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, 7 (3), e1002014

Polycomb Repressive Complex 2 Controls the Embryo-To-Seedling Phase Transition

Affiliations

Polycomb Repressive Complex 2 Controls the Embryo-To-Seedling Phase Transition

Daniel Bouyer et al. PLoS Genet.

Abstract

Polycomb repressive complex 2 (PRC2) is a key regulator of epigenetic states catalyzing histone H3 lysine 27 trimethylation (H3K27me3), a repressive chromatin mark. PRC2 composition is conserved from humans to plants, but the function of PRC2 during the early stage of plant life is unclear beyond the fact that it is required for the development of endosperm, a nutritive tissue that supports embryo growth. Circumventing the requirement of PRC2 in endosperm allowed us to generate viable homozygous null mutants for FERTILIZATION INDEPENDENT ENDOSPERM (FIE), which is the single Arabidopsis homolog of Extra Sex Combs, an indispensable component of Drosophila and mammalian PRC2. Here we show that H3K27me3 deposition is abolished genome-wide in fie mutants demonstrating the essential function of PRC2 in placing this mark in plants as in animals. In contrast to animals, we find that PRC2 function is not required for initial body plan formation in Arabidopsis. Rather, our results show that fie mutant seeds exhibit enhanced dormancy and germination defects, indicating a deficiency in terminating the embryonic phase. After germination, fie mutant seedlings switch to generative development that is not sustained, giving rise to neoplastic, callus-like structures. Further genome-wide studies showed that only a fraction of PRC2 targets are transcriptionally activated in fie seedlings and that this activation is accompanied in only a few cases with deposition of H3K4me3, a mark associated with gene activity and considered to act antagonistically to H3K27me3. Up-regulated PRC2 target genes were found to act at different hierarchical levels from transcriptional master regulators to a wide range of downstream targets. Collectively, our findings demonstrate that PRC2-mediated regulation represents a robust system controlling developmental phase transitions, not only from vegetative phase to flowering but also especially from embryonic phase to the seedling stage.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phenotypic analysis of postembryonic development.
Comparison between wild type (A–C) and fie mutant (C–L) development. (A) Wild type seedling at 5 DAS. (D) fie mutants resemble wild type plants at 5 DAS besides a reduction in growth. (B) Wild-type seedlings are in a vegetative phase at 15 DAS and under day-neutral growth conditions will start to bolt when one month old in contrast to (E) fie mutants that show flowers (arrow) already at 15 DAS. (C) Vegetative growth leads to major increase in size of a wild-type plant (left) after 40 DAS whereas a fie mutant plant (right) of the same age is much smaller (arrow). (F) 3 month-old fie mutant that has transformed into a callus-like structure. Examples of transformed and/or misplaced organs and cells in fie mutants (G–L). (G) Flower-like organs. (H) Leaves (arrow) develop from roots, which are able to transform into offshoots (I, arrow). (J) Somatic embryos are formed in high frequency. (K) Root hairs form at shoot tissue. (L) Leaf hairs (arrow) grow out from roots. Sugar-dependent lipid accumulation in WT and fie (M-O). Wild-type and fie mutant seedlings were stained with fat-red to visualize lipid accumulation at 5 DAS (M) or at 8 DAS (N). Whereas at early stages there is still a clear staining visible in wild type (M, left side), there is only a faint fed red signal detectable at 8 DAS (N, left). In contrast, fie shows strong staining in cotyledons and roots at both time points (M,N, right side). The lipid accumulation in fie is dependent on sugar, as mutants germinating on sugar-free medium show a strong reduction in staining (O).
Figure 2
Figure 2. Genome-wide distribution of H3K27me3 and H3K4me3 marks.
Genome browser view of Chromosome 4 with the H3K27me3 profile in wild type (first panel) and fie (second panel), boxed annotation of genes in blue (coding region) and grey (introns) (third row), boxed annotation of transposable elements and other heterochromatic regions in brown and orange (transposable element genes) (forth row), the H3K4me3 profile in wild type (fifth panel) and in fie (sixth panel). Enrichment in H3K27me3 or H3K4me3 marks is shown in green bars. (B) and (C) are close-ups and show the major loss of H3K27me3 in fie whereas H3K4me3 distribution is basically unchanged. Raw data have been deposited at GEO database (GSE24163, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24163).
Figure 3
Figure 3. Western blot detection of the H3K27me3 mark.
Western blot analysis of H3K27me3 in wild type and fie mutants. (A) Comparison between wild type and fie nuclear extracts revealed major loss of H3K27me3-signal in the mutant; however, after over-exposure (OE) a faint signal becomes visible in fie mutants. Detection of histone H3 was used a loading control. (B) Under less stringent conditions a weak signal in fie is detectable by the H3K27me3 antibody. Pre-incubating the antibody with a surplus of H3K27me3 peptides, reduces the signal intensity to only of faint band in wild-type extracts that roughly matches the intensity of the remaining signal seen in fie mutants. Detection of histone H3 was used as loading control. (C) Peptide competition assay using H3K27me3 antibody with increasing concentrations of H3K27me1- and H3K27me2-peptide in fie mutant resulted in a strong reduction of the remaining signal detected by the H3K27me3 antibody. (D) Both peptides could not reduce the H3K27me3 signal in wild-type extract indicating that the antibody works properly given that sufficient antigen is provided.
Figure 4
Figure 4. Overrepresented gene ontology categories in the set of H3K27me3 targets that are up-regulated in fie.
BiNGO (the Biological Network Gene Ontology tool) analysis representing over-represented categories of the ontology Biological Process among the genes that are marked by H3K27me3 in the wild type (20 DAS) and are significantly up-regulated in 7 DAS fie mutant seedlings. The size of the nodes is proportional to the number of genes in the test set which are annotated to that node. Colored nodes are significantly over-represented, with a color scale ranging from yellow (p-value = 0.05) to dark orange (p-value = 5.00E-7). Statistical testing was as described by Maere et al. (2005) .
Figure 5
Figure 5. PRC2 represses seed maturation and dormancy genes in the seedling.
All genes provided in this model have been identified as H3K27me3 targets and were significantly up-regulated in fie mutants. They include master regulators of seed development such as AGL15, LEC2, ABI3, FUS3, further downstream regulators such as WRI, integrators of environmental signals such as FLC and finally genes involved in seed storage and desiccation tolerance. * For detailed information which members of the oleosins (oil body coat proteins) and LEAs (late embryogenesis abundant proteins) are affected see Table S5. For LEC2 the up-regulation was only observed in qRT-PCR (Table S4). We find here that the ABA and GA hormonal signaling pathways that play a pivotal role in the transition form seed to seedling are under PRC2 control since genes playing a positive role in ABA signaling as well as genes with a negative role in GA signaling are H3K27me3 marked in wild type and up-regulated in fie. Large letters stand for high and small for low ABA and GA levels, respectively. Abscisic acid (ABA), Gibberellic acid (GA), AGAMOUS-Like 15 (AGL15, AT5G13790), LEAFY COTYLEDON 2 (LEC2, AT1G28300), ABA INSENSITIVE 3 (ABI3, AT3G24650), FUSCA 3 (FUS3, AT3G26790), WRINKLED1 (WRI, AT3G54320), FLOWERING LOCUS C (FLC, AT5G10140), CRUCIFERIN 3 (CRU3, AT4G28520), CRUCIFERINA (CRA1, AT5G44120), SEED STORAGE ALBUMIN 1 (2S1, AT4G27140), SEED STORAGE ALBUMIN 2, (2S2, AT4G27150), HYDROXYSTEROID DEHYDROGENASE 1 (AtHSD1, AT5G50600), Peroxiredoxin 1 (PER1, AT1G48130), ABA INSENSITIVE 4 (ABI4, AT2G40220), DELAY OF GERMINATION 1 (DOG1, AT5G45830), CHOTTO 1/AINTEGUMENTA-LIKE 5 (CHO1/AIL5, AT5G5739), SOMNUS (SOM, AT1G03790), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8, AT1G02065), AT-hook protein of GA feedback 1 (AGF1, AT4G35390), GIBBERELLIC ACID METHYLTRANSFERASE 2 (GAMT2, AT5G56300).
Figure 6
Figure 6. Dormancy and germination phenotypes.
(A) Both, homozygous fie and homozygous clf-swn mutants, show a similar delay in germination initially. However, clf swn germinates to 100% whereas 40% of fie seeds stay dormant within at least two weeks on plates. (B) Application of GA does not enhance the germination rate of fie mutant seeds. (C–N) Phenotypical comparison of wild type (C–E, I–K) and fie mutants (F–H, L–N). (C–H) Time series of post-embryonic development for dissected wild type (C–E) and fie (F–H) till day 3 after isolation. Initially, fie embryos isolated from dormant seeds (F) are undistinguishable from wild type embryos dissected 24h after imbibition (C). (D) Wild type embryos start to grow within 24h after the transfer to media. (E) Unfolding as well as greening of the cotyledons, root growth and root hair formation as well as anthocyanin accumulation takes place within 3 days. (G–H) Around ¼ of the fie seedlings break dormancy and display a similar developmental program as wild type from day 1 (G) to day 3 (H) after dissection. (I–K) Effect of ABA on wild type seedling growth. (I, J) Wild-type seedlings germinated on ABA-containing media are strongly affected in growth, appear yellowish, are delayed in greening and do not produce roots. (K) After the transfer from ABA plates to normal media, wild-type plants recover within 24h, e.g. they green, and show completely normal development further on. (L–N) The majority (around ¾) of the dissected fie mutant embryos do not develop similar to wild type, but resemble wild type plants germinated on high levels of exogenous ABA. (M,N) Typically, a strong delay in growth and greening with yellowish cotyledons and defects in root- and root hair growth and stunted leaves is observed, though the overall body plan is not affected (N) Greening is seen only after 10 days. (L) In some cases the mutant does not develop seedling traits at all.
Figure 7
Figure 7. The set of H3K4 trimethylated genes changes only marginally between wild type and fie and is under-represented among H3K27me3 targets.
VENN Diagram representing the number of genes marked by H3K27me3 in wild type (green) and H3K4me3 in wild type (purple) and fie (pink). The mutual overlap of the H3K4me3 targets in wild type and fie is larger than expected for two independent sets, while the overlap of the H3K4me3 marked genes with the H3K27me3 marked genes is significantly smaller (wild type H3K4me3 and fie H3K4me3: rf = 1.9, p<1.0e−99* wild type H3K4me3 and wild type H3K27me3: rf = 0.2, p<1.0e−99*; fie H3K4me3 and wild type H3K27me3: rf = 0.2, p<1.0e−99*). P-values marked by an asterisk (*) were below the calculation limits of the software (highly significant).

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