Embryonic epigenetic reprogramming by a pioneer transcription factor in plants

Abstract

Epigenetic modifications, including chromatin modifications and DNA methylation, have a central role in the regulation of gene expression in plants and animals. The transmission of epigenetic marks is crucial for certain genes to retain cell lineage-specific expression patterns and maintain cell fate1,2. However, the marks that have accumulated at regulatory loci during growth and development or in response to environmental stimuli need to be deleted in gametes or embryos, particularly in organisms such as plants that do not set aside a germ line, to ensure the proper development of offspring1,2. In Arabidopsis thaliana, prolonged exposure to cold temperatures (winter cold), in a process known as vernalization, triggers the mitotically stable epigenetic silencing of the potent floral repressor FLOWERING LOCUS C (FLC), and renders plants competent to flower in the spring; however, this silencing is reset during each generation3,4,5. Here we show that the seed-specific transcription factor LEAFY COTYLEDON1 (LEC1) promotes the initial establishment of an active chromatin state at FLC and activates its expression de novo in the pro-embryo, thus reversing the silenced state inherited from gametes. This active chromatin state is passed on from the pro-embryo to post-embryonic life, and leads to transmission of the embryonic memory of FLC activation to post-embryonic stages. Our findings reveal a mechanism for the reprogramming of embryonic chromatin states in plants, and provide insights into the epigenetic memory of embryonic active gene expression in post-embryonic phases, through which an embryonic factor acts to ‘control’ post-embryonic development processes that are distinct from embryogenesis in plants.

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Figure 1: FLC expression is de novo activated primarily by LEC1 in the pro-embryo and onwards.
Figure 2: LEC1 is required for FLC activation in embryogenesis following parental vernalization.
Figure 3: LEC1 activates FLC expression and promotes active chromatin modifications at the FLC locus.
Figure 4: The seed-specific LEC1 acts to promote FLC expression beyond seed development.

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Acknowledgements

We thank R. M. Amasino for providing the FLC::GUS plasmid and the seeds of FRI-Col, FRI flc-3 and FRI flc-2, I. E. Somssich for the pJawohl8-RNAi plasmid, and W. Yuan for technical assistance. This work was supported in part by the National Key Research and Development Program of China (2017YFA0503803), the Chinese Academy of Sciences (XDPB0404) and the Temasek Life Sciences Laboratory (Singapore).

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Contributions

Y.H. conceived the project; Z.T. performed most of the experiments; L.S. performed and, together with H.Y., analysed the in situ experiments; X.G. and Y.W. participated in the ChIP experiments with anti-Flag and anti-H3K27me3; Z.T. conducted all statistical analyses; Z.T. and Y.H. analysed most of the data; Y.H., Z.T. and H.Y. wrote the paper.

Corresponding author

Correspondence to Yuehui He.

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Reviewer Information Nature thanks R. Amasino and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 The vernalized FLC chromatin is fully reset in either a weak or a null elf6 mutant.

a, ELF6 gene structure. Filled boxes for exons, and the insertional T-DNA in the weak allele elf6-4 is indicated by a triangle. The primers used to amply ELF6 transcripts in b are indicated (forward (F), 5′-ATGGGTAATGTTGAAATTCCGAATT-3′; and reverse (R), 5′-CTCTGATTCTATTTTCCGCTTCT-3′). b, RT–qPCR analysis of ELF6 transcript levels in wild-type (Col) and elf6-4 seedlings (in the rapid-cycling or early-flowering Col background carrying a null fri allele). A low level of 3.5-kb 3′-truncated ELF6 transcripts, which is predicted to encode a protein with the H3K27 demethylase domain, was detected. Data are representative of two independent experiments. c, Flowering times of the indicated lines grown in long-day conditions. Box plots display median (line), interquartile range (box limits), whiskers (extending 1.5 times the interquartile range), and data points (red circles for plants and 12 plants per treatment). V, vernalized; NV, non-vernalized; NV(V), non-vernalized following vernalization in the previous generation. d, Analysis of FLC transcript levels in FRI-Col (FRI) and FRI elf6-3 and FRI elf6-4 seedlings. FLC mRNA levels were quantified by RT–qPCR and normalized to the constitutively expressed UBC21 gene4. Three biological repeats were conducted. c, d, All data are mean ± s.d. P values determined by two-tailed Student’s t-test with a 95% confidence interval. Discrete leaf number data were log-transformed.

Extended Data Figure 2 Analysis of FLC::GUS lines, FLC expression and lec1 mutants.

a, GUS staining of FLC::GUS embryos (in the FRI background). 5-DAP seeds from the reference line and nine independent T1 lines were stained. Top, schematic structure of the FLC::GUS fusion gene, as described previously13. The GUS coding sequence was inserted into a 16-kb genomic FLC clone (in frame into the sixth exon of FLC; filled boxes for exons, and the genes down- and upstream of FLC are delineated in grey. The experiment was repeated independently once with similar results. Scale bars, 100 μm. b, Segregation of selfed progeny from F1 of the reference FLC::GUS line crossed to a FRI line. An approximate segregation ratio of 3:1 (185 plants bearing FLC::GUS to 58 non-transgenic) was observed (P < 0.01, χ2 test), confirming a single transgene locus. c, FLC mRNA levels in 1-DAP FRI-Col seed tissues. Parental plants were vernalized or non-vernalized; FLC transcript levels were normalized directly to UBC21. Data are mean ± s.d. (three biological repeats). **P < 0.01, two-tailed t-test. d, RT–qPCR analysis of LEC1 mRNA expression in 3-DAP lec1 siliques (in the Col background). Top, schematic structure of LEC1. Insertional T-DNAs are indicated by a triangle. Data are representative of three independent experiments. e, lec1 mutations are fully recessive and cause early flowering. The number of leaves formed before flowering for each line was scored (18 plants per line). **P < 0.01, between Col and lec1 mutant (two-tailed t test with log-transformed data). Data are mean ± s.d.

Extended Data Figure 3 LEC1 is required for FLC activation in embryogenesis.

a, b, FLC::GUS expression is suppressed in 3-DAP (a) and 5-DAP (b) lec1 FRI embryos. FLC::GUS staining in most lec1 FRI embryos (118 out of 120 3-DAP and 76 out of 78 5-DAP) was reduced compared to the FRI embryos. Scale bars, 100 μm. c, Analysis of embryonic FLC mRNA levels in FRI lec1-4 by in situ hybridization. Notably, FLC expression in a majority of embryos examined was reduced. FRI (3 DAP), n = 8; FRI lec1-4 (3 DAP), n = 10; FRI (5 DAP), n = 3; FRI lec1-4 (5 DAP), n = 19. Scale bars, 50 μm. d, FLC mRNA levels in 5-DAP seeds of the indicated genotypes. FLC transcript levels were normalized directly to TUB2; data are mean ± s.d. (three biological repeats). **P < 0.01, between FRI and FRI lec1 (two-tailed t-test). e, Flowering times of the indicated lines grown in long-day conditions. Box plots display median (line), interquartile range (box limits), whiskers (extending 1.5 times the interquartile range), and data points (each red circle denotes one plant). FRI, n = 16; fri, n = 16; lec1-4 FRI, n = 32; lec1-4 l1l FRI, n = 32. Letters mark significant differences determined by one-way ANOVA (with log-transformed data) to compare means (P < 0.05). At a population level, lec1-4 l1l FRI appeared to flower moderately earlier than lec1-4 FRI (also see Fig. 1c).

Extended Data Figure 4 Alignment of LEC1 homologues and expression analysis of NF-YBs and FLC in NF-YB1,8,10-RNAi lines.

a, Sequence alignment of LEC1, L1L, NF-YB1, NF-YB2, NF-YB8 and NF-YB10. LEC1 and L1L C-terminal tails are not included in the alignment. b, NF-YB1, NF-YB8 and NF-YB10 expression were knocked down specifically in 5-DAP siliques of two NF-YB1,8,10-RNAi lines (homozygous single-locus T-DNA) in the lec1-4 l1l FRI background. The dsRNAi cassette was constructed using part of the NF-YB10 transcribed region. Transcript levels were normalized directly to TUB2. Data are mean ± s.d. (three biological repeats). *P < 0.05, **P < 0.01, between lec1 l1l FRI and an indicated line (two-tailed t-tests). c, Box plots of flowering times of FRI NF-YB1,8,10-RNAi-1 and RNAi-2 (in LEC1/lec1-4 L1L/l1l) grown in long-day conditions. Box plots display median (line), interquartile range (box), whiskers (extending 1.5 times the interquartile range), and data points (red dots; 16 plants per line were scored). Letters denote significant differences determined by one-way ANOVA with log-transformed data (P < 0.05). Error bars denote s.d. d, FLC mRNA levels in 5-DAP siliques of the indicated lines. Relative levels to FRI-Col are presented; data are mean ± s.d. (three biological repeats). e, FLC mRNA levels in single 5-DAP seeds (T3 homozygotes) of the NF-YB1,8,10-RNAi-1 line (in the lec1 l1l FRI background). Each seed is numbered (10 seeds were examined); the seeds of fri (Col), FRI or lec1 l1l FRI were pooled. Data points are technical replicates, and bars indicate means. Relative levels to FRI are presented.

Extended Data Figure 5 LEC1 is required for FLC activation in embryogenesis after parental vernalization.

a, A schematic illustration of the experimental procedure. Two single lec1-4 FRI plants flowered moderately earlier than FRI-Col were selected at G1, and plant I and plant II flowered after 55 and 58 leaves were formed, respectively. b, Analysis of FLC transcript levels in lec1-4 FRI seedlings before and after vernalization. Seedlings were vernalized (exposed to cold) for 5 weeks. The G3 NV(V) seedlings were derived from 15 randomly selected G2 V plants. Each sample (pooled seedlings) was quantified in triple replicates (technical), and bars indicate means. Relative levels to G2 NV (FRI) are presented.

Extended Data Figure 6 Functional analysis of LEC1–Flag and a LEC1-bound region at the FLC promoter.

a, LEC1–Flag is expressed specifically in siliques (bearing seeds) in five out of the six independent T1 transgenic lines, as revealed by western blotting analysis. LEC1pro-LEC1:Flag was introduced into lec1-4. Top, schematic drawing of the transgene; black boxes denote LEC1 exons. In five out of the six lines expressing LEC1–Flag, this protein is expressed only in siliques, but not in rosette leaves, consistent with the fact that the endogenous LEC1 is exclusively expressed in seed development14,15. In the remaining line (#3), LEC1–Flag was detected in leaves, most likely owing to a positional effect of the transgene. M, markers; NT, non-transgenic. The experiment was repeated independently once with similar results. b, Rescue of lec1-4 by the LEC1pro-LEC1:Flag transgene (T3 generation). Data are representative of three independent experiments. c, Western blotting analysis of LEC1–Flag in siliques from two lines (T3). Data are representative of two independent experiments. d, Mutations of the CCAAT motifs in the FLC promoter cause FLC-dependent early flowering. The #1 to #4 motifs or the #5 motif were each mutated into AACCG, and the wild-type and mutant FLC transgenes were subsequently introduced into FRI flc-2. FLC-II is a region examined by the ChIP assays presented in Fig. 3a. Flowering times of T1 plants were scored, and box plots display median (line), interquartile range (box limits) and whiskers (extending 1.5 times the interquartile range). Letters mark significant differences revealed by one-way ANOVA with log-transformed data (P < 0.05).

Extended Data Figure 7 LEC1 engages chromatin modifiers to activate FLC expression in embryogenesis.

a, ChIP analysis of H3K4me3 on FLC chromatin in 5-DAP siliques. Relative fold changes in FRI over lec1-4 FRI are shown. Data are mean ± s.d. (three biological repeats), and significant differences were observed between FRI and lec1 FRI in all examined regions (P < 0.01, two-tailed t-test). b, EFS is required for FLC expression in early embryogenesis. FLC transcript levels in 2-DAP and 5-DAP developing seeds were quantified by RT–qPCR and normalized directly to TUB2. Data are mean ± s.d. (three biological repeats). **P < 0.01, between efs (efs FLC) and Col (two-tailed t test). c, EFS–Flag rescued the efs mutant phenotype. Total leaf number (at flowering) for each indicated line (16 plants per line) grown in long-day conditions were examined. Data are mean ± s.d. Letters mark significant differences revealed by one-way ANOVA with log-transformed data (P < 0.05). d, Western blotting analysis of EFS–Flag levels in 5-DAP seeds of EFS:Flag (in efs)-1 and EFS:Flag (in lec1-4 efs)-1 lines. Both lines bear single-locus transgenes. The amount of Rubisco in each lane from a gel with duplicated samples serves as a loading control. Data are representative of two independent experiments. e, LEC1 does not regulate the expression of ELF6, FRI, EFS or ATX1. Transcript levels in 5-DAP FRI-Col and lec1-4 FRI siliques were quantified by RT–qPCR, and relative levels to FRI-Col are presented. Data are mean ± s.d. (three biological repeats). Notably, the ATX1 H3K4 methyltransferase catalyses H3K4 methylation on FLC chromatin11. f, SWC6 is epistatic to LEC1 in FLC activation during embryo development. FLC::GUS staining was examined in 3-DAP embryos of FRI-Col, swc6 FRI and swc6 lec1-4 FRI. Scale bars, 100 μm. The experiment was repeated independently once with similar results. SWC6 is a core subunit of SWR1c that deposits the histone variant H2A.Z into FLC chromatin23.

Extended Data Figure 8 LEC1 is exclusively expressed in seed development under normal growth conditions.

a, Schematic structure of LEC1pro-LEC1:GUS. 1.2-kb genomic LEC1 coding region (the first exon and intron) was included. The gene upstream of LEC1 is delineated in grey. b, LEC1:GUS expression patterns. Ten lines were examined and a representative line is shown. The expression patterns of FLC::GUS serve as control. Scale bars, 100 μm (embryos), and 2 mm (seedlings). Notably, GUS expression from a LEC1 promoter region only was detected in seedlings38, indicating a role of the genomic coding region for LEC1 regulation. c, RT–qPCR analysis of LEC1 mRNA expression in embryogenesis and post-embryo stages in FRI-Col. LEC1 transcripts (labelled as LEC1) were examined by a primer pair that amplifies only cDNA templates; gcLEC1 was amplified by a primer pair used previously38, which can amplify both genomic and cDNA templates (see lanes 1, 2 and 4). PCR cycle numbers are in parentheses. LEC1 mRNAs were not detected in seedlings grown under normal conditions (long days), but were weakly expressed in seedlings under constant dark stress for five days. The experiment was repeated independently once with similar results.

Extended Data Figure 9 Analysis of FLC::GUS staining, the FLC-PTGS-1 and FLC-PTGS-2 lines and individual lec1 FRI progeny.

a, Table showing the analysis of FLC::GUS activity in FRI and lec1-4 FRI embryos or germinated seeds. b, Embryonic FLC suppression by artificial miRNAs does not affect FLC-dependent late flowering. Total leaf number at flowering for T4 progeny grown in long days was scored for each line (18 plants per line). P values determined by two-tailed t-tests (with log-transformed data and a 95% confidence interval). Data are mean ± s.d. c, FLC transcript levels in individual 15-day-old FLC-PTGS-2 seedlings (T4 homozygotes) grown in long-day conditions. d, FLC mRNA levels in the second-pair rosette leaves of individual FRI-Col and lec1-4 FRI plants (from selfed LEC1/lec1 FRI) with 15 rosette leaves (grown in 12-h light/12-h dark), quantified by RT–qPCR. e, ChIP analysis of H3K36me3 at the FLC locus. Total chromatin was extracted from each plant examined in d, and an FLC promoter region (FLC-I) was examined. f, FLC mRNA levels in the second-pair rosette leaves of individual plants grown in 12-h light/12-h dark. g, ChIP analysis of H3K27me3 at FLC. Total chromatin was extracted from each plant examined in f, and FLC-I was examined. Data points in cg are technical replicates, and bars indicate means. Relative levels to FRI-Col are presented.

Extended Data Figure 10 Timed chemical induction of LEC1 and a model for FLC chromatin state transmission.

a, DEX induction of LEC1 expression using a stringent two-component glucocorticoid-inducible expression system. Top, schematic drawing of pOp-LEC1 p35S-GR:LhG4 construction. In this system, upon binding to DEX, the transcriptional activator GR:LhG4 recognizes the pOp6 promoter to activate LEC1 transcription. 10-DAP seeds were treated once with DEX as described in Fig. 4e. LEC1 mRNA levels were normalized directly to TUB2 and barely detected in the pOp-LEC1 p35S-GR:LhG4 seedlings. Data are mean ± s.d. (three biological repeats). P values determined by two-tailed t-tests with a 95% confidence interval are shown. b, The expression of LEC1, FLC or At2S3 is largely not affected by DEX treatment in the non-transgenic Col background. Transcript levels were measured in 2-day-old stratified seeds treated once with 10 μM DEX and 2-DAG seedlings grown from mock or DEX-treated seeds. Data are mean ± s.d. (three biological repeats). u.d., undetected. c, Schematic diagram to illustrate the ‘control’ of FLC expression by the seed-specific LEC1 NF-Y throughout the Arabidopsis life cycle. The LEC1 NF-Y de novo activates FLC expression and promotes initial establishment of the active chromatin state marked with H3K36me3 in early embryogenesis and consequently ‘resets’ the silenced state inherited from gametes. This active chromatin state is maintained in seed development and passed on to post-embryonic stages, leading to transmission of the embryonic memory of active FLC expression to post-embryonic life (seedling). Winter cold (vernalization) leads to an enrichment at FLC of a PRC2 H3K27 methyltransferase complex that catalyses the repressive H3K27me3, resulting in a silenced state in seedlings, and upon returning to warmth, this state is maintained. FLC remains to be silenced in sperm and egg cells at the reproductive stage.

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This file contains the uncropped gels and blots for Extended Data Figs. 1b, 2d, and 8c, and the uncropped western blots for Extended Data Figs. 6a, 6c and 7d.

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Tao, Z., Shen, L., Gu, X. et al. Embryonic epigenetic reprogramming by a pioneer transcription factor in plants. Nature 551, 124–128 (2017). https://doi.org/10.1038/nature24300

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