Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

Abstract

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.

Keywords: CRP; EC1; double fertilization; gamete fusion; polytubey; sperm activation.

Figure 1. Seed abortion phenotype in ec1-RNAi lines. (A) Quantification of the unfused sperm cell phenotype. The number of wild type and ec1-RNAi ovules containing unfused sperm nuclei was quantified 30 to 40 h after pollinating emasculated pistils with the sperm marker line HTR10-mRFP1. (B) Immature siliques of selfed wild type and ec1-RNAi plants. Undeveloped degenerating ovules (arrows) were frequently detected in the siliques of ec1-RNAi lines. Wild type ovules were normally developed. Bar = 500 µm. (C) Quantification of seed set in developing siliques of the wild type and of 3 independent ec1-RNAi lines. Mean values ± SEM (standard error of the mean) are shown. n = number of siliques (with 40 to 50 ovules, each); PT, pollen tube; WT, wild type.

Figure 2. Delayed fertilization detected in ec1-RNAi ovules at 2 to 3 DAP. (A–F) DIC microscopy analyses of cleared developing ovules, prepared from 5 mm long siliques. Visible nuclei of the egg cell, zygote, or embryo are artificially colored in red; endosperm or central cell nuclei are artificially colored in blue. (A) Wild type ovule with 4-celled embryo and developing endosperm. (B) Ovule of a heterozygous ec1-RNAi plant showing normally developed embryo and endosperm. (C) Delayed development of fertilized ec1-RNAi ovule. The elongated zygote is visible, with its nucleus in the center. Four endosperm nuclei are visible in this focus plane. (D) Delayed development of fertilized ec1-RNAi ovule. The 2-celled proembryo and 6 endosperm nuclei are visible in this focus plane. (E) ec1-RNAi ovule with detectable central cell and egg cell nuclei. (F) Degenerating ec1-RNAi ovule with collapsed female gametophyte. (G) Quantification of the various phenotypes shown in A-F in the wild type and in 3 independent ec1-RNAi lines. Mean values ± SEM (standard error of the mean) are shown, n, number of siliques (with 40 to 50 ovules, each); 2-celled/2-c, 2-celled proembryo; ccn, central cell nucleus; ecn, egg cell nucleus; emb, embryo; end, endosperm; FG (deg), female gametophyte (degenerated); WT, wild type; zyg, zygote. Bars = 20 μm.

Figure 3. Unfused sperm cells are present both in unfertilized and in fertilized ec1-RNAi ovules. (A–I) CLSM images after Feulgen staining. (A) Pollen grains from mature anthers showing fluorescent signals of the 2 sperm cell nuclei (arrowheads) and the vegetative cell nucleus. (B) Wild type ovule 2 d after emasculation (DAE). Fluorescent signals of the 2 synergid nuclei, the egg cell, and the central cell nucleus are visible. (C) Wild type ovule 8 HAP. Sperm cells have been delivered and the sperm nuclei are detectable (arrowheads) near the gamete fusion site. (D) Fertilized wild type ovule, 30 HAP. The nuclei of the apical and the basal cell of the proembryo and 3 endosperm nuclei are detectable in this focus plane. (E) ec1-RNAi ovule, 2 DAE. Nuclei of synergid cells, egg, and central cell are detectable. (F) ec1-RNAi ovule, 30 HAP. Egg and central cell are unfertilized, 2 sperm cell nuclei (arrowheads) are detectable in each synergid cell. (G and H) Fertilized ec1-RNAi ovules, 30 HAP. The nuclei of the developing zygote and the endosperm are visible. Two (G) and 4 (H) unfused sperm cells (arrowheads) are detectable within the region of the synergid cells. (I) Pollen tube-targeted (arrowhead) ovule with collapsed female gametophyte. (J) Quantification of fertilization and sperm cell phenotypes in Feulgen-stained ovules 30 HAP (n = 102 ovules from 5 different lines); Ac, apical cell; bc, basal cell; central cell; ccn, central cell nucleus; ec, egg cell; ecn, egg cell nucleus; en, endosperm nucleus; PT, pollen tube; sy, synergid cell; syn, synergid cell nucleus; vn, vegetative cell nucleus; zyg, zygote. Bars = 20 µm.