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. 2024 Mar 5;20(3):e1011203.
doi: 10.1371/journal.pgen.1011203. eCollection 2024 Mar.

An epigenetic timer regulates the transition from cell division to cell expansion during Arabidopsis petal organogenesis

Ruirui Huang  1 , Vivian F Irish  1   2
Affiliations

An epigenetic timer regulates the transition from cell division to cell expansion during Arabidopsis petal organogenesis

Ruirui Huang et al. PLoS Genet. .

Abstract

A number of studies have demonstrated that epigenetic factors regulate plant developmental timing in response to environmental changes. However, we still have an incomplete view of how epigenetic factors can regulate developmental events such as organogenesis, and the transition from cell division to cell expansion, in plants. The small number of cell types and the relatively simple developmental progression required to form the Arabidopsis petal makes it a good model to investigate the molecular mechanisms driving plant organogenesis. In this study, we investigated how the RABBIT EARS (RBE) transcriptional repressor maintains the downregulation of its downstream direct target, TCP5, long after RBE expression dissipates. We showed that RBE recruits the Groucho/Tup1-like corepressor TOPLESS (TPL) to repress TCP5 transcription in petal primordia. This process involves multiple layers of changes such as remodeling of chromatin accessibility, alteration of RNA polymerase activity, and histone modifications, resulting in an epigenetic memory that is maintained through multiple cell divisions. This memory functions to maintain cell divisions during the early phase of petal development, and its attenuation in a cell division-dependent fashion later in development enables the transition from cell division to cell expansion. Overall, this study unveils a novel mechanism by which the memory of an epigenetic state, and its cell-cycle regulated decay, acts as a timer to precisely control organogenesis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. RBE physically associates with the TPL protein.
(A) Diagram of native RBE protein and RBE with point mutations in the EAR motif (mRBE). Native and altered amino acid sequences are shown. (B) Yeast two-hybrid assays between RBE or mRBE with TOPLESS (TPL).—symbol indicates only the AD domain. Double Dropout (DDO) and Quadruple Dropout (QDO) were used as selective media. (C) Bimolecular fluorescence complementation assay (BiFC) to detect reconstitution of YFP fluorescence. YFP fluorescence shows interaction between RBE and TPL in the nuclei. Position of nuclei detected by fluorescence of H2B-mCherry. Panels (left to right): YFP; H2B-mCherry; merged. Scale bars, 50 um (D) Co-immunoprecipitation (Co-IP) of YFP-RBE and Myc-TPL in Agrobacterium-infiltrated leaves of N. benthamiana. Extracts were immuno-precipitated with anti-GFP antibody and detected using anti-GFP and anti-MYC antibodies. The experiment was repeated three times with similar results. IP, immunoprecipitation.
Fig 2
Fig 2. The EAR motif is necessary for RBE repressive effects during petal development.
(A) Overexpression lines of RBE, 35S::RBE (RBE-OX), 35S::RBE with an EAR motif mutation (mRBE-OX), 35S::RBE with an EAR motif deletion (dRBE-OX), TPL fusion to mRBE (mRBE-TPL-OX) and TPL fusion to dRBE (dRBE-TPL-OX). Scale bar, 0.5 cm. (B) Quantification of petal blade index (width/length) of representative overexpression lines. Overexpression lines with more than 5-fold gene expression changes were quantified (S5A Fig). Error bars represent mean ± SD of four biological replicates. (C) Quantification of petal epidermal cell size of representative overexpression lines. Error bars represent mean ± SD of four biological replicates. (D) Relative levels of TCP5 expression as assessed by qRT-PCR in seedlings of representative overexpression lines. Tip41-like was used as an internal control. Error bars represent mean ± SD of four biological replicates. t-test, **P<0.01, *P<0.05.
Fig 3
Fig 3. TPL and HDA19 repress TCP5 expression during petal development.
(A) Relative levels of TCP5 expression as assessed by qRT-PCR in wild type, rbe-1, tpl-1, hda19-1. Error bars represent mean ± SEM of three biological replicates; the differences were not statistically significant. ACT2 was used as the internal control. (B) The petal cell size of different genotypes. t-test, error bars represent mean ± SD of three biological replicates. (C, D) The repressive activity conferred by RBE in different genotypes; effectors and reporter diagrammed in (C), and relative activity (RBE/BD) shown in (D). Error bars represent mean ± SD of four biological replicates. t-test, **P< 0.01, *P <0.05.
Fig 4
Fig 4. TPL binds to TCP5 promoter regions.
(A) Schematic diagram showing the TCP5 locus and the genomic regions used for ChIP assays in (B). (B) ChIP assays using 35S::GR-RBE TPLp::TPL-HA one week old seedlings after 16h DEX treatment. Mu-like transposon served as the negative control and its value was set to 1. Error bars represent mean ± SD of three biological replicates. **P< 0.01, *P <0.05.
Fig 5
Fig 5. TCP5 chromatin dynamics after RBE transient overexpression.
The regions used for ChIP assays are diagrammed in Fig 4A. (A) H3K9ac analysis by ChIP assays using inflorescences 16h after a single 10μM DEX (grey bars) or mock (empty bars) treatment. The TA3 retrotransposon and EIF4 genes were used for negative controls. Error bars represent mean ± SEM of four biological replicates. (B) H3K27me3 analysis by ChIP assays using inflorescences 16h after a single 10μM DEX (grey bars) or mock (empty bars) treatment. Error bars represent mean ± SEM of two biological replicates. (C) DNA accessibility at the transcription start site (P1) of TCP5 assayed by FAIRE 16h after a single 10μM DEX (grey bars) or mock (empty bars) treatment. The TA3 retrotransposon was used a negative control and was set to 1. Error bars represent mean ± SD of four biological replicates. (D) ChIP assay for RNA Pol II binding using inflorescences 16h after a single 10μM DEX (grey bars) or mock (empty bars) treatment. The Mu-like transposon served as a negative control. Error bars represent mean ± SEM of four biological replicates. t-test, **P< 0.01, *P <0.05.
Fig 6
Fig 6. RBE transient expression induces the transcriptional delay of TCP5 expression.
(A) Nuclear accumulation of the GR-RBE fusion protein after a single 10μM DEX treatment. Inflorescence tissues were collected at 1d, 3d, 5d and 7d. The GR-RBE fusion protein and histone H3 as nuclear internal control were detected by immunoblotting. (B) Relative levels of TCP5 expression as assessed by qRT-PCR in floral tissues collected at 1d, 3d, 5d and 7d. Error bars represent mean ± SD of at least three biological replicates. The TCP5 genomic regions used for ChIP assays in (C) and (D) are diagrammed in Fig 4A. (C) H3K9ac analysis by ChIP assays using inflorescences 5d (light grey bars) and 7d (dark grey bars) after a single 10μM DEX treatment or mock treatment (empty bars). Relative enrichment indicates percent input normalized to that of TA3. Error bars represent mean ± SD of three biological replicates. (D) H3K27me3 analysis by ChIP assays using inflorescences 5d (light grey bars) and 7d (dark grey bars) after a single 10μM DEX treatment (grey bars) or mock treatment (empty bars). Relative enrichment indicates percent input normalized to that of TA3. Error bars represent mean ± SD of three biological replicates. t-test, **P< 0.01, *P <0.05.
Fig 7
Fig 7. Induction of TCP5 is cell division dependent and the temporal regulatory model.
(A) Relative levels of TCP5 expression as assessed by qRT-PCR in floral tissues collected 5d after mock treatment, 50μM gibberellic acid 3 (GA3) alone, 10μM DEX alone, and 10μM DEX with GA3. Tip41-like served as the internal control. Error bars represent mean ± SD of three biological replicates. t-test, **P< 0.01, *P <0.05. (B) A model of epigenetic timing mechanisms induced by RBE to regulate TCP5.
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Grants and funding

This project was supported by the National Science Foundation (https://www.nsf.gov/) through grant IOS-1354389 to VFI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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