ARTICLE https://doi.org/10.1038/s42003-019-0377-7 OPEN The EC-HDA9 complex rhythmically regulates histone acetylation at the TOC1 promoter in Arabidopsis 1234567890():,; Kyounghee Lee1, Paloma Mas2,3 & Pil Joon Seo 1,4,5 Circadian clocks are conserved time-keeper mechanisms in some prokaryotes and higher eukaryotes. Chromatin modification is emerging as key regulatory mechanism for refining core clock gene expression. Rhythmic changes in histone marks are closely associated to the TIMING OF CAB EXPRESSION 1 (TOC1) Arabidopsis clock gene. However, the chromatin- related modifiers responsible for these marks remain largely unknown. Here, we uncover that the chromatin modifier HISTONE DEACETYLASE 9 (HDA9) and the Evening complex (EC) component EARLY FLOWERING 3 (ELF3) directly interact to regulate the declining phase of TOC1 after its peak expression. We found that HDA9 specifically binds to the TOC1 promoter through the interaction with ELF3. The EC-HDA9 complex promotes H3 deacetylation at the TOC1 locus, contributing to suppressing TOC1 expression during the night, the time of EC function. Therefore, we have identified the mechanism by which the circadian clock inter- twines with chromatin-related components to shape the circadian waveforms of gene expression in Arabidopsis. 1 Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea. 2 Center for Research in Agricultural Genomics (CRAG), Consortium CSIC-IRTA-UAB-UB, Parc de Recerca Universitat Autònoma de Barcelona (UAB), Bellaterra (Cerdanyola del Vallés), Barcelona, Spain. 3 Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain. 4 Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. 5 Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Republic of Korea. Correspondence and requests for materials should be addressed to P.J.S. (email: pjseo1@snu.ac.kr) COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 C ircadian clocks generate biological rhythms with a period rhythmic oscillation in histone acetylation at the core clock of 24 h and control plant growth and development in promoters10,21,24. As pharmacological inhibition of the RPD3/ synchronization with the environmental cycles. Multiple HDA1 family class I HDAC activities25 with TSA (Trichostatin transcriptional feedback loops define the basic architecture of the A) affects the circadian oscillation20,25, we investigated their plant circadian clock. In Arabidopsis, two single-MYB transcrip- possible function within the circadian clock. Among the four tion factors, CIRCADIAN CLOCK–ASSOCIATED 1 (CCA1) and members of this family (HDA6, HDA7, HDA9, and HDA19)26, LATE ELONGATED HYPOCOTYL (LHY), repress transcription we focused on two members not previously studied, HDA7 and of the TOC1/PSEUDO RESPONSE REGULATOR 1 (PRR1) that in HDA9. turn represses CCA1 and LHY expression, constituting the central To that end, we obtained HDA7 and HDA9-deficient mutants, loop1,2. The central loop is interlocked with additional tran- hda7–2 and hda9–1, and analyzed rhythmic expression of the scriptional loops. In the morning, a transcriptional repressing circadian oscillator gene, CCA1. Quantitative real-time RT-PCR wave includes additional members of the PRR family including (RT-qPCR) analysis revealed that the circadian expression of PRR5, PRR7, and PRR9 and CCA1 and LHY3,4. Evening- CCA1 was unaffected in hda7–2 mutant compared with wild type expressed clock components such as TOC1 and the EC (Supplementary Fig. 1). However, the lack of HDA9 led to clear contribute to the repression of the morning-expressed genes5–8. alterations in CCA1 circadian oscillation (Supplementary Figs. 1 Additional transcriptional regulation also underlies circadian and 2). CCA1 phase appeared advanced, which suggests a possible frameworks9–11. Moreover, accumulating evidence suggests that shortening of the circadian period in hda9–1 (Supplementary multiple layers of regulation such as alternative splicing, con- Fig. 1). Consistently, expression of circadian output genes, COLD, trolled protein turnover, and posttranslational modification fur- CIRCADIAN RHYTHM, AND RNA BINDING (CCR2) and ther contribute to a precise rhythmic oscillation12–14. CHLOROPHYLL A/B BINDING–PROTEIN 2 (CAB2), and Chromatin conformation influences the accessibility of tran- several circadian oscillator genes displayed a similar pattern with scriptional regulator(s) to the associated DNA regions. Chemical a period shortening and advanced rhythmic phase (Fig. 1a, b; also modifications at histone N-terminal tails contribute to proper see Supplementary Figs. 3–5). Thus, HDA9 is important for packing of chromatin15. In particular, histone acetylation is proper circadian oscillation. implicated primarily in transcriptional activation16. Histone acetyltransferases (HATs) catalyze the addition of acetyl groups to lysine residues at histones, neutralizing positive charges and thus Binding of HDA9 to the TOC1 promoter. We next aimed to decreasing the affinity of histones to DNA17. This facilitates the identify whether HDA9 regulation of circadian gene expression accessibility of transcriptional regulators and other chromatin modifiers18. In contrast, histone deacetylases (HDACs) antagonize the action of HATs by removing the acetyl groups from histone19. a 50 The first study relating chromatin modification and the Ara- CCR2 bidopsis circadian clock identified the circadian changes in His- 40 Col-0 tone 3 acetylation (H3ac) at the TOC1 promoter. The antagonistic hda9-1 Relative expression action of CCA1 at dawn and another single MYB clock-related 30 transcription factor REVEILLE8/LHY-CCA1-LIKE5 (RVE8/ LCL5) was found to define the hypo-acetylated and hyper- acetylated states of H3 at the TOC1 promoter during the day10. 20 Indeed, CCA1 facilitates a repressive chromatin conformation at dawn either by interfering with HAT accessibility or by recruiting 10 HDAC activity20. During the day, RVE8/LCL5 antagonizes the CCA1 repressive function, favoring H3ac10. These counteracting 0 functions precisely shape the waveform of TOC1 expression10. The rhythmic accumulation of different histone marks was also b 4.5 observed at the promoters of other core clock components, CAB2 including CCA1, LHY, LUX ARRHYTHMO (LUX), and PRRs, 4 and correlates with their transcript accumulation21. 3.5 Relative expression Despite the clear rhythms in histone acetylation at the TOC1 3 locus, the chromatin remodeling factor(s) contributing to the rhythms of histone acetylation and its inner working mechanism 2.5 remain elusive. Here, we report that HDA9, a member of the 2 reduced potassium dependency 3 (RPD3)/HDA1 family class I 1.5 HDAC22,23, is involved in the circadian regulation of TOC1 by suppressing its expression. HDA9 specifically interacts with an 1 EC component, ELF3, and the physical interaction enables HDA9 0.5 to bind to the TOC1 promoter. HDA9 might facilitate a closed 0 chromatin structure at the TOC1 promoter, contributing to its ZT48 52 56 60 64 68 72 76 80 84 88 92 96 100 104 108 112 116 declining phase of expression during the night period. These results provide an insight into how temporal regulation of histone Fig. 1 Mutation of HDA9 alters circadian oscillation. Two-week-old acetylation is achieved in order to stably maintain circadian seedlings grown under neutral day conditions (ND) were transferred to activity. continuous light conditions (LL) at ZT0. Whole seedlings (n > 15) were harvested from ZT48 to ZT116 to analyze transcript accumulation of CCR2 (a) and CAB2 (b). eIF4a was used as the normalization control. Two Results technical replicates were averaged. Bars indicate the standard deviation. Altered circadian oscillation in hda9 mutant plants. Previous The white and gray boxes indicate the subjective day and night, studies have shown that histone deacetylation is important for the respectively 2 COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 ARTICLE occurs through direct binding to a core clock locus. To that end, revealed that HDA9 specifically interacted with ELF3 (Fig. 3a), we performed chromatin immunoprecipitation (ChIP) assays but not with other examined clock proteins (Fig. 3a). using 35 S:MYC-HDA9 transgenic plants. ChIP enrichment was To support the interaction of HDA9 with ELF3 in vivo, we examined by quantitative PCR (qPCR) in the promoter regions of performed bimolecular fluorescent complementation (BiFC) selected clock genes, which include key clock-related cis-elements, analysis using Arabidopsis protoplasts. The HDA9 cDNA such as CCA1-binding site (CBS), evening element (EE), G-box, sequence was fused in-frame to the 5’-end of a gene sequence and/or LUX binding site (LBS) (Fig. 2a)5,7,27,28, in which H3ac encoding the C-terminal half of YFP (cYFP), and the ELF3 gene levels are known to be rhythmically oscillating21. Our results was fused in-frame to the 5’-end of a sequence encoding the N- showed no significant ChIP amplification except at the TOC1 terminal half of YFP (nYFP). The fusion constructs were then promoter (Fig. 2b). HDA9 specifically associated with a promoter transiently co-expressed in Arabidopsis protoplasts. The HDA9- region at the TOC1 locus (Fig. 2b), but not within TOC1 coding ELF3 combination was able to visualize yellow fluorescence, region (Supplementary Fig. 6). which was exclusively detected in the nucleus (Fig. 3b). Moreover, To further explore the circadian function of HDA9, we in planta interactions of HDA9 and ELF3 were also verified examined its possible oscillatory expression, but found that the (Fig. 3c and Supplementary Fig. 10). These results indicate that transcript accumulation of HDA9 did not significantly oscillate HDA9 associates with ELF3 to repress TOC1 expression. throughout a circadian cycle (Supplementary Fig. 7). Thus, HDA9 mRNA expression is not circadianly regulated. We nevertheless asked whether HDA9 might rhythmically bind to the TOC1 The EC facilitates HDA9 binding to the TOC1 promoter. Since promoter. Notably, binding of HDA9 was observed at Zeitgeber ELF3 is a key component of the EC30, HDA9 could be associated Time 16 (ZT16) (Fig. 2b and Supplementary Fig. 8), when TOC1 with the EC. This is in agreement with the temporal association of expression is rather low1. No obvious amplification was observed HDA9 to TOC1, because EC function is highly relevant at at other time points examined (Supplementary Fig. 8). ZT1630. However, it is currently unknown whether the EC indeed HDA9 protein biochemically catalyzes the removal of acetyl associates with the TOC1 promoter. To test this possibility, we groups from histone H3 proteins22,23. Since HDA9 binds to the employed pELF3:ELF3-MYC/elf3–1, pELF4:ELF4-HA/elf4–2, and TOC1 promoter (Fig. 2b), we hypothesized that HDA9 might pLUX:LUX-GFP/lux-4 transgenic plants30. ChIP assays were contribute to changes in H3ac accumulation at the TOC1 locus. performed with epitope-tagged transgenic plants immunopreci- ChIP assays with an anti-H3ac antibody revealed that H3ac pitated with corresponding antibodies. DNA bound to epitope- accumulation at a distal region of the TOC1 promoter declined tagged proteins was analyzed by qPCR assays. The ChIP-qPCR from ZT12 to ZT16 in wild type (Fig. 2c), consistent with the analysis showed that the TOC1 promoter was enriched by ELF3, declined expression of TOC11. However, the reduction of H3ac at ELF4, and LUX (Figs. 4a–c), also in the regions where HDA9 is the TOC1 promoter was impaired in hda9–1 mostly at ZT16 recruited (Fig. 2a, b). Furthermore, binding of EC components to (Fig. 2c), when HDA9 binds to the TOC1 promoter (Fig. 2b and the TOC1 promoter occurred mainly at ZT16 (Fig. 4a–c), which is Supplementary Fig. 8). HDA9 might bind to the specific consistent with the timing of HDA9 binding (Fig. 2b). promoter regions and then catalyze H3 deacetylation of Direct binding of EC to the TOC1 promoter most likely allows neighboring sites. The spatial separation was consistent with the transcriptional repression of TOC1. To verify the possibility, we molecular function of other chromatin modifiers29. conducted transient expression assays with the TOC1 promoter Given that H3ac is associated with gene activation16, HDA9 fused to the 35S minimal promoter. The recombinant reporter function may facilitate TOC1 repression. To test this hypothesis, plasmid and the effector construct expressing ELF3, ELF4, or we performed transient expression assays using Arabidopsis LUX were co-transformed into Arabidopsis protoplasts. As protoplasts. The TOC1 promoter was fused to the 35S minimal expected, they bound to the TOC1 promoter and repressed its promoter. The recombinant reporter plasmid and the effector expression (Fig. 4d), although they had different transcriptional construct expressing HDA9 gene were co-transformed into repressive activity. Given that EC components are known to have Arabidopsis protoplasts. Co-expression of these constructs overlapped and separate biological functions, ELF3 and LUX may repressed the GUS activity by 40% (Fig. 2d). In addition, we play a particular role in TOC1 regulation. also examined TOC1 expression in HDA9-misexpressed plants. In Since the EC recruits HDA9 to repress gene expression through hda9–1 mutant plants, TOC1 expression was clearly advanced, H3 deacetylation, we measured H3ac levels at the TOC1 promoter resulting in higher expression during the day and just after in elf3–8, elf4–101, and lux-6 mutants. ChIP-qPCR analysis subjective dusk (Fig. 2e and Supplementary Fig. 9). Taken revealed that H3 deacetylation at a distal region of the TOC1 together, we propose that HDA9 is recruited to the TOC1 promoter was impaired at ZT16 in all mutant examined promoter after its peak time and represses TOC1 expression by (Fig. 5a–c), in a similar fashion to that observed for hda9–1 promoting H3 deacetylation. mutant (Fig. 2c). In addition, we also analyzed expression of TOC1 in elf3–8 and lux-6 and found that TOC1 expression was arrhythmic and elevated at some time points, including the Protein–protein interaction of HDA9 and ELF3. Our results subjective night (ZT64-ZT68) (Fig. 5d, e; also see Supplementary show that the HDA9 protein is recruited to the TOC1 promoter. Figs. 11 and 12). Consistently, CCA1 gene expression was also Given that HDA9 is not circadianly regulated, we hypothesized arrhythmic and suppressed during the subjective day (Supple- that additional molecular component(s) might contribute to the mentary Fig. 13), which correlates with the elevated expression of rhythmic function of HDA9. As HDA9 itself does not have its repressor TOC12. These results suggest that the EC and HDA9 binding selectivity on specific DNA regions, DNA-binding pro- act together in the control of histone deacetylation at the TOC1 teins may be required for specific HDA9 binding to the TOC1 promoter. promoter. To examine this hypothesis, we carried out yeast-two- The importance of EC function for HDA9 binding to the TOC1 hybrid assays with expression constructs containing core clock promoter was tested using 35S:MYC-HDA9 transgenic plants transcription factors. The HDA9-GAL4 DNA binding domain crossed with the elf3–8 mutant. ChIP assays using an anti-MYC fusion construct was co-expressed with a construct expressing a antibody showed that HDA9 binding to the TOC1 locus was clock gene fused in-frame to the 3’-end of GAL4 activation reduced in the elf3–8 mutant background compared with the domain in yeast cells. Cell growth on the selective medium wild-type background (Fig. 6a), although HDA9 protein similarly COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 a 1kb CCA1 ELF3 LHY LUX A B A B A B A B –1126~–976 –337~–132 –943~–869 –118~–44 –910~–836 –500~–403 –431~–165 –126~–18 PRR5 PRR7 PRR9 TOC1 A B A B A B A B C –1393~–1246 –375~–151 –1015~–935 –589~–475 –458~–309 –156~–57 –1627~–1469 –379~–245 –1205~–1123 Col-0 35S:MYC-HDA9 b 2 3 2 4 3 2 2 3 A B A B A B A B Relative binding 1 1.5 1 2 1.5 1 1 1.5 0 0 0 0 0 0 0 0 CCA1 ELF3 LHY LUX 3 3 3 3 3 4 6 3 A B A B A B B C * Relative binding 1.5 1.5 1.5 1.5 1.5 2 3 1.5 0 0 0 0 0 0 0 0 PRR5 PRR7 PRR9 TOC1 c e 9 2 2 2 Col-0 TOC1 A B C 8 Col-0 Relative expression hda9-1 hda9-1 7 * Relative H3ac level 6 * 5 1 1 1 4 * * 3 2 * 1 0 0 0 0 ZT48 52 56 60 64 68 ZT12 ZT16 ZT12 ZT16 ZT12 ZT16 Time in continuous light (h) d Reporter Min 35S GUS Nos-T pMin35S pTOC1 Min 35S GUS Nos-T pTOC1 * Effector CaMV 35S HDA9 Nos-T 0 0.5 1 1.5 Relative GUS activity Fig. 2 HDA9 associates with TOC1 promoter to catalyze H3 deacetylation. a Genomic structures of core clock genes. Exons are represented by black boxes. Underbars indicate the regions amplified by PCR after chromatin immunoprecipitation (ChIP). Red, blue, green, and yellow arrowheads represent CBS, EE, G-box, and LBS motifs, respectively. b Binding of HDA9 to clock gene promoters. Two-week-old plants entrained with ND cycles were subjected to LL. Plants were harvested at ZT16 for ChIP analysis with anti-MYC antibody. Enrichment of fragmented genomic regions was analyzed by ChIP-qPCR. Biological triplicates were averaged and statistically analyzed with Student’s t-test (*P < 0.05; difference between Col-0 and 35 S:MYC-HDA9 plants). Bars indicate the standard error of the mean. c H3ac levels at the TOC1 locus in hda9–1. Two-week-old plants entrained with ND cycles were subjected to LL. Plants were harvested at ZT12 and ZT16 for ChIP analysis with anti-H3ac antibody. Biological triplicates were averaged and statistically analyzed with Student’s t-test (*P < 0.05; difference between Col-0 and hda9–1 plants). Bars indicate the standard error of the mean. d Transient expression assays. The effector and reporter constructs were coexpressed into Arabidopsis protoplasts. The GUS activities were measured fluorimetrically. Biological triplicates were averaged and statistically analyzed with Student’s t-test (*P < 0.05). Bars indicate the standard error of the mean. e Expression of TOC1 in hda9–1. Two-week-old seedlings grown under ND were transferred to LL at ZT0. Whole seedlings were harvested from ZT48 to ZT68. eIF4a was used as the normalization control. Two technical replicates were averaged and statistically analyzed with Student’s t-test (*P < 0.05; difference between Col-0 and hda9–1 plants). Bars indicate the standard deviation. The white and gray boxes indicate the subjective day and night 4 COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 ARTICLE ELF3-nYFP a b nYFP ELF3-nYFP HDA9-cYFP HDA9-cYFP cYFP IDD14-RFP P LUX ELF4 ELF3 TOC1 PRR9 PRR7 PRR3 LCL5 LHY CCA1 AD BD DIC HDA9 –LW BD Autofluorescence HDA9 –LWAH c HDA9-MYC – + ELF3-GFP + + RFP α-MYC MYC-IP α-GFP YFP α-MYC Input α-GFP Merged Fig. 3 HDA9 interacts with EC. a Yeast-two-hybrid assays. Yeast-two-hybrid assays were performed with the HDA9 proteins fused to the DNA-binding domain (BD) of GAL4 and clock components fused with the transcriptional activation domain (AD) of GAL4 for analysis of interactions. Interactions were examined by cell growth on selective media. -LWHA indicates Leu, Trp, His, and Ade drop-out plates. -LW indicates Leu and Trp drop-out plates. GAL4 was used as a positive control. b BiFC assays. Partial fragments of YFP protein were fused with HDA9 and ELF3 and co-expressed in Arabidopsis protoplasts. IDD14-RFP was used as a nucleus marker. Scale bar, 10 µm. c Coimmunoprecipitation assays. Agrobacterium tumefaciens cells containing 35S: HDA9-MYC and 35S:ELF3-GFP constructs were coinfiltrated to 3-week-old N. benthamiana leaves. Epitope-tagged proteins were detected immunologically using corresponding antibodies. Full blot images were shown in Supplementary Fig. 10 accumulated in both backgrounds (Supplementary Fig. 14). Since oscillation of H3ac levels may be pervasive in core clock genes, HDA9 promotes H3 deacetylation at the cognate regions, we also ensuring circadian homeostasis. measured H3ac levels in the same plants. H3ac levels were reduced in 35S:MYC-HDA9 plants compared with wild type, but H3 deacetylation was compromised in 35S:MYC-HDA9xelf3–8 Discussion plants, which was equivalent to elf3–8 (Fig. 6b). Further, 35S: Rhythmic expression of core clock genes is intimately associated MYC-HDA9 transgenic plants displayed robust circadian expres- with the levels of H3ac, especially H3K56ac and H3K9/14ac, and sion of TOC1, while the amplitude was reduced compared with H3K4me3 at the gene promoters in Arabidopsis21,31. Despite the wild type (Supplementary Fig. 15). However, arrhythmic connection between chromatin modification and the circadian circadian expression was observed in 35S:MYC-HDA9xelf3–8 control, just a few responsible chromatin modifiers have been (Fig. 6c), similar to EC mutants (Fig. 5d, e). Indeed, HDA9 demonstrated to participate in circadian chromatin remodeling. function is most likely dependent on EC. Transient expression The SET DOMAIN GROUP 2 (SDG2)/ARABIDOPSIS assays using Arabidopsis protoplasts revealed that HDA9 binds to TRITHORAX–RELATED 3 (ATXR3) protein establishes the TOC1 promoter to inhibit expression (Fig. 2d). However, the H3K4me3 mark at core clock gene loci to promote expression. HDA9 function disappeared in elf3–8 and lux-6 mutants (Fig. 6d). The H3K4me3 mark inhibits clock repressor binding at the core These results suggest that HDA9 requires the ELF3, possibly EC, clock promoters, facilitating transcriptional repression to target for binding to the TOC1 promoter. genes only at specific time-of-day21. Accordingly, the In summary, TOC1 expression is regulated by rhythmic SDG2/ATXR3-deficient mutants exhibit a decrease in H3K4me3 changes in histone acetylation states. A-yet-unidentified HAT accumulation, advanced clock repressor binding, and reduced activity is required for the rising phase of TOC1 expression. After amplitude of core clock gene expression21. In addition, the JmjC its peak expression, HDA9 is recruited to the TOC1 promoter domain-containing histone demethylase JMJ30/JMJD5 is also through the interaction with ELF3, possibly the EC. The EC- involved in circadian homeostasis. The JMJ30/JMJD5 gene is HDA9 complex facilitates H3 deacetylation at the TOC1 locus to circadian-regulated and peaks at dusk32,33. The central oscillators reach its basal expression during night period (Fig. 7). Diurnal CCA1 and LHY directly bind to and repress JMJ30/JMJD5. Then, COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio 5 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 Col-0 a pELF3:ELF3-MYC/elf3-1 2 3 2 3 5 5 A B C A B ** C Relative binding * Relative binding 1 1.5 1 1.5 2.5 2.5 0 0 0 0 0 0 ZT12 ZT16 Col-0 b pELF4:ELF4-HA/elf4-2 3 5 3 9 4 2 A B * C A * B C * Relative binding Relative binding 1.5 2.5 1.5 4.5 2 1 0 0 0 0 0 0 ZT12 ZT16 Col-0 c pLUX:LUX-GFP/lux-4 3 2 2 14 4 3 A B C A B C * * Relative binding Relative binding 1.5 1 1 7 2 1.5 0 0 0 0 0 0 ZT12 ZT16 d 1.5 1.5 1.5 Reporter ELF3 ELF4 LUX Min 35S GUS Nos-T Relative GUS activity pTOC1 Min 35S GUS Nos-T 1 1 1 * Effector * CaMV 35S ELF3 Nos-T CaMV 35S ELF4 Nos-T 0.5 0.5 0.5 CaMV 35S LUX Nos-T 0 0 0 pMin35S pTOC1 pMin35S pTOC1 pMin35S pTOC1 Fig. 4 EC directly binds to the TOC1 promoter. a–c Binding of ELF3, ELF4, and LUX to the TOC1 locus. Two-week-old plants entrained with ND cycles were subjected to LL. Plants were harvested at ZT12 and ZT16 for ChIP analysis. Enrichment of fragmented genomic regions was analyzed by ChIP-qPCR (see Fig. 2a). Two biological replicates were averaged and statistically analyzed with Student’s t-test (*P < 0.05, **P < 0.01). Bars indicate the standard error of the mean. d Effects of ELF3, ELF4, and LUX on TOC1 repression. The effector and reporter constructs were transiently coexpressed in Arabidopsis protoplasts. The GUS activities were measured fluorimetrically. Biological triplicates were averaged and statistically analyzed with Student’s t-test (*P < 0.05). Bars indicate the standard error of the mean JMJ30/JMJD5 reciprocally regulates expression of CCA1 and impaired in the tpl-1 mutant, demonstrating that HDAC reg- LHY32. ulation is largely dependent on TPL34. A couple of HDACs have been shown to be implicated in the We here show that HDA9 regulates circadian oscillation by Arabidopsis circadian system. PRR5, PRR7, and PRR9 directly modulating TOC1 expression. HDA9 binds to the TOC1 pro- repress CCA1 and LHY34. Notably, PRRs interact with TOPLESS/ moter and stimulates H3 deacetylation to suppress TOC1 TOPLESS RELATED PROTEINS (TPL/TPRs), as well as HDA6 expression. HDA9 interacts with the EC component ELF3, and and HDA1934. Consistently, inhibition of the TPL activity com- rhythmic binding of HDA9 to the TOC1 promoter is defined by promises transcriptional repression activities of PRR5, PRR7, and the EC. Accordingly, although HDA9 has no obvious rhythmic PRR9, lengthening circadian period34. The potent HDAC inhi- expression pattern, the EC-HDA9 interactions facilitate temporal bitor TSA activates CCA1 expression, but this activation is regulation of TOC1. Consistent with the fact that the EC is 6 COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 ARTICLE Col-0 sometimes observed the anti-phase of binding regions and his- a elf3-8 tone modifications29,36. While HDA9 binds to a proximal region 2 2 2 A B C of the TOC1 promoter, changes in histone acetylation are dras- Relative H3ac level tically observed in a distal region. This may be due to propagation * and expansion of epigenetic modification of chromatin. Chro- 1 1 1 matin remodeling components that regulate epigenetic contexts on chromatin would also be involved, and thus the responsible protein components should be further elucidated. Future studies will give a comprehensive view of circadian-chromatin networks. 0 0 0 Altogether, these results exemplify a mechanism by which chro- ZT12 ZT16 ZT12 ZT16 ZT12 ZT16 matin factors engage with clock components to rhythmically Col-0 modulate chromatin conformation and hence transcriptional b elf4-101 oscillation. 2 2 2 A B C The HDA9 protein is implicated in plant developmental pro- Relative H3ac level cesses. HDA9 is highly expressed in dry seeds and represses seedling traits to maintain seed dormancy37. The hda9 mutants 1 * 1 1 show reduced seed dormancy with higher expression of genes responsible for photosynthesis and photoautotroph, such as RuBisCO and RuBisCO activase (RCA)37. The HDA9 action is antagonistic to HDA6 and HDA19, which repress embryonic 0 0 0 properties in germinating seeds37, balancing the proper transition ZT12 ZT16 ZT12 ZT16 ZT12 ZT16 from seed to autotropic seedling. Col-0 The HDA9-deficient mutants also exhibit early flowering c lux-6 phenotypes under short day conditions38,39. HDA9 binds to the 2 3 2 AGAMOUS–LIKE 19 (AGL19) promoter and negatively regulates A B C * Relative H3ac level its transcription through the alterations in H3K9 and H3K27 acetylation levels at the AGL19 locus39. Consequently, the HDA9- 1 1.5 1 AGL19 module mainly regulates FLOWERING LOCUS T (FT) expression, in parallel with photoperiod or autonomous pathways to prevent precocious flowering in short days39. The epigenetic regulation of AGL19 by HDA9 is also relevant, in part, during 0 0 0 vernalization process. Given that HDA9 is functional when ZT12 ZT16 ZT12 ZT16 ZT12 ZT16 AGL19 is actively expressed, such in the adult stage or after vernalization39, this HDAC protein is able to associate with d 4 e 7 transcriptionally active genes to reset the chromatin state. TOC1 TOC1 * 6 Accumulating evidence further supports that the HDA9- Relative expression Relative expression 3 * 5 POWERDRESS (PWR) complex stimulates H3 deacetylation at * * 4 * * a genome-wide level and is implicated in additional develop- 2 3 * mental processes, such as floral stem cell fate, stress responses, 2 and aging22,23. 1 Col-0 1 Col-0 Notably, HDA9 activity can be diurnally gated. HDACs often elf3-8 lux-6 form a massive protein complex containing for instance Sin3, 0 0 RPD3-type HDAC, SIN3-ASSOCIATED POLYPEPTIDE 18 ZT48 52 56 60 64 68 ZT48 52 56 60 64 68 (SAP18) and SAP3040. Homologs of each component are present Time in continuous light (h) Time in continuous light (h) in Arabidopsis: six SIN3-Like (SNL1-SNL6), four RPD3 HDACs, Fig. 5 EC represses TOC1 expression by promoting H3 deacetylation. one SAP18, and two SAP30 function-relateds (AFR1 and AFR2) 40. The Arabidopsis Sin3-HDAC complex is crucial for diurnal a–c Elevated H3ac levels at TOC1 locus in EC mutants. Two-week-old plants entrained with ND cycles were subjected to LL. Plants were harvested at control of FT40. This complex accumulates at dusk and is ZT12 and ZT16 for ChIP analysis with anti-H3ac antibody. Two biological recruited to FT promoter to ensure periodic histone deacetyla- replicates were averaged and statistically analyzed with Student’s t-test tion. Dynamic cycles of H3 acetylation and deacetylation ensure (*P < 0.05). Bars indicate the standard error of the mean. d and adequate levels of the gene transcription and thereby prevent e Expression of TOC1 in elf3–8 and lux-6. Two-week-old seedlings grown precocious flowering in the premature inductive conditions40. under ND were transferred to LL at ZT0. Whole seedlings were harvested This study further supports periodic functions of HDA9 in from ZT48 to ZT68. eIF4a was used as the normalization control. Two controlling circadian gene expression. H3ac states can be rever- technical replicates were averaged and statistically analyzed with Student’s sible and account for stably oscillating gene expression. Taken t-test (*P < 0.05). Bars indicate the standard deviation. The white and gray together, the HDA9 protein seems to integrate temporal infor- boxes indicate the subjective day and night mation to regulate oscillator gene expression and a variety of physiological output processes in order to optimize plant growth and development. expressed highly during night period35, the EC-HDA9 complex is relevant in the declining phase of TOC1 expression. Nonetheless, many questions remain to be answered. Considering the variable Methods alteration patterns of circadian gene expression in hda9–1 Plant materials and growth conditions. Arabidopsis thaliana (Columbia-0 eco- type) was used for all experiments unless otherwise specified. Plants were grown mutant, EC and HDA9 are extensively interconnected with other under neutral-day conditions (NDs; 12-h light/12-h dark cycles) with cool white circadian and chromatin-related components, accounting for fluorescent light (100 μmol photons m−2 s−1) at 22 °C. The elf3–8, elf4–101, dynamic contribution to circadian oscillation. Moreover, it is hda7–2, and hda9–1 were previously reported39,41–43. The lux-6 mutant (SALK- COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio 7 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 a TOC1-B b TOC1-A 8 2.5 * α-H3ac 2 Relative H3ac level Relative binding 1.5 4 n.s. 1 0.5 0 0 Col-0 35S: 35S:MYC-HDA9 Col-0 elf3-8 35S: 35S:MYC-HDA9 MYC-HDA9 xelf3-8 MYC-HDA9 xelf3-8 c d 50 Reporter Col-0 Min 35S GUS Nos-T 35S:MYC-HDA9xelf3-8 pTOC1 Min 35S GUS Nos-T 40 Effector CaMV 35S HDA9 Nos-T Relative expression 30 1.5 Min35S pTOC1 Relative GUS activity 20 1 10 0.5 *** 0 ZT48 52 56 60 64 68 0 Time in continuous light (h) Col-0 elf3-8 lux-6 Fig. 6 EC is required for HDA9 binding to the TOC1 promoter. a Binding of HDA9 to the TOC1 locus. b H3ac levels in 35S:MYC-HDA9xelf3–8. In a and b, two-week-old plants entrained with ND cycles were subjected to LL. Plants were harvested at ZT16 for ChIP analysis. Two biological replicates were averaged and statistically analyzed with Student’s t-test (*P < 0.05). Bars indicate the standard error of the mean. n.s., not significant. c Expression of TOC1 in 35S:MYC-HDA9xelf3–8. Two-week-old seedlings grown under ND were transferred to LL at ZT0. Whole seedlings were harvested from ZT48 to ZT68. eIF4a was used as the normalization control. Biological triplicates were averaged. Bars indicate the standard error of the mean. The white and gray boxes indicate the subjective day and night. d Transient expression assays. The GUS activities were measured fluorimetrically. Biological triplicates were averaged and statistically analyzed with Student’s t-test (***P < 0.001). Bars indicate the standard error of the mean 022315) was obtained from Arabidopsis Biological Resource Center (ABRC). To Yeast two-hybrid assays. Yeast two-hybrid assays were performed using the BD produce transgenic plants overexpressing the HDA9 gene, a full-length cDNA was Matchmaker system (Clontech, Mountain View, CA, USA). The pGADT7 vector subcloned into the binary pBA002 vector under the control of the CaMV 35 S was used for the GAL4 activation domain fusion, and the pGBKT7 vector was used promoter. Agrobacterium tumefaciens-mediated Arabidopsis transformation was for the GAL4 DNA binding domain fusion. The yeast strain AH109 harboring the then performed. LacZ and His reporter genes was used. PCR products were subcloned into the pGBKT7 and pGADT7 vectors. The expression constructs were co-transformed into yeast AH109 cells, and transformed cells were selected by growth on SD/-Leu/- Quantitative real-time RT-PCR analysis. Plants grown under conditions/ZT Trp medium and SD/-Leu/-Trp/-His/-Ade. indicated were harvested and ground in liquid nitrogen. Total RNA was extracted by mixing the tissue powder with 700 μl of TRI reagent (TAKARA Bio, Singa, Bimolecular fluorescence complementation (BiFC) assays. The HDA9 gene was Japan). The extraction mixture was incubated at 65 °C for 5 min. After mixing with fused in-frame to the 5′ end of a gene sequence encoding the C-terminal half of 700 μl of chloroform:isoamyl alcohol (24:1), the extraction mixture was centrifuged EYFP in the pSATN-cEYFP-C1 vector (E3082). The ELF3 cDNA sequences were for 20 min at room temperature. The RNA in the liquid phase was precipitated at 4 fused in-frame to the 5′ end of a gene sequence encoding the N-terminal half of °C in a final concentration of 2 M LiCl. RNA pellet was washed by 70% ethanol, EYFP in the pSATN-nEYFP-C1 vector (E3081). Expression constructs were co- briefly air dried, and suspended in distilled water. Reverse transcription (RT) was transformed into Arabidopsis protoplasts. Expression of the fusion constructs was performed using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase monitored by fluorescence microscopy using a Zeiss LSM510 confocal microscope (Dr. Protein, Seoul, South Korea) with oligo(dT18) to synthesize first-strand cDNA (Carl Zeiss, Jena, Germany). from 2 μg of total RNA. Total RNA samples were pretreated with an RNAse-free DNAse to remove DNA contamination. Quantitative RT-PCR reactions were performed on the Step-One Plus Real- Chromatin immunoprecipitation (ChIP) assays. The epitope-tagged transgenic Time PCR System (Applied Biosystems). The PCR primers used are listed in plant samples were cross-linked with 1% formaldehyde, ground to powder in liquid Supplementary Table 1. The comparative CT method was used to determine the nitrogen, and then sonicated (15-s ON/15-s OFF for seven times, each with 5 min). relative gene expression, with the expression of Eukaryotic translation initiation The sonicated chromatin complexes were bound with corresponding antibodies. factor 4A1 (eIF4A) gene (At3g13920) as the internal control. All RT-qPCR Anti-MYC (05–724, Millipore, Billerica, USA), anti-H3ac (06–599, Millipore, reactions were performed with biological triplicates using total RNA samples Billerica, USA), anti-HA (ab9110, Abcam, Cambridge, USA), and salmon sperm extracted from three independent replicate samples. DNA/protein A agarose beads (16–157, Millipore, Billerica, USA) were used for 8 COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 ARTICLE 4. Farre, E. M., Harmer, S. L., Harmon, F. G., Yanovsky, M. J. & Kay, S. A. On Overlapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock. Curr. Biol. 15, 47–54 (2005). Ac Ac 5. Helfer, A. et al. LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock. Curr. Biol. 21, 126–133 (2011). 6. Chow, B. Y., Helfer, A., Nusinow, D. A. & Kay, S. A. ELF3 recruitment to the Ac PRR9 promoter requires other evening complex members in the Arabidopsis Ac circadian clock. Plant Signal. Behav. 7, 170–173 (2012). ELF3 7. Dixon, L. E. et al. Temporal repression of core circadian genes is mediated HAT HDA9 through EARLY FLOWERING 3 in. Arab. Curr. Biol. 21, 120–125 (2011). Ac 8. Locke, J. C. et al. Experimental validation of a predicted feedback loop in the Ac multi-oscillator clock of Arabidopsis thaliana. Mol. Syst. Biol. 2, 59 (2006). Off 9. Xie, Q. et al. LNK1 and LNK2 are transcriptional coactivators in the A RN Arabidopsis circadian oscillator. Plant Cell 26, 2843–2857 (2014). 1m 10. Farinas, B. & Mas, P. Functional implication of the MYB transcription factor C TO RVE8/LCL5 in the circadian control of histone acetylation. Plant J. 66, 318–329 (2011). 11. Lau, O. S. et al. Interaction of Arabidopsis DET1 with CCA1 and LHY in mediating transcriptional repression in the plant circadian clock. Mol. Cell 43, ZT0 ZT12 ZT24 703–712 (2011). Fig. 7 Working diagram of the EC-HDA9 complex in circadian control. 12. Petrillo, E., Sanchez, S. E., Kornblihtt, A. R. & Yanovsky, M. J. Alternative splicing adds a new loop to the circadian clock. Commun. Integr. Biol. 4, Transcript accumulation of TOC1 correlates with H3ac levels at the gene 284–286 (2011). promoter. As-yet-unidentified HAT catalyzes H3ac at the TOC1 promoter 13. Mas, P., Kim, W. Y., Somers, D. E. & Kay, S. A. Targeted degradation of TOC1 during the daytime. After peak expression, the ELF3 protein, presumably by ZTL modulates circadian function in Arabidopsis thaliana. Nature 426, evening complex (EC), recruits HDA9. The EC-HDA9 complex binds to the 567–570 (2003). TOC1 promoter and removes H3ac at neighboring sites to decline its 14. Seo, P. J. & Mas, P. Multiple layers of posttranslational regulation refine circadian clock activity in Arabidopsis. Plant Cell 26, 79–87 (2014). expression during nighttime 15. Shu, H., Wildhaber, T., Siretskiy, A., Gruissem, W. & Hennig, L. Distinct modes of DNA accessibility in plant chromatin. Nat. Commun. 3, 1281 (2012). 16. Eberharter, A. & Becker, P. B. Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics. chromatin immunoprecipitation. The immunoprecipitated chromatin complexes EMBO Rep. 3, 224–229 (2002). were purified by incubating with 50 μl slurry of Protein-A Sepharose (GE, 17. Boycheva, I., Vassileva, V. & Iantcheva, A. Histone acetyltransferases in plant 17–5280–01) pre-equilibrated with 1 mg/ml salmon sperm DNA and 1 mg/ml BSA development and plasticity. Curr. Genom. 15, 28–37 (2014). for 3 h at 4 °C. The affinity beads bound chromatin complexes were washed with 18. Gorisch, S. M., Wachsmuth, M., Toth, K. F., Lichter, P. & Rippe, K. Histone 850 μl nuclei lysis buffer for three times, LNDET buffer (0.25 M LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, pH8.0) for three times and TE buffer (10 mM acetylation increases chromatin accessibility. J. Cell. Sci. 118, 5825–5834 Tris–HCl, 1 mM EDTA, pH8.0) for three times. Chromatin complexes were eluted (2005). from the beads with use of 200 μl elution buffer (1% SDS, 0.1 M NaHCO3) followed 19. Yang, X. J. & Seto, E. HATs and HDACs: from structure, function and by the digestion with 2.5 ml Proteinase-K (20 mg/ml, Invitrogen, Carlsbad, CA) for regulation to novel strategies for therapy and prevention. Oncogene 26, overnight at 65 °C. DNA was purified using phenol/chloroform/isoamyl alcohol 5310–5318 (2007). and sodium acetate (pH 5.2). The level of precipitated DNA fragments was 20. Perales, M. & Mas, P. A functional link between rhythmic changes in quantified by quantitative real-time PCR using specific primer sets (Supplementary chromatin structure and the Arabidopsis biological clock. Plant Cell 19, Table 2). Values were normalized to an internal control (eIF4a). Values for control 2111–2123 (2007). plants were set to 1 after normalization against eIF4a for quantitative PCR analysis. 21. Malapeira, J., Khaitova, L. C. & Mas, P. Ordered changes in histone modifications at the core of the Arabidopsis circadian clock. Proc. Natl Acad. Sci. USA 109, 21540–21545 (2012). Transient expression assays. For transient expression assays using Arabidopsis 22. Kim, Y. J. et al. POWERDRESS and HDA9 interact and promote histone H3 protoplasts, reporter and effector plasmids were constructed. The reporter plasmid deacetylation at specific genomic sites in. Arab. Proc. Natl Acad. Sci. USA 113, contains a minimal 35S promoter sequence and the GUS-coding gene. The TOC1 14858–14863 (2016). promoter was inserted into the reporter plasmid. The coding regions of HDA9, 23. Chen, X. et al. POWERDRESS interacts with HISTONE DEACETYLASE 9 to ELF3, ELF4, and LUX were subcloned into the effector vector for transient over- expression under the control of the CaMV 35 S promoter. An amount of 2.5 × 105 promote aging in Arabidopsis. eLife 5, https://doi.org/10.7554/eLife.17214 protoplasts was co-transfected with 4 μg reporter construct, 4 μg effector construct, (2016). and 2 μg CaMV 35 S promoter-luciferase construct as the internal transfection 24. Hung, F. Y. et al. The Arabidopsis LDL1/2-HDA6 histone modification control. Transfected cells were incubated at 22 °C overnight and harvested for LUC complex is functionally associated with CCA1/LHY in regulation of circadian and GUS reporter assays. clock genes. Nucleic Acids Res. https://doi.org/10.1093/nar/gky749 (2018). 25. Li, H. et al. The histone deacetylase inhibitor trichostatin a promotes totipotency in the male gametophyte. Plant Cell 26, 195–209, https://doi.org/ Reporting Summary. Further information on experimental design is available in 10.1105/tpc.113.116491 (2014). the Nature Research Reporting Summary linked to this article. 26. Liu, X. et al. Transcriptional repression by histone deacetylases in plants. Mol. Plant 7, 764–772 (2014). Data availability 27. Ezer, D. et al. The G-Box transcriptional regulatory code in. Arab. Plant All relevant data are available from the corresponding author on request. Physiol. 175, 628–640 (2017). 28. Michael, T. P. & McClung, C. R. Phase-specific circadian clock regulatory elements in Arabidopsis. Plant Physiol. 130, 627–638 (2002). Received: 22 September 2018 Accepted: 6 March 2019 29. Ko, J. H. et al. Growth habit determination by the balance of histone methylation activities in Arabidopsis. EMBO J. 29, 3208–3215 (2010). 30. Ezer, D. et al. The evening complex coordinates environmental and endogenous signals in Arabidopsis. Nat. Plants 3, 17087 (2017). 31. Song, H. R. & Noh, Y. S. Rhythmic oscillation of histone acetylation and References methylation at the Arabidopsis central clock loci. Mol. Cells 34, 279–287 1. Alabadi, D. et al. Reciprocal regulation between TOC1 and LHY/CCA1 within (2012). the Arabidopsis circadian clock. Science 293, 880–883 (2001). 32. Lu, S. X. et al. The Jumonji C domain-containing protein JMJ30 regulates 2. Gendron, J. M. et al. Arabidopsis circadian clock protein, TOC1, is a DNA- period length in the Arabidopsis circadian clock. Plant Physiol. 155, 906–915 binding transcription factor. Proc. Natl Acad. Sci. USA 109, 3167–3172 (2012). (2011). 3. Nakamichi, N. et al. PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are 33. Jones, M. A. et al. Jumonji domain protein JMJD5 functions in both the plant transcriptional repressors in the Arabidopsis circadian clock. Plant Cell 22, and human circadian systems. Proc. Natl Acad. Sci. USA 107, 21623–21628 594–605 (2010). (2010). COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio 9 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0377-7 34. Wang, L., Kim, J. & Somers, D. E. Transcriptional corepressor TOPLESS Foundation of Korea, from the European Commission Marie Curie Research Training complexes with pseudoresponse regulator proteins and histone deacetylases to Network (ChIP-ET) to P.M. and by the CERCA Programme/Generalitat de Catalunya. regulate circadian transcription. Proc. Natl Acad. Sci. USA 110, 761–766 We acknowledge financial support from the Spanish Ministry of Economy and Com- (2013). petitiveness, through the Severo Ochoa Programme for Centers of Excellence in R&D 35. Huang, H. & Nusinow, D. A. Into the evening: complex interactions in the 2016–2019 (SEV‐2015‐0533). Arabidopsis circadian clock. Trends Genet. 32, 674–686 (2016). 36. Yu, C. W. et al. HISTONE DEACETYLASE6 interacts with FLOWERING Author contributions LOCUS D and regulates flowering in Arabidopsis. Plant Physiol. 156, 173–184 P.J.S. and P.M. conceived and designed the experiments. P.J.S. and P.M. wrote the paper. (2011). K.L. conducted experiments and contributed to the study design under the supervision of 37. van Zanten, M. et al. HISTONE DEACETYLASE 9 represses seedling traits in P.J.S. Arabidopsis thaliana dry seeds. Plant J. 80, 475–488 (2014). 38. Kim, W., Latrasse, D., Servet, C. & Zhou, D. X. Arabidopsis histone deacetylase HDA9 regulates flowering time through repression of AGL19. Biochem. Additional information Biophys. Res. Commun. 432, 394–398 (2013). Supplementary information accompanies this paper at https://doi.org/10.1038/s42003- 39. Kang, M. J., Jin, H. S., Noh, Y. S. & Noh, B. Repression of flowering under a 019-0377-7. noninductive photoperiod by the HDA9-AGL19-FT module in. Arab. New Phytol. 206, 281–294 (2015). Competing interests: The authors declare no competing interests. 40. Gu, X., Wang, Y. & He, Y. Photoperiodic regulation of flowering time through periodic histone deacetylation of the florigen gene. Ft. PLoS Biol. 11, e1001649 Reprints and permission information is available online at http://npg.nature.com/ (2013). reprintsandpermissions/ 41. Hicks, K. A., Albertson, T. M. & Wagner, D. R. EARLY FLOWERING3 encodes a novel protein that regulates circadian clock function and flowering Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in in Arabidopsis. Plant Cell 13, 1281–1292 (2001). published maps and institutional affiliations. 42. Kikis, E. A., Khanna, R. & Quail, P. H. ELF4 is a phytochrome-regulated component of a negative-feedback loop involving the central oscillator components CCA1 and LHY. Plant J. 44, 300–313 (2005). Open Access This article is licensed under a Creative Commons 43. Cigliano, R. A. et al. Histone deacetylase AtHDA7 is required for female Attribution 4.0 International License, which permits use, sharing, gametophyte and embryo development in. Arab. Plant Physiol. 163, 431–440 adaptation, distribution and reproduction in any medium or format, as long as you give (2013). appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless Acknowledgements indicated otherwise in a credit line to the material. If material is not included in the We would like to thank Dr. Clara Conicella for providing hda7–2 mutant. This work was article’s Creative Commons license and your intended use is not permitted by statutory supported by the Basic Science Research (NRF-2016R1D1A1B03931139) and Basic regulation or exceeds the permitted use, you will need to obtain permission directly from Research Laboratory (NRF-2017R1A4A1015620) programs provided by the National the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Research Foundation of Korea and by the Next-Generation BioGreen 21 Program licenses/by/4.0/. (PJ01314501) provided by the Rural Development Administration, by research grants from the Spanish Ministry of Economy and Competitiveness, from the Generalitat de Catalunya (AGAUR), from the Global Research Network of the National Research © The Author(s) 2019 10 COMMUNICATIONS BIOLOGY | (2019)2:143 | https://doi.org/10.1038/s42003-019-0377-7 | www.nature.com/commsbio