Arabidopsis GEX1 Is a Nuclear Membrane Protein of Gametes Required for Nuclear Fusion During Reproduction

Abstract

During the life cycle of flowering plants, nuclear fusion, or karyogamy, occurs three times: once during female gametogenesis, when the two polar nuclei fuse in the central cell, and twice during double fertilization. In Arabidopsis thaliana , nuclear fusion events during sexual reproduction proceed without the breakdown of the nuclear envelope, indicating that nuclear membrane fusion is essential for the completion of this process. Arabidopsis gamete expressed 1 (GEX1) is a membrane protein that is conserved among plant species. GEX1 shares homology with the yeast karyogamy protein Kar5, which is primarily expressed in the nuclear membrane. The GEX1 family represents a putative karyogamy factor. Herein, we show that GEX1 is required for the nuclear fusion events in Arabidopsis reproduction. GEX1-deficient mature female gametophytes were found to contain two unfused polar nuclei in close proximity within the central cell. Electron microscopy showed that the outer membrane of the polar nuclei was connected via the endoplasmic reticulum, whereas the inner membrane remained unfused. These results indicate that GEX1 is involved in polar nuclear membrane fusion following the fusion of the outer nuclear membrane. Furthermore, sperm nuclear fusion events were defective in the fertilized egg and central cell following plasmogamy in the fertilization of gex1-1 female gametophytes by gex1-1 pollen. An analysis of GEX1 localization in the female gametophyte using a transgenic line expressing GFP-tagged GEX1 driven by the GEX1 promoter showed that GEX1 is a nuclear membrane protein in the egg and central cell. Time-lapse live-cell imaging showed that in developing female gametophytes, the nuclear GFP-GEX1 signal was first detectable in the central cell shortly before the polar nuclei came in close contact, and then in the egg cell. Thus, we suggest that the GEX1-family proteins are nuclear membrane proteins involved in karyogamy in the reproduction of eukaryotes including flowering plants.

The Physcomitrium patens egg cell expresses several distinct epigenetic components and utilizes homologues of BONOBO genes for cell specification

“…The GO term ‘karyogamy’ came up in cluster 3, mainly consisting of genes highly expressed in both egg and antheridia (Table S12). This includes GAMETE EXPRESSED1 ( GEX1 ), encoding a membrane protein homologous to the yeast karyogamy protein Kar5 (Beh et al ., 1997; Alandete‐Saez et al ., 2011; Ning et al ., 2013; Speijer et al ., 2015; Nishikawa et al ., 2020). GEX1 is localized to the nuclear membrane of the egg and central cell, and, upon double fertilization, it is required for sperm nuclear fusion in both cells.…”

The Molecular Life of Diatoms

“…Localization and knock-out studies will be necessary to further elucidate their role during gametangia recognition and gamete fusion. Successful gamete fusion in most eukaryotic lineages is mediated by the plasma membrane-associated Hap2 fusogen protein (Wong and Johnson 2010) while the Gex1/Kar5/Brambleberry nuclear membrane protein is involved in nuclear fusion (karyogamy) (Nishikawa et al 2020). Strikingly, homologs for both genes are missing from the gene repertoire of sexual diatoms P. multistriata and S. marinoi (Ferrante et al 2019), suggesting the presence of alternative fusogens or of highly diverged Gex1 and Hap2 genes.…”

Expression of GEX1 Orthologs of Brassica rapa and Oryza sativa Rescued the Nuclear Fusion Defect of the Arabidopsis GEX1 Mutant

“…We also showed that Arabidopsis gamete-expressed 1 (AtGEX1) is a nuclear membrane protein necessary for nuclear fusion during Arabidopsis reproduction [ 8 ]. The AtGEX1 gene was identified by screening genes that are expressed in the sperm cells [ 9 ].…”

“…AtGEX1 is also involved in sperm nuclear fusion in the fertilized egg and central cells. The resulting gex1-1 homozygous mutant seeds showed aberrant endosperm proliferation and defects in early embryo development, which resulted in seed abortion [ 8 , 13 ].…”

“…Orthologs of AtGEX1 have been identified in various land plants [ 8 ]. GEX1 family proteins are part of Kar5/GEX1/Brambleberry family, which includes budding yeast Kar5 and zebrafish Brambleberry proteins [ 14 ].…”