and Ceratopteris richardii , a fern, as a model species, we demonstrate that stomatal regulators can display multifunctionality and stage-specificity at the same time. We show that, within both fern and gymnosperm groups, stomatal regulators display capacity to the stage-specific functionality similar to angiosperm stomatal regulatory factors. However, they also exhibit a limited degree of multifunctionality, particularly within the early stomatal lineage, suggesting that strict stage-specificity may have evolved only in the angiosperm lineage. Our data suggests that transcriptional regulation plays a central role in guiding of developmental transitions within the stomatal lineage of gymnosperms. Although PaSM and PaFAMA show some aspects of multifunctional characteristics, their expression window is limited to early and late stomatal lineage within spruce, respectively. We hypothesize that duplication of a gene ancestral to PaSM may have enabled, for the first time, independent transcriptional regulation of the two genes carrying out SPCH/MUTE functions. It is possible that presence of separate, independently regulated expression may have enabled or accelerated the emergence of functional divergence, leading to the distinct SPCH and MUTE functions observed in angiosperms. Results Norway spruce genome contains two genes encoding bHLH1a members To identify bHLH1a coding genes in Picea abies (Norway spruce), we performed a phylogenetic analysis and constructed a corresponding phylogenetic tree. We found that spruce genome contains a single SPCH/MUTE -like gene ( PaSM ; MA_120602g0010) and a FAMA -like gene ( PaFAMA ; MA_57244g0010) ( Fig.1 a ). We next compared the domain structures of Arabidopsis thaliana and Picea abies SPCH, MUTE, and FAMA orthologues ( Fig. 1b ). Both PaSM and PaFAMA contain the conserved bHLH1a and C-terminal domains characterizing the subgroup 1a transcription factors 16 , 17 . Additionally, PaSM has a truncated MITOGEN-ACTIVATED PROTEIN KINASE target domain (MAPKTD), a region important for the regulation of AtSPCH protein stability 18 . Because AtSPCH variants lacking MAPKTD phosphorylation sites can promote guard mother cell (GMC) differentiation similar to MUTE 19 , we predicted that PaSM function may resemble that of MUTE. However, we noticed that PaSM lacks a MUTE unique domain, which function has not been defined. PaFAMA contains FAMA-specific domains as well as the LxCxE motif, which is essential for the interaction with RETINOBLASTOMA-RELATED (RBR) protein in Arabidopsis 20 . Based on the domain structures, we hypothesized that PaSM may functionally resemble both SPCH and MUTE or alternatively either SPCH or MUTE, whereas PaFAMA is likely to retain FAMA-like functions. Download figure Open in new tab Fig. 1. Phylogenetic tree showing single SPCH, MUTE, and FAMA orthologues in Norway spruce. a , A maximum-likelihood bHLH phylogenetic tree of subfamilies Ia. As, Alsophila spinulosa (fern); Ath, Arabidopsis thalinana (dicot); Atr, Amborella trichopoda (basal angiosperm); Cr, Ceratopteris richardii (fern); Gb, Gingko biloba (gymnosperm); Mp, Marchantia polymorpha (liverwort); Os, Oriza sativa (monocot); Pa, Picea abies (gymnosperm); Pl, Pinus lambertiana (gymnosperm); Pm, Pseudotsuga menziesii (gymnosperm); Pp, Physcomitrella patens (moss); Sm, Selaginella moellendorffii (lycophyte). Background color shows each clade. Red, FAMA/SMF clade; green, SPCH/MUTE clade; yellow, MYC67/70 clade; blue, TEC1/6 clade. The genes shown in red indicate the SPCH, MUTE, and FAMA orthologues identified in previous studies. b , Comparison of bHLH1a subfamily domain structures in tracheophyte. Each bHLH1a gene has bHLH domain (yellow) and C-terminus conserved domain (purple). AtSPCH and PaSM have mitogen-activated protein kinase target domain (MAPKTD) (red), although the domain in PaSM is truncated. Green shows the putative MAPK target motif and orange shows putative SnRK2 target motif. AtMUTE has MUTE specific domain (blue). CrSM has neither MAPKTD nor MUTE specific domain. FAMA orthologues have FAMA specific domain (pink) and extension of bHLH domain (brown). Conserved and distinct features of stomatal development in Norway spruce We next investigated stomatal development in Norway spruce needles. Stomatal development proceeds from the base towards the tip of the needle and stomata formation is restricted to the specific cell files ( Fig. S4a ). Spruce needles are rhombic in cross-section, with each of the four faces containing up to three stomata forming cell files. We observed occasional stomatal cell file switching. To analyze early developmental stages of stomatal development, we sampled young needles located near the vegetative meristem of spruce sapling. Because stomatal development was largely complete in needles that are longer than 10 mm, we focused on the needles that are shorter than 10 mm ( Fig. 2a ). In Norway spruce, protodermal cells underwent morphologically symmetric division ( Fig. 2b, g ). After the division, one daughter cell became elongated and squared while the other daughter became rounded ( Fig. 2c, h ). These observations suggest that the cell fate asymmetry is established right before, during or soon after the division. Tracing the stomata forming cell file from the maturing stomata towards younger cell stages, we reasoned that the elongated and squared cell was most likely GMC, whereas the rounded cell was a non-stomatal daughter cell. Previous study in pines has shown that protodermal cells undergo equal division and their daughter cells differentiate into a GMC and a polar subsidiary cell (SC), respectively 21 . Thus, we refer to non-stomatal daughter cells as polar SCs in spruce as well. During GMC differentiation, the cells in the non-stomatal row adjacent to the GMC differentiated into a lateral subsidiary mother cell (SMCs). Later, GMC and lateral SMCs divided symmetrically and asymmetrically, respectively, and created a pair of guard cells (GCs) and the SCs ( Fig. 2d, i ). In Brachypodium distachyon , the GMC division occurs after the lateral SC recruitment 22 ; therefore, we examined whether lateral SMCs divide earlier than GMCs in Norway spruce. There was no consistency in the order of the division: GMC divided earlier in 14 out of 30 observed stomatal complexes. After the GMC and lateral SMC divisions, GCs started to sink down into the hypodermal layer ( Fig. 2e, f, j, k ). In mature stomatal complex, GCs were completely sunken in the hypodermal layer similar to other conifers 21 . These observations suggest that spruce stomatal development includes both conserved features—fate asymmetry formation, GMC differentiation and division, and GC formation, however, there are also distinct features, such as recruitment of several SCs and translocation of GCs from epidermis to subepidermal position. Download figure Open in new tab Fig. S1. bHLH1a orthologues in C.fern and spruce have a capacity to promote entry division and enhance the crack between epidermal cells a–f , The abaxial surface of 10-day-old cotyledons of Col-0 ( a ), pSPCH:PaSM/spch-3 ( b ), pSPCH:CrSM/spch-3 ( c ), pSPCH:PaFAMA/spch-3 ( d ), pSPCH:CrFAMA/spch-3 ( e ), and pSPCH:PaSM/spch-3 × mute ( f ). The spch mutant background was homozygous in a, b, c, e , and f , and unknown in d . The mute mutant background was homozygous in f. g–n , Confocal images of 2-day-old abaxial cotyledons of Col-0 ( g ), spch-3 mutant ( h ), pSPCH:PaSM/spch-3 ( j ), pSPCH:PaFAMA/spch-3 ( k ), pSPCH:CrSM/spch-3 ( l ), and pSPCH:CrFAMA/spch-3 ( m, n ). o–u , DIC images of 10-day-old ( o ) and 5-day-old ( p–u ) abaxial cotyledons of pSPCH:CrSM/spch-3 , weakly expressing line ( o ), Col-0 ( p ), spch-3 mutant ( q ), pSPCH:PaSM/spch-3 ( r ), pSPCH:PaSM/spch-3 x mute ( s ), pSPCH:CrSM/spch-3 , strongly expressing line ( t ), and pSPCH:CrFAMA/spch-3 ( u ). White dashed lines indicate the boundary between epidermal and mesophyll cells. Magenta, propidium iodide; white: SR2200. Scale bar, 10 um ( g–n, o ), 500 µm ( p–u ). Download figure Open in new tab Fig. S2. Both C.fern and spruce bHLH1a orthologues partially complemented mute mutant a, b , Confocal images of 10-day-old ( a ) and 2-day-old ( b ) abaxial cotyledons of pMUTE: CrSM-3x venus-OCST/mute-1 ( a ) and pMUTE:AtMUTE-CFP (b). c , Number of pairs of stomata at 10-day-old abaxial cotyledon of pMUTE:CrSM /mute-1 . d–g , Ratio of cell types at 10-day-old abaxial cotyledon of pMUTE:PaSM /mute-1 ( d ), pMUTE:PaFAMA /mute-1 ( e ), pMUTE:CrSM /mute-1 ( f ) and pMUTE:CrFAMA /mute-1 ( g ). Download figure Open in new tab Fig. S3. FAMA orthologues effectively complemented fama mutant a, b , Confocal images of 10-day-old abaxial cotyledons of fama-1 ( a ) and pFAMA:CrSM /fama-1 ( b ). c , Stomatal index of abaxial cotyledon in 10-day-old pFAMA:CrFAMA/fama-1 seedlings. Both normal and abnormal stomata were quantified. d , e , Ratio of cell types at 10-day-old abaxial cotyledon of pFAMA:PaFAMA /fama-1 ( d ) and pFAMA:CrFAMA /fama-1 ( e ). f–g , Ratio of cell types of abnormal stomata at 10-day-old abaxial cotyledon of pFAMA:PaFAMA /fama-1 ( f ) and pFAMA:CrFAMA /fama-1 ( g ). c , Statistical significance was assessed using Steel-Dwass-Critchlow-Fligner test (* p < 0.05, ** p < 0.01, *** p < 0 . 001 ). Download figure Open in new tab Fig. S4. Spruce needles are rhombic in cross-section, with each of the four faces containing one to three stomatal cell files a , Confocal images of cross-section of 1 cm needle from 4.25-month-old Norway spruce sapling. Red circles indicate the stomatal cells. Download figure Open in new tab Fig. 2. Conserved and distinct features of stomatal development in Norway spruce. a , Longitudinal view of a young Norway spruce needle near the vegetative meristem. A 0.4-cm needle from a one-month-old sapling was imaged. Gray: SR2200. Scale bar: 1 mm. b–k , Detailed structures of young spruce needles. b– f , Cross-sections; g–k , lateral view. b, g , Symmetric division of stomatal precursor cells (pink). c, h , The divided protodermal cells differentiate into an elongated guard mother cell (GMC) and a round polar subsidiary cell (P). The cell adjacent to GMC will develop into lateral subsidiary mother cell (SMC, white asterisk). d, i , The GMC divides symmetrically to produce a pair of guard cells (GCs, yellow), while the SMC divides asymmetrically to generate lateral subsidiary cells (SCs, blue). e, j , After GMC division, GCs begin to sink into the hypodermal layer. f, k , GCs are located at the hypodermal layer. l–n , Characterization of developmental stages. Stage 1, 2 and 3 is characterized predominantly by asymmetric cell divisions (ACDs) in l , GMC differentiation and division in m and sinking stomata in n , respectively. o, p , qRT-PCR analysis at each developmental stage. Expression levels were normalized to Actin and presented relative to Stage 1. o , PaSM is preferentially expressed at Stage 1, with little or no expression at Stages 3 and 4 which is characterized by sinking and mature stomata. p , PaFAMA is predominantly expressed at Stage 2, with similar levels observed at Stages 1, 3, and 4. PaSM and PaFAMA show spatially distinct gene expression during Norway spruce stomatal development Our phylogenetic analyses suggested that PaSM and PaFAMA are candidate regulators controlling stomatal development in Norway spruce. To test whether their expression patterns correlate with specific stomatal developmental stages, we performed quantitative real time PCR (qRT-PCR) analysis. We cut spruce needles of similar sizes and grouped them accordingly. Using confocal microscopy, we then examined which developmental stages were predominant in each group. Stage 1 was characterized by prevalent asymmetric cell divisions (ACDs), although we observed also some differentiating and dividing GMCs in this region ( Fig. 2l ). Stage 2 contained mostly differentiating and dividing GMCs, however, some ACDs were also present ( Fig. 2m ). Stage 3 was dominated by sinking stomata; GMC divisions were occasionally observed, however, ACDs and GMC differentiation were completely absent ( Fig. 2n ). Stage 4 consisted of sinking guard cells. We noticed some morphological variation among needles of similar size, suggesting that needle length might not fully reflect the stomatal developmental stages. We measured expression levels of PaSM and PaFAMA across the stages 1-4 by using qRT-PCR. PaSM expression was most abundant at Stage 1 while its modest expression still persisted at Stage 2. Importantly, PaSM transcripts were not detected in Stages 3 and 4, with Ct values falling below the detection threshold ( Fig. 2o ). These results indicate that PaSM transcription is high during early stomatal development, particularly correlating with presence of ACDs and possibly also with GMC differentiation. In contrast, PaFAMA expression level was highest at Stage 2. Its transcripts were present at moderate levels during Stages 3 and 4. However, we noticed that some PaFAMA expressions were still present at the base of the needle, at Stage 1 ( Fig. 2p ). High PaFAMA expression peaks at Stage 2 and remains detectable in Stages 3 and 4 suggests that PaFAMA may act during GMC division and also may play a role in the regulation of guard cell maturation process. Taken together, these findings suggest that PaSM is expressed predominantly during early stages of stomatal development, whereas PaFAMA functions later, from GMC division through guard cell maturation. Considering also their domain structures, it seems possible that PaSM integrates the roles of both SPCH and MUTE or alternatively functionally substitute for one of them, whereas PaFAMA is likely to retain FAMA -like activity. Both PaSM and FAMA can act during early developmental stages To get more insight into the putative functions of PaSM and PaFAMA , we investigated whether they could replace AtSPCH, AtMUTE or AtFAMA . We introduced PaSM and PaFAMA in the Arabidopsis spch-3, mute-1 , and fama-1 mutants under the control of the respective AtSPCH, AtMUTE , or AtFAMA promoters and assessed their ability to restore stomata production and to rescue seedling lethality. Expression of PaSM under the AtSPCH promoter in spch -3, which normally produces an epidermis composed only of pavements cells 6 ( Fig. 3b ), resulted in frequent cell divisions, production of irregularly shaped stomata, occasional single GCs ( Fig. 3c ). Rescue of seedling lethality and seed production was also observed. However, we noticed that these lines displayed cracks between epidermal cells and occasionally partial detachment of the epidermis from inner layers ( Fig. S1b ), which prevented accurate quantification of stomatal number in 10-day-old seedlings. To mitigate this issue, we analyzed 5-day-old cotyledons, which exhibited fewer cracks ( Fig. S1r ) and verified that PaSM was sufficent to recover stomata production in the absence of AtSPCH ( Fig. 3i ). These results indicate that PaSM has ability to drive entry to stomatal lineage although with lower efficiency compared to AtSPCH . Download figure Open in new tab Fig. 3. Both PaSM and PaFAMA act during early developmental stages. a–d , DIC images of 10-day-old abaxial cotyledons of Col-0 ( a ), spch-3 mutant ( b ), pSPCH:PaSM/spch-3 ( c ), and pSPCH:PaFAMA/spch-3 ( d ). e–h , Confocal images of 10-day-old abaxial cotyledons of Col-0 ( e ), mute-1 mutant ( f ), pMUTE:PaSM/mute-1 ( g ), and pMUTE:PaFAMA/mute-1 ( h ). i , Stomatal number per 5-day-old abaxial cotyledon in pSPCH:PaSM/spch-3 seedlings. j , Stomatal number per 10-day-old abaxial cotyledon in pSPCH:PaFAMA/spch-3 seedlings. k–l , Stomatal density in 10-day-old abaxial cotyledons of pMUTE:PaSM/mute-1 ( k ) and pMUTE:PaFAMA/mute-1 ( l ). i–l , Statistical significance was assessed Steel-Dwass-Critchlow-Fligner test in i, k, l and Exact Wilcoxson rank sum test followed by Bonferroni multiple comparison correction in j (* p < 0.05, ** p < 0.01, *** p < 0 . 001 , different letters show statistically significant differences between two groups; p < 0.05). White, SR2200. Red asterisks indicate stomata; green triangles indicate single GC; blue asterisks indicate small cells. Scale bars, 10 µm ( a–c, e–h ), 500 µm ( d ). Next, we assessed whether PaFAMA could replace AtSPCH. PaFAMA expression under the control of AtSPCH promoter was sufficient to drive formation of stomata and stomatal lineage cells in the cotyledons of the spch-3 , albeit infrequently ( Fig. 3d ). Seedling lethality was occasionally rescued, however, the plant lines failed to produce seeds. We observed high variability in stomatal numbers across the cotyledon and between individual seedlings. To produce more accurate view of this phenotype we counted total number of stomata per cotyledon. Our analysis showed that three independent pSPCH:PaFAMA / spch-3 lines consistently produced stomata whereas spch-3 did not ( Fig. 3j ), indicating that PaFAMA can partially substitute AtSPCH . Because the unique function of AtSPCH among group 1a bHLHs is to promote ACDs, we next examined whether PaSM and PaFAMA can drive ACDs in the absence of AtSPCH . We observed abaxial epidermis of young cotyledons in the pSPCH:PaSM/spch-3 and pSPCH:PaFAMA/spch-3 lines and found that some of the divisions were physically asymmetric ( Fig. S1j, k ). These findings indicate that both PaSM and PaFAMA can promote fate asymmetry and in doing so, induce physically asymmetric stomatal lineage entry divisions. However, these two proteins show different ability to rescue seedling lethality, suggesting a difference in the functionality or structure of the stomatal complexes between these lines. We then introduced PaSM and PaFAMA into mute-1 mutant. While both pAtMUTE:PaSM/mute-1 and pAtMUTE:PaFAMA/mute-1 lines showed wild-type like stomata, they still retained some arrested meristemoids typical of mute mutant 5 ( Fig. 3f, g, h ). Additionally, we occasionally observed presence of small cells with unknown identity ( Fig. 3g, h ). In pAtMUTE:PaFAMA/mute-1 , we frequently found single guard cells, which were rarely observed in pAtMUTE:PaSM/mute-1 ( Fig. S2d, e ). We compared stomatal density (SD) between the wild-type, mute-1 , and mute-1 complemented by pAtMUTE:PaSM or pAtMUTE:PaFAMA ( Fig. 3k, l ). Two independent lines of both pAtMUTE:PaSM/mute-1 and pAtMUTE:PaFAMA/mute-1 showed significantly increased SD compared to the mute mutant, although SD remained lower than that of the wild-type ( Fig. 3k, l ). We defined relative proportions of stomatal cell types and found that pAtMUTE:PaSM/mute-1 showed higher relative number of stomata, whereas pAtMUTE:PaFAMA/mute-1 displayed higher relative numbers of meristemoids and single GCs ( Fig. S2d, e ). Both transgenes recovered defects in seedling viability and seed production observed in mute . Our data shows that both PaSM and PaFAMA can partially replace the function of AtMUTE , however, PaSM shows better rescue efficiency than PaFAMA , suggesting that PaSM is functionally more similar to AtMUTE than PaFAMA ,. PaSM can drive pore formation whereas PaFAMA promotes guard cell fate Finally, we explored whether PaSM and PaFAMA can drive the last cell fate transition in the stomatal lineage and replace the function of AtFAMA . Expression of PaSM in fama-1 under the control of AtFAMA promoter resulted in partially complemented seedlings that failed to produce seeds. We found clusters of small tumor-like cells resembling catepillar-like tumors present in the fama 7 ; however, their division orientation was altered compared to typical fama tumors ( Fig. 4c ). We also occasionally found stomata-like structures; they were composed of GC-like cells with additional divisions implying that their GC identity is unstable ( Fig. 4e ). We classified both fama tumor-like and stomata-like cells as “stomatal tumors” and quantified the proportion exhibiting pore-like characteristics. In fama mutant, tumors did not form pore-like characteristics whereas 15–20% of stomatal tumors in the pFAMA:PaSM / fama-1 lines displayed pore-like structures ( Fig. 4f ). These findings indicate that PaSM can initiate pore formation in the absence of AtFAMA , however, it cannot terminate guard cell divisions. Download figure Open in new tab Fig. 4. PaSM can drive pore formation whereas PaFAMA promotes guard cell fate a–e , Confocal images of 10-day-old abaxial cotyledons of Col-0 ( a ), fama-1 mutant ( b ), pFAMA:PaSM/fama-1 ( c ), pFAMA:PaSM/fama-1 ( d ), and an enlarged image of lectangel in c ( e ). Optical section of the z-stack image shows that stomatal tumor has a pore-like structure in e. f , Number of stomatal tumors having pores per total stomatal tumor in abaxial cotyledon in 10-day-old pFAMA:PaSM/fama-1 seedlings. g , h , Stomatal index of abaxial cotyledon in 10-day-old pFAMA:PaFAMA/fama-1 seedlings. Only normal stomata were quantified in g , and normal stomata and abnormal stomata were quantified in h. f–h , Statistical significance was assessed by Steel-Dwass-Critchlow-Fligner test (* p < 0.05, ** p < 0.01, *** p < 0 . 001 ). White, SR2200. Scale bars, 10 µm ( a–d ). In the pFAMA:PaFAMA/fama-1 lines, we observed both normal stomata and “abnormal stomata”, which is characterized by additional cell divisions within GCs ( Fig. 4d ). We first quantified the stomatal Index (SI) considering only normal stomata; one of three independent lines exhibited similar SI with wild-type ( Fig. 4g ). When both normal and abnormal stomata were included, two out of three lines displayed SI values that were not significantly different from wild-type ( Fig. 4h ). Abnormal stomata showed diverse structures; most abundant defects resembled the stomata-in-stomata phenotype 20 . Additional variants included: (i) parallel divisions along the stomatal pore axis within GCs, (ii) both parallel and vertical divisions in each GC within the same paired of GCs, and (iii) vertical divisions followed by extra divisions of the smaller daughter cell, producing structures reminiscent of fama -like tumors ( Fig. S3d, f ). This data shows that PaFAMA can replace function of AtFAMA , however, there are likely some species-specific differences causing the unstable differentiation in fraction of the guard cells. AtMUTE enhances ability of the PaSM to complement spch -3 Our cross-species complementation analysis shows that PaSM can partially replace AtSPCH or AtMUTE . To investigate genetic relationship of these genes, we generated spch-3 mute-1 double mutant and expressed PaSM under the control of AtSPCH promoter. Notably, the expression domain of the AtSPCH promoter largely overlaps with that of AtMUTE . The resulting plant line showed similar phenotype as pSPCH:PaSM/spch-3 lines, which showed aberrant stomata and cracks between epidermal cells ( Fig. S1f, s ), however, the pSPCH:PaSM/spch-3 mute-1 displayed fewer number of stomata per cotyledon than pSPCH:PaSM/spch-3 ( Fig. 3i ). This data shows that PaSM is indeed a multifunctional protein which can partially recover absence of both AtSPCH and AtMUTE . Since PaSM shares a common ancestral gene with AtSPCH or AtMUTE , it is likely that the ancestral gene already displayed some of the characteristics currently seen in AtSPCH and AtMUTE . Further, our analysis reveals that AtMUTE enhances the ability of the PaSM to complement spch -3 through unknown molecular mechanisms. Two C.fern orthologues also regulated stomatal development Our phylogenetic analysis revealed that, among gymnosperm, Pseudotsuga menziesii (Douglas fir) genome contains both SM and FAMA orthologues. Ginkgo biloba , which diverged early in the gymnosperm lineage, appears to lack an SM ortholog, and genome of Pinus lambertiana lacks both SM and F AMA orthologues, while it may reflect low-quality sequence data ( Fig. 1a ). To investigate the evolutionary origin of these two bHLH1a homologues, we searched for SM and FAMA orthologues in other taxa. We identified two bHLH1a orthologues in Ceratopteris richardii (C.fern): one that clustered with the SM clade, here called CrSM , and the other that clustered with the FAMA clade, here called CrFAMA . Domain structure analysis revealed that both genes contained the bHLH domain and the conserved C-terminal domain; CrFAMA additionally possessed the FAMA-specific domain and the LxCxE motif. We found neither the MAPKTD nor MUTE-specific domain in CrSM ( Fig. 1b ). We next tested whether CrSM and CrFAMA could functionally substitute for AtSPCH, AtMUTE , or AtFAMA in Arabidopsis through cross-species complementation. In some of the lines, expression of CrSM under the control of AtSPCH promoter in spch-3 mutants resulted in a phenotype resembling overexpression of AtMUTE ; nearly all epidermal cells were converted into stomata 23 ( Fig. 5c, m ). In other lines, cluster of stomata were observed ( Fig. S1o ). We interpret that former lines expressed CrSM at higher levels than in the latter lines. Especially in the lines with strong CrSM expression, we found that 10-day-old cotyledons were not suitable for quantification, as parts of the epidermis were peeled off ( Fig. S1c ). By contrast, CrFAMA expression in spch-3 led to an epidermis composed primarily of abnormal transdifferentiated cells, with only infrequent stomata ( Fig. 5d, 5n , Fig. S1u ). Loss of epidermal integrity was observed in pSPCH:CrFAMA/spch-3 seedlings as well ( Fig. S1e ). Since the loss of epidermal integrity became more severe as development progressed, both lines were quantified at 5-day-old, when less peeled off epidermis was observed. Both pSPCH :CrSM/spch-3 and pSPCH:CrFAMA/spch-3 complementation lines retained seeds production ability. Download figure Open in new tab Fig. 5. C.fern orthologues show similar complement ability to Norway spruce stomatal genes a–d , DIC images of 10-day-old abaxial cotyledons of Col-0 ( a ), spch-3 mutant ( b ), pSPCH:CrSM/spch-3 ( c ), and pSPCH:CrFAMA/spch-3 ( d ). e–l , Confocal images of 10-day-old abaxial cotyledons of Col-0 ( e, i ), mute-1 mutant ( f ), pMUTE:CrSM/mute-1 ( g ), pMUTE:CrFAMA/mute-1 ( f ), fama-1 mutant ( j ), pFAMA:CrSM/fama-1 ( k ), and pFAMA:CrFAMA/fama-1 ( l ). m , o , Stomatal density in 10-day-old abaxial cotyledons of pSPCH:CrSM/spch-3 ( m ) and pMUTE:CrSM/mute-1 ( o ). q , Number of cell division per fama tumor in 10-day-old adaxial cotyledons of pFAMA:CrSM/fama-1 . n , Number of stomata in 10-day-old abaxial cotyledons of pSPCH:CrFAMA/spch-3 . p , Stomatal density in 10-day-old abaxial cotyledons of pMUTE:CrFAMA/mute-1 . r , Stomatal index of abaxial cotyledon in 10-day-old pFAMA:CrFAMA/fama-1 seedlings. Only normal stomata were quantified. m-r , Statistical significance was assessed using Steel-Dwass-Critchlow-Fligner test (* p < 0.05, ** p < 0.01, *** p < 0 . 001 ). White, SR2200. Square bracket indicates a pair of stomata; blue asterisks indicate small cell; green triangle indicates single GC. Scale bars, 10 µm ( a–l ). To investigate whether these complementation lines exhibit asymmetric divisions, we observed young cotyledons. Analysis of young cotyledons revealed that most cell divisions in pSPCH:CrSM/spch-3 were symmetric, though occasional asymmetric divisions were also observed ( Fig. S1l ). In pSPCH:CrFAMA lines, we detected many epidermal cells were stained with propidium iodide possibly due to temporary changes in membrane permeability, differences in cell wall or membrane structure, or cell death ( Fig. S1m ), yet a few asymmetric divisions were still detectable ( Fig. S1n ). These results indicate that both CrSM and CrFAMA can promote asymmetric division. Next, we tested whether CrSM and CrFAMA can replace AtMUTE. CrSM expressions under the AtMUTE promoter produced normal stomata, however, they were often occurring in pairs ( Fig. 5g , Fig. S2c ). SD of these lines was either similar to wild-type or increased ( Fig. 5o ). We noticed that venus-tagged CrSM protein was detected in young GCs where the endogenous AtMUTE is typically not expressed ( Fig. S2a, b ). This accumulation suggests that CrSM is not degraded at the appropriate time point. This is likely to cause increased activity window leading to the additional round of symmetric division in young GCs, thus creating paired GCs. Expression of CrFAMA under the AtMUTE promoter partially rescued the mute-1 mutant ( Fig. 5h, p ), producing stomata as well as meristemoids, small cells, and single GCs ( Fig. S2g ). Rescue of seedling lethality and seed production was also observed in both pMUTE:CrMUTE and pMUTE:CrFAMA . These results suggest that CrSM and CrFAMA can partially substitute for AtMUTE . Finally, we introduced CrSM and CrFAMA into fama - 1 mutant under the control of AtFAMA promoter. The former line failed to produce stomata indicating that CrSM cannot substitute for FAMA in Arabidopsis ( Fig. 5k ). However, we noticed an increased number of cell divisions within fama tumors in pFAMA:CrSM/fama line compared to fama ( Fig. 5k, q , Fig. S3a, b ). This phenotype resembles effects of ectopic expression of CYCD5;1 under the control of AtMUTE promoter in fama-1 mutants 24 . Since AtFAMA is required for CYCD5;1 repression, we infer that CrSM is not able to repress CYCD5;1 expression in Arabidopsis or alternatively, CrSM can promote expression of other cell cycle related factors. Expression of CrFAMA under the AtFAMA promoter produced both normal and abnormal stomata, suggesting that CrFAMA can efficiently substitute AtFAMA despite the large phylogenetic distance between C.fern and Arabidopsis . Observed phenotypes were quantitatively and qualitatively similar between pFAMA:PaFAMA/fama and pFAMA:CrFAMA/fama complementation lines ( Fig. 5l, r , Fig. S3e, g ) suggesting striking similarity in CrFAMA and PaFAMA functions. Discussion While the morphology of conifer stomatal complex was described decades ago, the underlying molecular mechanisms have remained poorly understood. Bioinformatic analyses have revealed that gymnosperm genomes contain a single gene resembling both SPCH and MUTE , in addition to FAMA , unlike angiosperms, which carry three distinct genes identifiable as SPCH, MUTE , and FAMA 9 , 25 . However, experimental evidence for the functions of these genes in gymnosperms has been lacking. In this study, we identified one bHLH1a gene in each SPCH/MUTE and FAMA clade, consistent with previous analyses, and hypothesized that PaSM may possess functions corresponding to both SPCH and MUTE , or alternatively to either SPCH or MUTE , while PaFAMA may carry FAMA -like functions. Our cross-species complementation experiments demonstrated that PaSM can functionally substitute for both SPCH and MUTE . Previous work showed the limited ability of AtMUTE to compensate for AtSPCH function 19 , indicating in Arabidopsis SPCH and MUTE functions have diverged. In contrast, our complementation experiments demonstrated that PaSM can functionally substitute for both SPCH and MUTE , suggesting that PaSM is a multifunctional protein with capabilities of both SPCH and MUTE. We found that pSPCH:PaSM/spch-3 exhibited excessive cell divisions and aberrant stomata; similar phenotype has been observed when an AtSPCH variant with a disrupted MAPK phosphorylation site is expressed 26 . This observation is consistent with PaSM possessing a rudimentary MAPKTD domain. It is possible that PaSM protein is more stable than AtSPCH, perhaps due to differences in how protein stability is regulated via the MAPK pathway. However, it is noteworthy that fraction of the high stringency MAPK target sites important for the regulation of SPCH stability are already present in PaSM 18 . Similar to AtSPCH 12 , PaSM did not rescue the fama mutant. However, PaSM induced the pore formation within stomatal tumor-like structures, which has not been reported in AtSPCH complementation lines 12 . This result implies that PaSM retains a residual capacity for FAMA-like activity in promotion of stomata maturation. PaFAMA effectively complemented the fama mutant and partially rescued the mute mutant. These findings align with previous study show that AtFAMA and BdFAMA can effectively rescue both mute and fama mutant 15 . Further, bHLH1a ortholog from Marchantia polymorpha ( MpSETA ) and Physcomitrella patens ( PpSMF1 ) can partially rescue Arabidopsis mute and fama mutants 12 , 27 . Surprisingly, our analysis revealed that PaFAMA is able to partially substitute for AtSPCH , unlike AtFAMA, MpSETA , and PpSMF 12 , 15 , 27 . Our findings suggest that the primary function of PaFAMA is to regulate fate transition in the late stomatal lineage similar to AtFAMA . However, it seems possible that PaFAMA has also capacity to modify early stomatal lineage since it can partially replace AtSPCH function and its transcripts are present already in the early stages of stomatal development in spruce needles. Complementation assays using C. fern orthologues further suggests that stomatal development in ferns is regulated by two bHLH1a genes. While PaSM contains truncated MAPKTD, CrSM completely lacks this domain, allowing us to compare effect of the absence/presence of MAPKTD within SMs. pSPCH:CrSM lines, which displayed clusters of stomata, resembled lines with constitutively active AtSPCH variant lacking the MAPKTD domain 18 . Because CrSM completely lacks this domain, it may possess a more MUTE-like function than SPCH-like activity. Our study provides a snapshot of the evolutionary journey of stomatal development. Based on our data, it seems likely that a multi-functional gene was duplicated and diversified into two genes with specific functions, while still retaining some degree of multifunctionality. Our qRT-PCR analysis supports this view: PaSM was primarily expressed in regions of ACDs and GMC differentiation, but not, or weakly, expressed in the maturing stomata stage. This result, together with complementation analysis, supports the idea that single stomatal gene, PaSM , regulates ACDs and GMC differentiation in spruce and possibly also in other gymnosperms. PaFAMA showed wide expression window; its expression extended beyond GMC and young guard cell stages into maturing stomata—unlike AtFAMA , which is not expressed in mature guard cells 28 . This expression pattern may suggest additional roles for PaFAMA potentially in the formation of non-stomatal tissues, late-stage stomatal maturation, or immunity. Supporting this idea, FAMA has been shown to promote the myrosin cell formation in leaf vein in Arabidopsis 29 , and interact with the F-BOX STRESS-INDUCED (FBS) protein to regulate stomatal closure via ABA signaling during pathogen attack 30 . PaSM and CrSM show dual function in the early stomatal lineage. Therefore, it seems possible that they are active during the developmental window spanning ACDs to GMC differentiation. Given the clearly separated temporal domains of SPCH and MUTE, the stage specificity of bHLH1a proteins may have evolved in parallel with their divergence. Our study provides insight into the molecular evolution of stomatal development beyond the angiosperm clade. However, a limitation of this study is that functions of PaSM and PaFAMA are examined in Arabidopsis , therefore our analyses might not reflect their functions in their native species. It is possible that structural similarity between spruce/fern bHLHs and angiosperm regulators may allow these proteins to drive developmental transitions in Arabidopsis , even if they do not function identically in their native species. Although genetic study is challenging, future work is required to assess their functions in Norway spruce and C. fern. Because stomatal complex in Norway spruce contains several subsidiary cells, it will be intriguing to examine protein mobility of PaSM. In Brachpodium , BdMUTE moves from GMCs to adjacent cells to recruit lateral SMC 14 . While PaSM (461 aa, 50.6 kDa) is substantially larger than BdMUTE (237 aa, ∼25.5 kDa) 14 , it could be mobile or alternatively, induce intracellular signals that promote SC specification in the neighboring cells. Norway spruce stomata are sunken, a feature shared by many gymnosperms and some tropical species 21 , 31 , 32 . Our observations indicate that the GMC enlarges along not only the longitudinal and transverse axes but also the adaxial– abaxial axis of the needles ( Fig. 2c ), which is consistent with previous findings in pines 21 . However, unlike pines, Norway spruce stomata were not fully sunken immediately after GMC division, and the subepidermal chamber remained partially enclosed. These observations, together with previous reports, indicate that key aspects of stomatal development—including asymmetric fate acquisition, GMC differentiation, and guard cell formation—are conserved among conifers, but there might be species-specific differences, particularly in the timing of symmetric divisions and sinking of stomata into the hypodermal layer. Such differences may reflect environmental influences or as-yet-unidentified regulatory mechanisms. As we could not find the sunken stomata formation in our complementation lines, its underlying mechanisms remains to be studied in Norway spruce in future. Materials and Methods Phylogenetic analysis We performed Orthofinder 33 utilizing the amino acid sequence information from Phytozome ( https://phytozome-next.jgi.doe.gov/ ) and PlantGenIE ( https://plantgenie.org/ ), and selected the OrthoGroup which included AtSPCH, AtMUTE and AtFAMA as bHLH1a orthologues. We then aligned their sequences with MAFFT 34 and automatically trimmed the aligned sequences by trimAl 35 . A phylogenetic tree was constructed using the trimmed sequence by IQ-TREE 36 , and evaluated the reliability of its branches with both the bootstrap method and the approximate likelihood-ratio test. Each was performed 1000 times. Unless specified, default settings were used. Plant material and growth conditions spch-3, mute-1, fama-1 were described previously. Arabidopsis seeds were sown on half Murashige and Skoog (MS) (Prod. No: M0221.0100, Duchefa Biochemie) plates adjusted to pH 5.75 and solidified with 0.8% plant agar (Prod. No: P1001.1000, Duchefa Biochemie). After stratification for more than two days at 4 degrees, plates were incubated at 23 degrees in the long day condition (16 h light/8 h dark). Norway spruce seeds were sown on a mixture of peat and vermiculite (4:1) in plates and incubated more than one week at 4 degrees. Plates were then incubated at 20 degrees under the constitutive light condition. After germination, seedlings were transferred to pots individually. We defined the day when plates were moved to the growth chamber as a day 0. Plasmid construction and plant transformation PaSM and PaFAMA were amplified using the Picea abies cDNA from the needles. CrSM genomic sequence was amplified from gemomic DNA extracted from C.fern sporophyte leaf. CrFAMA cds sequence was synthesized using Biomatik service ( https://www.biomatik.com/ ). All the cross-species complementation analysis plasmid were produced utilizing the Multisite Gateway Technology. About 2.5 kb, 1.7 kb and 3 kb upstream sequences from the start codon were used as AtSPCH, AtMUTE and AtFAMA promoter, respectively. Sample preparation and imaging For DIC imaging, the plants were fixed with EtOH and Acetic acid (7:1) and kept at least overnight. The plants were rinsed with MQ-water several times and mounted in Hoyer’s solution (7.5 g arabic gum, 5 ml glycerol, 103 g chloral hydrate and 30 ml MQ-water). After plants became transparent, cotyledons were observed using a Leica DM6B light microscope. For confocal microscopy imaging, Arabidopsis seedlings were submerged in 0.1% SCRI Renaissance Stain 2200 (SR2200) in 1xPhosphate-buffered saline (PBS) using a centrifuge to stain cell wall. To stain with propidium iodide (PI), the seedlings were submerged into 1 mg/mL PI solution for several seconds followed by immediate wash with MQ water. Norway spruce needles were cut from the saplings and fixed with 4% paraformaldehyde (PFA) in 1xPBS with 0.05% Triton X-100 under vacuum. After one hour vacuum treatment, needles were washed with 1xPBS several times. For longitudinal imaging, needles were transferred to 0.1% SR2200 in ClearSee 37 . For cross-section imaging, needles were embedded in 4% agarose and 200 um-thick cross-sections were made using a vibratome. The sections were then transferred to 0.1% SR2200 in ClearSee. The stained samples were imaged with a confocal microscope (Leica Sterallis8). qRT-PCR Stage 1, 2, 3 and 4 were sampled from the bottom half of 0.2-0.3 cm needles, the bottom half of 0.4-0.6 cm needles, the distal half of 0.4-0.6 cm needles, and the distal one-third of 0.8-0.9 cm needles, respectively. RNA was extracted from each developmental stage, as described previously 38 . After DNase treatment (Thermo Scientific, 89836), cDNA was synthesized using First Strand cDNA Synthesis Kit (Thermo Scientific, K1612). qRT-PCR was performed with three biological replicates and three technical replicates. The data were normalized to the internal control (actin) and presented relative to stage 1. Author Contributions Conceptualization, funding acquisition, project administration, resources, supervision: AV; Data production and analysis: MZI, KE; Visualization: MZI; Writing AV, MZI; All authors have read and approved the final manuscript. Acknowledgments We would like to thank the gardeners L. Grönholm, D. Richterich. We thank M. Lyytikäinen, M. Herpola, K. Kainulainen and M. Mäkelä for technical assistance support, Vatén group members for comments and discussion. Confocal microscopy and histology experiment were performed at the Light Microscopy Unit and BI Histology Core Facility at the University of Helsinki, respectively. This work was supported by the Research Council of Finland grant 340443 “Control of stomatal development upon environmental change”. Funder Information Declared Research Council of Finland , 340443 References (1). ↵ Doll , Y. ; Koga , H. ; Tsukaya , H. 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Share Fern and gymnosperm SPCH/MUTE and FAMA can regulate multiple cell fate transitions during stomatal development Miki Zaizen-Iida , Kaotar Elhazzime , Anne Vatén bioRxiv 2025.11.10.687565; doi: https://doi.org/10.1101/2025.11.10.687565 Share This Article: Copy Citation Tools Fern and gymnosperm SPCH/MUTE and FAMA can regulate multiple cell fate transitions during stomatal development Miki Zaizen-Iida , Kaotar Elhazzime , Anne Vatén bioRxiv 2025.11.10.687565; doi: https://doi.org/10.1101/2025.11.10.687565 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 (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)