Cotyledon opening during seedling deetiolation is determined by ABA-mediated splicing regulation

Cotyledon opening during seedling deetiolation is determined by ABA-mediated splicing regulation | 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 Cotyledon opening during seedling deetiolation is determined by ABA-mediated splicing regulation Guiomar Martín , Alvaro Larran , Julia Qüesta , Paula Duque doi: https://doi.org/10.1101/2025.01.29.635410 Guiomar Martín 1 GIMM - Gulbenkian Institute for Molecular Medicine , Lisbon, Portugal 2 Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona , 08007 Barcelona, Spain 3 Centre for Research in Agricultural Genomics , 08193 Cerdanyola del Vallés, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: guiomar.martin{at}cragenomica.es paula.duque{at}gimm.pt Alvaro Larran 2 Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona , 08007 Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Julia Qüesta 2 Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona , 08007 Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paula Duque 1 GIMM - Gulbenkian Institute for Molecular Medicine , Lisbon, Portugal Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: guiomar.martin{at}cragenomica.es paula.duque{at}gimm.pt Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract During seedling deetiolation, plants adjust their development to expose photosynthetic tissues to sunlight, enabling the transition from heterotrophic to autotrophic growth. While various plant hormones are known to influence this process, the role of abscisic acid (ABA) remains unclear. Here, we reveal that ABA plays a major role in controlling the dynamics of cotyledon aperture during seedling deetiolation. In darkness, ABA accumulates in the cotyledons to effectively repress their opening. However, light exposure reverses this effect, allowing the cotyledons to open. Our findings indicate that the ABA-mediated regulation of cotyledon dynamics is accompanied by genome-wide rearrangements in both transcriptional and splicing patterns. We demonstrate that the ABA-dependent adjustments of cotyledon and splicing dynamics in response to light depend on the positive role of two splicing factors, RS40 and RS41. Moreover, we identify transcriptional and posttranscriptional mechanisms that repress the activity of these proteins in the dark. Altogether, this work sheds light on the interplay between light and ABA, highlighting a new developmental outcome: cotyledon opening, and identifying a novel layer of gene regulation: alternative splicing. Introduction Plants that germinate in subterranean darkness grow heterotrophically from seed reserves. Under these conditions, hypocotyls elongate rapidly to reach the soil surface and ensure survival, while the apical hook remains folded and the cotyledons closed to protect the apical meristem. This developmental program is known as skotomorphogenesis and, through a process called seedling deetiolation, it switches to photomorphogenesis once plants reach the soil surface and perceive sunlight ( Arsovski et al, 2012 ). In turn, photomorphogenic development is characterized by the arrest of hypocotyl elongation, the unfolding of the hook, the opening and expansion of cotyledons, and the synthesis of chlorophyl ( Gommers & Monte, 2018 ). These developmental adjustments are key to transitioning to phototrophic growth. Decades of extensive research have determined the molecular pathways that direct seedling deetiolation. These studies have mainly focused on the transcriptional control of gene expression ( Wu, 2014 ; Cheng et al, 2021 ). In the dark, the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), whose activity depends on its interaction with SUPPRESSOR OF PHYA-105 (SPA) proteins, favors the accumulation of PHYTOCHROME INTERACTING FACTORS (PIFs) while preventing the action of ELONGATED HYPOCOTYL 5 (HY5) ( Deng et al, 1991 ; Ponnu & Hoecker, 2021 ). PIFs and HY5 act respectively as negative ( Leivar et al, 2008 ; Leivar & Monte, 2014 ) and positive regulators of light responses ( Oyama et al, 1997 ; Gangappa & Botto, 2016 ). When seedlings reach the soil surface, light perceived by the different families of plant photoreceptors enhances the accumulation of positive regulators of photormophogenesis, which then reverts the transcriptional program established in darkness ( Lee et al, 2007 ; Leivar et al, 2009 ). During the transition from intron-containing precursor messenger RNA (pre-mRNA) to mature mRNA (mRNA), introns are removed and the flanking exons joined. Splice sites define the exon/intron boundaries and are recognized by the spliceosome, a large and dynamic protein complex consisting of five small nuclear ribonucleoprotein particles (snRNPs) and over 200 additional proteins ( Chen & Moore, 2015 ). However, not every potential splice site is recognized every time a gene is transcribed, resulting in the production of more than one mRNA from the same gene via a process known as alternative splicing. The splicing outcome is modulated by splicing regulators such as serine/arginine-rich (SR) proteins ( Long & Caceres, 2008 ) and heterogeneous nuclear ribonucleoproteins (hnRNPs) ( Han et al, 2010 ). These proteins bind to cis regulatory elements located in exonic or intronic sequences of the pre-mRNA, to either promote or inhibit spliceosome recognition of specific splice sites. SR proteins represent the most extensively studied splicing factors in plants. In Arabidopsis thaliana , they are encoded by 18 genes distributed among six subfamilies, three of which are plant-specific; in addition, there are two genes coding for SR-like proteins ( Barta et al, 2010 ). Most of these splicing factors show dynamic expression ( Shikata et al, 2014 ; Calixto et al, 2018 ) and phosphorylation ( van Bentem et al, 2006 ; Fluhr, 2008 ) patterns, being often regulated through alternative splicing ( Kalyna et al, 2006 ). Notably, recent genome-wide studies have revealed that splicing landscapes are subjected to light control during seedling deetiolation ( Shikata et al, 2014 ; Martín, 2023 ; Hartmann et al, 2016 ), suggesting an active role for this molecular process in deetiolation. In agreement, a few functional studies have demonstrated that mutations in splicing-related proteins ( Shikata et al, 2012 ; Xin et al, 2017 ; Yan et al, 2022 ; Kathare et al, 2022 ), as well as enhanced expression of particular splice variants ( Shikata et al, 2014 ; Hartmann et al, 2016 ; Dong et al, 2020 ; Huang et al, 2022 ), can alter plant responses to light signals at the seedling stage. Remarkably, among all light-induced physiological responses, these phenotypic studies have thus far only revealed defects in the regulation of hypocotyl length. Despite these significant lines of evidence, we are still far from understanding how alternative splicing transduces light signals during seedling deetiolation. Seedling deetiolation is known to be regulated by various plant hormones ( de Wit et al, 2016 ; Liu et al, 2017 ), yet the role of abscisic acid (ABA) in this process remains unclear ( Humplík et al, 2017 ). ABA is pivotal in many aspects of plant development, such as seed dormancy, and in implementing stress responses ( Cutler et al, 2010 ). Early ABA signal transduction involves the hormone’s perception by intracellular receptors, triggering derepression of SNF1-related kinases 2 (SnRK2) protein kinases, which then phosphorylate downstream effector proteins ( Umezawa et al, 2010 ). Given its accumulation in dormant seeds and plants facing stressful conditions, functional and molecular studies addressing the biological implications of ABA have focused mainly on these physiological contexts. Consequently, the endogenous regulation of ABA in controlling seedling deetiolation is not fully understood. The accumulation of ABA under stress is known to regulate the ABSCISIC ACID INSENSITIVE 5 (ABI5) transcription factor ( Lopez-Molina et al, 2001 ) to repress seedling post-germinative growth, a developmental stage during which light and ABA signaling pathways interact. First, light availability influences ABA sensitivity, which is enhanced in the dark, where COP1 and PIFs promote ABA-induced post-germinative arrest by positively regulating the activity of ABI5 ( Yadukrishnan et al, 2020 ; Qi et al, 2020 ). Moreover, the positive regulators of photomorphogenesis HY5, FHY3 and DET1 also influence ABA signaling ( Chen et al, 2008 ; Tang et al, 2013 ; Xu et al, 2020 ). These reports, which address the molecular interplay between ABA and light signaling, have used exogenously applied ABA, thus mimicking a physiological scenario in which plants are subjected to abiotic stress. In this study, we investigated the role of endogenous ABA during seedling deetiolation in non-stressful environments. Our findings reveal that etiolated Arabidopsis seedlings accumulate ABA in the cotyledons, contributing to their closure in the dark. Once the plant perceives a light stimulus, the levels of ABA decrease, resulting in the opening of the cotyledons. Importantly, we show that ABA control of cotyledon opening during seedling deetiolation relies on the regulation of the activity of two SR proteins, RS40 and RS41. These results not only uncover a new developmental role for ABA but also extend our knowledge of the molecular interplay between ABA and light signaling by introducing an additional layer of regulation: alternative splicing. Our work demonstrates that transcriptional and posttranslational processes control the activity of these proteins to enable cotyledon opening in response to light. Results Endogenous ABA represses cotyledon opening in etiolated seedlings Fluorescence analysis of transgenic lines expressing the green fluorescent protein ( GFP) reporter under the control of either 35S or the promoter of the RAB18 gene, a classical gene marker for ABA content ( Hauser et al, 2017 ), revealed that the RAB18:GFP signal in etiolated seedlings is restricted mainly to cotyledons and disappears after light exposure ( Fig. 1A ). This finding was supported by the quantification of RAB18 transcript levels in wild-type (WT) cotyledons during seedling deetiolation ( Fig. 1B ). Thus, our data reveal that ABA predominantly accumulates in the cotyledons of etiolated seedlings. In agreement, the expression levels of RAB18 and RD29B, another important ABA-responsive gene ( Hauser et al, 2017 ), are reduced in mutant seedlings with a constitutive photomorphogenic phenotype in the dark (Appendix Fig. S1; Pham et al., 2018 ). Download figure Open in new tab Fig. 1. ABA represses cotyledon aperture during seedling deetiolation. (A) Fluorescence images of seedlings expressing GFP under the control of either the 35S or the RAB18 promoter and grown for three days in the dark (left) and then exposed for 24 hours to continuous white light (WL) or kept in darkness (D) for the same amount of time (right). (B) RAB18 transcript levels analyzed by RT-qPCR in cotyledons of wild-type (WT) seedlings grown in the dark for 3 days (0h) and then transferred to WL for 4 or 8 hours (h). Data are the means ± SE of biological triplicates, with different letters indicating statistically significant differences between timepoints by Tukey’s multiple comparison test ( P < 0.05). (C) Quantification of cotyledon opening in the snrk2-2.3.6 , pyl1458 and abi1-1 ABA-hyposensitive mutants and their respective WT seedlings (Col-0 or L er ) grown in the dark for 2, 3, 4 or 7 days (d). At least 70 seedlings per genotype were analyzed. Different letters indicate statistical differences between medians (Dunn’s test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Quantification of cotyledon opening in at least 35 WT seedlings grown for 3 days in the dark and then exposed to light in the presence of different concentrations (µM) of ABA. (E) Representative images of 3-day-old Col-0 and snrk2 triple mutants exposed for 24 hours to WL in the absence or presence of 100 µM ABA. (F) Quantification of cotyledon opening in the three ABA-hyposensitive mutants and their respective WTs grown as in (D). At least 40 seedlings per genotype were analyzed. In C, D and F, thick lines and shaded areas represent respectively the median and the interquartile range, with the experiments repeated at least twice with similar results. Given these observations, we decided to investigate the functional role of ABA in etiolated cotyledons. We first examined the cotyledon phenotype of three well known ABA-signaling mutants: snrk2.236 ( Fujita et al, 2009 ), pyl1458 ( Gonzalez-Guzman et al, 2012 ) and abi1-1 ( Meyer et al, 1994 ), all of which are hyposensitive to the action of the hormone. Notably, all three mutants displayed more open cotyledons when compared to WT seedlings ( Fig. 1C ), indicating that the endogenous ABA in the cotyledons represses their aperture in the dark. In line with this conclusion, light-induced cotyledon opening was repressed by exogenously supplied ABA in a dose-dependent manner ( Fig. 1D ; Appendix Fig. S2), with this effect being nearly undetectable in the ABA-hyposensitive mutants ( Fig. 1E, F ). Together, these results show that during seedling deetiolation endogenous ABA levels are tissue and light-dependent. This pattern unveils a functional role in seedling development. In the dark, ABA accumulates in the cotyledons, preventing them from opening. However, after light exposure, ABA levels decline, allowing for photomorphogenic development of the cotyledons. ABA reduces light-mediated transcriptional changes in cotyledons To investigate how ABA affects light responsiveness at the transcriptional level during seedling deetiolation, we sequenced the mRNA of dark-grown cotyledons and cotyledons exposed to light for three hours in the absence or presence of exogenous ABA (Appendix Fig. S3). The comparison between etiolated cotyledons and those exposed to light in the absence of ABA (D vs WL) showed that 1,289 genes were downregulated in response to light (47%; WL-down) while 1,426 were upregulated (53%; WL-up) (Dataset EV1). Notably, around two thirds showed reduced responsiveness to light after 3 hours of ABA treatment ( Fig. 2A ). This indicates that ABA largely antagonizes the transcriptional changes triggered by light, which is in line with its repressive role in cotyledon opening (see Fig. 1 ). As exemplified in Appendix Fig. S4, not all of the light-mediated transcriptional changes were subjected to ABA control, indicating that ABA does not produce a universal disruption of light transcriptional regulation. Download figure Open in new tab Fig. 2. ABA reverses light-induced transcriptional changes. (A) Percentage of light-repressed or light-induced genes (respectively WL-down and WL-up) whose light responsiveness is lower (WL-ABA WL) in the presence of ABA. Fold changes (FC) were quantified from our RNA sequencing data for cotyledons extracted from wild-type (WT) seedlings grown for 3 days in the dark and then exposed to continuous white light (WL) for 3 hours in the absence or presence of ABA (100 µM). (B) Distribution of the expression values of genes whose light regulation is reversed by ABA (see Methods for details). (C) Enriched gene ontology categories of biological process (BP) and cellular component (CC) for genes defined as light-induced (left) or repressed (right) and reversed by ABA. DAVID p -value indicates significance (Fisher’s exact test; P < 0.05; Dataset EV2). (D) Transcript levels of genes involved in light-regulated cotyledon opening ( Dong et al., 2019 ) quantified from our RNA sequencing data. Data are the means ± SE of biological triplicates and relative to the dark timepoint. Different letters indicate statistically significant differences between conditions by Tukey’s multiple comparison test ( P < 0.05). To gain further insight into ABA control of light signaling, we focused our analysis on genes whose light responsiveness was suppressed by ABA (ABA-reversed; Fig. 2B ; see Methods for further details). A gene ontology (GO) analysis revealed that genes whose light induction was reversed by ABA are linked to biological processes and cellular compartments associated with organ growth, such as cell division and the cell wall ( Fig. 2C ; Dataset EV2). Accordingly, the expression of SAUR genes, which are established mediators of light-induced cotyledon opening ( Dong et al, 2019 ), aligns with this pattern ( Fig. 2D ). These results are consistent with our discovery that cotyledon photomorphogenic changes are suppressed by ABA ( Fig. 1 ; Appendix Fig. S2). On the other hand, genes whose light downregulation was reversed by ABA are enriched for ABA-responsive genes, such as the key transcription factors of the ABA-signaling pathway ABI5 and ABF3 ( Fig. 2C ; Appendix Fig. S5). The fact that ABA-responsive genes are enriched among our set of cotyledon light-repressed genes reversed by ABA supports our finding that ABA is functionally active in this tissue in the dark. ABA represses light-regulated splicing changes in cotyledons Previous studies have determined how light modifies the splicing landscape of whole etiolated seedlings ( Shikata et al, 2014 ; Hartmann et al, 2016 ; Martín, 2023 ). Our splicing analysis of cotyledon samples revealed that light regulation of alternative splicing in etiolated cotyledons coincides with that observed in whole seedlings. First, the 224 genes subjected to alternative splicing in response to light (Dataset EV3) were enriched for splicing-related GO categories (Appendix Fig. S6A; Dataset EV4; Shikata et al., 2014 ; Hartmann et al., 2016 ; Martín, 2023 ). Second, the number of splice variants with retained introns in the dark was higher than in the light (Appendix Fig. S6B; Hartmann et al., 2016 ; Martín, 2023 ). Finally, the mRNA splice forms whose abundance increases in the dark tend to produce unproductive transcripts, and most of them are targeted to the Nonsense-Mediated Decay (NMD) pathway (Appendix Fig. S6C-D; Hartmann et al., 2016 ; Martín, 2023 ). These data demonstrate that, as predicted for whole seedlings, the proportion of unproductive transcripts in dark-grown cotyledons is higher than upon light exposure. In the light, we identified 35 differential alternative splicing (DAS) events in response to the 3-hour ABA treatment (WL-ABA vs WL; Dataset EV5). Interestingly, ABA-regulated DAS events were also regulated by light exposure (D vs WL) and dark conditions mimic the percent of inclusion (PSI) values of ABA-treated light-grown seedlings ( Fig. 3A ). Moreover, similarly to what we observed for gene expression, the vast majority of the PSI value changes induced by light in cotyledons from dark-grown seedlings (D vs WL; 267 DAS events) were reduced in the presence of ABA ( Fig. 3B ). Figure 3C and Appendix Fig. S7 shows this ABA reversion of light-mediated splicing changes in two well-known light-regulated DAS events characterized by producing unproductive isoforms in the dark ( Petrillo et al, 2014 ; Hartmann et al, 2018 ). In agreement with the overlapping splicing patterns found between dark and ABA conditions, ABA-regulated DAS events in the light (WL-ABA vs WL) are enriched for events that disrupt the ORFs when ABA is present ( Fig. 3D ; unproductive in WL-ABA). Furthermore, the PSI values of these unproductive DAS events are higher in the upf1upf3 mutant ( Fig. 3E ), indicating that the mRNAs in which they occur are degraded through the NMD. Download figure Open in new tab Fig. 3. ABA controls light-regulated splicing changes. (A) Percent of inclusion (PSI) values, quantified from our RNA sequencing data, for the differential alternative splicing (DAS) events upregulated (left) and downregulated (right) by ABA in cotyledons from wild-type (WT) seedlings grown for 3 days in the dark and then exposed to continuous white light (WL) for 3 hours in the absence or presence of ABA (100 µM). (B) Percentage of light-induced or light-repressed DAS events (WL-up and WL-up, respectively) whose light responsiveness is lower (WL-ABA WL) in the presence of ABA. (C) RT-PCR analysis of SR30 (top) and RS31 (bottom) alternative transcript levels in seedlings grown as in (A) with the exception that seedlings were exposed to WL for 8 hours. The bar graphs present Percent of Splice In (PSI) values after quantification of the band intensities using the Image J software. Data are the means ± SE of biological triplicates and asterisks indicate statistically significant between WL and WL-ABA conditions (Student’s test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Percentage of DAS events located in gene coding sequence (CDS) regions that potentially produce unproductive mRNAs or alternative protein isoforms (see Methods for details) in three groups of DAS events: non-regulated (non-reg.), and up-, or downregulated by ABA. DAS events that have the ultimate effect of generating unproductive isoforms in response to ABA are highlighted. (E) PSI values of the CDS-located, light-regulated DAS events that generate unproductive transcripts in wild-type (WT), upf1 , upf3 , and upf1upf3 seedling samples. This quantification was conducted from RNA sequencing data obtained from GSE41432 ( Drechsel et al., 2013 ). These results thus indicate that, compared to light conditions, both ABA and darkness reduce the proportion of functional mRNAs in the cotyledons. Moreover, the observed splicing patterns ( Fig. 3A-C ) are in line with our phenotypical data showing that ABA blocks light-induced morphogenic changes. This correlation suggested that splicing dynamics are important for controlling cotyledon opening during seedling deetiolation. ABA controls cotyledon opening via regulation of RS40 and RS41 splicing factors To establish a functional link between light regulation of alternative splicing and cotyledon aperture, we screened the SR family of splicing regulators for mutants with defects in the dynamics of cotyledon opening during seedling deetiolation. Strikingly, this screen identified RS40 and RS41, a pair of paralogous SR splicing factors ( Kalyna & Barta, 2004 ), as positive regulators of this process. Cotyledon opening in two independent rs40 rs41 double mutant lines was markedly delayed during seedling deetiolation ( Fig. 4A ). Phenotyping of two single mutants for each SR protein determined that only the upstream mutation of RS41 produces a significant delay in cotyledon aperture and that this effect is milder than that of the respective double mutant ( Figs. 4A ; Appendix Fig. S8). We thus concluded that these two proteins act synergistically to positively regulate cotyledon opening during seedling deetiolation. Notably, light-induced alternative splicing changes in the cotyledons were barely perceptible in the rs40 rs41 double mutant (Appendix Fig. S9). Download figure Open in new tab Fig. 4. RS40 and RS41 splicing factors mediate light and ABA regulation of cotyledon opening. (A) Quantification of cotyledon opening in rs40 rs41 double mutants and their respective wild-type (WT) grown for 3 days in the dark and then exposed to white light (WL) for 3, 6, 9 or 24 hours (h). (B) RT-qPCR quantification of RS40 and RS41 transcript levels in cotyledons of WT seedlings grown in the dark for 3 days and then transferred to WL for 3 hours (h) in the absence or presence of ABA (100 µM). PP2A was used as a reference gene. For each gene, expression levels in the WT were set to 1. Data are the means ± SE of biological triplicates. Different letters indicate statistically significant differences between conditions by one-way ANOVA test ( P < 0.05). (C) Quantification of cotyledon opening in WT, RS40 -or RS41 -overexpressing seedlings grown in the dark for 2, 3 4 or 7 days (d). ( D-E ) Cotyledon angle values for seedlings of WT, RS40 - and RS41 -overexpressing lines (D) or of rs40 rs41 double mutant lines and their respective WTs (E) grown for 3 days in the dark and then exposed to WL for 3, 6, 9 or 24 hours (h) in the absence or presence of ABA (100 µM). In A and C-E , thick lines and shaded areas represent respectively the median and the interquartile range of at least 65 seedlings. The experiment was repeated at least twice with similar results. Given that RS40 and RS41 are positive regulators of light-induced cotyledon aperture and alternative splicing, both ABA-dependent processes, we hypothesized that the activity of these two proteins is regulated by light in an ABA-dependent manner. We found that in dark-grown cotyledons RS40 expression is reduced by light but unaffected by ABA treatment ( Fig. 4B ). However, the mRNA levels of RS41 were subtly but consistently enhanced by light only in the absence of ABA ( Figs. 4B ; Appendix Fig. S10). Therefore, the expression of the latter gene is controlled by both light and ABA. SR proteins have also long been known to be under extensive alternative splicing control ( Palusa et al, 2007 ; Kalyna & Barta, 2004 ). In particular, the mRNAs of the Arabidopsis RS40 and RS41 genes harbor alternative splicing events that encompass premature stop codons ( Kalyna et al, 2006 ; Iida & Go, 2006 ). The inclusion levels of these alternative splicing events in our cotyledon samples were also light-regulated; in the dark, the percentage of unproductive mRNAs was higher than in the light but ABA did not interfere with this regulation (Appendix Fig. S11), precluding a functional link between RS40 and RS41 splicing patterns and ABA regulation of cotyledon aperture. By contrast, our transcriptional data had suggested that the transcriptional control that light and ABA exert on RS41 , but not on RS40 , plays an important role in regulating cotyledon opening during seedling deetiolation. To confirm this, we disrupted the transcriptional regulation of RS41 and RS40 by stably expressing each gene under the control of the constitutive 35S promoter (Appendix Fig. S12). Interestingly, the cotyledons of transgenic lines overexpressing RS41 but not those of RS40 overexpressor lines were more open in etiolated seedlings ( Fig. 4C ). This suggests that RS41 overexpression overcomes the repressive role that endogenous ABA exerts on cotyledon opening. In accordance, RS41 -overexpressing seedlings were hyposensitive to exogenous ABA in the light, while seedlings overexpressing RS40 responded to ABA as WT seedlings ( Figs. 4D and Appendix Fig. S13). In line with the ABA-hyposensitive phenotype of RS41 -overexpressing plants, rs40 rs41 double mutants were hypersensitive to ABA at both the phenotypical ( Fig. 4E ) and molecular (Appendix Fig. S14) levels. Overall, our results indicate that RS40 and RS41 control light-regulated alternative splicing and cotyledon opening during seedling deetiolation in an ABA-dependent manner. Our data also show that the transcriptional control of RS41 is sufficient to regulate cotyledon aperture downstream of both the light and ABA signals. RS40 and RS41 phosphorylation represses cotyledon opening in etiolated seedlings Extensive data have demonstrated that in eukaryotes the activity of SR proteins is modulated by phosphorylation ( Long & Caceres, 2008 ). In vivo phosphorylation of SR proteins has also been detected in plants ( van Bentem et al, 2006 ; Wang et al, 2023 ), although little is known about the biological contexts of this regulation. Here, we examined a potential role for RS40 and RS41 phosphorylation in seedling deetiolation using kinases inhibitors, as previously established ( Lin et al, 2022 ; Haltenhof et al, 2020 ). To this end, we treated etiolated seedlings with the kinase inhibitor K252a, which is known to mimic light-induced splicing patterns in the dark (Appendix Fig. S15; Hartmann et al., 2016 ), although the molecular basis for K252a-induced alternative splicing changes remains unknown. Importantly, we found that at least the two K252a-induced alternative splicing changes that we profiled are dependent on the phosphorylation status of RS40 and RS41 (Appendix Fig. S15). Moreover, K252a caused the opening of etiolated cotyledons ( Fig. 5A, B ; Appendix Fig. S16), indicating that phosphorylation events are pivotal in maintaining cotyledon closure in the dark. Strikingly, the rs40 rs41 double mutants were insensitive to this effect ( Fig. 5A, B ), while single mutants were partially affected (Appendix Fig. S17). Download figure Open in new tab Fig. 5. Effect of the K252a kinase inhibitor on RS40- and RS41-regulated cotyledon opening. (A) Representative images of 3-day-old Col-3 wild-type (WT) and rs40rs41.1 seedlings exposed for 9 hours to the absence or presence of K252a (1 µM). (B-C) Quantification of cotyledon opening in rs40 rs41 double mutants (B) and RS40 - or RS41 -overexpresssing plants (C) as well as in their respective WTs (Col-0 or Col-3; see Methods for details) grown as in (A). Data represent the median of at least 50 seedlings, and asterisks indicate statistically significant differences between K252a-treated mutants and their respective WTs (Mann–Whitney test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., non-significant). The experiment was repeated at least twice with similar results. (D) Cotyledon angle values of WT seedlings grown as in (A) and in the presence of ABA (100 µM). Thick lines and shaded areas represent respectively the median of at least 25 seedlings and the interquartile range. These results demonstrate the requirement for phosphorylated RS40 and RS41 in etiolated cotyledons to maintain their distinct closure and splicing profile. Further supporting the significance of posttranslational regulation of RS40 and RS41 at this developmental stage, we observed that K252a enhances cotyledon opening in RS40 - and RS41 -overexpressing seedlings ( Fig. 5C ; Appendix Fig. S18). In fact, RS40OX plants behaved like the WT under control conditions, but their cotyledons were more open upon addition of the kinase inhibitor ( Fig. 5C ; Appendix Fig. S18). Finally, exogenously supplied ABA failed to fully block K252a-induced cotyledon opening ( Fig. 5D ), consistent with the notion that endogenous ABA in cotyledons acts via phosphorylation of RS40 and RS41 to repress cotyledon aperture in the dark ( Fig. 6 ). Download figure Open in new tab Fig. 6. Proposed model for SR protein regulation of cotyledon opening during seedling deetiolation. Cotyledon opening is central to the transition from heterotrophic to autotrophic growth during deetiolation. Our data show that in etiolated seedlings, endogenous ABA prevents the opening of cotyledons. Under these conditions, the RS40 and RS41 splicing factors remain phosphorylated, which inhibits their activity. This regulatory mechanism changes the pattern of alternative splicing in response to light, increasing the proportion of functional mRNAs in cotyledons. PTC, premature termination codon. Discussion A study conducted by Humplík et al. in 2015 revealed a spatio-temporal regulation of ABA content in tomato seedlings during seedling deetiolation ( Humplík et al, 2015b ). The authors found that in darkness, ABA accumulates predominantly in the cotyledons and to a lesser extent in the elongation zone of hypocotyls. This finding aligned with previous research indicating a decrease in ABA levels upon exposure to light in etiolated seedlings of pea and lentil ( Weatherwax et al, 1996 ; Symons & Reid, 2003 ). Based on these data, as well as on the experimental profiling of hypocotyl length in Arabidopsis and tomato ABA signaling mutants ( Barrero et al, 2008 ; Humplík et al, 2015a ), they concluded that in etiolated seedlings endogenous ABA positively affects hypocotyl elongation. This conclusion challenged the traditional view of ABA as a growth inhibitor ( Humplík et al, 2017 ), which is largely based on experiments involving externally supplemented ABA ( Lorrai et al, 2018 ). The authors also argued that the biological function of cotyledon-accumulated ABA might be to inhibit the maturation of chloroplasts and stomatal development, but no data was provided to support this claim. Apart from these initial results, the spatio-temporal regulation of ABA content during seedling deetiolation has not been addressed further. The present study shows that in Arabidopsis ABA levels are elevated in etiolated cotyledons until seedlings are exposed to light ( Fig. 1A ). This result is supported by the fact that Arabidopsis genes encoding ABA biosynthetic hormones ( AtNCED2 , 3 , 5 , and 9 ) are light-repressed ( Charron et al, 2009 ). Interestingly, our data demonstrate that in this tissue endogenous ABA represses cotyledon opening ( Fig. 1C-D ). This function is in line with the known repressive role of exogenously provided ABA on light-induced cotyledon greening ( Xu et al, 2020 ; Guan et al, 2014 ), adding biological insight into the longstanding debate about the relationship between light and ABA (Kraepiel & E, 1997). Different studies have implicated SR proteins in mediating ABA-related physiological processes during early seedling development ( Laloum et al, 2023 , 2018 ; Albuquerque-Martins et al, 2023 ; Carvalho et al, 2010 ; Chen et al, 2013 ). In particular, the rs40 and rs41 loss-of-function. mutants are hypersensitive to the repressive effect of externally supplied ABA on seed germination and root elongation ( Chen et al, 2013 ). Apart from the involvement of RS40 and RS41 in the splicing regulation of stress-related genes ( Chen et al, 2013 ) and miRNA biogenesis ( Chen et al, 2015 ), no other functional roles have been identified for these proteins. Here, we show that RS40 and RS41 play a prominent role in promoting light-induced cotyledon opening and alternative splicing during seedling deetiolation. While distinct splicing profiles of seedlings grown in either dark or light conditions had already been reported ( Shikata et al, 2014 ; Hartmann et al, 2016 ; Martín, 2023 ), the involvement of RS40 and RS41 in this regulation remained unknown. Our data demonstrate that both cotyledon opening and light-induced splicing changes in etiolated seedlings are reduced in mutant plants lacking functional RS40 and RS41, indicating that these two proteins play a positive role in mediating light responses. To date, few splicing-related proteins have been implicated in the control of seedling deetiolation. The SWAP1-SFPS-RRC1 ternary complex of RNA-binding proteins was found to coordinate alternative splicing and seedling development during deetiolation downstream of plant photoreceptors ( Xin et al, 2017 , 2019 ; Kathare et al, 2022 ). Notably, our study is the first to link the activity of plant splicing factors to the dynamics of cotyledon opening. Future work will uncover the functional contribution of RS40- and RS41-regulated alternative isoforms in determining cotyledon aperture. Our results also indicate that the regulation exerted by RS40 and RS41 on cotyledon opening and alternative splicing depends on basal ABA levels. The studies we conducted with loss- and gain-of-function plant lines support a model in which these proteins are inactive in the dark, when ABA content is higher and cotyledons remain closed, and active in the light. Interestingly, experimental data using the K252a kinase inhibitor show that these proteins are phosphorylated in the dark, which blocks their capacity to enhance cotyledon aperture ( Fig. 5 ). Previous studies of the ABA-responsive phosphoproteome identified RS41 among over 100 proteins that were differentially phosphorylated in response to ABA ( Wang et al, 2013 ; Umezawa et al, 2013 ). Umezawa et al. (2013) found evidence of a phosphopeptide in this protein that is downregulated by ABA, whereas Wang et al. (2013) described this protein as actively phosphorylated by the ABA-activated SnRK2.2, 2.3 and 2.6 kinases. Further work is required to precisely determine the ABA-mediated phosphorylation events that occur in this protein during seedling deetiolation. Importantly, in addition to the posttranslational regulation found for these proteins, our work demonstrates that transcriptional regulation of RS41 is also crucial for controlling cotyledon opening. Overall, our study underscores a novel role for endogenous ABA in controlling light-mediated cotyledon aperture during seedling deetiolation and links this biological function to the action of the RS40 and RS41 splicing factors. These findings significantly expand our current understanding of ABA and light signals and their molecular interplay, shedding new light on pathways that, so far, have been extensively studied only at the transcriptional level. Methods Plant materials The Arabidopsis thaliana seeds used in this study include the previously described snrk2-2.3.6 ( Fujita et al, 2009 ), pyl1458 ( Gonzalez-Guzman et al, 2012 ) and abi1-1 ( Meyer et al., 1994 ) mutants, which were kindly provided by E. Baena-González and P. Rodríguez. Seedlings expressing the green fluorescent protein (GFP) under the control of the RAB18 promoter ( Hauser et al., 2013 ) were obtained from the European Arabidopsis Stock Centre (NASC) in homozygosis (N68523). Single mutants rs40.1 (Sail_1055_C08; N841686) and rs41.2 (Salk_066799; N566799) were also obtained from NASC, propagated and genotyped to obtain homozygous mutants. Mutants rs40.2 (WiscDsLox382G12) and rs41.1 (Sail_64_C03) were previously reported ( Chen et al., 2015 ) and generously supplied by A. Watcher. As detailed in Appendix Fig. S7, rs40.1 and rs41.1 insertional mutants are in the Col-3 background, while rs40.2 and rs41.2 are in Col-0. The rs40rs41.1 and rs40rs41.2 double mutants were generated by crossing the two mutants of each genetic background and WT siblings from each cross were selected to be used as controls. As specified in the figure legends, each mutant line was always compared to the respective WT background. RS40OX and RS41OX lines were generated by cloning respectively a 1053- and 1074-bp fragment containing the coding sequence region of each gene under the control of the cauliflower mosaic virus (CaMV) 35S strong constitutive promoter in the eGFP-tagged version of the binary pBA002 vector using the XhoI/BamhI restriction sites. The resulting 35S::RS40-GFP ( OX40 ) and 35S::RS41-GFP ( OX41 ) constructs were transformed by agroinfiltration into Col-0 seedlings. Growth conditions and data representation Seeds were surface sterilized and sown on MS medium containing 1x Murashige and Skoog (MS) salts (Duchefa Biochemie), 2.5 mM MES (pH 5.7), 0.5 mM myo-inositol, and 0.8% agar (w/v). After stratification for 4 days at 4°C in darkness, seeds were submitted to a pulse of 3 hours of white light (90 mmol·m -2 ·s -1 ) to induce germination. For dark timecourse experiments, seedlings were then grown in continuous darkness for 2, 3, 4 and 7 days. When assessing cotyledon dynamics during deetiolation, seedlings were grown in the dark for 3 days and then exposed to white light (45 mmol·m -2 ·s -1 ) for 3, 6, 9 and 24 hours. Seedlings treated with ABA (Sigma) or the broad-range kinase inhibitor K252a (Cayman) were sown on top of filter paper placed on MS medium and, after 3 days of growth in the dark, transferred to plates supplemented with the respective reagent for different time periods: 3, 4, 6, 8, 9 or 24 hours of growth in either dark or white light conditions. The concentration and the time period used in each experiment is detailed in the figure legends. Phenotypic measurements of cotyledon opening were performed using the National Institutes of Health ImageJ software ( https://rsbweb.nih.gov/ij ), analyzing at least 30 seedlings per experiment. Phenotypic and microscopy experiments were repeated at least two times to validate reproducibility. For assessing gene expression or splicing, a minimum of three biological replicates were analyzed per condition and/or genotype tested, with the exception of the data shown in Appendix Fig. S11. Depending on the data distribution, these are presented as means and standard errors or medians and interquartile ranges. In accordance, statistical differences were determined by parametric (Unpaired t -test and Tukey) or non-parametric (Mann–Whitney and Dunn) tests using GraphPad Prism 8. Statistically significant differences were defined as indicated in the figure legends. RNA extraction Total RNA was extracted from dissected Arabidopsis thaliana cotyledons using the InnuPREP Plant RNA kit (Analytik Jena BioSolutions) and 1 µg treated with DNase I (Promega) to remove genomic DNA. cDNA synthesis using the oligo dT primer and the enzyme SuperScript III reverse transcriptase (Invitrogen) was conducted in the presence of RNase Out (Invitrogen). Darkness samples were collected in a dark room under green light. The cDNA was then used to quantify either gene expression or inclusion of alternatively spliced sequences. Gene expression and splicing analysis of individual genes Gene expression was measured by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) using a QuantStudioTM 7 Flex Real-Time PCR System 384-well format and the Absolute SYBR Green ROX Mix (Thermo Scientific) on 2.5 µL of cDNA (diluted 1:10) per 10 µL of reaction volume, containing 300 nM of each gene-specific primer (Dataset EV6). The PP2A gene was used for normalization ( Shin et al., 2007 ). Alternative splicing of SR30 , RS31 , RS40 and RS41 was quantified from RT-PCRs performed with the NZYTaq II 2x Green Master Mix (NZYtech). Reaction cycles were 95°C for 3 min (1X), 95°C for 30 s/58°C for 30 s/72°C for 5 min (35X), using primers flanking each alternative sequence (Dataset EV6). The PCR products were then loaded and run on a 2% agarose gel. GFP microscopy visualization Fluorescence images of the pRAB18-GFP and 35S-GFP transgenic plant lines were obtained from 3-day germinated seeds and then transferred to white light (45 mmol·m - 2 ·s -1 ) or kept in darkness for 24 hours, using the high sensitivity monochrome camera of a Leica DM6 epifluorescent microscope. Fluorescence images from 3-day old 35S overexpressing RS40-GFP and RS41-GFP etiolated seedlings were obtained using a Zeiss 980 Elyra 729 confocal microscope. A z-stack of 5 slices was captured, and merged images were generated and shown in Appendix Fig. S12. RNA sample preparation and sequencing RNA was extracted from 3-day-old wild-type etiolated seedlings treated or not with ABA (100 µM) for 3 hours in complete darkness, with a sample also collected before starting the treatment. These samples were collected in a dark room under green light. Oligo dT, strand-specific libraries from biological triplicates were built and sequenced using HiSeq2500 at the Centre for Genomic Regulation Genomics Unit (Barcelona). An average of 90 million 125-nucleotide single-end reads were generated per sample. Gene expression quantification from RNA sequencing data Quantification of Arabidopsis transcript expression from our RNA sequencing experiment (GSE273664) and public sequencing data (GSE112662, GSE164122, GSE41432) was performed using vast-tools v2.2.2 ( Tapial et al., 2017 ). This tool provides the cRPKM number (corrected-for-mappability reads per kbp of mappable sequence per million mapped reads) for each Arabidopsis transcript. This number is equivalent to the number of mapped reads per million mapped reads divided by the number of uniquely mappable positions of the transcript ( Labbé et al., 2012 ). To identify genes differentially expressed by light, we used the command vast-tools compare_expr with the option -norm to perform a quantile normalization of cRPKM values between dark and light samples. In addition, we filtered out the genes that were not expressed at cRPKM > 5 and had read counts > 50 across all the three replicates of at least one of two samples compared. Finally, light-regulated genes were defined as those with a fold change of at least 2 between each of the individual replicates from each condition. Alternative splicing quantification from RNA sequencing data We employed vast-tools v2.2.2 ( Tapial et al., 2017 ) to quantify alternative splicing from our sequencing data. This tool maps the RNA sequencing data to the araTha10 library, which is composed of an extended annotation of the Ensemble Plants v31, including all exon-exon and exon-intron junction sequences found in the Arabidopsis thaliana genome ( Martín et al., 2021 ). Derived from this mapping, vast-tools quantifies exon skipping (ES), intron retention (IR) as well as alternative donor (ALTD) and acceptor (ALTA) site choices. For all these types of events, vast-tools estimates the percent of inclusion (PSI) of the alternative sequence and associates a quality score to each alternative splicing event, based on the read coverage that sustains its PSI quantification (see https://github.com/vastgroup/vast-tools for details). To define differential alternative splicing (DAS) in response to light or ABA, we used the vast-tools compare command with the –min_ALT_use 25, -p_IR and -legacy_ALT filters (see https://github.com/vastgroup/vast-tools for details). The first ensures that ALT3 and ALT5 events are located in exons with a minimum PSI of 25 in each compared sample. The second filter eliminates those IR events with a significant imbalance between the two exon-intron junctions ( P < 0.05; binomial test; Braunschweig et al., 2014 ). Finally, -legacy_ALT forces up and down sequence inclusion evaluation of all ALT annotated events, not only of the most external spliced sites. Then, light- and ABA-regulated DAS events were defined as those with a |ΔPSI| > 15 between dark and light samples or ABA-treated and untreated light samples. In addition, to be selected as differentially regulated spliced events, the PSI distribution could not overlap between conditions (-min_range:5). Gene ontology enrichment analyses To identify significantly enriched biological processes, molecular functions and cellular components among the different sets of genes, analyses were performed using the functional annotation classification system DAVID ( Huang et al., 2007 ). For each comparison, only genes with transcripts or DAS events that passed equivalent filters than those used to define differentially expressed or spliced events were used as a background. Predicted impact of the alternative sequences on the gene coding sequence The impact predictions for all differentially spliced events were obtained from the Downloads section of PastDB ( http://pastdb.crg.eu/wiki/Downloads ). Briefly, this tool determines whether an alternative sequence preserves the frame and therefore generates an alternative protein, or whether it contains an in-frame stop codon or frame shift when included or excluded (see Martín et al., 2021 for details). Based on the presence of these disrupting sequences, we classified DAS events as unproductive when included or excluded. Author contributions GM and PD conceived the project. GM and AL performed experiments. All authors analyzed and discussed the data. GM and PD wrote the manuscript with inputs from all the authors. Disclosure and competing interests statement The authors declare no competing interests. Data availability Raw sequencing data were submitted to the Sequence Read Archive (accession number GSE273664). Supplementary Materials Appendix Fig. S1. RAB18 and RD29B expression levels in constitutive photomorphogenic mutants. Appendix Fig. S2. ABA repression of cotyledon aperture in the light. Appendix Fig. S3. RAB18 and RD29B expression levels in ABA-treated cotyledons during seedling deetiolation. Appendix Fig. S3. Expression levels of genes whose light responsiveness is unaffected by ABA. Appendix Fig. S4. ABI5 and ABF3 expression levels in ABA-treated cotyledons during seedling deetiolation. Appendix Fig. S5. Light-regulated splicing changes in cotyledons from etiolated seedlings. Appendix Fig. S7. Light and ABA regulation of AS events in SR30 and RS31 . Appendix Fig. S8. Light regulation of cotyledon opening in rs40 and rs41 single mutants. Appendix Fig. S9. Light and ABA regulation of AS in the rs40 rs41 double mutant. Appendix Fig. S10. ABA regulation of RS41 expression during seedling deetiolation. Appendix Fig. S11. Light and ABA regulation of AS events in RS40 and RS41 . Appendix Fig. S12. Generation of transgenic plants overexpressing RS40 and RS41 . Appendix Fig. S13. Light and ABA regulation of cotyledon opening in plants overexpressing RS40 or RS41 . Appendix Fig. S14. RAB18 and RD29B expression levels in ABA-treated rs40 rs41 double mutants. Appendix Fig. S15. K252a regulation of SR30 and RS31 light- and ABA-regulated AS events. Appendix Fig. S16. K252a regulation of cotyledon opening in etiolated seedlings. Appendix Fig. S17. K252a regulation of cotyledon opening in the rs40 and rs41 single mutants. Appendix Fig. S18. K252a regulation of cotyledon opening in plants overexpressing RS40 or RS41 . Acknowledgements We thank Andreas Wachter, Pedro Rodríguez, and Elena Baena-González for kindly providing the rs40-2 , rs41-1 , pyl1458 , snrk2-2.3.6 and abi1-1 mutants, Nil Veciana for technical assistance with the Leica DM6 epifluorescent microscope, and Vera Nunes for excellent plant care at the Instituto Gulbenkian de Ciência (IGC) Plant Facility. This work was funded by Fundação para a Ciência e a Tecnologia (FCT) through grants PTDC/BIA-BID/30608/2017, PTDC/ASP-PLA/2550/2021 (DOI: 10.54499/PTDC/ASP-PLA/2550/2021) and UIDB/ 04551/2020 (DOI: 10.54499/UIDB/04551/2020) as well as by the Spanish Ministry of Science and Innovation through grants RYC2020-030160-I and PID2021-125223NA-I00 (DOI: AEI/10.13039/501100011033/ FEDER, UE). G.M. was supported by an EMBO Long-Term Fellowship (ALTF 1576-2016) and a Marie Skłodowska-Curie Individual Postdoctoral Fellowship (EU project 750469). Footnotes https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE273664 References ↵ Albuquerque-Martins , R. , Szakonyi , D. , Rowe , J. , Jones , A. M. , and Duque , P . ( 2023 ). ABA signaling prevents phosphodegradation of the SR45 splicing factor to alleviate inhibition of early seedling development in Arabidopsis . Plant Commun 4 : 100495 . OpenUrl CrossRef PubMed ↵ Arsovski , A. A. , Galstyan , A. , Guseman , J. M. , and Nemhauser , J. L . ( 2012 ). Photomorphogenesis . Arabidopsis Book 10 : e0147 – e0147 . OpenUrl CrossRef PubMed ↵ Barrero , J. M. , Rodríguez , P. L. , Quesada , V. , Alabadí , D. , Blázquez , M. A. , Boutin , J.-P. , Marion-Poll , A. , Ponce , M. R. , and Micol , J. L . ( 2008 ). The ABA1 gene and carotenoid biosynthesis are required for late skotomorphogenic growth in Arabidopsis thaliana . Plant Cell Environ 31 : 227 – 234 . OpenUrl CrossRef PubMed Web of Science ↵ Barta , A. , Kalyna , M. , and Reddy , A. S. N . ( 2010 ). Implementing a Rational and Consistent Nomenclature for Serine/Arginine-Rich Protein Splicing Factors (SR Proteins) in Plants . Plant Cell 22 : 2926 – 2929 . OpenUrl Abstract / FREE Full Text ↵ Braunschweig , U. , Barbosa-Morais , N. L. , Pan , Q. , Nachman , E. N. , Alipanahi , B. , Gonatopoulos-Pournatzis , T. , Frey , B. , Irimia , M. , and Blencowe , B. J . ( 2014 ). Widespread intron retention in mammals functionally tunes transcriptomes . Genome Res 24 : 1774 – 1786 . OpenUrl Abstract / FREE Full Text ↵ Calixto , C. P. G. , Guo , W. , James , A. B. , Tzioutziou , N. A. , Entizne , J. C. , Panter , P. E. , Knight , H. , Nimmo , H. G. , Zhang , R. , and Brown , J. W. S . ( 2018 ). Rapid and Dynamic Alternative Splicing Impacts the Arabidopsis Cold Response Transcriptome . Plant Cell 30 : 1424 – 1444 . OpenUrl Abstract / FREE Full Text ↵ Carvalho , R. F. , Carvalho , S. D. , and Duque , P . ( 2010 ). The Plant-Specific SR45 Protein Negatively Regulates Glucose and ABA Signaling during Early Seedling Development in Arabidopsis . Plant Physiol 154 : 772 – 783 . OpenUrl Abstract / FREE Full Text ↵ Charron , J.-B. F. , He , H. , Elling , A. A. , and Deng , X. W . ( 2009 ). Dynamic Landscapes of Four Histone Modifications during Deetiolation in Arabidopsis . Plant Cell 21 : 3732 – 3748 . OpenUrl Abstract / FREE Full Text ↵ Chen , W. , and Moore , M. J . ( 2015 ). Spliceosomes . Current Biology 25 : R181 – R183 . OpenUrl CrossRef PubMed ↵ Chen , H. , Zhang , J. , Neff , M. M. , Hong , S.-W. , Zhang , H. , Deng , X.-W. , and Xiong , L . ( 2008 ). Integration of light and abscisic acid signaling during seed germination and early seedling development . Proceedings of the National Academy of Sciences 105 : 4495 – 4500 . OpenUrl Abstract / FREE Full Text ↵ Chen , T. , Cui , P. , Chen , H. , Ali , S. , Zhang , S. , and Xiong , L . ( 2013 ). A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis . PLoS Genet 9 : e1003875 . OpenUrl CrossRef PubMed ↵ Chen , T. , Cui , P. , and Xiong , L . ( 2015 ). The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis . Nucleic Acids Res 43 : 8283 – 8298 . OpenUrl CrossRef PubMed ↵ Cheng , M.-C. , Kathare , P. K. , Paik , I. , and Huq , E . ( 2021 ). Phytochrome Signaling Networks . Annu Rev Plant Biol 72 : 217 – 244 . OpenUrl CrossRef PubMed ↵ Cutler , S. R. , Rodriguez , P. L. , Finkelstein , R. R. , and Abrams , S. R . ( 2010 ). Abscisic Acid: Emergence of a Core Signaling Network . Annu Rev Plant Biol 61 : 651 – 679 . OpenUrl CrossRef PubMed Web of Science ↵ de Wit , M. , Galvão , V. C. , and Fankhauser , C. ( 2016 ). Light-Mediated Hormonal Regulation of Plant Growth and Development . Annu Rev Plant Biol 67 : 513 – 537 . OpenUrl CrossRef PubMed ↵ Deng , X. W. , Caspar , T. , and Quail , P. H . ( 1991 ). cop1: a regulatory locus involved in light-controlled development and gene expression in Arabidopsis . Genes & Development 5 : 1172 – 1182 . OpenUrl Abstract / FREE Full Text ↵ Dong , J. , Sun , N. , Yang , J. , Deng , Z. , Lan , J. , Qin , G. , He , H. , Deng , X. W. , Irish , V. F. , Chen , H. , et al. ( 2019 ). The Transcription Factors TCP4 and PIF3 Antagonistically Regulate Organ-Specific Light Induction of SAUR Genes to Modulate Cotyledon Opening during De-Etiolation in Arabidopsis . Plant Cell 31 : 1155 – 1170 . OpenUrl Abstract / FREE Full Text ↵ Dong , J. , Chen , H. , Deng , X. W. , Irish , V. F. , and Wei , N . ( 2020 ). Phytochrome B Induces Intron Retention and Translational Inhibition of PHYTOCHROME-INTERACTING FACTOR31 [OPEN] . Plant Physiol 182 : 159 – 166 . OpenUrl Abstract / FREE Full Text ↵ Drechsel , G. , Kahles , A. , Kesarwani , A. K. , Stauffer , E. , Behr , J. , Drewe , P. , Rätsch , G. , and Wachter , A . ( 2013 ). Nonsense-mediated decay of alternative precursor mRNA splicing variants is a major determinant of the Arabidopsis steady state transcriptome . Plant Cell 25 : 3726 – 3742 . OpenUrl Abstract / FREE Full Text ↵ Fluhr , R . ( 2008 ). Regulation of Splicing by Protein Phosphorylation - Nuclear pre-mRNA Processing in Plants . In (ed. Reddy , A. S. N .) and Golovkin , M .), pp. 119 – 138 . Berlin, Heidelberg : Springer Berlin Heidelberg . ↵ Fujita , Y. , Nakashima , K. , Yoshida , T. , Katagiri , T. , Kidokoro , S. , Kanamori , N. , Umezawa , T. , Fujita , M. , Maruyama , K. , Ishiyama , K. , et al. ( 2009 ). Three SnRK2 Protein Kinases are the Main Positive Regulators of Abscisic Acid Signaling in Response to Water Stress in Arabidopsis . Plant Cell Physiol 50 : 2123 – 2132 . OpenUrl CrossRef PubMed Web of Science ↵ Gangappa , S. N. , and Botto , J. F . ( 2016 ). The Multifaceted Roles of HY5 in Plant Growth and Development . Mol Plant 9 : 1353 – 1365 . OpenUrl CrossRef PubMed ↵ Gommers , C. M. M. , and Monte , E . ( 2018 ). Seedling Establishment: A Dimmer Switch-Regulated Process between Dark and Light Signaling . Plant Physiol 176 : 1061 – 1074 . OpenUrl FREE Full Text ↵ Gonzalez-Guzman , M. , Pizzio , G. A. , Antoni , R. , Vera-Sirera , F. , Merilo , E. , Bassel , G. W. , Fernández , M. A. , Holdsworth , M. J. , Perez-Amador , M. A. , Kollist , H. , et al. ( 2012 ). Arabidopsis PYR/PYL/RCAR Receptors Play a Major Role in Quantitative Regulation of Stomatal Aperture and Transcriptional Response to Abscisic Acid . Plant Cell 24 : 2483 – 2496 . OpenUrl Abstract / FREE Full Text ↵ Guan , C. , Wang , X. , Feng , J. , Hong , S. , Liang , Y. , Ren , B. , and Zuo , J . ( 2014 ). Cytokinin antagonizes abscisic acid-mediated inhibition of cotyledon greening by promoting the degradation of abscisic acid insensitive5 protein in Arabidopsis . Plant Physiol 164 : 1515 – 1526 . OpenUrl Abstract / FREE Full Text ↵ Haltenhof , T. , Kotte , A. , De Bortoli , F. , Schiefer , S. , Meinke , S. , Emmerichs , A.-K. , Petermann , K. K. , Timmermann , B. , Imhof , P. , Franz , A. , et al. ( 2020 ). A Conserved Kinase-Based Body-Temperature Sensor Globally Controls Alternative Splicing and Gene Expression . Mol Cell 78 : 57 – 69 .e4. OpenUrl CrossRef PubMed ↵ Han , S. P. , Tang , Y. H. , and Smith , R . ( 2010 ). Functional diversity of the hnRNPs: past, present and perspectives . Biochemical Journal 430 : 379 – 392 . OpenUrl Abstract / FREE Full Text ↵ Hartmann , L. , Drewe-Boß , P. , Wießner , T. , Wagner , G. , Geue , S. , Lee , H.-C. , Obermüller , D. M. , Kahles , A. , Behr , J. , Sinz , F. H. , et al. ( 2016 ). Alternative Splicing Substantially Diversifies the Transcriptome during Early Photomorphogenesis and Correlates with the Energy Availability in Arabidopsis . The Plant Cell 28 : 2715 – 2734 . OpenUrl Abstract / FREE Full Text ↵ Hartmann , L. , Wießner , T. , and Wachter , A . ( 2018 ). Subcellular Compartmentation of Alternatively Spliced Transcripts Defines SERINE/ARGININE-RICH PROTEIN30 Expression . Plant Physiol 176 : 2886 – 2903 . OpenUrl Abstract / FREE Full Text ↵ Hauser , F. , Chen , W. , Deinlein , U. , Chang , K. , Ossowski , S. , Fitz , J. , Hannon , G. J. , and Schroeder , J. I . ( 2013 ). A Genomic-Scale Artificial MicroRNA Library as a Tool to Investigate the Functionally Redundant Gene Space in Arabidopsis . Plant Cell 25 : 2848 – 2863 . OpenUrl Abstract / FREE Full Text ↵ Hauser , F. , Li , Z. , Waadt , R. , and Schroeder , J. I . ( 2017 ). SnapShot: Abscisic Acid Signaling . Cell 171 : 1708 – 1708 .e0. OpenUrl CrossRef PubMed ↵ Huang , D. W. , Sherman , B. T. , Tan , Q. , Collins , J. R. , Alvord , W. G. , Roayaei , J. , Stephens , R. , Baseler , M. W. , Lane , H. C. , and Lempicki , R. A . ( 2007 ). The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists . Genome Biol 8 : R183 . OpenUrl CrossRef PubMed ↵ Huang , C.-K. , Lin , W.-D. , and Wu , S.-H . ( 2022 ). An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development . Genome Biol 23 : 50 . OpenUrl CrossRef PubMed ↵ Humplík , J. F. , Bergougnoux , V. , Jandová , M. , Šimura , J. , Pěnčík , A. , Tomanec , O. , Rolčík , J. , Novák , O. , and Fellner , M . ( 2015a ). Endogenous Abscisic Acid Promotes Hypocotyl Growth and Affects Endoreduplication during Dark-Induced Growth in Tomato (Solanum lycopersicum L .). PLoS One 10 : e0117793 . OpenUrl CrossRef PubMed ↵ Humplík , J. F. , Turečková , V. , Fellner , M. , and Bergougnoux , V . ( 2015b ). Spatio-temporal changes in endogenous abscisic acid contents during etiolated growth and photomorphogenesis in tomato seedlings . Plant Signal Behav 10 : e1039213 . OpenUrl CrossRef PubMed ↵ Humplík , J. F. , Bergougnoux , V. , and Van Volkenburgh , E. ( 2017 ). To Stimulate or Inhibit? That Is the Question for the Function of Abscisic Acid . Trends Plant Sci 22 : 830 – 841 . OpenUrl CrossRef PubMed ↵ Iida , K. , and Go , M . ( 2006 ). Survey of Conserved Alternative Splicing Events of mRNAs Encoding SR Proteins in Land Plants . Mol Biol Evol 23 : 1085 – 1094 . OpenUrl CrossRef PubMed Web of Science ↵ Kalyna , M. , and Barta , A . ( 2004 ). A plethora of plant serine/arginine-rich proteins: redundancy or evolution of novel gene functions? Biochem Soc Trans 32 : 561 – 564 . OpenUrl Abstract / FREE Full Text ↵ Kalyna , M. , Lopato , S. , Voronin , V. , and Barta , A . ( 2006 ). Evolutionary conservation and regulation of particular alternative splicing events in plant SR proteins . Nucleic Acids Res 34 : 4395 – 4405 . OpenUrl CrossRef PubMed Web of Science ↵ Kathare , P. K. , Xin , R. , Ganesan , A. S. , June , V. M. , Reddy , A. S. N. , and Huq , E . ( 2022 ). SWAP1-SFPS-RRC1 splicing factor complex modulates pre-mRNA splicing to promote photomorphogenesis in Arabidopsis . Proceedings of the National Academy of Sciences 119 : e2214565119 . OpenUrl CrossRef PubMed Kraepiel , Y. , and E, M. ( 1997 ). Photomorphogenesis and phytohormones . Plant Cell Environ 20 : 807 – 812 . OpenUrl CrossRef ↵ Labbé , R. M. , Irimia , M. , Currie , K. W. , Lin , A. , Zhu , S. J. , Brown , D. D. R. , Ross , E. J. , Voisin , V. , Bader , G. D. , Blencowe , B. J. , et al. ( 2012 ). A Comparative Transcriptomic Analysis Reveals Conserved Features of Stem Cell Pluripotency in Planarians and Mammals . Stem Cells 30 : 1734 – 1745 . OpenUrl CrossRef PubMed ↵ Laloum , T. , Martín , G. , and Duque , P . ( 2018 ). Alternative Splicing Control of Abiotic Stress Responses . Trends Plant Sci 23 : 140 – 150 . OpenUrl CrossRef PubMed ↵ Laloum , T. , Carvalho , S. D. , Martín , G. , Richardson , D. N. , Cruz , T. M. D. , Carvalho , R. F. , Stecca , K. L. , Kinney , A. J. , Zeidler , M. , Barbosa , I. C. R. , et al. ( 2023 ). The SCL30a SR protein regulates ABA-dependent seed traits and germination under stress . Plant Cell Environ 46 : 2112 – 2127 . OpenUrl CrossRef ↵ Lee , J. , He , K. , Stolc , V. , Lee , H. , Figueroa , P. , Gao , Y. , Tongprasit , W. , Zhao , H. , Lee , I. , and Deng , X. W . ( 2007 ). Analysis of Transcription Factor HY5 Genomic Binding Sites Revealed Its Hierarchical Role in Light Regulation of Development . Plant Cell 19 : 731 – 749 . OpenUrl Abstract / FREE Full Text ↵ Leivar , P. , and Monte , E . ( 2014 ). PIFs: Systems Integrators in Plant Development . Plant Cell 26 : 56 – 78 . OpenUrl Abstract / FREE Full Text ↵ Leivar , P. , Monte , E. , Oka , Y. , Liu , T. , Carle , C. , Castillon , A. , Huq , E. , and Quail , P. H . ( 2008 ). Multiple Phytochrome-Interacting bHLH Transcription Factors Repress Premature Seedling Photomorphogenesis in Darkness . Current Biology 18 : 1815 – 1823 . OpenUrl CrossRef PubMed Web of Science ↵ Leivar , P. , Tepperman , J. M. , Monte , E. , Calderon , R. H. , Liu , T. L. , and Quail , P. H . ( 2009 ). Definition of Early Transcriptional Circuitry Involved in Light-Induced Reversal of PIF-Imposed Repression of Photomorphogenesis in Young Arabidopsis Seedlings . Plant Cell 21 : 3535 – 3553 . OpenUrl Abstract / FREE Full Text ↵ Lin , J. , Shi , J. , Zhang , Z. , Zhong , B. , and Zhu , Z. ( 2022 ). Plant AFC2 kinase desensitizes thermomorphogenesis through modulation of alternative splicing . iScience 25 . ↵ Liu , X. , Li , Y. , and Zhong , S . ( 2017 ). Interplay between Light and Plant Hormones in the Control of Arabidopsis Seedling Chlorophyll Biosynthesis . Frontiers in Plant Science 8 . ↵ Long , J. C. , and Caceres , J. F . ( 2008 ). The SR protein family of splicing factors: master regulators of gene expression . Biochemical Journal 417 : 15 – 27 . OpenUrl CrossRef ↵ Lopez-Molina , L. , Mongrand , S. , and Chua , N.-H . ( 2001 ). A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis . Proceedings of the National Academy of Sciences 98 : 4782 – 4787 . OpenUrl Abstract / FREE Full Text ↵ Lorrai , R. , Boccaccini , A. , Ruta , V. , Possenti , M. , Costantino , P. , and Vittorioso , P . ( 2018 ). Abscisic acid inhibits hypocotyl elongation acting on gibberellins, DELLA proteins and auxin . AoB Plants 10 : ply061 . OpenUrl PubMed ↵ Martín , G . ( 2023 ). Regulation of alternative splicing by retrograde and light signals converges to control chloroplast proteins . Frontiers in Plant Science 14 . ↵ Martín , G. , Márquez , Y. , Mantica , F. , Duque , P. , and Irimia , M . ( 2021 ). Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals . Genome Biol 22 : 35 . OpenUrl CrossRef PubMed ↵ Meyer , K. , Leube , M. P. , and Grill , E . ( 1994 ). A Protein Phosphatase 2C Involved in ABA Signal Transduction in Arabidopsis thaliana . Science (1979) 264 : 1452 – 1455 . OpenUrl Abstract / FREE Full Text ↵ Oyama , T. , Shimura , Y. , and Okada , K . ( 1997 ). The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl . Genes Dev 11 : 2983 – 2995 . OpenUrl Abstract / FREE Full Text ↵ Palusa , S. G. , Ali , G. S. , and Reddy , A. S. N . ( 2007 ). Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses . The Plant Journal 49 : 1091 – 1107 . OpenUrl CrossRef PubMed Web of Science ↵ Petrillo , E. , Herz , M. A. G. , Fuchs , A. , Reifer , D. , Fuller , J. , Yanovsky , M. J. , Simpson , C. , Brown , J. W. S. , Barta , A. , Kalyna , M. , et al. ( 2014 ). A chloroplast retrograde signal regulates nuclear alternative splicing . Science (1979) 344 : 427 – 430 . OpenUrl Abstract / FREE Full Text ↵ Pham , V. N. , Xu , X. , and Huq , E . ( 2018 ). Molecular bases for the constitutive photomorphogenic phenotypes in Arabidopsis . Development 145 :dev169870. ↵ Ponnu , J. , and Hoecker , U . ( 2021 ). Illuminating the COP1/SPA Ubiquitin Ligase: Fresh Insights Into Its Structure and Functions During Plant Photomorphogenesis . Frontiers in Plant Science 12 . ↵ Qi , L. , Liu , S. , Li , C. , Fu , J. , Jing , Y. , Cheng , J. , Li , H. , Zhang , D. , Wang , X. , Dong , X. , et al. ( 2020 ). PHYTOCHROME-INTERACTING FACTORS Interact with the ABA Receptors PYL8 and PYL9 to Orchestrate ABA Signaling in Darkness . Mol Plant 13 : 414 – 430 . OpenUrl CrossRef PubMed ↵ Shikata , H. , Shibata , M. , Ushijima , T. , Nakashima , M. , Kong , S.-G. , Matsuoka , K. , Lin , C. , and Matsushita , T . ( 2012 ). The RS domain of Arabidopsis splicing factor RRC1 is required for phytochrome B signal transduction . The Plant Journal 70 : 727 – 738 . OpenUrl CrossRef PubMed Web of Science ↵ Shikata , H. , Hanada , K. , Ushijima , T. , Nakashima , M. , Suzuki , Y. , and Matsushita , T . ( 2014 ). Phytochrome controls alternative splicing to mediate light responses in Arabidopsis Proceedings of the National Academy of Sciences 111 : 18781 LP – 18786. OpenUrl CrossRef ↵ Shin , J. , Park , E. , and Choi , G . ( 2007 ). PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis . The Plant Journal 49 : 981 – 994 . OpenUrl CrossRef PubMed Web of Science ↵ Symons , G. M. , and Reid , J. B . ( 2003 ). Hormone levels and response during de-etiolation in pea . Planta 216 : 422 – 431 . OpenUrl CrossRef PubMed Web of Science ↵ Tang , W. , Ji , Q. , Huang , Y. , Jiang , Z. , Bao , M. , Wang , H. , and Lin , R . ( 2013 ). FAR-RED ELONGATED HYPOCOTYL3 and FAR-RED IMPAIRED RESPONSE1 Transcription Factors Integrate Light and Abscisic Acid Signaling in Arabidopsis . Plant Physiol 163 : 857 – 866 . OpenUrl Abstract / FREE Full Text ↵ Tapial , J. , Ha , K. C. H. , Sterne-Weiler , T. , Gohr , A. , Braunschweig , U. , Hermoso-Pulido , A. , Quesnel-Vallières , M. , Permanyer , J. , Sodaei , R. , Marquez , Y. , et al. ( 2017 ). An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms . Genome Res 27 : 1759 – 1768 . OpenUrl Abstract / FREE Full Text ↵ Umezawa , T. , Nakashima , K. , Miyakawa , T. , Kuromori , T. , Tanokura , M. , Shinozaki , K. , and Yamaguchi-Shinozaki , K . ( 2010 ). Molecular Basis of the Core Regulatory Network in ABA Responses: Sensing, Signaling and Transport . Plant Cell Physiol 51 : 1821 – 1839 . OpenUrl CrossRef PubMed Web of Science ↵ Umezawa , T. , Sugiyama , N. , Takahashi , F. , Anderson , J. C. , Ishihama , Y. , Peck , S. C. , and Shinozaki , K . ( 2013 ). Genetics and Phosphoproteomics Reveal a Protein Phosphorylation Network in the Abscisic Acid Signaling Pathway in Arabidopsis thaliana . Sci Signal 6 : rs8 . OpenUrl Abstract / FREE Full Text ↵ van Bentem , S. de la F ., Anrather , D. , Roitinger , E. , Djamei , A. , Hufnagl , T. , Barta , A. , Csaszar , E. , Dohnal , I. , Lecourieux , D. , and Hirt , H . ( 2006 ). Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism . Nucleic Acids Res 34 : 3267 – 3278 . OpenUrl CrossRef PubMed Web of Science ↵ Wang , P. , Xue , L. , Batelli , G. , Lee , S. , Hou , Y.-J. , Van Oosten , M. J. , Zhang , H. , Tao , W. A. , and Zhu , J.-K. ( 2013 ). Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action . Proceedings of the National Academy of Sciences 110 : 11205 – 11210 . OpenUrl Abstract / FREE Full Text ↵ Wang , T. , Wang , X. , Wang , H. , Yu , C. , Xiao , C. , Zhao , Y. , Han , H. , Zhao , S. , Shao , Q. , Zhu , J. , et al. ( 2023 ). Arabidopsis SRPKII family proteins regulate flowering via phosphorylation of SR proteins and effects on gene expression and alternative splicing . New Phytologist 238 : 1889 – 1907 . OpenUrl CrossRef PubMed ↵ Weatherwax , S. C. , Ong , M. S. , Degenhardt , J. , Bray , E. A. , and Tobin , E. M . ( 1996 ). The Interaction of Light and Abscisic Acid in the Regulation of Plant Gene Expression . Plant Physiol 111 : 363 – 370 . OpenUrl Abstract ↵ Wu , S.-H . ( 2014 ). Gene Expression Regulation in Photomorphogenesis from the Perspective of the Central Dogma . Annu Rev Plant Biol 65 : 311 – 333 . OpenUrl CrossRef PubMed ↵ Xin , R. , Zhu , L. , Salomé , P. A. , Mancini , E. , Marshall , C. M. , Harmon , F. G. , Yanovsky , M. J. , Weigel , D. , and Huq , E . ( 2017 ). SPF45-related splicing factor for phytochrome signaling promotes photomorphogenesis by regulating pre-mRNA splicing in Arabidopsis . Proc Natl Acad Sci U S A 114 : E7018 – E7027 . OpenUrl Abstract / FREE Full Text ↵ Xin , R. , Kathare , P. K. , and Huq , E . ( 2019 ). Coordinated Regulation of Pre-mRNA Splicing by the SFPS-RRC1 Complex to Promote Photomorphogenesis . Plant Cell 31 : 2052 – 2069 . OpenUrl Abstract / FREE Full Text ↵ Xu , D. , Wu , D. , Li , X. , Jiang , Y. , Tian , T. , Chen , Q. , Ma , L. , Wang , H. , Deng , X. W. , and Li , G . ( 2020 ). Light and Abscisic Acid Coordinately Regulate Greening of Seedlings . Plant Physiol 183 : 1281 – 1294 . OpenUrl Abstract / FREE Full Text ↵ Yadukrishnan , P. , Rahul , P. V. , Ravindran , N. , Bursch , K. , Johansson , H. , and Datta , S . ( 2020 ). CONSTITUTIVELY PHOTOMORPHOGENIC1 promotes ABA-mediated inhibition of post-germination seedling establishment . The Plant Journal 103 : 481 – 496 . OpenUrl CrossRef PubMed ↵ Yan , T. , Heng , Y. , Wang , W. , Li , J. , and Deng , X. W . ( 2022 ). SWELLMAP 2, a phyB-Interacting Splicing Factor, Negatively Regulates Seedling Photomorphogenesis in Arabidopsis . Front Plant Sci 13 . View the discussion thread. Back to top Previous Next Posted January 31, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Cotyledon opening during seedling deetiolation is determined by ABA-mediated splicing regulation Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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Share Cotyledon opening during seedling deetiolation is determined by ABA-mediated splicing regulation Guiomar Martín , Alvaro Larran , Julia Qüesta , Paula Duque bioRxiv 2025.01.29.635410; doi: https://doi.org/10.1101/2025.01.29.635410 Share This Article: Copy Citation Tools Cotyledon opening during seedling deetiolation is determined by ABA-mediated splicing regulation Guiomar Martín , Alvaro Larran , Julia Qüesta , Paula Duque bioRxiv 2025.01.29.635410; doi: https://doi.org/10.1101/2025.01.29.635410 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)