EGG CELL 1 function and stability during double fertilization in Arabidopsis thaliana

During double fertilization of angiosperms, the two sperm cells are transported conjointly to the female gametophyte, where one sperm cell fuses with the egg cell and the other sperm cell with the central cell. These fusions have to take place in a controlled manner to avoid undesired gamete fusion events and prevent polyspermy. Only little is known about gamete recognition and coordination of gamete fusion in plants. The aim of this work was to characterize the function of the Arabidopsis egg cell-expressed EGG CELL 1 (EC1) gene family during gamete recognition and to identify putative interaction partners. The EC1 gene family comprises five members that encode cysteine-rich proteins, which are secreted from the egg cell during fertilization. Triple knockout mutants were additionally transformed with an RNAi construct targeting the remaining two genes. In these plants (ec1+/-) the fusion of the sperm cells with the female gametes was impaired resulting in a reduced seed set. Detailed analyses of ec1+/- plants showed that in 45% of the ovules of ec1+/- plants sperm cells did not fuse with the female gametes and that non-fused sperm cells were always observed as pairs, which indicated that EC1 might function in sperm cell separation. This hypothesis was supported by the observation that single sperm cells of mutant pollen seemed to be able to fuse in ec1 ovules. With the aim to identify interaction partners of EC1, a pollen cDNA library was screened using a yeast-two-hybrid approach. Two putative interactors were found: (i) a protein containing two ubiqutin-like (UBL) domains (UbDKγ3), which is probably involved in substrate delivery to the 26S proteasome and (ii) a regulatory subunit of the Phosphatase 2A (PP2A B’θ). The putative interaction with a PP2A subunit and predicted phosphorylation sites at the C-terminus of EC1 indicated that phosphorylation might play a role in EC1. The transient expression of a phospho-mimicking variant of EC1 fused to eGFP in N. benthamiana leaves was more stable, i.e. showed fluorescence, compared to the wild type form of EC1. Moreover, a proteasome inhibitor experiment with plants expressing EC1.1 fused to eGFP under control of the 35S promoter suggested that misexpressed EC1 is rapidly degraded via the ubiquitin-proteasome pathway. Based on these findings, it was hypothesized that the pollen tube delivers the regulatory subunit of PP2A, which triggers dephosphorylation of the secreted EC1 and thereby marks it for degradation. This was supported by the observation that misexpressed PP2A B’θ in the synergid cell partially phenocopied the ec1 phenotype. This work shows that EC1 is essential during double fertilization probably for gamete recognition or sperm cell separation. After fertilization and in all other cells, EC1 is unstable, its degradation is highly regulated and any protein accumulation is avoided

Egg cell-secreted EC1 triggers sperm cell activation during double fertilization

Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple-sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions

A General G1/S-Phase Cell-Cycle Control Module in the Flowering Plant <em>Arabidopsis thaliana</em>

<div><p>The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant <em>Arabidopsis thaliana</em>, which, as a member of the <em>Plantae</em> supergroup, is phylogenetically only distantly related to <em>Opisthokonts</em> such as yeast and animals. This module comprises the <em>Arabidopsis</em> homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb)-related 1 (RBR1), the plant-specific F-box protein F-BOX-LIKE 17 (FBL17), the plant specific cyclin-dependent kinase (CDK) inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2⁺/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network.</p> </div

Pollen phenotypes of mutants for components of the G1/S phase control module.

<p>(A) Tricellular mature wild-type DAPI-stained pollen at anthesis (one vegetative cell enclosing two sperm cells). (B) DAPI-stained pollen at anthesis from heterozygous <i>cdka;1</i> mutant plants (similar to pollen from heterozygous <i>fbl17</i> mutants, data not shown) containing approximately 43% bicellular pollen (one vegetative cell and one sperm-cell-like cell) and 57% tricellular, wild-type-like pollen. (C) DAPI-stained pollen at anthesis from double heterozygous <i>cdka;1 fbl17</i> mutant plants carrying a hemizygous <i>Pro_CDKA;1:CDKA;1:YFP</i> rescue construct (similar to pollen from <i>e2fa^−/− fbl17^+/−</i> mutants, data not shown) and containing single-celled pollen grains (only one vegetative-like cell), in addition to bicellular (<i>cdka;1</i>/<i>fbl17</i>-like) and tricellular (wild-type-like) pollen. (D) Close-up of bicellular pollen as found in <i>cdka;1</i> or <i>fbl17</i> heterozygous plants. (E) Close-up of monocellular pollen grains as found in <i>cdka;1 fbl17</i> or <i>e2fa fbl17</i> double heterozygous mutants. (F) Quantification of DAPI-stained pollen. The DNA content of the single-celled pollen from <i>cdka;1^+/− fbl17^+/−</i> or <i>e2fa^−/− fbl17^+/−</i> double mutants reaches 1C, similarly to the vegetative nucleus in wild-type pollen and, thus, resides in a G1 phase.</p

General G1/S phase cell-cycle control module.

<p>(A) The transcription factor E2F activates the expression of <i>FBL17</i>, which is repressed by RBR1. FBL17 targets the CDKA;1 inhibitors KRP1, KRP3, KRP4, KRP6 and KRP7 for proteasome-dependent degradation, enabling the germ cell to progress through S phase. Phosphorylation of RBR by the CDKA;1-cyclin complex will relieve the inhibition of the S-phase genes and allows transcription of the <i>FBL17</i> gene. Promoters and genes are depicted in light sand color; proteins in dark sand; transcription is indicated by a grey arrow; negative regulation, i.e. at the transcriptional or protein level, is shown by rust-colored lines with a blunt end; positive regulation by a green line with a green arrowhead. Receiving input is placed above and executing output under the respective gene/protein. (B) The model presented in A gives rise to a bistable switch controlling the G1-to-S transition in the plant cell cycle. KRPs inhibit the CDKA;1-cyclin complexes, which in turn downregulate the levels of KRPs by phosphorylating and inhibiting RBR1, thereby activating E2F-dependent FBL17 synthesis leading to the degradation of KRPs. The antagonistic interaction between CDKA;1 and KRP is illustrated by the two curves (red and green for KRP and for CDKA;1, respectively) along which the rates of synthesis and degradation of KRPs and the CDKA;1-cyclin complexes are exactly balanced. The KRP balance curve has an inverse S-shape with high and low levels, depending on the CDKA;1-cyclin values. The dashed branch of the balance curve represents unstable steady states. At low CDKA;1-cyclin levels, only one steady state exists with high KRP levels and low CDKA;1 activity. At intermediate CDKA;1 levels, the system is bistable with three steady states. At high CDKA;1-cyclin values, the steady-state level of KRP is low and the CDKA;1-cyclin complexes are fully active. The transition from high to low KRP values corresponds to the G1-to-S transition. (C) Quantitative expression analyses of <i>CDKA;1</i> and <i>FBL17</i> in wild type and heterozygous <i>cdka;1</i> mutants. The mean plus standard deviation of the normalized relative quantities (NRQ) of three biological replicates are shown. The stars indicate statistically significant differences based on a t-test of log-transformed data with a p<0.05. As expected, the expression of <i>CDKA;1</i> drops by approximately 50% in the mutant. In addition, <i>FBL17</i> expression declines, consistent with a prediction of the model presented in A and B.</p

Interaction assays of dominant negative versus wild-type CDKA;1 variants.

<p>Positive BiFC assays showing the interaction of CDKA;1 with KRP1 under bright field (A) and epifluorescence (B). (C) The dominant-negative CDKA;1 variant CDKA;1^D146N interacting with both positive (cyclins) and negative (KRPs) regulators similar to the wild-type CDKA;1 form. The CDKA;1^PSTAIRE-dead variant shows neither interaction with cyclins nor with KRPs, but can still bind to the cofactor CKS. The interactions are presented in a semiquantitative manner based on the number of positive cells obtained and the observed fluorescent intensities. All interactions were at least three times independently tested.</p

Mature ovules and seed development in wild type and <i>cdka;1 fbl17</i> double mutant.

<p>(A–C) Wild-type embryo sac and seed development; (D–F) aberrant development in a class of <i>cdka;1 fbl17</i> mutant plants. (A) Wild type showing a typical cellular morphology (arrowheads pointing to the central cell nucleus and the egg cell nucleus from top to bottom, respectively). Aberrant morphologies in the <i>cdka;1^+/− fbl17^+/−</i> double mutant with one (D), or two (G) nuclei in the absence of a cellularized egg apparatus. (B) While the wild-type seed 3 days after pollination has a normal embryo and endosperm development, the double mutant seed development collapsed after pollination, with only one (E) or two (H) nuclei staying in the middle of the empty embryo sac. (C) Wild type showing normal seed development 6 days after pollination, while <i>cdka;1^+/− fbl17^+/−</i> double mutants show approximately 24% seed abortion (F) after pollination with the wild-type pollen.</p

Pollen phenotypes.

<p>Pollen from anthers just before flowering of wild type and the indicated genotypes was stained with DAPI and epifluorescence was observed under UV illumination. n = total number of pollen analyzed.</p