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A robust mechanism for resetting juvenility during each generation in Arabidopsis

Abstract

Multicellular organisms undergo several developmental transitions during their life cycles. In contrast to animals, the plant germline is derived from adult somatic cells. As such, the juvenility of a plant must be reset in each generation. Previous studies have demonstrated that the decline in the levels of miR156/7 with age drives plant maturation. Here we show that the resetting of plant juvenility during each generation is mediated by de novo activation of MIR156/7 in Arabidopsis. Blocking this process leads to a shortened juvenile phase and premature flowering in the offspring. In particular, an Arabidopsis plant devoid of miR156/7 flowers even without formation of rosette leaves in long days. Mechanistically, we find that different MIR156/7 genes are reset at different developmental stages through distinct reprogramming routes. Among these genes, MIR156A, B and C are activated de novo during sexual reproduction and embryogenesis, while MIR157A and C are reset upon seed germination. This redundancy generates a robust reset mechanism that ensures accurate restoration of the juvenile phase in each plant generation.

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Fig. 1: Expression of the MIR156/7 genes during the plant life cycle.
Fig. 2: Generation of the mir156/7 multiple mutants by CRISPR/Cas9.
Fig. 3: Characterization of high-order mir156/7 mutants.
Fig. 4: The embryonic effect of MIR156A–C on developmental transitions.
Fig. 5: Identification of the JRR for the embryonic activation of MIR156A/C.
Fig. 6: LEC2 triggers the embryonic activation of MIR156A/C.
Fig. 7: MIR156A/C are direct targets of LEC2.
Fig. 8: Model of the resetting of juvenility and MIR156/7 genes during each generation in Arabidopsis.

Data availability

All materials are available from the corresponding author on request. The ATAC-seq, ChIP-seq, RNA-seq and sRNA-seq data (BioProject PRJCA003872) are deposited in the Beijing Institute of Genomics Data Center (http://bigd.big.ac.cn). Source data are provided with this paper.

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Acknowledgements

We thank X. Hou (South China Botanical Garden, CAS) for the lec2 seeds; J. Zhu (Shanghai Center for Plant Stress Biology, CEPMS/SIPPE, CAS) and Q. Chen (China Agricultural University) for CRISPR/Cas9 plasmids; H. Liu (CEPMS/SIPPE, CAS) for the Dual-LUC system; L. Liu and S.-N. Yin (Core Facility Center of CEPMS/SIPPE, CAS) for technical support on FACS; members of the J.-W.W. lab for discussion and comments on the manuscript. This work was supported by grants from the National Key Research and Development Program (2016YFA0500800) to J.-W.W., National Natural Science Foundation of China (31788103; 31721001) to J.-W.W., Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27030101) to J.-W.W., Science and Technology Commission of Shanghai Municipality (18JC1415000) to J.-W.W., and Shanghai Post-doctoral Excellence Program (2019029) to Y.-J.C.

Author information

Authors and Affiliations

Authors

Contributions

J.G., K.Z., Y.-J.C. and J.-W.W. designed the research. J.G., Y.-J.C., S.Y. and D.Z. generated the mir156/7 high-order mutants; K.Z. performed the lec2-related experiments; Y.-J.C. generated and analysed MIR156/7 reporters; Y.-J.C. and H.L. performed in situ hybridization assay on miR156; J.G. performed in situ hybridization assay on FUL; J.G., K.Z. and F.-X.W. generated the ATAC-seq and ChIP-seq dataset; F.-X.W. generated p35S:LEC2-GR, p35S:RKD4-GR and pABI3:MIM156 plants; J.G., G.-D.S and Z.-G.X. analysed the ATAC-seq, ChIP-seq and sRNA-seq data; L.-Y.W. established the ATAC-seq protocol; Y.-X.M. helped with FACS; X.-Y.Z. prepared the LEC2-DBD protein; J.G., K.Z., Y.-J.C. and J.-W.W. analysed the data; and J.-W.W. wrote the article.

Corresponding author

Correspondence to Jia-Wei Wang.

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The authors declare no competing interests.

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Nature Plants thanks Remko Offringa, Tony Millar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression of miR156/7 in Arabidopsis.

a, Expression of miR156/7 and pMIR156C:GFP-N7 reporter in the shoot apex and embryos. Plants were grown under LD conditions. The shoot apices of 6- or 18-day-old plants and the embryos at the bent cotyledon stage were harvested and examined. For the shoot apices samples, all the leaves larger than 0.5 mm in length were removed. The expression level in the shoot apex at 6 d was set to 1.0. The results were represented by mean ± sem, n = 4. The statistical analysis is shown in Supplementary Table 1. b, Schematic diagram of miR156/7 sensor. The GFP-N7 was driven by the promoter of RIBO (AT2G18020), and followed by the 3’ UTR of SPL3 which contains a miR156/7 target site (red line). c, Periodic expression of miR156/7* during alternation of generations. The abundance of miR156a*-d*, miR156f*, miR157a*/b* and miR157c* was used to infer the periodic expression of miR156/7 during alternation of generations. miR157a/b have the same miRNA* sequence (miR157a*/b*). The star sequence for miR156e was not available. The results were represented by mean ± sd. For juvenile leaf, adult leaf and pollen samples, n = 3; for other tissue samples, n = 2. Please note that miR156a-c are highly abundant within the miR156 family. See also Fig. 1c. The original data for miR156/7 abundance are shown in Supplementary Table 2.

Source data

Extended Data Fig. 2 Expression of pMIR156C:GFP-N7.

a, Expression of the pMIR156C:GFP-N7 reporter in cotyledon and leaves in long days. Dashed lines mark the successive rosette leaves (the 1st to 4th leaf). GFP-N7 fluorescence was examined on the day as indicated in the left corner of each panel. DAS, days after sowing. Scale bar, 1.0 mm. See also ref. 23. b,c, Expression of pMIR156C:GFP-N7 in developing embryos. The embryos at different development stage as indicated in (b) were examined. Dashed lines mark the outline of embryos. Selfed homozygous plants were examined in (b) and reciprocal crossed plants between pMIR156C:GFP-N7 and pRIBO:H2B-mCherry were examined in (c). The fluorescence of H2B-mCherry is not shown. Scale bar, 20 µm.

Extended Data Fig. 3 Expression of pMIR156A:GFP-N7 and pMIR157A:GFP-N7.

a, Expression of the pMIR156A:GFP-N7 reporter in cotyledon and leaves in long days. Dashed lines mark the successive rosette leaves (the 1st to 4th leaf). Scale bar, 0.5 mm. b, Expression of the pMIR156A:GFP-N7 reporter in pollen and mature ovule. Scale bar, 20 µm. c, Expression of pMIR156A:GFP-N7 in developing embryos. The embryos at different development stage were examined. Dashed lines mark the outline of embryos. Scale bar, 20 µm. d, Expression of pMIR157A:GFP-N7 in pollen, embryo (left) and seedling (right). Dashed lines mark the outline of a globular stage embryo, cotyledon and leaves. pMIR157A:GFP-N7 was not expressed in pollen and embryo, which were examined using a wide-field fluorescence microscope. Scale bars, 50 μm for pollen and embryo, and 0.5 mm for cotyledon and leaves.

Extended Data Fig. 4 Phenotypes and complementation of the mir156/7 multiple mutants.

a, Phenotypes of WT and mir156/7 multiple mutants under LD conditions. Plants were grown for 14 days. Scale bar, 1 cm. See also Fig. 2c. b, Appearance of the 1st abaxial trichome in the mutants under LD conditions. Data were represented by violin plots with median and quartiles (n ≥ 11). c, Phenotypes of the T1-generation of mir156oct and mir156/7duodec transformed with the genomic fragment of MIR156C. Plants were grown under LD conditions. Scale bar, 2 cm. d, Appearance of the 1st abaxial trichome in the T1-generation of mir156oct and mir156/7duodec transformed with a genomic fragment of MIR156C. Plants were grown under LD conditions. Data were represented by violin plots with median and quartiles (n ≥ 4). Letters indicate significant differences as determined by one-way ANOVA, p < 0.01. e, Flowering time indicated by the number of rosette leaves after bolting. The T1-generation plants were grown under LD conditions. Data were represented by violin plots with median and quartiles (n ≥ 4). Letters indicate significant differences as determined by one-way ANOVA, p < 0.01. The sample size and statistical analysis results are given in Supplementary Table 5 (b-e).

Source data

Extended Data Fig. 5 Leaf shape and flowering time analyses of the mir156/7 multiple mutants.

a, Leaf shapes of WT, mir156abc, mir156oct, and mir156/7duodec under LD conditions when flowering. Scale bar, 1 cm. b, Cotyledon length/width ratio of WT, mir156abc, mir156oct, and mir156/7duodec under LD conditions. The results were shown as mean ± sd, n ≥ 12, with 2 cotyledons from each plant. Letters indicate significant differences as determined by one-way ANOVA, p < 0.01. The sample size and statistical analysis results are given in Supplementary Table 5. c, Flowering time phenotype of WT, mir156abc, mir156oct, and mir156/7duodec under SD conditions. Please note that the mir156/7duodec mutant had already bolted. Scale bar, 5 cm. d, Flowering time measured by the total number of rosette leaves after bolting. The WT, mir156abc, mir156oct, and mir156/7duodec mutants were grown under SD conditions. Data were represented by violin plots with median and quartiles (n ≥ 12). Letters indicate significant differences as determined by one-way ANOVA, p < 0.05. The sample size and statistical analysis results are given in Supplementary Table 5. See also Fig. 3c. e, Temporal expression of AP1 and SOC1 in the shoot apices of WT, mir156abc, mir156oct, and mir156/7duodec mutants under LD conditions. The results were represented by mean ± sd, n = 3. Two biological replicates were performed. One representative replicate with three technical repeats was shown. The statistical analysis is shown in Supplementary Table 1. See also Fig. 3e.

Source data

Extended Data Fig. 6 Comparison of the embryos of WT and the mir156/7duodec mutant.

a, Volcano plots showing differentially expressed genes based on RNA-seq. The embryos of WT and the mir156/7duodec mutant were compared. Ns, no significant difference between two samples. The known genes involved in flowering time and floral patterning were indicated. The full names of the selected genes are given in Supplementary Table 6. b, Gene ontology (GO) term analyses based on the ATAC-seq (left) and RNA-seq (right) datasets. The selected 17 highly enriched up-regulated GO biological processes based on the ATAC-seq dataset are indicated. The -log10(FDR) were given. See also Supplementary Table 7. c, The ATAC-seq tracks (left panel) and RNA-seq data (right panel) for representative flowering time genes. RNA counts were normalized by DESeq2 based on size factors and were shown as mean ± sd, n = 2. The values in brackets represent normalized ATAC-seq reads count. Blue, the coding region of selected genes; gray, adjacent genes. The directions of genes were shown by white arrowheads. d,e, Embryonic phenotype of WT and mir156/7duodec mutant. The representative embryos at heart or mature stage were shown in (d). Please note that the development of some mir156/7duodec embryos was delayed. The proportion of normal embryos was shown in (e). Three biological replicates were performed (n = 100 for each replicate). The results were shown as mean ± sd. Two tailed Student’s t test, p < 0.05; ns, no significant difference. Scale bar, 50 µm.

Source data

Extended Data Fig. 7 Identification of a role of the JRR in transgenerational reset of juvenility.

a, The ATAC-seq tracks (upper panel) and H3K27me3 ChIP-seq data (lower panel) for MIR156B, MIR157A and MIR157C loci at different developmental stage. Blue arrows show the position and direction of the hairpin region of the MIRNA gene; adjacent genes are shown as gray boxes with transcription direction indicated by white arrowheads. The values in brackets represent normalized reads count. See also Fig. 5a. b, Schematic diagram of the MIR156C reporter and its JRR-deletion form. c, Schematic diagram of the MIR156C genomic construct and its JRR-deletion form. d, Phenotype of transgenic plants. The mir156ac double mutant (as in Fig. 6c) was transformed with MIR156CWT or MIR156C-JRR construct. Plants were grown under LD conditions. Please note that the mutant phenotype was only rescued by MIR156CWT construct. Scale bar, 1 cm. e, Appearance of the 1st abaxial trichome in WT, mir156ac, and transgenic plants grown under LD conditions. The mir156ac mutants were transformed with MIR156CWT or MIR156C-JRR constructs. Four independent T2 lines for each construct are shown (n ≥ 16). Data are represented by violin plots showing the median and quartiles. Letters indicate significant differences as determined by one-way ANOVA, p < 0.01. The statistical analysis is shown in Supplementary Table 5.

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Extended Data Fig. 8 MIR156C and FLC are direct targets of LEC2.

a, Leaf length/width ratio of WT, mir156ac and lec2 under LD conditions. The results were shown as mean ± sd. n ≥ 12. Dash lines represented the lack of rosette leaves in some plants caused by early flowering. The data of WT were the same as Fig. 3d. The statistical analysis is shown in Supplementary Table 5. b, Leaf shapes of WT, mir156ac and lec2 under LD conditions when flowering. Scale bar, 1 cm. c, Transcript abundance of selected genes by RNA-seq. RNA counts were normalized by DESeq2 based on size factors and were shown as mean ± sd, n = 2. d, The ChIP-PCR data of H3K27me3 for MIR156A and MIR156C gene loci in WT and lec2. The symbols are as described in Extended Data Fig. 7. Two biological replicates were performed. One representative replicate with three technical repeats is shown. The results were represented by mean ± sd. Two-tailed Student’s t test, **, p < 0.01; *, p < 0.05; ns, no significant difference. The statistical analysis is shown in Supplementary Table 1. e, Competitive EMSA showing binding of LEC2-DBD (DNA-binding domain of LEC2) to the RY motif of MIR156C promoter. The relative amount of unlabeled competitive oligonucleotide is indicated on the top. Schematic of the MIR156C genomic region and the position of the RY motif are shown in Fig. 5a. The assays without GST-LEC2-DBD protein (-) or with mutated probes (mut) were performed as controls. The probe sequences are listed in Supplementary Table 3. f, ATAC-seq tracks and LEC2 ChIP data for MIR156B gene locus in WT and lec2. The ChIP-seq data of p35S:LEC2-3xFLAG-GR seedlings and ChIP-PCR data of pLEC2:LEC2-3xFLAG showed similar results. The symbols are as described in Extended Data Fig. 7. The results were represented by mean ± sem, n = 3. Two-tailed Student’s t test, *, p < 0.05; ns, no significant difference. The statistical analysis is shown in Supplementary Table 1. See also Fig. 6h. g, ATAC-seq tracks and p35S:LEC2-3xFLAG-GR ChIP-seq data for MIR157A and MIR157C and FLC gene loci. Symbols were described in Extended Data Fig. 7. See also Fig. 6h. h, Expression levels of MIR156B and miR156 in the embryos (3 days after pollination) of WT and rkd4 mutant. n = 3 independent biological replicates. The results are shown as mean ± sem. Two-tailed Student’s t test, **, p < 0.01. The statistical analysis is shown in Supplementary Table 1. i, Induction of MIR156B and miR156 by RKD4. We generated an inducible line for RKD4 based on the GR-DEX system. The 10-day-old p35S:RKD4-GR seedlings were treated with DMSO (mock) or 10 μM DEX for 4 h. n = 3 independent biological replicates. The results are shown as mean ± sem. Two-tailed Student’s t test, **, p < 0.01; ****, p < 0.0001. The statistical analysis is shown in Supplementary Table 1.

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Supplementary information

Reporting Summary.

Supplementary Table 1

Quantification statistics.

Supplementary Table 2

Small RNA-seq data.

Supplementary Table 3

Oligos used in the study.

Supplementary Table 4

Mutants of mir156/7 generated by CRISPR/Cas9.

Supplementary Table 5

Phenotype statistics.

Supplementary Table 6

Differential analysis of ATAC and RNA-seq for mir156/7duodec mutant and WT embryo.

Supplementary Table 7

GO analysis of ATAC and RNA-seq for mir156/7duodec mutant and WT embryo.

Supplementary Table 8

Differential analysis of ATAC-seq for embryos, juvenile leaf and adult leaf.

Supplementary Table 9

Differential analysis of ATAC and RNA-seq for lec2 mutant and WT embryo.

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Unprocessed EMSA blot.

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Gao, J., Zhang, K., Cheng, YJ. et al. A robust mechanism for resetting juvenility during each generation in Arabidopsis. Nat. Plants 8, 257–268 (2022). https://doi.org/10.1038/s41477-022-01110-4

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