Membrane proteins involved in flowering plant gamete interactions

Flowering plants have evolved a unique mode of sexual reproduction termed double fertilization, where two female gametes, the egg cell and the central cell, get fertilized by one sperm cell each. The fertilized egg cell gives rise to the zygote, whereas fertilization of the central cell results in formation of the nutrient-storing and embryo-nourishing endosperm. Successful seed formation requires both fertilization events, but molecular mechanisms regulating gamete recognition, attachment and fusion are poorly understood. Prior to this work, only three proteins residing or acting on plant gamete surfaces were known: The sperm membrane protein GAMETE EXPRESSED2 (GEX2) is involved in pre-fusion attachment whereas HAPLESS2 (HAP2) functions as membrane fusogen. Cysteine-rich EGG CELL1 (EC1) peptides are secreted by the egg cell and act as sperm activating molecules by inducing HAP2 re-localization from endomembrane compartments to the sperm surface. In order to identify novel sperm cell surface proteins with a role in gamete interactions, a protocol for bulk sperm cell isolation from maize was established in this work. Sperm cells were used for RNA-seq transcriptomic profiling and proteomics approaches. The protein composition of membrane-enriched sperm microsomal fractions was analyzed by high-throughput LC-MS/MS. Combined with publicly available Arabidopsis sperm transcriptome data, the maize sperm cell proteome and transcriptome data were used to select candidate genes, or gene families, for functional studies in Arabidopsis. Translational reporter activity in sperm cells was detected for 5 candidates. Assuming functional redundancies, CRISPR/Cas9 was established and genome edited mutants were screened for fertilization defects. Loss-of-function mutants of two sperm-expressed DOMAIN OF UNKNOWN FUNCTION 679 Membrane Proteins (DMP8 and DMP9) displayed severe reproductive defects. dmp8,9 double mutants showed impaired male fertility and up to four unfused dmp8,9 sperm cells were frequently observed close to the female gametes. Beside fertilization failure, also aborted seeds were observed in dmp8,9 siliques, caused by single fertilization events. Quantification revealed that dmp8,9 sperm cells preferentially fertilize the central cell. To address whether gamete adhesion or fusion is impaired in sperm cells lacking functional DMP8 and DMP9, in situ cell adhesion assays were performed and showed that dmp8,9 sperm cells frequently manage to attach to the egg cell, suggesting DMP8 and DMP9 act after gamete attachment but before or during fusion. DMPs are short four-span transmembrane proteins conserved in Viridiplantae (green plants). To gain insight about functionally important DMP9 regions and evolutionary conserved protein function, complementation of the dmp8,9 mutant was performed. 8 Deletion constructs expressing truncated DMP9 versions failed to rescue the mutant phenotype. Complementation with most ancient DMP from Chlamydomonas reinhardtii (CrDMP) was not successful, however a putative DMP9 ortholog from Amborella trichopoda (AmTrDMP) managed to partially rescue the fusion-defective dmp8,9 phenotype, suggesting functional conservation at least in flowering plants. The biochemical function of DMPs is not yet known, but Arabidopsis thaliana DMP1 was suggested to be involved in membrane remodeling. Transient overexpression of DMP9-GFP in tobacco leaf epidermis cells induced DMP1-like membrane remodeling events, which are likely artificial. Mutant dmp8,9 sperm cells were furthermore used as a tool to investigate sperm-induced events in the egg cell. Upon sperm cell arrival, the Arabidopsis egg cell secretes EC1 proteins to render the sperm cells competent for fusion but the precise moment of triggered EC1 secretion is not yet known. Quantification of EC1-GFP signals in unfertilized egg cells and in those with adjacent, unfused dmp8,9 sperm cells gave evidence that the secretion of EC1-GFP by the egg cell does not depend on DMP8/9 and is upstream of sperm adhesion. Furthermore, in mammals two plasma membrane-localized TETRASPANINS (TETs) are crucial for gamete interactions and accumulate at the egg-sperm contact site to form fusion competent membrane patches. To investigate whether a similar scenario holds true during flowering plant gamete interactions, the subcellular distribution of egg- and central cell-expressed Arabidopsis thaliana TET9-GFP was monitored during interaction with dmp8,9 sperm cells. A role for TET9 in the formation of fusion-competent sites seems unlikely, as TET9-GFP remained uniformly distributed at the egg- and central cell plasma membrane with attached but unfused dmp8,9 sperm cells. This suggests that TETs expressed in angiosperm gametes might fulfil different, yet unknown functions. In summary, two sperm-expressed membrane proteins were discovered that function after gamete attachment to facilitate gamete fusion. It will be an important task for the future to unravel the biochemical-mechanistic function of DMP8 and DMP9, which may involve membrane remodeling, supporting the activity of the fusogen HAP2, or the activation or delivery of HAP2 to the sperm cell surface. The maize sperm transcriptomic and proteomic datasets that have been generated in this work will serve as valuable resources for the identification of more membrane-localized proteins with a role during gamete interaction and fusion

Gamete fusion is facilitated by two sperm cell-expressed DUF679 membrane proteins

Successful double fertilization in flowering plants relies on two coordinated gamete fusion events, but the underlying molecular processes are not well understood. We show that two sperm-specific DOMAIN OF UNKNOWN FUNCTION 679 membrane proteins (DMP8 and DMP9) facilitate gamete fusion, with a greater effect on sperm-egg fusion than on sperm-central cell fusion. We also show that sperm adhesion and sperm cell separation depend on egg cell-secreted EGG CELL 1 proteins

Maternal ENODLs Are Required for Pollen Tube Reception in Arabidopsis

During the angiosperm (flowering-plant) life cycle, double fertilization represents the hallmark between diploid and haploid generations [1]. The success of double fertilization largely depends on compatible communication between the male gametophyte (pollen tube) and the maternal tissues of the flower, culminating in precise pollen tube guidance to the female gametophyte (embryo sac) and its rupture to release sperm cells. Several important factors involved in the pollen tube reception have been identified recently [2-6], but the underlying signaling pathways are far from being understood. Here, we report that a group of female-specific small proteins, early nodulin-like proteins (ENODLs, or ENs), are required for pollen tube reception. ENs are featured with a plastocyanin-like (PCNL) domain, an arabinogalactan (AG) glycomodule, and a predicted glycosylphosphatidylinositol (GPI) anchor motif. We show that ENs are asymmetrically distributed at the plasma membrane of the synergid cells and accumulate at the filiform apparatus, where arriving pollen tubes communicate with the embryo sac. EN14 strongly and specifically interacts with the extracellular domain of the receptor-like kinase FERONIA, localized at the synergid cell surface and known to critically control pollen tube reception [6]. Wild-type pollen tubes failed to arrest growth and to rupture after entering the ovules of quintuple loss-of-function EN mutants, indicating a central role of ENs in male-female communication and pollen tube reception. Moreover, overexpression of EN15 by the endogenous promoter caused disturbed pollen tube guidance and reduced fertility. These data suggest that female-derived GPI-anchored ENODLs play an essential role in male-female communication and fertilization.Natural Science Foundation of China [31230006, 31370344]; National Basic Research Program of China [2012CB944801]; German Research Council (DFG Collaborative Research Center) [SFB924]SCI(E)[email protected]

SUPPRESSOR OF FRIGIDA (SUF4) Supports Gamete Fusion via Regulating Arabidopsis EC1 Gene Expression

The EGG CELL1 (EC1) gene family of Arabidopsis (Arabidopsis thaliana) comprises five members that are specifically expressed in the egg cell and redundantly control gamete fusion during double fertilization. We investigated the activity of all five EC1 promoters in promoter-deletion studies and identified SUF4 (SUPPRESSOR OF FRIGIDA4), a C2H2 transcription factor, as a direct regulator of the EC1 gene expression. In particular, we demonstrated that SUF4 binds to all five Arabidopsis EC1 promoters, thus regulating their expression. The down-regulation of SUF4 in homozygous suf4-1 ovules results in reduced EC1 expression and delayed sperm fusion, which can be rescued by expressing SUF4-b-glucuronidase under the control of the SUF4 promoter. To identify more gene products able to regulate EC1 expression together with SUF4, we performed coexpression studies that led to the identification of MOM1 (MORPHEUS' MOLECULE1), a component of a silencing mechanism that is independent of DNA methylation marks. In mom1-3 ovules, both SUF4 and EC1 genes are down-regulated, and EC1 genes show higher levels of histone 3 lysine-9 acetylation, suggesting that MOM1 contributes to the regulation of SUF4 and EC1 gene expression