Chemistry

The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor

JMJ13 particularly demethylates H3K27me3

We beforehand recognized 21 JmjC domain-containing proteins within the Arabidopsis thaliana genome and predicted JMJ13 as one of many 15 doubtlessly lively histone demethylases19. JMJ13 is a homolog of ELF6/JMJ11 and REF6/JMJ12, the 2 Arabidopsis KDM4 subfamily H3K27me3 demethylases (Supplementary Desk 1)25,26. To find out whether or not JMJ13 is an lively demethylase, we carried out enzymatic exercise assays in vivo utilizing a Nicotiana benthamiana leaf-based assay25,27 (Fig. 1a). In cells the place JMJ13-GFP was over-expressed, H3K27me3, however not H3K27me2 and H3K27me1, was markedly diminished (Fig. 1b, c). In distinction, there have been no important variations within the tri-, di- and mono-methylation ranges of H3K4, H3K9, or H3K36 websites (Supplementary Fig. 1). The H3K27me3 demethylase exercise of JMJ13-GFP was abolished when His293 and Glu295, the 2 conserved iron-binding amino acids, had been changed by alanine (Fig. 1d, e).

Fig. 1Fig. 1

JMJ13 is an H3K27me3 demethylase in vivo. a Schematic illustration of GFP-tagged JMJ13 and JMJ13-H293A-E295A-GFP constructs. HD, helical area. b, c Over-expression of JMJ13-GFP reduces the degrees of H3K27me3 however not H3K27me2 and H3K27me1 in vivo. d, e Over-expression of JMJ13-H293A-E295A-GFP has no impact on H3K27 methylation. In b, d, the white arrows level to the transfected nuclei stained by methylation-specific histone antibodies (crimson, proper panels), DAPI (blue, left panels), and the GFP sign from the JMJ13-GFP or JMJ13-H293A-E295A-GFP (inexperienced, center panels), respectively. Scale bars, 2 μm. In c, e, greater than 20 pairs of transfected nuclei versus non-transfected nuclei in the identical subject of view had been noticed and quantifications statistical analyzed. Error bars characterize imply ± SE. Pupil’s t take a look at was used to calculate the P worth between JMJ13-GFP and WT. ****P worth < zero.0001. The dots denote the person information factors. Supply information are supplied as a Supply Information file.

We additional carried out a MALDI-TOF mass-spectrometry-based in vitro demethylase assay24. The recombinant expressed JMJ13 catalytic area (JMJ13CD, residues 90–578, Fig. 2a) displayed unambiguous demethylation exercise towards H3K27me3 peptides, however not H3K4me3, H3K9me3, or H3K36me3 peptides, confirming that JMJ13 is an H3K27me3 site-specific histone demethylase (Supplementary Fig. 2a–d). Additional assays present that JMJ13 has excessive H3K27me3 demethylase exercise, however no important exercise on H3K27me2 and H3K27me1 (Supplementary Fig. second–f). Collectively, the in vivo and in vitro outcomes exhibit that JMJ13 is predominately an H3K27me3-specific demethylase.

Fig. 2Fig. 2

Constructions of JMJ13-α-KG and JMJ13-NOG-H3K27me3 complexes. a A schematic illustration of the area structure of the JMJ13 (higher panel) and the JMJ13 catalytic area assemble utilized in crystallization (decrease panel). b Total construction of JMJ13CD-α-KG complicated in ribbon illustration with the jumonji, helical, and zinc finger domains coloured in inexperienced, magenta and cyan, respectively. The α-KG, nickel ion and zinc ions are proven in stick, orange ball, and silver balls, respectively. The zinc coordination residues are highlighted in stick illustration. c Total construction of JMJ13CD-NOG-H3K27me3 peptide complicated with JMJ13 in ribbon and NOG and H3K27me3 peptide in space-filling representations, respectively. d An electrostatics floor view of JMJ13CD in complicated with the H3K27me3 peptide in stick illustration displaying that the peptide suits right into a negatively cost pocket of JMJ13CD. CD catalytic area, HD helical area

Crystal construction of JMJ13CD

To know the mechanism of H3K27me3-specific demethylation by JMJ13, we carried out structural research. JMJ13 has a central catalytic area flanked by versatile areas on each N- and C-termini (Fig. 2a). The crystal construction of the JMJ13 catalytic area (JMJ13CD), together with the anticipated jumonji, helical, and zinc finger domains, in complicated with the co-factor α-ketoglutarate (α-KG), was decided to 2.four Å decision (Fig. 2b and Supplementary Desk 2). Regardless of being in numerous subfamilies, the general construction of JMJ13CD resembles the beforehand reported buildings of human KDM5A/B/C and Arabidopsis JMJ14, that are composed of two elements: the jumonji area and the helical plus zinc finger domains24,28,29 (Fig. 2b). The jumonji area adopts a typical α-KG-dependent oxygenase fold with a double-stranded β-helix within the middle surrounded by a number of α-helices (Fig. 2b). A Ni2+ ion, which changed the endogenous Fe2+ ion throughout nickel column purification, and the co-factor α-KG are situated within the lively middle of the jumonji area (Fig. 2b). The helical area consists of 4 lengthy α-helices forming a helical bundle (Fig. 2b).

JMJ13 and different members of the KDM5 subfamily of histone demethylases are predicted to own a C5HC2-type (5 cysteine residues adopted by a histidine and a pair of further cysteine residues) zinc finger area to coordinate two Zn2+ ions. Nevertheless, our structural evaluation confirmed that the anticipated C5HC2 zinc finger of JMJ13 in truth adopts a C4HCHC association, which consists of a CCCH-type zinc finger (Cys500, Cys503, Cys522, and His525) and a CCHC-type zinc finger (Cys514, Cys516, His519, and Cys534) (Fig. 2b). Reasonably than the anticipated Cys507, the His525 contributes to zinc coordination within the C4HCHC-type zinc finger area in JMJ13. Though the C4HCHC zinc finger area is embedded within the main sequence of the helical area, structurally it kinds an unbiased area that interacts with the helical area (Fig. 2a–b) in the same method to the helical and C5HC2 domains of JMJ1424.

Recognition of the H3K27me3 peptide by JMJ13

To analyze the substrate recognition and catalytic mechanism of JMJ13, we decided the crystal construction of JMJ13CD in complicated with the α-KG analog N-oxalylglycine (NOG) and an H3K27me3 peptide, at 2.6 Å decision (Fig. 2c and Supplementary Desk 2). Total the construction carefully resembles the JMJ13-α-KG complicated with a root-mean-square deviation (RMSD) of zero.49 Å (Supplementary Fig. 3a). The peptide might be traced from H3A24 to H3A31 (Supplementary Fig. 3b). The peptide binds in a negatively charged cleft with the residues H3K27me3 to H3P30 situated on the backside of the cleft and the opposite flanking residues extending out from the cleft (Fig. second). The aspect chain of H3K27me3 inserts right into a deep binding pocket inside the lively website cleft (Fig. second). An NOG molecule and a Ni2+ ion are deeply buried within the middle of the lively website (Fig. 2c).

The interactions between JMJ13 and the H3K27me3 peptide are restricted to the area between H3R26 and H3P30. H3R26 kinds salt bridge and hydrogen bonding interactions with Asp236 of JMJ13 (Fig. 3a). H3S28 kinds a side-chain hydrogen bond with Asp296 of JMJ13 (Fig. 3a). H3P30 positions its aspect chain prolyl ring such that the aircraft of the propyl ring is parallel with and stacks on high of the phenyl ring of Phe179 of JMJ13 (Fig. 3a), leading to hydrophobic stacking and CH-π interactions. In contrast with the JMJ13 peptide-free construction, the aspect chain of Phe179 undergoes a major rotation to permit stacking with H3P30, indicative of a peptide binding-induced conformational change (Fig. 3b).

Fig. threeFig. 3

Structural foundation for the popularity of H3K27me3 by JMJ13. a H3R26 and H3S28 type hydrogen bonds (dashed silver strains) with JMJ13 Asp236 and Asp296, respectively. The prolyl ring of H3P30 stacks with the phenyl ring of JMJ13 Phe179. b The superposition of JMJ13-α-KG complicated (in cyan) and JMJ13-NOG-H3K27me3 complicated (in inexperienced) reveals that the binding of H3K27me3 peptide induces a considerably conformational change of the aspect chain of Phe179. The SIGMAA weighted 2Fo-Fc maps at 1 sigma stage of the Phe179 within the two complexes are proven in meshes. c The methyl teams are particularly anchored by surrounding CH–O hydrogen bonds. The nickel ion is coordinated by NOG, a water molecule, and surrounding residues. d In vitro H3K27me3 demethylation exercise assay of MBP-tagged JMJ13CD and its mutants displaying the mutations of key residues concerned in peptide binding and catalysis are reducing the exercise of JMJ13. The MBP protein was used as a unfavourable management. The chances of the product peptide are proven as means ± SD (n = three). Inexperienced dots denote the person information factors. Supply information are supplied as a Supply Information file

The lively website of JMJ13 shows options typical of α-KG-dependent oxygenases noticed for different jumonji area histone demethylases (Fig. 3c)20,30. The trimethyllysine docks into the deep binding pocket with the three methyl teams anchored by an intensive CH–O hydrogen-bonding community (Fig. 3c), which is important for fixing the conformation of the pinnacle group of the sure trimethyllysine. A Ni2+ ion and an NOG molecule are coordinated by surrounding residues (Fig. 3c).

To dissect the catalytic mechanism, we carried out a structure-based mutagenesis research. To that finish, we produced the JMJ13D236A and JMJ13D296A proteins, which have alanine replacements of the important thing residues concerned in recognition of H3R26 and H3S28, respectively. These proteins confirmed considerably diminished demethylase exercise (Fig. 3d). Mutation of Phe179 to serine, a small hydrophilic residue, produced a average lower of exercise, and alternative by glutamine, a hydrophilic residue with a bigger aspect chain, considerably impaired exercise (Fig. 3d), confirming the significance of the hydrophobic interplay between Phe179 and H3P30. Mutations of trimethyllysine or the nickel ion-binding residues fully abolished the exercise of JMJ13, revealing their important position in catalysis (Fig. 3d).

The H3K27me3 mark resides inside the identical ARKme3S consensus motif because the H3K9me3 mark, making the 2 marks chemically troublesome to tell apart. Within the JMJ13 construction, the precise stacking interplay between H3P30, on the n + three place, and Phe179 selects towards the H3K9me3 substrate, which has an H3G12 on the n + three place, thereby guaranteeing substrate specificity.

Structural comparability with different histone demethylases

JMJ13 belongs to the KDM4 subfamily of histone demethylases based mostly on phylogenetic sequence evaluation of the catalytic area19. Nevertheless, JMJ13 possesses a site structure that doesn’t resemble the 2 recognized KDM4 subfamily H3K27me3 demethylases, REF6 and ELF619, which comprise 4 tandem DNA-binding C2H2 zinc fingers on the finish of the C-terminus (Supplementary Desk 1). As an alternative, JMJ13 resembles the H3K4me3-specific KDM5 subfamily histone demethylases, resembling human KDM5/JARID1 and Arabidopsis JMJ1424,28,29, which comprise a fused helical-zinc finger cassette to the C-terminus of the jumonji area (Supplementary Desk 1). In human, KDM4 subfamily demethylases are H3K9me3/H3K36me3 bi-specific enzymes; furthermore, all recognized human H3K27me3 demethylases belong to the KDM6 subfamily, which is absent in crops (Supplementary Desk 1)19,20. Thus, it’s intriguing plant KDM4 subfamily demethylase employs a KDM5 subfamily-like area structure to conduct the features of human KDM6 subfamily demethylases.

We superposed our JMJ13–H3K27me3 complicated and the human UTX–H3K27me3 complicated (PDB code: 3AVR) based mostly on the jumonji area (Fig. 4a)31,32. The jumonji plus helical domains of the 2 buildings are comparable (Fig. 4a). The area in UTX akin to the JMJ13 C4HCHC area is the zinc binding area, which coordinates just one zinc ion (Fig. 4b)31. The zinc finger domains of JMJ13 and UTX are of comparable topology (Fig. 4b)31. The catalytic facilities of the 2 buildings share very comparable conformations (Fig. 4a)31. The 2 substrate peptides from the 2 complexes possess the identical directionality and have comparable conformations (Fig. 4a and c). Asp1089 of UTX occupies the equal place of Asp236 in JMJ13, which is concerned in H3R26 recognition, indicating the same H3R26 recognition mechanism (Fig. 4c)31. H3S28 is acknowledged by Asp296 in JMJ13, however UTX has no particular interplay with H3S28 (Fig. 4c)31. H3P30 stacks with Phe179 in JMJ13, however with Professional1144 in UTX, which is in a unique and non-homologous place (Fig. 4d)31. The popularity of H3P30 is important for distinguishing between H3K27me3 and H3K9me3, indicating that JMJ13 and UTX have independently developed completely different particular stacking interactions with H3P30 to make sure substrate specificity31.

Fig. fourFig. 4

Comparability of JMJ13 and different histone demethylases. a A superposition of the JMJ13-NOG-H3K27me3 complicated (in inexperienced) and UTX-NOG-H3K27me3 (in orange, PDB code: 3AVR) reveals the same total buildings and peptide-binding websites. b The buildings of the C4HCHC twin zinc finger of JMJ13 (left panel, in inexperienced) and the C4 single zinc finger of UTX (proper panel, in orange) displaying completely different zinc coordination however comparable folding topology. c The superposition of the JMJ13-NOG-H3K27me3 complicated (in inexperienced) and the UTX-NOG-H3K27me3 (in orange) reveals that the 2 proteins make use of correspondent residues Asp236 of JMJ13 and Asp1089 of UTX to acknowledge H3R26 within the two complexes, respectively. In distinction, JMJ13 acknowledge H3S28 by a aspect chain hydrogen bond with Asp296, however UTX lacks this recognition. d The superposition of the JMJ13-NOG-H3K27me3 complicated (in inexperienced) and UTX-NOG-H3K27me3 (in orange) reveals that JMJ13 acknowledges H3P30 by Phe179, whereas UTX acknowledges H3P30 by Professional1144 at completely different positions. e A superposition of JMJ13-NOG-H3K27me3 complicated (in inexperienced) and JMJ14-NOG-H3K4me3 complicated (in magenta, PDB code: 5YKO) reveals virtually similar total buildings. f An in depth view of the comparability of the zinc finger domains of JMJ13 (in inexperienced) and JMJ14 (in magenta). The 2 zinc finger domains possess comparable total buildings with the primary zinc ions occupying the identical place however the second zinc ions situated at completely different positions. g The detailed buildings of the zinc coordination of two zinc ions from JMJ13 (higher panel, in inexperienced) and the JMJ14 (decrease panel, in magenta) present that the primary zinc ions have the identical coordination however the second ones have completely different coordination. h The schematic illustration of the sequence motif and coordination of the zinc ions of the JMJ13 C4HCHC area (higher panel) and JMJ14 C5HC2 area (decrease panel). The spacing residues are denoted as X

The KDM4 subfamily member JMJ13 possesses a jumonji-helical-zinc finger domain-like association of the catalytic fragment, just like the KDM5 subfamily member JMJ14 (Supplementary Desk 1)24. The 2 proteins have comparable area preparations and total buildings with an RMSD of 1.99 Å (Fig. 4e)24. In addition to the conserved jumonji and helical domains, the peptide spine of the C4HCHC zinc finger of JMJ13, and the C5HC2 zinc finger of JMJ14 are fairly comparable (Fig. 4f)24. Nevertheless, the zinc coordination topologies differ. The primary Zn2+ ion occupies a similar place in each JMJ13 and JMJ14. Nevertheless, the second zinc ion occupies completely different websites within the two proteins and every is coordinated in a different way (Fig. 4f–h), but the proteins preserve very comparable total zinc finger area topologies (Fig. 4f–h). Whether or not this relationship is indicative of comparable operate is presently not recognized.

Our structure-based sequence alignment recognized the important thing residues concerned in H3K4me3 recognition by JMJ14 and H3K27me3 recognition by JMJ13. These key residues are usually not conserved (Supplementary Fig. four), though usually the JMJ13 and JMJ14 present comparable total buildings (Fig. 4e). It means that the conserved jumonji-helical-zinc finger cassette could operate as a basic scaffold for histone demethylases, with particular options evolving inside particular person demethylases to allow differential regulation and substrate specificity.

Ectopic JMJ13 expression causes pleiotropic defects

H3K27me3 features as a repressive histone mark18,33. To analyze the operate of JMJ13, which removes H3K27me3, we generated JMJ13-GFP (JMJ13ox) over-expressing transgenic crops. JMJ13ox crops had pleiotropic phenotypes, with dwarf seedlings, early flowering, and upward curling leaves (Supplementary Fig. 5a), all of which elevated in severity with rising JMJ13 expression ranges (Supplementary Fig. 5b). The JMJ13-GFP seedlings confirmed phenotypes just like H3K27me3 silencing-deficient mutants lhp134 or REF6 over-expression crops, with diminished dimension of leaf floor cells (Supplementary Fig. 5c, d)25.

Some H3K27me3 goal genes, together with APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), AGAMOUS (AG), and SEPALLATA3 (SEP3), that are usually expressed in flowers, had been ectopically activated within the JMJ13ox seedlings. (Supplementary Fig. 5e). Different genes, for instance, FT and SOC1, that are expressed at low ranges in 10-day-old seedlings, and the meristem operate genes KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 1 (KNAT1) and TOUCH four (TCH4), all of that are targets of REF6, had been additionally upregulated in JMJ13ox seedlings (Supplementary Fig. 5e). According to these modifications in gene expression, H3K27me3 however not H3K4, H3K9, and H3K36 confirmed a robust world discount within the two robust JMJ13ox transgenic strains (Supplementary Fig. 5f).

JMJ13 represses flowering

We recognized jmj13, a T-DNA insertion within the second intron of JMJ13 (GABI116B03, At5g46910) (Supplementary Fig. 6a)35. No JMJ13 transcript was detected within the jmj13 mutant (Supplementary Fig. 6b–c). The plant Polycomb-group (Computer-G) protein CURLY LEAF (CLF), which features as an H3K27me3 methyltransferase, is required to repress targets resembling AG and SHOOTMERISTEMLESS (STM). CLF, thereby controls flowering time, leaf morphology, and floral organogenesis36,37. The jmj13 clf double mutant phenotype was just like however weaker than that of clf when it comes to flowering time, curling of leaves (Supplementary Fig. 6d–g) and fertility, indicating that jmj13 partially suppresses the clf phenotype. Persistently, the expression ranges of some H3K27me3 goal genes that had been ectopically activated in clf had been diminished within the jmj13 clf double mutant (Supplementary Fig. 6h). Nevertheless, the expression of SEP3 and AG shouldn’t be diminished in jmj13 clf relative to clf, though the leaf curling and early flowering phenotypes are weakly suppressed within the jmj13 clf double mutant. This may very well be as a result of different H3K27me3 goal genes, or as a result of practical redundancy with REF6, ELF6 or different doable H3K27me3 demethylases. Collectively, these genetic interactions exhibit that JMJ13 features as an H3K27me3 demethylase that partially antagonizes the H3K27 methyltransferase CLF in vivo, though additional investigation can be required to disclose the small print of crosstalk between CLF and JMJ13 and whether or not the phenotypic suppression is because of direct antagonistic operate at specific loci.

We additional measured flowering time and scored whole leaf quantity at bolting, in numerous photoperiod situations (LD and SD) and underneath completely different ambient temperature situations (low, 16 °C; and excessive, 28 °C). The jmj13 mutants displayed early flowering in LD situations38, no matter low or excessive temperatures. Nevertheless, jmj13 crops flowered early at 28 °C, however not at 16 or 22 °C, when grown in SD situations (Supplementary Fig. 7).

To confirm that lack of JMJ13 operate is accountable for the early flowering phenotype of jmj13, we remodeled jmj13 with a 7.zero kb genomic assemble (gJMJ13) together with 2.6 kb upstream and zero.5 kb downstream of the coding area. All of the transgenic crops exhibited comparable flowering time to Col crops at 22 °C in LD situations (Fig. 5a). In contrast with the totally rescued pJMJ13:JMJ13-HA jmj13 strains, strains containing a assemble with level mutations within the two conserved iron-binding amino acids, H293A and E295A (pJMJ13:JMJ13mu-HAjmj13), confirmed comparable flowering time to jmj13 mutants. This means that the enzyme exercise is critical for JMJ13 operate, along with the jumonji and zinc finger domains (Fig. 5a, b).

Fig. 5Fig. 5

JMJ13 negatively modulates flowering time in a temperature- and photoperiod-dependent method. a Three complementary strains Comp −1, −2 and −three (pJMJ13:JMJ13-HAjmj13) confirmed comparable flowering time with wild sort in 22 °C LD situations, however the complementary strains Comp-Mu −1, −2 and −three with website mutation within the two conserved iron-binding amino acids (pJMJ13:JMJ13mu-HAjmj13), the complementary strains with truncation in JmjC Comp-ΔJmjc −1,−2, −three (pJMJ13:JMJ13-ΔJmjc-HAjmj13) and zinc finger area Comp-ΔZF −1, −2, −three (pJMJ13:JMJ13-ΔZF-HAjmj13) stay early flowering. The plant photos had been created by the authors on this analysis. b The flowering time of complementary strains (Comp −1, −2, −three); (Comp-Mu −1, −2, −three); (Comp-ΔJmjc −1, −2, −three); (Comp-ΔZF 1, −2, −three) had been assessed by counting rosette leaf numbers in bolting seedlings grown underneath 22 °C, 28 °C LD, and 22 °C, 28 °C SD situations. Values are means ± SE of indicated variety of crops for 3 unbiased organic repeats. N quantity was marked within the backside of the column. Pupil’s t exams was used to calculate the P worth between Col and different strains. ****P worth < zero.0001; ***P < zero.zero01; **P < zero.01; *P worth < zero.05; Bar = 1 cm. The person information factors are proven as dots. Supply information are supplied as a Supply Information file

In Col crops, LD situations and elevated ambient temperatures promote flowering. Nevertheless, when grown in SD situations, elevated temperature may, to some extent, overcome the unfavorable day-length situation to advertise flowering. According to these phenotypic observations, the reproductive improvement of jmj13 mutants was hypersensitive to, and promoted by, each LD situations and elevated ambient temperatures (Fig. 5b). Collectively, these outcomes recommend that JMJ13 negatively modulates flowering time underneath LD and in SD underneath increased temperature.

Crosstalk between JMJ13 and different flowering time regulators

To analyze the mechanism underlying JMJ13′s regulation of flowering in response to temperature, we crossed jmj13 with flm and svp. The flm jmj13 and svp jmj13 double mutants flowered earlier in all situations, together with 22 °C and SD, whereas jmj13 confirmed a wild-type flowering phenotype in 22 °C and SD (Supplementary Fig. 8a). Over-expression of FLM-β however not FLM-δ repressed early flowering in jmj13 (Supplementary Fig. 8b–d). These genetic information are in step with the likelihood that FLM and SVP could act downstream or in parallel with JMJ13 within the regulation of flowering.

Within the photoperiodic pathway, the circadian clock-regulated GIGANTEA (GI) protein positively regulates the oscillating expression of CO2,5. The transcript stage and protein exercise of CO are coordinately managed by the sunshine signaling pathway and the circadian clock. FT expression is activated by CO and repressed by FLM and SVP. Thus, transcriptional regulation of FT is a key output ensuing from integration of photoperiodic cues with temperature alerts. The flowering time of double mutants of ft jmj13, co jmj13, and gi jmj13 had been assessed by counting rosette and cauline leaf numbers in bolting seedlings (Supplementary Fig. 9a–d). We discovered that every one three double mutant strains displayed comparable rosette leaf numbers to the ft, co, and gi single mutants (Supplementary Fig. 9a–d), and FT expression was upregulated in jmj13 in all early flowering situations (Supplementary Fig. 9e). The mutations of CO, GI, and FT repressed early flowering in jmj13. These genetic interactions point out that JMJ13 could crosstalk with GI/CO in flowering time regulation.

Moreover, we carried out RNA-seq for Col and jmj13 grown underneath 22 °C LD situations. The expression of SVP decreased in jmj13 in contrast with wild sort (Supplementary Fig. 10a, b), which is in step with the genetic information. Against this, the expression of CO, Late Elongated Hypocotyl (LHY) and Circadian Clock Related 1 (CCA1) was upregulated in jmj13 (Supplementary Fig. 10a, b and Supplementary information 1), indicating that many genes are regulated straight or not directly by further mechanisms, resembling circadian rhythm. Curiously, we noticed that top temperature and long-day photoperiod induced JMJ13 expression and enhance JMJ13 protein accumulation in 7-day-old seedlings (Supplementary Figs. 10c, d and 11). These outcomes advised that JMJ13 could have an effect on flowering time by means of temperature- and photoperiod-mediated pathways.

We additional analyzed the H3K27me3 ranges in Col and jmj13 by chromatin immunoprecipitation adopted by sequencing (ChIP-Seq) in numerous day-length and temperature situations. Nevertheless, we didn’t observe the over-accumulation of H3K27me3 in jmj13 at FLM/SVP and CO/GI and no important variations between Col and jmj13 had been noticed at these loci (Supplementary Fig. 12). This may increasingly because of the practical redundancy of ELF6, REF6 or different doable H3K27me3 demethylases. These observations are in in step with latest report that world H3K27me3 ranges had been strongly elevated solely within the triple elf6 ref6 jmj13 mutant in Arabidopsis38.


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