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DNA methylation and histone modifications regulate de novo shoot regeneration in Arabidopsis by modulating WUSCHEL expression and auxin signaling - PubMed

DNA methylation and histone modifications regulate de novo shoot regeneration in Arabidopsis by modulating WUSCHEL expression and auxin signaling

Wei Li et al. PLoS Genet. 2011 Aug.

Abstract

Plants have a profound capacity to regenerate organs from differentiated somatic tissues, based on which propagating plants in vitro was made possible. Beside its use in biotechnology, in vitro shoot regeneration is also an important system to study de novo organogenesis. Phytohormones and transcription factor WUSCHEL (WUS) play critical roles in this process but whether and how epigenetic modifications are involved is unknown. Here, we report that epigenetic marks of DNA methylation and histone modifications regulate de novo shoot regeneration of Arabidopsis through modulating WUS expression and auxin signaling. First, functional loss of key epigenetic genes-including METHYLTRANSFERASE1 (MET1) encoding for DNA methyltransferase, KRYPTONITE (KYP) for the histone 3 lysine 9 (H3K9) methyltransferase, JMJ14 for the histone 3 lysine 4 (H3K4) demethylase, and HAC1 for the histone acetyltransferase-resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Second, we showed that regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications through bisulfite sequencing and chromatin immunoprecipitation. Third, DNA methylation in the regulatory regions of WUS was lost in the met1 mutant, thus leading to increased WUS expression and its localization. Fourth, we did a genome-wide transcriptional analysis and found out that some of differentially expressed genes between wild type and met1 were involved in signal transduction of the phytohormone auxin. We verified that the increased expression of AUXIN RESPONSE FACTOR3 (ARF3) in met1 indeed was due to DNA demethylation, suggesting DNA methylation regulates de novo shoot regeneration by modulating auxin signaling. We propose that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role in this process.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mutation in key epigenetic genes alters the rate of Arabidopsis shoot regeneration in vitro.

A) Frequency of shoot regeneration from calli of the wild type (Ws) and the mutant met1. B) Frequency of shoot regeneration from calli of the wild type (Ler) and the mutant kyp-2. C) Frequency of shoot regeneration from calli of the wild type (Col) and the mutants jmj14-1 and hac1-3. Calli were induced from pistils on CIM, and were then transferred onto SIM for shoot induction. Data are presented as mean values from three sets of biological replicates. In each replicate, at least 60 calli were examined. D) Calli of the wild type (Ws) and the mutant met1 cultured on SIM for 0 day, 4 days, 6 days, 10 days and 14 days. Scale bars, 1 mm.

Figure 2
Figure 2. DNA methylation and histone modifications regulate WUS transcript levels.

A) Transcript levels of WUS in calli of the wild type (Ws) and the mutant met1. B) Transcript levels of WUS in calli of the wild type (Ler) and the mutant kyp-2. C) Transcript levels of WUS in calli of the wild type (Col) and the mutants, hac1-3, hac1-5, jmj14-1 and jmj14-2. Total RNAs were isolated from calli of wild type (Ws, Ler and Col) and various mutants (met1, kyp-2, jmj14-1, jmj14-2, hac1-3 and hac1-5) cultured on SIM at the indicated time points, respectively. WUS transcript levels were quantified by qRT-PCR. The results are shown as mean values of three biological replicates with standard errors. The relative expression level of WUS gene, corresponding to the expression level of TUBULIN2, was calculated using the comparative C(T) method. After incubating on CIM for 20 days (S0), some of the calli were transferred onto SIM for further induction for 4 days (S4) and 6 days (S6), other calli were still cultured on CIM as controls (C24, C26). C16, C24, C26 indicated that pistils as explants were cultured on CIM for 16 days, 24 days and 26 days, respectively.

Figure 3
Figure 3. Regulation of WUS expression in met1 mutant.

A) By roots as explants, pWUS::GUS transgenic calli in the wild type transferred onto SIM for 6 days, 8 days, 10 days and 14 days, and pWUS::GUS transgenic calli in the met1 mutant transferred onto SIM for 6 days, 8 days, 10 days and 14 days. Arrowheads indicate pWUS::GUS signals. Scale bars, 1 mm. B) Longitudinal sections of pWUS::GUS transgenic calli in both the wild type and the met1 mutant transferred onto SIM for 6 days, 8 days, 10 days and 14 days, respectively. Scale bars, 50 µm.

Figure 4
Figure 4. Analysis of WUS methylation through bisulfite genomic sequencing.

A) A diagram of WUS structure, with +1 as the transcription start site and rectangles representing the methylated region I, II and III. B) Cytosine methylation at region I, II and III of WUS was determined by bisulfite genomic sequencing. Genomic DNA methylation status of WUS is shown in calli of the wild type on CIM for 16 days (WT, C16) and for 20 days (WT, S0), and on SIM for 6 days (WT, S6). Calli of met1 are incubated on CIM for 16 days (met1, C16) and for 20 days (met1, S0), and on SIM for 6 days (met1, S6). H represents A, T or C.

Figure 5
Figure 5. ChIP assays of calli of wild type on SIM at the WUS locus.

A) A diagram of WUS structure, with +1 as the transcription start site, and bars representing the regions examined by ChIP. B) ChIP analysis using antibodies against trimethyl H3K4 and dimethyl H3K9 at 5′ and 3′ regions of WUS in calli of wild type for 20 days on CIM (S0) and 6 days on SIM (S6). C) ChIP analysis using antibodies against H3 acetyl Lys 9 at 5′ and 3′ regions of WUS in calli of wild type (S0, S6). ACTIN was used as a control. The input was chromatin before immunoprecipitation. ‘No AB’ corresponds to chromatin treated with normal mouse IgG as the negative control. Three biological replicates were analyzed and each was tested by three technical replicates, and similar results were obtained. Representative data were shown.

Figure 6
Figure 6. Identification of the candidate genes regulated by DNA methylation.

A) The overlap between differentially-expressed genes of S6 versus S0 (Table S3) and M0 versus S0 (Table S4) were identified as candidate genes, and were listed in Table S5. A two-fold difference in the expression level of genes with a q value≤0.05 between S6 versus S0 and M0 versus S0 was set as the threshold for the selection of differentially-expressed genes. B)–E) Cytosine methylation levels of ARF3, ARF4, IAA18 and BLH7 genes in calli of wild type (S0, S6), and calli of met1 (M0) were determined by bisulfite genomic sequencing. H represents A, T or C.

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