BiP-mediated polar nuclei fusion is essential for the regulation of endosperm nuclei proliferation in Arabidopsis thaliana

Abstract

Nuclear fusion is an essential process in the sexual reproduction of animals and plants. In flowering plants, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse to produce the diploid central cell nucleus, and twice during double fertilization. The yeast Ig binding protein (BiP) is a molecular chaperone Hsp70 in the endoplasmic reticulum that regulates nuclear membrane fusion during mating. Here we report that in Arabidopsis thaliana, BiP is involved in the fusion of polar nuclei during female gametophyte development. BiP-deficient mature female gametophytes contain two unfused polar nuclei, in spite of their close contact. This indicates a surprising conservation of BiP function in nuclear fusion between plants and yeasts. We also found that endosperm nuclear division becomes aberrant after fertilization of the BiP-deficient female gametophytes with wild-type pollen. This is experimental evidence for the importance of fusion of the polar nuclei in the proliferation of endosperm nuclei.

Fig. 1.

The bip1 bip2 double mutation causes defects in the fusion of polar nuclei during female gametophyte development. (A and B) Siliques of wild-type (A) and b1/+ b2/b2 (B) plants at 8 DAP. Half of the seeds in the b1/+ b2/b2 siliques aborted during development. (Scale bars, 0.5 mm.) (C–F) CLSM analysis of ovules in wild-type (C), b2/b2 (D), and b1/+ b2/b2 (E and F) plants. Half of the ovules in b1/+ b2/b2 plants are morphologically normal (E), and in the other half, the polar nuclei remain unfused (F). syn, synergid nuclei; ecn, egg nucleus; scn, secondary nucleus; upn, unfused polar nuclei. (Scale bars, 50 μm.) (G and H) Electron micrographs of the central cell of a b1 b2 female gametophyte containing unfused polar nuclei. The region indicated by a box in G is magnified in H. The arrows indicate the nuclear envelopes of the two polar nuclei. pn1, polar nucleus 1; pn2, polar nucleus 2. (Scale bars, 1 μm.)

Fig. 2.

The bip1 bip2 female gametophyte is not defective in fertilization. (A and B) The bip1 bip2 double mutation does not affect pollen tube guidance. Wild-type (A) or b1/+ b2/b2 (B) pistils were pollinated using pollen from a pBiP1::GUS transgenic plant. Pollen tube entry into the female gametophytes was visualized by GUS staining at 1 DAP (blue color). The percentages of GUS-positive ovules are shown below the panels. (Scale bars, 0.5 mm.) (C–E) Pistils of wild-type (3 pistils) or b1/+ b2/b2 (4 pistils) plants were pollinated with wild-type pollen. Ovules were fixed at 7 h after pollination, cleared, and observed by CLSM. Representative images of ovules containing degenerated synergid cells are shown. Each wild-type ovule contains a endosperm nucleus in the central cell (C), whereas the b1/+ b2/b2 pistils have two types of ovules, one type (b2) containing a secondary nucleus (D) and the other (b1 b2) containing unfused polar nuclei (E) in the central cell. The numbers of fertilized ovules (containing degenerated synergid cells) and total ovules observed are shown below the panel. (F–K) Pistils of a b1/+ b2/b2 plant expressing histone H2B-GFP from the ACTIN11 promoter were pollinated with wild-type pollen expressing HTR10-mRFP1. Ovules were dissected from the pistils and observed by CLSM at 8 h after pollination. (F–H) Images of a fertilized b2 ovule. (I–K) Images of a b1 b2 ovule. (F and I) GFP fluorescence (green), (G and J) mRFP fluorescence (magenta), and (H and K) merged images. The dashed lines indicate the edges of the embryo sacs. syn, synergid nucleus; dsc, degenerated synergid cell; en, endosperm nucleus; zn, zygote nucleus; upn, unfused polar nucleus. (Scale bars, 50 μm.)

Fig. 3.

Fusion of polar nuclei during female gametogenesis is essential for normal proliferation of endosperm nuclei. (A–H) Embryo development after b2 female gametophytes were fertilized with wild-type pollen (b2 seeds, A–D) and after b1 b2 female gametophytes were fertilized with wild-type pollen (b1 b2 seeds, E–H). (A and E) 2 DAP embryo sacs. (B and F) 3 DAP embryo sacs. (C and G) 4 DAP embryo sacs. (D and H) 5 DAP embryo sacs. (I–N) Endosperm development after b2 female gametophytes were fertilized with wild-type pollen (b2 seeds, I–K), and after b1 b2 female gametophytes were fertilized with wild-type pollen (b1 b2 seeds, L–N). (I) and (L) 1 DAP embryo sacs. (J and M) 2 DAP embryo sacs. (K and M) 3 DAP embryo sacs. Arrowheads and arrows indicate endosperm nuclei that are larger and smaller than the ones observed in wild-type seeds, respectively. (O–R) Expression of paternally derived AGL62-GFP in the b2 (O and P) or b1 b2 seeds (Q and R). Pistils of the b1/+ b2/b2 plants were pollinated with pollen from the AGL62-GFP transgenic plant, and the GFP fluorescence (green) was observed at 1 DAP (O and Q) and 2 DAP (P and R). Chlorophyll autofluorescence appears as a magenta color. The arrow indicates a nuclear cytoplasmic domain without a GFP signal in the nucleus. (S and T) The DNA contents of embryo nuclei (blue bars) and peripheral endosperm nuclei (red bars) in the b2 (S, data from 2 seeds) and b1 b2 seeds (T, data from 3 seeds). The DNA contents of the nuclei were examined in optical sections of the seeds stained with propidium iodide. The DNA contents of at least 17 diploid sporophytic nuclei in the integument cells in the same seeds were used as references for the diploid DNA content. (U and V) Expression of a paternally derived BiP1::GUS transgene in the b2 (U) and b1 b2 (V) seeds at 3 DAP. (Scale bars, 20 μm.)

Fig. 4.

Size variation in the endosperm nuclei of the b1 b2 seeds without fertilization of the central cell. (A–D) Endosperm development after b1/+ b2/b2 pistils were pollinated with pollen of the CDC2A/cdc2a-1 plant. Four types of seeds were observed at 2 DAP. (A) Type A seed. (B) Type B seed. (C) Type C seed. (D) Type D seed. The number of each type of seed observed in two siliques is shown in each panel. (E) Wild-type seed produced by fertilization of a wild-type female gametophyte with CDC2A pollen at 2 DAP. (F) The b1 b2 seed produced by fertilization of a b1 b2 female gametophyte with wild-type pollen at 2 DAP. (G) The cdc2a seed produced by fertilization of a wild-type female gametophyte with cdc2a-1 pollen. (B, D and F Insets) Enlarged images of the framed areas. (Scale bars, 20 μm.)