CRK2 modulates flowering in Arabidopsis together with GLYCINE- RICH RNA BINDING PROTEIN 7 (GRP7)

CRK2 modulates flowering in Arabidopsis together with GLYCINE- RICH RNA BINDING PROTEIN 7 (GRP7) | 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 CRK2 modulates flowering in Arabidopsis together with GLYCINE- RICH RNA BINDING PROTEIN 7 (GRP7) View ORCID Profile Francisco J. Colina , View ORCID Profile Julia Krasensky-Wrzaczek , View ORCID Profile Alegría Pérez-Guillén , View ORCID Profile Michael Wrzaczek doi: https://doi.org/10.1101/2025.08.05.668606 Francisco J. Colina 1 Institute of Plant Molecular Biology, Biology Centre, Czech Academy of Sciences , 370 05 České Budějovice, Czech Republic 3 Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (EEZ-CSIC) , 18008 Granada, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Francisco J. Colina Julia Krasensky-Wrzaczek 1 Institute of Plant Molecular Biology, Biology Centre, Czech Academy of Sciences , 370 05 České Budějovice, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Julia Krasensky-Wrzaczek Alegría Pérez-Guillén 1 Institute of Plant Molecular Biology, Biology Centre, Czech Academy of Sciences , 370 05 České Budějovice, Czech Republic 3 Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (EEZ-CSIC) , 18008 Granada, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alegría Pérez-Guillén Michael Wrzaczek 1 Institute of Plant Molecular Biology, Biology Centre, Czech Academy of Sciences , 370 05 České Budějovice, Czech Republic 2 Faculty of Science, University of South Bohemia , 370 05 České Budějovice, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael Wrzaczek For correspondence: michael.wrzaczek{at}umbr.cas.cz Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Timely flowering is critical for the reproductive success of plants. Plants adjust flowering time through integration of multiple internal and environmental cues. A complex signalling system lies behind this integration, including multiple proteins like the GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), linked to the modulation of stress and flowering in Arabidopsis. Available data show the interaction between GRP7 and the receptor-like kinase CYSTEINE-RICH RECEPTOR-LIKE KINASE 2 (CRK2). Like GRP7, CRK2 plays multifaceted roles in the coordination of stress responses and development, but its role in development remains unclear. Phenotypic analyses of the crk2 and grp7-1 single mutants as well as the crk2/grp7-1 double mutant supported the interaction between CRK2 and GRP7. Moreover, exogenous gibberellic acid did not recover the rosette phenotype of crk2 mutants but as in sensitive plants, inhibited GA-biosynthesis transcripts. The pseudo insensitivity of crk2 mutant was recovered in the crk2/grp7-1 line pointing to a common role of CRK2 and GRP7 in the modulation of gibberellin signalling. Together, CRK2 and GRP7 integrate hormonal and spatial signals required for floral transition. These insights highlight a previously unrecognized layer of regulation in GA signalling and open new avenues to understand how membrane-localized kinases and RNA-binding proteins intersect to control developmental timing. Introduction In this article, we explored how the interaction with the GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7) may allow CYSTEINE-RICH RECEPTOR-LIKE KINASE 2 (CRK2) to modulate flowering time in Arabidopsis. Our model points to a functional interaction between CRK2 and GRP7 that allows the RLK to introduce inputs into signalling mechanisms which, mediated by the plant phytohormone gibberellic acid (GA), might modulate flowering and environmental responses in Arabidopsis. Timely flowering is crucial for plants, as it ensures production of viable seeds and thereby the next generation. Plants adjust flowering time through the integration of internal and environmental cues, including energy status, temperature and photoperiod to optimize reproductive output in a changing environment ( Andrés & Coupland, 2012 ; Wahl et al ., 2013 ; Song et al ., 2015 ; Yu et al ., 2015 ; Kazan & Lyons, 2016 ). This regulation involves pathways responsive to light (photoperiod) and temperature (vernalization) as well as endogenous signals (autonomous pathway, sugar, age). Among these, GA plays central roles in flowering control ( Langridge, 1957 ; Wilson et al ., 1992 ; Porri et al ., 2012 ; Mateos et al ., 2015 ; Bao et al ., 2020 ), but also have roles in environmental responses ( Colebrook et al ., 2014 ). Despite extensive knowledge of the core flowering pathways, the full molecular network that coordinates them and integrates flowering with other responses to the environment remains incomplete ( Kazan & Lyons, 2016 ). Receptor-like kinases (RLKs) are emerging as integrators of developmental transitions, including flowering, and environmental stimuli ( Wang et al ., 2020 ). Among them, CRK2 plays multifaceted roles in stress signalling ( Hunter et al ., 2019 ; Kimura et al ., 2020 ) and development, as suggested by the late flowering and small rosette size of the crk2 mutant ( Bourdais et al ., 2015 ). Interestingly, CRK2 has previously been associated with GA signalling ( Cao et al ., 2006 ; Bassel et al ., 2011 ). However, the molecular mechanism of the integration of CRK2 with GA signalling has so far not been elucidated. GRP7, also known as SMALL RNA-BINDING PROTEIN 1 (SRBP1), a diurnally modulated RNA-binding protein ( Staiger et al ., 2003 ), has been identified as an in planta interaction partner of CRK2 ( Hunter et al ., 2019 ). Like CRK2, GRP7 modulates stress responses and developmental processes, including flowering time ( Nicaise et al ., 2013 ; Wang et al ., 2020 ). GRP7 negatively regulates its own expression ( Staiger et al ., 2003 ) as well as the expression of the central flowering repressor FLOWERING LOCUS C ( FLC ) ( Streitner et al ., 2008 ; Steffen et al ., 2019 ) within the autonomous pathway (AP). Moreover, phenotypes of GRP7 overexpression plants suggest a role as negative modulator of GA signalling ( Löhr et al ., 2014 ). By participating in AP and GA signalling, GRP7 provides a possible mechanism for its partner, CRK2 to input signals into the GA and/or AP flowering pathways. Results and discussion CRK2 modulates development in Arabidopsis through endogenous pathways Under long day (LD) conditions, crk2 exhibits a late flowering phenotype ( Bourdais et al ., 2015 ). Interestingly, crk2 showed an even more severe flowering delay in short day (SD) ( Figure 1a ; Figure S1; Table S1; Table S2a). In SD-grown Arabidopsis, flowering transition relies on endogenous pathways, including the AP and GA pathways. Mutants in these pathways, frequently show a more severe flowering delay in SD compared to LD ( Macknight et al ., 2002 ; Ausín et al ., 2004 ; Streitner et al ., 2008 ; Steffen et al ., 2019 ), suggesting that CRK2 might act via similar mechanisms. Notably, crk2 plants also had fewer rosette leaves compared to Col-0 at the time of bolting ( Figure 1b , Table S2b). Similar phenotypes where flowering time does not correlate with leaf number are rare ( Koornneef et al ., 1991 ; Méndez-Vigo et al ., 2010 ). Download figure Open in new tab Figure 1. CRK2 and GRP7 modulate Arabidopsis flowering time under non inductive conditions. (a) Flowering time (in days) of Arabidopsis thaliana Col-0, crk2, grp7-1, crk2/grp7-1 double mutant, and two complementation lines ( CRK2p::CRK2YFP/crk2 #1-14 and #1-22) grown under short-day (SD) conditions. Statistical significance was assessed by Kruskal–Wallis followed by Wilcoxon post hoc test. Letters indicate statistically significant differences between genotypes (p < 0.05). (b) Number of rosette leaves at flowering for the same genotypes under SD conditions. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Letters indicate statistically significant differences between genotypes (p < 0.05). (c) Representative rosettes of 75-day-old plants grown under SD conditions. (a, b) Data are presented as boxplots showing the median, interquartile range (IQR), and whiskers extending to 1.5×IQR. Individual data points are overlaid. CRK2 interacts functionally with the RNA binding protein GRP7 Interestingly, under SD crk2 flowered even later than grp7-1 and crk2/grp7-1 , which flowered later than Col-0 ( Figure 1a , Table S2a). As previously described, the rosette size of crk2 was small compared to Col-0 ( Kimura et al ., 2020 ) while rosettes of grp7-1 and crk2/grp7-1 showed a similar size to Col-0 ( Figure 1c ). This partial rescue of the crk2 developmental phenotypes by grp7-1 adds to previous evidence on CRK2-GRP7 interaction ( Hunter et al ., 2019 ), suggesting that GRP7 and CRK2 interact functionally. This links CRK2 with the GA and AP pathways, which are both modulated by GRP7 ( Kim et al ., 2008 ; Löhr et al ., 2014 ; Xiao et al ., 2015 ; Wang et al ., 2020 ). CRK2 modulates GA signalling with the involvement of GRP7 Both CRK2 and GRP7 are linked to GA biosynthesis and signalling ( Cao et al ., 2006 ; Kim et al ., 2008 ; Bassel et al ., 2011 ; Löhr et al ., 2014 ; Wang et al ., 2020 ). Exogenous GA treatment reduces CRK2 transcript abundance in the GA biosynthesis deficient ga1-3 mutant ( Bassel et al ., 2011 ). Like crk2 , GA insensitive mutants like gai display small rosette size and a severe flowering delay under SD ( Wilson et al ., 1992 ). The gai mutant carries a GA-resistant allele of the GAI DELLA, a repressor of GA responses degraded upon GA perception ( Peng et al ., 1997 ). Interestingly, DELLAs positively regulate CRK2 transcript levels ( Cao et al ., 2006 ). However, whether CRK2 regulates DELLAs is an open question, which can be tested in the future by evaluating DELLA stability in crk2 . Besides the CRK2 link with GA signalling, CRK2 transcript abundance also correlates with the GA biosynthesis-related transcript GIBBERELLIN 3-BETA-DIOXYGENASE 1 ( GA3ox1 ) ( Bassel et al ., 2011 ). GRP7 overexpression plants are sensitive to exogenous GA but contain reduced levels of bioactive GAs and reduced transcript abundance of GA biosynthesis and response genes like ENT-KAURENE SYNTHASE ( GA2 ; GA biosynthesis) and GIBBERELLIC ACID STIMULATED ARABIDOPSIS 9 ( GASA9 ; GA response) ( Löhr et al ., 2014 ). To evaluate whether CRK2 and GRP7 genetically interact in the GA pathway we investigated the impact of GA treatment on crk2, grp7-1 , the crk2/grp7-1 double mutant, and crk2 complementation lines ( Kimura et al ., 2020 ) in comparison to Col-0. As previously described, treatment with GA increased the rosette size of Col-0 compared to mock treatment ( Figure 2a ) ( Ribeiro et al ., 2012 ; Löhr et al ., 2014 ) while crk2 was insensitive to the phytohormone ( Figure 2a ). By contrast, GA treatment increased rosette size in grp7-1 and crk2/grp7-1 , similar to Col-0 ( Figure 2a ), suggesting a functional rescue of the GA sensitivity by grp7-1 . In addition, crk2 showed higher abundance of GA biosynthesis related GA2 and GA3ox1 transcripts compared to Col-0 under mock conditions ( Figure 2b Figure S2, Table S3a, Table S4). GA3ox1, involved in the final steps of bioactive GA synthesis, is subject to feedback repression by GA ( Chiang et al ., 1995 ). Surprisingly, while GA treatment did not affect crk2 rosette size, transcript abundance of GA3ox1 was reduced following GA treatment in crk2 similar to Col-0 ( Figure 2b ; Table S3a). The change in GA3ox1 abundance in crk2 showed functionally negative feedback and suggested that in crk2 , GA signalling is impaired downstream of perception. Transcript abundance of GA3ox1 in grp7-1 and crk2/grp7-1 was similar to Col-0 under mock conditions. In our experiment, all included genotypes but crk2 and grp7-1 showed a downward trend in the abundance of the GA-response transcript GASA9 ( Figure 2c ; Table S3b). Addition of GA reduces GASA9 abundance in Col-0 ( Zhang & Wang, 2008 ), suggesting altered transcriptional responses to GA in crk2 and grp7-1 . Download figure Open in new tab Figure 2. Effect of GA treatment. (a) Representative rosettes of two-month-old plants grown under SD conditions and under weekly spraying with mock or GA 3 containing solutions. (b) Abundance of GA-biosynthesis transcript GA3ox1 . Pairwise comparisons were performed within each genotype using Wilcoxon rank-sum test. P-values were adjusted for multiple testing using the Benjamini– Hochberg (BH) method (* p<0.1, ** p<0.05). (c) Abundance of GA-responsive transcript GASA9 . Pairwise comparisons were performed within each genotype using Wilcoxon rank-sum test. P-values were adjusted for multiple testing using the Benjamini–Hochberg (BH) method (* p<0.1). (b, c) Data are presented as boxplots showing the median, interquartile range (IQR), and whiskers extending to 1.5×IQR. Individual data points are overlaid. (d) Hypothetical model for the CRK2-GRP7-based modulation of flowering time in Arabidopsis. CRK2 is likely a flowering modulator in Arabidopsis. Our data points to a functional interaction between CRK2 and GRP7 focused on GA signalling and independent of the roles of GRP7 in the modulation of FLC . In our hypothesis, the RLK acts over the GA signalling pathway via phosphorylation-based modulation (likely inhibition) of GRP7 but might also exhibit a GRP7-independent influence on GA signalling. The phenotype of the crk2 / grp7-1 double mutant indicates a genetic interaction between CRK2 and GRP7 in GA signalling. However, whether they act in parallel or at least partially through the same pathway is still an open question. GRP7 likely is a phosphorylation substrate for CRK2 ( Figure 2d ), which carries a functional kinase domain ( Hunter et al ., 2019 ; Kimura et al ., 2020 ). The recombinant cytosolic region of CRK2 (CRK2cyt) phosphorylates recombinant GRP7, but not the GRP7 interactor JACALIN-LECTIN LIKE1 (JAC1), in vitro (Figure S3). Moreover, previous works found that the RLK FERONIA (FER) phosphorylates GRP7 in its RGG domain ( Wang et al ., 2020 ). This phosphorylation impacts GRP7 subcellular location and liquid-liquid phase separation (LLPS) ability which in turn impacts its affinity for RNA ( Wang et al ., 2020 ; Xu et al ., 2024 ; Lühmann et al ., 2024 ). Phosphorylation-mediated changes in GRP7 likely impact the splicing-mediated maturation of stress- and development-related transcripts ( Wang et al ., 2020 ). If GRP7 is phosphorylated by CRK2 in vivo it would be thus intriguing to describe the phosphorylation sites and their relevance in the presented genetic dependency between CRK2 and GRP7 at GA signalling. Interestingly, available RNA immunoprecipitation data shows that GRP7 targets multiple GA biosynthesis and response ( GASA ) transcripts ( Meyer et al ., 2017 ; Wang et al ., 2020 ) suggesting a direct effect of GRP7 on the maturation of GA response transcripts and providing a promising starting point for future approaches. It will be thus interesting to analyze whether CRK2, potentially through phosphorylation of GRP7, modulates the interaction of GRP7 with GA-related transcripts and their processing. Using in vivo phosphoproteomic analyses of Col-0 and crk2 and RNA immunoprecipitation (RIP)-seq of plants expressing non-phosphorylatable variants of GRP7 could provide these insights in the future. Despite a possible direct role of CRK2 on the modulation of GA signalling via GRP7, the functional interaction between CRK2 and GRP7 here described also provides potential links between CRK2 and the AP. The AP converges with the cold-associated vernalization flowering pathway at the repression of FLC . Therefore, the flowering delay of AP mutants like grp7-1 is reversed by cold treatment ( Streitner et al ., 2008 ; Steffen et al ., 2019 ). Interestingly, vernalization at 4ºC for 45 days rescued the flowering delay of grp7-1 and crk2/grp7-1 , but only partially for crk2 (Figure S4a; Table S5. Despite the vernalization effect, Col-0 and crk2 showed a similar abundance of FLC (Figure S4b, c; Table S6) and vernalization reduced FLC transcript levels similarly in Col-0 and crk2 (Figure S4c; Table S6c). By contrast, the crk2/grp7-1 double mutant showed high FLC abundance similar to grp7-1 (Figure S4a; Table S6a). Together with the similar flowering phenotypes of grp7-1 and crk2/grp7-1 ( Figure 1 ; Table S2, S6) this suggests that GRP7 modulates FLC independently of CRK2 ( Figure 2d ). Despite this intriguing finding, future works should determine whether the identified lack of effect on FLC in crk2 at the whole-plant level is explained by a targeted effect of GRP7 phosphorylation on a specific subset of its targets or is associated with a spatial functional compartmentalization of the CRK2-GRP7 interaction. Are CRK2 and GRP7 involved in the modulation of symplastic transport of flowering signals? Despite the many open questions on how CRK2 contributes to flowering via GA and whether GRP7 is involved, their known roles may contribute to lead future approaches towards characterizing these mechanisms. CRK2 has previously been linked to regulation of callose deposition ( Hunter et al ., 2019 ). Callose has important roles in controlling plasmodesmal connectivity and contributes to regulating transport of mobile protein flowering signals like the flowering promotor FLOWERING LOCUS T (FT) to the SAM ( Murata et al ., 2025 ). Interestingly, altered callose deposition has been observed in grp7-1 after pathogen infection ( Fu et al ., 2007 ). Therefore, CRK2 and GRP7 may modulate symplastic transport via callose deposition. Moreover, GRP7 has been linked to the transport of short RNAs (sRNAs) via plasmodesmata ( Yan et al ., 2020 ), and has been identified in phloem exudates ( Batailler et al ., 2012 ) and plasmodesmata enriched fractions ( Fernandez-Calvino et al ., 2011 ), suggesting that GRP7 might be a cell-to-cell mobile protein. Mobile GRP7 could be able to shuttle its RNA load between cells. RNA transport between cells, especially when this transport involves long-distance movement via phloem, is currently matter of active debate following recent reanalysis of mobile RNA datasets, which showed that RNA transport is likely less common than previously suggested ( Paajanen et al ., 2025 ). However, targeted approaches have successfully identified the mRNA of the DELLA protein GAI , to move through the phloem and induce dwarfism in distal tissues ( Haywood et al ., 2005 ). It is thus tempting to speculate on a role of CRK2 and GRP7 in the modulation of the transport and/or the maturation of GA-related transcripts. CRK2 might modulate GRP7 function to coordinate RNA processing and intercellular transport, thereby integrating hormonal, spatial, and post-transcriptional regulatory layers essential for developmental transitions such as flowering. In summary, our results position CRK2 as a key modulator of GA signalling, acting partly through GRP7 to control development ( Fig. 2d ). Competing interests The authors declare no competing interests. Author contribution FC and MW conceived and designed the project. FC, AP, and JKW carried out experiments. FC analyzed the data. FC, JKW and MW wrote the article. All authors read and contributed to the final article. Data availability All data is available in the supplementary files provided alongside this manuscript, including: flowering and transcript abundance data, used methodologies, statistical procedures, reagents (including oligonucleotides) and instruments. Sequence data used in this manuscript can be found in the TAIR platform ( https://www.arabidopsis.org/ ) under the following accession nos.: AT1G22690, AT1G15550, AT1G79460, AT5G10140, AT5G08290, AT5G25760. Supplementary figure legends Figure S1 . The flowering delay of the crk2 mutant is more severe under SD. Figure S2 . Expression of the GA biosynthesis genes GA2 and GA3ox1 is altered in crk2 . Figure S3 . The recombinant cytosolic domain of CRK2 (CRK2cyt) phosphorylates recombinant GRP7, but not the GRP7-interactor JACALIN-LECTIN LIKE1 (JAC1), in vitro . Figure S4 . The crk2 mutant is sensitive to vernalization but does not show increased FLC expression. Supplementary table legends Table S1 . Photoperiod experiment. Raw data and statistical analysis. Table S2 . Flowering time and rosette leaf number at flowering. Raw data and statistical analysis. Table S3 . Relative transcript abundance of GA3ox1 & GASA9 . Table S4 . Relative transcript abundance of GA biosynthesis-related genes. Raw data and statistical analysis. Table S5 . Vernalization experiment. Normalized data and statistical analysis. Table S6 . Relative transcript abundance of FLC. Raw data and statistical analysis. Table S7 . List of oligonucleotides used for gene expression analysis. Acknowledgments The authors would like to thank Sunita Jindal, María Carbó and Adam Zeiner ( Institute of Plant Molecular Biology, Biology Centre, Czech Academy of Sciences, Czechia ), and to Ainhoa Martínez Medina, Iván Fernández López and Ana María Jiménez Jiménez ( Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Spain ) for critical comments on the article. This work was supported by the Czech Academy of Sciences (startup grant to MW) and by the Czech Science Foundation (grant 23-04866S). FC was funded by EMBO (award number: ALTF 1115-2021; 2022-2024) and the Czech Ministry of Youth and Education MSCA Fellowships Interfellows (Project number: CZ.02.01.01/00/22_010/0003414; 2024-2025). This work was carried out with the support of the Growth Facility BC Core Facilities, Institute for Plant Molecular Biology, Biology Centre CAS. Funder Information Declared Czech Science Foundation, https://ror.org/01pv73b02 , 23-04866S EMBO , ALTF-1115-2021 Czech Ministry of Youth and Education MSCA Fellowships Interfellows , CZ.02.01.01/00/22_010/0003414 Footnotes The manuscript has been revised to clarify the data and state the hypothetical nature of the interaction between CRK2 and GRP7. An in vitro kinase assay showing that CRK2 phosphorylates GRP7, but not the GRP7-interacting protein JAC1, has been added to the manuscript. References ↵ Andrés F , Coupland G. 2012 . The genetic basis of flowering responses to seasonal cues . Nature Reviews Genetics 13 : 627 – 639 . OpenUrl CrossRef PubMed ↵ Ausín I , Alonso-Blanco C , Jarillo JA , Ruiz-García L , Martínez-Zapater JM . 2004 . Regulation of flowering time by FVE, a retinoblastoma-associated protein . Nature Genetics 36 : 162 – 166 . OpenUrl CrossRef PubMed Web of Science ↵ Bao S , Hua C , Shen L , Yu H. 2020 . New insights into gibberellin signaling in regulating flowering in Arabidopsis . Journal of Integrative Plant Biology 62 : 118 – 131 . OpenUrl PubMed ↵ Bassel GW , Glaab E , Marquez J , Holdsworth MJ , Bacardit J. 2011 . Functional network construction in Arabidopsis using rule-based machine learning on large-scale data sets . The Plant Cell 23 : 3101 – 3116 . OpenUrl Abstract / FREE Full Text ↵ Batailler B , Lemaître T , Vilaine F , Sanchez C , Renard D , Cayla T , Beneteau J , Dinant S. 2012 . Soluble and filamentous proteins in Arabidopsis sieve elements . Plant, Cell & Environment 35 : 1258 – 1273 . OpenUrl CrossRef PubMed ↵ NM Springer , Ed Bourdais G , Burdiak P , Gauthier A , Nitsch L , Salojärvi J , Rayapuram C , Idänheimo N , Hunter K , Kimura S , Merilo E , et al. 2015 . Large-scale phenomics identifies primary and finetuning roles for CRKs in responses related to oxidative stress ( NM Springer , Ed.). PLOS Genetics 11 : e1005373 . OpenUrl ↵ Cao D , Cheng H , Wu W , Soo HM , Peng J. 2006 . Gibberellin mobilizes distinct DELLA-dependent transcriptomes to regulate seed germination and floral development in Arabidopsis . Plant Physiology 142 : 509 – 525 . OpenUrl Abstract / FREE Full Text ↵ Chiang HH , Hwang I , Goodman HM . 1995 . Isolation of the Arabidopsis GA4 locus . The Plant Cell 7 : 195 – 201 . OpenUrl Abstract / FREE Full Text ↵ Colebrook EH , Thomas SG , Phillips AL , Hedden P. 2014 . The role of gibberellin signalling in plant responses to abiotic stress . Journal of Experimental Biology 217 : 67 – 75 . OpenUrl Abstract / FREE Full Text ↵ Fernandez-Calvino L , Faulkner C , Walshaw J , Saalbach G , Bayer E , Benitez-Alfonso Y , Maule A. 2011 . Arabidopsis plasmodesmal proteome . PLoS ONE 6 : e18880 . OpenUrl CrossRef PubMed ↵ Fu ZQ , Guo M , Jeong B , Tian F , Elthon TE , Cerny RL , Staiger D , Alfano JR . 2007 . A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity . Nature 447 : 284 – 288 . OpenUrl CrossRef PubMed Web of Science ↵ Haywood V , Yu T , Huang N , Lucas WJ . 2005 . Phloem long□distance trafficking of GIBBERELLIC ACID□INSENSITIVE RNA regulates leaf development . The Plant Journal 42 : 49 – 68 . OpenUrl CrossRef PubMed Web of Science ↵ Hunter K , Kimura S , Rokka A , Tran HC , Toyota M , Kukkonen JP , Wrzaczek M. 2019 . CRK2 enhances salt tolerance by regulating callose deposition in connection with PLDα1 . Plant Physiology 180 : 2004 – 2021 . OpenUrl Abstract / FREE Full Text ↵ Kazan K , Lyons R. 2016 . The link between flowering time and stress tolerance . Journal of Experimental Botany 67 : 47 – 60 . OpenUrl CrossRef PubMed ↵ Kim JS , Jung HJ , Lee HJ , Kim KA , Goh C-H , Woo Y , Oh SH , Han YS , Kang H. 2008 . Glycine-rich RNA-binding protein7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana . The Plant Journal 55 : 455 – 466 . OpenUrl CrossRef PubMed Web of Science ↵ Kimura S , Hunter K , Vaahtera L , Tran HC , Citterico M , Vaattovaara A , Rokka A , Stolze SC , Harzen A , Meißner L , et al. 2020 . CRK2 and C-terminal phosphorylation of NADPH oxidase RBOHD regulate reactive oxygen species production in Arabidopsis . The Plant Cell 32 : 1063 – 1080 . OpenUrl Abstract / FREE Full Text ↵ Koornneef M , Hanhart CJ , Van Der Veen JH . 1991 . A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana . Molecular and General Genetics 229 : 57 – 66 . OpenUrl CrossRef Web of Science ↵ Langridge J. 1957 . Effect of Day-length and Gibberellic Acid on the Flowering of Arabidopsis . Nature 180 : 36 – 37 . OpenUrl CrossRef Web of Science ↵ Löhr B , Streitner C , Steffen A , Lange T , Staiger D. 2014 . A glycine-rich RNA-binding protein affects gibberellin biosynthesis in Arabidopsis . Molecular Biology Reports 41 : 439 – 445 . OpenUrl CrossRef PubMed ↵ Lühmann KL , Seemann S , Martinek N , Ostendorp S , Kehr J. 2024 . The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation . Scientific Reports 14 : 16018 . OpenUrl PubMed ↵ Macknight R , Duroux M , Laurie R , Dijkwel P , Simpson G , Dean C. 2002 . Functional significance of the alternative transcript processing of the Arabidopsis floral promoter FCA . The Plant Cell 14 : 877 – 888 . OpenUrl Abstract / FREE Full Text ↵ Mateos JL , Madrigal P , Tsuda K , Rawat V , Richter R , Romera-Branchat M , Fornara F , Schneeberger K , Krajewski P , Coupland G. 2015 . Combinatorial activities of SHORT VEGETATIVE PHASE and FLOWERING LOCUS C define distinct modes of flowering regulation in Arabidopsis . Genome Biology 16 : 31 . OpenUrl CrossRef PubMed ↵ Méndez-Vigo B , De Andrés MT , Ramiro M , Martínez-Zapater JM , Alonso-Blanco C. 2010 . Temporal analysis of natural variation for the rate of leaf production and its relationship with flowering initiation in Arabidopsis thaliana . Journal of Experimental Botany 61 : 1611 – 1623 . OpenUrl CrossRef PubMed Web of Science ↵ Meyer K , Köster T , Nolte C , Weinholdt C , Lewinski M , Grosse I , Staiger D. 2017 . Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 . Genome Biology 18 : 204 . OpenUrl CrossRef PubMed ↵ Murata Y , Nagata K , Abe M. 2025 . Cell-to-cell translocation of florigen is inhibited by low ambient temperature through abscisic acid signaling in Arabidopsis thaliana . Proceedings of the National Academy of Sciences USA 122 ( 28 ): e2507987122 . OpenUrl PubMed ↵ Nicaise V , Joe A , Jeong B , Korneli C , Boutrot F , Westedt I , Staiger D , Alfano JR , Zipfel C. 2013 . Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7 . The EMBO Journal 32 : 701 – 712 . OpenUrl Abstract / FREE Full Text ↵ Paajanen P , Tomkins M , Hoerbst F , Veevers R , Heeney M , Thomas HR , Apelt F , Saplaoura E , Gupta S , Frank M , et al. 2025 . Re-analysis of mobile mRNA datasets raises questions about the extent of long-distance mRNA communication . Nature Plants 11 : 977 – 984 . OpenUrl PubMed ↵ Peng J , Carol P , Richards DE , King KE , Cowling RJ , Murphy GP , Harberd NP . 1997 . The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses . Genes & Development 11 : 3194 – 3205 . OpenUrl Abstract / FREE Full Text ↵ Porri A , Torti S , Romera-Branchat M , Coupland G. 2012 . Spatially distinct regulatory roles for gibberellins in the promotion of flowering of Arabidopsis under long photoperiods . Development 139 : 2198 – 2209 . OpenUrl Abstract / FREE Full Text ↵ Ribeiro DM , Araújo WL , Fernie AR , Schippers JHM , Mueller-Roeber B. 2012 . Action of gibberellins on growth and metabolism of Arabidopsis plants associated with high concentration of carbon dioxide . Plant Physiology 160 : 1781 – 1794 . OpenUrl Abstract / FREE Full Text ↵ Song YH , Shim JS , Kinmonth-Schultz HA , Imaizumi T. 2015 . Photoperiodic flowering: Time measurement mechanisms in leaves . Annual Review of Plant Biology 66 : 441 – 464 . OpenUrl CrossRef PubMed ↵ Staiger D , Zecca L , Kirk DAW , Apel K , Eckstein L. 2003 . The circadian clock regulated RNA□binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre□mRNA . The Plant Journal 33 : 361 – 371 . OpenUrl CrossRef PubMed Web of Science ↵ Steffen A , Elgner M , Staiger D. 2019 . Regulation of Flowering Time by the RNA-Binding Proteins AtGRP7 and AtGRP8 . Plant and Cell Physiology 60 : 2040 – 2050 . OpenUrl CrossRef PubMed ↵ Streitner C , Danisman S , Wehrle F , Schöning JC , Alfano JR , Staiger D. 2008 . The small glycine-rich RNA binding protein At GRP7 promotes floral transition in Arabidopsis thaliana: At GRP7 and the floral transition in Arabidopsis thaliana . The Plant Journal 56 : 239 – 250 . OpenUrl CrossRef PubMed Web of Science ↵ Wahl V , Ponnu J , Schlereth A , Arrivault S , Langenecker T , Franke A , Feil R , Lunn JE , Stitt M , Schmid M. 2013 . Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana . Science 339 : 704 – 707 . OpenUrl Abstract / FREE Full Text ↵ Wang L , Yang T , Wang B , Lin Q , Zhu S , Li C , Ma Y , Tang J , Xing J , Li X , et al. 2020 . RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants . Science Advances 6 : eaaz1622 . OpenUrl FREE Full Text ↵ Wilson RN , Heckman JW , Somerville CR . 1992 . Gibberellin is required for flowering in Arabidopsis thaliana under short days . Plant Physiology 100 : 403 – 408 . OpenUrl Abstract / FREE Full Text ↵ Xiao J , Li C , Xu S , Xing L , Xu Y , Chong K. 2015 . JACALIN-LECTIN LIKE1 regulates nuclear accumulation of GRP7, influencing RNA processing of FLC Antisense transcripts and flowering time in Arabidopsis . Plant Physiology 169 : 2102 – 2117 . OpenUrl Abstract / FREE Full Text ↵ Xu F , Wang L , Li Y , Shi J , Staiger D , Yu F. 2024 . Phase separation of GRP7 facilitated by FERONIA-mediated phosphorylation inhibits mRNA translation to modulate plant temperature resilience . Molecular Plant 17 : 460 – 477 . OpenUrl CrossRef PubMed ↵ Yan Y , Ham B-K , Chong YH , Yeh S-D , Lucas WJ . 2020 . A plant SMALL RNA-BINDING PROTEIN 1 family mediates cell-to-cell trafficking of RNAi signals . Molecular Plant 13 : 321 – 335 . OpenUrl PubMed ↵ Yu S , Lian H , Wang J-W. 2015 . Plant developmental transitions: the role of microRNAs and sugars . Current Opinion in Plant Biology 27 : 1 – 7 . OpenUrl CrossRef PubMed ↵ Zhang S , Wang X. 2008 . Expression pattern of GASA, downstream genes of DELLA, in Arabidopsis . Science Bulletin 53 : 3839 – 3846 . OpenUrl View the discussion thread. Back to top Previous Next Posted November 09, 2025. Download PDF Supplementary Material 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 CRK2 modulates flowering in Arabidopsis together with GLYCINE- RICH RNA BINDING PROTEIN 7 (GRP7) 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. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share CRK2 modulates flowering in Arabidopsis together with GLYCINE- RICH RNA BINDING PROTEIN 7 (GRP7) Francisco J. Colina , Julia Krasensky-Wrzaczek , Alegría Pérez-Guillén , Michael Wrzaczek bioRxiv 2025.08.05.668606; doi: https://doi.org/10.1101/2025.08.05.668606 Share This Article: Copy Citation Tools CRK2 modulates flowering in Arabidopsis together with GLYCINE- RICH RNA BINDING PROTEIN 7 (GRP7) Francisco J. Colina , Julia Krasensky-Wrzaczek , Alegría Pérez-Guillén , Michael Wrzaczek bioRxiv 2025.08.05.668606; doi: https://doi.org/10.1101/2025.08.05.668606 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 (7640) Biochemistry (17706) Bioengineering (13902) Bioinformatics (41978) Biophysics (21465) Cancer Biology (18611) Cell Biology (25528) Clinical Trials (138) Developmental Biology (13387) Ecology (19920) Epidemiology (2067) Evolutionary Biology (24332) Genetics (15615) Genomics (22519) Immunology (17747) Microbiology (40424) Molecular Biology (17194) Neuroscience (88662) Paleontology (667) Pathology (2839) Pharmacology and Toxicology (4827) Physiology (7650) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9826) Zoology (2271)