FIE, a nuclear PRC2 protein, forms cytoplasmic complexes in Arabidopsis thaliana

Abstract

Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers that regulate developmental pathways in plants. PcGs form nuclear multi-subunit Polycomb Repressive Complexes (PRCs). The PRC2 complex mediates gene repression via methylation of lysine 27 on histone H3, which consequently leads to chromatin condensation. In Arabidopsis thaliana, several PRC2 complexes with different compositions were identified, each controlling a particular developmental program.The core subunit FIE is crucial for PRC2 function throughout the plant life cycle, yet accurate information on its spatial and temporal localization was absent. This study focused on identifying FIE accumulation patterns, using microscopy and biochemical approaches. Analysing endogenous FIE and transgenic gFIE-green fluorescent protein fusion protein (gFIE-GFP) showed that FIE accumulates in the nuclei of every cell type examined. Interestingly, gFIE-GFP, as well as the endogenous FIE, also localized to the cytoplasm in all examined tissues. In both vegetative and reproductive organs, FIE formed cytoplasmic high-molecular-mass complexes, in parallel to the nuclear PRC2 complexes. Moreover, size-exclusion chromatography and bimolecular fluorescence complementation assays indicated that in inflorescences FIE formed a cytoplasmic complex with MEA, a PRC2 histone methyltransferase subunit. In contrast, CLF and SWN histone methyltransferases were strictly nuclear. Presence of PRC2 subunits in cytoplasmic complexes has not been previously described in plants. Our findings are in agreement with accumulating evidence demonstrating cytoplasmic localization and function of PcGs in metazoa. The cytosolic accumulation of PRC2 components in plants supports the model that PcGs have alternative non-nuclear functions that go beyond chromatin methylation.

Keywords: Cytoplasm; FIE; MEA; PRC2.; PcG; Polycomb complex.

Fig. 1.

FIE protein accumulates in all Arabidopsis tissues and organs. (A) Detection of endogenous FIE protein in Arabidopsis tissues. A similar amount of nuclear chromatin protein extracts from rosette, stems, cauline, and inflorescence of 20-day-old wild-type plants and siliques from 6–10 days after pollination (DAP) were separated by SDS-PAGE and then immunodetected using αFIE antibodies. Protein size marker is indicated on the left. Ponceau staining was used to assess equal sample loading. (B) A scheme of the ProFIE:FIE_gDNA-GFP transgene construct (not drawn to scale). Red boxes represent exons. (C) gFIE-GFP chimeric protein from line no. TH142-1 is observed in reproductive and vegetative tissues and organs. Each image was taken from a single focal plane, unless otherwise indicated. Each panel shows a merge of GFP epifluorescence (green) and the corresponding chloroplast autofluorescence (red) and/or a bright-field image. (1) Rosette leaf, abaxial epidermis; scale bar (SB): 50 μm. (2) Rosette leaf, mesophyll cells; SB: 25 μm. (3) Trichome on the surface of a rosette leaf, Z-stack overlay; SB: 50 μm. (4) Hypocotyl, Z-stack overlay; SB: 50 μm. (5) Cauline leaf, abaxial epidermis; SB: 25 μm. (6) Petal; SB: 25 μm. (7) Sepal; SB: 50 μm. (8) Carpel; SB: 250 μm. (9) Pollen; SB: 20 μm. (10) Unfertilized ovules; SB: 50 μm. (11) Developing seed containing a four cell-stage embryo; SB: 50 μm. (12) Developing seed containing a heart-stage embryo; SB: 100 μm. (13) Main root, root hairs; SB: 50 μm. (14) Main and lateral roots; SB: 500 μm. (15) Budding lateral root; SB: 50 μm. (16) Main root tip, quiescent center; SB: 50 μm.

Fig. 2.

gFIE-GFP is localized to the cytoplasm. Laser scanning confocal microscopy imaging was employed following plasmolysis of Arabidopsis rosette leaf cells to determine the subcellular localization of the gFIE-GFP fusion protein (green). To visualize the cell walls, leaf samples were stained with propidium iodide (red). Each image was taken from a single focal plane. (A) Cells before plasmolysis. (B) Plasmolysed cells. Arrowheads indicate cell wall. Scale bar: 25 μm.

Fig. 3.

Endogenous FIE protein accumulates in the cytoplasm. (A) Nuclear (Nuc) and soluble cytoplasmic (Cyt) proteins were extracted from inflorescences of FIE-GFP plants, and analysed by SDS-PAGE followed by immunoblotting with αFIE and αGFP (Covance) antibodies. (B) Native cytoplasmic proteins were extracted from various tissues of wild-type Arabidopsis plants and equal amounts of samples were analysed by SDS-PAGE. The blot was probed with αFIE antibody. FIE was detected as a double band in all tissues, aside from rosette leaves. (C) Non-denaturated cytosolic protein extracts from rosette leaf tissue were sedimented by ultracentrifugation. Equal volumes from supernatant soluble fraction before and after (−/+) sedimentation were analysed by SDS-PAGE and probed with αFIE antibody. Absence of RuBisCO large chain protein band at ~53 kDa (marked with asterisk) following ultracentrifugation demonstrated successful sedimentation of large protein complexes. Ponceau staining in panels (A–C) was used to assess equal loading of samples.

Fig. 4.

FIE forms high-molecular-mass complexes in the cytosol. Size-exclusion chromatography analysis of cytosolic proteins extracted from rosette leaves of 17-day- old seedlings of WT and TH142 (ProFIE:FIE_gDNA-GFP) transgenic plants. The blots were immunoprobed with αGFP (top panel) and αFIE (middle and bottom panels) antibodies. Size markers (kDa) are indicated above relevant fractions. Large amounts of gFIE-GFP protein in fractions 10–14 above and below the main band are probably the result of large amounts of polypeptide. Migration of FIE protein in fractions 6–10 to an apparent lower molecular size band, as compared with other fractions, may result from the presence of a large amount of RuBisCO large chain (~53 kDa), as seen in Fig. 3C.

Fig. 5.

Subcellular localization of the interaction between FIE and PcG-SET domain proteins. (A) Equal amounts of nuclear (Nuc) and cytoplasmic (Cyt) protein extracts from WT inflorescences were analysed by SDS-PAGE and probed with αMEA antibodies. Equal loading was based on the presence of non-specific low-molecular-mass proteins in both samples (marked with asterisk). (B) Nuclear and cytoplasmic protein extracts of 14-day-old wild-type seedlings analysed by SDS-PAGE and probed with αCLF, αSWN and αFIE antibodies. CLF and SWN proteins were detected only in the nuclear fraction, while FIE was detected in both fractions. (C) Size-exclusion chromatography analysis of cytosolic proteins extracted from inflorescences (flowers after anthesis) of ProFIE:FIE_gDNA-GFP transgenic plants. The blots were immunoprobed with αMEA (top lane) and αGFP (lower lane) antibodies. Arrows point to a 97 kDa MEA polypeptide (upper arrow), and non-specific faster migrating polypeptides (lower arrow). Size markers (kDa) are indicated above relevant fractions. (D) BiFC assay in Arabidopsis cotyledon leaves using YC-HA-FIE/YN-GG-MEA, YC-HA-FIE/YN-GG-CLF and YC-HA-FIE/YN-GG-SWN. Red signal, chloroplast autofluorescence; green signal, reconstituted-YFP fluorescence, resulting from the interaction. Scale bar: 20 μm.