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A genetic network mediating the control of bud break in hybrid aspen

A genetic network mediating the control of bud break in hybrid aspen ARTICLE DOI: 10.1038/s41467-018-06696-y OPEN A genetic network mediating the control of bud break in hybrid aspen 1 1 1,2 1 1,3 1 Rajesh Kumar Singh , Jay P. Maurya , Abdul Azeez , Pal Miskolczi , Szymon Tylewicz , Katja Stojkovič , 1 2 1 Nicolas Delhomme , Victor Busov & Rishikesh P. Bhalerao In boreal and temperate ecosystems, temperature signal regulates the reactivation of growth (bud break) in perennials in the spring. Molecular basis of temperature-mediated control of bud break is poorly understood. Here we identify a genetic network mediating the control of bud break in hybrid aspen. The key components of this network are transcription factor SHORT VEGETATIVE PHASE-LIKE (SVL), closely related to Arabidopsis floral repressor SHORT VEGETATIVE PHASE, and its downstream target TCP18, a tree homolog of a branching reg- ulator in Arabidopsis. SVL and TCP18 are downregulated by low temperature. Genetic evi- dence demonstrates their role as negative regulators of bud break. SVL mediates bud break by antagonistically acting on gibberellic acid (GA) and abscisic acid (ABA) pathways, which function as positive and negative regulators of bud break, respectively. Thus, our results reveal the mechanistic basis for temperature-cued seasonal control of a key phenological event in perennial plants. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 87 Umeå, Sweden. 2 3 School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931, USA. Department of Plant and Microbial Biology, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland. Correspondence and requests for materials should be addressed to R.P.B. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 1 1234567890():,; ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y he survival of perennial plants in boreal and temperate dormancy as mediators of temperature controlled bud break. We ecosystems is dependent on seasonally synchronized show that SVL is a positive regulator of TCP18/BRC1 and toge- Tannual growth cycles. In long-lived trees, growth ceases ther they form a temperature-responsive transcriptional module and dormancy is established prior to the onset of winter. Dor- that mediates control of bud break. We demonstrate that com- mancy is maintained during the winter and gradual release from ponents of antagonistic ABA and GA hormonal pathways are dormancy is followed by reactivation of growth in the spring . downstream targets of SVL in bud break regulation. Thus, our Photoperiod and temperature are the main environmental cues results reveal a temperature responsive genetic network mediating regulating the seasonal synchronization of the key developmental bud break, a major phenological process in perennial plants. transitions in the annual growth cycle . The timing of growth cessation in the autumn, governed primarily by photoperiod Results signals, is mediated by the FLOWERING LOCUS T/CONSTANS Transcription factor SVL participates in bud break control.We 2,3 (FT/CO) module in the model plant hybrid aspen . Interaction screened transgenic hybrid aspen lines overexpressing transcrip- of FT2 with FD-LIKE1 (FDL1) promotes growth under long tion factors to identify genes involved in bud break. The results photoperiods by ensuring high expression of the transcription showed that relative to wild-type hybrid aspen plants, bud break factor LIKE-AP1 (LAP1) . LAP1 is a positive regulator of was significantly delayed in transgenic lines overexpressing a AINTEGUMENTA-LIKE1 (AIL1), a transcriptional regulator of poplar MADS box named SVL (SVP-LIKE), highly similar to 5,6 key cell cycle genes, including D-type cyclins . When a reduc- Arabidopsis floral repressor SVP (Fig. 1a–d, i, Supplementary tion in day length below a critical threshold permitting growth Fig. 1a). Sequence analysis indicated that SVL is similar to Ara- (short days/SD) is sensed, downregulation of FT2 results in bidopsis SVP and DAM genes described in several tree growth cessation and formation of a bud structure at the apex . 25,28 species . However, phylogenetic analysis shows that hybrid Buds enclose arrested leaf primordia and shoot apical meristems aspen SVL is more similar to SVP from Arabidopsis and apple within protective bud scales . (Supplementary Fig. 2) than to poplar MADS-box genes 27–29 After growth cessation, continuation of short days induce 29 that are homologs of DAM genes in peach . To confirm the role dormancy in the buds before winter . Recently, plant hormone of SVL in bud break, transgenic hybrid aspen plants with reduced abscisic acid (ABA) has been shown to mediate photoperiodic SVL expression (SVLRNAi) were also generated and scored for 7,9,10 control of bud dormancy . Once dormancy is established, bud break (Supplementary Fig. 1b). In contrast with SVL over- 11,12 buds no longer respond to growth-promotive signals . Hence expressers (SVLoe), SVLRNAi lines showed early bud break dormancy must first be released before growth can be reactivated compared to the control wild-type hybrid aspen plants (Fig. 1e–h, in the buds. Dormancy release is induced by prolonged exposure j). As SVLoe and SVLRNAi react similarly under short days, to low temperature (LT) following which growth can be reacti- (Supplementary Fig. 3a and b), results indicate that SVL, has a vated as visibly manifested by bud break, i.e., emergence of new negative role in bud break in hybrid aspen. 11,13–16 leaves from the buds . The mechanisms underlying dormancy release and bud break LT and ABA antagonistically modulate SVL expression. Our are intimately linked but the underlying molecular mechanisms data indicated that bud break was affected in SVL transgenics are not well understood. However, physiological and tran- being delayed in SVL overexpressers and occurring earlier in SVL scriptomic approaches have noted that increase in expression of downregulated plants (Fig. 1) indicating that SVL mediates in bud gibberellic acid (GA) biosynthesis related genes and FT2 homolog break, a process regulated by temperature signal. Therefore, we FT1, that are potent growth promoters and simultaneous down- investigated the temperature-responsiveness of SVL expression. regulation of the components of ABA pathway coincides tem- 17–19 SVL expression was significantly downregulated by exposure to porally with dormancy release and transition to bud break . low temperature and remained lower than its levels in dormant Moreover, exogenous applications of GA and ABA respectively 19,20 buds prior to low temperature treatment (Fig. 1k). SVL expres- promote and delay bud break . However, the endogenous role sion marginally increased somewhat following buds’ exposure to of these components in bud break remains uncharacterized so far. warm temperatures, but nevertheless remained lower than in the Earlier studies have also drawn parallels between vernalization, dormant buds. Thus, SVL expression is negatively regulated by flowering promotion by low temperature in Arabidopsis and bud 21,22 low temperature. It has been reported that upon low temperature, break . For example, low temperature induces changes in the there is an increase in the repressive marks like histone H3 lysine expression and chromatin status of DORMANCY ASSOCIATED 23,24 27 trimethylation (H3K27me3) in the promoters of SVL like MADS-BOX (DAM) genes . Interestingly, overexpression of 23,24 DAM genes in peach and pear . However, we did not observe DAM genes can delay bud break . While informative, these any significant increase of H3K27me3 marks at the SVL locus primarily based on gain-of-function approaches have not upon low temperature treatment (Supplementary Fig. 4) indi- addressed the endogenous roles of DAM genes, and the cating that in contrast with other DAM genes, SVL suppression is mechanism(s) whereby they mediate control of bud break. In not due to increase in H3K27 trimethylation at the SVL locus. addition, an AP2-family transcription factor designated EARLY Like temperature signal, ABA has been implicated in bud break BUD BREAK 1 (EBB1) has been identified by activation tagging in with exogenous application of ABA delaying bud break , hybrid poplar, which is clearly relevant as EBB1 overexpression phenocopying SVL overexpresseors. Therefore we investigated and downregulation results in early and late bud break, respec- 26 whether ABA, mediates in the control of SVL expression. ABA tively . Nevertheless, whether EBB1 acts directly in temperature application induces SVL expression (Supplementary Fig. 5) and control of bud break and its downstream targets remain moreover in the buds of transgenic hybrid aspen plants with uncharacterised. Thus, the molecular basis for translation of reduced response to ABA, SVL expression is significantly reduced temperature signals into promotion of bud break remains poorly (Supplementary Fig. 6). Thus, ABA in contrast with low understood. temperature acts positively in control of SVL expression. Here we report on identification of transcription factors, SHORT VEGETATIVE PHASE-LIKE (SVL) similar to Arabi- dopsis SHORT VEGETATIVE PHASE (SVP) a flowering FT1 and GA20 oxidase genes are negatively regulated by SVL. time regulator and TEOSINTE BRANCHED1, CYCLOIDEA, Low temperature enhances the expression of FT1 and compo- PCF/BRANCHED1 (TCP18/BRC1) , involved in axillary bud nents of GA biosynthesis e.g. GA20 oxidases in buds mirroring 2 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE a b ef WT SVLoe_2 WT SVLRNAi_2 cd gh SVLoe_4 SVLoe_8 SVLRNAi_10 SVLRNAi_12 ij k 1.5 1.2 2.0 * 1.0 1.5 A 1.0 0.8 1.0 0.6 0.5 0.5 0.4 0.0 0.2 0.0 10WSD 2WC 5WC 2WLD Fig. 1 Delayed and early bud break in plants over- and under-expressing SVL. a–d Bud break is earlier in a WT plants than in three independent SVL overexpressing lines, designated SVLoe2 b, SVLoe4 c, and SVLoe8 d. e–h In contrast, bud break is later in WT plants h than in independent SVLRNAi lines 2 f,10 g, and 12 h. I, j Time to bud break relative to wild type controls in SVLoe i and SVLRNAi j lines. Average time taken to bud break ± standard error mean (SEM), with respect to WT considered as 1, is shown with data from 10 plants from each line. Asterisks (*) indicate significant differences (P < 0.05) with respect to WT. k Low temperatures suppress SVL expression. Relative expression of SVL after 10 weeks of SD, followed by 2 and 5 weeks of low temperature (2WC, 5WC at +4 °C) and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). SVL expression from three independent biological replicates ± SEM is shown relative to the reference gene UBQ with 10WSD time point set to 1. Different letters A–D over the bars indicate significant differences at P < 0.001. Statistical analysis was done using one way ANNOVA implying Dunnett’s/Tukey’s multiple comparison test 6,19 the downregulation of SVL . Given the growth promotive role buds after exposure to low temperatures. In contrast, dormant 5,9,19 of FT and GA’s , we investigated whether SVL could parti- buds of SVLoe plants expressed NCED3 at a higher level and did cipate in bud break by affecting FT1 and/or GA pathway. In not downregulate it, relative to WT buds, in response to low agreement with previous findings, low temperature-induced temperature (Fig. 3a). Whereas in SVLRNAi buds, NCED3 expression of FT1 and GA20 oxidases in WT buds. In contrast, expression was lower and downregulated to a higher extent by induction of FT1 and GA20 oxidases was reduced in SVLoe buds low temperature than in the WT buds (Fig. 3b). Additionally, the (Fig. 2a) and, conversely, FT1 and GA20 oxidases induction was expression of genes encoding RCAR/PYL1 and RCAR/PYL2, enhanced in SVLRNAi plants relative to the wild type, after highly similar to ABA receptors, which activate downstream exposure to low temperature (Fig. 2b). These findings suggest a signaling responses after binding ABA, is consistently higher in negative role for SVL in the induction of FT1 and GA20 oxidases SVL overexpressers than in wild-type buds, but lower in SVL expression by low temperature in hybrid aspen buds. downregulated plants (Fig. 3a, b). Thus, ABA upregulates SVL, and in turn, SVL positively regulates ABA biosynthesis and signaling-related genes forming a feedback loop. SVL modulates ABA biosynthesis and signaling gene expres- sion. ABA induces SVL expression as shown before and exo- genous application of ABA delays bud break . Therefore, we SVL acts upstream of the TCP18/BRC1 transcription factor. investigated the transcriptional regulation of ABA biosynthesis TCP18/BRC1 a transcription factor that regulates axillary bud and response machinery during bud break in the wild type and outgrowth, has been recently shown to control ABA signaling in the SVL transgenic plants. The expression of NCED3, which Arabidopsis . The expression of the hybrid aspen gene homo- encodes a key enzyme in ABA biosynthesis, decreased in WT logous to TCP18/BRC1 is downregulated following exposure to NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 3 WT SVLoe_2 SVLoe_4 SVLoe_8 WT SVLRNAi_2 SVLRNAi_10 SVLRNAi_12 Time to bud break relative to WT Time to bud break relative to WT SVL relative expression ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y 8 10 WT WT WT ** ** ** SVLoe SVLoe SVLoe ** ** ** ** 0 0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD b 10 40 WT ** WT WT SVLRNAi SVLRNAi SVLRNAi ** *** *** ** ** 0 0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD Fig. 2 SVL negatively regulates expression of FT1 and GA20 oxidases during dormancy release and bud break. Expression patterns of genes encoding FT1, GA20 oxidases (GA20ox_1 and GA20ox_2) in apices of a WT and SVLoe and b WT and SVLRNAi after 10 weeks of SDs(10WSD), at the time of dormancy release (i.e. after 2weeks of LT, 2WC), and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). Expression values of the cited genes shown are averages for three biological replicates ± SEM, relative to the reference gene UBQ and with 10WSD time point set to 1. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using multiple t-tests low temperature (Supplementary Fig. 7). Therefore, we investi- site for MADS-box transcription factors. In contrast, no evidence gated whether hybrid aspen TCP18/BRC1 homolog could be a of SVL binding to promoters of GA20 oxidase (1 and 2) or RCAR/ target of SVL, by examining TCP18/BRC1 expression in SVL PYL genes was detected indicating that SVL indirectly affects the transgenics. TCP18/BRC1 expression was downregulated by low expression of these genes (Supplementary Fig. 8). temperature treatment in WT buds, whereas this downregulation was attenuated in SVLoe buds and TCP18/BRC1 expression was Reduction of GA represses early bud break in SVLRNAi lines. consistently higher in SVL overexpressing transgenic plants than Early bud break in SVLRNAi plants is correlated with enhanced in WT plants at all-time points (Fig. 3a). Conversely, TCP18/ expression of GA biosynthesis in these plants relative to wild type BRC1 expression was lower in SVL downregulated (SVLRNAi) suggesting that the GA pathway could be a downstream target of lines, suggesting that TCP18/BRC1 could be a downstream target SVL in bud break regulation. We tested this hypothesis by gen- of SVL (Fig. 3b). erating SVLRNAi plants overexpressing a poplar GA2 oxidase (Supplementary Fig. 9) with known ability to reduce GA levels . We then compared the bud break of SVLRNAi with SVLRNAi SVL regulates FT1, NCED3, and TCP18/BRC1 expression expressing GA2 oxidase (Fig. 5). Our data show that GA2 oxidase directly. The presented results showed that SVL mediates in expression repressed the early bud break phenotype of SVLRNAi temperature control of bud break and expression of growth transgenic lines. These results along with gene expression data promoters and repressors, including FT1, GA20 oxidases, NCED3, strongly support that GA pathway is a downstream target of SVL RCAR/PYL, and TCP18/BRC1.As SVL is a transcriptional reg- in temperature controlled bud break. ulator, we investigated which of these genes are direct down- stream targets of SVL by chromatin immunoprecipitation (ChIP)-RT–PCR experiments on DNA isolated from shoot apices Overexpression of RCAR/PYL1 and TCP18/BRC1 delays bud of transgenic hybrid aspen plants expressing Myc-tagged SVL break. The expression of hybrid aspen TCP18/BRC1 and RCAR/ (Myc-SVL) and WT control. We found clear evidence for binding PYL ABA receptors is affected in SVL transgenics and like SVL, of SVL in the promoters of FT1, NCED3, and TCP18/BRC1 TCP18/BRC1 and RCAR/PYLs are downregulated in the buds (Fig. 4) all of which contain a CArG motif known to be a target after exposure to low temperature. Therefore we hypothesized 4 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications FT1 relative expression FT1 relative expression GA20ox_1 relative expression GA20ox_1 relative expression GA20ox_2 relative expression GA20ox_2 relative expression NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE 2.0 2.5 2.0 WT SVLoe WT SVLoe WT SVLoe WT SVLoe *** *** ** *** ** ** 2.0 1.5 1.5 *** *** 1.5 ** 1.0 1.0 6 ** 1.0 *** 0.5 0.5 0.5 0.0 0.0 0 0.0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD 1.2 2.0 WT SVLRNAi WT SVLRNAi WT SVLRNAi WT SVLRNAi 1.5 1.2 ** ** *** *** 1.0 1.0 1.5 ** 0.8 1.0 ** 0.8 ** *** 0.6 1.0 ** 0.6 ** 0.4 0.5 0.4 0.5 *** 0.2 0.2 0.0 0.0 0.0 0.0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD Fig. 3 SVL positively regulates expression of the ABA biosynthesis gene NCED3, ABA receptors (RCAR/PYLs), and TCP18/BRC1-like transcription factors during dormancy release and bud break. Expression patterns of NCED3, RCAR/PYL1, RCAR/PYL2, and TCP18 in apices of a WT and SVLoe and b WT and SVLRNAi after 10 weeks of SDs (10WSD), at the time of dormancy release (i.e. after 2weeks of LT, 2WC), and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). Expression values of the cited genes shown are averages for three biological replicates ± SEM, relative to the reference gene UBQ and with 10WSD time point set to 1. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using multiple t-test ATG a F1 R1 F1 R1 F2 R2 F1 R1 F2 R2 ATG F2 R2 ATG 3 kb 2 kb 1 kb 3 kb 2 kb 1 kb 3 kb 2 kb 1 kb 3 10 8 *** *** *** 0 0 0 WT Myc-SVL WT Myc-SVL WT Myc-SVL Fig. 4 SVL binds to FT1, NCED3, and TCP promoters in vivo in chromatin immunoprecipitation (ChIP) assays. a Diagrammatic representation of FT1, NCED3, and TCP18/BRC1 promoters showing the CArG motif and their positions within a 3 kb region. F1–R1 indicates positions of DNA fragments used to assess DNA–protein interactions in ChIP assays, and F2–R2 indicates positions of DNA fragments with no CArG motif used as negative controls in the assays. b Enrichment of the DNA fragments containing the CArG motif quantified by ChIP-q-PCR. Presented values were first normalized by their respective input values, then fold enrichments in WT and Myc-SVL plants relative to negative controls were calculated. Bars show an average values from three independent biological replicates ± SEM. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using t-test that bud break involves the downregulation of TCP18/BRC1 and (Supplementary Fig. 10). In both, RCAR/PYL1 and TCP18/BRC1 RCAR/PYLs. We tested this hypothesis by generating transgenic overexpressers, bud break was significantly delayed compared to plants that would maintain high levels of RCAR/PYL1 and wild type control plants (Fig. 6) indicating that RCAR/PYL1 and TCP18/BRC1 then investigated bud break in these genotypes TCP18/BRC1 have repressive roles in bud break regulation. NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 5 NCED3 relative expression NCED3 relative expression FT1 promoter fold enrichment/control RCAR1/PYL1 relative expression RCAR1/PYL1 relative expression NCED3 promoter fold enrichment/control RCAR2/PYL2 relative expression RCAR2/PYL2 relative expression TCP18/BRC1 promoter fold enrichment/control TCP18/BRC1 relative expression TCP18/BRC1 relative expression ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y 1.5 ab C C 1.0 0.5 cd 0.0 Fig. 5 Overexpression of GA2 oxidase represses early bud break in SVLRNAi lines. a–d Bud break is earlier in a WT and b SVLRNAi plants than in c, d two independent lines overexpressing GA2 oxidase in a SVLRNAi background, designated GA2oxoe/SVLRNAi_1 and 6, respectively. e Time to bud break relative to WT controls for SVLRNAi plants, and lines overexpressing GA2 oxidase in a SVLRNAi background. Average time taken to bud break ± SEM, with respect to WT considered as 1, is shown with data from 10 plants from each line. Different letters A–C over the bars indicate significant differences at P < 0.001. Statistical analysis was done using one way ANNOVA implying Tukey’s multiple comparison test ab 2.5 2.0 2.0 * 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Fig. 6 RCAR/PYL1 or TCP18/BRC1 overexpression delays bud break in hybrid aspen. Time to bud break relative to WT controls for a RCAR/PYL1oe and TCP18/BRC1oe b plants. Average time taken to bud break ± SEM, with respect to WT considered as 1, is shown with data from 10 plants from each line. Asterisks (*) indicate significant differences (P < 0.01), with respect to WT. Statistical analysis was done using t-test Discussion hybrid aspen or peach DAM genes. Nevertheless, high degree of The timing of bud break in spring is critical for the survival of similarity between SVP, SVL, and DAM suggest that they com- perennial trees growing in temperate and boreal ecosystems as prise a larger sub-family of MADS box genes. Our results indicate premature bud break can lead to fatalities from cold snaps that SVL expression is downregulated in hybrid aspen buds after occurring early in the spring. Conversely, later than optimal bud low temperature treatment. Application of both gain- and loss-of- break reduces the competitiveness of these trees. Here we present function approaches confirmed SVL’s role as an endogenous molecular framework underlying the regulation of bud break by mediator of temperature signals and its function as a negative temperature signal. regulator of bud break. SVL is a member of MADS box family of By screening for mediators of temperature regulation of bud transcription factors that often form homo and heteromeric break, we identified SVL, a MADS box transcription factor. complexes. Loss-of-individual MADS box proteins results in Although SVL shows similarity to previously described DAM perturbation of these complexes leading to various phenotypes genes, it clusters closer to SVP in Arabidopsis and apple than to and this maybe the case in SVL downregulated hybrid aspen 6 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications WT TCP18oe_2 TCP18oe_11 TCP18oe_12 WT RCARoe_10 RCARoe_18 WT SVLRNAi GA2oxoe/SVLRNAi_1 GA2oxoe/SVLRNAi_6 Time to bud break relative to WT Time to bud break relative to WT Time to bud break relative to WT NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE plants as well. Downregulation of SVL described here is similar to downregulation of SVL and TCP18/BRC1 by low temperature. As 3,5 the downregulation of the floral repressor FLC, also a MADS-box FT1 can act as a positive regulator of seasonal growth ,we 33,34 protein, in Arabidopsis during vernalization (promotion of propose that suppression of TCP18/BRC1 by low temperature flowering by prolonged exposure to low temperature) and those would serve to prevent TCP18/BRC1 from antagonizing FT- 35–37 of other DAM genes associated with bud break . In vernali- mediated promotion of bud break. SVL can directly bind to the zation, FLC is silenced by increases in histone H3 lysine tri- FT1 and TCP18/BRC1 promoters, and SVL has opposite effects methylation, resulting in flowering . Similarly, exposure to low on FT1 and TCP18/BRC1 expression (as it does on GA and ABA temperature results in increases in H3k27 trimethylation of DAM pathways). SVL regulation of FT1 described here has similarities loci that have been implicated in bud break in some plants , with the proposed regulation of FT homolog by SVL related DAM highlighting the resemblance between bud break and vernaliza- genes in leafy spurge indicating the conservation of this tion. However, we have not detected any significant change in mechanism . Thus, by downregulating SVL, low temperature H3K27me3 marks at the SVL locus of hybrid aspen following low would concomitantly induce FT1 expression and downregulate temperature treatment (Supplementary Fig. 4). Instead, down- TCP18/BRC1, resulting in a positive feedforward loop that regulation of ABA pathway, a positive regulator of SVL, upon enhances the potential for bud break. exposure to extended low temperature, could underlie down- In contrast with photoperiodically controlled growth cessation, 25,36 regulation of SVL expression. Thus, the expression of SVL during temperature signals control bud break. Although DAM genes bud break is distinct from that shown for FLC in Arabidopsis and EBB1 have been implicated in this process, their roles and during vernalization or other DAM like genes during bud break. modes of action in bud break are not entirely clear and a mole- SVL regulates hormonal pathways that act antagonistically in cular framework underlying bud break has not emerged so far. bud break. SVL directly and indirectly promotes expression of We identified transcription factor SVL and its several targets: genes encoding NCED3 (a key ABA biosynthesis enzyme) and TCP18 and components of the antagonistically acting ABA and RCAR/PYL ABA receptors, respectively. Both of these genes are GA signaling pathways and elucidated their role in bud break. We downregulated in response to low temperature, like SVL, and propose that SVL and its downstream targets form a genetic their response to low temperature is modulated in SVL trans- network underlying the temperature-mediated control of bud genics. Interestingly, while SVL positively regulates ABA pathway, break in hybrid aspen as summarized in the model (Fig. 7). ABA itself promotes SVL expression. Thus ABA and SVL form a According to this model, the extended cold temperature signal re-enforcing loop that acts to delay bud break. In contrast with its down regulates SVL and its targets (e.g. TCP18/BRC1 and positive effects on ABA synthesis and signaling-related genes, we RCAR/PYL) together with simultaneous upregulation of FT1 and obtained clear indications that SVL represses GA pathway. Inter the GA pathway could enhance the potential for bud break. alia, low temperature induces the expression of GA20 oxidase,a The antagonistic roles of ABA and GA in bud break identified key GA biosynthesis gene. The low temperature effect on here are reminiscent of their similar antagonistic actions in GA20 oxidase expression is modulated by SVL since SVL over- control of seed germination; a process inhibited by ABA and expression and silencing respectively weakened and strengthened its induction in response to low temperature. These observations suggested that control of bud break is mediated by SVL acting Low antagonistically on ABA and GA pathways. This hypothesis was temperature supported by the subsequent genetic analysis of bud break in plants in which ABA or GA pathway were modulated. Bud break was delayed in plants overexpressing RCAR/PYL and enhancing GA2 oxidase (which catalyzes degradation of GA) expression ABA suppresses the early bud break phenotype of SVLRNAi plants. Taken together, these results explain why SVL overexpression delays bud break and its downregulation has the opposite effect of promoting early bud break. Thus, extended low temperature SVL promotes bud break by downregulating SVL expression thereby relieving the repressive effect of ABA and promoting the GA GA pathway’s positive effect downstream. NCED3 In Arabidopsis and other plants e.g. pea, signals or events e.g. RCARs FT TCP18 /PYL decapitation, that activate axillary bud outgrowth also induce 16,39 downregulation of TCP18/BRC1 . Moreover, TCP18/BRC1 transcription factor has been demonstrated to act as a negative regulator of axillary bud outgrowth by controlling bud activation 16,40,41 potential . Transcriptional analysis indicated that hybrid aspen homolog of Arabidopsis TCP18/BRC1 was downregulated upon exposure to low temperature, like SVL. Moreover, our data Bud break indicated that TCP18/BRC1 was a direct target of SVL and its expression was altered in SVL transgenics indicating a role as a Fig. 7 Hypothetical model integrating components involved in bud break. negative regulator of bud break, a hypothesis supported by ana- Low temperature reduces ABA levels and suppresses SVL expression, lysis of TCP18/BRC1 transgenics. Although in Arabidopsis, SVP leading to induction of FT1 expression and GA biosynthesis, which has not been implicated in bud dormancy or in regulation of promotes bud break. In the absence of low temperatures, high levels of SVL TCP18/BRC1, our data now reveals a role for TCP18/BRC1 in expression induce NCED3 and RCAR/PYL, thereby maintaining high ABA SVL mediated control of seasonal growth in tree. levels and sensitivity in buds, ensuring that they remain dormant. SVL In Arabidopsis, TCP18/BRC1 has an additional role in subsequently induces TCP18/BRC1, suppressing bud break. Low repressing FT-mediated promotion of flowering in axillary buds temperatures trigger reduction in SVL expression and its suppressive by binding FT . It is noteworthy that the expression of FT1 effects, followed by bud break in hybrid aspen buds is induced simultaneously with the NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y promoted by GA . Moreover, low temperature treatment pro- Plant transformation and screening of transgenic lines. Hybrid aspen was transformed with the vectors pK2GW7- Myc-SVL, SVL-pK7GWIWG2, pK2GW7- motes germination of seeds as well as bud break . Thus, antag- RCAR, and pK2GW7-TCP18 via Agrobacterium-mediated transformation to onistic action between ABA and GA has been harnessed through generate transgenic lines. SVLRNAi lines were used as background to transform evolution as a regulatory module in the control of dormancy and pH2GW7-GA2 oxidase. For screening of transgenic lines leaf samples were taken post-dormancy processes mediated by temperature signals in from each line and used for Protein/RNA isolation, which were further used for western blotting and q-PCR analysis. To check expression of off targets in SVL both seeds and buds that are crucial lifecycle adaptations to downregulated lines (SVLRNAi) expression of selected genes was checked by q- seasonal climatic changes. PCR (Supplementary Fig. 1d). Putative off target genes were selected on basis of The Arabidopsis homologs of SVL and TCP18/BRC1 tran- protein/nucleotide homology. scription factors, SVP and TCP18/BRC1, respectively, are 16,40,44 involved in floral transition and axillary bud outgrowth . ABA treatment of buds. For analysis of ABA response, apices were cut and placed Floral transition is morphogenetically distinct from phenological in MS medium with or without 50 mM ABA and were sampled and used for traits, such as apical bud dormancy and bud break. However, analysis . there are commonalities in environmental cues that regulate these developmental events which may explain why similar genetic RNA isolation and quantitative real-time PCR analysis. Total RNA was extracted from samples of tissues, all taken at 2 pm, using a Sigma Spectrum Total pathways appear to have been harnessed in the adaptive reg- 35,45 Plant RNA isolation kit. Portions (10 µg) of total RNA were treated with RNase- ulatory machinery involved . Our results provide an example Free DNase (Qiagen) and cleaned using an RNeasy® Mini Kit (Qiagen). One of the utilization of flowering regulators in phenological events by micrograms of the RNA from each sample was used to generate cDNA using an external cues. We have previously shown that tree orthologs of iScript cDNA synthesis kit (BioRad). Selected (UBQ-like) reference genes were validated using GeNorm Software. qRT–PCR analyses were carried out with a APETALA1 (AP1) are mediators of photoperiodic control of 5 Roche LightCycler 480 II instrument and relative expression values were calculated seasonal growth and show here that SVL-TCP18/BRC1 plays a using the ΔCt method . A complete list of primers used in the RT–PCR analysis is similar role in temperature control of bud break. Notably, in presented in Supplementary Table 1. addition to vegetative cycles, trees undergo floral transition and floral buds are also subject to dormancy and bud break. Thus, the Chromatin immunoprecipitation assays. The chromatin immunoprecipitation use of signaling components homologous to regulators of floral (ChIP) assays were carried out as previously described with some 50,51 transition in bud break control may allow perennials to integrate modifications . Briefly, apices from actively growing hybrid aspen plants were collected and immersed in cross-linking buffer containing 1% formaldehyde and the two processes, flowering and bud break, in floral buds (when kept under vacuum for 20 min, and then glycine was added to a final concentration trees eventually acquire flowering competence at maturity) by of 0.125 M to stop the cross-linking. Cross-linked samples were rinsed 3–4 times using common signaling components, a possibility that warrants with water, frozen in liquid nitrogen and stored at −80 °C. Tissue samples (c.a. 1 g) further analysis. were ground into fine powder and suspended in precooled nuclei isolation buffer, gently vortex-mixed to visual homogeneity then filtered through two layers of Miracloth. The homogenized, filtered mixtures were then centrifuged and the Methods pellets obtained were re-suspended in nuclei lysis buffer. Chromatin was sheared to Plant material and growth conditions. Hybrid aspen (Populus tremula x tre- about 0.3–0.5 kb fragments by sonication (Bioruptor UCD-300, Diagenode). After muloides) clone T89 (wild type/WT) and the transgenic plants described below sonication, the samples were centrifuged again to remove cell debris and each were cultivated in half-strength MS medium (Duchefa) under sterile conditions for supernatant was transferred to a new tube (after retaining 10% of each sonicated 5 weeks then transferred to soil and grown for another 4 weeks in the greenhouse sample used as Input DNA control in the Q-PCR analyses. After centrifugation, the (16 h photoperiods, 22 °C and 66% relative humidity). Further plants were grown supernatant was precleared with 40 µl protein A-magnetic beads for 60 min at 4 °C in growth chambers and initially grown under long day (LD) conditions (16 h, 20 ° with gentle agitation and shaking. Fifteen micrograms of anti-MYC monoclonal C light/8 h, 15 °C cycles) for 1 week for acclimatization and subsequently under antibody (Abcam, Cambridge, UK, Cat no. ab32; GR255064) was added to each short day (SD) conditions (8 h, 20 °C light/16 h, 15 °C dark cycles) for 10 weeks. supernatant and the resulting mixtures were further incubated overnight at 4 °C. Responses to SDs were determined by monitoring bud set and plant growth. After Protein A-magnetic beads (Dynabeads, Invitrogen) were then added again and 10 weeks of SDs, plants were exposed to low temperatures (4 °C) for 5 weeks to incubation was continued for 2 h. The magnetic beads were washed two times each break dormancy and subsequently to the warm LD conditions (LD/WT). Bud with low salt buffer, high salt buffer, LiCl buffer, and TE buffer. The immuno- break was scored when bud swelling and emergence of green leaves were observed. complexes were collected from beads with 250 µl of elution buffer and incubated at Apex samples were taken for expression analysis: after plants had ceased growth 65 °C for 20 min with agitation. 0.3 M NaCl was added to each tube (and Input and developed dormancy, i.e. after 10 weeks of short days (10WSD) and after; both DNA control) and cross-linking was reversed by incubation at 65 °C overnight. 2 weeks (2WC) and 5 weeks (5WC) of exposure to low temperature (4 °C) to Residual protein was degraded by incubation with 20 mg of Proteinase K in 10 mM induce dormancy release; and followed by two weeks after the transfer to long day/ EDTA and 40 mM Tris-HCl, pH 8.0, at 45 °C for 1 h. After proteinase treatment warm temperature conditions (2WLD). Pictures of apices were taken using a precipitated DNA was purified using a ChIP DNA clean and concentrator kit Canon EOS digital camera to monitor bud burst. according to the manufacturer’s protocol (Zymo Research Corp.). Both immuno- precipitated and input DNA were analyzed by real-time PCR using a Light Cycler instrument (Roche Applied Science). All buffers used were prepared following Generation of plasmid constructs. Full-length Myc-SVL (containing 3X Myc previous report . sequence), RCAR/PYL, TCP18/BRC1, and GA2-oxidase cDNAs were amplified using cDNA prepared from mRNA extracted from hybrid aspen apices as tem- ChIP-seq experiment. For the ChIP-seq experiment the apical buds of three plates and primers listed in Supplementary Table 1. SVL, RCAR/PYL, and TCP18/ biological replicates were collected from WT plants after 10 weeks in SD (10WSD) BRC1 cDNAs were cloned into the pENTR/D-TOPO donor vector (Invitrogen) 46 and after an additional 4 week cold (4WC) treatment. ChIP assays were carried and transferred into the pK2GW7/pH2GW7 plant transformation vectors , which described above. Anti-Trimethyl-Histone H3 (LysK27) (Millipore, Cat No. #07- contains a CaMV3S promoter to generate plasmids designated pK2GW7-Myc- 449) and Anti-Histone H3 (Abcam, Cat No. ab1791) antibodies were used for SVL, pK2GW7-RCAR, pK2GW7-TCP18, and pH2GW7-GA2 oxidase, respectively, chromatin immunoprecipitation. Ovation Ultralow IL Multiplex System I (Part No. which were subsequently transformed into Agrobacterium strain 0304, NuGEN) was used to generate the sequencing libraries according to the GV3101pmp90RK . To generate a SVL-RNAi construct, a 156 bp fragment was product instructions. Pair end sequencing was done by BGI-Tech. Sequencing amplified using primers listed in Supplementary table and full-length SVL cDNA as reads were processed following the guidelines described at http://www.epigenesys. template. The amplified fragment was cloned into pENTR/D-TOPO then trans- eu/en/protocols/bio-informatics/1283-guidelines-for-rna-seq-data-analysis. Briefly, ferred into the plant transformation vector pK7GWIWG2 (I) containing a reads quality was first assessed using FastQC (http://www.bioinformatics. CaMV3S promoter to generate a SVL-pK7GWIWG2 (I) construct. ABA insensitive babraham.ac.uk/projects/fastqc/), v0.11.4. Reads mapping to ribosomal RNA transgenic plants (abi1-1) developed earlier were used in the study. (rRNA) were quantified and filtered using SortMeRNA (v2.1;settings --log --paired_in --fastx--sam --num_alignments 1) using the rRNA sequences provided Phylogenetic analysis. Protein sequences collected by the best BLAST match for with SortMeRNA. Reads were then filtered to remove adapters and trimmed for SVP from the Popgenie database (http://popgenie.org) or NCBI were aligned and a quality using Trimmomatic (v0.36; settings TruSeq3-PE-2.fa:2:30:10 SLI- phylogenetic tree developed using MEGA7 . The evolutionary history was inferred DINGWINDOW:5:20 MINLEN:50). After every filtering step, FastQC was run using the neighbor-joining method with 1000 bootstrap replicates. The evolu- again to ensure that no technical artefacts were introduced. Reads were then tionary distances were computed using the Poisson correction method with units of mapped to the hybrid aspen genome (Populus tremula × tremuloides, clone T89) the number of amino acid substitutions per site. using STAR with settings --outQSconversionAdd −31 --outReadsUnmapped 8 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE Fastx. 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V., Lippman, Z., Martienssen, R. & Colot, V. Profiling histone modification patterns in plants using genomic tiling microarrays. Nat. Methods 2, 213–218 (2005). 52. Kopylova, E., Noe, L. & Touzet, H. SortMeRNA: fast and accurate filtering of Open Access This article is licensed under a Creative Commons ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 Attribution 4.0 International License, which permits use, sharing, (2012). adaptation, distribution and reproduction in any medium or format, as long as you give 53. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for appropriate credit to the original author(s) and the source, provide a link to the Creative Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). Commons license, and indicate if changes were made. The images or other third party 54. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, material in this article are included in the article’s Creative Commons license, unless 15–21 (2013). indicated otherwise in a credit line to the material. If material is not included in the 55. Li, H. Aligning sequence reads, clone sequences and assembly contigs with article’s Creative Commons license and your intended use is not permitted by statutory BWA-MEM. Preprint at http://arXiv:1303.3997 (2013). regulation or exceeds the permitted use, you will need to obtain permission directly from 56. Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, the copyright holder. To view a copy of this license, visit http://creativecommons.org/ R137–R137 (2008). licenses/by/4.0/. 57. R Core Team. R: A language and environment for statistical computing. 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A genetic network mediating the control of bud break in hybrid aspen

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Abstract

ARTICLE DOI: 10.1038/s41467-018-06696-y OPEN A genetic network mediating the control of bud break in hybrid aspen 1 1 1,2 1 1,3 1 Rajesh Kumar Singh , Jay P. Maurya , Abdul Azeez , Pal Miskolczi , Szymon Tylewicz , Katja Stojkovič , 1 2 1 Nicolas Delhomme , Victor Busov & Rishikesh P. Bhalerao In boreal and temperate ecosystems, temperature signal regulates the reactivation of growth (bud break) in perennials in the spring. Molecular basis of temperature-mediated control of bud break is poorly understood. Here we identify a genetic network mediating the control of bud break in hybrid aspen. The key components of this network are transcription factor SHORT VEGETATIVE PHASE-LIKE (SVL), closely related to Arabidopsis floral repressor SHORT VEGETATIVE PHASE, and its downstream target TCP18, a tree homolog of a branching reg- ulator in Arabidopsis. SVL and TCP18 are downregulated by low temperature. Genetic evi- dence demonstrates their role as negative regulators of bud break. SVL mediates bud break by antagonistically acting on gibberellic acid (GA) and abscisic acid (ABA) pathways, which function as positive and negative regulators of bud break, respectively. Thus, our results reveal the mechanistic basis for temperature-cued seasonal control of a key phenological event in perennial plants. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 87 Umeå, Sweden. 2 3 School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931, USA. Department of Plant and Microbial Biology, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland. Correspondence and requests for materials should be addressed to R.P.B. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 1 1234567890():,; ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y he survival of perennial plants in boreal and temperate dormancy as mediators of temperature controlled bud break. We ecosystems is dependent on seasonally synchronized show that SVL is a positive regulator of TCP18/BRC1 and toge- Tannual growth cycles. In long-lived trees, growth ceases ther they form a temperature-responsive transcriptional module and dormancy is established prior to the onset of winter. Dor- that mediates control of bud break. We demonstrate that com- mancy is maintained during the winter and gradual release from ponents of antagonistic ABA and GA hormonal pathways are dormancy is followed by reactivation of growth in the spring . downstream targets of SVL in bud break regulation. Thus, our Photoperiod and temperature are the main environmental cues results reveal a temperature responsive genetic network mediating regulating the seasonal synchronization of the key developmental bud break, a major phenological process in perennial plants. transitions in the annual growth cycle . The timing of growth cessation in the autumn, governed primarily by photoperiod Results signals, is mediated by the FLOWERING LOCUS T/CONSTANS Transcription factor SVL participates in bud break control.We 2,3 (FT/CO) module in the model plant hybrid aspen . Interaction screened transgenic hybrid aspen lines overexpressing transcrip- of FT2 with FD-LIKE1 (FDL1) promotes growth under long tion factors to identify genes involved in bud break. The results photoperiods by ensuring high expression of the transcription showed that relative to wild-type hybrid aspen plants, bud break factor LIKE-AP1 (LAP1) . LAP1 is a positive regulator of was significantly delayed in transgenic lines overexpressing a AINTEGUMENTA-LIKE1 (AIL1), a transcriptional regulator of poplar MADS box named SVL (SVP-LIKE), highly similar to 5,6 key cell cycle genes, including D-type cyclins . When a reduc- Arabidopsis floral repressor SVP (Fig. 1a–d, i, Supplementary tion in day length below a critical threshold permitting growth Fig. 1a). Sequence analysis indicated that SVL is similar to Ara- (short days/SD) is sensed, downregulation of FT2 results in bidopsis SVP and DAM genes described in several tree growth cessation and formation of a bud structure at the apex . 25,28 species . However, phylogenetic analysis shows that hybrid Buds enclose arrested leaf primordia and shoot apical meristems aspen SVL is more similar to SVP from Arabidopsis and apple within protective bud scales . (Supplementary Fig. 2) than to poplar MADS-box genes 27–29 After growth cessation, continuation of short days induce 29 that are homologs of DAM genes in peach . To confirm the role dormancy in the buds before winter . Recently, plant hormone of SVL in bud break, transgenic hybrid aspen plants with reduced abscisic acid (ABA) has been shown to mediate photoperiodic SVL expression (SVLRNAi) were also generated and scored for 7,9,10 control of bud dormancy . Once dormancy is established, bud break (Supplementary Fig. 1b). In contrast with SVL over- 11,12 buds no longer respond to growth-promotive signals . Hence expressers (SVLoe), SVLRNAi lines showed early bud break dormancy must first be released before growth can be reactivated compared to the control wild-type hybrid aspen plants (Fig. 1e–h, in the buds. Dormancy release is induced by prolonged exposure j). As SVLoe and SVLRNAi react similarly under short days, to low temperature (LT) following which growth can be reacti- (Supplementary Fig. 3a and b), results indicate that SVL, has a vated as visibly manifested by bud break, i.e., emergence of new negative role in bud break in hybrid aspen. 11,13–16 leaves from the buds . The mechanisms underlying dormancy release and bud break LT and ABA antagonistically modulate SVL expression. Our are intimately linked but the underlying molecular mechanisms data indicated that bud break was affected in SVL transgenics are not well understood. However, physiological and tran- being delayed in SVL overexpressers and occurring earlier in SVL scriptomic approaches have noted that increase in expression of downregulated plants (Fig. 1) indicating that SVL mediates in bud gibberellic acid (GA) biosynthesis related genes and FT2 homolog break, a process regulated by temperature signal. Therefore, we FT1, that are potent growth promoters and simultaneous down- investigated the temperature-responsiveness of SVL expression. regulation of the components of ABA pathway coincides tem- 17–19 SVL expression was significantly downregulated by exposure to porally with dormancy release and transition to bud break . low temperature and remained lower than its levels in dormant Moreover, exogenous applications of GA and ABA respectively 19,20 buds prior to low temperature treatment (Fig. 1k). SVL expres- promote and delay bud break . However, the endogenous role sion marginally increased somewhat following buds’ exposure to of these components in bud break remains uncharacterized so far. warm temperatures, but nevertheless remained lower than in the Earlier studies have also drawn parallels between vernalization, dormant buds. Thus, SVL expression is negatively regulated by flowering promotion by low temperature in Arabidopsis and bud 21,22 low temperature. It has been reported that upon low temperature, break . For example, low temperature induces changes in the there is an increase in the repressive marks like histone H3 lysine expression and chromatin status of DORMANCY ASSOCIATED 23,24 27 trimethylation (H3K27me3) in the promoters of SVL like MADS-BOX (DAM) genes . Interestingly, overexpression of 23,24 DAM genes in peach and pear . However, we did not observe DAM genes can delay bud break . While informative, these any significant increase of H3K27me3 marks at the SVL locus primarily based on gain-of-function approaches have not upon low temperature treatment (Supplementary Fig. 4) indi- addressed the endogenous roles of DAM genes, and the cating that in contrast with other DAM genes, SVL suppression is mechanism(s) whereby they mediate control of bud break. In not due to increase in H3K27 trimethylation at the SVL locus. addition, an AP2-family transcription factor designated EARLY Like temperature signal, ABA has been implicated in bud break BUD BREAK 1 (EBB1) has been identified by activation tagging in with exogenous application of ABA delaying bud break , hybrid poplar, which is clearly relevant as EBB1 overexpression phenocopying SVL overexpresseors. Therefore we investigated and downregulation results in early and late bud break, respec- 26 whether ABA, mediates in the control of SVL expression. ABA tively . Nevertheless, whether EBB1 acts directly in temperature application induces SVL expression (Supplementary Fig. 5) and control of bud break and its downstream targets remain moreover in the buds of transgenic hybrid aspen plants with uncharacterised. Thus, the molecular basis for translation of reduced response to ABA, SVL expression is significantly reduced temperature signals into promotion of bud break remains poorly (Supplementary Fig. 6). Thus, ABA in contrast with low understood. temperature acts positively in control of SVL expression. Here we report on identification of transcription factors, SHORT VEGETATIVE PHASE-LIKE (SVL) similar to Arabi- dopsis SHORT VEGETATIVE PHASE (SVP) a flowering FT1 and GA20 oxidase genes are negatively regulated by SVL. time regulator and TEOSINTE BRANCHED1, CYCLOIDEA, Low temperature enhances the expression of FT1 and compo- PCF/BRANCHED1 (TCP18/BRC1) , involved in axillary bud nents of GA biosynthesis e.g. GA20 oxidases in buds mirroring 2 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE a b ef WT SVLoe_2 WT SVLRNAi_2 cd gh SVLoe_4 SVLoe_8 SVLRNAi_10 SVLRNAi_12 ij k 1.5 1.2 2.0 * 1.0 1.5 A 1.0 0.8 1.0 0.6 0.5 0.5 0.4 0.0 0.2 0.0 10WSD 2WC 5WC 2WLD Fig. 1 Delayed and early bud break in plants over- and under-expressing SVL. a–d Bud break is earlier in a WT plants than in three independent SVL overexpressing lines, designated SVLoe2 b, SVLoe4 c, and SVLoe8 d. e–h In contrast, bud break is later in WT plants h than in independent SVLRNAi lines 2 f,10 g, and 12 h. I, j Time to bud break relative to wild type controls in SVLoe i and SVLRNAi j lines. Average time taken to bud break ± standard error mean (SEM), with respect to WT considered as 1, is shown with data from 10 plants from each line. Asterisks (*) indicate significant differences (P < 0.05) with respect to WT. k Low temperatures suppress SVL expression. Relative expression of SVL after 10 weeks of SD, followed by 2 and 5 weeks of low temperature (2WC, 5WC at +4 °C) and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). SVL expression from three independent biological replicates ± SEM is shown relative to the reference gene UBQ with 10WSD time point set to 1. Different letters A–D over the bars indicate significant differences at P < 0.001. Statistical analysis was done using one way ANNOVA implying Dunnett’s/Tukey’s multiple comparison test 6,19 the downregulation of SVL . Given the growth promotive role buds after exposure to low temperatures. In contrast, dormant 5,9,19 of FT and GA’s , we investigated whether SVL could parti- buds of SVLoe plants expressed NCED3 at a higher level and did cipate in bud break by affecting FT1 and/or GA pathway. In not downregulate it, relative to WT buds, in response to low agreement with previous findings, low temperature-induced temperature (Fig. 3a). Whereas in SVLRNAi buds, NCED3 expression of FT1 and GA20 oxidases in WT buds. In contrast, expression was lower and downregulated to a higher extent by induction of FT1 and GA20 oxidases was reduced in SVLoe buds low temperature than in the WT buds (Fig. 3b). Additionally, the (Fig. 2a) and, conversely, FT1 and GA20 oxidases induction was expression of genes encoding RCAR/PYL1 and RCAR/PYL2, enhanced in SVLRNAi plants relative to the wild type, after highly similar to ABA receptors, which activate downstream exposure to low temperature (Fig. 2b). These findings suggest a signaling responses after binding ABA, is consistently higher in negative role for SVL in the induction of FT1 and GA20 oxidases SVL overexpressers than in wild-type buds, but lower in SVL expression by low temperature in hybrid aspen buds. downregulated plants (Fig. 3a, b). Thus, ABA upregulates SVL, and in turn, SVL positively regulates ABA biosynthesis and signaling-related genes forming a feedback loop. SVL modulates ABA biosynthesis and signaling gene expres- sion. ABA induces SVL expression as shown before and exo- genous application of ABA delays bud break . Therefore, we SVL acts upstream of the TCP18/BRC1 transcription factor. investigated the transcriptional regulation of ABA biosynthesis TCP18/BRC1 a transcription factor that regulates axillary bud and response machinery during bud break in the wild type and outgrowth, has been recently shown to control ABA signaling in the SVL transgenic plants. The expression of NCED3, which Arabidopsis . The expression of the hybrid aspen gene homo- encodes a key enzyme in ABA biosynthesis, decreased in WT logous to TCP18/BRC1 is downregulated following exposure to NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 3 WT SVLoe_2 SVLoe_4 SVLoe_8 WT SVLRNAi_2 SVLRNAi_10 SVLRNAi_12 Time to bud break relative to WT Time to bud break relative to WT SVL relative expression ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y 8 10 WT WT WT ** ** ** SVLoe SVLoe SVLoe ** ** ** ** 0 0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD b 10 40 WT ** WT WT SVLRNAi SVLRNAi SVLRNAi ** *** *** ** ** 0 0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD Fig. 2 SVL negatively regulates expression of FT1 and GA20 oxidases during dormancy release and bud break. Expression patterns of genes encoding FT1, GA20 oxidases (GA20ox_1 and GA20ox_2) in apices of a WT and SVLoe and b WT and SVLRNAi after 10 weeks of SDs(10WSD), at the time of dormancy release (i.e. after 2weeks of LT, 2WC), and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). Expression values of the cited genes shown are averages for three biological replicates ± SEM, relative to the reference gene UBQ and with 10WSD time point set to 1. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using multiple t-tests low temperature (Supplementary Fig. 7). Therefore, we investi- site for MADS-box transcription factors. In contrast, no evidence gated whether hybrid aspen TCP18/BRC1 homolog could be a of SVL binding to promoters of GA20 oxidase (1 and 2) or RCAR/ target of SVL, by examining TCP18/BRC1 expression in SVL PYL genes was detected indicating that SVL indirectly affects the transgenics. TCP18/BRC1 expression was downregulated by low expression of these genes (Supplementary Fig. 8). temperature treatment in WT buds, whereas this downregulation was attenuated in SVLoe buds and TCP18/BRC1 expression was Reduction of GA represses early bud break in SVLRNAi lines. consistently higher in SVL overexpressing transgenic plants than Early bud break in SVLRNAi plants is correlated with enhanced in WT plants at all-time points (Fig. 3a). Conversely, TCP18/ expression of GA biosynthesis in these plants relative to wild type BRC1 expression was lower in SVL downregulated (SVLRNAi) suggesting that the GA pathway could be a downstream target of lines, suggesting that TCP18/BRC1 could be a downstream target SVL in bud break regulation. We tested this hypothesis by gen- of SVL (Fig. 3b). erating SVLRNAi plants overexpressing a poplar GA2 oxidase (Supplementary Fig. 9) with known ability to reduce GA levels . We then compared the bud break of SVLRNAi with SVLRNAi SVL regulates FT1, NCED3, and TCP18/BRC1 expression expressing GA2 oxidase (Fig. 5). Our data show that GA2 oxidase directly. The presented results showed that SVL mediates in expression repressed the early bud break phenotype of SVLRNAi temperature control of bud break and expression of growth transgenic lines. These results along with gene expression data promoters and repressors, including FT1, GA20 oxidases, NCED3, strongly support that GA pathway is a downstream target of SVL RCAR/PYL, and TCP18/BRC1.As SVL is a transcriptional reg- in temperature controlled bud break. ulator, we investigated which of these genes are direct down- stream targets of SVL by chromatin immunoprecipitation (ChIP)-RT–PCR experiments on DNA isolated from shoot apices Overexpression of RCAR/PYL1 and TCP18/BRC1 delays bud of transgenic hybrid aspen plants expressing Myc-tagged SVL break. The expression of hybrid aspen TCP18/BRC1 and RCAR/ (Myc-SVL) and WT control. We found clear evidence for binding PYL ABA receptors is affected in SVL transgenics and like SVL, of SVL in the promoters of FT1, NCED3, and TCP18/BRC1 TCP18/BRC1 and RCAR/PYLs are downregulated in the buds (Fig. 4) all of which contain a CArG motif known to be a target after exposure to low temperature. Therefore we hypothesized 4 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications FT1 relative expression FT1 relative expression GA20ox_1 relative expression GA20ox_1 relative expression GA20ox_2 relative expression GA20ox_2 relative expression NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE 2.0 2.5 2.0 WT SVLoe WT SVLoe WT SVLoe WT SVLoe *** *** ** *** ** ** 2.0 1.5 1.5 *** *** 1.5 ** 1.0 1.0 6 ** 1.0 *** 0.5 0.5 0.5 0.0 0.0 0 0.0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD 1.2 2.0 WT SVLRNAi WT SVLRNAi WT SVLRNAi WT SVLRNAi 1.5 1.2 ** ** *** *** 1.0 1.0 1.5 ** 0.8 1.0 ** 0.8 ** *** 0.6 1.0 ** 0.6 ** 0.4 0.5 0.4 0.5 *** 0.2 0.2 0.0 0.0 0.0 0.0 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD 10WSD 2WC 2WLD Fig. 3 SVL positively regulates expression of the ABA biosynthesis gene NCED3, ABA receptors (RCAR/PYLs), and TCP18/BRC1-like transcription factors during dormancy release and bud break. Expression patterns of NCED3, RCAR/PYL1, RCAR/PYL2, and TCP18 in apices of a WT and SVLoe and b WT and SVLRNAi after 10 weeks of SDs (10WSD), at the time of dormancy release (i.e. after 2weeks of LT, 2WC), and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). Expression values of the cited genes shown are averages for three biological replicates ± SEM, relative to the reference gene UBQ and with 10WSD time point set to 1. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using multiple t-test ATG a F1 R1 F1 R1 F2 R2 F1 R1 F2 R2 ATG F2 R2 ATG 3 kb 2 kb 1 kb 3 kb 2 kb 1 kb 3 kb 2 kb 1 kb 3 10 8 *** *** *** 0 0 0 WT Myc-SVL WT Myc-SVL WT Myc-SVL Fig. 4 SVL binds to FT1, NCED3, and TCP promoters in vivo in chromatin immunoprecipitation (ChIP) assays. a Diagrammatic representation of FT1, NCED3, and TCP18/BRC1 promoters showing the CArG motif and their positions within a 3 kb region. F1–R1 indicates positions of DNA fragments used to assess DNA–protein interactions in ChIP assays, and F2–R2 indicates positions of DNA fragments with no CArG motif used as negative controls in the assays. b Enrichment of the DNA fragments containing the CArG motif quantified by ChIP-q-PCR. Presented values were first normalized by their respective input values, then fold enrichments in WT and Myc-SVL plants relative to negative controls were calculated. Bars show an average values from three independent biological replicates ± SEM. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using t-test that bud break involves the downregulation of TCP18/BRC1 and (Supplementary Fig. 10). In both, RCAR/PYL1 and TCP18/BRC1 RCAR/PYLs. We tested this hypothesis by generating transgenic overexpressers, bud break was significantly delayed compared to plants that would maintain high levels of RCAR/PYL1 and wild type control plants (Fig. 6) indicating that RCAR/PYL1 and TCP18/BRC1 then investigated bud break in these genotypes TCP18/BRC1 have repressive roles in bud break regulation. NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 5 NCED3 relative expression NCED3 relative expression FT1 promoter fold enrichment/control RCAR1/PYL1 relative expression RCAR1/PYL1 relative expression NCED3 promoter fold enrichment/control RCAR2/PYL2 relative expression RCAR2/PYL2 relative expression TCP18/BRC1 promoter fold enrichment/control TCP18/BRC1 relative expression TCP18/BRC1 relative expression ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y 1.5 ab C C 1.0 0.5 cd 0.0 Fig. 5 Overexpression of GA2 oxidase represses early bud break in SVLRNAi lines. a–d Bud break is earlier in a WT and b SVLRNAi plants than in c, d two independent lines overexpressing GA2 oxidase in a SVLRNAi background, designated GA2oxoe/SVLRNAi_1 and 6, respectively. e Time to bud break relative to WT controls for SVLRNAi plants, and lines overexpressing GA2 oxidase in a SVLRNAi background. Average time taken to bud break ± SEM, with respect to WT considered as 1, is shown with data from 10 plants from each line. Different letters A–C over the bars indicate significant differences at P < 0.001. Statistical analysis was done using one way ANNOVA implying Tukey’s multiple comparison test ab 2.5 2.0 2.0 * 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Fig. 6 RCAR/PYL1 or TCP18/BRC1 overexpression delays bud break in hybrid aspen. Time to bud break relative to WT controls for a RCAR/PYL1oe and TCP18/BRC1oe b plants. Average time taken to bud break ± SEM, with respect to WT considered as 1, is shown with data from 10 plants from each line. Asterisks (*) indicate significant differences (P < 0.01), with respect to WT. Statistical analysis was done using t-test Discussion hybrid aspen or peach DAM genes. Nevertheless, high degree of The timing of bud break in spring is critical for the survival of similarity between SVP, SVL, and DAM suggest that they com- perennial trees growing in temperate and boreal ecosystems as prise a larger sub-family of MADS box genes. Our results indicate premature bud break can lead to fatalities from cold snaps that SVL expression is downregulated in hybrid aspen buds after occurring early in the spring. Conversely, later than optimal bud low temperature treatment. Application of both gain- and loss-of- break reduces the competitiveness of these trees. Here we present function approaches confirmed SVL’s role as an endogenous molecular framework underlying the regulation of bud break by mediator of temperature signals and its function as a negative temperature signal. regulator of bud break. SVL is a member of MADS box family of By screening for mediators of temperature regulation of bud transcription factors that often form homo and heteromeric break, we identified SVL, a MADS box transcription factor. complexes. Loss-of-individual MADS box proteins results in Although SVL shows similarity to previously described DAM perturbation of these complexes leading to various phenotypes genes, it clusters closer to SVP in Arabidopsis and apple than to and this maybe the case in SVL downregulated hybrid aspen 6 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications WT TCP18oe_2 TCP18oe_11 TCP18oe_12 WT RCARoe_10 RCARoe_18 WT SVLRNAi GA2oxoe/SVLRNAi_1 GA2oxoe/SVLRNAi_6 Time to bud break relative to WT Time to bud break relative to WT Time to bud break relative to WT NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE plants as well. Downregulation of SVL described here is similar to downregulation of SVL and TCP18/BRC1 by low temperature. As 3,5 the downregulation of the floral repressor FLC, also a MADS-box FT1 can act as a positive regulator of seasonal growth ,we 33,34 protein, in Arabidopsis during vernalization (promotion of propose that suppression of TCP18/BRC1 by low temperature flowering by prolonged exposure to low temperature) and those would serve to prevent TCP18/BRC1 from antagonizing FT- 35–37 of other DAM genes associated with bud break . In vernali- mediated promotion of bud break. SVL can directly bind to the zation, FLC is silenced by increases in histone H3 lysine tri- FT1 and TCP18/BRC1 promoters, and SVL has opposite effects methylation, resulting in flowering . Similarly, exposure to low on FT1 and TCP18/BRC1 expression (as it does on GA and ABA temperature results in increases in H3k27 trimethylation of DAM pathways). SVL regulation of FT1 described here has similarities loci that have been implicated in bud break in some plants , with the proposed regulation of FT homolog by SVL related DAM highlighting the resemblance between bud break and vernaliza- genes in leafy spurge indicating the conservation of this tion. However, we have not detected any significant change in mechanism . Thus, by downregulating SVL, low temperature H3K27me3 marks at the SVL locus of hybrid aspen following low would concomitantly induce FT1 expression and downregulate temperature treatment (Supplementary Fig. 4). Instead, down- TCP18/BRC1, resulting in a positive feedforward loop that regulation of ABA pathway, a positive regulator of SVL, upon enhances the potential for bud break. exposure to extended low temperature, could underlie down- In contrast with photoperiodically controlled growth cessation, 25,36 regulation of SVL expression. Thus, the expression of SVL during temperature signals control bud break. Although DAM genes bud break is distinct from that shown for FLC in Arabidopsis and EBB1 have been implicated in this process, their roles and during vernalization or other DAM like genes during bud break. modes of action in bud break are not entirely clear and a mole- SVL regulates hormonal pathways that act antagonistically in cular framework underlying bud break has not emerged so far. bud break. SVL directly and indirectly promotes expression of We identified transcription factor SVL and its several targets: genes encoding NCED3 (a key ABA biosynthesis enzyme) and TCP18 and components of the antagonistically acting ABA and RCAR/PYL ABA receptors, respectively. Both of these genes are GA signaling pathways and elucidated their role in bud break. We downregulated in response to low temperature, like SVL, and propose that SVL and its downstream targets form a genetic their response to low temperature is modulated in SVL trans- network underlying the temperature-mediated control of bud genics. Interestingly, while SVL positively regulates ABA pathway, break in hybrid aspen as summarized in the model (Fig. 7). ABA itself promotes SVL expression. Thus ABA and SVL form a According to this model, the extended cold temperature signal re-enforcing loop that acts to delay bud break. In contrast with its down regulates SVL and its targets (e.g. TCP18/BRC1 and positive effects on ABA synthesis and signaling-related genes, we RCAR/PYL) together with simultaneous upregulation of FT1 and obtained clear indications that SVL represses GA pathway. Inter the GA pathway could enhance the potential for bud break. alia, low temperature induces the expression of GA20 oxidase,a The antagonistic roles of ABA and GA in bud break identified key GA biosynthesis gene. The low temperature effect on here are reminiscent of their similar antagonistic actions in GA20 oxidase expression is modulated by SVL since SVL over- control of seed germination; a process inhibited by ABA and expression and silencing respectively weakened and strengthened its induction in response to low temperature. These observations suggested that control of bud break is mediated by SVL acting Low antagonistically on ABA and GA pathways. This hypothesis was temperature supported by the subsequent genetic analysis of bud break in plants in which ABA or GA pathway were modulated. Bud break was delayed in plants overexpressing RCAR/PYL and enhancing GA2 oxidase (which catalyzes degradation of GA) expression ABA suppresses the early bud break phenotype of SVLRNAi plants. Taken together, these results explain why SVL overexpression delays bud break and its downregulation has the opposite effect of promoting early bud break. Thus, extended low temperature SVL promotes bud break by downregulating SVL expression thereby relieving the repressive effect of ABA and promoting the GA GA pathway’s positive effect downstream. NCED3 In Arabidopsis and other plants e.g. pea, signals or events e.g. RCARs FT TCP18 /PYL decapitation, that activate axillary bud outgrowth also induce 16,39 downregulation of TCP18/BRC1 . Moreover, TCP18/BRC1 transcription factor has been demonstrated to act as a negative regulator of axillary bud outgrowth by controlling bud activation 16,40,41 potential . Transcriptional analysis indicated that hybrid aspen homolog of Arabidopsis TCP18/BRC1 was downregulated upon exposure to low temperature, like SVL. Moreover, our data Bud break indicated that TCP18/BRC1 was a direct target of SVL and its expression was altered in SVL transgenics indicating a role as a Fig. 7 Hypothetical model integrating components involved in bud break. negative regulator of bud break, a hypothesis supported by ana- Low temperature reduces ABA levels and suppresses SVL expression, lysis of TCP18/BRC1 transgenics. Although in Arabidopsis, SVP leading to induction of FT1 expression and GA biosynthesis, which has not been implicated in bud dormancy or in regulation of promotes bud break. In the absence of low temperatures, high levels of SVL TCP18/BRC1, our data now reveals a role for TCP18/BRC1 in expression induce NCED3 and RCAR/PYL, thereby maintaining high ABA SVL mediated control of seasonal growth in tree. levels and sensitivity in buds, ensuring that they remain dormant. SVL In Arabidopsis, TCP18/BRC1 has an additional role in subsequently induces TCP18/BRC1, suppressing bud break. Low repressing FT-mediated promotion of flowering in axillary buds temperatures trigger reduction in SVL expression and its suppressive by binding FT . It is noteworthy that the expression of FT1 effects, followed by bud break in hybrid aspen buds is induced simultaneously with the NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y promoted by GA . Moreover, low temperature treatment pro- Plant transformation and screening of transgenic lines. Hybrid aspen was transformed with the vectors pK2GW7- Myc-SVL, SVL-pK7GWIWG2, pK2GW7- motes germination of seeds as well as bud break . Thus, antag- RCAR, and pK2GW7-TCP18 via Agrobacterium-mediated transformation to onistic action between ABA and GA has been harnessed through generate transgenic lines. SVLRNAi lines were used as background to transform evolution as a regulatory module in the control of dormancy and pH2GW7-GA2 oxidase. For screening of transgenic lines leaf samples were taken post-dormancy processes mediated by temperature signals in from each line and used for Protein/RNA isolation, which were further used for western blotting and q-PCR analysis. To check expression of off targets in SVL both seeds and buds that are crucial lifecycle adaptations to downregulated lines (SVLRNAi) expression of selected genes was checked by q- seasonal climatic changes. PCR (Supplementary Fig. 1d). Putative off target genes were selected on basis of The Arabidopsis homologs of SVL and TCP18/BRC1 tran- protein/nucleotide homology. scription factors, SVP and TCP18/BRC1, respectively, are 16,40,44 involved in floral transition and axillary bud outgrowth . ABA treatment of buds. For analysis of ABA response, apices were cut and placed Floral transition is morphogenetically distinct from phenological in MS medium with or without 50 mM ABA and were sampled and used for traits, such as apical bud dormancy and bud break. However, analysis . there are commonalities in environmental cues that regulate these developmental events which may explain why similar genetic RNA isolation and quantitative real-time PCR analysis. Total RNA was extracted from samples of tissues, all taken at 2 pm, using a Sigma Spectrum Total pathways appear to have been harnessed in the adaptive reg- 35,45 Plant RNA isolation kit. Portions (10 µg) of total RNA were treated with RNase- ulatory machinery involved . Our results provide an example Free DNase (Qiagen) and cleaned using an RNeasy® Mini Kit (Qiagen). One of the utilization of flowering regulators in phenological events by micrograms of the RNA from each sample was used to generate cDNA using an external cues. We have previously shown that tree orthologs of iScript cDNA synthesis kit (BioRad). Selected (UBQ-like) reference genes were validated using GeNorm Software. qRT–PCR analyses were carried out with a APETALA1 (AP1) are mediators of photoperiodic control of 5 Roche LightCycler 480 II instrument and relative expression values were calculated seasonal growth and show here that SVL-TCP18/BRC1 plays a using the ΔCt method . A complete list of primers used in the RT–PCR analysis is similar role in temperature control of bud break. Notably, in presented in Supplementary Table 1. addition to vegetative cycles, trees undergo floral transition and floral buds are also subject to dormancy and bud break. Thus, the Chromatin immunoprecipitation assays. The chromatin immunoprecipitation use of signaling components homologous to regulators of floral (ChIP) assays were carried out as previously described with some 50,51 transition in bud break control may allow perennials to integrate modifications . Briefly, apices from actively growing hybrid aspen plants were collected and immersed in cross-linking buffer containing 1% formaldehyde and the two processes, flowering and bud break, in floral buds (when kept under vacuum for 20 min, and then glycine was added to a final concentration trees eventually acquire flowering competence at maturity) by of 0.125 M to stop the cross-linking. Cross-linked samples were rinsed 3–4 times using common signaling components, a possibility that warrants with water, frozen in liquid nitrogen and stored at −80 °C. Tissue samples (c.a. 1 g) further analysis. were ground into fine powder and suspended in precooled nuclei isolation buffer, gently vortex-mixed to visual homogeneity then filtered through two layers of Miracloth. The homogenized, filtered mixtures were then centrifuged and the Methods pellets obtained were re-suspended in nuclei lysis buffer. Chromatin was sheared to Plant material and growth conditions. Hybrid aspen (Populus tremula x tre- about 0.3–0.5 kb fragments by sonication (Bioruptor UCD-300, Diagenode). After muloides) clone T89 (wild type/WT) and the transgenic plants described below sonication, the samples were centrifuged again to remove cell debris and each were cultivated in half-strength MS medium (Duchefa) under sterile conditions for supernatant was transferred to a new tube (after retaining 10% of each sonicated 5 weeks then transferred to soil and grown for another 4 weeks in the greenhouse sample used as Input DNA control in the Q-PCR analyses. After centrifugation, the (16 h photoperiods, 22 °C and 66% relative humidity). Further plants were grown supernatant was precleared with 40 µl protein A-magnetic beads for 60 min at 4 °C in growth chambers and initially grown under long day (LD) conditions (16 h, 20 ° with gentle agitation and shaking. Fifteen micrograms of anti-MYC monoclonal C light/8 h, 15 °C cycles) for 1 week for acclimatization and subsequently under antibody (Abcam, Cambridge, UK, Cat no. ab32; GR255064) was added to each short day (SD) conditions (8 h, 20 °C light/16 h, 15 °C dark cycles) for 10 weeks. supernatant and the resulting mixtures were further incubated overnight at 4 °C. Responses to SDs were determined by monitoring bud set and plant growth. After Protein A-magnetic beads (Dynabeads, Invitrogen) were then added again and 10 weeks of SDs, plants were exposed to low temperatures (4 °C) for 5 weeks to incubation was continued for 2 h. The magnetic beads were washed two times each break dormancy and subsequently to the warm LD conditions (LD/WT). Bud with low salt buffer, high salt buffer, LiCl buffer, and TE buffer. The immuno- break was scored when bud swelling and emergence of green leaves were observed. complexes were collected from beads with 250 µl of elution buffer and incubated at Apex samples were taken for expression analysis: after plants had ceased growth 65 °C for 20 min with agitation. 0.3 M NaCl was added to each tube (and Input and developed dormancy, i.e. after 10 weeks of short days (10WSD) and after; both DNA control) and cross-linking was reversed by incubation at 65 °C overnight. 2 weeks (2WC) and 5 weeks (5WC) of exposure to low temperature (4 °C) to Residual protein was degraded by incubation with 20 mg of Proteinase K in 10 mM induce dormancy release; and followed by two weeks after the transfer to long day/ EDTA and 40 mM Tris-HCl, pH 8.0, at 45 °C for 1 h. After proteinase treatment warm temperature conditions (2WLD). Pictures of apices were taken using a precipitated DNA was purified using a ChIP DNA clean and concentrator kit Canon EOS digital camera to monitor bud burst. according to the manufacturer’s protocol (Zymo Research Corp.). Both immuno- precipitated and input DNA were analyzed by real-time PCR using a Light Cycler instrument (Roche Applied Science). All buffers used were prepared following Generation of plasmid constructs. Full-length Myc-SVL (containing 3X Myc previous report . sequence), RCAR/PYL, TCP18/BRC1, and GA2-oxidase cDNAs were amplified using cDNA prepared from mRNA extracted from hybrid aspen apices as tem- ChIP-seq experiment. For the ChIP-seq experiment the apical buds of three plates and primers listed in Supplementary Table 1. SVL, RCAR/PYL, and TCP18/ biological replicates were collected from WT plants after 10 weeks in SD (10WSD) BRC1 cDNAs were cloned into the pENTR/D-TOPO donor vector (Invitrogen) 46 and after an additional 4 week cold (4WC) treatment. ChIP assays were carried and transferred into the pK2GW7/pH2GW7 plant transformation vectors , which described above. Anti-Trimethyl-Histone H3 (LysK27) (Millipore, Cat No. #07- contains a CaMV3S promoter to generate plasmids designated pK2GW7-Myc- 449) and Anti-Histone H3 (Abcam, Cat No. ab1791) antibodies were used for SVL, pK2GW7-RCAR, pK2GW7-TCP18, and pH2GW7-GA2 oxidase, respectively, chromatin immunoprecipitation. Ovation Ultralow IL Multiplex System I (Part No. which were subsequently transformed into Agrobacterium strain 0304, NuGEN) was used to generate the sequencing libraries according to the GV3101pmp90RK . To generate a SVL-RNAi construct, a 156 bp fragment was product instructions. Pair end sequencing was done by BGI-Tech. Sequencing amplified using primers listed in Supplementary table and full-length SVL cDNA as reads were processed following the guidelines described at http://www.epigenesys. template. The amplified fragment was cloned into pENTR/D-TOPO then trans- eu/en/protocols/bio-informatics/1283-guidelines-for-rna-seq-data-analysis. Briefly, ferred into the plant transformation vector pK7GWIWG2 (I) containing a reads quality was first assessed using FastQC (http://www.bioinformatics. CaMV3S promoter to generate a SVL-pK7GWIWG2 (I) construct. ABA insensitive babraham.ac.uk/projects/fastqc/), v0.11.4. Reads mapping to ribosomal RNA transgenic plants (abi1-1) developed earlier were used in the study. (rRNA) were quantified and filtered using SortMeRNA (v2.1;settings --log --paired_in --fastx--sam --num_alignments 1) using the rRNA sequences provided Phylogenetic analysis. Protein sequences collected by the best BLAST match for with SortMeRNA. Reads were then filtered to remove adapters and trimmed for SVP from the Popgenie database (http://popgenie.org) or NCBI were aligned and a quality using Trimmomatic (v0.36; settings TruSeq3-PE-2.fa:2:30:10 SLI- phylogenetic tree developed using MEGA7 . The evolutionary history was inferred DINGWINDOW:5:20 MINLEN:50). After every filtering step, FastQC was run using the neighbor-joining method with 1000 bootstrap replicates. The evolu- again to ensure that no technical artefacts were introduced. Reads were then tionary distances were computed using the Poisson correction method with units of mapped to the hybrid aspen genome (Populus tremula × tremuloides, clone T89) the number of amino acid substitutions per site. using STAR with settings --outQSconversionAdd −31 --outReadsUnmapped 8 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06696-y ARTICLE Fastx. 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V., Lippman, Z., Martienssen, R. & Colot, V. Profiling histone modification patterns in plants using genomic tiling microarrays. Nat. Methods 2, 213–218 (2005). 52. Kopylova, E., Noe, L. & Touzet, H. SortMeRNA: fast and accurate filtering of Open Access This article is licensed under a Creative Commons ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 Attribution 4.0 International License, which permits use, sharing, (2012). adaptation, distribution and reproduction in any medium or format, as long as you give 53. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for appropriate credit to the original author(s) and the source, provide a link to the Creative Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). Commons license, and indicate if changes were made. The images or other third party 54. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, material in this article are included in the article’s Creative Commons license, unless 15–21 (2013). indicated otherwise in a credit line to the material. If material is not included in the 55. Li, H. Aligning sequence reads, clone sequences and assembly contigs with article’s Creative Commons license and your intended use is not permitted by statutory BWA-MEM. Preprint at http://arXiv:1303.3997 (2013). regulation or exceeds the permitted use, you will need to obtain permission directly from 56. Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, the copyright holder. To view a copy of this license, visit http://creativecommons.org/ R137–R137 (2008). licenses/by/4.0/. 57. R Core Team. R: A language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria, 2018. https://www.R- project.org/ URL. © The Author(s) 2018 10 NATURE COMMUNICATIONS | (2018) 9:4173 | DOI: 10.1038/s41467-018-06696-y | www.nature.com/naturecommunications

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