AFC kinases function as thermosensors that regulate warm temperature-responsive growth inArabidopsis

AFC kinases function as thermosensors that regulate warm temperature-responsive growth in Arabidopsis | 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 AFC kinases function as thermosensors that regulate warm temperature-responsive growth in Arabidopsis View ORCID Profile Benjamin Dimos-Röhl , Felix Ostwaldt , Jannik Bäsmann , Paula Hausmann , View ORCID Profile Philipp Kreisz , View ORCID Profile Markus Krischke , Christoffer Lutsch , Philipp C. Müller , Miriam Strauch , View ORCID Profile Christoph Weiste , View ORCID Profile Tingting Zhu , View ORCID Profile Ive De Smet , View ORCID Profile Florian Heyd , View ORCID Profile Daniel Maag doi: https://doi.org/10.1101/2024.06.21.600040 Benjamin Dimos-Röhl 1 Laboratory of RNA Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin , 14195 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Benjamin Dimos-Röhl Felix Ostwaldt 1 Laboratory of RNA Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin , 14195 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jannik Bäsmann 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paula Hausmann 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Philipp Kreisz 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Philipp Kreisz Markus Krischke 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Markus Krischke Christoffer Lutsch 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Philipp C. Müller 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Miriam Strauch 1 Laboratory of RNA Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin , 14195 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christoph Weiste 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christoph Weiste Tingting Zhu 3 Department of Plant Biotechnology and Bioinformatics, Ghent University , 9052 Ghent, Belgium 4 VIB Center for Plant Systems Biology , 9052 Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tingting Zhu Ive De Smet 3 Department of Plant Biotechnology and Bioinformatics, Ghent University , 9052 Ghent, Belgium 4 VIB Center for Plant Systems Biology , 9052 Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ive De Smet Florian Heyd 1 Laboratory of RNA Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin , 14195 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Florian Heyd For correspondence: florian.heyd{at}fu-berlin.de daniel.maag{at}uni-wuerzburg.de Daniel Maag 2 Department of Pharmaceutical Biology, Faculty of Biology, Julius-von-Sachs-Institute of Biosciences, Julius-Maximilians-Universität Würzburg , 97082 Würzburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel Maag For correspondence: florian.heyd{at}fu-berlin.de daniel.maag{at}uni-wuerzburg.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Plants respond to elevated temperatures with enhanced elongation growth and an upward movement of their leaves. These adaptive growth responses depend on a rapid transcriptional, post-transcriptional and post-translational reprogramming. It is unclear, however, how temperature information is sensed and integrated with the cellular splicing machinery to establish warm-temperature dependent splicing patterns. In animals, CDC2-LIKE KINASES (CLKs) function as body temperature sensors that control temperature-dependent alternative splicing through the phosphorylation of serine/arginine-rich (SR) proteins. Here, we demonstrate that the CLK-homologous ARABIDOPSIS FUS3-COMPLEMENTING (AFC) kinases function as temperature sensors that regulate post-transcriptional RNA processing to control warm temperature-dependent growth responses in Arabidopsis. We show that the contrasting temperature-activity profiles of the three Arabidopsis AFCs depend on specific structural elements including a conserved activation segment within the kinase domain. By combining protein structure prediction with site-directed mutagenesis, we provide insights into structural features that determine different temperature-activity profiles of the three AFC paralogs. Subsequent analyses of afc mutant plants demonstrate their requirement for hypocotyl elongation growth and thermonastic leaf movement at elevated temperature. Impaired hypocotyl elongation of afc triple mutant seedlings was accompanied by defects in temperature-dependent splicing especially affecting the post-transcriptional regulation of transcripts encoding splicing factors. Finally, based on transcriptomics, immunodetection and mutant analyses our data indicate SR34 and SR34a as phosphorylation targets that mediate temperature-dependent post-transcriptional RNA processing downstream of AFCs. In conclusion, the characterisation of Arabidopsis AFC kinases as thermosensors provides compelling evidence that temperature-controlled AFC activity is evolutionarily conserved between plants and animals. Introduction Plants exhibit a substantial morphological and developmental plasticity in response to varying temperature conditions. Accordingly, a moderate temperature increase within the physiological range already has profound effects on plant architecture including enhanced hypocotyl, petiole, and root elongation growth as well as thermonastic leaf movement ( 1 ). These warm temperature-dependent morphological alterations have been grouped under the term thermomorphogenesis ( 2 , 3 ). At the cellular level, thermomorphogenic growth responses are accompanied by a substantial rearrangement of the proteome that depends on epigenetic ( 4 ), transcriptional ( 5 ), post-translational ( 6 , 7 ) and hormonal regulation ( 8 , 9 ). The basic helix-loop-helix proteins PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF7 have been identified as the central transcriptional regulators in thermomorphogenesis signalling ( 10 - 12 ). Upon exposure to elevated temperatures, PIF4 is derepressed and induces the transcription of temperature-responsive genes including that of auxin-biosynthetic enzymes, thereby positively regulating elongation growth ( 12 ). Downstream of auxin, functional brassinosteroid signalling is required for thermomorphogenic growth responses involving the transcription factors BRASSINAZOLE-RESISTANT 1 (BZR1) and BZR2 ( 3 ). However, PIF4- and auxin-independent regulation of BZR1 activity at elevated temperatures has also been described ( 7 ). While the hormonal and transcriptional regulation underlying thermomorphogenesis is relatively well understood, the contribution of additional regulatory layers such as post-transcriptional regulation only came into view in recent years ( 13 , 14 ). For example, seedlings defective in the spliceosomal protein SNW/SKI-INTERACTING PROTEIN (SKIP), which display global defects in pre-mRNA splicing ( 15 ), show reduced hypocotyl elongation growth in response to elevated temperature ( 14 ). At the genome-wide level, it has been estimated that 30% to 60% of all intron-containing genes undergo alternative splicing in response to various high temperature treatments in Arabidopsis ( 16 ). Thus, warm temperature-induced alternative splicing emerges as central regulatory mechanism that allows for fast adjustments of the proteome in response to changing temperature conditions ( 13 ). Notably, all these responses require the accurate perception of the surrounding temperature. Three major thermosensory mechanisms for the perception of moderately elevated temperatures have been identified thus far, consisting of the thermal reversion of active phytochrome B (phyB) ( 17 , 18 ), liquid-liquid phase separation of EARLY FLOWERING 3 (ELF3) ( 19 ), and thermosensory RNA secondary structures ( 10 ). Remarkably, all known thermosensory pathways converge on PIF4/PIF7-dependent signalling further underscoring their importance as an integrative hub in thermomorphogenesis signalling ( 20 ). However, none of the known thermosensors have been linked to warm temperature-induced alternative splicing and it is thus unclear how an increase in ambient temperature is being perceived and integrated with the cellular splicing machinery. Splicing of precursor mRNA is catalysed by the spliceosome, a large complex in the nucleus consisting of five small nuclear ribonucleoprotein (snRNP) complexes and various splicing factors. Among them, evolutionarily conserved serine/arginine-rich (SR) proteins govern splice-site selection by the recognition of cis -regulatory elements within the pre-mRNA sequence, thereby regulating both constitutive and alternative splicing ( 21 ). SR proteins consist of one or two N-terminal RNA-binding domains and a C-terminal RS domain, which contains multiple arginine/serine dipeptide repeats ( 22 ). The expression of SR proteins is regulated in a temperature-dependent manner at the transcriptional and post-transcriptional level ( 23 , 24 ). In addition, they possess several phosphorylation sites that determine their activity and subnuclear localisation ( 25 ). Notably, in response to an increase in temperature several SR proteins are rapidly phosphorylated, suggesting warm temperature-dependent post-translational regulation of SR protein activity ( 26 ). In animals, these phosphorylation sites are targeted by CDC2-LIKE KINASES (CLKs). Recently, it has been shown that CLK1 and CLK4 regulate temperature-dependent alternative splicing in human cells within the physiological temperature range through phosphorylation of SR proteins ( 27 ). Moreover, in vitro kinase assays using homologous mouse CLK1 and CLK4 demonstrated that temperature sensitivity is an intrinsic property of these proteins that depends on subtle conformational changes of a conserved activation segment consisting of 25 amino acid within the active centre of the kinase domain ( 27 ). Altogether, these observations suggest that, in animals, CLKs act as conserved temperature sensors that facilitate context-dependent gene expression through their regulatory effect on the splicing machinery. The Arabidopsis genome contains three genes that are homologous to human CLK1 , i.e., ARABIDOPSIS FUS3-COMPLEMENTING 1 ( AFC1), AFC2 , and AFC3 ( 28 ). Both, in vitro and in vivo data support their involvement in pre-mRNA processing through binding and phosphorylation of SR proteins ( 29 - 32 ). Overexpression of the tobacco AFC2 homolog in Arabidopsis led to reduced growth and late flowering, suggesting a role of AFC2 in plant development ( 31 ). More recently, functional roles during low temperature acclimation and thermomorphogenesis have been proposed for AFC3 and AFC2, respectively ( 30 , 33 ). However, the mechanistic link between AFC activity, RNA processing and temperature responsiveness remained elusive. Moreover, it is unknown whether plant AFC kinases, alike their animal counterparts, also possess thermosensory properties. In this study, we aimed at unravelling a potential function of AFC kinases as molecular temperature sensors in Arabidopsis. Using in vitro kinase assays, we first demonstrate that Arabidopsis AFC1, AFC2, and AFC3 show distinct temperature-activity profiles within the physiological temperature range of Arabidopsis that depend on the conserved activation segment and the unstructured N-terminal region of the protein. We then used CRISPR/Cas9 to generate single and higher order afc mutants to resolve the functional role of AFCs in thermomorphogenic growth responses and signalling. Our data show that AFCs are required for thermoresponsive hypocotyl elongation as well as the establishment of temperature-dependent splicing patterns. Finally, we identify SR34 and SR34a as likely targets of AFC activity at elevated temperatures contributing to thermomorphogenic hypocotyl elongation growth. Therefore, we propose that AFC kinases function as molecular thermosensors that translate ambient temperature information into a cellular signal through phosphorylation of SR proteins thereby ultimately governing thermoresponsive growth in Arabidopsis. Results AFC1, AFC2 and AFC3 show distinct temperature-activity profiles mediated by the unstructured N-terminus and the activation segment Our recent work showed that animal CLKs display highly temperature-dependent activities with an off-switch at the upper limit of the physiologically relevant temperature range in various endothermic species ( 27 ). Based on this finding, we aimed at investigating whether the activities of the homologous Arabidopsis kinases, AFC1 (At3g53570), AFC2 (At4g24740), and AFC3 (At4g32660) (Fig. S1A), are also temperature-dependent. A comparison of the predicted three-dimensional structures of the three AFC kinase domains with the experimentally resolved structure of human CLK1 yielded a high degree of structural homology with all major sequence elements being conserved and only minor differences, mainly in unstructured regions ( Fig. 1A ). Subsequent in vitro kinase assays revealed a prominent temperature sensitivity of all three kinases with distinct temperature-activity profiles. While AFC1 activity was highest at lower temperatures between 4 °C and 20 °C, AFC3 had its highest activity between 24 °C and 32 °C ( Fig. 1B and C, Fig. S1B). By contrast, AFC2 showed largely stable activity between 4 °C and 28 °C. Notably, the in vitro activity of all three kinases declined rapidly at temperatures above 32 °C. Download figure Open in new tab Figure 1. AFC1, AFC2 and AFC3 show distinct temperature-activity profiles mediated by the unstructured N-terminus and the activation segment. A , Superimposition of the predicted kinase domains of AFC1 (P51566, residues 102-467), AFC2 (P51567, residues 85-427) and AFC3 (P51568, residues 58-400) with the crystal structure of human CLK1 kinase domain (PDB: 6TW2). B , Temperature-dependent phosphorylation of a GST-SR substrate by AFC1, AFC2 and AFC3. After separation by SDS-PAGE,phosphorylation of synthetic GST-SR was detected by autoradiography (top, 32 P). Equal loading was confirmed by Coomassie staining (bottom). Loading controls for the three kinases are shown in Fig. S1B. C , Quantification of temperature-dependent substrate phosphorylation activity of AFC1, AFC2 and AFC3. For quantification, the highest signal intensity within an assay was set to 100 and used for normalisation. Shown are mean values ± SE; n = 3 for AFC1 and n = 6 for AFC2 and AFC3, respectively. D-F , Temperature-dependent activity of chimeric AFC1 containing the AFC3 N-terminus (D), chimeric AFC3 containing the AFC2 activation segment (E), and an AFC3 H257Q point mutant (F). Reaction temperatures are indicated on top. In all cases, representative images of synthetic GST-SR phosphorylation (autoradiography, 32 P, top) and the respective substrate loading controls (Coomassie staining, bottom) are shown. Displayed are mean values ± SE from n = 3 (D), n = 3 (E), and n = 6-7 (F) replicates. Statistically significant differences were determined by Student’s t -tests (*: p < 0.05, **: p < 0.01, ***: p < 0.001). Note that data for AFC1 depicted in (D) and AFC3 depicted in (F) are the same as presented in (C). WT: wild type. To address the molecular details underlying the opposing temperature-activity profiles of AFC1 and AFC3, we created several chimeric proteins and mutants focusing on the unstructured N-terminus and specific amino acids within the activation segment, which are known to set the specific temperature-activity profile of animal CLKs ( 27 ). We first generated a chimeric kinase consisting of the AFC3 N-terminus and the AFC1 kinase domain. This chimera showed a strikingly different temperature-activity profile rather resembling that of AFC3 than of AFC1, showing that the N-terminus is involved in setting the temperature-controlled activity. In addition, the chimeric kinase remained active at higher temperatures above 28 °C ( Fig. 1D ), suggesting that the N-terminus plays a role in stabilising the kinase domain or the active centre. Furthermore, this stabilising effect seems to depend on the exact sequence context, as it was absent in full-length AFC3. The kinase domains alone displayed an activity similar to the full-length kinases (Fig. S1C), showing that the altered activity of the AFC1-AFC3 chimera was mediated by the presence of the AFC3 N-terminus and not the absence of the AFC1 N-terminus. We then created a chimeric isoform of AFC3 containing the AFC2 activation segment ( Fig. 1E ). This chimera showed a temperature-activity profile reminiscent of the behaviour of both WT kinases, as the activity remained higher in the temperature range between 12 °C and 24 °C, as seen in AFC2, and decreased at lower temperatures, as seen in AFC3. Finally, two amino acid residues, R343 and H344, are instrumental in mediating the temperature sensitivity of animal CLK1, as they change their conformation in a temperature-dependent manner potentially blocking substrate access to the active centre at higher temperature ( 27 ). These amino acids are conserved in all three AFCs (Fig. S1A). We thus tested whether they were also involved in the temperature-dependent regulation of AFC activity. While an AFC1 H304Q mutation had little effect on the temperature-activity profile (Fig. S1D), the corresponding AFC2 H285Q (Fig. S1E) and AFC3 H257Q ( Fig. 1F ) mutants showed strongly altered temperature-activity profiles compared to the respective WT isoforms. This suggests that a mechanism similar to the one that regulates temperature-controlled activity of CLKs also controls AFC2 and AFC3 activity. Altogether, these data establish AFC activity as highly temperature-responsive in the range between 4 °C and 40 °C with striking differences between AFC1, AFC2, and AFC3. Furthermore, we show that at least two protein domains, the unstructured N-terminus and the activation segment, are involved in mediating temperature sensitivity and that the temperature-activity profile can be modulated by mutating specific amino acids. A glutamine centred H-bond network mediates AFC3 activity at high temperatures To further understand the molecular and structural basis underlying the different temperature-activity profiles of AFC1 and AFC3, we examined the predicted structures in more detail. Superposition of the AFC1 and AFC3 kinase domains revealed a high similarity with a root mean square deviation (RMSD) of 1.25 Å and a TM-score of 0.92 with 69% identity ( Fig. 2A ). Structural differences were restricted to a flexible loop in the C lobe and the also flexible, but catalytically relevant activation segment. Download figure Open in new tab Figure 2. A glutamine centred H-bond network mediates AFC3 activity at high temperatures. A , Superimposition of the predicted structures of the kinase domains of AFC1 (P51566, residues 102-467, grey) and AFC3 (P51568, residues 58-400, teal) with the AFC3 activation segment coloured in light orange. Residues of AFC3 involved in the formation of the H-bond network around AFC3 Q254 are shown as stick models. B,C , Enlarged view of the H-bond network around AFC3 Q254 (B) and AFC1 S301 (C). Interacting residues of AFC3 and the corresponding AFC1 residues are depicted as sticks with the formed H-bonds (distance cut-off = 3.2 Å) coloured in orange (AFC3) or grey (AFC1). D,E , Representative images of temperature-dependent auto- and substrate phosphorylation of AFC3 WT (D) and an AFC3 Q254A mutant (E). Phosphorylation of GST-tagged AFCs and synthetic GST-SR was detected by autoradiography (top, 32 P). Equal loading was confirmed by Coomassie staining (bottom). F,G , Quantification of temperature-dependent auto-phosphorylation (F) and substrate phosphorylation (G) activity of AFC3 WT and AFC3 Q254H mutant. For quantification, the highest signal intensity within an assay was set to 100 and used for normalisation. Shown are mean values ± SE from n = 4 (AFC3 WT) and n = 3 (AFC3 Q254A) replicates. Statistically significant differences were determined by Student’s t -tests (*: p < 0.05, **: p < 0.01, ***: p < 0.001). For the activation segment of AFC3, we observed an H-bond network involving Q254 and the sidechains of S277 from the αF-helix and S260, which is also part of the activation segment, and the backbones of C273, S260 and Y258 ( Fig. 2B ). These interactions may stabilise the region of the activation segment containing R256 and H257 in an active conformation. As discussed above, the homologous residues in human CLK1 are essential for defining the temperature-activity optimum ( 27 ) and an H257Q mutation also resulted in a strongly altered temperature-activity profile of AFC3 ( Fig. 1F ). In AFC1, which is not active at temperatures above 32 °C ( Fig. 1B and C ), the amino acids corresponding to AFC3 Q254 and S260 are S301 and A307, respectively. Since S301 only forms a single H-bond in AFC1, this part of the activation segment lacks stabilising interactions ( Fig. 2C ). Thus, we hypothesised, that the H-bond network around AFC3 Q254 stabilises R256 and H257 in an active conformation at higher temperatures, thereby allowing AFC3 activity at temperatures where AFC1 is already inactive. To test this hypothesis, we examined an AFC3 Q254A mutant in in vitro kinase assays ( Fig. 2D and E ). Indeed, temperature-responsive autophosphorylation activity was shifted towards lower temperatures by several degrees for the AFC3 Q254A mutant compared to WT AFC3 including a substantial decline in activity at temperatures above 32 °C ( Fig. 2F ). Concerning substrate phosphorylation, no significant differences were observed between both variants between 4 °C and 32 °C ( Fig. 2G ). However, the Q254A mutation led to a more pronounced decrease in substrate phosphorylation above 32 °C than was observed for WT AFC3, yielding a similar effect as for autophosphorylation. In conclusion, these data show that residue Q254 is required for the activity of AFC3 at temperatures above 32 °C. AFCs are required for thermoresponsive growth Based on the observed temperature-sensitive activity of the three AFC kinases we hypothesised that they are involved in the regulation of temperature-dependent growth responses in vivo . Initial support for this hypothesis came from the observation that warm temperature-dependent hypocotyl elongation in eight-day-old Arabidopsis Columbia-0 (Col-0) seedlings was negatively affected by the CLK1/4 inhibitor TG003 ( 34 ) in a dose-dependent manner ( Fig. 3A , Fig. S2A-D). We then generated afc single and higher order mutant lines using CRISPR/Cas9 (Fig. S3A-C, Fig. S4A-C and Fig. S5A). The afc single mutants did not show any differences in temperature-dependent hypocotyl elongation ( Fig. 3B ). However, hypocotyl growth was significantly reduced in warm temperature-exposed seedlings of two independent afc1 afc2 afc3 triple mutant lines designated as afc1/2/3 #1 and afc1/2/3 #2 ( Fig. 3C and D ). At the same time no difference in hypocotyl length between the two mutant lines and the wild type (WT) was observed at 17 °C and 21 °C suggesting a warm temperature-specific contribution of AFCs to hypocotyl elongation. In addition, we observed a strongly reduced thermonastic leaf movement response in afc triple mutant plants (Fig. S5B-D) as well as a modest reduction in warm temperature-dependent petiole elongation (Fig. S5E). We thus concluded that AFCs are required for warm temperature-dependent vegetative growth responses in Arabidopsis. Download figure Open in new tab Figure 3. AFCs are required for thermoresponsive growth. A , Hypocotyl lengths of eight-day-old Col-0 seedlings supplemented with the AFC kinase inhibitor TG003 and exposed to 17 °C or 28 °C. Seedlings were grown on non-supplemented medium for three days and then transferred to TG003-containing or DMSO-containing (solvent control) medium. On day five half of the plates were shifted to 28 °C while the other half remained at 17 °C ( n = 39-48 seedlings per treatment and temperature). Inhibition of AFC activity by TG003 was confirmed in vitro for recombinant AFC3 (Fig. S2A). B , Hypocotyl lengths of seven-day-old Col-0, afc1, afc2 and afc3 single mutant seedlings at 17 °C, 21 °C and 28 °C. Seedlings were grown at 17°C for four days and then exposed to the indicated temperatures for three days ( n = 43-59 seedlings per genotype and temperature). C , Overview of the genetic modifications in the two independent afc1 afc2 afc3 triple mutant lines generated by CRISPR/Cas9 and designated as afc1/2/3 #1 and afc1/2/3 #2 . D , Hypocotyl lengths of seven-day-old seedlings of Col-0, afc1/2/3 #1 and afc1/2/3 #2 at 17 °C, 21 °C and 28 °C. Seedlings were grown at 17 °C for four days and then exposed to the indicated temperatures for three days ( n = 29-40 seedlings per genotype and temperature). Statistically significant differences were determined by two-way ANOVAs followed by Tukey HSD post-hoc tests and are indicated by different letters above boxes ( p < 0.05). AFCs regulate temperature-dependent splicing of genes involved in RNA processing Next, we aimed at investigating the temperature-dependent splicing patterns in the afc triple mutant lines in more detail. To this end, we performed RNA-sequencing (RNA-seq) on seven-day old afc1/2/3 #1 and afc1/2/3 #2 seedlings following exposure to 28 °C for 8 and 24 hours, respectively, and compared their splicing patterns to those of WT seedlings ( Fig. 4A ). Subsequent principal component analysis on all detected splice events yielded a clear separation between the different genotypes and temperature conditions indicating a genotype-dependent effect of temperature on the global splicing patterns ( Fig. 4B ). We then assessed whether AFCs are involved in the regulation of a specific type of alternative splicing in response to a temperature increase. Therefore, we first identified genes that were differentially alternatively spliced (DAS) after the temperature shift for each of the three genotypes by pairwise comparisons and subsequently analysed the relative distribution of the four types of alternative splicing. However, we did not observe any alterations concerning the frequencies of the different alternative splicing types between the two mutant lines and the WT. Irrespectively of plant genotype, intron retention (IR) was the dominant type of temperature-dependent alternative splicing, followed by exon skipping (ES), and alternative usage of 3’ (Alt 3’) and 5’ (Alt 5’) splice sites ( Fig. 4C ). Download figure Open in new tab Figure 4. AFCs regulate the temperature-dependent splicing of genes that are involved in RNA processing. A , Schematic overview of the experimental conditions used for the analysis of temperature-dependent splicing patterns. Seedlings of Col-0, afc1/2/3 #1 and afc1/2/3 #2 were grown at 17 °C and then exposed to 28 °C for 24 hours starting at the end of day 7 or exposed to 28 °C for 8 hours starting on day 8. Control seedlings remained at 17 °C. All samples were taken at Zeitgeber Time (ZT) 11 of day 8, i.e., 11 hours after the onset of light. B , Principal component analysis of all detected splice events. The different sample groups are indicated by different colours. C, Relative frequencies of the different alternative splicing types among the differentially alternatively spliced genes in Col-0, afc1/2/3 #1 and afc1/2/3 #2 following exposure to 28 °C for 8 or 24 hours. D , Intersection of genes that were differentially alternatively spliced in both afc1/2/3 #1 and afc1/2/3 #2 compared to Col-0 at the different temperature conditions. E , Gene ontology term enrichment analysis for genes that were DAS in both afc1/2/3 #1 and afc1/2/3 #2 compared to Col-0 at the different temperature conditions. F , Temperature-dependent splicing of SR45a exon 5 in Col-0, afc1/2/3 #1 and afc1/2/3 #2 . Representative Shashimi plots for the indicated temperatures (top) and the rMATS-derived quantification of exon 5-inclusion (bottom) are shown with the functional consequence of exon 5-inclusion indicated on the right ( n = 3-4). G , Temperature-dependent splicing of RS40 exon 4 in Col-0, afc1/2/3 #1 and afc1/2/3 #2 . Inclusion of exon 4 produces a poison isoform containing two premature termination codons. Representative Shashimi plots for the indicated temperatures are shown on top. rMATS-derived percentages of exon 4 inclusion (orange) and RS40 normalised read counts (blue) are shown below ( n = 3-4). AS: alternative splicing, ES: exon skipping, IR: intron retention, Alt 5’: alternative 5’ splice site, Alt 3’: alternative 3’ splice site. Next, we were interested in the genes that showed genotype-dependent differences in their splicing patterns at the different temperatures ( Fig. 4D , Supplemental Table 1). Considering only genes that were affected in both mutant lines compared to WT yielded 111 differentially alternatively spliced genes (DASGs) under control conditions (Supplemental Table 2). At 28 °C, 222 and 205 DASGs were detected after 8 and 24 hours, respectively, suggesting a higher impact of AFCs on alternative splicing at warm temperatures. Out of the 308 unique genes that were DAS in afc1/2/3 #1 and afc1/2/3 #2 at 28 °C, 41% were spliced in a temperature-dependent manner in the WT, including several genes coding for SR proteins (Supplemental Table 2). Subsequent gene ontology (GO) term enrichment analysis of the DASGs likewise yielded a significant enrichment of several terms associated with RNA metabolism and processing, especially at the early time point of the 28 °C treatment ( Fig. 4E , Supplemental Table 3) Since the alternative splicing of SR protein genes may result in altered SR protein expression and/or activity and thus be instrumental in the establishment of global temperature-dependent alternative splicing patterns, we examined the impact of AFC activity on the temperature-dependent splicing of SR protein genes and the resulting consequences in more detail. As one example we chose the non-classical SR protein SR45a (At1g07350). Under control conditions nearly all detected transcripts of SR45a included exon 5, which contains a premature termination codon (PTC) and gives rise to a truncated protein isoform that lacks the C-terminal RS domain ( Fig. 4F ). Exposure to 28 °C, however, induced skipping of exon 5 in ∼50% of the detected SR45a transcripts in WT but not in afc1/2/3 #1 or afc1/2/3 #2 seedlings. Exon 5-skipping produces the transcript isoform that encodes full-length SR45a containing the C-terminal RS domain. Thus, warm temperature-dependent switching between truncated and full-length SR45a in WT seedlings depends on AFCs. In another example, we observed genotype-dependent differences in the temperature-regulated splicing of RS40 (At4g25500), In WT seedlings, exposure to elevated temperature elicited an increase in the abundance of a nonsense-mediated mRNA decay-sensitive RS40 transcript isoform via inclusion of exon 4, which correlated with a decrease in total RS40 transcript levels ( Fig. 4G ). By contrast, inclusion of PTC-containing exon 4 was already enhanced in both afc1/2/3 #1 and afc1/2/3 #2 under control conditions and increased even further at 28 °C. This also resulted in lower total RS40 transcript levels, suggesting AFC-dependent post-transcriptional regulation of RS40 expression, which is likely further controlled through retention of intron 3 (labelled in bold in Fig. 4F ). In conclusion, our data provide evidence that AFCs are involved in the regulation of temperature-dependent alternative splicing and that they particularly control the correct splicing of a subset of genes that are involved in RNA processing themselves. SR proteins contribute to temperature-induced hypocotyl elongation Since SR proteins serve as potential targets of AFC kinases ( 29 , 35 ), we next aimed at investigating their role during seedling thermomorphogenesis in more detail. To identify potential SR proteins that are involved in mediating AFC-dependent temperature signalling we first investigated the temperature-dependent expression of all 20 genes coding for SR proteins using our RNA-seq data (Supplemental Table 4). While only two SR genes were significantly downregulated after exposure to 28 °C in WT seedlings, six SR genes were significantly upregulated (|log 2 FC| > 1, p < 0.05) ( Fig. 5A , Supplemental Table 4) confirming the previously reported temperature-dependent regulation of SR protein expression ( 23 ). Moreover, seven SR genes were differentially expressed (DE) in afc1/2/3 #1 and afc1/2/3 #2 compared to the WT with six of them showing reduced transcript levels in the mutant seedlings ( Fig. 5A , Supplemental Table 4). Notably, there was little overlap between SR genes that were DE and DAS in afc triple mutants compared to the WT. Download figure Open in new tab Figure 5. SR proteins contribute to warm temperature-dependent hypocotyl elongation. A , Temperature-dependent expression of SR proteins in seven-day-old seedlings of Col-0, afc1/2/3 #1 and afc1/2/3 #2 . Seedlings were exposed to 28 °C for the indicated time periods and transcript levels determined by RNA-sequencing. Genes that were differentially expressed (DE) or differentially alternatively spliced (DAS) in both afc triple mutants compared to Col-0 under at least one of the three temperature conditions are indicated next to the gene name. Y: yes, N: no. B , Analysis of SR protein phosphorylation status in nine-day-old seedlings of Col-0 and afc1/2/3 #2 that were exposed to 28 °C for one hour or remained at 17 °C as control. Phosphorylation was assayed using an α-pan-phosphoepitope SR-specific antibody and an α-histone H3 antibody for normalisation. C , Hypocotyl lengths of seven-day-old seedlings of Col-0, sr34, sr34a and afc1/2/3 #2 at 17 °C, 21 °C and 28 °C. Seedlings were grown at 17 °C for four days and then exposed to the indicated temperatures for three days ( n = 25-43 seedlings per genotype and temperature). Statistically significant differences were determined by two-way ANOVA followed by a Tukey HSD post-hoc test and are indicated by different letters above boxes ( p < 0.05). D , Current working model on the function of AFC kinases as temperature sensors during thermomorphogenic growth responses. Upon exposure to a temperature increase, AFCs phosphorylate specific SR proteins, likely SR34 and SR34a. This in turn leads to the post-transcriptional regulation of splicing-related genes including genes coding for SR proteins, thereby contributing to the establishment of global warm temperature-dependent alternative splicing patterns. Correct splicing at elevated temperatures is required for proper thermomorphogenic growth responses. Consistently, afc triple mutant seedlings show altered temperature-dependent alternative splicing patterns and reduced hypocotyl elongation growth at 28 °C. To further narrow down the list of potential AFC targets we identified SR proteins that are rapidly phosphorylated in WT seedlings after exposure to 28 °C in two datasets, a first one from Arabidopsis seedlings exposed to elevated temperature during the night ( 26 ) (Supplemental Table 5) and a second one from Arabidopsis seedlings exposed to elevated temperature during the day (Supplemental Table 5). Comparing these two datasets revealed common regulation of SR34, SR34a and SCL30, but only for SR34 (S134) and SR34a (S273) the same phosphosites were targeted in both datasets (Fig. S6) making these good candidates for further investigation. To establish SR34 and SR34a as targets of warm temperature-dependent AFC activity, we assessed SR protein phosphorylation in wild-type and afc1/2/3 #2 seedlings using an α-pan-phosphoepitope SR-specific antibody ( 27 ). In agreement with temperature-dependent SR34 and SR34a phosphorylation, we observed an increased signal intensity at approximately 32 kDa in wild-type seedlings that were exposed to 28 °C for one hour ( Fig. 5B , Fig. S7A-E). By contrast, signal intensity was strongly reduced in warm temperature-treated afc triple mutant seedlings compared to the 17 °C control. Finally, we assessed thermoresponsive hypocotyl elongation of sr34 knockout and sr34a knockdown mutant seedlings (Fig. S8A and B) and observed a significant reduction for both genotypes ( Fig. 5C ). Taken together, these data indicate a functional role of SR34 and SR34a in seedling thermomorphogenesis downstream of AFC activity. Discussion Plants are able to actively adjust their growth and development to the prevalent temperature conditions ( 2 ). This, however, requires the accurate perception and integration of temperature information with cellular processes and signalling ( 36 ). Here, we have shown that AFC kinases serve as temperature sensors that control temperature-dependent alternative splicing patterns and thermomorphogenic growth responses. Thermosensory properties had been demonstrated previously for the human homologs CLK1 and CLK4 ( 27 ). Our data thus provide compelling evidence that temperature-controlled AFC activity is evolutionarily conserved between plants and animals. In contrast to their mammalian counterparts, the three plant AFCs showed distinct temperature-activity profiles indicating a functional diversification that is likely adapted to the broader temperature range plants are exposed to, and which contrasts with the tightly controlled core body temperature of endotherms ( 27 ). This hypothesised temperature adaptation is further supported by the observation that CLKs from poikilothermic animals change their activity within the physiological temperature range of the respective host organism ( 27 ). From a mechanistic point of view the structural elements that determine the temperature-responsive activity of animal CLKs and plant AFCs are identical, including the unstructured N-terminus and the activation segment. However, our data suggest that additional, so far unknown, mechanisms contribute to AFC temperature sensitivity. This is supported by the observation that the targeted mutation of H257 within the RH motif of AFC3 led to a shift of maximum kinase activity towards lower temperatures while the opposite was observed for the corresponding mutant of AFC2 (H285Q). By contrast, the corresponding mutation in AFC1 (H304Q) had little effect on the temperature-activity profile. Taken together, these data start to unravel structural differences that allow distinct temperature-activity profiles of different AFCs and provide a framework for targeted mutations to alter the precise temperature response of the individual kinases. A recent study suggested a negative regulatory role for AFC2 during thermomorphogenic growth in Arabidopsis ( 30 ). According to this study, afc2 single mutants displayed a slightly hyperresponsive hypocotyl elongation at 28 °C. In our hands, however, neither one of the T-DNA insertion lines that were used in that study (Fig. S8C), nor our CRISPR/Cas9-generated afc2 single mutant ( Fig. 3B ) displayed hyperresponsive hypocotyl elongation. By contrast, the growth suppression caused by genetic perturbation of AFC kinase activity ( Fig. 3D ) was similar to the effect exerted by pharmacological inhibition ( Fig. 3A ) both supporting a clear positive regulatory function of AFCs during thermomorphogenesis. Downstream of AFCs, our data indicate SR34 and SR34a as phosphorylation targets at elevated temperature that mediate hypocotyl elongation growth at 28 °C. Notably, the reduction in hypocotyl elongation growth in sr34 and sr34a was less pronounced than in the afc triple mutants indicating an at least partial functional redundancy. This is the first report on a functional role of SR proteins in plant responses towards elevated temperatures. Thus far, SR34 and SR34a were only implicated in the context of ABA-dependent responses ( 37 , 38 ). For human SR proteins, a complex network of post-transcriptional auto- and cross-regulation has been shown ( 39 - 41 ). Accordingly, SR proteins contribute to the post-transcriptional regulation of other SR protein transcripts ( 39 ), but also regulate the splicing of their own pre-mRNA ( 40 , 41 ). In plants, several SR proteins were shown to regulate the splicing of other SR protein transcripts ( 37 , 42 , 43 ) while post-transcriptional autoregulation has only been demonstrated for SCL33 and RS2Z33 ( 44 , 45 ). In line with this, several SR protein transcripts were differentially spliced in the afc triple mutants indicating that AFCs regulate thermomorphogenic growth responses through altering the alternative splicing and expression of specific members of the SR protein family constituting a complex combinatorial network. Prospective work needs to address the interaction between individual AFCs and SR proteins in more detail and determine their precise role in the regulation of warm temperature-dependent alternative splicing. Based on these observations, we propose a model by which AFCs serve as molecular temperature sensors that integrate environmental temperature information with the cellular splicing machinery to control thermomorphogenic growth responses ( Fig. 5D ). Upon a temperature increase, AFCs phosphorylate specific SR proteins. This in turn leads to the post-transcriptional regulation of splicing-related genes including genes coding for SR proteins, which is required for the establishment of global warm temperature-dependent splicing patterns and hypocotyl elongation growth. Thus far, all known thermosensory mechanisms converge on PIF4/7-dependent signalling ( 20 ). While we did not observe any enrichment of PIF4- or auxin-regulated genes among the differentially spliced genes in afc triple mutants ( Fig. 4E , Supplemental Table 3), treatment of mutant seedlings with the synthetic auxin picloram resulted in a complete restoration of WT-like hypocotyl elongation growth at 28 °C (Fig. S8D) suggesting that AFCs act upstream of auxin signalling. Moreover, our analyses of temperature-dependent growth responses in afc single and higher order mutants indicate a functional redundancy between the different AFCs during warm temperature-mediated hypocotyl elongation that seems counterintuitive given their contrasting temperature-responsiveness in vitro . This contradiction may be due to the fact that for the kinase assays an SR-repeat-rich peptide was used as target. Therefore, it cannot be excluded that AFCs display slightly different temperature-dependent activities in vivo or that they even display contrasting substrate specificities at different temperatures ( 46 , 47 ). Future studies should thus address the contribution of individual AFCs to temperature-mediated growth responses in more detail and determine how AFC-dependent alternative splicing integrates with known thermomorphogenesis-related signalling pathways. To conclude, our detailed understanding of the molecular mechanism underlying AFC temperature sensitivity provides a starting point for the targeted engineering of AFC isoforms with altered temperature-responsive properties. Given that AFCs are conserved across all land plants ( 28 ), this may ultimately aid in the generation of crops with enhanced thermal resistance. Furthermore, the generation of temperature-switchable kinases might become a valuable tool for thermogenetics ( 48 ). Methods Plant material and growth conditions Mutant lines of Arabidopsis thaliana that were used in this study are listed in Supplemental Table 6. All mutants were in the Columbia-0 (Col-0) background. Seeds were surface sterilised using chlorine gas and sown on 0.5x Murashige and Skoog (MS) medium (pH 5.7, 1% (w/v) sucrose, 1.2% (w/v) phytoagar). Arabidopsis seeds were stratified for 24 to 48 hours at 4 °C in the dark and then transferred to a growth chamber at long-day conditions (16 h light/8 h darkness) set to 17 °C at 100-120 µmol photons m -2 s -1 (LED lights, Polyklima PK-520). For experiments with soil-grown plants, surface sterilised seeds were directly sown on soil, stratified for 48 hours at 4 °C and grown under the same conditions as described above. Three-dimensional protein structure analysis To assess structural differences in the AFC family, predictions of full-length A. thaliana AFC1, AFC2 and AFC3 structures by AlphaFold ( 49 ) published in the AlphaFold protein structure database ( 50 ) as entries P51566 (AFC1), P51567, AFC2) and P51568 (AFC3) were used. For analysis only the kinase domains, AFC1 residues 102-467, AFC2 residues 85-427 and AFC3 residues 58-400, were considered. Structure data was analysed with PyMOL 2.3.4 and Coot 0.9.6 ( 51 ). Similarity of structures was calculated with the rigid jFATCAT algorithm ( 52 , 53 ) via the RCSB PDB pairwise structure alignment tool. In vitro kinase assays On-bead in vitro kinase assays were carried out as described previously ( 27 ). 0.5 µM of the purified recombinant AFCs were pre-incubated with 2 µM GST-RS in reaction buffer (50 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 5 mM DTT, 0.1 mM spermidine) for 20 min at the indicated temperatures. After addition of an ATP mixture (γ- 32 P-ATP:ATP, 1:20k, ca. 0.3 Ci/mmol) to a final concentration of 22 µM the samples were incubated for 5 min at the same temperatures. The reaction was stopped by addition of 6x SDS sample buffer and incubation at 95°C for 5 min. Samples were analysed on 12% SDS-PAGE gels. Gels were stained with Coomassie, de-stained with 10% (v/v) acetic acid, 40% (v/v) ethanol and imaged. Subsequently gels were placed on filter paper, dried in a gel drier for 45 min and exposed on phosphoscreens overnight. The phosphoscreens were detected in a Typhoon FLA 7000 phosphoimager (650 nm laser, latitude L4, PMT = 500, 100 µm pixel size) and bands quantified using ImageQuant TL software. Details on the heterologous expression and purification of AFCs can be found in SI Appendix . Generation of afc mutant lines Genome-edited afc mutant lines were generated using CRISPR/Cas9 ( 54 ) as described previously ( 55 ). Homozygous mutants exhibiting frame shifts and premature stop codons were identified by PCR amplification and sequencing of the targeted genomic regions. All primers used for the generation and sequencing of afc mutant lines are listed in Supplemental Table 6. More detailed information can be found in SI Appendix . Hypocotyl length measurements For hypocotyl elongation assays, Arabidopsis seedlings were grown at 17 °C for four days before they were subjected to the indicated temperature treatments for another three days. For TG003 treatments, Arabidopsis seedlings were grown for three days on 0.5x MS plates at 17 °C and then transferred to 0.5x MS plates containing 50 µM TG003 (Sigma-Aldrich, Catalogue No.: T5575) or an equivalent volume of DMSO as solvent control and grown for additional two days at 17 °C to allow for uptake of the compound. On day six half of the plates were subjected to a three-day treatment at the indicated temperatures, whereas the other half remained at 17 °C as control. For all hypocotyl length measurements, plates were scanned at the end of the experiment using a flatbed scanner and hypocotyl lengths were quantified using the software Agnes Roots Measurements, Version 1.2. RNA-sequencing To assess warm temperature-dependent splicing patterns of Col-0, afc1/2/3 #1 and afc1/2/3 #2 , seedlings were grown at 17 °C and then exposed to 28 °C for 24 hours starting at the end of day seven or exposed to 28 °C for 8 hours starting on day 8. Control seedlings remained at 17 °C. Samples were taken at Zeitgeber Time (ZT) 11, i.e., 11 hours after the onset of light. Twenty seedlings were pooled per replicate and total RNA was extracted from homogenised plant tissue using a phenol-based RNA isolation method (NucleoZol, Macherey-Nagel, Catalogue No.: 740404). PolyA+ libraries were prepared by and sequenced at BGI Genomics (Hong Kong, China). Raw RNA sequence data was obtained in the FASTQ file format. For gene expression analysis, reads of each sample were mapped to the Arabidopsis genome (Col-0 TAIR10 genome release) using SALMON (v1.8.0) ( 56 ) and quantified using DEseq2 (v1.28.1) ( 57 ). For differential splicing analysis, raw reads were aligned to the Arabidopsis reference genome (Col-0 TAIR10 genome release) using STAR (version 2.7.5b) ( 58 ). Quantification was done using rMATS (4.1.2) ( 59 ). Pairwise comparisons were performed to identify differentially alternatively spliced transcripts between different genotypes or temperature conditions. Raw data from RNA-seq of this article can be found in Gene Expression Omnibus (GSE269859). Immunodetection of phosphorylated SR proteins To assess warm temperature-dependent phosphorylation of SR proteins in Col-0 and afc1/2/3 #2 , seedlings were grown at 17 °C for nine days and then exposed to 28 °C for one hour. Control seedlings remained at 17 °C. Forty seedlings were pooled per replicate. Total protein was extracted from homogenised tissue, separated by SDS-PAGE and blotted onto PVDF membranes for immunodetection. Phosphorylated SR proteins were detected using a mouse monoclonal α-pan-phosphoepitope SR-specific antibody (1H4, Merck, Catalogue-No.: MABE50) at 0.025 µg/mL. Detection of histone H3 using a rabbit polyclonal α-H3 antibody (Agrisera, Catalogue No: AS10 710) at a dilution of 1:5000 served as loading control and was used for normalisation. Primary antibodies were detected using HRP-linked goat α-mouse IgG (Thermo Scientific, Catalogue No.: G-21040) and goat α-rabbit IgG (Merck, Catalogue No.: AP307F) antibodies at dilutions of 1:5000. Signal detection and quantification was achieved using the Clarity Western ECL substrate kit (Bio-Rad, Catalogue No.: 1705061), a ChemiDoc MP imaging system (Bio-Rad) and Image Lab, Version 6.1 (Bio-Rad laboratories). Statistical analyses Hypocotyl elongation was analysed using two-factorial analysis of variance (ANOVA) followed by a TukeyHSD post-hoc test for pairwise comparisons. Normal distribution of the residuals was tested using the ‘plotresid’ function of the R package ‘RVAideMemoire’, Version 0.9-80 ( 60 ). Equality of variances was tested using Levene’s test. In case data did not meet the required assumptions, analyses were done on log-transformed data. All analyses were done using RStudio, Version 1.4.1717 and R, Version 4.1.1. Acknowledgments We thank Prof. Dr. Dr. Martin J. 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Share AFC kinases function as thermosensors that regulate warm temperature-responsive growth in Arabidopsis Benjamin Dimos-Röhl , Felix Ostwaldt , Jannik Bäsmann , Paula Hausmann , Philipp Kreisz , Markus Krischke , Christoffer Lutsch , Philipp C. Müller , Miriam Strauch , Christoph Weiste , Tingting Zhu , Ive De Smet , Florian Heyd , Daniel Maag bioRxiv 2024.06.21.600040; doi: https://doi.org/10.1101/2024.06.21.600040 Share This Article: Copy Citation Tools AFC kinases function as thermosensors that regulate warm temperature-responsive growth in Arabidopsis Benjamin Dimos-Röhl , Felix Ostwaldt , Jannik Bäsmann , Paula Hausmann , Philipp Kreisz , Markus Krischke , Christoffer Lutsch , Philipp C. Müller , Miriam Strauch , Christoph Weiste , Tingting Zhu , Ive De Smet , Florian Heyd , Daniel Maag bioRxiv 2024.06.21.600040; doi: https://doi.org/10.1101/2024.06.21.600040 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 (7647) Biochemistry (17728) Bioengineering (13921) Bioinformatics (42047) Biophysics (21490) Cancer Biology (18637) Cell Biology (25555) Clinical Trials (138) Developmental Biology (13403) Ecology (19942) Epidemiology (2067) Evolutionary Biology (24368) Genetics (15625) Genomics (22549) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88761) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9835) Zoology (2272)