Maternal control of RNA decay safeguards embryo development

Maternal control of RNA decay safeguards embryo 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 Maternal control of RNA decay safeguards embryo development View ORCID Profile Gerardo Del Toro-De León , Maria Sofia Trenti , Varsha Vasudevan , Ursula Krause , View ORCID Profile Claudia Köhler doi: https://doi.org/10.1101/2025.05.28.656625 Gerardo Del Toro-De León 1 Department of Plant Reproductive Biology and Epigenetics, Max Planck Institute of Molecular Plant Physiology , Potsdam, 14476, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gerardo Del Toro-De León For correspondence: koehler{at}mpimp-golm.mpg.de deltoro{at}mpimp-golm.mpg.de Maria Sofia Trenti 1 Department of Plant Reproductive Biology and Epigenetics, Max Planck Institute of Molecular Plant Physiology , Potsdam, 14476, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Varsha Vasudevan 1 Department of Plant Reproductive Biology and Epigenetics, Max Planck Institute of Molecular Plant Physiology , Potsdam, 14476, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ursula Krause 1 Department of Plant Reproductive Biology and Epigenetics, Max Planck Institute of Molecular Plant Physiology , Potsdam, 14476, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Claudia Köhler 1 Department of Plant Reproductive Biology and Epigenetics, Max Planck Institute of Molecular Plant Physiology , Potsdam, 14476, Germany 2 Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology , Uppsala 75007, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Claudia Köhler For correspondence: koehler{at}mpimp-golm.mpg.de deltoro{at}mpimp-golm.mpg.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract As in mammals, the plant embryo is surrounded by maternal tissues that provide protection from the external environment. In angiosperms, a double fertilization process results in the formation of a diploid embryo and a triploid endosperm, both of which develop within maternal sporophytic integuments. Thus, the seed of angiosperms is a combined structure of three genetically distinct components: embryo, endosperm, and maternal integuments. It has long been assumed that the maternal plant influences embryo development, but there is limited molecular evidence for a direct pathway through which the maternal sporophyte aYects embryogenesis. Here we show that secondary small interfering RNAs (siRNAs) generated upon exosome impairment lead to embryo abortion through a maternal sporophytic eYect. Depletion of the core subunit of the RNA-processing exosome RRP45B (CER7) causes globular embryo arrest connected to ectopic post-transcriptional gene silencing (PTGS). Seed coat expression of CER7 suppresses seed abortion, demonstrating a maternal sporophytic control of embryo development through an RNA decay safeguard pathway. Our data support a model in which a primary siRNA trigger originates in the maternal integuments of the cer7 seed coat and drives PTGS amplification in reproductive tissues after fertilization, ultimately leading to seed abortion. In addition, our genetic and molecular data suggest that overloading of AGO1 with siRNAs impairs miRNA function, likely leading to embryo arrest. Our data highlight the complex interplay between maternal and embryonic gene regulation, reinforcing the importance of controlled RNA decay in plant development. Introduction In plants, as in mammals, embryos develop within specialized maternal tissues that provide protection and facilitate nutrient transfer. From basal bryophytes to angiosperms, plants have evolved structures of diverse complexity that support embryo development potentially exerting varying degrees of control over embryogenesis 1 . In angiosperms, the embryo develops surrounded by the endosperm and the seed coat, three genetically distinct components that together constitute the seed. The endosperm is typically a triploid tissue resulting from a second fertilization event, an adaptative reproductive strategy unique to angiosperms. The seed coat is a multilayered envelope of maternal sporophytic origin, derived from the ovule integuments. The complex composition of the seed in angiosperms strongly presumes that both the endosperm and the seed coat influence embryo development. In the angiosperm model plant Arabidopsis thaliana , several lines of evidence indicate that the endosperm is key for embryogenesis, as defects in its development result in embryo arrest and seed abortion 2 – 4 . For example, embryo patterning requires the peptide molecule EMBRYO SURROUNDING FACTOR 1 (ESF1), which is maternally derived from the central cell, the female gametic precursor cell of the endosperm 5 . Transition from the coenocytic to the cellular stage of endosperm development is crucial for embryo survival and implicated to establish desiccation tolerance in the embryo 2 . Furthermore, regulated endosperm breakdown by the endosperm-specific ZHOUPI gene is important for embryonic epidermal development 6 . In contrast, little is known about how the maternal sporophytic tissues impact embryogenesis. Besides being a physical protective structure, little evidence implicates the maternal integuments in a more direct role during embryo development. Recent reports suggest that the seed coat could potentially work as a communication barrier regulating the movement of molecules across seed compartments. For example, auxin is actively transported from the integuments to the suspensor 7 , the ephemeral basal cell of the embryo and the only known symplastic connection to the surrounding maternal tissues. Other molecules have been suggested to be transferred from the seed coat to the fertilization products, but the evidence is scarce or indirect 8 – 10 . In genetics, the influence of the integuments on embryogenesis implies a sporophytic maternal genetic eYect. Sporophytic maternal mutations with embryo defects reveal the function of the impaired gene in maternal tissues relevant to embryogenesis, which translates into a non-Mendelian penetrance of the mutant phenotypes. Only a handful of reports have provided genetic evidence supporting sporophytic maternal control over embryo development 11 – 14 . However, the mechanisms are poorly understood. Here we report the discovery of a sporophytic maternal pathway surveilling embryo development through an RNA processing decay mechanism. Our genetic and molecular data demonstrate that mutations in ECERIFERUM7 ( CER7 )/ RRP45B , a core component of the RNA exosome, are embryo lethal with a sporophytic maternal eYect. The exosome complex is an evolutionarily conserved holoenzyme in eukaryotes responsible for 3′-5′ RNA decay. Nuclear and cytoplasmic forms of the exosome share a core complex constituted by a barrel-shaped structure of nine subunits associated to the ribonucleolytic subunit DIS3/RRP44 15 , 16 . Mutations in the core components of the exosome are often lethal in Arabidopsis thaliana . Disruption of the RRP41L subunit delays germination and aYects early seedling development 17 , while mutations in RRP41 , RRP42 , DIS3 / RRP44A and RRP4 have been reported to cause female gametophyte and embryo defects 18 – 21 . Interestingly, mutations in the core exosome subunit encoding gene CER7 result in severe seed inviability 22 , 23 , but the underlying cause of this defect remains unknown. The cer7 mutant was initially identified by its wax-deficient outer cuticular surfaces, which result in glossier or brighter stem phenotypes 24 . This phenotype is caused by the accumulation of secondary small interfering RNAs (siRNAs) targeting the key wax biosynthetic gene CER3 , causing reduced CER3 RNA levels 22 , 25 – 27 . Disruption of RNA metabolic processes often results in aberrant transcripts entering the post-transcriptional gene silencing (PTGS) pathway, causing reduced transcript levels of endogenous genes 28 – 30 . We found that the exosome prevents the accumulation of aberrant transcripts in the seed coat, capable of triggering RNA silencing in the fertilization products impairing embryo development. Our data suggest that in the cer7 mutant, RNA molecules derived from sporophytic maternal tissues induce embryo arrest through ectopic RNA silencing, implying RNA transport from the maternal sporophyte to the embryo. Results cer7 mutants arrest at the globular stage of embryo development To characterize the reduced seed viability observed in CER7/RRP45B mutants, we analyzed two independent loss-of-function alleles, cer7-3 (SAIL_747_B08) and cer7-4 (GK_089C02) ( Supplementary Fig. 1 ) 22 , 23 . Homozygous mutants of both cer7 alleles produced strongly wrinkled seeds ( Supplementary Fig. 2A ). To accurately determine seed abortion rates, we performed manual crosses and quantified seed abortion at 14 days after pollination (DAP). We found that over 90% of developing seeds displayed a size comparable to wild-type (WT) siblings within the same silique but lacked green pigmentation, suggesting embryonic arrest; we refer to these as ‘aborted seeds’. ( Supplementary Fig. 2 ). To further characterize defects associated with seed abortion, we assessed embryo development at 2, 3, 5, and 7 DAP. At 2 DAP most developing seeds were at the 1-2 cell embryo stage in WT and both cer7 mutant alleles. At 3 DAP ∼25% of WT embryos were at the preglobular and 75% at the globular stage, while >85% of the cer7 embryos of both alleles remained at the preglobular stage ( Fig. 1A-B ), indicating a slight delay in development compared to WT. At 5 and 7 DAP, over 95% of wild-type embryos had reached the heart and torpedo stage, respectively, whereas embryos from both cer7 mutants remained at the globular stage with no apparent cell division defects. These findings show that cer7 mutant seeds exhibit a strong developmental arrest at the early to mid-globular stages of embryogenesis ( Fig. 1A-B ). We did not detect defects in endosperm development in both cer7 alleles, proliferation and cellularization occurred at same speed as in WT, indicating that defects in cer7 are primarily restricted to embryo development ( Fig. 1C ). We therefore concluded that cer7 shows seed inviability mainly due to a globular embryo arrest. Download figure Open in new tab Fig. 1: Phenotypic and genetic characterization of cer7 mutants. ( A ) Nomarsky images of embryos in whole mount cleared seeds of wild-type (WT) Col-0 and cer7-4 at 2, 3, 5 and 7 days after pollination (DAP). ( B ) Quantification of embryo stages in WT and cer7 alleles. ( C ) Endosperm cellularization in WT and cer7-4 determined by Feulgen staining at 5 and 7 DAP. ( D ) Quantification of seed abortion in hand self-pollinated heterozygous plants at 14 DAP (top panel), representative images of siliques (bottom panel). ( E ) Quantification of seed abortion at 14 DAP in indicated genetic crosses to determine the maternal eYect in cer7 mutants. ( F ) Representative images of siliques quantified in (E). Bar color code for D and E: dark gray denotes sporophytic maternal plant homozygous for cer7 ; light gray denotes WT and heterozygous cer7 sporophytic maternal plant. Pairwise t-test, **P-value < 0.01; * P-value < 0.05; ns, not significant. Scale bar 50 µm (A and B). cer7 mutants show a sporophytic maternal eOect on embryo lethality To determine whether the seed phenotype of cer7 follows a recessive Mendelian segregation, we first quantified seed abortion in heterozygous plants. Surprisingly, heterozygous plants did not exhibit seed abortion in either of the two independent alleles tested ( Fig. 1D ). Segregation analysis of the progeny showed the expected 1:2:1 genotypic ratio of a classic recessive mutation (16 +/+ :49 +/- :16 -/- , P -value >0.05, Chi-square test), arguing against gametophytic defects biasing phenotypic proportions. We therefore hypothesized that cer7 embryo arrest is caused by a sporophytic maternal eYect. To test this hypothesis, we reciprocally crossed homozygous cer7 mutants with WT or pollen from heterozygous mutants and scored abortion at 14 DAP. We found that cer7 homozygous mutants crossed with WT pollen resulted in 17% and 40% of seed abortion for cer7-4 and cer7-3 , respectively. In contrast, WT maternal plants crossed with cer7 pollen showed normal seed viability ( Fig. 1E-F ). Interestingly, when pollinating homozygous cer7 plants with heterozygous pollen we observed 60% of aborted seeds for both alleles tested, while the genetically identical progeny derived from crossing heterozygous plants with homozygous pollen showed 11% and 7% of aborted seeds for cer7-4 and cer7-3 , respectively ( Fig. 1E-F ). Collectively, these data reveal that homozygous and heterozygous cer7 embryos abort at high rates only when developing within homozygous maternal cer7 mutants, demonstrating that cer7 is a sporophytic maternal eYect mutant with embryo defects. Importantly, our genetic analysis showed 60% of seed abortion when homozygous maternal cer7 mutants were pollinated with pollen from heterozygous mutants, compared to >90% of seed abortion from self-pollinated homozygous plants, indicating an additional zygotic component on seed abortion. Since both cer7 alleles consistently exhibited equivalent phenotypes, subsequent experiments were conducted using cer7-4 . cer7 embryo lethality is triggered by maternally controlled PTGS Disruption of CER7 has been implicated to cause aberrant transcripts entering the PTGS pathway, causing reduced transcript levels of endogenous genes 22 , 25 – 27 . We therefore hypothesized that seed abortion in cer7 could also be explained by ectopic PTGS. To test this idea, we introduced cer7 into sgs3 and rdr6 mutant backgrounds, two key players in RNA silencing 31 . We indeed found that cer7;sgs3 and cer7;rdr6 double mutants fully rescued seed abortion ( Fig. 2A-B ), demonstrating that embryo arrest in cer7 is dependent on aberrant PTGS. Download figure Open in new tab Fig. 2: Seed abortion in cer7 is dependent on maternally controlled PTGS. ( A ) Quantification of seed abortion at 14 days after pollination (DAP) in the indicated genetic crosses. Numbers of analyzed seeds are indicated on the right side of the graph. ( B ) Representative images of siliques quantified in (A). ( C ) Scheme depicting the expected PTGS amplification in reciprocal crosses. Dark grey indicates active PTGS amplification; light grey indicates impaired amplification. Top panel: cer7;SGS3 sporophytic maternal plant and progeny amplify PTGS. Bottom panel: cer7;sgs3 maternal plant with PTGS impairment; progeny retains amplification. ( D ) Quantification of seed abortion at 14 DAP in the indicated genetic crosses. Numbers of analyzed seeds are indicated on the right side of the graph. ( E ) Representative images of siliques quantified in (C). Bar color code for B and E: Dark grey indicates PTGS amplification by the cer7 homozygous sporophytic maternal plant; light grey indicates impaired PTGS amplification in the sporophytic maternal plant. ( F ) As in (A), scheme depicting the expected PTGS amplification in selfed cer7;ago1-27/+ plants. The sporophytic maternal plant and the majority of the progeny amplify PTGS (dark grey). A subset of progeny ( ago1-27 homozygotes, ∼25%) is expected to show impaired PTGS amplification, indicated in light grey in the accompanying pie chart. Pairwise t-test, **P-value < 0.01; * P-value < 0.05; ns, not significant. Amplification of siRNAs requires the synthesis of double-stranded RNAs (dsRNAs) mediated by SGS3-RDR6, which are later processed by DCL4/2 to produce 21/22-nt siRNAs 31 , 32 . Since we determined that the cer7 seed phenotype is established by a sporophytic maternal eYect, we aimed to analyze whether PTGS-mediated seed collapse was likewise maternally controlled. To test this idea, we reciprocally crossed homozygous cer7 mutants with cer7;sgs3 and cer7;rdr6 double mutants to deplete PTGS amplification in either the sporophytic maternal or zygotic tissues and scored seed abortion at 14 DAP. Embryos resulting from crosses between maternal cer7 plants and cer7;sgs3 pollen develop within a sporophyte capable of amplifying siRNAs, as SGS3 is functional in the maternal sporophytic tissues ( Fig. 2C ). Accordingly, we observed an average of 85% of aborted seeds in this cross. In contrast, embryos resulting from the reciprocal cross develop within a cer7;sgs3 maternal sporophyte unable of amplifying PTGS, but the fertilization products retain this ability given the functional SGS3 copy inherited from the paternal parent ( Fig. 2C ). This cross resulted in 59% aborted seeds, which was significantly less than in the reciprocal cross. Similar results were obtained with cer7 and cer7;rdr6 reciprocal crosses ( Fig. 2A-B ). These observations indicate that cer7;sgs3/rdr6 maternal sporophytes are capable of inducing PTGS-mediated embryo arrest, meaning that siRNA amplification can also occur in the fertilization products. These results suggest that the trigger for PTGS in the maternal sporophytes is upstream of SGS3-RDR6, likely an aberrant RNA resulting from deficient exosome function. Our observations imply that the trigger that induces aberrant PTGS is generated in the sporophytic maternal tissues in alignment with the sporophytic maternal-eYect, and is capable to reach the embryo for subsequent RNA silencing amplification, leading to cer7 embryo arrest. In agreement with this notion, we observed higher seed abortion rates in cer7 x cer7;sgs3 compared to cer7;sgs3 x cer7 (85% versus 59%), suggesting that PTGS amplification in the cer7 maternal sporophyte contributes to an increased dosage of ectopic siRNAs resulting in higher seed abortion frequency. We therefore concluded that cer7 seed abortion is the result of PTGS, which is maternally triggered in sporophytic tissues and amplified in both the maternal sporophyte and the fertilization products likely through RNA movement across seed compartments. AGO1 is required for PTGS-mediated cer7 embryo arrest AGO1 is the main eYector of PTGS. We therefore asked whether cer7 PTGS-dependent seed abortion was mediated by AGO1. To test this idea we introduced cer7 into the ago1-27 mutant, a weak allele specifically impaired in sense PTGS (S-PTGS) 33 . Indeed, the cer7 ; ago1-27 double mutant fully suppressed seed abortion, implicating RNA silencing through AGO1 ( Fig. 2D-E ). We then assessed whether ago1-27 is required in the sporophyte or in the fertilization products to rescue cer7 seed abortion. To test this, we scored seed abortion in cer7 ; ago1-27/+ heterozygous plants. We anticipated that if ago1-27 suppresses seed abortion zygotically, at least 25% of the progeny should be viable ( Fig. 2F ). Indeed, we found that self-pollinated cer7 ; ago1-27/+ plants showed an average of 32 % viable seeds, in comparison to 2% in cer7 homozygous plants ( Fig. 2D-E ). These data indicate that AGO1 acts in the fertilization products to induce cer7 seed abortion. Finally, we explored whether aberrant PTGS in cer7 was triggered by microRNA activity. Little is known about the mechanisms by which PTGS is initiated in endogenous genes. However, similar to TAS genes, a subset of endogenous transcripts prone to PTGS amplification are targets of microRNAs 34 , 35 . To test this idea, we introduced cer7 into the dcl1-14 background, a viable hypomorphic allele of DCL1 which encodes a key RNase III enzyme required for miRNA biogenesis. The cer7;dcl1-14 double mutant showed equivalent abortion levels to the cer7 single mutant, arguing against microRNA-triggered PTGS ( Fig. 2D-E ). Nevertheless, dcl1-14 mutant is a weak allele and retains low levels of microRNA activity 36 ; therefore, we cannot exclude the possibility that leaky microRNA activity in dcl1-14 is suYicient to induce PTGS amplification in cer7 . CER7 activity in the seed coat rescues seed abortion Our data strongly suggest that an aberrant transcript or a subset of transcripts accumulate in cer7 sporophytic maternal tissues and reach the embryo, leading to PTGS amplification and seed abortion. We reasoned that by expressing CER7 in the seed coat we could prevent the generation and movement of aberrant transcripts entering PTGS, suppressing embryo arrest. To test this hypothesis, we generated complementation lines expressing CER7-GFP under the control of promoters that are specifically active in the seed coat ( Fig. 3A-E ). Constitutive expression of CER7-GFP under the RPS5A promoter fully suppressed both the glossy stem phenotype and seed abortion in all lines tested, indicating that CER7-GFP is functional ( Fig. 3A, E, F ). We then expressed CER7-GFP under control of the BANYULS ( BAN ) promoter, which is specifically active in the endothelium, the innermost cell layer of the inner integument (ii1) 37 , 38 . We found that BAN::CER7-GFP expression caused a significant reduction of seed abortion in 27% of the lines tested ( Fig. 3B, G ). Since all the three layers of the inner integument are symplastically connected 9 , we reasoned that aberrant transcripts could easily move from the outermost layers and potentially overload endothelium CER7-GFP activity. We therefore expressed CER7 under the deltaVPE ( dVPE ) promoter, which is specifically active in the outer two cell layers of the inner integument (ii2 and ii3) 39 . Expression of dVPE::CER7-GFP resulted in a significant reduction of seed abortion in 40% of the lines tested ( Fig. 3C, H ). We then analyzed the combined complementation eYect of CER7-GFP under control of dVPE and BAN (ii1, ii2, ii3) by crossing those lines that showed little to no rescue. We observed a strong additive eYect, as all tested plants expressing both constructs showed a significant reduction of seed abortion ( Fig. 3D, I ). Importantly, neither the dVPE nor the BAN promoter CER7-GFP expression rescued the glossy stem phenotype ( Fig. 3E ). These results demonstrate that the activity of CER7 is required in the seed coat to prevent formation of aberrant transcripts, consistent with the sporophytic maternal-eYect of cer7 . Download figure Open in new tab Fig. 3: Seed coat expression of CER7 suppresses cer7 seed abortion. ( A–D ) CER7-GFP expression driven by the constitutive RPS5A promoter (A), the seed coat-specific dVPE promoter (B), the BAN promoter (C), or a combination of both dVPE and BAN promoters (D). Green represents GFP expression, blue shows autofluorescence. Scale bar 50 µm. ( E ) Stems of 6 week old cer7;pRPS5A::CER7-GFP (left) and cer7;pBAN::CER7-GFP;pdVPE::CER7-GFP (right). White arrows indicate glossy stem phenotype. Scale bar 200 µm. ( F-I ) Quantification of seed abortion in mature siliques in cer7 mutants expressing CER7-GFP under the constitutive RPS5A promoter (F), the seed coat-specific inner integument (ii1) BAN promoter (G), the ii2-3 specific dVPE promoter (H) and the combined expression of d VPE and BAN promoters (ii1-3) (I). Representative images of the indicated siliques depicted in the bottom panels. Numbers of analyzed seeds are indicated on the right side of the graph. DAP, days after pollination. L, individual transgenic line. Pairwise t-test, **P-value < 0.01; * P-value < 0.05; ns, not significant. PTGS response in cer7 developing seeds We have shown that cer7 embryo lethality is dependent on maternally controlled PTGS, which implies RNA silencing mediated by ectopic accumulation of siRNAs. We therefore hypothesized that a gene or group of genes relevant for embryo development were targeted by siRNAs produced both in sporophytic and fertilization products leading to embryo arrest. We thus aimed to identify downregulated genes targeted by siRNAs in cer7 seeds. For this purpose, we generated sRNA-seq and RNA-seq libraries in triplicates of WT, cer7 , sgs3, and cer7;sgs3 seeds at 3 DAP ( Supplementary Fig. 3-4, Supplementary Table 1-2 ). First, we asked whether coding genes were indeed ectopically generating siRNAs in cer7 . Because PTGS is mediated by 21-nt and 22-nt siRNAs, we fragmented the genome in 100bp windows and identified significantly enriched regions with 21/22-nt siRNAs in cer7 samples (LFC > 0; FDR <0.05). As expected, over 90% of the 21/22-nt sRNA-enriched windows in cer7 overlapped with coding genes. Since SGS3 is required for PTGS and sgs3 suppressed the cer7 mutant phenotype, we then selected those loci that lost siRNAs in the cer7;sgs3 double mutant ( Supplementary Fig. 5A-B ). We analyzed whether the distribution of 21/22-nt siRNA was present on both strands of coding sequences, a hallmark of PTGS silencing, and identified 19 genes targeted by secondary siRNAs in cer7 seeds ( Fig. 4A , Supplementary Fig. 5 and Supplementary Dataset 1 ). Next, we tested whether ectopic 21/22-nt siRNA accumulation correlated with reduced gene expression. We conducted diYerential gene expression analysis (LFC ±0.58; FDR <0.05, Supplementary Fig. 6-7, Supplementary Dataset 2 ), but found only three genes that were downregulated and accumulated secondary 21/22-nt siRNAs in cer7 seeds: AGO1 , XYLOGLUCAN ENDOTRANSGLUCOSYLASE 9 ( XTH9 ) and BETA GALACTOSIDASE 18 ( BGLU18 ) ( Fig. 4B ). To evaluate the potential relevance for early embryo development, we analyzed gene expression in seed compartments using public datasets 40 . AGO1 and XTH9 genes are expressed in embryonic tissues, whereas BGLU18 showed no detectable expression, making its involvement in embryo arrest in cer7 mutants unlikely ( Fig. 4C ). XTH9 encodes an enzyme involved in cell wall remodeling and expansion. However, since xth9 mutants have no reported seed defects 41 , reduced XTH9 expression is unlikely to contribute to the cer7 phenotype. Notably, ago1 null mutants show strong pleiotropic phenotypes and infertility, demonstrating essential functions in development 42 , 43 . However, early globular embryo arrest has not been reported in ago1 mutants to date. Therefore, we concluded that PTGS directed on individual genes alone is not suYicient to explain the early globular embryo arrest in cer7 , suggesting that addi-onal factors contribute to seed abor-on. Download figure Open in new tab Fig. 4: PTGS in cer7 developing seeds at 3 DAP. ( A ) sRNA read counts (RPM) for 21/22-nt siRNA accumulating genes in cer7 at 3 DAP seeds of cer7 . ( B ) RNA-seq of 3 DAP seeds showing diYerential expression of genes accumulating 21/22-nt siRNAs in cer7 . Pink color indicates genes with reduced transcript levels and grey indicated genes with no significant change. ( C ) Expression level of 21/22-nt siRNA accumulating genes in diYerent seed compartments along development. ‡ Genes with low or no expression in the embryo. Letters within heatmap boxes indicate MAS5 detection calls: P, present; A, absent; M, marginal. Letters above heatmap boxes indicate developmental stages: pg, preglobular; g, globular; h, heart-stage; lc, linear cotyledon; m, mature green. WS, whole seed; SC, seed coat; CZSC, chalazal seed coat; MCE, micropylar endosperm; PEN, peripheral endosperm; CZE, chalazal endosperm; EP, embryo proper; SUS, suspensor (Belmonte et al. 2013). Pairwise t-test, **P-value < 0.01; * P-value < 0.05; ns, not significant. AGO1 loading of a subset of miRNAs is compromised in cer7 We showed that PTGS-mediated cer7 embryo arrest requires AGO1 function ( Fig. 2D-F ) and that AGO1 transcripts were targeted by siRNAs in cer7 , resulting in a 50% reduction of transcript levels compared to WT ( Supplementary Fig. 7A ). Previous work revealed that ectopically accumulating 21-nt siRNAs compete with miRNAs for loading into AGO1 44 . Because miRNAs are instrumental for embryo development 45 , we hypothesized that cer7 mutants are compromised in miRNA function due competing ectopic siRNAs. Supporting this idea, the sporophytic maternal eYect observed in cer7 echoes the phenotype of the short integuments1 ( sin1 ) mutant, an allele of DCL1 that like cer7 shows maternal sporophytic embryo arrest 13 . Moreover, loss of MIR167 function also causes a sporophytic maternal eYect with embryonic defects, strikingly similar to those observed in cer7 14 . To test whether indeed miRNA loading was compromised in cer7 , we analyzed the populations of 21-nt siRNAs in our sRNA-seq seed samples and found a significant reduction of miRNAs in cer7 compared to the other genotypes ( Fig. 5A ). We found a higher proportion of ribosomal RNA-derived 21-nt sRNAs (ri-siRNAs) in cer7, in line with previous reports showing increased accumulation of 21-nt ri-siRNAs in mutants impaired in RNA degradation 14 ( Fig. 5A ). To directly test which populations of sRNAs were loaded into AGO1 in the cer7 mutant, we performed small RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) of AGO1 from 2-3 DAP WT and cer7 siliques in duplicates ( Supplementary Fig. 8, Supplementary Table 3 ). We first analyzed the size distribution of our RIP-seq libraries and indeed found an enrichment of 21-nt sRNAs and a depletion of 24-nt sRNA as expected for sRNAs associated with AGO1 ( Fig. 5B ). The great majority of the 21-nt sRNAs corresponded to miRNAs in all samples; however, we observed a higher proportion of 21-nt siRNAs derived from protein coding genes but not ri-siRNAs in cer7 samples and a proportional reduction of miRNAs ( Fig. 5C ). To examine diYerences in AGO1 loading of sRNAs, we fragmented the genome into 100bp windows and identified regions significantly enriched or depleted of 21/22-nt sRNA in cer7 compared to WT (LFC ±0; FDR <0.05). Consistent with our sRNA-seq analysis from seeds, all genes identified as enriched for 21/22-nt sRNAs were also enriched in our AGO1 IP data ( Supplementary Fig. 9, Supplementary Dataset 3 ). This observation confirms that a subset of genes generates ectopic siRNAs that compete for AGO1 loading with miRNAs. To analyze whether specific miRNAs were depleted from AGO1 in cer7 , we analyzed the identified windows significantly depleted of 21/22-nt sRNAs in cer7 compared to WT. Indeed, we found that these regions correspond to a subset of miRNAs that were significantly less abundant in AGO1-IP samples from cer7 ( Supplementary Dataset 4 ). To confirm whether these miRNAs were recovered in cer7;sgs3 , we analyzed their relative abundance across our sRNA-seq seed samples and indeed found that cer7 showed a significant reduction of these miRNAs compared to the other genotypes ( Fig. 5D ). We therefore concluded that in cer7 ectopic accumulation of 21/22-nt siRNAs derived from PTGS impairs loading of a subset of miRNAs onto AGO1, compromising its function. Download figure Open in new tab Fig. 5: AGO1 loading of a subset of miRNAs is compromised in cer7 . ( A ) Overview of 21-nt sRNA classes accumulating in 3 DAP seeds of the indicated genotypes. ( B ) Size distribution of sRNAs after AGO1 RIP-seq ( C ) Overview of sRNA classes identified by AGO1 RIP-seq in 2-3 DAP siliques in WT and cer7 . ( D ) Heat map of miRNAs that overlap with a significantly depleted window of 21-nt sRNAs in AGO1 RIP-seq sRNA-seq cer7 samples. ( E ) DiYerential gene expression (DGE) of predicted genes targeted by miRNAs depleted in cer7 . Pink and green color indicate downregulated and upregulated genes respectively, grey indicate genes with no significant change. ( F ) Genome-browser screen-shot depicting 21-nt siRNAs along AGO1 gene. Interestingly, one of the miRNAs significantly depleted from AGO1 is MIR167 , whose knockout in Arabidopsis was recently shown to cause a maternal sporophytic eYect on globular embryo arrest, strikingly similar to cer7 14 . This observation suggests that impairment of miRNAs and specifically MIR167 could explain the globular arrest of cer7 embryos. To further examine eYects on gene expression, we analyzed the transcript abundance of AGO1-depleted miRNA target genes in cer7 RNA-seq. We found only few miRNA target genes with increased transcript levels in cer7 ( Fig. 5E ), likely a consequence for the eYect being restricted to the embryo, which comprises only a small proportion of the seed. Nevertheless, among the upregulated miRNA target genes we found SLP2 and AGO2 , which are regulated by miR156 and miR403, respectively. Both miRNAs were significantly depleted from AGO1 in cer7 seeds ( Fig. 5D ). Interestingly, we also observed decreased loading of miR165/166 and miR168 , which regulate PHABULOSA / PHAVOLUTA ( PHB / PHV ) and AGO1 , respectively; however, these miRNA target mRNAs did not increase in abundance, instead, their levels were reduced. A subset of genes targeted by miRNAs are prone to induce secondary siRNAs, including AGO1 and PHB 46 , 47 . We indeed found enrichment of 21/22-nt siRNAs in PHB and AGO1 in our AGO1 RIP-seq samples of cer7, explaining their reduced abundance ( Fig. 5E-F ). These observations substantiate the idea of miRNA depletion through ectopic siRNA competition for AGO1 loading, suggesting that miR168 and miR165/166 are likely outcompeted by ectopic 21-nt siRNAs derived from their own target loci. Discussion In this study, we discovered that RNA surveillance in the maternal sporophyte is important to ensure embryo survival, suggesting that the exchange of RNAs between the maternal sporophyte and the embryo is substantially more relevant than what has been generally recognized. PTGS-dependent seed abortion in cer7 We demonstrated that cer7 developmental defects are linked to ectopic PTGS, as suppression of PTGS in sgs3 and rdr6 mutant backgrounds fully restored seed viability ( Fig. 2A-B ). Despite this suppression, introducing wild-type SGS3 and RDR6 alleles through pollen reinstated the seed abortion phenotype, suggesting that PTGS amplification can still occur in the embryo ( Fig. 2A-C ). These findings indicate that a PTGS trigger—likely an aberrant RNA fragment—is generated in the cer7 sporophyte and subsequently transported to the embryo, where it serves as a template for RDR6-SGS3-mediated siRNA amplification ( Fig. 6 ). Validating this idea, expression of CER7 in the seed coat was suYicient to suppress the seed abortion phenotype of cer7 , in line with the sporophytic maternal eYect in embryo development ( Fig. 3 ). Further supporting the role of PTGS in cer7 embryo lethality, we found that the cer7;ago1-27 double mutant fully suppressed seed abortion. Analysis of self-pollinated cer7;ago1-27 /+ heterozygous plants confirmed that AGO1 acts in the embryo to suppress cer7 -associated embryo arrest ( Fig. 2D-E ). Given that ago1-27 is a hypomorphic allele deficient in PTGS but not miRNA function 33 , we conclude that maternal PTGS is the primary driver of cer7 seed abortion. Download figure Open in new tab Fig. 6: Model of sporophytic-maternally dependent PTGS–mediated embryo arrest in cer7 . In WT, the exosome clears aberrant transcripts (depicted in blue) preventing their channeling to the PTGS pathway. In cer7, aberrant transcripts accumulate in the seed coat and are the templates of RDR6;SGS3 for dsRNA synthesis which are processed by DCL4/2 to produce ectopic 21/22-nt siRNAs (depicted in red). Ectopic siRNAs are loaded into AGO1 and outcompete a subset of miRNAs (depicted in green) for loading onto AGO1, including MIR167 , which c ontributes to the sporophytic maternally controlled embryo arrest in cer7 . Our data support a model in which an aberrant transcript or subset of transcripts accumulates in cer7 sporophytic maternal tissues and reaches the embryo, leading to PTGS amplification and seed abortion. Our data also show that PTGS amplification similarly occurs in the sporophytic tissues, likely contributing to an increase in the dosage of ectopic siRNAs reaching the embryo, consistent with the sporophytic maternal eYect of cer7 on embryo development. As a result, genes such as PHB and AGO1 are targeted for ectopic siRNA-mediated silencing, undermining embryonic development. Nevertheless, RNA-seq and sRNA-seq analyses suggest that mRNA depletion due to PTGS alone does not fully account for the cer7 phenotype. While only three genes enriched in 21/22-nt siRNAs ( AGO1 , XTH9 , BGLU ) exhibited reduced mRNA abundance in cer7 seeds, AGO1 IP sRNA-seq identified additional targets, including CER3 , BGLU37 , and PHB . Notably, PHB and AGO1 are expressed in embryonic tissues, and PHB mRNA levels were depleted in cer7 RNA-seq data. Since null ago1 mutants are inviable but do not exhibit severe early embryo defects 42 , our findings suggest that ectopic siRNA accumulation targeting essential genes, such as PHB , contributes to the cer7 embryo arrest. Weak ago1 alleles exacerbate embryo defects when PHB , PHV , and REV —all members of the class III HD-Zip transcription factor family—are lost 48 , supporting this hypothesis. Consistently, AGO1 RIP-seq from cer7 seeds revealed 21-nt secondary siRNAs for PHB , PHV , and REV , as well as ectopic siRNA accumulation for SAMDC1 , a gene crucial for embryo patterning 49 . Defective miRNA loading in AGO1 contributes to cer7 embryo arrest Our data furthermore suggest that defective miRNA loading into AGO1 is an additional component of the cer7 embryo arrest. Supporting this hypothesis, our findings include: 1) a reduction in the proportion of miRNAs in cer7 sRNA-seq samples from developing seeds; 2) an increase in 21-nt siRNAs from protein-coding genes in cer7 AGO1 RIP-Seq; 3) significant depletion of key miRNAs crucial for embryo development in cer7 AGO1 RIP-Seq; 4) mir167 mutants exhibiting an identical globular embryo arrest with a sporophytic maternal eYect 14 ; and 5) dcl1 ( sin1 ) mutants, which disrupt miRNA biogenesis, displaying similar maternal-eYect embryo arrest 50 , 51 . These observations suggest that ectopic siRNA accumulation disrupts AGO1 function, leading to impaired miRNA loading and subsequent embryo developmental arrest. Maternal sporophyte as a regulator of embryogenesis The angiosperm seed structure implies a direct maternal influence on embryo development, beyond its mechanical properties 52 . However, the only known symplastic connection between seed coat and zygotic tissues is the suspensor 7 , facilitating auxin transport to the embryo. Given the strong embryo arrest phenotype in cer7 , along with the absence of observable endosperm defects, our findings support a model in which PTGS precursors and siRNAs move from the maternal seed coat to the embryo via the suspensor, imposing a dosage-sensitive PTGS-dependent arrest ( Fig. 6 ). This model is consistent with the observed suppression of cer7 seed defects upon CER7 expression in the seed coat. Alternatively, RNA movement could also occur through vesicular traYicking, suggesting that RNA-based maternal control over embryogenesis may involve additional, unexplored transport mechanisms. Determining the nature of these transported RNA molecules and their specific mechanisms of movement represents an exciting avenue for future research. Conclusion This study provides evidence that the RNA exosome plays a crucial role in seed development by preventing the accumulation of aberrant transcripts in maternal seed coat tissues. If left unchecked, these transcripts trigger the production of secondary siRNAs, with deleterious consequences for embryo development. Our findings further indicate that the maternal integuments of the seed function not only as a physical protective structure but also as a permeability barrier where RNA molecules can cross seed compartments, exerting maternal control over embryogenesis. The discovery of CER7 as a key regulator in sporophytic maternal tissues highlights the complex interplay between maternal and embryonic gene regulation, revealing the importance of RNA decay in plant development. Methods Plant material and growth conditions Seeds were surface-sterilized using 70% ethanol, followed by three washes with sterile water. They were then sown on ½ MS medium supplemented with 0.5% sucrose and 0.8% agar. After stratification at 4 °C for 2 days, seeds were germinated under long-day conditions (16 h light/8 h dark) at 21 °C. Seedlings were transferred to soil after 10–12 days and grown in phytotrons under long-day conditions (21 °C day/20 °C night, 70% relative humidity, 150 µE light intensity). All Arabidopsis thaliana plants used in this study were in the Columbia-0 (Col-0) genetic background. Mutant alleles were obtained from the Arabidopsis Biological Resource Center ( www.arabidopsis.org ) and include: cer7-3 (SAIL_747_B08) 22 , cer7-4 (GK_089C02) 23 , rdr6-15 (SAIL_617_H07) 53 , sgs3-14 (SALK_001394) 54 , dcl1-14 (SALK_056243) 36 , and ago1-27 33 . Phenotypic analysis of embryo development and DIC microscopy Homozygous cer7 and wild-type (WT) plants were grown in parallel, emasculated, and pollinated two days later. Siliques were harvested at 2, 3, 5, and 7 days after pollination (DAP), and developing seeds were cleared using a modified Hoyer’s solution consisting of 7.5 g gum Arabic, 100 g chloral hydrate, 5 ml glycerol, and 30 ml water, which was diluted 1:1 with a solution containing 7.5 g gum Arabic, 5 ml glycerol, and 30 ml water. Embryos were visualized using a Leica DM2500 microscope equipped with Nomarski diYerential interference contrast (DIC) optics. Seed abortion and segregation analysis cer7 was introgressed into the rdr6-15 , sgs3-14 , dcl1-14, and ago1-27 mutant backgrounds by crossing. Homozygous and heterozygous cer7 plants and double mutants were identified by PCR genotyping. Crosses were performed by emasculating flowers and pollinating them two days later. Seed abortion was evaluated at 14 DAP using a dissecting microscope, when aborted seeds could be distinguished from WT siblings. At this stage, aborted seeds appeared white, while WT seeds in the same silique were green. All crosses were performed with at least two biological and two technical replicates, using diYerent parent plants and independent experimental batches. Pictures were taken with a Keyence Digital Microscope. Genetic segregation analysis was conducted on heterozygous cer7 plants identified by PCR genotyping and hand pollinated. Progeny was sown, and individual plants were genotyped by PCR. Segregation ratios were tested against the expected Mendelian ratio for a recessive allele using a Chi-square test. All genotyping primers are listed in Supplementary Table 4 . Feulgen staining Sample preparation and embedding for Feulgen staining was performed as previously described (Braselton, Wilkinson, and Clulow 1996). Briefly, whole siliques were fixed in ethanol:acetic acid (3:1) overnight, then washed three times in water for 15 minutes each. Samples were incubated in freshly prepared 5 N HCl for 1 hour, followed by three additional 15-minute water washes. Staining was performed in SchiY’s reagent for 3 hours, followed by three washes in cold water and sequential 10-minute washes in increasing concentrations of ethanol (10%, 30%, 50%, 70%, 90%). The samples were then incubated in 100% ethanol overnight. Embedding was performed using a graded ethanol:LR White resin series (3:1, 1:1, 1:3) for 1 hour each, followed by overnight incubation in pure LR White. Samples were mounted in LR White with accelerator and polymerized overnight at 60°C. Samples were imaged using a LEICA Stellaris 8 DIVE microscope in multiphoton mode, with excitation at 800 nm and detection in the 563– 668 nm emission range. Construction of CER7-GFP transgenic plants Regions upstream of the translation start sites of dVPE (-2,976 bp; AT3G20210), BAN (-191 bp), and RPS5A (-1,660bp; AT3G11940) were cloned into the pB7FWG.2 vector 55 , replacing the CaMV 35S promoter. The pB7FWG.2 vector was linearized with Sac I and Spe I, and the upstream fragments were inserted using the Takara In-Fusion Snap Assembly Kit (TAKARA, 638945) to generate new destination vectors. The CER7 coding sequence (1317 bp) was amplified from cDNA and recombined into pDONR221 using the Gateway BP Clonase II Enzyme Mix (ThemoFisher, 11789100). Final constructs, pdVPE::CER7-GFP , pBAN::CER7-GFP , and RPS5A::CER7-GFP , were obtained via LR recombination (ThermoFisher, 11791020) between CER7_pDONR221 and the corresponding promoter-containing destination vectors. See Supplementary Table 4 for the list of promoters used for cloning. Heterozygous cer7 plants were transformed with the final constructs using the floral dip method 56 . Transgenic plants were selected based on Basta resistance and PCR. GFP fluorescence was assessed using a Leica DM2500 fluorescence microscope. Transgenic heterozygous and homozygous cer7 lines were identified by PCR genotyping. Complementation analysis was carried out in homozygous cer7 backgrounds using at least seven independent transgenic lines. Confocal microscopy Imaging of pdVPE::CER7-GFP, pBAN::CER7-GFP , and RPS5A::CER7-GFP expression was done using a LEICA Stellaris 8 Dive microscope with an excitation of 488 nm and an emission range of 493–551 nm. Developing seeds were mounted in 10% glycerol and fluorescence was observed. RNA-seq Wild-type (WT), cer7-4 , sgs3-14 , and cer7-4;sgs3-14 double mutant plants were manually self-pollinated. Siliques were dissected at 3 days after pollination (DAP), and approximately 500 seeds per genotype were collected and stored in RNAlater solution (ThermoFisher, AM7021). Total RNA was extracted using TRIzol Reagent (Invitrogen, 15596018) according to the manufacturer’s instructions. RNA samples were treated with DNase I at 37 °C for 1 hour (ThermoFisher, EN0521), followed by heat inactivation at 65 °C for 10 minutes and DNase removal by TRIzol extraction. mRNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645S) in combination with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S). Sequencing was performed on a NextSeq 1000 platform using 150-bp paired-end (PE) mode. PE reads were trimmed using Trim Galore by removing 5 bp from the 5′ end and 20 bp from the 3′ end. Trimmed reads were aligned to the Arabidopsis thaliana reference genome (TAIR10) using HISAT2 57 . Mapped reads were sorted and indexed with SAMtools 58 . Gene-level read counts were obtained using HTSeq-count, and expression values were normalized to transcripts per million (TPM) using StringTie. DiYerential gene expression analysis was performed with DESeq2 (v1.42.0) 59 in R (v4.3.0). Genes with an absolute log2 fold change (LFC) ≥ 0.8 and a false discovery rate (FDR) < 0.05 were considered significantly diYerentially expressed. Data visualization was carried out using the ggplot2 package in R. sRNA-seq sRNA libraries were generated for wild-type (WT), cer7-4 , sgs3-14 , and cer7-4;sgs3-14 using same DNAse-treated RNA used for mRNA-seq libraries referred above. 1-2ug of DNAse-treated RNA was loaded into a denaturing acrylamide gel at 16% and gels bands between 16- and 30-nt were excised and small RNA enriched samples were recovered using DNA gel elution buYer from NEB and a Cellulose Acetate Membrane column. Libraries were made using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) following the manufacturer’s instructions. Sequencing was performed on a NovaSeq 6000 platform using 150-bp paired-end (PE) mode. Only one read of the pair was used in further analyses. Adapters were removed from the first read of each 150-bp PE library using Cutadapt, and reads between 19–26 nt in length were retained for further analysis. Processed reads were aligned to the Arabidopsis thaliana reference genome (TAIR10), and small RNA loci were annotated using ShortStack 60 , 61 with the parameters --mincov 2 and --mmap f. To identify regions with diYerential small RNA accumulation, 21-nt and 22-nt reads were extracted from BAM files using SAMtools 58 , and coverage across 100-bp genomic windows was calculated using the bedtools multicov function. DiYerential accumulation of small RNAs across windows was assessed with DESeq2 59 in R (v4.3.0). Windows with an absolute log2 fold change (LFC) ≥ 0 and a false discovery rate (FDR) < 0.05 were considered significant and merged if they were within 100 bp of each other using the bedtools merge function. Significant windows were annotated to TAIR10 genome features using bedtools intersect, and visually inspected in a genome browser to confirm genotype-specific enrichment or depletion. Data visualization was performed using the ggplot2 package in R. AGO1 RIP-seq Siliques closest to the inflorescences of homozygous cer7 and wild-type (WT) plants were removed, and newly formed siliques were harvested five days later, corresponding to an estimated 2–3 days after self-pollination. For each replicate, 500 mg of siliques were collected and flash-frozen in liquid nitrogen. Endogenous AGO1 immunoprecipitation was performed as previously described 62 . Briefly, tissue was ground to a fine powder using a mortar and pestle, resuspended in three volumes of crude extract buYer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10% glycerol; 5 mM MgCl₂; 0.1% IGEPAL; 5 mM DTT; and 1× cOmplete™ Protease Inhibitor Cocktail), and incubated at 8 rpm for 20 minutes at 4 °C. The crude extract was centrifuged at 20,000 × g for 15 minutes, repeated 2–3 times until clarified. Equal amounts of clarified extract were incubated with AGO1 antibody (Agrisera, AS09527) pre-bound to PureProteome Protein A magnetic beads (Millipore, LSKMAGA02) for 3 hours at 4 °C on a rotating carousel (10–12 rpm). Immune complexes were washed four times with crude extract buYer, and AGO1-associated RNA was extracted directly from the beads using TRIzol Reagent (Invitrogen, 15596018) following the manufacturer’s protocol. Small RNA library preparation and downstream analysis were performed as described above, omitting the initial small RNA enrichment step. Sequencing was performed on a NextSeq 1000 platform. Data availability Sequencing data generated in this study are available in the Gene Expression Omnibus in NCBI under the accession numbers GSE295374 for RNA-seq and GSE295375 for sRNA-seq. Author contributions G.D.T.D.L. and C.K. conceptualized the project, developed the methodology and provided supervision. G.D.T.D.L., M.S.T., V.V., and U.K. conducted experiments, G.D.T.D.L. performed bioinformatic analyses. C.K. acquired funding and administered the project. G.D.T.D.L. and C.K. wrote the original paper draft. All authors reviewed and edited the paper. Competing interests Authors declare that they have no competing interests. Funding Knut and Alice Wallenberg Foundation grant 2018-0206 (C.K.), Knut and Alice Wallenberg Foundation grant 2019-0062 (C.K.), Max Planck Society, Germany. Acknowledgments We thank Daniela Barro-Trastoy and Kai Wang for helpful comments on the manuscript. Funder Information Declared Knut and Alice Wallenberg Foundation, https://ror.org/004hzzk67 , grant 2018-0206 (C.K.) , grant 2019-0062 (C.K.) Max Planck Society, Germany References 1. ↵ Woudenberg , S. , Hadid , F. , Weijers , D. & Borassi , C . The maternal embrace: the protection of plant embryos . J. Exp. Bot . 75 , 4210 – 4218 ( 2024 ). OpenUrl CrossRef PubMed 2. ↵ Xu , W. , Sato , H. , Bente , H. , Santos-González , J. & Köhler , C . Endosperm cellularization failure induces a dehydration-stress response leading to embryo arrest . Plant Cell 35 , 874 – 888 ( 2023 ). OpenUrl CrossRef PubMed 3. Hehenberger , E. , Kradolfer , D. & Köhler , C . Endosperm cellularization defines an important developmental transition for embryo development . Development 139 , 2031 – 2039 ( 2012 ). OpenUrl Abstract / FREE Full Text 4. ↵ Weijers , D. , Van Hamburg , J.-P. , Van Rijn , E. , Hooykaas , P. J. J. & OYringa , R. Diphtheria toxin-mediated cell ablation reveals interregional communication during Arabidopsis seed development . Plant Physiol . 133 , 1882 – 1892 ( 2003 ). OpenUrl Abstract / FREE Full Text 5. ↵ Costa , L. M. et al. Central Cell-Derived Peptides Regulate Early Embryo Patterning in Flowering Plants . Science 344 , 168 – 172 ( 2014 ). OpenUrl Abstract / FREE Full Text 6. ↵ Yang , S. et al. The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development . Development 135 , 3501 – 3509 ( 2008 ). OpenUrl Abstract / FREE Full Text 7. ↵ Robert , H. S. et al. Maternal auxin supply contributes to early embryo patterning in Arabidopsis . Nat. Plants 4 , 548 – 553 ( 2018 ). OpenUrl CrossRef PubMed 8. ↵ Morley-Smith , E. R. et al. The transport of sugars to developing embryos is not via the bulk endosperm in oilseed rape seeds . Plant Physiol . 147 , 2121 – 2130 ( 2008 ). OpenUrl Abstract / FREE Full Text 9. ↵ Stadler , R. , Lauterbach , C. & Sauer , N . Cell-to-cell movement of green fluorescent protein reveals post-phloem transport in the outer integument and identifies symplastic domains in Arabidopsis seeds and embryos . Plant Physiol . 139 , 701 – 712 ( 2005 ). OpenUrl Abstract / FREE Full Text 10. ↵ Grover , J. W. et al. Abundant expression of maternal siRNAs is a conserved feature of seed development . Proc. Natl. Acad. Sci. U. S. A . 117 , 15305 – 15315 ( 2020 ). OpenUrl Abstract / FREE Full Text 11. ↵ Mizzotti , C. et al. The MADS box genes SEEDSTICK and ARABIDOPSIS B sister play a maternal role in fertilization and seed development . Plant J . 70 , 409 – 420 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 12. Zhang , M. et al. Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis . Plant J . 92 , 1005 – 1019 ( 2017 ). OpenUrl CrossRef PubMed 13. ↵ Ray , S. , Golden , T. & Ray , A . Maternal eYects of the short integument mutation on embryo development in Arabidopsis . Dev. Biol . 180 , 365 – 369 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 14. ↵ Yao , X. et al. An essential role for miRNA167 in maternal control of embryonic and seed development . Plant Physiol . 180 , 453 – 464 ( 2019 ). OpenUrl Abstract / FREE Full Text 15. ↵ Dziembowski , A. , Lorentzen , E. , Conti , E. & Séraphin , B. A single subunit, Dis3, is essentially responsible for yeast exosome core activity . Nat. Struct. Mol. Biol . 14 , 15 – 22 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 16. ↵ Lebreton , A. , Tomecki , R. , Dziembowski , A. & Séraphin , B . Endonucleolytic RNA cleavage by a eukaryotic exosome . Nature 456 , 993 – 996 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 17. ↵ Yang , M. et al. RRP41L, a putative core subunit of the exosome, plays an important role in seed germination and early seedling growth in Arabidopsis . Plant Physiol . 161 , 165 – 178 ( 2013 ). OpenUrl Abstract / FREE Full Text 18. ↵ Chekanova , J. A. et al. Genome-wide high-resolution mapping of exosome substrates reveals hidden features in the Arabidopsis transcriptome . Cell 131 , 1340 – 1353 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 19. Kumakura , N. , Otsuki , H. , Tsuzuki , M. , Takeda , A. & Watanabe , Y . Arabidopsis AtRRP44A is the functional homolog of Rrp44/Dis3, an exosome component, is essential for viability and is required for RNA processing and degradation . PLoS One 8 , e79219 ( 2013 ). OpenUrl CrossRef PubMed 20. Yan , X. , Yan , Z. & Han , Y . RRP42, a subunit of exosome, plays an important role in female gametophytes development and mesophyll cell morphogenesis in Arabidopsis . Front. Plant Sci . 8 , 981 ( 2017 ). 21. ↵ Zhang , W. , Murphy , C. & Sieburth , L. E . Conserved RNaseII domain protein functions in cytoplasmic mRNA decay and suppresses Arabidopsis decapping mutant phenotypes . Proc. Natl. Acad. Sci. U. S. A . 107 , 15981 – 15985 ( 2010 ). OpenUrl Abstract / FREE Full Text 22. ↵ Hooker , T. S. , Lam , P. , Zheng , H. & Kunst , L . A core subunit of the RNA-processing/degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis . Plant Cell 19 , 904 – 913 ( 2007 ). OpenUrl Abstract / FREE Full Text 23. ↵ Lange , H. et al. RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis . Nat. Commun . 10 , ( 2019 ). 24. ↵ Koornneef , M. , Hanhart , C. J. & Thiel , F . A Genetic and Phenotypic Description of Eceriferum (cer) Mutants in Arabidopsis thaliana . J. Hered . 80 , 118 – 122 ( 1989 ). OpenUrl CrossRef Web of Science 25. ↵ Lam , P. et al. The exosome and trans-acting small interfering RNAs regulate cuticular wax biosynthesis during Arabidopsis inflorescence stem development . Plant Physiol . 167 , 323 – 336 ( 2015 ). OpenUrl Abstract / FREE Full Text 26. Lam , P. et al. RDR1 and SGS3, components of RNA-mediated gene silencing, are required for the regulation of cuticular wax biosynthesis in developing inflorescence stems of Arabidopsis . Plant Physiol . 159 , 1385 – 1395 ( 2012 ). OpenUrl Abstract / FREE Full Text 27. ↵ Zhao , L. & Kunst , L . SUPERKILLER complex components are required for the RNA exosome-mediated control of cuticular wax biosynthesis in Arabidopsis inflorescence stems . Plant Physiol . 171 , 00450.2016 ( 2016 ). 28. ↵ Martinez de Alba , A. E. , et al. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs . Nucleic Acids Res . 43 , 2902 – 2913 ( 2015 ). OpenUrl CrossRef PubMed 29. Zhang , X. et al. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis . Science 348 , ( 2015 ). 30. ↵ Moreno , A. B. et al. Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants . Nucleic Acids Res . 41 , 4699 – 4708 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 31. ↵ Vaucheret , H. & Voinnet , O . The plant siRNA landscape . Plant Cell 36 , 246 – 275 ( 2024 ). OpenUrl CrossRef PubMed 32. ↵ Yoshikawa , M. et al. Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis . Proc. Natl. Acad. Sci. U. S. A . 118 , 1 – 9 ( 2021 ). OpenUrl CrossRef PubMed 33. ↵ Morel , J.-B. et al. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance . Plant Cell 14 , 629 – 639 ( 2002 ). OpenUrl Abstract / FREE Full Text 34. ↵ Chen , H.-M. et al. 22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants . Proc. Natl. Acad. Sci. U. S. A . 107 , 15269 – 15274 ( 2010 ). OpenUrl Abstract / FREE Full Text 35. ↵ Cuperus , J. T. et al. Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis . Nat. Struct. Mol. Biol . 17 , 997 – 1003 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 36. ↵ Zheng , B. , Chen , X. & McCormick , S . The anaphase-promoting complex is a dual integrator that regulates both MicroRNA-mediated transcriptional regulation of cyclin B1 and degradation of Cyclin B1 during Arabidopsis male gametophyte development . Plant Cell 23 , 1033 – 1046 ( 2011 ). OpenUrl Abstract / FREE Full Text 37. ↵ Debeaujon , I. et al. Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of diYerentiation and role in seed development . Plant Cell 15 , 2514 – 2531 ( 2003 ). OpenUrl Abstract / FREE Full Text 38. ↵ Devic , M. et al. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development . Plant J . 19 , 387 – 398 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 39. ↵ Nakaune , S. et al. A vacuolar processing enzyme, deltaVPE, is involved in seed coat formation at the early stage of seed development . Plant Cell 17 , 876 – 887 ( 2005 ). OpenUrl Abstract / FREE Full Text 40. ↵ Belmonte , M. F. et al. Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed . Proceedings of the National Academy of Sciences 1222061110 - ( 2013 ). 41. ↵ Varapparambath , V. et al. Mechanical conflict caused by a cell-wall-loosening enzyme activates de novo shoot regeneration . Dev. Cell 57 , 2063 – 2080 .e10 ( 2022 ). OpenUrl CrossRef PubMed 42. ↵ Bohmert , K. et al. AGO1 defines a novel locus of Arabidopsis controlling leaf development . EMBO J . 17 , 170 – 180 ( 1998 ). OpenUrl Abstract / FREE Full Text 43. ↵ Fagard , M. , Boutet , S. , Morel , J. B. , Bellini , C. & Vaucheret , H . AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals . Proc. Natl. Acad. Sci. U. S. A . 97 , 11650 – 11654 ( 2000 ). OpenUrl Abstract / FREE Full Text 44. ↵ You , C. et al. FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis . Nat. Commun . 10 , 4424 ( 2019 ). OpenUrl CrossRef PubMed 45. ↵ Plotnikova , A. , Kellner , M. J. , Schon , M. A. , Mosiolek , M. & Nodine , M. D . MicroRNA dynamics and functions during Arabidopsis embryogenesis . Plant Cell 31 , 2929 – 2946 ( 2019 ). OpenUrl Abstract / FREE Full Text 46. ↵ Vigh , M. L. , BressendorY , S. , ThieYry , A. , Arribas-Hernández , L. & Brodersen , P . Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis . Nucleic Acids Res . 50 , 1396 – 1415 ( 2022 ). OpenUrl CrossRef PubMed 47. ↵ Branscheid , A. et al. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis . Nucleic Acids Res . 43 , 10975 – 10988 ( 2015 ). OpenUrl CrossRef PubMed 48. ↵ Grigg , S. P. et al. Repression of apical homeobox genes is required for embryonic root development in Arabidopsis . Curr. Biol . 19 , 1485 – 1490 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 49. ↵ Ge , C. et al. BUD2, encoding an S-adenosylmethionine decarboxylase, is required for Arabidopsis growth and development . Cell Res . 16 , 446 – 456 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ Schwartz , B. , Yeung , E. & Meinke , D . Disruption of morphogenesis and transformation of the suspensor in abnormal suspensor mutants of Arabidopsis . Development 120 , 3235 – 3245 ( 1994 ). OpenUrl Abstract 51. ↵ Nodine , M. D. & Bartel , D. P . MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis . Genes Dev . 24 , 2678 – 2692 ( 2010 ). OpenUrl Abstract / FREE Full Text 52. ↵ Bauer , A. et al. Spatiotemporally distinct responses to mechanical forces shape the developing seed of Arabidopsis . EMBO J . 43 , 2733 – 2758 ( 2024 ). OpenUrl CrossRef PubMed 53. ↵ Allen , E. et al. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana . Nat. Genet . 36 , 1282 – 1290 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 54. ↵ Peragine , A. , Yoshikawa , M. , Wu , G. , Albrecht , H. L. & Poethig , R. S . SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis . Genes Dev . 18 , 2368 – 2379 ( 2004 ). OpenUrl Abstract / FREE Full Text 55. ↵ Karimi , M. , Inze , D. & Depicker , A . GATEWAY TM vectors for Agrobacterium -mediated plant transformation . Trends Plant Sci . 7 , 193 – 195 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 56. ↵ Clough , S. J. & Bent , A. F . Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana . Plant J . 16 , 735 – 743 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 57. ↵ Kim , D. , Paggi , J. M. , Park , C. , Bennett , C. & Salzberg , S. L . Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype . Nat. Biotechnol . 37 , 907 – 915 ( 2019 ). OpenUrl CrossRef PubMed 58. ↵ Danecek , P. et al. Twelve years of SAMtools and BCFtools . Gigascience 10 , giab008 ( 2021 ). OpenUrl CrossRef PubMed 59. ↵ Love , M. I. , Huber , W. & Anders , S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biol . 15 , 550 ( 2014 ). OpenUrl CrossRef PubMed 60. ↵ Axtell , M. J . ShortStack: Comprehensive annotation and quantification of small RNA genes . RNA 19 , 740 – 751 ( 2013 ). OpenUrl Abstract / FREE Full Text 61. ↵ Johnson , N. R. , Yeoh , J. M. , Coruh , C. & Axtell , M. J . Improved placement of multi-mapping small RNAs . G3: Genes, Genomes, Genetics 6 , 2103 – 2111 ( 2016 ). OpenUrl CrossRef 62. ↵ Hacquard , T. et al. The Arabidopsis F-box protein FBW2 targets AGO1 for degradation to prevent spurious loading of illegitimate small RNA . Cell Rep . 39 , 110671 ( 2022 ). 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Share Maternal control of RNA decay safeguards embryo development Gerardo Del Toro-De León , Maria Sofia Trenti , Varsha Vasudevan , Ursula Krause , Claudia Köhler bioRxiv 2025.05.28.656625; doi: https://doi.org/10.1101/2025.05.28.656625 Share This Article: Copy Citation Tools Maternal control of RNA decay safeguards embryo development Gerardo Del Toro-De León , Maria Sofia Trenti , Varsha Vasudevan , Ursula Krause , Claudia Köhler bioRxiv 2025.05.28.656625; doi: https://doi.org/10.1101/2025.05.28.656625 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 (7617) Biochemistry (17633) Bioengineering (13856) Bioinformatics (41841) Biophysics (21399) Cancer Biology (18529) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24281) Genetics (15582) Genomics (22461) Immunology (17700) Microbiology (40295) Molecular Biology (17140) Neuroscience (88413) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4813) Physiology (7632) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)