Analysis of polycomb repressive complex 2 (PRC2) subunits in Picea abies with a focus on embryo development

Abstract

Background: Conserved polycomb repressive complex 2 (PRC2) mediates H3K27me3 to direct transcriptional repression and has a key role in cell fate determination and cell differentiation in both animals and plants. PRC2 subunits have undergone independent multiplication and functional divergence in higher plants. However, relevant information is still absent in gymnosperms.

Results: To launch gymnosperm PRC2 research, we identified and cloned the PRC2 core component genes in the conifer model species Picea abies, including one Esc/FIE homolog PaFIE, two p55/MSI homologs PaMSI1a and PaMSI1b, two E(z) homologs PaKMT6A2 and PaKMT6A4, a Su(z)12 homolog PaEMF2 and a PaEMF2-like fragment. Phylogenetic and protein domain analyses were conducted. The Esc/FIE homologs were highly conserved in the land plant, except the monocots. The other gymnospermous PRC2 subunits underwent independent evolution with angiospermous species to different extents. The relative transcript levels of these genes were measured in endosperm and zygotic and somatic embryos at different developmental stages. The obtained results proposed the involvement of PaMSI1b and PaKMT6A4 in embryogenesis and PaKMT6A2 and PaEMF2 in the transition from embryos to seedlings. The PaEMF2-like fragment was predominantly expressed in the endosperm but not in the embryo. In addition, immunohistochemistry assay showed that H3K27me3 deposits were generally enriched at meristem regions during seed development in P. abies.

Conclusions: This study reports the first characterization of the PRC2 core component genes in the coniferous species P. abies. Our work may enable a deeper understanding of the cell reprogramming process during seed and embryo development and may guide further research on embryonic potential and development in conifers.

Keywords: H3K27me3; Norway spruce; Polycomb group; Seed development; Somatic embryogenesis.

Fig. 1

Phylogenetic tree of Esc/FIE homologs. The tree was made using maximum likelihood (ML) and amino acid data. Branch lengths are indicated by the number on the respective branch. Bootstrap support values are indicated by the number beneath the respective branch. Esc/FIE is highly conserved between gymnosperm and dicots. The tree contains sequences from green algae (Ostreococcus lucimarinus, Ol; Volvox carteri, Vc; and Chlamydomonas reinhardtii, Cr. ), bryophytes (Physcomitrella patens, Pp), lycophytes (Selaginella moellendorffii, Sm), ferns (Ceratopteris richardii, Cer), gymnosperms (P. abies, Pa., Pseudotsuga menziesii, Pm, Taxus baccata, Tb, Gnetum montanum Gm, Pinus sylvestris, Ps and Ginkgo biloba, Gb), and angiosperms (dicotyledons: A. thaliana, At, Nymphaea colorata, Nc, Citrus sinensis, Cs, Populus trichocarpa. Pt and Vitis vinifera, Vv; monocots: Zea mays, Zm, Oryza sativa, Os, Brachypodium distachyon, Bd.). Drosophila melanogaster (Dm) was used as an outgroup. The P. abies sequences are indicated by red bold font. Most Esc/FIE proteins contain two or three WD40 domains

Fig. 2

Phylogenetic tree of p55/MSI homologs. The tree was made using maximum likelihood (ML) and amino acid data. Branch lengths are indicated by the number on the respective branch. Bootstrap support values are indicated by the number beneath the respective branch. The MSI1 homolog has been duplicated in gymnosperm. The tree contains sequences from green algae (Ostreococcus lucimarinus, Ol; Volvox carteri, Vc; and Chlamydomonas reinhardtii, Cr. ), bryophytes (Physcomitrella patens, Pp), lycophytes (Selaginella moellendorffii, Sm), ferns (Ceratopteris richardii, Cer), gymnosperms (P. abies, Pa., Pseudotsuga menziesii, Pm, Taxus baccata, Tb, Gnetum montanum Gm, Pinus sylvestris, Ps and Ginkgo biloba, Gb), and angiosperms (dicotyledons: A. thaliana, At, Nymphaea colorata, Nc, Citrus sinensis, Cs, Populus trichocarpa. Pt and Vitis vinifera, Vv; monocots: Zea mays, Zm, Oryza sativa, Os, Brachypodium distachyon, Bd.). Drosophila melanogaster (Dm) was used as an outgroup. The P. abies sequences are indicated by red bold font. Most of the p55/MSI proteins contain subunit C of the CAF1 complex (CAF1C-H4-bd) and three or five WD40 domains

Fig. 3

Phylogenetic tree of E(z) homologs. The tree is made using maximum likelihood (ML) and amino acid data. Branch lengths are indicated by the number on the respective branch. Bootstrap support values are indicated by the number beneath the respective branch. The phylogenetic analyses showed that E(z) homologs underwent independent radiation after the split between angiosperms and gymnosperms. The tree contains sequences from green algae (Ostreococcus lucimarinus, Ol; Volvox carteri, Vc; and Chlamydomonas reinhardtii, Cr. ), bryophytes (Physcomitrella patens, Pp), lycophytes (Selaginella moellendorffii, Sm), ferns (Ceratopteris richardii, Cer), gymnosperms (P. abies, Pa., Pseudotsuga menziesii, Pm, Taxus baccata, Tb, Gnetum montanum Gm, Pinus sylvestris, Ps and Ginkgo biloba, Gb), and angiosperms (dicotyledons: A. thaliana, At, Nymphaea colorata, Nc, Citrus sinensis, Cs, Populus trichocarpa. Pt and Vitis vinifera, Vv; monocots: Zea mays, Zm, Oryza sativa, Os, Brachypodium distachyon, Bd.). Drosophila melanogaster (Dm) was used as an outgroup. The P. abies sequences are indicated by red bold font. Most E(z) proteins have SET, SANT and CXC domains

Fig. 4

Phylogenetic tree of Su(z)12 homologs. The tree is made using maximum likelihood (ML) and amino acid data. Branch lengths are indicated by the number on the respective branch. Bootstrap support values are indicated by the number beneath the respective branch. The phylogeny of Su(z)12 homologs was similar to that of known plant evolution. The tree contains sequences from green algae (Ostreococcus lucimarinus, Ol; Volvox carteri, Vc; and Chlamydomonas reinhardtii, Cr.), bryophytes (Physcomitrella patens, Pp), lycophytes (Selaginella moellendorffii, Sm), ferns (Ceratopteris richardii, Cer), gymnosperms (P. abies, Pa., Pseudotsuga menziesii, Pm, Gnetum montanum Gm, Pinus sylvestris, Ps and Ginkgo biloba, Gb), and angiosperms (dicotyledons: A. thaliana, At, Nymphaea colorata, Nc, Citrus sinensis, Cs, Populus trichocarpa. Pt and Vitis vinifera, Vv; monocots: Zea mays, Zm, Oryza sativa, Os, Brachypodium distachyon, Bd.). Drosophila melanogaster (Dm) was used as an outgroup. The P. abies sequences are indicated by red bold font. All Su(z)12 proteins have a ZnF-C2H2 domain and the VEFS box

Fig. 5

Expression analysis of the P. abies PRC2 subunit genes in seeds. Bars = 250 μm. (A) Early seed, inset: longitudinal section of the early seed. (B) Longitudinal section of seed contains late zygotic embryo (ZE). ep, embryo proper; sus, suspensor. Inset: late ZE (left) and endosperm (right). (C) Seed contains mature ZE. (D) Relative transcript level of the P. abies PRC2 genes (± SEM). Seedling needles were used as a somatic control. All data were relative to the transcript level of PaMSI1a in early seed. Error bars indicate standard deviations (n = 3) between biological replicates. Means that do not share a letter are significantly different according to Duncan test (p-value < 0.05). All of the PRC2 genes, except PaFIE, showed higher transcript abundance in the samples at late stage than it at mature stage. The transcript level of PaFIE, PaMSI1a and PaKMT6A2 were relatively stable among the seed samples. The higher transcript level of PaKMT6A2 in late endosperm might be due to mix of samples from earlier stage. The transcript level of PaMSI1b and PaKMT6A4 were higher in ZEs than in endosperms. The PaEMF2-like fragment was dominantly detected in endosperms and early seed, which also contained endosperm

Fig. 6

Expression analysis of the P. abies PRC2 subunit genes during somatic embryo (SE) development and germination. Bar = 250 μm. A-D) Somatic embryogenesis of the nonembryonic callus (NEC), including (A) proliferating NEC; (B) cultures after one week on medium lacking auxin and cytokinin to stimulate differentiation of early SE; (C) cultures on maturation medium supplemented with ABA for one week; (D) cultures on maturation medium for three weeks. Of Note, no SE developed in NEC. E-L) Somatic embryogenesis and germination of embryonic callus (ECs), including (E) proliferating ECs; (F) cultures after one week on medium lacking auxin and cytokinin to stimulate differentiation of early SE; (G) cultures on maturation medium supplemented with ABA for one week, note the intermediate SE pointed by the white arrows; (H) cultures on maturation medium for 2 weeks, note the late SE; (I) maturing SE after 4 weeks on maturation medium; (J) Fully matured SE after 6 weeks on the maturation medium; K) desiccated SE after partial desiccation treatment; L) germinated SE after 4 weeks of germination. M) Relative transcript level of the P. abies PRC2 genes (± SEM). All data were relative to the transcript level of PaMSI1a in the callus of NEC. Error bars indicate standard deviations (SD) (n = 3) between biological replicates. Means that do not share a letter are significantly different according to Duncan test (p-value < 0.05). Asterisks indicate the significant difference between NEC and EC samples according to least significance difference test (‘*’, p-value < 0.05; ‘**’, p-value < 0.01; and ‘nd’, no difference)

Fig. 7

H3K27me3 immunolocalization in Picea abies seeds. Bars = 250 μm. A-C) H3K27me3 is widespread in the endosperm (en). (A) A seed contains an early zygotic embryo (ZE). Lower-left inset, control. Note that H3K27me3 is widespread in the embryonal proper (ep). (B) A seed contains a late ZE. Upper-left inset, enlarged late ZE with higher resolution; lower-left inset, control. Note that H3K27me3 is deposited at the shoot apical meristem (SAM) and root apical meristem (RAM) region. (C) A seed contains mature ZE (upper, anti-H3K27me3; lower, control). (D) Enlarged mature ZE with higher resolution. Note that H3K27me3 is particularly deposited at SAM, RAM, cytoledon primordia (cp.) and around the pith

Fig. 8

Sketch shows the coniferous embryogenesis and the developmental processes in which the PRC2 subunits participate in. In conifer, the fertilized zygotic nucleus divides into four free nuclei without cellularization. Then, after several round cell division, the basal tiers elongate to form a functional suspensor and the apical tiers form the embryonal proper. During somatic embryogenesis, the elimination of auxin and cytokinin stimulate the differentiation of early somatic embryos. Early embryogeny begins with the elongation of the embryonal suspensor. The zygotic and somatic embryogenesis are morphologically similar from early embryogeny phase. Embryo patterning, including the establishment of root apical meristem, shoot apical meristem, procambium, cotyledon primordia and cortex etc., are performed during early and late embryogeny. After maturation, embryos can germinate under suitable conditions. Our data suggested that PaEMF2-like, together with the generally expressed PaMSI1a and PaFIE, involve in the development of endosperm; no endosperm specific E(z) homolog was charactered in our study; PaKMT6A4, PaMSI1s and PaFIE were related with embryogenesis and might contribute to embryo patterning; no Su(z)12 homolog was upregulated in early SE in our study; PaKMTA2 and PaEMF2, together with PaMSI1a and PaFIE, might participate in the embryo to seedling transition. In addition, PaKMT6A2, PaEMF2, PaMSI1a and PaMSI1b might be antagonistic regulators of somatic embryogenesis