Jumonji C Domain Protein JMJ705-Mediated Removal ofHistone H3 Lysine 27 Trimethylation Is Involved inDefense-Related Gene Activation in RiceC W

Tiantian Li,a Xiangsong Chen,a Xiaochao Zhong,a Yu Zhao,a Xiaoyun Liu,a Shaoli Zhou,a Saifeng Cheng,a

and Dao-Xiu Zhoua,b,1

a National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, Chinab Institut de Biologie des Plantes Unité Mixte de Recherche 8618 Université Paris-sud 11, 91405 Orsay, France

ORCID ID: 0000-0002-1540-0598 (D-X. Z.).

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation.Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmentalprocesses and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 duringgene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histonelysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and duringpathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation ofH3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogenXanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealedthat JMJ705 is involved in methyl jasmonate–induced dynamic removal of H3K27me3 and gene activation. The results suggest thatJMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genesand increase their basal and induced expression during pathogen infection.

INTRODUCTION

Histone modifications such as acetylation and methylation areimportant epigenomic information for gene regulation and genomeactivity. Acetylation of histone Lys residues induces permissivechromatin structure for gene activation, whereas histone Lysmethylation may have a positive or a negative effect on gene ex-pression, depending on the Lys position and the degree of meth-ylation (Berger, 2007). For instance, dimethylation of histone H3lysine 9 (H3K9me2) is almost exclusively associated with hetero-chromatin regions and is required for repression of repetitive ge-nomic sequences in plants, whereas trimethylation of H3 lysine 27(H3K27me3) is negatively correlated with gene expression. Con-versely, trimethylation of H3 lysine 4 (H3K4me3) and H3 lysine 36(H3K36me3) are associated with active genes (Liu et al., 2010).

Genome-wide analysis in Arabidopsis thaliana and rice (Oryzasativa) indicates that H3K4me3 is preferentially associated withactively transcribed genes (Zhang et al., 2007; Li et al., 2008; Huet al., 2012), whereas H3K27me3 is found mostly on repressedgenes (Turck et al., 2007; He et al., 2010; Hu et al., 2012). Howthese histone modifications affect gene expression remains un-clear. It is suggested that the methylation marks are recognized

and bound by specific proteins that may act as effectors to reg-ulate transcription (Shi et al., 2006; Vermeulen et al., 2007). How-ever, these marks can also be a consequence of a gene activationor repression process to mark and possibly to memorize geneexpression states (Bonasio et al., 2010; Muramoto et al., 2010).Histone methylation marks are established by evolutionarily con-served SET-domain proteins (named after three Drosophila mela-nogaster genes: Su[var]3-9, Enhancer of zeste, and Trithorax,which methylate H3K9, H3K27, and H3K4, respectively). During thepast few years, plant SET-domain proteins involved in H3K4me3and H3K27me3 have been shown to play important roles in plantdevelopmental gene expression (Pien and Grossniklaus, 2007; Liuet al., 2010; Berr et al., 2011). Changes of histone methylationpatterns also occur on inducible genes under stress conditions,suggesting that histone methylation is dynamic and may play a rolein inducible gene expression. For instance, H3K4me3 was foundto be increased on responsive genes upon stress treatment(Kim et al., 2008a; van Dijk et al., 2010). Accordingly, ArabidopsisTrithorax1, which is involved in H3K4 trimethylation, was found tobe necessary for gene induction by stress signals (Alvarez-Venegasand Avramova, 2005; Alvarez-Venegas et al., 2007; Ding et al.,2011). Conversely, H3K27me3 was removed from several induciblegenes upon application of the inductive signals (Kwon et al., 2009;Kim et al., 2010), implying that active removal of this histonemethylation mark may be associated with gene induction. How-ever, the role of H3K27me3 demethylation in stress-responsivegene activation and stress tolerance has not been clarified.Two protein groups are known to be involved in histone Lys

demethylation. The Lys- specific demethylase 1 group deme-thylases are flavin-dependent amine oxidases that reverse mono-methylated or dimethylated histone Lys (Shi et al., 2004). The

1Address correspondence to dao-xiu.zhou@u-psud.fr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Dao-Xiu Zhou (dao-xiu.zhou@u-psud.fr).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.113.118802

The Plant Cell, Vol. 25: 4725–4736, November 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

Figure 1. JMJ705 Is a Histone H3K27me2/3 Demethylase.

(A) Schematic presentation of the vector 35S-JMJ705-FLAG-HA. The relative positions of JmjN, JmjC, and zinc-finger (ZnF) domains are indicated.Arrow indicates the position of the substitution mutation H244A.(B) In vitro demethylase activity of JMJ705. Bulk histone was incubated with (+) or without (2) tobacco cell-expressed JMJ705-FLAG-HA fusion proteinand analyzed by protein gel blots using antibodies against specific histone modification modules indicated on the left. The same blots were analyzed byanti-H3. JMJ705-FLAG-HA was revealed by anti-HA.(C) In vivo histone demethylase activity of JMJ705. The 35S-JMJ705-FLAG-HA construct was transfected into tobacco leaf cells. Nuclei isolated fromleaves were inspected for expression of the fusion protein (stained with anti-HA and indicated by arrows) and then examined for histone methylationlevels (stained by DAPI) by using antibodies against specific histone H3 methylation modules indicated on the left. The H244A substitution mutation wastested similarly with anti-H3K27me2. At least 30 nuclei expressing JMJ705 fusion per transfection were observed and imaged. Bar = 25 mm.(D) Histone H3K27me2/3, H3K4me3 and H3K9me3methylation levels in wild type (ZH11, HY), JMJ705 overexpression (OX-5) and T-DNAmutant (jmj705) plantsrevealed by protein gel blots. Only one set of several repeated data is shown. H3 was detected as loading control. Mean signals 6 SD (from three replicates)relative to the wild type (set at 1) are indicated below the bands. Significance of differences was determined using t tests. *P < 0.05; **P < 0.01.

4726 The Plant Cell

Jumonji C (jmjC) group demethylases remove preferentially di-methylated and trimethylated histone lysines through ferrous ion[Fe(II)] and a-ketoglutaric acid–dependent oxidative reactions(Tsukada et al., 2006). JmjC proteins from yeast (Saccharomycescerevisiae) and animal cells are classified into seven phylogeneticsubgroups, each of which demethylates specific Lys residues(Mosammaparast and Shi, 2010). Plant jmjC proteins are generallyconserved with yeast and animal homologs, while there exista subgroup of more divergent jmjC proteins (Sun and Zhou, 2008).Members of this subgroup (i.e., Arabidopsis JMJ14, JMJ15, andJMJ18 and rice JMJ703) have been reported to be H3K4 deme-thylases and to regulate diverse aspects of chromatin function andplant development (Deleris et al., 2010; Lu et al., 2010; Searle et al.,2010; Yang et al., 2012a, 2012b; Chen et al., 2013; Cui et al., 2013).Conversely, the JMJD3/UTX (ubiquitously transcribed tetra-tricopeptide repeat, X chromosome) subgroup proteins, whichexhibit H3K27 demethylase activities in mammalian cells, are notfound in plants. Recent results showed that a member of theJMJD2 subgroup from Arabidopsis, known as RELATIVE OFEARLY FLOWERING6 (REF6/JMJ12) (Noh et al., 2004), can de-methylate H3K27 in Arabidopsis (Lu et al., 2011). However, otherJMJD2 members have been shown to demethylate H3K9me2/3and/or H3K36me3 (Shin and Janknecht, 2007; Sun and Zhou,2008). These results suggest that plant jmjC proteins may havediversified demethylation specificities to histone lysines.

In this work, we have studied the enzymatic activity and func-tion of gene regulation and plant growth of a rice JMJD2 member,namely JMJ705. Our results demonstrate that JMJ705 is anH3K27 demethylase preferentially involved in biotic stress-responsive gene expression. An increase in JMJ705 expressionin transgenic plants removes H3K27me3 from defense-relatedgenes, induces their expression, and enhances plant resistanceto the rice bacterial blight disease pathogen Xanthomonas oryzaepathovar oryzae (Xoo). Mutation of the gene reduced the level ofresistance. Importantly, we show that the expression of JMJ705is induced by stress signals including methyl jasmonate (MeJA)and Xoo infection and that JMJ705 is involved in MeJA-induceddynamic removal of H3K27me3 from responsive genes and theiractivation. The results suggest that JMJ705-mediated H3K27me3demethylation may contribute to a more sustained activation ofdefense-related genes by potentiating their basal and inducedexpression levels during biotic stress.

RESULTS

JMJ705 Is an H3K27 Demethylase

We previously showed that the rice JMJD2 protein JMJ706 is anH3K9 demethylase (Sun and Zhou, 2008). To determine the de-methylase activity of other rice JMJD2 proteins, we expressed theFLAG-hemagglutinin (HA)-tagged JMJ705 protein in transfectedtobacco (Nicotiana benthamiana) cells (Figure 1A). The demethy-lated histones were then analyzed by protein gel blots using spe-cific antibodies against histone H3 modification modules. Theanalysis revealed that the presence of JMJ705 clearly reduced thelevels of H3K27me3 and H3K27me2, but not that of H3K27me1,H3K4me3, H3K9me3, or H3K36me3 (Figure 1B), suggesting that

the demethylase activity of JMJ705 was specific to H3K27me3 andH3K27me2.To confirm the in vitro enzymatic activity, we transiently

expressed JMJ705-FLAG-HA in tobacco leaf cells and testedthe demethylase activity in vivo. Transfected cells were immu-nostained first with anti-HA to identify cells expressing theJMJ705 fusion protein, and then with antibodies against specificmethylated histone modules. In nuclei expressing the JMJ705fusion protein, H3K27me2 was shown to be clearly reduced, butH3K9me2, H3K4me3, H3K36me1, and H3K36me2 did not dis-play any discernible change compared with nontransfectednuclei (Figure 1C). We tested similarly for H3K27me3, but thecommercial antibodies that we tested could not produce anysignal in the immunostaining assays. The Fe(II) and a-ketoglutaricacid binding residues are highly conserved in jmjC demethy-lases and are critical for the demethylation activity in plant cells

Figure 2. JMJ705 Overexpression Produces a Leaf Lesion–MimicPhenotype at the Mature Stage.

(A) Leaf phenotype and JMJ705 transcript levels revealed by RNA gelblot in the wild type and five transgenic lines. rRNAs are shown asloading controls.(B) Comparison of wild-type (left) and overexpression (right) plants atthe mature stage.(C)mRNA levels of defense-related genes detected by real-time RT-PCRin wild-type (ZH11) and overexpression (OXJMJ705-5 and OXJMJ705-14)flag leaves. Bar indicates mean 6 SD from three biological repeats.[See online article for color version of this figure.]

H3K27me3 Removal in Disease Resistance 4727

(see Supplemental Figure 1 online; Lu et al., 2011; Chen et al.,2013). To confirm the results, we made a substitution mutation ofthe key Fe(II) binding residue His244 by Ala, and tested it in thein vivo assays. The substitution eliminated the H3K27me2 de-methylase activity of the protein (Figure 1C). The in vitro and invivo data together indicated that JMJ705 was a histone deme-thylase specific to H3K27me2/3.

JMJ705 Is Involved in Rice Defense-RelatedGene Expression

RT-PCR analysis indicated that JMJ705 was expressed in alltested tissues/organs, with a relatively higher level in rice leaves,and was found to be induced by NaCl, abscisic acid, ethylene(ACC), and JA (see Supplemental Figure 2 online), suggesting thatJMJ705 might be involved in stress responses. To study whetherJMJ705 expression was responsive to biotic stress, we inoculatedtwo rice varieties (IR24, a susceptible variety, and BB13, a resistantvariety) with Xoo (PXO99) and measured JMJ705 transcripts atdifferent time points. In both varieties, JMJ705 mRNA levels in-creased to 8- to 10-fold at 12 h after inoculation (see SupplementalFigure 2 online). This suggested that JMJ705 might be involved in

biotic stress response. To study whether increased expression ofJMJ705 could reduce the resting level of H3K27me3 and affectgene expression and plant growth, we produced transgenic riceplants overexpressing JMJ705-FLAG under the strong maizeubiquitin promoter in the Zhonghua11 (ZH11) background (Figure2A). Protein gel blot analysis indicated that the overexpression ofthe gene resulted in a reduction of the overall H3K27me2/3 levelsin the transgenic plants compared with wild type (Figure 1D). Theoverexpression transgenic lines displayed a leaf lesion-mimicphenotype at the mature stage (Figures 2A and 2B). The leaf le-sion-mimic phenotype is usually a result of activation of bioticstress responses in plant tissues (Lorrain et al., 2003). To testthis hypothesis, we analyzed the expression of biotic stress-responsive marker genes in the flag leaves of mature over-expression plants. Genes involved in oxidative stresses such asclass III peroxidase genes (POX5, POX8, and POX22), JA bio-synthesis and signaling pathway genes (LIPOXYGENASE [LOX],ALLENE OXIDE SYNTHASE2 [AOS2], OXOPHYTODIENOATEREDUCTASE7 (OPR7), and jasmonic acid–inducibleMYB (JAMYB)],and pathogenesis-related genes (PR5 and PR10) were selected foranalysis. These genes have been used as biotic stress-responsivemarker genes in rice (Chittoor et al., 1997; Lee et al., 2001; van Loon

Figure 3. JMJ705 Overexpression Enhances Rice Resistance to the Bacterial Pathogen Xoo.

(A) and (B) Three wild-type (ZH11) plants and the T2 segregates of line 5 (OXJMJ705-5) and line 14 (OXJMJ705-14) were inoculated with the Xoo strainPXO99 that causes rice blight disease. Genotyping of the T2 plants for the presence of the transgene is shown for each line. The percentages of the leaflesion areas were measured 14 d after inoculation. Bar indicates mean 6 SD from four to five replicates for the lesion area.(C) Leaf phenotype after PXO99 inoculation.(D) Bacterial growth rate (log [COLONY-FORMING UNITS/leaf]) measured at 0 (2 h post inoculation) and 3 to 12 d postinoculation (dpi). Significance ofbacterial growth differences between wild-type and overexpression plants was determined by Student’s t tests. Bar indicates mean 6 SD from fiveinoculated plants. *P < 0.05; **P < 0.01.[See online article for color version of this figure.]

4728 The Plant Cell

et al., 2006). In the overexpression flag leaves, the tested genesshowed higher expression levels than in the wild type (Figure 2C),indicating that elevated JMJ705 expression led to de-repression ofthe biotic stress-responsive genes under normal growth con-ditions. To test whether the overexpression of JMJ705 alteredplant resistance to pathogens, we inoculated wild-type (ZH11) andT1 generation (segregating) plants of line 5 (17 individuals) and line

14 (13 individuals) at the mature stage with the PXO99 strain ofXoo, which causes rice blight disease. Fourteen days after in-oculation, lesion areas were surveyed. In wild type and transgenicnegative segregates, 35 to 45% of the leaf areas showed necrosis,whereas only 10 to 20% (for line 14) or less than 30% (for line 5)of the leaf areas in the transgenic positive segregates displayednecrosis (Figures 3A and 3B). The observations were furtherconfirmed by inoculation with another Xoo isolate, PXO347 (seeSupplemental Figure 3 online). PXO99 growth rates were sig-nificantly slower in the overexpression leaves (Figure 3C). Theseresults suggest that elevated expression of JMJ705 inducesdefense-related gene expression, which may lead to enhancedplant resistance to the bacterial pathogen.To study the effect of loss of function of JMJ705, we char-

acterized a T-DNA insertion mutant line of the gene inHwayoung (HY) background. Analysis of the genomic sequenceconfirmed that the T-DNA was inserted in the zinc-finger motifregion of the gene (Figures 4A and 4B). A single copy of the

Figure 4. Characterization of a T-DNA Insertion Mutation of JMJ705.

(A) Schematic representation of the gene structure and position of theT-DNA insertion (open triangle). The positions of the primers used forgenotyping and RT-PCR are indicated.(B) Genotyping of nine segregates and the wild type (HY) using the twoprimer sets as indicated.(C) DNA gel blot analysis of copy number of the T-DNA insertion.(D) RT-PCR detection of JMJ705 transcripts.(E) Mature stage and panicle phenotype comparison between the wildtype (left) and mutant plant (right).(F) Leaf lesion area (%) in three wild-type (HY) and nine T-DNA (jmj705)plants 14 d after inoculation with the Xoo strain PXO99. Bar indicatesmean 6 SD from four to five replicates.(G) Leaf phenotype.(H) Bacterial growth rate on jmj705 mutant leaves compared with HY asdescribed in Figure 3.[See online article for color version of this figure.]

Figure 5. H3K27me3 and H3K4me3 ChIP-seq Intensities of Genes thatAre Upregulated or Downregulated in JMJ705 Overexpression PlantsCompared with the Genome-Wide Levels.

Numbers of sequenced tags (y-axis) per each 5% of the genic region(black box) or per 100-bp intervals in the 2 kb upstream and 2 kbdownstream regions (line, x-axis) are shown. Arrow indicates the di-rection of transcription.

H3K27me3 Removal in Disease Resistance 4729

T-DNA was detected in the mutant (Figure 4C). RT-PCR analysisindicated that the insertion truncated the full-length transcript(Figure 4D). Protein gel blot analysis did not reveal any clear changeof H3K27me2/3 levels compared with the wild type (Figure 1D). It ispossible that JMJ705-dependent demethylation might have littleeffect on the overall level of H3K27me3 in the seedlings undernormal growth conditions. The mutant displayed reduced plantheight and partial sterility (Figure 4E; see Supplemental Table 1online). When challenged with the Xoo strain PXO99, the mutantplant leaves showed larger necrotic areas and faster bacterial growththan the wild type (Figures 4F and 4H), suggesting that the mutantmight be more susceptible to the pathogen than the wild type.

JMJ705 Preferentially Activates H3K27me3-MarkedStress-Responsive Genes

To evaluate genome-wide effects of JMJ705 overexpression ongene expression, we used the Affymetrix microarray platform toanalyze transcriptomes of the wild type (ZH11 and HY), homozygousoverexpression (line 14 and line 5 combined with the same seedlingnumber/fresh weight), and the T-DNA mutant plants 14 d after ger-mination under normal growth conditions. Two biological repeatswere performed. In the overexpression lines, 301 genes showed >2fold upregulation and 105 genes showed >2 fold downregulationcompared with wild type (ZH11) (P < 0.05) (see Supplemental DataSet 1 online). Gene ontology (GO) analysis revealed that upregulatedgenes were significantly enriched for the categories of stress-responsive genes (89 of 301; P < 0.001) (see Supplemental Table 2online), while downregulated genes did not show any enrichment. Inthe T-DNA mutant 640 were upregulated, and 332 were down-regulated (>2-fold; P < 0.05), compared with HY (see SupplementalData Set 2 online). GO analysis revealed that upregulated genesin the mutant were also enriched for stress-responsive genes(see Supplemental Table 2 online). However, very few geneswere found to be deregulated in both overexpression and T-DNAplants, implying that the effect of the mutation (which might notaffect the overall levels of H3K27me2/3) and that of overexpressionof the gene (which likely decreased the overall H3K27me2/3)might affect the expression of different target genes under normalgrowth conditions.

To study whether the overexpression of JMJ705 preferentiallyaffected the expression of H3K27me3-marked genes, we com-pared the microarray data with genome-wide H3K27me3 chro-matin immunoprecipitation sequencing (ChIP-seq) data that wereobtained from rice seedlings grown under the same conditions (Huet al., 2012). Because H3K4me3 and H3K27me3 are antagonisthistone methylation markers for gene activity, we also analyzedH3K4me3 ChIP-seq data obtained at the same growth conditions(Hu et al., 2012). The analysis revealed that upregulated genes inthe overexpression plants were significantly enriched for H3K27me3(P = 3.596e-10), because 103 of the 301 (38.9%) upregulated genesin JMJ705 overexpression plants were marked by H3K27me3compared with 10,831 of 56,797 genes genome-wide (19%) (seeSupplemental Table 3 online). For downregulated genes, only 16of 105 (12.5%) were marked by H3K27me3 (see SupplementalTable 3 online). Conversely, downregulated, but not upregulated,genes were found to be enriched for H3K4me3 (P = 2.2e-16) (seeSupplemental Table 3 online).

Analysis of ChIP-seq read intensity (i.e., number of sequencedtags per each 5% of the genic region or per 100-bp intervals inthe 2 kb upstream and downstream regions) over the deregu-lated genes revealed that H3K27me3 intensity was much higherin upregulated genes than the genome-wide average, whereasthat of downregulated genes was close to the average level(Figure 5). The analysis indicated that the upregulated genesdisplayed a lower than average level of H3K4me3 in wild-typeplants, whereas the downregulated genes showed a higher thanaverage level of H3K4me3 (Figure 5). The analysis suggestedthat increased JMJ705 expression preferentially activated silentor underexpressed genes that were marked by a relatively highlevel of H3K27me3 and a relatively low level of H3K4me3.To validate the microarray data, we randomly selected 27

upregulated genes in the overexpression plant for real-time RT-PCR analysis (see Supplemental Table 4 online). The tested geneswere clearly upregulated in the overexpression lines comparedwith wild type (see Supplemental Figure 4 and SupplementalTable 4 online). In addition, many of the 27 genes were found tobe highly induced by MeJA (see Supplemental Figure 4 andSupplemental Table 4 online), which suggested that JA-induciblegenes might be among the preferential targets of JMJ705. Tostudy whether the upregulation of these genes was correlated toH3K27 demethylation, we performed ChIP assays to analyzeH3K27me3, H3K27me2, H3K4me3, H3K9me3, and H3K36me3

Figure 6. H3K27me2/3 Removal Is Related to JMJ705-Mediated GeneUpregulation.

Chromatin fragments isolated from wild-type (ZH11) and overexpressionplants (OXJMJ705) were immunoprecipitated with antibodies againstH3K4me3, H3K9me3, H3K36me3, H3K27me2, and H3K27me3 as in-dicated, and analyzed by real-time PCR using primer sets correspondingto transcriptional start site regions of 12 upregulated genes (numberedas in Supplemental Table 4 online). Chromatin fragments of JMJ705-FLAG overexpression plants were immunoprecipitated by anti-FLAG andanalyzed by PCR using the same primer sets (right bottom).

4730 The Plant Cell

levels near the transcriptional start site of 12 of the 27 genes inwild-type and overexpression plants. In the overexpression plants,H3K27me3 and H3K27me2 levels were clearly decreased in mostof the tested genes, while H3K4me3, H3K9me3, and H3K36me3levels were not clearly changed on most of the tested genes (Figure6). This analysis suggested that upregulation of most the testedgenes in the overexpression plants was correlated to demethyla-tion of H3K27me2/3, suggesting that the removal of H3K27me2/3might be related to the activation of the associated genes.

Elevated JMJ705 Enhances MeJA-Induced Gene Expression

To study whether JMJ705 plays a role in the process of JA-inducedgene activation, 14-d-old seedlings of JMJ705 overexpression and

wild-type (ZH11) plants were treated by MeJA (0.2 mM) for 0, 2, 4,12, and 24 h. Transcript levels of two JA-induced genes TERPENESYNTHASE3 (TPS3) and Os07g11739 (see Supplemental Table 4online), and the JA-inducible marker genes PR10 and JAMYB,weredetermined by quantitative RT-PCR (Figure 7). Before induction,the mRNA levels of TPS3 and Os07g11739 were ;5 to 20 timeshigher in the overexpression lines than in wild type, confirming themicroarray data (see Supplemental Table 4 online). By contrast, themRNA levels of JAMYB in the overexpression seedlings were notdifferent from that in wild type seedlings, suggesting that increasedJMJ705 expression had different effects on the basal expression ofthe two types of genes. However, increases of JAMYBmRNA weredetected in the overexpression plants at the mature stage (flagleaves, Figure 2C). A possible explanation is that the increases

Figure 7. JMJ705 Enhances JA Induction of Gene Expression.

mRNA levels of JA-responsive genes JAMYB, PR10, TPS3, and Os07g11739 (genes 5 and 7 in Supplemental Table 4 online) in wild type, two lines ofoverexpression and one line of RNAi plants (all in ZH11 background) treated by MeJA (0.2 mM) for 0 to 24 h were analyzed by quantitative RT-PCR.Relative mRNA levels are presented with wild type at 0 h set as 1. Because of great variation between induction experimental repeats, data from threebiological repeats are presented individually. Insets show relative mRNA levels during the first hours of induction for TPS3 and 07g11739. Bar indicatesmean 6 SD from three technical repeats.

H3K27me3 Removal in Disease Resistance 4731

might be due to a general activation of defense genes during ne-crotic phenotype production at the mature stage. The highest in-duction of TPS3, Os07g11739, and PR10 by MeJA occurred at12 h after treatment, whereas that of JAMYB seemed to occurearlier. JMJ705 overexpression had no clear effect on the inductionof TPS3 and Os07g11739 during the first (2 to 4) hours, but highlyincreased the induction levels at 12 h after treatment comparedwith wild type. In a lesser extent, a similar effect was observed forthe induction of PR10. The induction of JAMYBwas also enhancedin the overexpression plants. To confirm the results, we obtainedJMJ705 RNA interference (RNAi) plants in the ZH11 background(see Supplemental Figure 5 online). The MeJA induction of the fourgenes was reduced in the RNAi plants compared with the wild type(Figure 7). These data indicate that JMJ705 was involved in theinduction process of JA-responsive genes.

JMJ705 Is Involved in MeJA-Induced Removal of H3K27me3from Responsive Genes

To study whether JMJ705-mediated demethylation of H3K27me3was implicated in JA induction of gene activation, we measuredH3K27me3 levels by ChIP on JA-responsive genes in wild type,JMJ705 overexpression, and RNAi 14-d-old seedlings treated withJA for 0, 8, and 12 h. The analysis revealed the following ob-servations (Figure 8). First, before MeJA treatment, the level ofH3K27me3 on TPS3 and Os07g11739 was ;8 to 16 times higherthan that on PR10 or JAMYB in the wild type, suggesting that TPS3and Os07g11739 might be more subjected to regulation byH3K27me3. Second, in JMJ705 overexpression plants, H3K27me3levels were clearly reduced from OsSTP3 and Os07g11739 (con-firming the data in Figure 6), but not from JAMYB or PR10 beforetreatment. These data were consistent with the increased basalexpression of OsSTP3 and Os07g11739 in the overexpressionseedlings (Figure 7; see Supplemental Table 4 online), suggestingthat the removal of H3K27me3 was associated with the increaseof basal expression of genes. Finally, MeJA treatment reduced

H3K27me3 levels from the four genes in wild type plants, sug-gesting that the removal of H3K27me3 was associated with MeJA-induced gene activation. The reduction of H3K27me3 was morepronounced in the overexpression plants but was attenuated in theRNAi plants at 8 or 12 h. These observations suggest that JMJ705was implicated in MeJA-induced removal of H3K27me3.

DISCUSSION

JMJ705 Is a Stress-Responsive H3K27me2/3 Demethylase

In this work, we have provided in vitro and in vivo evidence thatthe rice JMJD2 protein JMJ705 specifically demethylates histoneH3K27me2/3 (Figures 1B and 1C). The results are corroboratedby the observations that overexpression of the gene reduced theoverall levels of H3K27me2/3 and resulted in preferential activa-tion of H3K27me3-marked genes (Figures 1D and 5A). The stressresponsiveness of JMJ705 expression and the enrichment ofH3K27me3-marked stress-related genes among the upregulatedgenes in JMJ705 overexpression plants (Figure 5A; see SupplementalFigure 2 and Supplemental Table 2 online) suggest that JMJ705is a histone demethylase involved in stress-responsive H3K27me3demethylation and gene expression.Previous results showed that a closely related Arabidopsis

JMJD2 protein, REF6 (JMJ12), demethylates H3K27me2/3 (Luet al., 2011). Mutation of REF6 delays flowering time in Arabidopsis(Noh et al., 2004), whereas its overexpression produces similarphenotypes to mutants of genes involved in H3K27me3 depositionand recognition (Noh et al., 2004; Lu et al., 2011). Although over-expression of JMJ705 did not produce any severe developmentalabnormality, several important developmental regulatory genessuch as OsMADS1, homeobox gene OSH71, and homeobox geneRough Sheath1 showed clear decreases of H3K27me3 and werehighly induced (see Supplemental Table 4 online). These ob-servations, together with the reduced plant height and partial ste-rility phenotypes of the T-DNA mutant, suggest that JMJ705 may

Figure 8. JA-Induced H3K27me3 Removal from Responsive Genes Is Dependent on JMJ705.

Anti-H3K27me3 ChIP assays were performed to analyze transcriptional start site of JA-responsive genes PR10, JAMYB, TPS3, and Os07g11739 inwild-type (ZH11) plants, overexpression (OXJMJ705), and JA-treated RNAi plants during 0, 8, and 12 h. Bar indicates mean 6 SD from three biologicalrepeats.

4732 The Plant Cell

also be involved in H3K27me3 removal from genes involved indevelopmental processes.

JMJ705-Mediated H3K27me3 Removal inStress-Inducible Gene Activation

H3K27 methylation is an important epigenetic mark involved ingene regulation in plants. Genome-wide profiling has identified;10to 20% of genes that are marked by H3K27me3 in Arabidopsis andrice, depending on the analyzed plant organs or developmentalstages (Turck et al., 2007; He et al., 2010; Hu et al., 2012). Unlike inanimal cells, in which H3K27me3 occupies large genomic regions,in plants, H3K27me3 is mostly restricted to the transcribed regionof a large number of genes with a strong bias toward the 59 tran-scribed regions (Figure 5) (Roudier et al., 2011), implying that thismark may be involved in the repression of transcriptional initiation.H3K27me3 is deposited by the polycomb group–repressive com-plex 2 (PRC2). Resetting of this chromatin modification mark maybe a necessary step for activation of the PRC2-repressed genes inplants. Because H3K27me3 is associated with many transcriptionfactor genes that are not expressed in the examined organs,H3K27me3-mediated gene silencing is suggested to be mainlyinvolved in developmental decision in plants (Zheng and Chen,2011). It has been shown that reduction of H3K27me3 from thedevelopmentally regulated gene KNUCKLES (KNU) precedes itsactivation, which is dependent on the transcription factor AG-AMOUS (AG) (Sun et al., 2009), suggesting that the removal ofH3K27me3 may be required for developmental gene activation.

However, many other genes, including stress-inducible genes,are also marked by this modification. H3K27me3 may play a role inmaintaining stress-responsive genes at silent or basal expressionlevels under normal conditions and should be reset upon receivinginductive signals. Reducing the resting levels of H3K27me3 frommarked genes may change the chromatin environment facilitatinggene transcriptional activation. The data showing that over-expression of JMJ705 activates mostly H3K27me3-marked andH3K4me3-depleted stress-responsive genes suggest that JMJ705at elevated levels removed H3K27me3 from these genes and in-creased their basal expression levels. The data showing that thedynamic removal of H3K27me3 from JA-responsive genes duringMeJA induction was correlated with their activation and was en-hanced by overexpression and impaired by RNAi of JMJ705(Figures 7 and 8) indicate that JMJ705-mediated H3K27me3 de-methylation is also involved in the defense signal-induced geneactivation process. Therefore, JMJ705-mediated H3K27me3 de-methylation may play a role in increasing both the basal and in-duced expression of defense-related genes, leading to a moresustained activation, which may possibly contribute to the eventualdevelopment of the necrotic phenotype and resistance to Xoo atthe mature stage (Figures 2 and 3) observed in the overexpressionplants. Because JMJ705 demethylates H3K27me2, the possibilitythat the phenotype is related to a reduction of H3K27me2 insteadof H3K27me3 is, however, not excluded.

Histone Modification in Plant Disease Resistance

Chromatin modification and remodeling have emerged as a keyprocess in the gene expression reprogramming of plant biotic

stress responses. Histone acetylation and H3K4 and H3K36methylation dynamics has been implicated in salicylic acid (SA)-signaling and JA-signaling pathway–mediated plant defense inArabidopsis (Tian et al., 2005; Zhou et al., 2005; Mosher et al., 2006;Kim et al., 2008b; Wu et al., 2008; Berr et al., 2010; Jaskiewiczet al., 2011). H3K4me3 is present on the PR1 chromatin before anystimulation and its level increases upon SA treatment (Mosher et al.,2006). H3K4me3 and H3K36me3 on the downstream genes seemto be readily in place as a permissive mark providing the basalexpression level of the marked genes or establishing the poisedchromatin state for efficient induction when stimulated (Berr et al.,2012). In addition, histone of H3K9 and H3K14 acetylation andH3K4me3 are synergistically set during the priming of diseasedefense-related genes (Jaskiewicz et al., 2011). The present dataprovide evidence that H3K27me3 homeostasis also plays an im-portant role for defense-related gene expression and support thehypothesis that H3K27me3 may be involved in maintaining theresting state of defense genes under normal conditions.Histone modification genes have been reported to regulate

plant defense pathways. Arabidopsis SET-domain gene8, ahistone H3K36 methyltransferase, mediates induction of the JA/ethylene pathway genes in the plant defense response to necroticfungi (Berr et al., 2010). Arabidopsis histone deacetylase HDA19plays a positive role in basal resistance to pathogens (Zhou et al.,2005). HDA9 physically interacts with and represses the activity oftwo WRKY family transcription factors, WRKY38 and WRYK62,both of which act as negative regulators in plant basal defense(Kim et al., 2008b). A recent report showed that HDA19 was in-volved in the repression of SA-mediated defense responses inArabidopsis (Choi et al., 2012). Recent studies in rice showed thatoverexpression of HDT701 (OsHDT1), a histone H4 deacetylasegene, in transgenic rice increases susceptibility to the rice patho-gens Magnaporthe oryzae and Xoo. By contrast, knockdown ofHDT701 in transgenic rice causes elevated levels of histone H4acetylation and elevated transcription of defense-related genesand enhanced resistance to both M. oryzae and Xoo (Ding et al.,2012). Thus, HDT701-dependent histone H4 deacetylation playsa negative role in rice immunity to pathogen attack. The enhancedresistance conferred by overexpression of JMJ705 and the in-creased susceptibility in the T-DNA mutant to the bacterial path-ogen Xoo suggest that JMJ705 may play a positive role in riceimmunity. JMJ705 may enhance plant defense by reducing basalH3K27me3 levels over defense-related genes, thereby augment-ing their basal expression and/or potentiating their higher ex-pression upon biotic stress. Because histone acetylation andmethylation play an important role in plant defense pathways, itremains unknown whether there is any functional interaction be-tween JMJ705 and HDT701 in the interplay of histone mod-ifications involved in rice disease resistance.

METHODS

Plant Materials

Rice cultivars (Oryza sativa spp japonica) ZH11 and HY were used in thisstudy. The T-DNA insertion line of JMJ705 (1C-05110.L) was obtainedfrom the Postech rice mutant database (http://www.postech.ac.kr/life/pfg/risd/). Insertion was confirmed by PCR using the primers F1 and R1and a T-DNA specific primer 2707L1 (see Supplemental Table 5 online).

H3K27me3 Removal in Disease Resistance 4733

The primers used for RT-PCR analysis presented in Figure 4 are listed inSupplemental Table 5 online.

Overexpression and RNAi of JMJ705 and Sequence Alignment

For the overexpression experiment, the JMJ705 full-length cDNA withouta stop codon was amplified from total cDNA of ZH11 using primers OXJ5-Fand OXJ5-R, and then cloned into the overexpression vector pU1301 in framewith 3 3 FLAG tag at the 39 end with KpnI and BamHI sites (Sun and Zhou,2008). For RNAi, cDNA from2584 bp to 3126 bp relative to the translation startsite was amplified from JMJ705 cDNA using primers RiJ5-F and RiJ5-R, andthen cloned into the RNAi vector pds1301 (Huang et al., 2007). The over-expression and RNAi constructs were transferred into Agrobacterium tume-faciens strainEHA105 and then transformed intoZH11aspreviously described(Huang et al., 2007). For sequence alignment, we retrieved JMJ protein se-quences from the ChromDB database (http://www.chromdb.org) and usedClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) for alignment.

Histone Demethylation Assays and Western Blot Analyses

For the in vivohistonedemethylation assay, the JMJ705 full-length cDNAusedin the overexpression eventwas cloned into pFA121 vector (Chen et al., 2013).For production of the substitution mutation of JMJ705, we used the FastMutagenesis System (Trans Gen Biotech FM111) with primers J5mut-F andJ5mut-R (see Supplemental Table 5 online) according to the instructions. TheJMJ705 mutant cDNA was then cloned into the vector pFA121. Tobacco(N. icotiana benthamiana) transfection, nuclei isolation, and immunostainingwere described previously (Chen et al., 2013). Slides were incubated withanti-HA at 1:200 dilution (mouse, M20003M; Ab-mart) and antimethylatedhistone antibodies (rabbit) at 1:150 dilution, followed by Alexa Fluor 488 goatanti-mouse (green fluorescence) (A-11029; Invitrogen) and Alexa Fluor568 goat anti-rabbit (red fluorescence) (A-11036; Invitrogen) secondary anti-bodies at 1:200 dilution. For presentation, native colors of fluorescence-labeledsecondary antibodies were changed using confocal software, so that JMJ705-FLAG-HA protein was displayed in red and methylated histone in green.JMJ705-FLAG-HA protein was affinity purified from pFA121-JMJ705 trans-fected tobacco leaves with anti-FLAG M2 magnetic beads (Sigma) incubatedwith bulk histones (Sigma) for the in vitro histone demethylation assay ac-cording to previously described methods (Whetstine et al., 2006). For proteingel blot analysis, histone-enriched fractions were extracted from wild-type,mutant, and transgenic rice leaves as described previously (Huang et al., 2007).

Other antibodies used in this study were as follows: anti-H3K27me3(07-449; Millipore), anti-H3K27me2 (ab24684; Abcam), anti-H3K27me1(ab113671,;Abcam), anti-H3 (ab1791; Abcam), anti-H3K4me3 (07-473;Millipore), anti-H3K9me3 (ab8898; Abcam), anti-H3K9me2 (07-441; Mil-lipore), H3K36me1 (ab9048; Abcam), anti-H3K36me2 (ab9049; Abcam),anti-H3K36me3 (ab9050; Abcam), and anti-FLAG (F3165; Sigma).

ChIP

The ChIP experiment was performed as described (Huang et al., 2007). Twograms of 14-d-old seedlings were harvested and crosslinked in 1% form-aldehyde under vacuum. Chromatin was extracted and fragmented to 200 to750bpby sonication, andChIPwasperformedusing the following antibodies:H3K4me3, H3K9me3, H3K36me3, H3K27me2, H3K27me3, and FLAG(F3165; Sigma). The precipitated and input DNAs were then analyzed by real-time PCR with gene-specific primers listed in Supplemental Table 5 online.

Pathogen Inoculation

To examine the resistance of plants to bacterial blight disease, plantswere inoculated with the Philippine Xoo strains PXO99 (race 6) andPXO347 (race 9c) at the booting stage by the leaf clipping method (Chen

et al., 2002). Disease was scored (3 to 5 leaves for each plant) as thepercent lesion area (lesion length/leaf length) at 2 weeks after inoculation.

For Xoo bacterial growth rate analysis, rice plants at the mature stagewere inoculated by the leaf clipping method, and the bacterial populationin the infected leaves was determined by counting colony-forming units.Three leaves were harvested separately from inoculated plants at 0 (2 hafter inoculation), 3, 6, 9, and 12 d postinoculation. After sterilization with75% alcohol for 1 min, samples were air-dried and ground into homo-genates in 1 mL sterilized distilled water. Homogenates were seriallydiluted (10-fold each time) to 107 with sterilized distilled water, and eachdilution was then spread on potato culture medium [boiled fresh potato30% (w/v), Suc 1.5% (w/v), Ca(NO3)2 0.05% (w/v), Na2HPO4$12H2O2% (w/v), tryptone 5% (w/v), agar 2% (w/v)]. Colonies were counted after2 to 3 d in the dark at 25°C. Average colony numbers from three sampleswere calculated.

Treatment with MeJA

For MeJA treatment, 14-d-old seedlings of wild-type, mutant, andtransgenic rice were treated with 0.2 mM MeJA. MeJA solution wasapplied to leaves by spraying. Samples were collected 2, 4, 8, 12, and 24 hafter treatment.

Microarray and ChIP-seq Analysis

For microarray analysis, 14-d-old seedlings of transgenic, mutant, andwild type plants, grown in one-half-strength Murashige and Skoog me-dium under a 16-h-light/8-h-dark cycle at 30°C for 14 d, were harvestedfor microarray analysis. RNA samples were extracted using TRIzol ac-cording to the manufacturer’s instructions (Invitrogen). Hybridization withAffymetrix GeneChip Rice Genome Arrays was performed at CapitalBioCorporation. Two biological repeats were performed. Gene expressionchanges between the samples were analyzed by the AffylmGUI packagefrom R software. For GO analysis of microarrays data, we used singularenrichment analysis (http://bioinfo.cau.edu.cn/agriGO/analysis.php), andchoose P < 0.001 as the cutoff for significant GO terms.

Expression Analysis by RNA Gel Blot and RT-PCR

For RNA gel blot analysis, 15 mg total RNA samples extracted from field-grown rice leaves was separated in 1.2% (w/v) formamide-denaturingagarose gels, and then transferred to nylon membranes. Gene-specificprobes were labeled with 32P-dCTP using the Random Primer kit (In-vitrogen) and hybridized to the RNA gel blots. The probe of JMJ705 wasamplified from JMJ705 cDNA using primers F2 and R2 (see SupplementalTable 5 online), resulting in a fragment of 433 bp of the cDNA.

For gene expression analysis, total RNA isolation and RT-PCR wereperformed. Real-time PCR analysis was performed using gene-specificprimers and SYBR Premix Ex Taq on a real-time PCR 7500 system(Applied Biosystems). At least three biological replicates and three technicalrepeats for every biological replicate were tested.

Accession Numbers

Sequence data from this article can be found in the Rice Genome An-notation Project website (http://rice.plantbiology.msu.edu/) under thefollowing accession numbers: JMJ705 (Os01g67970), POX5 (Os07g48040),POX8 (Os07g48010), POX22 (Os07g48020), LOX (Os08g39840), AOS2(Os03g12500), OPR7 (Os08g35740), JAMYB (Os11g45740), PR5(Os12g43430), PR10 (Os12g36860), and TPS3 (Os07g11790). The micro-array data described in this article have been deposited into the GEOdatabase (GSE48069). The ChIP-seq data were retrieved from the GEOdatabase (GSE30490).

4734 The Plant Cell

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Mutated Iron Binding Residue within theCatalytic Domain of JMJ705.

Supplemental Figure 2. Quantitative RT-PCR Analysis of JMJ705Transcripts in Different Tissues of ZH11 Plants and under Stress Conditions.

Supplemental Figure 3. Inoculation of Overexpression Plants with theXoo Strain PXO347.

Supplemental Figure 4. Quantitative RT-PCR Verification of 27Randomly Selected Genes from among Upregulated Genes in theMicroarray of the Overexpression Plants and their Induction by MeJA.

Supplemental Figure 5. Production of JMJ705 RNAi Plants.

Supplemental Table 1. Phenotype Comparisons between jmj705Mutant and the Wild-Type HY.

Supplemental Table 2. GO Analysis of Upregulated and Down-regulated Genes in JMJ705 Overexpression Plants and T-DNA MutantDetected with Affymetrix Microarray Analysis.

Supplemental Table 3. Number of Upregulated and DownregulatedGenes and Their Enrichment for H3K27me3 and H3K4me3 in JMJ705Overexpression and T-DNA Plants.

Supplemental Table 4. Validation by Quantitative RT-PCR of 27Randomly Selected Upregulated Genes from JMJ705 OverexpressionPlants and Induction Tests of Their Expression by MeJA.

Supplemental Table 5. Nucleotide Sequences of Primers Used in ThisStudy.

Supplemental Data Set 1. Upregulated and Downregulated Genes inJMJ705 Overexpression Plants.

Supplemental Data Set 2. Upregulated and Downregulated Genes inthe T-DNA Mutant.

ACKNOWLEDGMENTS

We thank Shiping Wang’s group for advice on Xoo inoculation and ricedisease evaluation, as well as Caiguo Xu and Xianghua Li for help in fieldexperiments and management. This work was supported by the RiceFunctional Genomics 863 Key Project of the Chinese Ministry of Scienceand Technology (2012AA10A303), the special transgenic program of theChinese Ministry of Agriculture (2014ZX0800902B), the FundamentalResearch Funds for the Central Universities (2011PY051), and grantsfrom the Bill and Melinda Gates Foundation.

AUTHOR CONTRIBUTIONS

T.L., X.C., X.L., Y.Z., S.Z., S.C. performed research; T.L., X.Z., D.X.Z.analyzed data; DXZ, T.L. wrote the article.

Received September 23, 2013; revised October 23, 2013; acceptedOctober 31, 2013; published November 26, 2013.

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4736 The Plant Cell

DOI 10.1105/tpc.113.118802; originally published online November 26, 2013; 2013;25;4725-4736Plant Cell

Dao-Xiu ZhouTiantian Li, Xiangsong Chen, Xiaochao Zhong, Yu Zhao, Xiaoyun Liu, Shaoli Zhou, Saifeng Cheng and

Involved in Defense-Related Gene Activation in RiceJumonji C Domain Protein JMJ705-Mediated Removal of Histone H3 Lysine 27 Trimethylation Is

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